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Intestinal barrier leakage constitutes a potential therapeutic target for many inflammatory diseases and represents a disease progression marker during chronic viral infections. However, the causes of altered gut barrier remain mostly unknown. Using murine infection with lymphocytic choriomeningitis virus, we demonstrate that, in contrast to an acute viral strain, a persistent viral isolate leads to long-term viral replication in hematopoietic and mesenchymal cells, but not epithelial cells (IECs), in the intestine. Viral persistence drove sustained intestinal epithelial barrier leakage, which was characterized by increased paracellular flux of small molecules and was associated with enhanced colitis susceptibility. Type I IFN signaling caused tight junction dysregulation in IECs, promoted gut microbiome shifts and enhanced intestinal CD8 T cell responses. Notably, both type I IFN receptor blockade and CD8 T cell depletion prevented infection-induced barrier leakage. Our study demonstrates that infection with a virus that persistently replicates in the intestinal mucosa increases epithelial barrier permeability and reveals type I IFNs and CD8 T cells as causative factors of intestinal leakage during chronic infections., Competing Interests: Disclosures: R. Knight reported, "Biota Technology (salary/stock), Commence (consulting fee), Prometheus (consulting fee), CoreBiome (stock), GenCirq (stock), DayTwo (consulting fee), Cybele Microbiome (stock), BiomeSense (consulting fee), and IBM (grant)." No other disclosures were reported., (© 2020 Labarta-Bajo et al.)

Infections elicit immune adaptations to enable pathogen resistance and/or tolerance and are associated with compositional shifts of the intestinal microbiome. However, a comprehensive understanding of how infections with pathogens that exhibit distinct capability to spread and/or persist differentially change the microbiome, the underlying mechanisms, and the relative contribution of individual commensal species to immune cell adaptations is still lacking. Here, we discovered that mouse infection with a fast-spreading and persistent (but not a slow-spreading acute) isolate of lymphocytic choriomeningitis virus induced large-scale microbiome shifts characterized by increased Verrucomicrobia and reduced Firmicute/Bacteroidetes ratio. Remarkably, the most profound microbiome changes occurred transiently after infection with the fast-spreading persistent isolate, were uncoupled from sustained viral loads, and were instead largely caused by CD8 T cell responses and/or CD8 T cell-induced anorexia. Among the taxa enriched by infection with the fast-spreading virus, Akkermansia muciniphila , broadly regarded as a beneficial commensal, bloomed upon starvation and in a CD8 T cell-dependent manner. Strikingly, oral administration of A. muciniphila suppressed selected effector features of CD8 T cells in the context of both infections. Our findings define unique microbiome differences after chronic versus acute viral infections and identify CD8 T cell responses and downstream anorexia as driver mechanisms of microbial dysbiosis after infection with a fast-spreading virus. Our data also highlight potential context-dependent effects of probiotics and suggest a model in which changes in host behavior and downstream microbiome dysbiosis may constitute a previously unrecognized negative feedback loop that contributes to CD8 T cell adaptations after infections with fast-spreading and/or persistent pathogens., Competing Interests: The authors declare no competing interest.

Appropriate immune responses require a fine balance between immune activation and attenuation. NLRC3, a non-inflammasome-forming member of the NLR innate immune receptor family, attenuates inflammation in myeloid cells and proliferation in epithelial cells. T lymphocytes express the highest amounts of Nlrc3 transcript where its physiologic relevance is unknown. We show that NLRC3 attenuated interferon-γ and TNF expression by CD4 + T cells and reduced T helper 1 (Th1) and Th17 cell proliferation. Nlrc3 -/- mice exhibited increased and prolonged CD4 + T cell responses to lymphocytic choriomeningitis virus infection and worsened experimental autoimmune encephalomyelitis (EAE). These functions of NLRC3 were executed in a T-cell-intrinsic fashion: NLRC3 reduced K63-linked ubiquitination of TNF-receptor-associated factor 6 (TRAF6) to limit NF-κB activation, lowered phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and diminished glycolysis and oxidative phosphorylation. This study reveals an unappreciated role for NLRC3 in attenuating CD4 + T cell signaling and metabolism., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We retrospectively reviewed microbiological data from a tertiary care hospital in Botswana, and found that Cryptococcus neoformans was cultured from 15% (193/1307) of all cerebrospinal fluid (CSF) specimens submitted for analysis, making it the most common diagnosed cause of meningitis in this population. Moreover, almost 70% of CSF samples with significant lymphocytosis did not yield a pathogen, suggesting that many causes of lymphocytic meningitis go undiagnosed.

Memory CD8 T cells play a critical role in protective immunity against intracellular pathogens. In addition to their ability to specifically recognize and lyse infected targets, activated CD8 T cells secrete cytokines that induce phagocytic cells to engulf and kill bacterial pathogens. In this study, we asked whether activation of Ag-specific CD8 T cells results in nonspecific killing of bystander bacteria during a mixed infection. Mice with epitope-specific memory CD8 T cells were coinfected with two isogenic strains of recombinant Listeria monocytogenes that differ in the cognate epitope. Recall responses by epitope-specific CD8 T cells rapidly inhibited the growth of epitope-bearing bacteria, impeding the course of infection within 6 h after challenge. This rapid inhibition was highly specific and did not affect the growth of coinfecting bacteria without the epitope. CTL recall did not enhance activation of innate immune cells, as evidenced by the absence of inducible NO synthase production in infectious foci. Our observations demonstrate the remarkable specificity of the bactericidal mechanisms of CTL and reveal the possibility for escape mutants to prevail in the hostile environment of a specific immune response. This implication has a bearing on subunit vaccine design strategies and understanding failure of immunization against bacterial infection.

Naive Ag-specific CD8(+) T cells expand, contract, and become memory cells after infection and/or vaccination. Memory CD8(+) T cells provide faster, more effective secondary responses against repeated exposure to the same pathogen. Using an adoptive transfer system with low numbers of trackable nontransgenic memory CD8(+) T cells, we showed that secondary responses can be comprised of both primary (naive) and secondary (memory) CD8(+) T cells after bacterial (Listeria monocytogenes) and/or viral (lymphocytic choriomeningitis virus) infections. The level of memory CD8(+) T cells present at the time of infection inversely correlated with the magnitude of primary CD8(+) T cell responses against the same epitope but directly correlated with the level of protection against infection. However, similar numbers of Ag-specific CD8(+) T cells were found 8 days postinfection no matter how many memory cells were present at the time of infection. Rapid contraction of primary CD8(+) T cell responses was not influenced by the presence of memory CD8(+) T cells. However, contraction of secondary CD8(+) T cell responses was markedly prolonged compared with primary responses in the same host mice. This situation occurred in response to lymphocytic choriomeningitis virus or L. monocytogenes infection and for CD8(+) T cell responses against multiple epitopes. The delayed contraction of secondary CD8(+) T cells was also observed after immunization with peptide-coated dendritic cells. Together, the results show that the level of memory CD8(+) T cells influences protective immunity and activation of naive precursors specific for the same epitope but has little impact on the magnitude or program of the CD8(+) T cell response.

LPS is the major active agent in the pathogenesis of Gram-negative septic shock. In this report we have studied the influence of concurrent viral infection on the outcome of LPS-induced shock. We find that infection with vesicular stomatitis virus sensitizes mice to LPS at an early time point following infection. Treatment of mice with the chemical IFN inducer, polyinosinic:polycytidylic acid, has a similar effect. This hypersensitivity to LPS correlated with hyperproduction of TNF-alpha in vivo. The cellular and molecular mechanisms underlying this phenomenon were investigated using Ab-depleted and gene-targeted mice. Our results revealed that while NK cell depletion and elimination of IFN-gamma partially protected against the sensitizing effects of vesicular stomatitis virus and polyinosinic:polycytidylic acid, the most striking effect was observed in IFN-alphabetaR-deficient mice. Thus hyperproduction of TNF-alpha was completely abrogated in IFN-alphabetaR-deficient mice, indicating that the principal mechanism underlying rapid virus-induced sensitization to LPS is an IFN-alphabeta-mediated priming of mice for an augmented production of TNF-alpha in response to LPS. This conclusion was further supported by the finding that pretreatment of mice with rIFN-alphabeta mimicked the effect of viral infection. In conclusion, our results reveal a previously unrecognized proinflammatory effect of IFN-alphabeta and point to a new pathway through which viral infection may influence the outcome of concurrent bacterial infection.

To elucidate the possible involvement of the inducible nitric oxide synthase (iNOS) and NO in the development of lymphocytic choriomeningitis (LCM), the consequences of inhibition of iNOS by the inhibitor aminoguanidine was examined in mice following intracerebral infection with LCM virus (LCMV). Aminoguanidine administration to mice infected with LCMV completely blocked increased plasma nitrate/nitrite levels and led to increased proinflammatory cytokine gene expression at early stages of lesion development in the brain, enhanced clinical severity and decreased survival time. The levels of LCMV recovered from the brain of aminoguanidine treated mice did not differ from those in infected control mice. These findings argue against either an anti-viral or pathogenic role of NO in LCM but rather suggest a possible protective action of this mediator.

IFN-alpha/beta have been shown to play a central role in the development of lymphocytic choriomeningitis and increasing attention has been focused on this group of cytokines as early regulatory factors directing T lymphocyte responses. In the present study, injection of antiserum to IFN-alpha/beta prevented the development of lymphocytic choriomeningitis, was associated with the absence of detectable expression of early 2'-5' oligo-adenylate synthetase mRNA and coincided with viremia of lymphocytic choriomeningitis virus (LCMV) followed by establishment of a persistent infection. The LCMV-specific cytotoxic T lymphocyte response was unchanged in cervical lymph nodes but decreased in the spleen of anti-IFN-alpha/beta-treated animals. The expression of cytokine mRNA (particularly IFN-gamma) in organs of LCMV-infected mice treated with anti-IFN-alpha/beta coincided with infiltration of lymphocytes and tissue destruction. Furthermore, a reduced number of infiltrating leukocytes in the brain and cervical lymph nodes and a low expression of cytokine mRNA in the brain was observed in anti-IFN-alpha/beta-treated animals. In total, the findings support the view that neutralization of IFN-alpha/beta leads to extensive LCMV replication in the viscera. The therapeutic effects of anti-IFN-alpha/beta antiserum seem to be independent of the functional capacity of T cells but probably result in a dispersion of activated T cells throughout the body of LCMV-infected mice. Absence of IFN-alpha/beta expression in the central nervous system is proposed as the mechanism behind the IFN-alpha/beta-dependent targeting of T cells to the brain.

The path that the arenavirus lymphocytic choriomeningitis virus (LCMV) uses to enter rodent fibroblastic cell lines was dissected by infectivity and inhibition studies and immunoelectron microscopy. Lysosomotropic weak bases (chloroquine and ammonium chloride) and carboxylic ionophores (monensin and nigericin) inhibited virus entry, assessed as virus nucleoprotein expression at early times post-infection, indicating that the entry process involved a pH-dependent fusion step in intracellular vesicles. That entry occurred in vesicles rather than by direct fusion of virions with the plasma membrane was confirmed by immunoelectron microscopy. The vesicles involved were large (150-300 nm diameter), smooth-walled, and not associated with clathrin. Unlike classical phagocytosis, virus uptake in these vesicles was a microfilament-independent process, as it was not blocked by cytochalasins. LCMV entry into rodent fibroblast cell lines thus involves viropexis in large smooth-walled vesicles, followed by a pH-dependent fusion event inside the cell.

This study documents that the immunosuppressive lymphocytic choriomeningitis virus (LCMV) variant, clone 13, shows a specific predilection for enhanced infection of macrophages both in vitro and in vivo and that single amino acid changes in the viral polymerase and glycoprotein are responsible for macrophage tropism. The growth difference seen between variant clone 13 and the parental Armstrong strain was specific for macrophages, since both clone 13 and Armstrong grew equally well in fibroblasts and neither isolate infected lymphocytes efficiently. Complete sequencing of the clone 13 genome, along with genetic analysis, showed that a single amino acid change in the polymerase (K-->Q at position 1079) was the major determinant of virus yield in macrophages. This was proven unequivocally by comparing the sequences of parental and reassortant viruses, which were identical at all loci except for the single mutation in the polymerase gene. This finding was further strengthened by showing that reversion at this site back to lysine (Q-->K) resulted in loss of macrophage tropism. In addition, an independently derived macrophage-tropic variant of LCMV, clone 28b, had a K-->N mutation at the same position. Thus, these results show that substitution of the positively charged amino acid K with a neutral amino acid (either Q or N) at residue 1079 of the polymerase resulted in enhanced viral replication in macrophages. In addition to the polymerase change, a mutation in the glycoprotein was also associated with macrophage tropism. This single amino acid change in the glycoprotein (F-->L at position 260) did not affect virus yield per macrophage but was critical in determining the number of macrophages infected. Our previous studies have shown that the same two mutations in the polymerase and glycoprotein are essential for establishing a chronic infection in adult mice. Since the same mutations confer macrophage tropism and ability to persist in vivo, these studies provide compelling evidence that infection of macrophages is a critical determinant of viral persistence and immune suppression.

Viruses that are non- or poorly cytopathic have developed various strategies to avoid elimination by the immune system and to persist in the host. Acute infection of adult mice with the noncytopathic lymphocytic choriomeningitis virus (LCMV) normally induces a protective cytotoxic T-cell response that also causes immunopathology. But some LCMV strains (such as DOCILE (LCMV-D) or Cl-13 Armstrong (Cl-13)) derived from virus carrier mice tend to persist after acute infection of adult mice without causing lethal immunopathological disease. Tendency to persist correlates with tropism, rapidity of virus spread and virus mutations. We report here that these LCMV isolates may persist because they induce most of the specific antiviral CD8+ cytotoxic T cells so completely that they all disappear within a few days and therefore neither eliminate the virus nor cause lethal immunopathology. The results illustrate that partially and sequentially induced (protective) immunity or complete exhaustion of T-cell immunity (high zone tolerance) are quantitatively different points on the scale of immunity; some viruses exploit the latter possibility to persist in an immunocompetent host.

Viral persistence depends on a virus having a non-lytic strategy of replication and the ability to escape immune surveillance. Cells of the immune system (lymphocytes/monocytes/macrophages) and central nervous system (neurons) are most often infected by DNA and RNA viruses that persist. Cytotoxic T lymphocytes (CTL) are the primary host defense that aborts or prevents viral persistence. Viral interaction with these specialized cells and of such infected cells with CTL is explored in this paper.

Objective: After an employee at a cancer research institute was diagnosed with lymphocytic choriomeningitis, an investigation was performed to determine the extent of lymphocytic choriomeningitis virus (LCMV) infections among the institute's employees and to identify risk factors for infection., Design: Retrospective cohort study., Setting: A US cancer research institute., Participants: Eighty-two of 90 institute employees., Main Outcome Measures: Serum LCMV antibodies., Results: Seven workers (9%) with definite LCMV infection (LCMV IgG antibody titer greater than or equal to 16) and one worker (1%) with probable infection (IgG titer = 8) were identified (10% overall seroprevalence). All infected employees handled animals or animal tissues and were more likely than other animal handlers to have worked with nude mice (Mus musculus) (P less than .02). Among the 31 employees who worked with nude mice at the institute, infected workers were more likely to clean the cages of nude mice (P much less than .001), change their bedding (P less than .01), and change their water (P less than .001). The institute had been injecting nude mice with LCMV-infected tumor cell lines and had recently increased the nude mouse population and the duration of experiments. These changes would have increased the LCMV burden at the facility and were temporally associated with the cluster of LCMV infections in employees., Conclusions: This LCMV outbreak, the first reported since 1974, is the first associated with nude mice. It illustrates the ongoing hazard LCMV poses in research laboratories. Since the symptoms of LCMV infection can be nonspecific, clinicians should consider this diagnosis in ill patients who report laboratory rodent exposure.

In (B10.BR----B10) chimeras infected with lymphocytic choriomeningitis (LCM) virus higher titers were attained in spleens and livers than in organs of the mice used for their construction, and the subsequent elimination was retarded, but eventually the virus was cleared. The numbers of LCM virus-specific CTL and their precursors as quantitated with chromium-release assay and limiting dilution method, respectively, were lower in chimeras than in B10.BR or C57BL/10J mice, and fewer were restricted for the haplotypes of the donors than of the recipients. The same was true with regard to antiviral effector cells, which were determined by adoptive immunization. The numbers of spleen cells releasing IgM and IgG antiviral antibodies were virtually as high in chimeras as they were in C57BL/10J and B10.BR mice. Transfer of immune splenocytes from either B10.BR or C57BL/10J mice resulted in incomplete virus elimination from the spleens of infected chimeras, whereas injection of a mixture of the two types of immune cells led to efficient clearance. We conclude that in the chimeras cells of both donor and recipient haplotypes participate in the infection, which is terminated by H-2k- and H-2b-restricted T lymphocytes that these animals are capable of generating. We conclude, furthermore, that clearance of the LCM virus from the tissues requires contact between effector and target cells.

Lymphocytic choriomeningitis virus (LCMV) was isolated from 14 out of 35 wild house mouse samples captured on two piers of Osaka port in Japan. Four of them were isolated from were isolated from 18 antibody positive mice while 10 were from 17 antibody negative mice. This is the first report of the isolation of LCMV from wild mice in Japan.

Viral variants of different phenotypes are present in the central nervous system (CNS) and lymphoid tissues of carrier mice infected at birth with the Armstrong strain of lymphocytic choriomeningitis virus. The CNS isolates are similar to the parental virus and cause acute infections in adult mice, whereas the lymphoid isolates cause chronic infections associated with suppressed T-cell responses. In this study, we provide a molecular basis for this organ-specific selection and identify a single amino acid change in the viral glycoprotein that correlates with the tissue specific selection and the persistent and immunosuppressive phenotype of the variants. This phenylalanine (F)-to-leucine (L) change at position 260 of the viral glycoprotein was seen in the vast majority (43 of 47) of the lymphoid isolates, and variants with L at this residue were selected in spleens of persistently infected mice. In striking contrast, isolates with the parental sequence (F at residue 260) predominated (48 of 59 isolates) in the CNS of the same carrier mice. Complete nucleotide sequence analysis of the major structural genes of several independently derived (from different mice) spleen isolates showed that these variants were greater than 99.8% identical to the parental virus. In fact, the only common change among these spleen isolates was the F----L mutation at residue 260 of the glycoprotein. These results show that an RNA virus can exhibit minimal genetic drift during chronic infection in its natural host, and yet a single or few mutations can result in the organ-specific selection of variants that are markedly different from the parental virus.

By the application of in situ hybridization to thin sections of paraffin-embedded tissues we have been able to determine with high resolution the cell types containing lymphocytic choriomeningitis virus nucleic acid in the tissues of persistently infected mice. We confirm and extend previous observations of virus persistence in the brain, lung, liver, kidney, pancreas, thyroid and reticuloendothelial system. In addition, we demonstrate for the first time persistence of viral nucleic acid in specific cell types in the thymus, lymph nodes, testes and bladder, and the adrenal, parathyroid and salivary glands; the cell types infected were observed in several animals. In lymphoid tissue, viral nucleic acid was predominantly located in the T cell-dependent areas of the spleen and lymph nodes; it was also present in cells of the thymic medulla. This has important implications for the deficiency in T cell function observed in persistently infected mice. In the testes, viral nucleic acid was detected in spermatogonia but not differentiating spermatocytes and therefore, in this tissue at least, persistence is related to the state of differentiation of the cell. Endocrine and exocrine dysfunctions have been described in persistently infected mice and we report that the highest levels of viral nucleic acid were found in the adrenal gland. The infection of endocrine and exocrine tissue was not pantropic, specific cell types expressed viral nucleic acid in each tissue. In the adrenal cortex, cells of the zona reticularis and zona fasciculata but not the zona glomerulosa were positive, whereas in the adrenal medulla viral nucleic acid was predominantly localized to adrenalin-secreting cells. Infection of the renal tubules, transitional epithelium of the bladder and the ducts of the salivary gland indicates the likely sites of virus production for the dissemination of arenavirus infections.

Nonherpetic encephalitis outside the newborn period is usually a self-limited disease. The majority of patients will recover without significant sequelae, and require only supportive therapy during the acute illness. Though the underlying viral etiology frequently will escape detection, identification of the infecting agent has considerable prognostic value which can complement clinical measures of severity of disease. The most important initial task of the clinician faced with a case of presumptive viral encephalitis is to eliminate the possibility of a treatable illness. Once this has been done, the diagnosis of viral encephalitis can be supported by documenting the characteristic slow-wave background activity on EEG, and a mild lymphocellular pleocytosis in the CSF. Because viral encephalitis can be caused by such a large number of organisms, the search for an etiology can be daunting. Realizing that all the agents described above can, at times, cause encephalitis without any clue to their identity, one nevertheless may use several pieces of historical information to narrow the possibilities. Travel history, animal exposures, immunization history, and seasonality all may help to steer the search in a particular direction and, indeed, may point to a nonvirologic cause as well. In addition, detection of extraneurological signs and symptoms may strongly indicate a specific virologic diagnosis. Finally, knowledge of concurrent community epidemic patterns, and of surveillance data routinely collected by local and state health departments, can help to increase or decrease the likelihood of a given pathogen. The causative viral agent usually can be identified by serological testing and viral culture. Occasionally, single serological determinations are diagnostic: in rabies (when the patient has not received immune prophylaxis), eastern equine encephalitis, and HIV, since seropositivity is strongly associated with symptomatic illness; and in Epstein-Barr virus, if a panel of antibody determinations which can time the infection is available. In addition, high CSF: serum titers for antibody against any neurotropic agent is usually diagnostic, though the absence of a high central nervous system antibody titer does not eliminate any potential viral pathogen. With these few exceptions, a single serological determination for a given pathogen is almost always impossible to interpret; paired sera (one obtained upon diagnosis, and one obtained 10 to 14 days later, either just prior to hospital discharge or at a follow-up visit) are far more helpful. Many viruses that directly infect the central nervous system are difficult to recover from the CSF; therefore, viral isolation from the nasopharynx and stool also should be sought.(ABSTRACT TRUNCATED AT 400 WORDS)

This study demonstrates cell-specific selection of viral variants during persistent lymphocytic choriomeningitis virus infection in its natural host. We have analyzed viral isolates obtained from CD4+ T cells and macrophages of congenitally infected carrier mice and found that three types of variants are present in individual carrier mice: (i) macrophage-tropic, (ii) lymphotropic, and (iii) amphotropic. The majority of the isolates were amphotropic and exhibited enhanced growth in both lymphocytes and macrophages. However, some of the lymphocyte-derived isolates grew well in lymphocytes but poorly in macrophages, and a macrophage-derived isolate replicated well in macrophages but not in lymphocytes. In striking contrast, the original wild-type (wt) Armstrong strain of lymphocytic choriomeningitis virus that was used to initiate the chronic infection and from which the variants are derived grew poorly in both lymphocytes and macrophages. These three types of variants also differed from the parental virus in their ability to establish a chronic infection in immunocompetent hosts. Adult mice infected with the wt Armstrong strain cleared the infection within 2 weeks, whereas adult mice infected with the variants harbored virus for several months. These results suggest that the ability of the variants to persist in adult mice is due to enhanced replication in macrophages and/or lymphocytes. This conclusion is further strengthened by the finding that the variants and the parental wt virus grew equally well in mouse fibroblasts and that the observed growth differences were specific for cells of the immune system.

Viruses persist in an immune population, as in the case of influenza, or in an individual, as postulated for human immunodeficiency virus, when they are able to escape existent neutralizing antibody responses by changing their antigens. It is now shown that viruses can in principle escape the immunosurveillance of virus-specific cytotoxic T cells by mutations that alter the relevant T-cell epitope.

In order to study the lesions produced by lymphocytic choriomeningitis virus (LCMV) adult guinea-pigs were inoculated intraperitoneally with 1000 PFU of the WE or the Armstrong strain of LCMV. The animals were sacrificed at 3, 7, 10 and 14 days p.i. and samples were taken for hematological, histopathological and virological studies. In the guinea-pigs infected with the WE strain there was destruction of the splenic red pulp with high titers of virus and different degrees of pneumonitis. The hematological studies showed lymphopenia from day 7 p.i. onwards and focal bone marrow necrosis. In the animals infected with the Armstrong strain there was a very moderate neutropenia and virus multiplication was less than 2 logs, comparing with WE, in every case. The presence of large numbers of polymorphonuclear neutrophil (PMN) in the splenic red pulp and in the lung infiltrate previous to the mentioned lesions, encourage the interpretation that the damage is somehow mediated by these cells. The guinea-pigs infected with the WE strain could represent a very convenient model to study the role of PMN in viral diseases, as well as the non-immune pathogenetic mechanisms for LCMV.

An increase in the virus dose from 10(2) LD50 (low dose) to 10(4) LD50 (high dose) of lymphocytic choriomeningitis virus (LCMV) results in markedly delayed virus clearance, in spite of a potent cytotoxic T-cell (TC) response. However, virus-specific delayed-type hypersensitivity (DTH) reactivity is markedly depressed in high-dose mice, suggesting an association between DTH and virus clearance. When virus-primed memory cells are transferred, DTH reactivity as well as virus-clearing capacity is restored in high-dose mice, indicating that the virus is not present in a changed or concealed form. The role of T-cells mediating DTH (TD cells) in virus clearance was also studied by adoptive transfer to naive recipients. Here the high-dose primed cells did mediate virus clearance, although no DTH reaction was detectable 24-72 h after transfer. However, when footpad swelling was measured 96 h or more after transfer a DTH response emerged, indicating that TD priming had taken place in high-dose animals. Pre-irradiation of high-dose primed cells markedly inhibited the antiviral activity as well as DTH, suggesting that upon transfer to naive recipients TD precursors from high-dose mice would proliferate into effector cells capable of mediating both functions. Treatment with anti-Lyt2+C' abrogated the capacity to induce virus-specific DTH, thus confirming that the cells involved are not helper T (TH) cells. We conclude that the DTH unresponsiveness in high-dose mice reflects a defective differentiation of TD precursor into effector cells which is reversible upon transfer to a less antigen loaded environment. Furthermore, it is suggested that TD function is crucial to the process of virus clearance.

A severe inflammatory process can be induced by transferring lymphocytic choriomeningitis virus (LCMV)-immune spleen cell populations into cyclophosphamide (Cy)-suppressed, LCMV-infected recipients. The extent of cellular invasion can be quantified accurately by counting cells obtained from the cisterna magna. The underlying lymphocyte-target cell interactions are apparently very specific, with massive cellular invasion being dependent on the donor and recipient sharing class I major histocompatibility complex glycoproteins. Unlike the situation found by others for delayed-type hypersensitivity in the mouse footpad, the inflammatory process in the cisterna magna was induced to a similar extent by unmanipulated LCMV-immune spleen cells and by lymphocyte populations from mice that were pretreated with 150-200 mg/kg of Cy. This dose of Cy did not eliminate cytotoxic T lymphocytes (CTL) in the mouse strains used. However, immune spleen cells from Cy-pretreated mice showed a reduced capacity to clear virus from the brain. Virus persisted even though the inflammatory process resolved. These results support earlier conclusions that immune CTL are directly responsible for the induction of severe meningitis in LCM, although CTL obviously do not constitute the sole effectors involved in eliminating virus from the central nervous system.

A method for rapid detection of virus carrier state in natural infection with lymphocytic choriomeningitis in house mice (Mus musculus) has been developed. The method consists in virus antigen detection in the brain, spleen, and feces of mice by the indirect hemagglutination test with immunoglobulin erythrocyte diagnostic agents.

Reassortant viruses of different strains of lymphocytic choriomeningitis viruses cause lethal disease after inoculation into neonatal BALB/c WEHI mice, but, in contrast, parental strains or reciprocal reassortants do not cause lethal disease. The disease is characterized by inhibition of growth and death. The pathogenic mechanism is the induction of interferon combined with higher virus titers and subsequent liver necrosis. The generation of lethal reassortants from nonlethal parent viruses likely has implications for understanding the outbreaks of unanticipated virulent disease within a viral family.

Exposure of weaned hamsters to an environment contaminated with LCM virus shed by tolerantly infected mice led to short subclinical infections. If infection occurred in early pregnancy, the young appeared normal at birth but their tissues were highly infective. For two to three months their bites and urine were also highly infective. A viraemia did not persist long enough for successive vertical transmissions of the infection to be likely. However, the viruria persisted in most prenatally infected hamsters for at least eight months and under simulated field conditions was a potent virus source for contact infections, leading to further generations of prenatally infected young. In the absence of the natural reservoir host, such long-term carriers could have been a major factor in causing the build-up of infection in colonies of hamsters which, when purchased as household pets, led to a recent spate of human clinical infections in Germany and the U.S.A.

Lymphocytic choriomeningitis virus (LCMV) persistent infection that results from the inoculation of C3H/St newborn mice causes growth hormone (GH) deficiency and associated disease characterized by both reduced weight and serum glucose levels. Molecular analysis of pituitary nucleic acids shows GH deficient mice have, on average, fivefold reduced levels of GH mRNA although the histopathology of such GH producing cells is normal. Northern blots indicate that the length of GH mRNA is comparable in the GH deficient, virus infected mice and the GH normal, uninfected age-matched controls. Hence, truncated GH mRNA cannot account for hormonal defect. Mice infected congenitally through mating of persistently infected parents have normal growth and blood glucose levels. GH mRNA levels in pituitaries of these mice are equivalent to those of uninfected age-matched controls but significantly greater than those seen in neonatally infected GH deficient mice. Although infectious virus titers in the sera are equivalent in congenitally and neonatally infected age- and sex-matched mice, virus titers are significantly lower in pituitaries and brains of the congenitally infected mice when compared to neonatally inoculated mice. Additionally, the number of GH-producing pituitary cells expressing viral proteins is less in congenitally infected mice relative to those in neonatally inoculated mice. Hence there is a direct association between viral replication in GH-producing cells, lowered GH mRNA, and GH deficiency.

Virus-induced disease occurs both through direct destruction of cells by viruses and/or secondarily through lysis of infected cells by immunological assault. We asked whether viruses could also cause injury and disease by altering the cell's normal or expected functions without destroying the cells. Here we show that a relatively noncytopathic virus can perturb endocrine functions by disordering the synthesis of a hormone needed for growth and glucose regulation, while replicating in a specialized cell making that hormone. Yet, despite viral replication and alteration in synthesis of the specialized cell's homeostatic product, the infected cell remains free from structural injury.

In different strains of inbred Syrian golden hamsters (Mesocricetus auratus), the lymphocytic choriomeningitis virus (LCMV) strains WE and Armstrong (ARM) produced systemic infection with infective virus and viral antigens detected predominantly in reticuloendothelial organs. Host and virus strain-dependent fatal wasting disease also occurred. After infection with WE, all MHA and PD4 hamsters died of a progressive wasting disease and infectivity persisted in organs at relatively high titres. LSH and CB strain hamsters resisted lethal disease and totally eliminated infection. LVG and LHC strain hamsters were intermediate in susceptibility to WE; some died of wasting and had persistently infected organs, while others cleared infection and survived. ARM was avirulent causing an inapparent infection in all hamsters. LCMV antibody responses were temporally comparable for all hamsters with either lethal or non-lethal infection. Histologically, lymphoid hyperplasia and low-grade systemic perivascular mononuclear leukocyte infiltration were found in all LCMV-infected hamsters. However, non-necrotic segmental ileal changes, which included vascular congestion, minimal haemorrhage and crypt epithelial growth extension into the intestinal wall, were found in susceptible hamsters when infected with the lethal WE strain.

Depending on passage history, strain WE infectious LCM virus either damages L cells more or less severely or leaves them morphologically intact. Correspondingly, the plaques which are formed on L cell monolayers are of different appearance, ranging from intensely turbid to clear. Multiplication of LCM virus in certain mouse organs profoundly affects plaque characteristics. The brain, for instance, favours lytic variants while the spleen supports the replication of virus which forms turbid plaques. This statement holds if virus taken from organs of persistently infected mice or virus passaged from mouse to mouse is analysed and is true also if the initial preparation contains virus forming predominantly either clear or turbid plaques on L cell monolayers. Selection is not rapid and not absolute. It may take months of multiplication before a final state is reached, and even then the number of characteristic plaques is usually in great excess of the rest but never reaches 100%. Cloning procedures may alter the proportions, but with our experimental conditions no plaque has ever been isolated which would retain its characteristics upon passage. Differences of plaque type morphology were not reflected in differences of pathogenic properties, and both clear and turbid variants caused persistent infection if used to infect newborn mice and led to disease with signs of neurological involvement and death if inoculated intracerebrally into adult animals.

The data presented confirm previous evidence of delayed hypersensitivity in mice following infection with Junin virus. Adaptive transfer of sensitized cells from adult mice which had received 5 Junin virus injections into preinfected newborn mice shortened their survival by 72 hours. It was clear, however, that the development of the immunological mechanism in adult mice occurred under certain conditions. This evidence was confirmed by the observations that the induction of cell-mediated immunity and the response of sensitized lymphocytes to viral antigens appeared to be related to multiple injections of the virus. On the other hand, the sensitized cells were present in adult mice by day 9 and vanished 60 days after the infection. These results revealed the difference between the lymphoid cells from adult mice in the early and the late stages of immunity. The implications of these results in the development of the fatal neurological disease induced by the Junin virus in newborn mice are discussed.

Swiss mice infected at birth with lymphocytic choriomeningitis virus develop glomerulonephritis. Injection of potent anti-mouse interferon globulin at the time of viral infection inhibited the development of these renal lesions. We conclude that the production of endogenous interferon by this virus in the first few days of life plays an important role in the pathogenesis of this glomerulonephritis.

We describe a patient with meningitis caused by lymphocytic choriomeningitis virus. Oligoclonal IgG bands were found in the patient's cerebrospinal fluid during acute and convalescent stages of the illness, and results of liver function tests were abnormal. Acute attacks of lymphocytic choriomeningitis virus infection in humans can be added to the list of diseases associated with cerebrospinal fluid oligoclonal IgG.

Lymphocytic choriomeningitis virus (LCMV) produces a persistent infection of the nervous system in susceptible mice. To map the localization of viral antigens in the central nervous system (CNS), the authors studied, by means of ultrastructural immune peroxidase techniques, 4-6-month-old mice persistently infected with LCMV following an intracerebral inoculation at birth. The greatest number of infected neurons was observed in the cortex, particularly of the limbic system, and certain nuclei of the hypothalamus. In the cerebellum, Purkinje cells selectively expressed viral antigens. Moderate numbers of infected neurons were found in the anterior horns of the spinal cord, basal ganglia, and thalamus. The immunoperoxidase technique using monoclonal antibodies showed that persistently infected neurons primarily expressed the nucleocapsid protein antigens of LCMV. Glycopeptide antigens were minimally expressed. Electron-microscopic examination of selected individual infected neurons showed viral antigens exclusively associated with ribosomes. No staining was seen on cell surfaces. Glutaraldehyde-fixed CNS tissue studied by electron microscopy did not reveal morphologic abnormalities or mature viral particles. This study demonstrates that LCMV persistently infects specific neuronal populations. Infected neurons express viral antigens in association with host ribosomes and show no significant morphologic alterations.

Current laboratory methods for the diagnosis of lymphocytic choriomeningitis are discussed, mainly from the point of view of recent improvements. The use of sentinel guinea-pigs is suggested for the detection of lymphocytic choriomeningitis infection in animal colonies.

The prevention of hamster-associated outbreaks of lymphocytic choriomeningitis virus infection in human beings requires rapid and reliable testing of large numbers of hamsters for the infection. To select the most effective test, the antibody response of infected hamsters was determined by the indirect fluorescent antibody and complement-fixation techniques. The indirect fluorescent antibody technique required less than 2 hours to complete, was the first to become positive after infection, and remained positive for at least several months. Infection in hamsters was also readily detected by the inoculation of mice with infected hamster tissues; virus could be isolated from several organs as early as postinoculation day (PID) 3, and all organs tested contained high concentrations of virus by PID 5. After PID 40, virus was detectable only in the kidney; this organ remained positive for over 3 months.

Persistence of lymphocytic choriomeningitis (LCM) virus in mice infected in utero or neonatally is due to impairment of the specific subsets of thymus-dependent lymphocytes which, in the adult normal mouse, are involved in elimination of LCM virus. Virus-thymocyte interactions were studied since it was likely that this impairment takes place in the thymus. Using an infectious centre assay, we found that about 1% of the thymocytes from foetal and neonatal mice were productively infected by the virus while thymocytes from older mice were refractory to infection. The infected cells were Thy 1-positive and agglutinated by peanut lectin together with immature lymphocytes. Later, when virus persistence was established, the number of infected thymocytes declined to about 0.1% and these cells were not agglutinated by lectin. The results are compatible with the assumption that thymic precursor T-cells capable of elimination LCM virus are chronically infected by the virus and rendered non-functional.

The efficiency of cloned class I MHC restricted CTL specific for the nucleoprotein or glycoprotein of lymphocytic choriomeningitis virus in either mediating virus clearance or immunopathologic disease in mice during acute infection was quantitated. Cloned CTL specific for either an internal (nucleoprotein) or surface (glycoprotein) protein of lymphocytic choriomeningitis virus, when administered intracerebrally 5 days after the initiation of infection induced fatal immunopathology, indicating that both internal and surface viral Ag play a role in immune mediated disease in vivo. Dose-response analysis indicated that only 10(2) to 10(3) cloned CTL injected intracerebrally were required to induce mortality in 50% of inoculated syngeneic mice. Thus relatively few virus-specific CTL are required to induce an acute immunopathologic disease in the central nervous system. In contrast, if cloned CTL are adoptively transferred at the time of initiation of viral infection they provide protection as demonstrated by their ability to eliminate virus from the brain and thus terminate the acute infection.

After intravenous infection of mice, lymphocytic choriomeningitis virus multiplied in spleens and livers, attaining highest concentrations on days 4 to 6. The subsequent clearance was as rapid, and 8 to 10 days after inoculation, infectivity was usually below detectability. During the effector phase of virus elimination, both cytotoxic T-cell (CTL) activity and the number of cells producing antiviral antibodies were high. Monoclonal antibodies directed against T lymphocytes and T-lymphocyte subsets were inoculated once intravenously 5, 6, or 7 days after infection of the animals, and the effects on antiviral immune responses, as well as on elimination of virus from the organs, were determined. Treatment with anti-Thy-1 and anti-Lyt-2 antibodies blocked elimination of the virus and profoundly diminished the activity of spleen CTLs but reduced the antibody response partially (anti-Thy-1) or increased it (anti-Lyt-2). In contrast, treatment with the anti-L3T4 antibody had essentially no effect on either virus elimination or CTL response but abolished antibody production. We conclude that Lyt-2+ (cytotoxic-suppressive) T lymphocytes are needed for elimination of the virus and also regulate the humoral response but that antiviral antibodies are not essential for control of the infection.

Death occurred earlier and its rate was higher in one-week-old mice treated with phytohaemagglutinin (PHA) and subsequently inoculated intracerebrally with LCM virus than in their virus infected but untreated littermates. Thus PHA treatment contributed to the outcome of LCM virus infection in the form of lethal meningitis. The course of LCM virus infection in 1-week-old PHA treated mice was similar as in the untreated 2-week-old mice. This indicates that PHA treatment accelerated the development of cell mediated immunological capacity in suckling mice.