Immune conflicts in lymphocytic choriomeningitis virus

Conclusions In this chapter, we have reviewed the immunopathology of LCMV disease and the possible mechanisms involved. While much of what has been learned about LCMV immunopathology is unique to this system, many of the basic principles can be used to increase our understanding of viral immunopathology in other systems. For example, knowledge of the immune mechanisms mediating LCMV hepatitis may provide insights into understanding and managing infections such as chronic active hepatitis B. Chronic active hepatitis B is likely immune mediated, given that the majority of chronic hepatitis B carriers do not have ongoing liver inflammation. As monoclonal antibody and cytokine immunotherapy becomes increasingly available, development of successful treatment modalities will depend upon a greater understanding of the effector arms of the immune system mediating hepatitis B liver damage.Immune-complex disease similar to that seen in LCMV carrier mice has been seen in other chronic viral infections. These include chronic hepatitis B, HIV, and lactate dehydrogenase virus in mice [13, 18, 44, 47, 54, 61]. Therapy of most immunecomplex disease has focused on treatment of the underlying disease or attending to the disease complications, such as end-stage nephritis. Studies of LCMV immunecomplex glomerulohephritis have shown that the disease severity varies depending on the genetic background of the host. Understanding the host factors involved in the development of immune-complex disease may provide insight into identifying those at risk and intervening prior to the development of the disease.Perhaps the most intriguing and challenging aspects of the study of LCMV immunopathology is its possible implications for better understanding infection with HIV. During HIV infection, as CD4 numbers decline, CD8⁺ CTL activity is lost and virus levels increase. As was seen during clearance of a chronic LCMV infection, the presence of CD4⁺ cells may be crucial in controlling viral spread through sustaining viral-specific CD8⁺ CTL activity. Infection of mice with the macrophage-tropic strain LCMV clone 13 results in immune suppression and susceptiblity to opportunistic infection with H. capsulatum. Studies of the mechanisms leading to susceptabilty to histoplasmosis may have implications for AIDS, since in endemic areas, disseminated histoplasmosis is a frequently seen complication in AIDS. Thus, studies of LCMV immunopathology serve to not only expand our knowledge of viral immunopathology in general, but also have practical implications for understanding and treating human disease.     

Antibody-complement interactions with purified lymphocytic choriomeningitis virus

Immune complex formation and immune virolysis of the Armstrong strain of lymphocytic choriomeningitis virus (LCMV) were examined. LCMV, harvested from acutely infected BHK cells propagated in the absence of serum, was purified by methanol precipitation and sucrose density gradient centrifugation. The 31- and 23-S RNA species of LCMV were labeled under conditions of actinomycin D treatment of cells that prevented the labeling of cell ribosomes in the virion. The addition of guinea pig antibody (Ab) to LCMV lowered viral infectivity and enhanced the sedimentation rate but not the density of [U-³H]LCMV and ¹²⁵iodine-surface protein-labeled LCMV. This suggested that Ab aggregated the virions into faster sedimenting complexes. The addition of complement (C) to the LCMV-Ab complex further reduced the infectivity and increased both the sedimentation rate and density of [¹²⁵I]LCMV. When [U-³H]LCMV was reacted with Ab and C and subjected to rate zonal sedimentation, the RNA label remained at the top of the gradient rather than sedimenting with the ¹²⁵I-surface protein-membrane label. This observation, which indicated that Ab and C lysed the virus, was confirmed by electron microscopy showing first coating of the viral membrane with electron dense material, then swelling and rupture of the membrane and release of viral core material. Using sera deficient in specified C components, it was found that the C-mediated inactivation proceeded via the classical C pathway. Sera deficient in latter C components (beyond C3) augmented the inactivation of LCMV-Ab complexes, but this inactivation was far more extensive when a complete C source was used. Evidence suggesting that immune and persistently infected mice produce C-fixing Ab to the surface of the LCM virion was documented.     

Identification of African epidemic icterus virus as lymphocytic choriomeningitis virus

By means of complement fixation, virus neutralization and fluorescent antibody tests, the virus known as African epidemic icterus virus is shown to be a strain of lymphocytic choriomeningitis virus.     

Biophysical and biochemical characterization of lymphocytic choriomeningitis virus

Summary The infectivity of LCM virus suspended in solutions buffered with Tris(hydroxymethyl)aminomethane and its hydrochloride has been found to be greatly stabilized. This stabilization (studied at 4° and 37° C), however, was markedly dependent on the protein content of the solution. If the protein content became too low (by dilution and/or partial purification of the virus), the Tris effect would disappear, but could be reversed if bovine serum albumin was added before inactivation studies were begun. The sensitivity of the virus to ultrasonic irradiation also followed the pattern established above (i.e. — diluted or purified virus was inactivated to a much greater extent than virus suspended in Eagle's medium plus serum). The data also indicated that sonication could break up aggregates of virus particles.Presented in part at the IXth International Congress for Microbiology, Moscow, USSR, 24–30 July 1966.     

Infectious lymphocytes in lymphocytic choriomeningitis virus carrier mice.

The infectivity of blood and lymphoid organs of mice persistently infected with lymphocytic choriomeningitis virus was found to be predominantly associated with lymphocytes and both T and B cells were infectious. A hypothesis is presented in which it is assumed that lymphocytes in carrier mice are infected via their LCM virus-specific antigen receptors, thereby leading to their antigen-triggered clonal expansion followed by infection and functional inactivation.     

Persistent infection of cultivated cells with lymphocytic choriomeningitis virus: Regulation of virus replication

Archives of Virology - L cells were infected with lymphocytic choriomeningitis virus (LCM virus). They were subcultivated and infectious virus and interfering virus were quantified at intervals....     

Protein structure of lymphocytic choriomeningitis virus: Identification of the virus structural and cell associated polypeptides

Lymphocytic choriomeningitis virus (LCMV) has been found to contain 3 major structural polypeptides. The largest of these was shown to be a nonglycosylated nucleoprotein (NP) of approximately 63,000 daltons which was found internally in the virion and was associated with a dense ribonucleoprotein complex. Two glycopeptides, termed GP-1 and GP-2, of approximately 54,000 and 35,000 daltons, respectively, were shown to be localized on the virion surface by proteolytic digestion of intact virus. Peptide maps of GP-1 and GP-2 indicated that they were independent polypeptides and that GP-2 was not a cleavage product of GP-1. Furthermore, the tryptic peptide map of NP was distinct from the two glycopeptides. Pulse-labeling studies of the intracellular synthesis of LCMV specific polypeptides in infected BHK-21 cells demonstrated synthesis of the nucleoprotein by 6 hr after infection at a multiplicity of infection of 10, corresponding to the beginning of log phase of viral replication. In cytosol extracts of LCMV-infected cells enhanced by immune precipitation an additional apparently nonstructural glycopeptide with a molecular weight of 74–75,000 was observed. This cell associated glycopeptide, designated GP-C, was readily detectable after 24–48 hr of infection, and appeared to accumulate with increasing time of incubation. While the relationship of GP-C to the virion glycopeptides GP-1 and GP-2 has not been determined, immune precipitation studies have shown that GP-C does not share antigenic determinants with NP. The probable relationships between the polypeptides of lymphocytic choriomeningitis virus both to the pathogenesis of virus induced immune diseases and to the structure of other arenaviruses have been discussed.     

Analysis of the genomic L RNA segment from lymphocytic choriomeningitis virus

The arenavirus genomic L RNA segment represents approximately 70% of the viral genetic material but current understanding of the organization, regulation, and functioning of the viral L products remains limited. Biological studies with reassortant viruses have implicated the L RNA segment in the lethal infection of adult guinea pigs produced by LCMV-WE but no further explanation of the pathogenic process is presently available. We have initiated a detailed molecular analysis of LCMV L products based on construction and characterization of L-specific cDNA clones and synthesis of L-specific hybridization probes. Nucleotide sequencing studies have allowed the derivation of a partial amino acid sequence for a predicted L protein and antisera raised against synthetic peptides have demonstrated an L protein in Western blotting experiments. Using this approach we have identified a single high molecular weight protein (approximately 200,000 Da) in purified virions and in viral ribonucleoprotein complexes extracted from acutely infected tissue culture cells. This L protein is translated from a nonpolyadenylated, genomic complementary L mRNA and potentially represents part or all of the viral RNA-dependent RNA polymerase.     

Analytical validation of a lymphocytic choriomeningitis virus real-time RT-PCR assay

Lymphocytic choriomeningitis virus (LCMV) is a rare cause of central nervous system disease in humans. Screening by real-time RT-PCR assay is of interest in the case of aseptic meningitis of unknown etiology.A specific LCMV real-time RT-PCR assay, based on the detection of genomic sequences of the viral nucleoprotein (NP), was developed to assess the presence of LCMV in cerebrospinal fluids (CSF) sent for viral screening to a Swiss university hospital laboratory.A 10-fold dilution series assay using a plasmid containing the cDNA of the viral NP of the LCMV isolate Armstrong (Arm) 53b demonstrated the high sensitivity of the assay with a lowest detection limit of ≤50 copies per reaction. High sensitivity was confirmed by dilution series assays in a pool of human CSF using four different LCMV isolates (Arm53b, WE54, Traub and E350) with observed detection limits of ≤10 PFU/ml (Arm53b and WE54) and 1 PFU/ml (Traub and E350).Analysis of 130 CSF showed no cases of acute infection. The absence of positive cases was confirmed by a published PCR assay detecting all Old World arenaviruses.This study validates a specific and sensitive real-time RT-PCR assay for the diagnosis of LCMV infections. Results showed that LCMV infections are extremely rare in hospitalized patients western in Switzerland.     

Isolation and characteristics of low molecular weight recombinant peptides, representing antigenic domains in the NP protein of lymphocytic choriomeningitis virus

High- and low-molecular recombinant peptides of LCM virus nucleoprotein, representing individual immunodominant antigenic sites, were obtained in pJC40 expressing vector and studied in solid-phase enzyme immunoassay. 2-7% proteins resultant from total cellular synthesis are recombinant peptides. Comparative analysis of antigenic properties of recombinant peptides and native viral protein showed that recombinant peptides are virtually not inferior to native viral protein in antigenic properties.