Energy parasites trigger oncogene mutation.

PURPOSE: Cancer initialization can be explained as a result of parasitic virus energy consumption leading to randomized genome chemical bonding. MATERIALS AND METHODS: Analysis of experimental data on cell-mediated immunity (CMI) containing about 12,000 cases of healthy humans, cancer patients and patients with precancerous cervical lesions disclosed that the specific cancer and the non-specific lactate dehydrogenase-elevating (LDH) virus antigen elicit similar responses. The specific antigen is effective only in cancer type of its origin but the non-specific antigen in all examined cancers. CMI results of CIN patients display both healthy and cancer state. The ribonucleic acid (RNA) of the LDH virus parasitizing on energy reduces the ratio of coherent/random oscillations. Decreased effect of coherent cellular electromagnetic field on bonding electrons in biological macromolecules leads to elevating probability of random genome reactions. RESULTS: Overlapping of wave functions in biological macromolecules depends on energy of the cellular electromagnetic field which supplies energy to bonding electrons for selective chemical bonds. CMI responses of cancer and LDH virus antigens in all examined healthy, precancerous and cancer cases point to energy mechanism in cancer initiation. CONCLUSIONS: Dependence of the rate of biochemical reactions on biological electromagnetic field explains yet unknown mechanism of genome mutation.

Diseases caused by defects of energy level and loss of coherence in living cells.

Human and animal diseases are brought about by pathological alterations of production, composition, and conformation of macromolecules and structures in cells. Additional contributing factors include changes in physiological states caused by disturbances of energy supply, energy transduction, energy dissipation in moving or oscillating parts, and parasitic energy consumption. Disturbances of energy states may endanger existence of the system. The cell-mediated immunity (CMI) response of T lymphocytes correlating with their adherence properties was examined using antigen prepared from the serum of inbred laboratory mice strain C3H H(2k) infected with lactate dehydrogenase elevating (LDH) virus. LDH virus is a parasite on the cellular energy system. Significant CMI response was elicited in T lymphocytes prepared from the blood of patients with cancer of different phenotypes, acute myocardial infarctions, schizophrenia, and recurrent spontaneous abortions in early pregnancy from unknown reasons. The CMI response is assumed to monitor transferred information about decreased levels of energy states and decoherence in the cells caused by mitochondrial malfunction, parasitic consumption, production of lactate, and possibly other disturbances. The LDH virus infection or similar pathological processes caused by different agents might be connected with the diseases and monitored by the examined CMI response. A large amount of mitoses with chromosome defects in aborted fetuses suggest increased mutability of genomes caused by defective energy states.

Modulation of lipopolysaccharide receptor expression by lactate dehydrogenase-elevating virus.

Lactate dehydrogenase-elevating virus (LDV) exacerbates mouse susceptibility to endotoxin shock through enhanced tumour necrosis factor (TNF) production by macrophages exposed to lipopolysaccharide (LPS). However, the in vivo enhancement of TNF production in response to LPS induced by the virus largely exceeds that found in vitro with cells derived from infected animals. Infection was followed by a moderate increase of Toll-like receptor (TLR)-4/MD2, but not of membrane CD14 expression on peritoneal macrophages. Peritoneal macrophages from LDV-infected mice unresponsive to type I interferons (IFNs) did not show enhanced expression of TLR-4/MD2 nor of CD14, and did not produce more TNF in response to LPS than cells from infected normal counterparts, although the in vivo response of these animals to LPS was strongly enhanced. In contrast, the virus triggered a sharp increase of soluble CD14 and of LPS-binding protein serum levels in normal mice. However, production of these LPS soluble receptors was similar in LDV-infected type I IFN-receptor deficient mice and in their normal counterparts. Moreover, serum of LDV-infected mice that contained these soluble receptors had little effect if any on cell response to LPS. These results suggest that enhanced response of LDV-infected mice to LPS results mostly from mechanisms independent of LPS receptor expression.

Lactate dehydrogenase-elevating virus induces systemic lymphocyte activation via TLR7-dependent IFNalpha responses by plasmacytoid dendritic cells.

BACKGROUND: Lactate dehydrogenase-elevating virus (LDV) is a natural infectious agent of mice. Like several other viruses, LDV causes widespread and very rapid but transient activation of both B cells and T cells in lymphoid tissues and the blood. The mechanism of this activation has not been fully described and is the focus of the current studies. PRINCIPAL FINDINGS: A known inducer of early lymphocyte activation is IFNalpha, a cytokine strongly induced by LDV infection. Neutralization of IFNalpha in the plasma from infected mice ablated its ability to activate lymphocytes in vitro. Since the primary source of virus-induced IFNalpha in vivo is often plasmacytoid dendritic cells (pDC's), we depleted these cells prior to LDV infection and tested for lymphocyte activation. Depletion of pDC's in vivo eradicated both the LDV-induced IFNalpha response and lymphocyte activation. A primary receptor in pDC's for single stranded RNA viruses such as LDV is the toll-like receptor 7 (TLR7) pattern recognition receptor. Infection of TLR7-knockout mice revealed that both the IFNalpha response and lymphocyte activation were dependent on TLR7 signaling in vivo. Interestingly, virus levels in both TLR7 knockout mice and pDC-depleted mice were indistinguishable from controls indicating that LDV is largely resistant to the systemic IFNalpha response. CONCLUSION: Results indicate that LDV-induced activation of lymphocytes is due to recognition of LDV nucleic acid by TLR7 pattern recognition receptors in pDC's that respond with a lymphocyte-inducing IFNalpha response.

Lactate dehydrogenase-elevating virus induces apoptosis in cultured macrophages and in spinal cords of C58 mice coincident with onset of murine amyotrophic lateral sclerosis.

Age-dependent poliomyelitis (ADPM) or murine amyotrophic lateral sclerosis (ALS) is a murine paralytic disease triggered in immunosuppressed genetically-susceptible mice by infection with the arterivirus lactate dehydrogenase-elevating virus (LDV). This disease provides an animal model for ALS, affecting anterior horn neurons and resulting in neuroparalysis 2-3 weeks after LDV infection. We have tested the hypothesis that spinal cord apoptosis is a feature of the LDV-induced murine ALS, since apoptosis is postulated to be a causal factor in human ALS. Gene microarray analyses of spinal cords from paralyzed animals revealed upregulation of several genes associated with apoptosis. Spinal cord apoptosis was investigated further by TUNEL and activated caspase-3 assays, and was observed to emerge concurrent with paralytic symptoms in both neuronal and non-neuronal cells. Caspase-3-dependent apoptosis was also triggered in cultured macrophages by neurovirulent LDV infection. Thus, virus-induced spinal cord apoptosis is a pre-mortem feature of ADPM, which affects both neuronal and support cells, and may contribute to the pathogenesis of this ALS-like disease.

Glucocorticoid regulation of lactate dehydrogenase-elevating virus replication in macrophages.

Lactate dehydrogenase-elevating virus (LDV) is a macrophage-tropic arterivirus which generally causes a persistent viremic infection in mice. LDV replication in vivo seems to be primarily regulated by the extent and dynamics of a virus-permissive macrophage population. Previous studies have shown that glucocorticoid treatment of chronically LDV-infected mice transiently increases viremia 10-100-fold, apparently by increasing the productive infection of macrophages. We have further investigated this phenomenon by comparing the effect of dexamethasone on the in vivo and in vitro replication of two LDV quasispecies that differ in sensitivity to immune control by the host. The single neutralizing epitope of LDV-P is flanked by two N-glycans that impair its immunogenicity and render LDV-P resistant to antibody neutralization. In contrast, replication of the neuropathogenic mutant LDV-C is suppressed by antibody neutralization because its epitope lacks the two protective N-glycans. Dexamethasone treatment of mice 16 h prior to LDV-P infection, or of chronically LDV-P infected mice, stimulated viremia 10-100-fold, which correlated with an increase of LDV permissive macrophages in the peritoneum and increased LDV infected cells in the spleen, respectively. The increase in viremia occurred in the absence of changes in total anti-LDV and neutralizing antibodies. The results indicate that increased viremia was due to increased availability of LDV permissive macrophages, and that during a chronic LDV-P infection virus replication is strictly limited by the rate of regeneration of permissive macrophages. In contrast, dexamethasone treatment had no significant effect on the level of viremia in chronically LDV-C infected mice, consistent with the view that LDV-C replication is primarily restricted by antibody neutralization and not by a lack of permissive macrophages. beta-Glucan, the receptor of which is induced on macrophages by dexamethasone treatment, had no effect on the LDV permissiveness of macrophages.

Chimeric arteriviruses generated by swapping of the M protein ectodomain rule out a role of this domain in viral targeting.

Arteriviruses are enveloped, positive-strand RNA viruses for which the two major envelope proteins GP(5) and M occur as disulfide-linked heterodimers. These were assumed to serve the viral targeting functions, but recent ectodomain swapping studies with equine arteritis virus (EAV) indicate that the GP(5) protein does not determine arteriviral tropism. Here, we focused on the short, 13- to 18-residue ectodomain of the M protein. Using an infectious cDNA clone of the Lelystad virus isolate of porcine reproductive and respiratory syndrome virus (PRRSV), we substituted the genomic sequence encoding the M ectodomain by that of murine lactate dehydrogenase-elevating virus, EAV, and the US PRRSV-isolate, VR2332. Viable viruses with a chimeric M protein were obtained in all three cases, but for the latter two only after removal of the genomic overlap between the M and GP(5) genes. Characterization of the chimeric viruses revealed that they could be distinguished immunologically from wild-type virus, that they were genetically stable in vitro, but that they were impaired in their growth, reaching lower titers than the parental virus. The latter appeared to be due to an increased particle-to-infectivity ratio of the chimeric virus particles. Interestingly, the chimeric viruses had retained their ability to infect porcine cells and had not acquired tropism for cells susceptible to the viruses from which the foreign ectodomains were derived. We conclude that the surface structures composed by the arterivirus M and GP(5) ectodomains do not determine viral tropism.

Transplacental lactate dehydrogenase-elevating virus (LDV) transmission: immune inhibition of umbilical cord infection, and correlation of fetal virus susceptibility with development of F4/80 antigen expression.

Maternal-to-fetal transmission of the murine lactate dehydrogenase-elevating virus (LDV) has been previously shown to be regulated by maternal immunity as well as gestational age. For the present study, the role of maternal immunity in placental and umbilical cord virus protection was studied, and virus targeting of umbilical cord and fetal macrophages was correlated with expression of the F4/80 macrophage phenotypic marker. The results showed that LDV-infected macrophages appeared in umbilical cord by 24 h post-infection of pregnant mice, and some LDV-infected macrophages displayed the F4/80 phenotype. This potential reservoir of virus for the fetus was inhibited by passive immunization of pregnant mice with IgG anti-LDV antibodies, which rapidly concentrated in the placenta and umbilical cord. Probing of umbilical cord cells with antibodies directed at MHC genetic markers demonstrated the presence of both maternal and fetal cells in umbilical cords. A strong developmental correlation was observed between fetal F4/80 expression and LDV susceptibility, at about 13.6 days of gestation. These results demonstrate immune suppression of free and cell-associated virus in umbilical cord, thus defining a potentially important mechanism for immune protection of the fetus from transplacental virus infection. The results also clarify the developmental basis for fetal susceptibility to LDV infection.

Replication competition between lactate dehydrogenase-elevating virus quasispecies in mice. Implications for quasispecies selection and evolution.

The common quasispecies of lactate dehydrogenase-elevating virus (LDV), LDV-P and LDV-vx, are highly resistant to the humoral host immune response because the single neutralization epitope on the ectodomain of the primary envelope glycoprotein, VP-3P, carries three large N-glycans. Two laboratory mutants, LDV-C and LDV-v, have lost two of the N-glycans on the VP-3P ectodomain, thereby gaining neuropathogenicity for AKR/C58 mice but at the same time, becoming susceptible to the humoral immune response of the host. In attempts to further assess the origins and evolution of these LDVs we have determined their competitiveness by monitoring their fate in mixed infections of wild type, SCID, nude, and cyclophosphamide-treated mice by reverse transcription/polymerase chain reaction assays that distinguish between them. In mixed infections with LDV-P and LDV-vx, LDV-C and LDV-v became rapidly lost even when present initially in large excess over the former. In mixed infections of mice unable to generate neutralizing antibodies, LDV-C and LDV-v also became replaced by LDV-P and LDV-vx as predominant quasispecies but more slowly than in immunocompetent mice. The results indicate that the humoral immune response plays an important role in the displacement of LDV-C and LDV-v by LDV-P and LDV-vx but that in addition, LDV-C and LDV-v possess an impaired ability to compete with LDV-P and LDV-vx in the productive infection of the subpopulation of macrophages that represents the host for all these LDVs. In addition, LDV-v outcompeted LDV-C in mixed infections and the same was the case for neutralization escape mutants of LDV-v and LDV-C which had regained all three N-glycosylation sites on the VP-3P ectodomain. Thus a hierarchy exists in replication fitness: LDV-P/LDV-vx>LDV-v>LDV-C, which is unrelated to the number of N-glycans on the VP-3P ectodomain. The implications of the results in relation to the evolution and selection of the LDV-quasispecies is discussed. LDV-P and LDV-vx are genetically highly stable and thus seem to have achieved evolutionary stasis with optimum ability to establish viremic persistent infections of mice that are unimpeded by the host immune responses.

Reduction of serum interferon (IFN)-gamma concentration and lupus development in NZBxNZWF(1)mice by lactic dehydrogenase virus infection.

The complexity of cytokine regulation and the imbalance of helper T (Th)1 and Th2 subsets in systemic lupus erythematosus (SLE) animal models and human SLE are well recognized. In this study in NZBxNZWF(1)mice, the effects of lactic dehydrogenase virus (LDV) infection on the production of interferon (IFN)-gamma in the serum and the development of autoimmune disease were examined. The progress of the disease (the development of glomerulonephritis, formation of glomerular IgG and C3 deposits, increase in the blood urea nitrogen values, and mortality) was parallel with an increase in serum IFN-gamma in uninfected NZBxNZWF(1)mice. These changes were inhibited in LDV-infected NZBxNZWF(1)mice. Our findings suggest that increase in serum IFN-gamma may be associated with the active disease in NZBxNZWF(1)mice.

Isolation of lactate dehydrogenase-elevating viruses from wild house mice and their biological and molecular characterization.

Lactate dehydrogenase-elevating virus (LDV) was first identified as a contaminant of transplantable mouse tumors that were passaged in laboratory mice. It has been assumed that these LDVs originated from LDVs endemic in wild house mouse populations. In order to test this hypothesis and to explore the relationships between LDVs from wild house mice among each other and to those isolated from laboratory mice, we have isolated LDVs from wild house mice and determined their biological and molecular properties. We have screened for LDV tissues of 243 wild house mice that had been caught in various regions of North, Central and South America between 1985 and 1994. We were able to isolate LDVs from the tissues of four mice, three had been caught in Baltimore, MD and one in Montana. We demonstrate that the phenotypic properties (ability to establish a long-term viremic infection, low immunogenicity of the neutralization epitope, high resistance to antibody neutralization and lack of neuropathogenicity) of the four wild house mouse LDVs are identical to those of the primary LDVs isolated from transplantable tumors (LDV-P and LDV-vx), which are distinct from those of the neuropathogenic LDV-C. Furthermore, ORF 5 and ORF 2 and their protein products (the primary envelope glycoprotein VP-3P, and the minor envelope glycoprotein, respectively) of the wild house mouse LDVs were found to be closely related to those of LDV-P and LDV-vx. The LDVs caught in Baltimore, MD were especially closely related to each other, whereas the LDV isolated in Montana was more distantly related, indicating that it had evolved independently. The ectodomain of VP-3P of all four wild house mouse LDVs, like those of LDV-P and LDV-vx, possess the same three polylactosaminoglycan chains, two of which are lacking in the VP-3P ectodomain of LDV-C. These results further strengthen the conclusion that the three polylactosaminoglycan chains are the primary determinants of the phenotypic properties of LDV-P/vx.

Regulation of immune complexes during infection of mice with lactate dehydrogenase-elevating virus: studies with interferon-gamma gene knockout and tolerant mice.

Mice persistently infected with lactate dehydrogenase-elevating virus (LDV) develop circulating IgG-containing hydrophobic immune complexes, with a molecular mass of 150 to 300 kd, which bind to the surfaces of high-capacity enzyme-linked immunosorbent assay (ELISA) plates. LDV infection also stimulates polyclonal B-cell activation and autoimmunity. For this study, interferon-gamma gene knockout (GKO) mice were utilized to study circulating immune complexes and other parameters of LDV infection. The kinetics of LDV viremia, formation of plasma IgG anti-LDV antibodies, and LDV replication in the spleen and liver were essentially normal in GKO mice. Polyclonal activation of B cells, as reflected by increased total plasma IgG concentration during LDV infection, was found to be intact in GKO mice, although at a lower magnitude than in control mice. The plasma concentration of IgG-containing hydrophobic immune complexes was reduced about 75% in LDV-infected GKO mice relative to normal LDV-infected controls. Allogeneic tissue responses were also found to be reduced in LDV-infected GKO mice relative to those in normal LDV-infected controls. These results dissociate specific anti-LDV immunity from formation of hydrophobic immune complexes, show that the IgG anti-LDV response as well as LDV replication in the spleen and liver are insensitive to physiological levels of interferon (IFN)-gamma, and suggest that IgG-containing immune complexes stimulated by LDV infection are a marker for autoimmunity.

Coexistence in lactate dehydrogenase-elevating virus pools of variants that differ in neuropathogenicity and ability to establish a persistent infection.

Neuropathogenic isolates of lactate dehydrogenase-elevating virus (LDV) differ from nonneuropathogenic isolates in their unique ability to infect anterior horn neurons of immunosuppressed C58 and AKR mice and cause paralytic disease (age-dependent poliomyelitis [ADPM]). However, we and others have found that neuropathogenic LDVs fail to retain their neuropathogenicity during persistent infections of both ADPM-susceptible and nonsusceptible mice. On the basis of a segment in open reading frame 2 that differs about 60% between the neuropathogenic LDV-C and the nonneuropathogenic LDV-P, we have developed a reverse transcription-PCR assay that distinguishes between the genomes of the two LDVs and detects as little as 10 50% infectious doses (ID50) of LDV. With this assay, we found that LDV-P and LDV-C coexist in most available pools of LDV-C and LDV-P. For example, various plasma pools of 10(9.5) ID50 of LDV-C/ml contained about 10(5) ID50 of LDV-P/ml. Injection of such an LDV-C pool into mice of various strains resulted in the rapid displacement in the circulation of LDV-C by LDV-P as the predominant LDV, but LDV-C also persisted in the mice at a low level along with LDV-P. We have freed LDV-C of LDV-P by endpoint dilution (LDV-C-EPD). LDV-C-EPD infected mice as efficiently as did LDV-P, but its level of viremia during the persistent phase was only 1/10,000 that observed for LDV-P. LDV-permissive macrophages accumulated and supported the efficient replication of superinfecting LDV-P. Therefore, although neuropathogenic LDVs possess the unique ability to infect anterior horn neurons of ADPM-susceptible mice, they exhibit a reduced ability to establish a persistent infection in peripheral tissues of mice regardless of the strain. The specific suppression of LDV-C replication in persistently infected mice is probably due in part to a more efficient neutralization of LDV-C than LDV-P by antibodies to the primary envelope glycoprotein, VP-3P. Both neuropathogenicity and the higher sensitivity to antibody neutralization correlated with the absence of two of three N-linked polylactosaminoglycan chains on the ca. 30-amino-acid ectodomain of VP-3P, which seems to carry the neutralization epitope(s) and forms part of the virus receptor attachment site.

Cytotoxic T cells are elicited during acute infection of mice with lactate dehydrogenase-elevating virus but disappear during the chronic phase of infection.

Lactate dehydrogenase-elevating virus (LDV) invariably establishes a life-long viremic infection in mice, which is maintained by replication of LDV in a renewable subpopulation of macrophages and escape from all host immune responses. We now demonstrate that cytotoxic T lymphocytes (CTLs) that specifically lyse LDV-infected macrophages and 3T3 cells producing the nucleocapsid protein of LDV were elicited in Swiss, B10.A, and (Swiss x B10.A)F1 mice. To detect target cell lysis, splenocytes needed to be expanded by a 5-day in vitro culture in the presence of recombinant interleukin 2 and syngeneic LDV protein-expressing cells. In vitro culture resulted in the specific expansion of CD8+ cells which mediated the lysis of target cells in a major histocompatibility complex class I-restricted manner. When CTLs were added to macrophage cultures at 1 h after infection with LDV, the lysis of the infected macrophages by the CTLs started about 5 h postinfection (p.i.) and, at an effector cell/target cell ratio of 25:1, resulted in the lysis of all LDV-infected macrophages in a culture by about 7 h p.i. However, lysis of the LDV replication in a culture was not rapid enough to significantly suppress the LDV yield in the culture. LDV replication in mice was also little affected by the presence of CTLs which were induced by immunization with 3T3 cells expressing the LDV nucleocapsid protein. Furthermore, all CTL precursor cells in infected mice had disappeared by 30 days p.i. Loss of CTL precursor cells in infected mice probably reflected high-dose clonal exhaustion, since LDV infection of a mouse results in massive production of LDV in all tissues of the mouse, but especially in lymphoidal tissues, and accumulation of LDV in newly formed germinal centers. Furthermore, slow LDV replication continues in the thymus and other lymphoidal organs.

Lactate dehydrogenase-elevating virus replication persists in liver, spleen, lymph node, and testis tissues and results in accumulation of viral RNA in germinal centers, concomitant with polyclonal activation of B cells.

Lactate dehydrogenase-elevating virus (LDV) replicates primarily and most likely solely in a subpopulation of macrophages in extraneuronal tissues. Infection of mice, regardless of age, with LDV leads to the rapid cytocidal replication of the virus in these cells, resulting in the release of large amounts of LDV into the circulation. The infection then progresses into life-long, asymptomatic, low-level viremic persistence, which is maintained by LDV replication in newly generated LDV-permissive cells which escapes all antiviral immune responses. In situ hybridization studies of tissue sections of adult FVB mice revealed that by 1 day postinfection (p.i.), LDV-infected cells were present in practically all tissues but were present in the highest numbers in the lymph nodes, spleen, and skin. In the central nervous system, LDV-infected cells were restricted to the leptomeninges. Most of the infected cells had disappeared at 3 days p.i., consistent with the cytocidal nature of the LDV infection, except for small numbers in lymph node, spleen, liver, and testis tissues. These tissues harbored infected cells until at least 90 days p.i. The results suggest that the generation of LDV-permissive cells during the persistent phase is restricted to these tissues. The continued presence of LDV-infected cells in testis tissue suggests the possibility of LDV release in semen and sexual transmission. Most striking was the accumulation of large amounts of LDV RNA in newly generated germinal centers of lymph nodes and the spleen. The LDV RNA was not associated with infected cells but was probably associated with virions or debris of infected, lysed cells. The appearance of LDV RNA in germinal centers in these mice coincided in time with the polyclonal activation of B cells, which leads to the accumulation of polyclonal immunoglobulin G2a and low-molecular-weight immune complexes in the circulation.

Pseudotype virions formed between mouse hepatitis virus and lactate dehydrogenase-elevating virus (LDV) mediate LDV replication in cells resistant to infection by LDV virions.

Infection of cultures of peritoneal macrophages with both lactate dehydrogenase-elevating virus (LDV) and mouse hepatitis virus (MHV) resulted in the formation of pseudotype virions containing LDV RNA which productively infected cells that are resistant to infection by intact LDV virions but not to infection by MHV. These cells were mouse L-2 and 3T3-17Cl-1 cells as well as residual peritoneal macrophages from persistently LDV-infected mice. Productive LDV infection of these cells via pseudotype virions was inhibited by antibodies to the MHV spike protein or to the MHV receptor, indicating that LDV RNA entered the cells via particles containing the MHV envelope. Simultaneous exposure of L-2 cells to both LDV and MHV resulted in infection by MHV but not by LDV. The results indicate that an internal block to LDV replication is not the cause of the LDV nonpermissiveness of many cell types, including the majority of the macrophages in an adult mouse. Instead, LDV permissiveness is restricted to a subpopulation of mouse macrophages because only these cells possess a surface component that acts as an LDV receptor.

Lactate dehydrogenase-elevating virus entry into the central nervous system and replication in anterior horn neurons.

The initial replication of lactate dehydrogenase-elevating virus (LDV) in mice, its invasion of the central nervous system (CNS) and infection of anterior horn neurons in C58 and AKXD-16 mice were investigated by Northern and in situ hybridization analyses. Upon intraperitoneal injection, LDV replication in cells in the peritoneum was maximal at 8 h post-infection (p.i.). Next, LDV infection was detected in bone marrow cells and then in macrophage-rich regions of all tissues investigated (12 to 24 h p.i.). By 2 to 3 days p.i., LDV RNA-containing cells had largely disappeared from all non-neuronal tissues due to the cytocidal nature of the LDV infection of macrophages. In the CNS at 24 h p.i. LDV replication was very limited and confined to cells in the leptomeninges. LDV replication in the cells of the leptomeninges should result in the release of progeny LDV into the cerebrospinal fluid and thus its dissemination throughout the CNS. However, in C58 and AKXD-16 mice, which are susceptible to paralytic LDV infection, only little LDV RNA and few LDV-infected cells were detectable in the spinal cord until at least 10 days p.i. Extensive cytocidal infection of anterior horn neurons occurred only shortly before the development of paralytic symptoms between 2 and 3 weeks p.i. The reason for the relatively long delay in LDV infection of anterior horn neurons is not known. No LDV RNA or LDV RNA-containing cells were detected in the brain, except in the leptomeninges at early times after infection.

Infection of central nervous system cells by ecotropic murine leukemia virus in C58 and AKR mice and in in utero-infected CE/J mice predisposes mice to paralytic infection by lactate dehydrogenase-elevating virus.

Certain mouse strains, such as AKR and C58, which possess N-tropic, ecotropic murine leukemia virus (MuLV) proviruses and are homozygous at the Fv-1n locus are specifically susceptible to paralytic infection (age-dependent poliomyelitis [ADPM]) by lactate dehydrogenase-elevating virus (LDV). Our results provide an explanation for this genetic linkage and directly prove that ecotropic MuLV infection of spinal cord cells is responsible for rendering anterior horn neurons susceptible to cytocidal LDV infection, which is the cause of the paralytic disease. Northern (RNA) blot hybridization of total tissue RNA and in situ hybridization of tissue sections demonstrated that only mice harboring central nervous system (CNS) cells that expressed ecotropic MuLV were susceptible to ADPM. Our evidence indicates that the ecotropic MuLV RNA is transcribed in CNS cells from ecotropic MuLV proviruses that have been acquired by infection with exogenous ecotropic MuLV, probably during embryogenesis, the time when germ line proviruses in AKR and C58 mice first become activated. In young mice, MuLV RNA-containing cells were found exclusively in white-matter tracts and therefore were glial cells. An increase in the ADPM susceptibility of the mice with advancing age correlated with the presence of an increased number of ecotropic MuLV RNA-containing cells in the spinal cords which, in turn, correlated with an increase in the number of unmethylated proviruses in the DNA extracted from spinal cords. Studies with AKXD recombinant inbred strains showed that possession of a single replication-competent ecotropic MuLV provirus (emv-11) by Fv-1n/n mice was sufficient to result in ecotropic MuLV infection of CNS cells and ADPM susceptibility. In contrast, no ecotropic MuLV RNA-positive cells were present in the CNSs of mice carrying defective ecotropic MuLV proviruses (emv-3 or emv-13) or in which ecotropic MuLV replication was blocked by the Fv-1n/b or Fv-1b/b phenotype. Such mice were resistant to paralytic LDV infection. In utero infection of CE/J mice, which are devoid of any endogenous ecotropic MuLVs, with the infectious clone of emv-11 (AKR-623) resulted in the infection of CNS cells, and the mice became ADPM susceptible, whereas littermates that had not become infected with ecotropic MuLV remained ADPM resistant.

Detection of negative-stranded subgenomic RNAs but not of free leader in LDV-infected macrophages.

The mechanism of synthesis of the seven subgenomic mRNAs of lactate dehydrogenase-elevating virus (LDV) was explored. One proposed mechanism, leader-primed transcription, predicts the formation of free 5'-leader in infected cells which then primes reinitiation of transcription at specific complementary sites on the antigenomic template. No free LDV 5'-leader of 156 nucleotides was detected in LDV-infected macrophages. Another mechanism, independent replication of the subgenomic mRNAs, predicts the presence of negative complements to all subgenomic mRNAs in infected cells which might be generated from subgenomic mRNAs in virions. Full-length antigenomic RNA was detected in LDV-infected macrophages by Northern hybridization at a level of < 1% of that of genomic RNA, but no negative polarity subgenomic RNAs. Negative complements to all subgenomic mRNAs, however, were detected by reverse transcription of total RNA from infected macrophages using as primer an oligonucleotide complementary to the antileader followed by polymerase chain reaction amplification using this sense primer in combination with various oligonucleotide primers complementary to a segment downstream of the junction between the 5' leader and the body of each subgenomic RNA. It is unclear whether these minute amounts of negative subgenomic RNAs function in the replication of the subgenomic mRNAs. They could also be by-products of the RNA replication process. Finally, no subgenomic mRNAs were detected in LDV virions.

Cytokine regulation of lactate dehydrogenase-elevating virus: inhibition of viral replication by interferon-gamma.

The mechanisms which regulate the replication of lactate dehydrogenase-elevating virus (LDV), a persistent murine model virus which infects macrophages, are unclear. For this study, the effects of murine recombinant interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on LDV replication were examined. LDV permissiveness was reduced in macrophages obtained from uninfected mice treated with IFN-gamma prior to cell harvest and in vitro LDV infection. Virus inhibition by IFN-gamma was also observed when neonatal LDV-infected mice were injected with this cytokine prior to macrophage harvest and analysis of LDV replication-positive cells. Persistently LDV-infected mice demonstrated an increase in viremia levels following treatment with TNF-alpha. Neither IFN-gamma nor TNF-alpha had any direct in vitro effect on LDV replication in cultured macrophages, suggesting that the actions of these cytokines required secondary or accessory in vivo events. These results provide evidence for cytokine-mediated regulation of LDV infection and support a role for the immune system in the LDV-host relationship.