Raymond R.R. Rowland

Lactate dehydrogenase-elevating virus : an ideal persistent virus ?

ConclusionsLDV contradicts all commonly held views about mechanisms of virus persistence, namely that persistence is primarily associated with noncytopathic viruses, or the selection of immune escape variants or other mutants, or a decrease in expression of certain viral proteins by infected cells, or replication in “immune-privileged sites”, or a general suppression of the host immune system, etc. [1, 2, 5, 54, 77, 78]. LDV is a highly cytocidal virus that invariably establishes a life-long, viremic, persistence in mice, in spite of normal anti-viral immune responses.One secret of LDVs success in persistence is its specificity for a renewable, nonessential population of cells that is continuously regenerated, namely a subpopulation of macrophages. Since the continuous destruction of these cells is not associated with any obvious health effects, this macrophage population seems nonessential to the well-being of its host. The only function identified for this subpopulation of macrophages is clearance of the muscle type of LDH and some other enzymes [59, 67, 68]. Furthermore, the effects of LDV infection on the host immune system, namely the polyclonal activation of B cells and its associated production of autoantibodies, and the slight impairment of primary and secondary antibody responses also do not seem to be severe enough to cause any clinical consequences.But how does LDV replication in macrophages escape all host defenses? Persistence is not dependent on the seletion of immune escape variants or other mutants ([58] and Palmer, Even and Plagemann, unpublished results). Also, LDV replication is not restricted to immune-privileged sites [5]. LDV replication persists in the liver, lymphoidal tissues and testis [66]. Only the latter could be considered a site not readily accessible to immune surveillance.Most likely, resistance of LDV replication to antiviral immune responses is related to the unique structure of its envelope proteins and the production of large quantities of viral antigens. High titers of anti-LDV antibodies are generated in infected mice but they neutralize LDV infectivity only very inefficiently and, even though the antiviral antibodies are mainly of the IgG2a and IgG2b isotypes, they do not mediate complement lyses of virions [31]. Interaction of the antibodies and complement with the VP-3/VP-2 heterodimers in the viral envelope may be impeded by the exposure of only very short peptide segments of these proteins at the envelope surface and the presence of large oligosaccharide side chains. Furthermore, since LDV maturation is restricted to intracytoplasmic cisternae [59, 71], the question arises of whether any of the viral proteins are available on the surface of infected cells for ADCC.CTLs also fail to control LDV replication. Altough CTLs specific for N/VP-1 are rapidly generated, these have disappeared by 30 days p.i. [26]. The reasons for this loss are unknown, but high-dose clonal exhaustion [41, 51, 77, 78] is a reasonable possibility since, regardless of the infectious dose, large amounts of LDV proteins are present in all the lymphoidal tissues at the time of the induction of the CTL response. Furthermore, after exhaustion of CTLs in the periphery, continuous replication of LDV in the thymus [65] assures that the mice become permanently immunologically tolerant with respect to LDV antigen-specific CTLs as a result of negative selection in the thymus. LDV might be a primary example for the effectiveness of a permanent clonal CTL deletion in adult animals under natural conditions of infection.The presumed modes of transmission of LDV in nature and the events associated with its infection of mice are strikingly similar to those observed during the acute and asymptomatic phases of infection with human immunodeficiency virus (HIV) [24, 29, 74, 78]. These include: (1) primary inefficient transmission via sexual and transplacental routes but effective transmission via blood; (2) primary replication in renewable populations of lymphoidal cells with production of large amounts of virus after the initial infection of the host followed by persistent low level of viremia in spite of antiviral immune responses; (3) persistence, reflecting continuous rounds of productive, cytocidal infection of permissive cells [59, 74] and the rate of generation of permissive cells which may be the main factor in determining the level of virus production (in the case of HIV, the rate of activation of CD4+ T cells to support a productive HIV replication might be the factor determining the rate of virus production and the progression of the disease); (4) rapid antibody formation but delayed production of neutralizing antibodies with limited neutralizing capacity; (5) rapid but transient generation of virus-specific CTLs; and (6) accumulation of large amounts of virus in newly formed germinal centers in the spleen and lymph nodes concomitant with an initiation of a permanent polyclonal activation of B cells resulting in an elevation of plasma IgG2a.The events described under points 2–6 might be generally associated with natural viremic persistent virus infections. Such persistent viruses, by necessity, have evolved properties that allow them to escape all host defenses and control of their infection by immunological processes is, therefore, difficult, if not impossible. Prevention of infection and chemotherapy may be the only approaches available to combat such virus infections.

Neonatal Infection of Mice With Lactate Dehydrogenase-elevating Virus Results in Suppression of Humoral Antiviral Immune Response but does not Alter the Course of Viraemia or the Polyclonal Activation of B Cells and Immune Complex Formation

Neonatal infection of FVB mice with lactate dehydrogenase-elevating virus (LDV) prevented the normal formation of anti-LDV antibodies observed in mice infected at 5 days of age or older. Even 22 weeks post-infection, the concentration of circulating anti-LDV antibodies in neonatally infected mice was insignificant. However, the time course and level of persistent viraemia were the same in neonatally infected mice lacking anti-LDV antibodies as in mice infected at 5 or 15 days of age which developed normal antiviral immune responses. The results support the view that LDV replication in mice is unaffected by antiviral immune responses and instead is primarily dependent on the rate of regeneration of LDV-permissive macrophages. This view is further supported by the following findings. Treatment of mice with cyclophosphamide or dexamethasone, which are known to increase plasma LDV levels, increased the proportion of LDV-permissive macrophages in the peritoneum. Injection of mice with interleukin-3, which is known to stimulate macrophage development, increased plasma LDV levels in persistently infected mice 10- to 100-fold. During the first month of age when mice possess a higher proportion of LDV-permissive macrophages than older mice and peritoneal macrophages exhibit self-sustained growth, the persistent plasma LDV titres were also 10- to 100-fold higher than in older mice. The polyclonal activation of B cells induced by LDV that results in a permanent elevation of IgG2a or IgG2b in the circulation, and the formation of 180K to 300K immune complexes containing IgG2a or IgG2b were also the same in neonatally infected mice and mice infected 5 or 15 days after birth. Thus, the polyclonal activation of B cells occurs in the absence of an antiviral humoral immune response and the immune complexes do not contain anti-LDV antibodies. The immune complexes probably consist of autoantibodies formed in the course of the polyclonal activation of B cells and their cellular antigens.

C58 and AKR mice of all ages develop motor neuron disease after lactate dehydrogenase-elevating virus infection but only if antiviral immune responses are blocked by chemical or genetic means or as a result of old age

Age-dependent poliomyelitis is a paralytic disease of C58 and AKR mice caused by cytocidal infection of anterior horn neurons with neuropathogenic strains of lactate dehydrogenase-elevating virus (LDV). The motor neurons are rendered LDV-permissive via an unknown mechanism through the expression of ecotropic murine leukemia virus (MuLV) in central nervous system (CNS) glial cells. Only old mice develop paralytic disease after LDV infection, but mice 5-6 months old or older can be rendered susceptible by suppression of anti-LDV immune responses by a single treatment with cyclophosphamide or X-irradiation before LDV infection. Younger mice appeared to be resistant in spite of this immunosuppresive treatment. The present results confirm that mice as young as 1 month of age possess CNS cells expressing ecotropic MuLV and show that these mice are susceptible to paralytic LDV infection provided their anti-LDV immune responses are blocked for an extended period of time by repeated cyclophosphamide treatments or by a genetic defect. Furthermore, old mice become naturally susceptible to paralytic LDV infection because of an impaired ability to mount a motor neuron protective anti-LDV immune response.

Regulation of transplacental virus infection by developmental and immunological factors: studies with lactate dehydrogenase-elevating virus.

Placental and fetal infections with lactate dehydrogenase-elevating virus (LDV) were determined by virus titration, indirect fluorescence antibody (IFA), and in situ hybridization with cDNA probes. Experiments were designed to determine the effects of gestational age, timing of maternal LDV infection, and immunological (antibody and cytokine) factors on mouse placental and fetal LDV infection. Virus infection of the placenta was detected at high levels (almost all placentas infected) within 24 h post-maternal infection (p.m.i.), whereas fetal LDV infection was detected only at a low level by 24 h p.m.i. The percentage of fetuses becoming LDV infected progressively increased between 24 and 72 h p.m.i. When fetal infection was studied at 72 h p.m.i., earlier gestational ages (9-11 days) were associated with fetal resistance to infection, whereas between 12.5 and 15 days of gestation, virus infection was detected in 50-71% of fetuses. Maternal treatment with interferon-gamma (IFN-gamma) or anti-LDV monoclonal antibodies was associated with reduced rates of fetal, but not placental, LDV infection. These results demonstrate that both developmental and immunological factors are important in the regulation of transplacental LDV infection.

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.

Lactate Dehydrogenase-Elevating Virus Variants: Cosegregation of Neuropathogenicity and Impaired Capability for High Viremic Persistent Infection

Neuropathogenic isolates of lactate dehydrogenase virus (LDV) differ from non-neuropathogenic isolates in their unique ability to cause a paralytic disease (age-dependent poliomyelitis, ADPM) in immunosuppressed C58 and AKR mice by cytocidally infecting their anterior horn neurons. We have recently reported that an original neuropathogenic LDV isolate, LDV-C-BR, contained a low level of a coexisting non-neuropathogenic LDV which, in a mixed infection of mice, rapidly outcompeted the former resulting in apparent loss of neuropathogenicity of the reisolated LDV. This correlated with an impaired ability of the neuropathogenic LDV to establish a viremic persistent infection. In the present study we identified the presence of three different quasispecies in another original neuropathogenic LDV by sequence analysis of cDNA clones of ORF 5 (encoding the primary envelope glycoprotein VP-3P) obtained from the isolate. Successful development of differential reverse transcription-polymerase chain reaction assays allowed us to biologically clone all three quasispecies through repeated end point dilutions. Only one of the quasispecies (LDV-v) was neuropathogenic. The other two, LDV-vP (probably the same as LDV-P) and LDV-vx (a novel LDV quasispecies that had not been previously identified), were non-neuropathogenic and found to be the common LDV quasispecies associated with almost all LDVs originally isolated from mice carrying various other transplantable tumors. The neuropathogenic LDV-v became selectively amplified in the spinal cords of paralyzed mice, but possessed an impaired ability to establish a persistent viremic infection and was rapidly out-competed by LDV-vP and LDV-vx in mixed infections, just as reported previously for LDV-C-BR. The results further support our hypothesis that neuropathogenicity and impaired capability for viremic persistence of LDV are determined by the same molecular feature. The only consistent and biologically relevant molecular difference we have observed between neuropathogenic and non-neuropathogenic LDVs is the number of polylactosaminoglycan chains associated with the ectodomain of VP-3P.

Mouse hepatitis virus infection of mice causes long-term depletion of lactate dehydrogenase-elevating virus-permissive macrophages and T lymphocyte alterations.

Abstract Intraperitoneal injection of pathogen-free B10.A mice with mouse hepatitis virus (MHV)-A59 resulted in a short subclinical infection which was terminated by a rapid antiviral immune response. The infection resulted in a rapid, but transient, about 10-fold increase in the number of macrophages and total cells in the peritoneum of the mice. This increase was preceded by a complete depletion of the peritoneum of the subpopulation of macrophages that supports a productive infection by lactate dehydrogenase-elevating virus (LDV). The depletion of LDV-permissive macrophages was a long-term effect; at 50 days post-infection with MHV, the proportion of LDV-permissive macrophages in the peritoneum had reached only 20% of that observed in the peritoneum of uninfected mice, whereas the total number of macrophages in the peritoneum had returned to normal. Furthermore, MHV infection resulted in a long-term alteration in the proliferative response of spleen T cells to concanavalin A (ConA) and in their ability to produce interferon γ; several times higher concentrations of ConA were required to induce a maximum proliferative response in spleen T cell populations from 5-week MHV-infected B10.A mice than in spleen T cell populations from infected companion mice but the former produced 5 times more interferon γ than the T cells from unifected mice.

Corticosteroid and immune responses of mice following mini-osmotic pump implantation

Serum corticosteroid (CS) levels were measured in (B6xA)F1 mice following the implantation of ALZET mini-osmotic pumps. Studies of shipment stress were used as a basis for comparison. At 24 and 48 h following the implantation of pumps CS levels were moderately elevated. However, immunization of animals at 24 h postsurgery with sheep erythrocytes showed normal immune responses. The results of these experiments indicate that the use of mini-osmotic pumps for the delivery of immunomodulatory drugs induces moderate and short-term increases in CS levels, which do not appear to interfere with normal immune function.