Chen Even

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is not dependent on the seletion of immune escape variants or other mutants ([58] and Palmer, Even and Plagemann, unpublished results).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.

Persistent infection of mice by lactate dehydrogenase-elevating virus: effects of immunosuppression on virus replication and antiviral immune responses.

Maximum plasma titers (10(9)-10(10) ID50/ml) of lactate dehydrogenase-elevating virus (LDV) in mice are observed one day after infection, but then decrease 4-5 log during the next 5 weeks to attain a persistent steady-state level for the remainder of the life of the animal. The decrease in plasma LDV level during the first 5 weeks after infection and long-term viremia were not affected by lethal X-irradiation of the mice, daily injections of cyclosporin A or depletion of the mice of T cells by treatment with anti-CD4, anti-CD8, or anti-Thy1.2 monoclonal antibodies, although these treatments inhibited the formation of anti-LDV antibodies. LDV viremia was also the same in nu/nu and nu/+ Swiss mice, though the former did not mount an anti-LDV immune response, while the latter did. The appearance of anti-LDV neutralizing antibodies in infected mice 1-2 months after infection or the injection of infected mice with high doses of anti-LDV neutralizing monoclonal antibodies also did not affect the level of LDV viremia. Repeated treatments of infected mice with either cyclophosphamide or dexamethasone caused 1-2 log increases in plasma LDV titers. Although cyclophosphamide treatment prevented the formation of anti-LDV antibodies, dexamethasone caused an increase in plasma LDV levels without affecting anti-LDV antibody formation. We conclude that an anti-LDV immune response does not play a significant role in controlling LDV replication in mice. The data support the view that within 1 day after infection of a mouse, all LDV-permissive macrophages, which appear to be the only cells supporting LDV replication in the mouse, are destroyed as a result of a cytocidal infection by LDV. Subsequently, LDV replication is limited by the rate of generation of new permissive macrophages. The steady-state viremia attained about 5 weeks after infection reflects a balance between LDV replication in permissive macrophages as they arise and LDV inactivation and clearance.

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.

Differential glycosylation of the ectodomain of the primary envelope glycoprotein of two strains of lactate dehydrogenase-elevating virus that differ in neuropathogenicity.

ORF 5 encoding the primary envelope glycoprotein, VP-3P, of a highly neuropathogenic isolate of lactate dehydrogenase-elevating virus (LDV-v) has been sequenced. It exhibits 92% nucleotide identity with the ORF 5 of an LDV isolate that lacks neuropathogenicity, LDV-P, and the amino acid identities of the predicted VP-3Ps of the two strains is 90%. Most striking, however, is the absence in the ectodomain of LDV-v VP-3P of two out of three potential N-glycosylation sites present in the ectodomain of VP-3P of LDV-P. The ectodomain of VP-3P has been implicated to play an important role in host receptor interaction. VP-3P of another neuropathogenic LDV strain, LDV-C, lacks the same two N-glycosylation sites (Godeny et al., 1993). In vitro transcription/translation of the ORFs 5 of LDV-P and LDV-v indicated that all three N-glycosylation sites in the ectodomain of LDV-P VP-3P became glycosylated when synthesized in the presence of microsomal membranes, whereas the glycosylation of the ORF 5 proteins of LDV-v and LDV-C was consistent with glycosylation at a single site. No other biological differences between the neuropathogenic and non-neuropathogenic strains have been detected. They replicate with equal efficiency in mice and in primary macrophage cultures.

Mode of neutralization of lactate dehydrogenase-elevating virus by polyclonal and monoclonal antibodies

SummaryNeutralization of the infectivity of [3H]uridine-labeled lactate dehydrogenase-elevating virus (LDV) by polyclonal mouse or rabbit antibodies to the envelope glycoprotein of LDV, VP-3, or by neutralizing monoclonal antibodies (mAb) that recognize a different epitope on VP-3 than the polyclonal antibodies correlated with an increase in the sedimentation rate of LDV from 230 S to ≧270 S. Incubation of LDV with normal mouse plasma or nonneutralizing mAbs to LDV VP-3 had no effect on its sedimentation rate. Similarly, incubation of a neutralization escape variant of LDV with the mAb used in its selection had no effect on its sedimentation rate, whereas neutralization of this variant by polyclonal mouse or rabbit anti-VP 3 antibodies increased the sedimentation rate. Neutralization of LDV infectivity was only observed at high antibody/virion ratios and often was followed by loss of the viral RNA. The results suggest that neutralization of LDV infectivity results from binding of multiple antibody molecules that recognize specific epitopes on the viral envelope glycoprotein and ultimately leads to disintegration of the virions.

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.

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.