Ectopic expression of murine CD163 enables cell-culture isolation of lactate dehydrogenase-elevating virus 63 years after its discovery.

IMPORTANCE: Mouse models of viral infection play an especially large role in virology. In 1960, a mouse virus, lactate dehydrogenase-elevating virus (LDV), was discovered and found to have the peculiar ability to evade clearance by the immune system, enabling it to persistently infect an individual mouse for its entire lifespan without causing overt disease. However, researchers were unable to grow LDV in culture, ultimately resulting in the demise of this system as a model of failed immunity. We solve this problem by identifying the cell-surface molecule CD163 as the critical missing component in cell-culture systems, enabling the growth of LDV in immortalized cell lines for the first time. This advance creates abundant opportunities for further characterizing LDV in order to study both failed immunity and the family of viruses to which LDV belongs, Arteriviridae (aka, arteriviruses).

Age-dependent poliomyelitis in mice is associated with respiratory failure and viral replication in the central nervous system and lung.

Age-dependent poliomyelitis (ADPM) is a virally induced neuroparalytic disease of mice and a model for amyotrophic lateral sclerosis (ALS). ADPM is triggered in genetically susceptible mice by immunosuppression and infection with lactate dehydrogenase-elevating virus (LDV). Both ADPM and ALS are characterized by progressive degeneration of anterior horn motor neurons, and death in ALS is usually associated with respiratory failure. To assess respiratory function in ADPM, we investigated ventilation in conscious control and LDV-infected C58/J mice breathing air and then 6.5% CO(2) in O(2). Three days after LDV infection, ventilation in response to CO(2) was half of that compared to the uninfected state, but become normalized by 10 days. Administration of cyclophosphamide alone (200 mg/kg, ip), an immunosuppressant, had no effect on ventilation. Induction of ADPM by concomitant administration of LDV to cyclophosphamide-treated mice resulted in altered gait, hindlimb paralysis, wasting, decreased metabolism, and decreased body temperature by 4 degrees C relative to controls. Compared to baseline values, mice with ADPM had decreased tidal volume and ventilation while breathing air, and while exposed to the CO(2) challenge they were unable to increase tidal volume, frequency of breathing, or ventilation. Using in situ hybridization, LDV replication was noted within the spinal cord, brain, and lung, but not in the diaphragm. Thus, respiratory failure is a contributory mechanism leading to death in ADPM and is associated with LDV replication in the CNS and lung. This animal model may be useful to investigate physiological and molecular mechanisms associated with the development of respiratory failure in neurodegenerative diseases.

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.

Replication of lactate dehydrogenase-elevating virus in various species cell lines infected with dual-, ampho- and xenotropic murine leukaemia viruses in vitro.

Lactate dehydrogenase-elevating virus (LDV) replicates in mouse macrophages in vivo and in vitro. It has been shown that LDV infects and replicates in motorneurons of the spinal cord of old, immunosuppressed C58 mice, which results in an acute poliomyelitis. In spite of extensive study, cells or cell lines other than macrophages which could support LDV infection and replication in vitro have not yet been detected. We have shown that LDV can replicate in mouse or rat cell lines which were previously infected with ecotropic murine leukaemia virus (MuLV). It was examined in this study whether other types of MuLV (dualtropic, amphotropic and xenotropic viruses) can also render the mouse cells or cells of other species susceptible to LDV infection as well as the ecotropic viruses. LDV infection and replication were seen in mouse cells infected with ecotropic, dual-tropic and amphotropic viruses. These were also seen in mink, rabbit and human cell lines infected with dual-, ampho- and xenotropic viruses. These results suggested that virtually all four classes of MuLV have the ability to elicit, in mouse cells or cells from heterologous species, permissiveness to LDV infection. The percent of LDV-infected cells increased up to approximately 80% in concentrated neurovirulent LDV-C-infected ecotropic MuLV-infected-mouse cells. The susceptibility of the cells gradually declined when they were maintained for more than one month. The LDV antigen-positive cells appeared as early as 6-8 h p.i., when a large amount of LDV and MuLV were added simultaneously. The replication of LDV was inhibited in MuLV-infected cells which had been treated previously with actinomycin D and cycloheximide, but not with zidovudine (AZT). A small percent of mouse cells became susceptible to LDV, when the cells were treated with iododeoxyuridine. This suggested that the induction of endogenous MuLV or part(s) of its genome from mouse chromosomes resulted in cells that were permissive to LDV.

Susceptibility of C58 mice to paralytic disease induced by lactate dehydrogenase-elevating virus correlates with increased expression of endogenous retrovirus in motor neurons.

The induction of poliomyelitis by lactate dehydrogenase-elevating virus (LDV) in C58 mice is dependent upon several host factors including old age, loss of immune competence and genetic predisposition. Two genetic components segregate with susceptibility to this neurological disease: the presence of multiple proviral copies of N-tropic endogenous murine leukemia viruses (MuLV) and homozygosity of the permissive allele for N-tropic viral replication (Fv-1n/n). We have quantified the levels of RNA for several endogenous retroviruses, using virus specific oligonucleotide probes, in various tissues of C58 mice in relation to age and immunosuppression. A tissue specific increase in expression of 3.0 kb AKR MuLV RNA in the spinal cords of mice occurred with increasing age of the mice and was enhanced several-fold by immunosuppression in old mice. Susceptibility to LDV-induced poliomyelitis occurs in the same age dependent manner as AKR MuLV expression and is also enhanced by immunosuppression. In contrast, the mink cell focus forming virus (MCF) RNA levels in the spinal cord remained constant despite apparent variations in MCF RNA expression in other tissues, and no xenotropic retrovirus RNA was detectable in spinal cords or brains of the C58 mice. The increased AKR MuLV RNA in the spinal cord was shown by in situ hybridization to be mainly located in the same motor neurons that become infected with LDV in these mice and are destroyed as paralysis develops. These results support a novel dual virus virus hypothesis for LDV-induced poliomyelitis in which increased endogenous retroviral expression in motor neurons renders these cells susceptible to cytocidal replication of LDV and hence to the development of LDV-induced poliomyelitis.

Genotypic variation among six isolates of lactate dehydrogenase-elevating virus.

In the present study, six lactate dehydrogenase-elevating virus (LDV) isolates obtained independently from inbred mice were compared by RNA oligonucleotide fingerprint analysis. The genome RNAs of four of the isolates gave unique fingerprint patterns. The patterns obtained for the other two isolates were similar, but not identical to one of the four unique patterns. These results indicate that more than one genotype of LDV exists and that virus isolates can be grouped by genotype. We have also demonstrated the presence of a 3'-terminal poly(A) tract by direct sequencing of 3'-32P-labelled LDV genome RNA. The presence of a 3'-terminal poly(A) tract distinguishes LDV from the members of the family Flaviviridae, which lack a 3'-poly(A), and justifies the current classification of LDV within the family Togaviridae.

Immune response to lactate dehydrogenase-elevating virus: isolation of infectious virus-immunoglobulin G complexes and quantitation of specific antiviral immunoglobulin G response in wild-type and nude mice.

Lactate dehydrogenase-elevating virus (LDV) causes a normally benign persistent infection of mice, resulting in a life-long viremia characterized by the presence of circulating infectious immune complexes, impaired clearance of certain enzymes from the blood, and modification of the host immune response to various heterologous antigens. In this study, we isolated infectious immunoglobulin G (IgG)-LDV complexes in the plasma of persistently infected mice by adsorption to and elution from protein A-Sepharose CL-4B. We found that practically all infectious LDV in the plasma of persistently infected mice is complexed to IgG. LDV infectivity in these complexes was partially neutralized, but could be reactivated by treatment with 2-mercaptoethanol. We also quantitated total plasma IgG and anti-LDV IgG in wild-type and nude Swiss and BALB/c mice as a function of the time after infection with LDV by radial immunodiffusion and an enzyme-linked immunosorbent assay, respectively. Total plasma IgG levels nearly doubled in BALB/c mice during 150 days of infection. IgG levels in uninfected nude mice were only 20% of those in uninfected BALB/c mice, but during infection with LDV increased to approximately those found in uninfected BALB/c mice. Anti-LDV IgG levels were almost as high in nude mice as in normal BALB/c mice. Isoelectric focusing of purified IgG from BALB/c mice showed that LDV infection resulted in the enhanced synthesis of all 16 normal IgG fractions that we could separate by this method, which suggests that LDV infection results in polyclonal activation of IgG-producing lymphocytes.

Immune response to lactate dehydrogenase-elevating virus: serologically specific rabbit neutralizing antibody to the virus.

A rabbit was immunized with large amounts of the lactate dehydrogenase-elevating virus (LDV) over a 9-month period. The plasma from this rabbit possessed an anti-LDV IgG titer of 1:80,000 as measured by the enzyme-linked immunosorbent assay (ELISA) method and a neutralizing titer of 1:1,000 for the homologous strain of LDV. LDV neutralization at 4 degrees C followed single-hit kinetics. In contrast, mouse anti-LDV IgG in plasma of chronically LDV-infected mice failed to neutralize LDV at 4 degrees C and neutralization at 37 degrees C was slow, biphasic, and inefficient compared with the neutralization caused by rabbit anti-LDV IgG, even though high levels of anti-LDV IgG were detectable in mouse plasma by the ELISA method. Rabbit anti-LDV IgG neutralized one heterologous strain of LDV as rapidly as it did the homologous strain, but failed to significantly neutralize five other strains of LDV, all of which were originally isolated from different mouse strains bearing transplantable tumors. The results indicate clear serological differences between LDV strains. Cross-reactions between the strains, however, were observed by ELISA, using the antibody induced during persistent infection of mice with each LDV strain. Immunoglobulin M (IgM) from mice infected for 15 days with the various strains bound equally to our LDV strain. IgG obtained from 2-month-infected mice also cross-reacted, but to a varying extent which partly correlated with the specificity detected by neutralization. Both rabbit and mouse anti-LDV IgG enhanced the infectivity of LDV at a low multiplicity of infection for primary cultures of peritoneal mouse macrophages.

Replication of lactate dehydrogenase-elevating virus in macrophages. 1. Evidence for cytocidal replication.

Cultures of starch-elicited peritoneal mouse macrophages in medium containing macrophage growth factor (MGF) were infected with lactate dehydrogenase-elevating virus (LDV) and, after various times in culture, LDV production was monitored as a function of time by infectivity titrations in mice, by measuring [3H]uridine incorporation into LDV RNA and extracellular LDV, by autoradiographic analysis of the proportion of productively infected cells and by electron microscopy. Regardless of the age of the cultures when infected with LDV, only a small proportion of the macrophages (generally between 3 and 20% of the total) became productively infected after a primary infection; maximum virus RNA synthesis and virus production occurred during the first 24 h after infection and then decreased precipitously. Productively infected macrophages could be readily recognized in electron micrographs of 24-h infected macrophage cultures and in sections of spleens from 24-h infected mice by characteristic morphological alterations. These consisted of formation of clusters of double-membrane vesicles with a diameter of 100 to 300 mumol, budding of nucleocapsids into vesicles with single membranes and accumulation of mature virions in these vesicles. One to 4 days later, however, such cells were no longer found in infected cultures or spleens of infected mice and superinfection did not restimulate LDV replication. Cultures established with macrophages from 1-day LDV-infected mice also did not support LDV replication. We conclude that LDV replication in cultures or mice is limited to a single cycle in a subpopulation of macrophages and that infection leads to cell death and rapid phagocytosis of the dead cells by the resistant, uninfected macrophages.

Actinomycin D cytotoxicity for mouse peritoneal macrophages and effect on lactate dehydrogenase-elevating virus replication.

Treatment of lactate dehydrogenase-elevating virus(LDV)-infected mouse peritoneal macrophage cultures with actinomycin D (1 microgram/ml) resulted in a progressive reduction in the formation of LDV-specific RNA and mature virus as the time of incubation with actinomycin D increased beyond 2-3 h. This effect, however, seemed to reflect an unusual sensitivity of macrophages to toxic effects of actinomycin D. Macrophage cytotoxicity and lysis became apparent 3-4 h after addition of actinomycin D; the initiation of synthesis of Sindbis virus RNA, which is insensitive to inhibition by actinomycin D in other cell culture systems, was also reduced in actinomycin-D-treated macrophages. Macrophages propagated in L-cell-conditioned medium were found to be less sensitive to actinomycin D cytotoxicity and, correspondingly, the initiation and synthesis of LDV RNA were less affected.

Autoradiographic method for detection of lactate dehydrogenase-elevating virus-infected cells in primary mouse macrophage cultures.

Peritoneal cells from starch-injected Swiss mice were propagated in plastic petri dishes and on cover slips in a mouse L-cell-conditioned medium for 12 to 24 h and then infected with various multiplicities of lactate dehydrogenase-elevating virus (LDV). Over 95% of the cells in these cultures phagocytosed latex particles and were, therefore, considered macrophages. Infected and mock infected macrophage cultures were supplemented with [3H]uridine at various times after infection and with actinomycin D 30 min before addition of the [3H]uridine. After 1 or 2 h of further incubation, plate cultures were analyzed for LDV-specific RNA, and cover slip cultures were investigated by autoradiography. Other cultures were labeled in the absence of actinomycin D, and the culture fluid was analyzed for labeled LDV. There was a good correlation between the production of LDV-specific RNA and LDV and the number of heavily labeled cells in these cultures. The labeled cells in these cultures. The labeled cells, therefore, were equated with productively infected cells. Only a maximum of about 20% of the macrophages, however, became heavily labeled regardless of the multiplicity of infection or the time, after infection, at which the cells were exposed to [3H]uridine. Only background labeling was observed in the remainder of the cells and in mock-infected cells treated with actinomycin D. The highest proportion of labeled cells was observed when the cells were infected with a multiplicity of infection of about 2,000 mouse infectious units per cell and labeled from 6 to 8 h after infection. Thereafter, the proportion of productively infected cells decreased progressively, concomitant with a decrease in the amounts of viral specific RNA and of LDV produced by the cultures. The results indicate that the majority of the macrophages in primary macrophage cultures do not support LDV replication. Their nonpermissiveness may depend on the physiological state of the cells or reflect the presence of subpopulations of macrophages, but no morphological differences between productively infected an uninfected cells were detectable.