Lactate Dehydrogenase-elevating Virus RNA Research Articles

Inhibition of virus-induced age-dependent poliomyelitis by interferon- γ

Age-dependent poliomyelitis (ADPM) is a neuroparolytic disease which results from combined infection of susceptible mice with lactate dehydrogenase-elevating virus (LDV) and murine leukemia virus (MuLV). The present study examined the effects of interferon- γ (IFN- γ) treatment on the incidence of ADPM, replication of LDV and MuLV and anti-LDV immunity. IFN- γ treatment of ADPM-susceptible C58/M mice protected them from paralytic disease, but had no detectable effect on the IgG anti-LDV response or LDV viremia. IFN- γ-mediated protection from ADPM correlated with reduced expression of LDV RNA, but not MuLV RNA, in the spinal cords of C58/M mice. These results confirm that spinal cord LDV replication is the determinant of ADPM and demonstrate that cytokine-mediated inhibition of LDV replication in the central nervous system prevents neuroparalytic disease.

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.

Determination of the 5' end of the lactate dehydrogenase-elevating virus genome by two independent approaches.

We have determined the 5' end of the lactate dehydrogenase-elevating virus (LDV) genome (strain LDV-P) using two independent approaches. In one approach, methylmercuric hydroxide-denatured genomic RNA was reverse-transcribed using as primer an oligonucleotide complementary to the 5' end of open reading frame (ORF) 1a. The first-strand cDNA was ligated with T4 RNA ligase to an oligonucleotide of which the 3' end was blocked. The ligated product was amplified by PCR, cloned and sequenced. In the second approach, untreated or decapped genomic RNA was ligated between the 3' and 5' ends, reverse-transcribed across the ligation junction and the product was amplified by PCR, cloned and sequenced. Both approaches yielded the same results, indicating that the 5' leader of LDV-P is 156 nucleotides long, inclusive of the 5' UAUAACC 3' sequence involved in the linkage of the 5' leader to the bodies of the seven subgenomic mRNAs of LDV. The 5' leader of LDV is about 50 nucleotides shorter than those of the related viruses, equine arteritis virus and Lelystad virus, but at least twice as long as the leaders of the coronaviruses. The finding that untreated LDV RNA was ligated 5' to 3' end as efficiently as RNA treated with decapping enzyme suggests that genomic LDV RNA may not possess a 5' cap but terminates with 5' phosphoryl-A.

Lactate dehydrogenase-elevating virus (LDV): subgenomic mRNAs, mRNA leader and comparison of 3'-terminal sequences of two LDV isolates

The 3'-terminal 1314 nucleotides of the genome of one isolate of lactate dehydrogenase-elevating virus, LDV-P, has been derived by sequence analyses of cDNAs from several genomic libraries and compared to that of another LDV isolate, LDV-C (Godeny et al. (1990) Virol. 177, 768–771). The 3'-non-coding segment of 80 nucleotides of the two LDV genomes is identical, whereas marked, but varying nucleotide and amino acid divergence is apparent in the three upstream overlapping open reading frames (ORF). The third ORF from the 3'-end exhibits only 82% nucleotide and 90% amino acid identity, whereas the 3'-terminal ORF, which encodes the nucleocapsid protein, exhibits approximately 99% amino acid identity. The second 3'-terminal ORF encodes an 18.8 kDa protein which lacks N-glycosylation sites but possesses 2 or 3 potential transmembrane helices in the N-terminal half of the molecule. A similar membrane organization is observed for the corresponding protein of equine arteritis virus and the M protein of mouse hepatitis virus. The sequence analyses combined with Northern hybridization analyses of RNA from LDV-infected macrophages and spleens of LDV-infected mice indicate that the three ORFs encoded by the 3'-terminal end of the LDV genome are expressed via the three smallest mRNAs (mRNAs 6–8) of the seven subgenomic mRNAs of LDV (mRNAs 2–8), which range in size from about 0.8 to 3.6 kb. All mRNAs have been shown to carry poly(A)-tracts and a common leader sequence. The seven mRNAs were produced in infected macrophage cultures concomitantly with genomic LDV RNA. Maximum LDV RNA synthesis was observed between 6 and 8 h post-infection. The same seven subgenomic mRNAs were detected in macrophages infected with three different isolates of LDV, but different relative amounts of some of the mRNAs were produced. The relative proportions of molecules of mRNAs 1–8 present in 6 h LDV-P-infected macrophages were about 13, 5, 5, 8, 6, 11, 11 and 27% of the total, respectively.

Persistent infection of mice by lactate dehydrogenase-elevating virus: transient virus replication in macrophages of the spleen

In order to assess whether the spleen is the major site of replication of lactate dehydrogenase-elevating virus (LDV) in mice during the acute phase of infection, LDV replication in the spleen was measured by electron microscopy and fluorescent antibody staining of tissue sections and northern hybridization of total spleen RNA with an LDV-specific cDNA probe, and the effect of splenectomy on LDV replication was determined. LDV RNA and antigens and infected cells, presumably macrophages, were present in the spleen in high concentrations 18–25 h post infection, but then rapidly disappeared to undetectable levels during the next 1–2 days. Thus, LDV clearly replicates in the spleen during the initial phase of infection, but LDV replication in the spleen is transient due to the cytocidal nature of LDV replication and destruction of all permissive macrophages in the spleen. Furthermore, spleen macrophages do not seem to represent the major source of LDV released into the circulation, since LDV viremia as well as anti-LDV antibody production were the same in splenectomized and control animals for at least 28 days postinfection.

Extensive cytocidal replication of lactate dehydrogenase-elevating virus in cultured peritoneal macrophages from 1–2-week-old mice

Indirect fluorescent antibody staining was used to examine the replication of lactate dehydrogenase-elevating virus (LDV) in primary cultures of peritoneal macrophages from BALB/c mice of different ages. Up to 80% of the total peritoneal macrophages from 1–2-week-old mice were susceptible to productive infection by LDV, though only 1–2% of the cells expressed detectable levels of IA antigen. The proportion of LDV-permissive peritoneal macrophages progressively decreased to 5–15% between 2 and 5 weeks of age of the mice. Macrophages from 9-day-old mice, when cultured in the presence of L cell conditioned medium, retained undiminished LDV permissiveness for at least 10 days in culture. The maximum proportion of LDV antigen-positive cells was detected between 8–10 h post infection of macrophages cultured from both 1–2-week-old and adult mice, concomitant with maximum LDV RNA synthesis. The LDV antigen positive macrophages disappeared between 12 and 48 h post infection. In cultures of macrophages from 9–10-day old mice, the loss of infected cells was clearly due to cell killing, proving unequivocally that LDV replication is cytocidal. Disintegration of LDV-infected macrophages or phagocytosis of killed macrophages by surviving macrophages must be very sudden and complete since infected cells disappeared without the appearance of trypan blue-stainable cells in the culture. Ten cell lines established from macrophages of 2, 9, and 10-day-old mice all contained a small proportion of LDV-permissive cells (1–4%). Individual clones of one of the lines contained a similar small proportion of LDV-permissive cells.

Protection of C58 mice from lactate dehydrogenase-elevating virus-induced motor neuron disease by non-neutralizing antiviral antibodies without interference with virus replication

The paralytic poliomyelitis induced in old, immunosuppressed C58 mice by a primary infection with the lactate dehydrogenase-elevating virus (LDV) is prevented by the presence of anti-LDV antibodies in the virus inoculum or by passive transfer of LDV-free plasma from chronically LDV-infected mice one day before infection. Non-neutralizing antibodies were protective and specifically directed to the lowest molecular weight form of the envelope glycoprotein of LDV (VP-3), which seems to exist in virions in at least ten molecular forms ranging from 24 to 44 kDa. The antibodies did not prevent the productive infection of the subpopulation of macrophages that represents the primary permissive cell type in the mouse as evidenced by normal plasma LDV levels nor the spread of LDV to the central nervous system. Many non-neuronal cells containing LDV RNA were detected by in situ hybridization in the spinal cords of mice that had been infected with LDV in the presence of protective antibodies. However, no LDV RNA-positive neurons were detected, which are normally found coincidental with the development of paralytic symptoms in LDV-infected C58 mice. We propose that an early even after infection is critical for the infection of neurons and is inhibited by the presence of non-neutralizing antibodies to the LDV glycoprotein.

Correlation between presence of lactate dehydrogenase-elevating virus RNA and antigens in motor neurons and paralysis in infected C58 mice

Lactate dehydrogenase-elevating virus (LDV) induces poliomyelitis in immunosuppressed C58 mice resulting in fatal paralysis. We have synthesized and cloned cDNA complementary to the LDV genome, and used the cDNA clones as in situ hybridization probes for the detection of LDV RNA in tissue sections. Direct fluorescent antibody staining using IgG from chronically infected mice was used for the detection of LDV antigens. Using these methods, we have detected LDV RNA and antigens in anterior horn neurons of paralyzed mice. The appearance of LDV RNA and antigen positive motor neurons and their location in the spinal cord correlated with the development of paralytic symptoms. No positive neurons were detected in LDV-infected, susceptible mice without signs of paralysis, but some glial cells of the white and gray matter in the spinal cords of these mice were found to contain LDV RNA. These analyses broaden the host cell range of LDV to include neuronal and other cells in the CNS and support the hypothesis of LDV replication in neurons as the cause of poliomyelitis and paralysis.

Cell surface receptors for lactate dehydrogenase-elevating virus on subpopulation of macrophages.

We examined the binding and internalization of unlabeled and 125I-labeled, purified lactate dehydrogenase-elevating virus (LDV) by peritoneal macrophages cultured in vitro. Upon incubation of the cells at 4 degrees C with greater than 100 ID50/cell, the virus was surface-bound on a small subpopulation of macrophages (about 5% of the total cells) as determined by electron microscopy, fluorescent antibody staining, and autoradiography of cells incubated with 125I-labeled LDV. At 37 degrees C, LDV particles were seen in intracellular endocytic vesicles also in about 5% of the cells, and the proportion of cells with virus-containing vesicles correlated with the proportion of cells which became productively infected with LDV as assessed by determining LDV RNA synthesis in individual cells and by fluorescent antibody staining. Pretreatment of the resident peritoneal macrophages with trypsin inhibited the binding of 125I-labeled LDV and the productive infection of the cells with the virus. After removal of the trypsin and incubation in complete medium, permissiveness for LDV reappeared after an 8-12 h lag, whereas Fc and C3 receptors reappeared more rapidly after trypsin treatment. Populations of resident peritoneal macrophages, starch-elicited peritoneal macrophages, splenic macrophages, and bone marrow macrophages contained a similar proportion of cells that could be productively infected with LDV. Little, if any, LDV replication was detected in cultures of lung, liver and peripheral blood macrophages as well as in thioglycollate-elicited and BCG-activated macrophages. We conclude that the permissiveness for LDV of resident peritoneal macrophages correlates with the presence of a trypsin-sensitive receptor present on a subpopulation of these cells. The identity of the receptor has not been definitively established. Treatment of macrophages with neuraminidase or various sugars had no significant effect on LDV replication. Lysis of I-A-positive macrophages with a monoclonal antibody and complement reduced the number of macrophages which could be productively infected by 50%, which suggests that macrophages lacking surface Ia can be productively infected with LDV in vitro.

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.