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.

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.

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.

Inhibition of virus-induced age-dependent poliomyelitis by interferon-gamma.

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-gamma (IFN-gamma) treatment on the incidence of ADPM, replication of LDV and MuLV and anti-LDV immunity. IFN-gamma 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-gamma-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.

Trojan Horse macrophages: studies with the murine lactate dehydrogenase-elevating virus and implications for sexually transmitted virus infection.

Previous studies have suggested that monocytes or macrophages may mediate internal virus spread. For the present study, the tissue distribution and infectious potential of dye-labelled and/or lactate dehydrogenase-elevating virus (LDV)-infected murine macrophages were determined. Murine peritoneal macrophages were labelled with the fluorescent carbocyanine tracking dye Dil, injected into mice, and the tissue distribution of Dil-labelled cells was determined by fluorescence analysis of frozen sections. Mice receiving intravenous (i.v.) or intraperitoneal injections of Dil-labelled macrophages displayed rapid and broad tissue distribution of the labelled cells. Intravaginal injection of Dil-labelled macrophages resulted in penetration into the placentas, but not the fetuses, of pregnant mice. When macrophages were LDV-infected and Dil-labelled prior to i.v. injection into pregnant mice, they homed to various tissues including the placenta, but were not found in fetuses. Intravaginal injection of LDV-infected macrophages resulted in systemic LDV infection, even though the free-virus dose was less than the minimum infectious dose by this route. Neither polyclonal nor monoclonal IgG anti-LDV antibodies protected mice from vaginal infection with cell-associated virus, and LDV-immune complexes were themselves infectious by the vaginal route. These results show that exogenous macrophages are widely distributed following parenteral injection, penetrate locally to placentas after intravaginal injection, and are capable of acting vaginally as relatively efficient virus infection-delivery vehicles. Thus, 'Trojan Horse' macrophages are potentially infectious vehicles both for internal virus spread and for animal-to-animal transmission.

Determination of the viremia threshold for dental cross-infection in a mouse model.

An animal model of dental virus transmission was developed using the lactate dehydrogenase-elevating virus (LDV) of mice to study cross infection. Mouse-to-mouse cross-infection was carried out by scaling the teeth of LDV-infected donor mice with dental instruments, immediately prior to using the contaminated instruments on the teeth of recipient indicator mice. The level of donor viremia was found to correlate with the rate of virus cross-infection, with a viremia threshold level of 10(7.5) ID50/ml observed for dental cross-infection. The blood volume transferred during dental cross-infection was approximately 10(-4) to 10(-5) ml, demonstrating the inefficiency of virus cross-infection, since deposition of about 1000 virions on dental instruments was associated with the threshold limit. Virus transferred during dental cross-infection rapidly entered the blood circulation, showing that dental cross-infection was not dependent on an oral infection. The results from these model studies predict the general inefficiency of dental instrument virus cross-infection, and a further reduced likelihood of dental cross-infection with appropriately cleaned instruments.

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.

Effectiveness of ultrasonic cleaning of dental instruments.

PURPOSE: To quantitate blood contamination present on dental instruments used for routine prophylaxis and to assess the effectiveness of ultrasonic decontamination in reducing blood and virus contamination on dental instruments. MATERIALS AND METHODS: Human blood contamination present on dental instruments obtained after routine prophylaxis was analyzed using IgG as a blood marker. RESULTS: The estimated contaminating blood volume was found to normally range between 1.4 x 10(-6) to 2.0 x 10(-4) ml. Attempts to saturate the instruments with blood contamination suggested that the maximum possible retained blood volume was about 10-fold higher than the normal levels of contamination. Hand scrubbing of contaminated instruments was both relatively ineffective and inconsistent in removing blood contamination. Decontamination in an ultrasonic cleaner was more effective than hand washing, resulting in greater than a 100-fold reduction of blood contamination. Using a mouse model virus (lactate dehydrogenase-elevating virus, LDV), high levels of virus contamination of dental instruments and dental handpieces were achieved, as determined by assay of residual virus. Ultrasonic treatment reduced the level of virus contamination present on dental instruments by one million-fold, and virus contamination present in dental handpieces was reduced by one thousand-fold. These results provide quantitative estimations of the infection threat and its reduction by ultrasonication, posed by human-exposed dental instruments.

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.

Immunoglobulin transfer from immune-reconstituted SCID mice to nursing neonates: blood distribution of antibody and association with perinatal virus protection.

Previous work in our laboratory has shown that fetal protection from maternal transmission of the murine lactate dehydrogenase-elevating virus (LDV) infection is mediated by adoptive transfer of maternal anti-viral immunity. In the present report, we have characterized reconstitution of immunity in immunodeficient SCID mice following transplantation with BALB/c spleen cells, and studied the fate and distribution of maternally-derived antibodies after passage to neonatal SCID mice by nursing. Immune-reconstituted SCID mice maintained stable immunity for up to 7 months post-transplantation, during which time they produced nonneutralizing IgG anti-LDV antibodies and protected their offspring from maternally-derived LDV infection. Using IgG isotype and allotype assays, it was found that maternal IgG antibodies transferred from breast milk to nursing neonatal mice and appeared in their circulation. Weaning of SCID mice from immunocompetent mothers permitted the determination of blood immunoglobulin isotype half-lives (3.6-10.6 days) in the absence of endogenous antibody production. LDV infection was transferred to nursing mice by nonimmune LDV-infected mothers, but protection from nursing-acquired LDV infection was associated with maternal viral immunity, breast milk transfer of IgG anti-LDV to nursing mice, and reduced breast milk virus titers. These findings show a nursing pathway for LDV infection, and demonstrate the potential of immune protection from this infection pathway.

Virucidal effect of murine duodenal extracts: studies with lactate dehydrogenase-elevating virus.

Mucosal resistance to infection with lactate dehydrogenase-elevating virus (LDV) has been previously demonstrated, and the LDV system presents an important murine model for the study of mucosal barriers to viral infection. In the present study, duodenal molecules were isolated from normal mice which had potent virucidal activity, when tested against LDV as well as canine herpes, canine hepatitis, Semliki forest, and visna viruses. The virucidal activity was demonstrated to be non-immune in nature, and was present in apparently non-enzymatic protein molecules, having a molecular mass of between 10-100 kDa by membrane filtration and 10-17 kDa by gel filtration. The anti-LDV activity of these molecules was suppressed by anti-duodenum antibodies in vitro, and in vivo studies suggested a possible protective role for the anti-viral molecules. We conclude that the normal mouse duodenum contains potent virucidal molecules, which are of interest to the study of biological and molecular mechanisms of viral resistance.

Regulation of maternal-fetal virus transmission in immunologically reconstituted SCID mice infected with lactate dehydrogenase-elevating virus.

Immunodeficient SCID (C.B-17 scid/scid) mice with persistent lactate dehydrogenase-elevating virus (LDV) infection failed to produce IgG anti-LDV antibodies, and during chronic infection transmitted virus infection to 95% of their offspring. In contrast, normal mice infected 15 or more days prior to giving birth produced IgG anti-LDV antibodies and transmitted LDV infection to only 0-46% of their fetuses. Transplacental transmission of LDV infection was dependent on the timing of maternal infection. Adoptive transfer of immune competence to LDV-infected SCID mice resulted in fetal protection from maternally transmitted virus infection. Fetal protection correlated with the presence of maternal IgG anti-LDV but not with fetal levels of IgG anti-LDV, and the levels of viremia in nonimmune SCID mice did not affect transplacental virus transmission. These results demonstrate the importance of maternal immunity in protecting the fetus from infection, and validate the use of this mouse model for investigation of immune mechanisms of transplacental virus transmission.

Infection of SCID mice with lactate dehydrogenase-elevating virus stimulates B-cell activation.

Mice of the C.B-17 strain homozygous for the scid mutation (SCID mice) were infected with lactate dehydrogenase-elevating virus (LDV), and plasma samples obtained at intervals up to 42 days postinfection were analyzed for total immunoglobulins, anti-LDV antibodies, virus-specific immune complexes, and viremia levels. The mice responded to LDV infection with transient increases in total blood IgM, production of IgM-antigen complexes and IgM anti-LDV, as well as increased blood IgG2a. However, SCID mice failed to make a specific IgG2a anti-LDV immune response, and their blood LDV levels were elevated about 100-fold relative to those of control mice. The results suggest a role for IgG antibodies in the regulation of viremia and demonstrate a viral pathway of B-cell differentiation in SCID mice.

Enhancement of murine susceptibility to oral lactate dehydrogenase-elevating virus infection by non-steroidal anti-inflammatory agents, and antagonism by misoprostol.

The murine lactate dehydrogenase-elevating virus (LDV) was used to study the effects of prostaglandin-acting agents on mucosal resistance to virus infection. Mice treated with non-steroidal anti-inflammatory drugs (NSAIDs) prior to oral exposure to LDV demonstrated a reduction in the mucosal barrier to LDV infection. Histological studies indicated that these NSAID effects were not a result of gross or microscopic tissue damage. The effects of two NSAIDs, indomethacin and diclofenac, were inhibited by co-treatment of mice with misoprostol, a synthetic PGE1 analog. The ability of misoprostol to modulate NSAID effects was not due to direct antiviral activity or to actions on LDV-permissive macrophages. These results show that the mammalian mucosal barrier to virus infection is prostaglandin-sensitive, and provide a model for the study of resistance to viral infection.

Analysis of immunoglobulin isotype blood levels, splenic B-cell phenotypes, and spleen cell immunoglobulin gene expression in mice infected with lactate dehydrogenase-elevating virus.

B-lymphocyte activation was studied in mice infected with lactate dehydrogenase-elevating virus (LDV). ELISA determinations of blood total immunoglobulin levels demonstrated that, at 10 days post-infection (p.i.) with LDV, only the IgG2a isotype was elevated. DNA-excess dot-blot hybridization showed that RNA specific for IgG2a and IgA immunoglobulin isotypes was increased in the spleens of mice at 10 days p.i. with LDV. Immunoglobulin surface phenotype analysis of spleen cells at 8-10 days p.i. with LDV revealed that there was no alteration in immunoglobulin isotype-bearing cell proportions, although total spleen mass and number of cells increased during LDV infection. When blood immunoglobulins from LDV-infected mice were analyzed by two-dimensional isoelectric focusing gels, followed by specific immunoblotting for immunoglobulin isotype, the presence of new IgG2a species was observed at 10 days p.i.

The relationship between route of infection and minimum infectious dose: studies with lactate dehydrogenase-elevating virus.

At present there is incomplete knowledge concerning the relationship of route of infection to minimum infectious dose (MID) for viruses of humans or other animals. The present work has used lactate dehydrogenase-elevating virus (LDV) as a mouse model for this relationship. The data establish a relative mucosal barrier to LDV transmission, which is more effective at oral, ocular and vaginal sites, than at the rectal site of inoculation.

Infection of mice with lactate dehydrogenase-elevating virus leads to stimulation of autoantibodies.

The development of autoimmunity was investigated in BALB/c and C58 mice infected with lactate dehydrogenase-elevating virus (LDV). Autoantibodies reactive by ELISA with syngeneic central nervous system antigens appeared early during LDV infection of both strains of mice, and were maintained for many months. Western blot analysis indicated that the LDV-induced autoantibodies reacted with a variety of different brain antigens, and mouse strain differences in the pattern of autoreactivity were observed. LDV infection of C58 and BALB/c mice also stimulated antibodies reactive with syngeneic liver-, kidney- and spleen-derived antigens, and in Swiss outbred mice heart-reactive antibodies were observed following LDV infection. These results show that autoimmunity is a feature of the deregulation of the immune system which occurs during LDV infection.

Antibody response of mice to lactate dehydrogenase-elevating virus during infection and immunization with inactivated virus.

BALB/c and Swiss mice were infected with lactate dehydrogenase-elevating virus (LDV) or immunized with glutaraldehyde-inactivated or ether-extracted virus and their plasma was monitored for anti-LDV IgG and IgM levels by ELISA and indirect fluorescent antibody staining, for neutralizing antibodies, for sensitized antibody-virus complexes, for immune complexes, and for total plasma IgG and IgM. In infected mice, anti-LDV IgM was transiently formed during the first 2 weeks post infection (p.i.) but only at a low level. Anti-LDV IgG was produced in a biphasic manner with an initial peak at about 10 days p.i. and a secondary rise reaching a maximum level 30-80 days p.i. which was retained throughout the persistent phase of infection. The concomitant appearance of comparable levels of low molecular weight immune complexes suggests that most anti-LDV IgG was complexed with LDV proteins. Also, as early as 10 days p.i., infectious antibody-LDV complexes developed, which were neutralizable by rabbit anti-mouse IgG, whereas antibodies that neutralize the infectivity of exogenously added LDV appeared only 1-2 months p.i. Throughout infection, most of the anti-LDV IgG was directed to VP-3, the envelope glycoprotein of LDV, which was found to exist in at least 10 distinct forms ranging in molecular weight from 24 to 42 kDa. Anti-LDV IgG levels as high as those observed in infected mice developed in mice immunized with inactivated LDV. Antibodies to glutaraldehyde-inactivated LDV were also mainly directed to VP-3, but exhibited no neutralizing activity. The polyclonal B cell activation associated with a persistent LDV infection and the formation of immune complexes were not observed in mice immunized with inactivated virus.