William A. Cafruny

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.

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.

Replication of lactate dehydrogenase-elevating virus in macrophages. 2. Mechanism of persistent infection in mice and cell culture.

SUMMARY A primary infection of peritoneal macrophage cultures with the lactate dehydrogenase-elevating virus (LDV) results in productive infection of 3 to 20% of the cells. When cultures were incubated in the absence of macrophage growth factor (MGF), LDV production ceased after a single cycle, but in cultures in which macrophage replication was stimulated by the presence of MGF LDV production continued for several weeks at a low level, representing not more than 1% of that observed during the acute phase. Significant amounts of interferon were not present in either acutely or persistently infected cultures, and treatment of persistently infected cultures with anti-interferon globulin or superinfection with LDV did not significantly stimulate LDV replication. Macrophage cultures established with peritoneal macrophages from LDV-infected mice also showed only a low level of LDV replication and were resistant to superinfection by LDV. Mouse hepatitis virus, Semliki Forest virus and vesicular stomatitis virus, on the other hand, replicated normally in LDV-persistently infected macrophage cultures. LDV replication was relatively resistant to interferon whether added to the cultures or generated endogenously by infection with Newcastle disease virus or defective-interfering (DI) particles of vesicular stomatitis virus. Temperature-sensitive mutants or DI particles of LDV were not detected in LDV-persistently infected cultures or chronically infected mice. The results support our hypothesis that the decrease in LDV production in mice or macrophage cultures at the end of the acute phase results from the destruction of the subpopulation of macrophages that is permissive for LDV, and that the low level persistent infection involves the passage of the virus to new permissive cells that are generated continuously, although at a low rate, from non-permissive precursor cells.

Acute infection of mice with lactate dehydrogenase-elevating virus enhances Fc and complement receptor activity of peritoneal macrophages.

Peritoneal macrophages isolated from Balb/c mice 1 day after infection with lactate dehydrogenase-elevating virus (LDV) exhibited a 5- to 10-fold enhancement of attachment and ingestion of sheep red blood cells coated with immunoglobulin (EAIgG) or immunoglobulin plus complement (EAIgMC). Macrophages isolated from mice 7 days after LDV infection or macrophages infected with LDV in culture were also slightly more active than macrophages from uninfected mice, but the differences were not significant. The results indicate that a specific increase in the number of Fc and C3 receptors on macrophages occurs during the acute phase of infection. This increase correlates with the transient appearance of interferon in acutely infected mice. We postulate that during the acute phase the productive infection of a subpopulation of macrophages that is permissive for LDV results in the synthesis of sufficient interferon to cause activation of the remaining non-permissive macrophages in the animal.

Replication of lactate dehydrogenase-elevating virus in C58 mice and quantification of antiviral antibodies and of tissue virus levels as a function of development of paralytic disease.

Infection with the lactate dehydrogenase-elevating virus (LDV) triggers a generally fatal paralytic disease in old immunosuppressed C58 mice, but not in comparable mice of many other strains. We have compared the replication of LDV and the humoral immune response to it in C58 mice and mice of various resistant strains. Plasma LDV titres of persistently infected C58 mice were about tenfold higher than in other strains of mice and the proportion of LDV-permissive macrophages in peritoneal exudates of C58 mice was about twice as high as that observed in other mouse strains. C58 mice developed normal levels of anti-LDV IgG, as measured by ELISA, and normal levels of IgG that sensitized LDV to neutralization by rabbit anti-mouse IgG. C58 mice also developed normal IgM and IgG responses to human gamma-globulin and sheep erythrocytes. The antibody responses to LDV were similarly inhibited by cyclophosphamide in C58 and resistant strains of mice, which enhanced the incidence of signs of paralysis only in C58 mice. Thus, the sensitivity of C58 mice to LDV-induced paralytic disease is not due to an inherent inability of the mice to mount a humoral antibody response to LDV, and a suppression of the antibody response by cyclophosphamide is not the only prerequisite for development of the disease. We have quantified LDV in various tissues of immunosuppressed and non-immunosuppressed, 8- or 9-month-old C58 mice as a function of time after LDV infection and in relation to the development of paralytic disease. Changes in tissue LDV titres as a function of time after infection paralleled those found in the plasma; LDV titres were highest 1 day post-infection, and then decreased to a lower persistent level during the next 1 to 2 weeks. Tissue LDV titres, including those of the spinal cord, were lower than those in the plasma, and our results indicate that most of the LDV in tissue homogenates may be attributable to blood contamination, even though the animals were extensively perfused before removal of the tissues.

Hydrophobic IgG-Containing Immune Complexes in the Plasma of Autoimmune MRL/lpr Mice, Lactate Dehydrogenase-Elevating Virus-Infected Mice, and Pigs: Association with Transforming Growth Factor-β and pH-Dependent Amplification

Persistent infection of mice with lactate dehydrogenase-elevating virus (LDV) is associated with polyclonal B cell activation, autoimmunity, and circulating hydrophobic IgG-containing immune complexes (ICs), which bind to the surfaces of uncoated ELISA plates in the presence of 0.05% Tween 20. We demonstrate here that hydrophobic IgG-containing ICs also appear naturally in the plasma of autoimmune MRL/lpr mice. These and the similar hydrophobic ICs of LDV-infected mice as well as pigs coincide on ELISA plate surfaces with TGF-beta, apparently in the form of an IgG-TGF-beta complex. Circulating hydrophobic IgG-containing ICs are also susceptible to considerable amplification in vitro by exposure to alkaline conditions. By this latter method, the fraction of in vivo hydrophobic IgG, relative to the maximum in vitro chemically inducible IgG, was found to be about 20% in the plasma of LDV-infected mice, 5% in normal mouse plasma, and less than about 2% in pig plasma. These results indicate the potential for both chemically induced and protein-binding contributions to the generation of hydrophobic IgG-containing molecules, and have implications for immunopathological mechanisms in autoimmunity and persistent virus infections.

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.

Polyclonal Activation of B Cells by Lactate Dehydrogenase-Elevating Virus is Mediated by N-Glycans on the Short Ectodomain of the Primary Envelope Glycoprotein

The common strains of lactate dehydrogenase-elevating virus (LDV-P and LD-vx) are primary examples for viruses that cause a permanent polyclonal activation of B cells that results in IgG2a hyper-gammaglobulinemia and the generation of autoantibodies and circulating immune complexes in their host, the mouse (Notkins et al., 1966; Coutelier and van Snick, 1985; Li et al, 1990). Plasma IgG2a levels increase from generally below 0.5 mg/ml to 2-6 mg/ml by two weeks post infection (p.i.) and remain elevated thereafter (see later). LDV-P/vx cause life-long persistent viremic infections (see Fig. 1A) which are maintained by continuous rounds of replication in a renewable subpopulation of macrophages and resistance to host immune responses (Plagemann, 1996). Previous results have shown that the single neutralization epitope located on the short (about 30 amino acids long) ectodomain of the primary envelope glycoprotein, VP-3P, carries three large N-glycan chains in LDV-P and LDV-vx (see Fig. 1A insert) that suppress the immunogenicity of the epitope and impair antibody neutralization of the virions of these quasispecies (Chen et al, 2000; Plagemann et al., 1999). The present results indicate that the three N-glycans on the VP-3P ectodomains of LDV-P/vx also play a critical role in the polyclonal activation of B cells by these LDVs.

Antibiotic-Mediated Inhibition of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Infection: A Novel Quinolone Function Which Potentiates the Antiviral Cytokine Response in MARC-145 Cells and Pig Macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically significant agent for which there currently are no effective treatments. Development of antiviral agents for PRRSV as well as many other viruses has been limited by toxicity of known antiviral compounds. In contrast, antibiotics for non-virus microbial infections have been widely useful, in part because of their acceptable toxicity in animals. We report here the discovery that the quinolone-containing compound Plasmocin™, as well as the quinolones nalidixic acid and ciprofloxacin, have potent anti-PRRSV activity in vitro. PRRSV replication was inhibited by these antibiotics in both cultured MARC-145 cells and cultured primary alveolar porcine macrophages (PAMs). Furthermore, sub-optimal concentrations of nalidixic acid synergized with antiviral cytokines (AK-2 or IFN-γ) to quantitatively and qualitatively inhibit PRRSV replication in MARC-145 cells or PAMs. The antiviral activity of Plasmocin and nalidixic acid correlated with reduced actin expression in MARC-145 cells. Replication of the related lactate dehydrogenase-elevating virus (LDV) was also inhibited in primary mouse macrophages by Plasmocin. These results are significant to the development of antiviral strategies with potentially reduced toxicity, and provide a model system to better understand regulation of arterivirus replication.