SUMMARYCultures of starch-elicited peritoneal mouse macrophages in medium containing macrophage growth factor (MGF) were infected with lactate dehydrogenaseelevating 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 ~m, 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.

SUMMARYA 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.

Mouse fibroblasts (L-929) and Vero (green monkey kidney) cells were infected with the rickettsia Coxiella burnetii, and persistent infections developed and were studied over a 6to 10-month period. Ultrastructural comparisons were made between the two infected cell types, and both were tested cytochemically for the presence of acid phosphatase, a marker enzyme of lysozymes. Rickettsiae were always observed within vacuoles, and some infected L cells showed flattened endoplasmic reticulum as compared with uninfected cells. Rickettsiae in Vero cells were most often seen in vacuoles containing whorls of membranes ("myelin configurations") which were also seen in uninfected cells. Rickettsiae in Vero cells were pleomorphic, with acid phosphatase reaction product in their periplasmic space. This suggests either rickettsial degradation by lysosomal enzymes which penetrated the cell envelope or a penetration after the rickettsiae were dead. Vacuoles of infected Vero cells showed much more reaction product than that in infected L cells, and most rickettsiae in L cells had a normal appearance and showed no reaction product in their periplasmic space.

Liver glycogen is depleted in guinea pigs infected with Coxiella burneti. Syntheses of the glycogen precursors uridine triphosphate and uridine diphosphate glucose are unaffected during Q fever, but glycogen synthetase activity is inhibited. Exogenous cortisol relieves this inhibition in infected animals. Orotate and amino acids are more rapidly incorporated into ribonucleic acid and protein during infection. It is proposed that the biochemical defect in the synthesis of glycogen lies in the inactivation of glycogen synthetase.

Surface layers of Coxiella burneti studied at a high resolution reveal a plasma membrane and an outer surface membrane 6 to 7 nm thick, and a thin, moderately electron-dense intermediate layer associated with the inner surface of the outer membrane of many cells. This layer appears to be unaffected by lysozyme treatment. Ruthenium red staining was used to delineate a layer of filamentous material external to the outer membrane; this fuzzy layer has a mean thickness of 20 nm and is not often seen on the surface of cells prepared by conventional means. Both antigenic phase I and II cells show a ruthenium red-binding surface layer. It is suggested that this fuzzy layer may be, among other possibilities, a highly branched mucopolysaccharide.