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

Abstract

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.

Lactate dehydrogenase-elevating virus: an ideal persistent virus?

“…Upon primary infection of a mouse, LDV productively infects all the permissive macrophages (estimated at about 106 cells/mouse) within the first day p.i. [50,59,71,73]. Plasma LDV titers reach up to 105~ IDs0/ml 1 day p.i.…”

“…Infections of primary cell cultures showed that only subpopulations of macrophages from the peritoneum and various tissues of mice support LDV replication to a significant extent [71][72][73]. LDV-infected cells were recognized by the presence of viral RNA or viral proteins in infected cells (see Fig.…”

“…and maximum virus is produced by about 16 h p.i. with a yield of about 10 4 50% infectious units (IDso)/cell [73]. Cell death ensues soon thereafter [56].…”

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

“…6A-E /ml) 0.5 h before incubation for 2 h with ['Hluridine (10 kCi/ml). After labeling the medium was discarded, the cells were lysed in a buffered solution containing 1% SDS and the lysates analyzed by zone centrifugation in 0.15-0.9 M sucrose density gradients containing SDS as described previously (Tong et al, 1977). Fractions from the gradients were analyzed for radioactivity in acid-insoluble material.…”

“…This possibility needed serious consideration since defective interfering RNAs in the molecular weight range of the subgenomic mRNAs arise rapidly in cells during undiluted passage of coronaviruses and toroviruses (van der Most et al, 1991;Makino et al, 1991;Snijder et al, 1991a) and since only six subgenomic RNAs have been detected in cells infected with EAV (den Boon et al, 1991) rather than the seven we find in LDV-P-infected cells. Furthermore, for an efficient infection with LDV, cultured macrophages must be exposed to very high multiplicities of infection (100-1000 mouse ID,,/macrophage; Tong et al, 1977;Onyekaba et al, 1989) and stocks of LDV generally consist of plasma harvested from mice 20-24 h p.i. with lo6 ID,,/mouse.…”