Much of the mystery of pathogenesis of animal virus disease lies in the realm of specificity of virus effect on cell type and in the differing effect of a given virus on different hosts. Tissue culture, despite its great contributions to other aspects of cytopathology, has not so far furnished significant clues to these basic problems. We have recently found that an acute virus disease of mice, mouse hepatitis," 2 has a selective destructive effect for cells derived from the reticulo-endothelial system (macrophages) and that the apparent genetic difference in susceptibility of different strains of mice is reflected in the behavior of macrophages from these strains in tissue culture. Tests of hybrids resulting from crosses between resistant and susceptible strains indicate that susceptibility is inherited and that genetic segregation of susceptibility and resistance occurs in the F2 and backeross generations.Materials and Methods.-The macrophages were obtained by explanting fragments of liver from newborn (1-3 days old) mice into roller tubes either directly onto the glass or onto a reconstituted collagen substrate.3' 4 The collagen was prepared according to the method of Ehrmann and Gey by extraction of 0.1 per cent acetic acid, dialysis against distilled water, and reconstitution to an agar-like slant with ammonium hydroxide vapors. The supernatant medium, except in otherwise specified cases; consisted of 60 per cent Gey's balanced salt solution, 10 per cent chick embryo extract (50 per cent), and 30 per cent horse serum (obtained from Microbiological Associates), with 0.004 per cent phenol red, 100 units of penicillin, and 10 micrograms of streptomycin. In some experiments, as designated in the text, chicken serum or a combination of chicken serum and horse serum was used. The chicken serum was obtained from White Leghorns kept for routine bleeding in our laboratory. The cultures were incubated in a roller drum and maintained in this manner at 370C throughout the experiments.The cultures were inoculated with the virus three to four days following explantation. The medium was renewed every two to three days until the end of the particular experiment. Supernatant fluids were frozen at -40'C and reserved for titration in mice. The cells were observed directly in the roller tube and their

SummaryWhen blood is withdrawn from Limulus, the horseshoe crab, a cellular clot composed of amebocytes quickly forms. Amebocytes are the only type of cell in Limulus blood. During coagulation, Limulus amebocytes undergo morphological changes that are strikingly similar to those seen when mammalian platelets undergo aggregation and viscous metamorphosis. The clottable protein in Limulus blood is derived entirely from amebocytes, and gelation does not require extracellular factors. Cell free Limulus plasma is incoagulable. The cellular localization of clottable protein in Limulus blood provides a precedent for the presence of fibrinogen in mammalian platelets.The clottable protein in lysates of amebocytes has a spectral absorption pattern with a maximum at 270-275 mμ, appears to have a low sedimentation coefficient, is stable at –20° C for 1 week, and is destroyed by heating at 56° C for 30 min. The protein gels upon exposure to endotoxin; and the rate of gelation is related to the concentration of endotoxin but is independent of the concentration of protein in the range tested. The kinetics of this reaction are consistent with the concept that an enzymatic system mediates the conversion of the cellular protein into a gel by endotoxin. Increase in light scattering during the reaction detects as little as 0.004 μg of E. coli endotoxin/ml. This conversion will perhaps provide insight into one of the biological activities of endotoxins.

PLATES 45 ~o 50(Received for publication, January 9, 1963) It is now well established that a number of animal virus diseases (1) as well as plant diseases (2) are directly affected by the genetic makeup of the host. In some cases resistance is dominant; in at least one (mouse hepatitis) susceptibility seems dominant (3). The demonstration that the macrophage is the cell which is paramount in susceptibility to mouse hepatitis virus, MItV, (4) was followed by a demonstration that cultures of newborn mouse liver from susceptible mice yielded susceptible macrophages and from resistant mice yielded resistant cells (3). The susceptibility factor was manifested in the hybrids, F~, and back-cross generations of cultures from young mice as well as in whole mice. It was, therefore, inferred that genetic susceptibility resides in the cells--in particular in the macrophages. In order to test this definitively, four kinds of tests have been carried out directly in the mice and in their cells, and the four methods compared: (a) individual mouse susceptibility (phenotype), (b) production of susceptible offspring (genotype), (c) susceptibility of macrophages from liver cultures, and (d) susceptibility of macrophages from peritoneal washings of adult mice. The first part of this paper reports the results of these correlations, describes the susceptibility of several back-crosses, and confirms the cellular nature of genetic susceptibility to this virus. The second part deals with the susceptibility of peritoneal macrophages in culture and the conversion of resistant to susceptible cells by the addition of extracts of the susceptible cells to cultures of resistant cells (5). If there is a correlation between the susceptibility of macrophages and the susceptibility'of the individual animal to MHV infection, then different kinds of cross and back-cross generations of susceptible and resistant animals can be studied by examining their macrophages without sacrificing the mouse, and the progeny can be used for selective breeding.

Peritoneal macrophages from genetically resistant C3H mice and genetically susceptible Princeton (PRI) mice adsorbed the MHV (PRI) strain of mouse hepatitis virus equally well. The difference between the permissive cells and the nonpermissive ones seems to reside in the ability of the former to "eclipse" the virus and, subsequently, support virus replication. C3H cells exposed to low multiplicities of the virus remained intact with no demonstrable viral replication. Virus, taken up by the resistant cells, was protected from heat and underwent slow inactivation while few or no virus particles were released into the medium.

lOs We are indebted to Dr. Norton Zinder of the Rockefeller Institute for performing the experiments on bacteriophage production. Levallorphan, a nonnarcotic analogue of levorphanol, was used in these experiments because of the legal intricacies involved in the transfer of narcotics from one laboratory to another. We found levallorphan to be a somewhat more potent inhibitor of RNA synthesis than levorphanol.

No abstract

No abstract

No abstract