SUMMARYFactor I of the anthrax toxin was isolated and showed one major component in the ultracentrifuge and on paper electrophoresis; it contained less than 5.5% of extraneous antigens detectable by serological precipitation in gels. The final preparation contained all the usual amino acids (N = 10*1%) and some carbohydrate (6 yo, calculated as glucose) and phosphorus (0.7 yo). The most striking aspects of its analysis were a high ash (10-13y0) and a light absorption a t 260 mp. The high ash was not due to one element but to a highly variable metal content (mainly Ca, Mg, Ni, Cu) indicating a powerful and indiscriminate chelating action of factor I. This chelating action might have been due to the chemical entity which absorbed light a t 260 mp and which was not RNA or DNA.The final preparation of factor I was not toxic when injected alone but when mixed with purified factor I1 it evoked oedema in the skin of a rabbit and killed mice. However, the concentration of this mixture which killed mice formed a much larger skin reaction in rabbits than a comparable dose (based on mouse LD50) of either crude toxin or a mixture of crude factors I and 11. An investigation of this fact led to the demonstration and partial purification of a third factor (111) of the anthrax toxin which: (1) was different serologically from factors I and 11; (2) was present in anthrax toxin produced in vivo; (3) was non-toxic when injected alone; (4) was lethal for mice when mixed with factor I1 but not with factor I ; ( 5 ) increased the lethality of mixtures of factors I and I1 for mice and decreased their capacity to produce oedema in the skin of rabbits. A mixture of factors I, I1 and I11 showed synergic action in toxicity tests in mice; the mixture killed guinea pigs which showed signs of oligaemic secondary shock (as did guinea pigs killed by anthrax infection).

Visna lentiviruses have a natural tropism for cells of the macrophage lineage of sheep and goats, but virus replication in these cells in vivo is restricted so that only small quantities of virus are produced. One restricting factor suggested in previous studies is that virus replication is dependent on the maturity of the cells: the more mature the cell, the less restrictive the replication of the virus. Since monocytes in peripheral blood are precursors of macrophages, we investigated the effect of cell maturation on virus replication under limited control conditions in vitro by inoculating blood leukocytes with virus and retarding the maturation of monocytes to macrophages during cultivation in serum-free medium. Using enzyme markers that identified the cells in their resting monocytic stage (peroxidase) and mature macrophage stage (acid phosphatase) along with quantitative in situ hybridization and immunocytochemistry with viral reagents to trace the efficiency of virus replication, we correlated virus replication with cell maturation. Only a few monocytes were susceptible to infection, and virus replication did not extend beyond a low level of transcription of viral RNA. In the acid phosphatase-positive, maturing macrophage, susceptibility of the cells to infection was increased and virus replication was greatly amplified to the level of translation of viral polypeptides. However, virus maturation was delayed by 3 days until further cell maturation had occurred. Thus, the entire life cycle of the virus, from its attachment to the target cell to its maturation in the cell, was dependent on the level of maturation/differentiation of the monocytic cell.

Wild-type Sindbis virus strain AR339 (SV) and a neurovirulent mutant (NSV), derived by neonatal and weanling mouse brain passage, both cause acute fatal encephalitis in neonatal mice, but NSV alone kills adult mice. NSV cannot be distinguished from SV by immune sera or simple biochemical tests. To localize the molecular changes associated with neuroadaptation, we used a new array of 30 anti-SV monoclonal antibodies to probe for differences between SV and NSV in four tests: immunoprecipitation, enzyme-linked immunosorbent assay binding, neutralization, and hemagglutination inhibition. Seventeen monoclonal antibodies detected differences. Both El and E2 glycoprotein gene products were altered during neuroadaptation, but the preponderance of changes was clustered on E2. The capsid protein C was not measurably altered. Mapping of both viruses with these monoclonal antibodies showed that during neuroadaptation SV topography substantially shifted, masking and unmasking biologically important neutralization and hemagglutination inhibition sites. These conformational rearrangements, predominantly on E2, coincided with the acquisition of increased neurovirulence and new lethality for adult mice.

SUMMARY:Highly labile factors (I and 11) of anthrax toxin were purified from the toxic plasma of guinea-pigs just dead from anthrax by fractionation methods which involved the xninimuxn of inaetivation. The final preparations of factors I and 11, which still contained constituents of guinea-pig plasma, were toxic when injected together but innocuous when injected separately.The specific, lethal and oedema-producing toxin of Bm*Zlw ar&hraciS found in the plasma of guinea-pigs dying of anthrax (Smith, Keppie & Stanley, 1955) has two components which form a synergic mixture (Smith et al. Further purification of these factors was hampered by their extreme lability. The present paper describes fractionations whereby they were considerably purified without appreciable loss of toxicity. A brief report of some of this work has appeared elsewhere (Stanley & Smith, 1958; Sargeant & Smith, 1958). METHODSAnthrax toxin produced in infected guinea-pigs was obtained as described by Smith et aZ. (1055). It was imperative that the plasma of guinea-pigs should be collected within a few minutes of death from anthrax. When delay occurred here, or in the subsequent removal of bacteria, the tendency for factor I to lose activity and to aggregate and become insoluble during subsequent fractionation was more pronounced.This was described by Peterson & Sober (1956). It was made in the laboratory from I.C.I. solka floc S.W. 40.B or bought from Eastman Kodak Co.Assay of factors I and I I of arzthrax toxin. Serial descending dilutions (0.1 ml.) of the test sample (factor I or factor 11) were injected intradermally into the shaven side of a rabbit, immediately after mixing each dilution with a fixed amount (0.1 ml.) of the complementary factor (either crude factor I1 or crude factor I; for preparation see below) a t a concentration which just failed to produce a skin lesion when 0.2 ml. was injected alone. The oedematous lesions formed by these mixtures of factors I and I1 were observed 16 hr. after injection and the null point, i.e. the first dilution of the test sample not giving a positive reaction, was noted as a measure of factor I or factor 11. The assay would detect twofold differences in activity.

hemagglutination, and antiserum to E2 neutralizes infectivity (11), but monoclonal antibodies (MAbs) to either glycoprotein can inhibit hemagglutination, neutralize infectivity, or both (9, 35, 36, 38). E2 may be an important determinant of virulence (30, 38). Recently Schmaljohn et al. (35) showed that neutralizing