Morris Pollard

Replication pattern of psittacosis virus in a tissue culture system

Abstract The growth curve of psittacosis virus in McCoy cells has been determined. There is a 17-hour latent period before virus production is demonstrable and maximum virus levels are achieved by 48 hours. Addition of either FU or FUDR had an inhibitory effect on the amount of virus subsequently produced, and this effect was reversible by uracil and thymidine (for FUDR only). These drugs probably act by immediately stopping the production of viable viral DNA, and the amount of virus ultimately synthesized by a culture may be a measure of the amount of viral DNA which had already been synthesized at the time of drug addition. By varying the time of drug addition it was found that there was a 10-hour lag between DNA synthesis and its incorporation into particles. This strongly suggests that psittacosis virus replicates by an assembly of subunits, not by binary fission, and hence is a true virus.

Electron and Fluorescence Microscopy of Mouse Hepatitis Virus.

Summary Livers of mice infected with mouse hepatitis virus (MHV 1) were examined by conventional staining procedures and by electron and fluorescence microscopy using acridine orange fluorochrome and fluorescein-tagged antibody. Preparations stained with acridine orange showed abnormal accumulations of RNA-staining material in cytoplasm of hepatic cells. Inclusion bodies of similar size, shape, and location were stained selectively with fluorescein-tagged antibody. Observations by electron microscopy showed virus particles in cytoplasm which measured 90 ± 20 mμ in diameter. The authors are indebted to Dr. R. H. Rigdon and his staff for invaluable technical assistance.

Ornithosis in Sea-Shore Birds

Summary Serological evidence of ornithosis-like infections among several species of sea-shore birds is presented. Over 40% of the serums of sea gulls, willets, and skimmers which were examined showed complement-fixing antibodies for ornithosis. One strain of an ornithosis-like agent has been isolated from the willet species.

CYTOCHEMISTRY OF TRACHOMA VIRUS REPLICATION IN TISSUE CULTURES

As psittacosis virus replicates, a cytochemical sequence has been observed with acridine orange stain and ultraviolet illumination.’ s 2 The viral deoxyribonucleic acid (DNA) initiates a reaction, in the course of which it is surrounded by a ribonucleic acid (RNA) matrix (red). Each red sphere (“red ball”) enlarges, multiplies, and maturates. In the course of maturation, each “red ball” goes through a sequence of color changes from red to orange to yellow to yellow-green. Only at the latter stage is the agent infective to animals and to fresh cells. Only at the latter stage will it absorb specific fluorescein-tagged antibody. This cytochemical sequence is dynamic and predictable. I t has been used in studies on the role of nucleic acids, in replication of psittacosis virus.3 z 4 Fluorinated pyrimidines have been incorporated into the process of viral biosynthesis with ensuing visual evidence of “fraudulent” RNA and “incomplete” DNA, neither of which were infectious. Addition of aminopterin to the nutrient fluid of newly infected cells interrupted development of psittacosis virus a t the “red ball” stage.6 Some antibiotics have induced cytochemically defined alterations in the pattern of viral maturation, thereby providing a tool for screening of drugs for psittacosis and related viruses.%S Thus, organizational stages in the chronology of virus replication have been demonstrated by means of inserting metabolic analogs at time intervals, which affected specific facets of the biosynthetic process. The sequence of participants thus demonstrable were folic acid, DNA, RNA, and protein. Interference at any point in this sequence interfered with the evolution of mature virus in the cultures. Most of the experimental procedures described above with psittacosis virus have been extended to trachoma virus, and the results support the familial interrelationship that has been demonstrated by other procedures. Materials and Methods

Propagation of duck hepatitis virus in tissue culture

Summary Duck hepatitis virus (DHV) survived storage for at least 21 days at 37°C. It survived treatment with di-ethyl ether and with fluorocarbon. DHV has been propagated through 25 consecutive passages in tissue cultures prepared with chick embryo explants. It was not propagated in cultures of trypsinized chick embryo cells or in cell lines of mammalian origin.

Susceptibility of Cortisone-treated Mice to Infection with Mouse Hepatitis Virus.

Summary A small proportion of mice inoculated with cortisone alone died or developed ascites and/or liver lesions. Bacteria were isolated from some of the livers showing cortisone-induced lesions. Homogenates of such livers were not infective for normal suckling Swiss mice. Cortisone acetate reduced susceptibility of normally resistant weanling Swiss mice to infection with the Gledhill strain of mouse hepatitis virus. The effectiveness of cortisone in decreasing host resistance was enhanced as dosage of the steroid was increased. There was a decrease in this cortisone effect as the time interval between virus and cortisone administration was increased from 1 to 5 days.

Oncolytic Effect of Viruses in Tissue Cultures.

Summary Tumor implants, growing in roller tube cultures, demonstrated a marked oncolytic response to St. Louis encephalitis infection. Two human and 2 rodent tumors were destroyed completely by SLE virus, less completely by neurotropic influenza virus. Infections with Lansing poliomyelitis, Newcastle disease, herpes and chick embryo adapted influenza A viruses did not interfere with growth of tissue implants. Rabies virus appeared to stimulate the growth of such tumor implants. The tissue culture technic can be used as a survey tool in rapidly determining the cytopathogenic effect of viral agents on tumors.

Infra Red Absorption Spectra of Viruses.

Summary Infra red absorption spectra have been determined of meningopneumonitis, ornithosis, mumps, and Newcastle disease viruses. The spectra of mumps and Newcastle disease are each distinctly different from that of meningopneumonitis and ornithosis, whereas the spectra of the latter two agents seem to indicate a close interrelationship.

Preparation of specific complement-fixing antigen with Lansing poliomyelitis virus.

Summary Specific complement-fixing antigens were prepared from cotton rat tissues infected with Lansing poliomyelitis, rabies, and St. Louis encephalitis viruses. The antigens were prepared by concentration of the viruses through high speed centrifugation. Lansing antigens prepared from lyophilized tissues were of little value as CF antigens. A relatively rapid rise and fall in poliomyelitis complement-fixing antibody levels in cotton rats is described. The Lansing antigen fixed complement with serum from Lansing-immune monkeys, but not with serum frommonkeys similarly immunized against Brun-hilde and Leon viruses.

Gytochemical Effects of Substituted Pyrimidines on Psittacosis Virus.

Summary Cytochemical changes associated with psittacosis virus infection of tissue cultures were studied by acridine orange staining and fluorescence microscopy. Normal virus development was interrupted by the addition of 5-fluorouracil and 5-fluoroorotic acid which caused the accumulation of “fraudulent” RNA. It is suggested that the substituted pyrimidines interfered with development of intact virus through inhibition of protein synthesis.