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In previous reports of this series, it was shown that persistent infection of MCN cultures with certain myxoviruses rendered the cells insusceptible to superinfection by several cytopathogenic viruses. It was thought that production of an interferon might be the cause of this resistance and efforts to confirm this suggestion have been presented. Addition of ultraviolet-inactivated myxoviruses (mumps, Newcastle disease, influenza A, and Sendai) to MCN cultures for periods of 2 to 3 hours, followed by washing and refeeding of the cells, led to the subsequent release into the media of a substance which induced in fresh MCN cells a transitory resistance to infection by vesicular stomatitis virus, and prevented incomplete reproductive cycles of influenza A and Sendai viruses. Media containing this substance were free of detectable hemagglutinating activity and viral complement-fixing antigens. The substance was not neutralized by specific antiviral sera; it was not sedimentable by high speed centrifugation; it was not adsorbed onto red cells; but it was inactivated by trypsin. Thus, its properties matched those of the interferon described by Isaacs and his associates. A comparison of the extent of resistance induced in MCN cells by decreasing doses of ultraviolet-inactivated myxoviruses (interference test) and the protection afforded by the media removed from the cultures prior to challenge and transferred to fresh MCN tubes (interferon test) revealed that wherever interference became detectable in the cells, the media of the corresponding cultures contained some interferon. Interferon was obtained by inactivated myxoviruses also from primary cell cultures by the same techniques, but not from HeLa cells. Interferons derived from one type of culture may protect others equally well or show a certain degree of host specificity in that resistance in homologous cells may be somewhat more pronounced than in heterologous cultures. No resistance could be induced in HeLa cells by the interferon preparations employed. Interferon was detected also in MCN cultures, persistently infected with mumps virus. Its concentration was apparently too small in carrier cultures maintained as routine to be measurable. However, when the cells were grown in heavy sheets in roller bottles, and especially when the volume of medium was reduced for several days prior to harvest, interferon became readily detectable. These results strengthen the suggestion that interferon may play a decisive role in the establishment and maintenance of persistent infections in the system under study. Its nature, source, mode of action, and exact role in persistent infection remains to be elucidated.

Ribonucleic acid extracted with phenol from Type 1 poliovirus, Coxsackie A-9, Coxsackie B-1, and ECHO 8 viruses infected non-primate cells and animals insusceptible to whole virus as such. Viral RNA was proved infectious for insusceptible cells in test systems of established cell lines, primary monolayer cultures, Maitland type cultures, and living animals inoculated intracerebrally. Cells of rabbit, swine, mouse, guinea pig, chicken, and hamster were infected. Each virus produced was identical with the virus donating RNA, in (a) neutralization by homotypic antiserum, (b) resistance to ribonuclease treatment, and (c) failure to be adsorbed or replicated by nonprimate cells, even of the strain producing the virus from RNA. Produced virus was adsorbed and replicated by susceptible primate cells as usual. Virus in RNA-infected cell cultures was produced in a single cycle unaccompanied by overt cytopathic effect on non-primate cells or disease of intracerebrally inoculated animals. By drastic elution of infective poliovirus associated with rabbit cells exposed to massive inocula of intact virus, intact poliovirus was shown to infect insusceptible non-primate cells to produce progeny indistinguishable from the parent virus population. Under these conditions, infection was accomplished by about 10 virus plaque-forming units per billion inoculated.

Inoculation of the MCN and Lung-To lines of human cells in continuous culture with Newcastle disease (NDV), mumps, or 6-6 viruses led to slight cytopathic effects (CPE) if the multiplicity of infection exceeded one. On second passage or with smaller initial inocula no CPE became apparent. The viruses multiplied, however, as determined by titrations in HeLa cultures or chick embryos. Indeed, persistently infected sublines of MCN and Lung-To were readily established without resort to special manipulations and some of these have been carried now for over 18 months on the same media and schedules as the uninfected parent strains. The viruses were found to be associated mainly with the cells and only 1, or at most 10 per cent of it was detectable in the media. The titers obtained were always low in relation to the available cell population. Reduction or even omission of the horse serum component in the media or ultraviolet irradiation of the cultures did not increase the yield of virus, and CPE became apparent only when similarly treated, uninfected cultures were, likewise, affected by the manipulations. The persistently infected cultures differed from their uninfected counterparts in that they exhibited (a) decreased cellular growth rates and ultimate yields; (b) increased aerobic glycolysis; and (c) a high degree of resistance to cytopathogenic viruses, influenza A (PR8), herpes simplex and, especially vesicular stomatitis (VSV) viruses. Prolonged treatment of persistently infected cultures by addition of specific antiviral immune sera to the media reduced significantly the amount of virus present and the degree of resistance to VSV. However, upon removal of the sera after as many as 187 days of treatment the viruses reappeared in all but one instance. The cured culture, on reinfection, became again persistently infected. No evidence was obtained to indicate genetic inhomogeneity of the cell populations. Of 50 cloned MCN lines none was destroyed by NDV and all became persistently infected. None were initially resistant to VSV but all after establishment of persistent NBV infection. All 39 cloned lines derived from MCN(NDV) cultures in the presence of anti-NDV serum, were free of virus and susceptible to VSV, and all acquired persistent infections and with it resistance to VSV following inoculation of NDV. NDV maintained in MCN cultures differed from the parent, chick embryo-adapted strain with respect to its plaque morphology. Whereas the former yielded only plaques on monolayers of chick embryo fibroblasts which were of pin-point size and hazy, those obtained with the latter were rarely of this type and mostly large and clear. This apparent selection of virus particles did not alter significantly their behavior with respect to cytopathogenicity for uninfected MCN cultures.

Hahon, Nicholas (U. S. Army Chemical Corps, Frederick, Md.) and James J. Friel. Comparative studies of the multiplication of antigenically related poxviruses on the chorioallantoic membrane of the chick embryo. J. Bacteriol. 83:837-843. 1962.-A comparison was made of the general growth curves of several poxviruses, variola, alastrim, vaccinia, cowpox, rabbitpox, and monkeypox, on the chorioallantoic membrane of the chick embryo. All curves showed a lag period, followed by a tenfold increase of infectivity each 5 hr for approximately 20 to 25 hr, a peak growth level at 48 to 52 hr (except monkeypox), and a gradual decline after 72 hr. Closer examination of the lag period revealed a rapid decline of virus infectivity within 2 to 4 hr after inoculation and the manifestation of an eclipse phase for 4 and 6 hr with all poxviruses. The lag period was terminated with the detection of intracellular virus at 8 to 12 hr, followed by the appearance of extracellular virus 2 to 6 hr later. The proportion of extracellular to intracellular virus was 0.1 or less throughout the different stages of poxvirus growth. In contrast to findings with the other poxviruses, however, extracellular virus equaled or exceeded the quantity of intracellular virus during the latter stages of the growth curves of variola and alastrim viruses.

The establishment of two strains of rhesus monkey kidney cells in continuous tissue culture, the development of a subline adapted to chemically defined medium, and the isolation of several clonal derivatives were described. Growth characteristics, chromosome numbers, malignant potentiality, and freeze storage data are presented. The cells were studied for their sensitivity to a large number of viruses and were extensively compared with primary cultures of monkey kidney cells for sensitivity to poliovirus. The cell strains were not sensitive to all the viruses which could be grown in primary cultures of the same tissue but were susceptible to most of them. In some instances an advantage to the use of the cell strain for certain viruses was noted.