- Volume 20, Issue 1, 1973

We have studied the synthesis of virus RNA in human embryo lung cells infected with rhinovirus type 2. The three species of RNA in extracts of infected cells are, in order of decreasing electrophoretic mobility, single-stranded RNA, replicative form and replicative intermediate. The kinetics of synthesis of these RNA species were investigated. The electrophoresis of mixtures of single-stranded RNA synthesized early and late in infection showed no differences in size. This observation was confirmed with a bovine enterovirus. The native form of poliovirus type 1 single-stranded RNA migrated faster on polyacrylamide electrophoresis than rhinovirus single-stranded RNA.

The neural spread of pseudorabies virus in calves is described. Twenty-four calves were infected subcutaneously with the virus and killed from 48 h after inoculation until the terminal stages of the disease at 120 h post-inoculation. Virus tissue isolations and histological, fluorescent and electron microscopic techniques were employed.

Virus multiplied in the fasciae at the inoculation site for 60 h and subsequently appeared almost simultaneously in the entire 75 cm length of peripheral nerve, and related spinal ganglion and spinal cord segment. As the disease progressed virus spread cranially and caudally along the spinal cord so that by death virus was present throughout the central nervous system. The findings were indicative of a centripetal spread of virus along the peripheral nerve.

The studies established that a cellular progression of virus to the central nervous system was not possible. The pathway of the virus must have involved a fluid medium since it travelled over 75 cm in less than 72 h. The media available were the perineurial fluid, endoneurial fluid and axoplasm. No evidence of virus transport in the perineurial fluid was observed, and although some movement probably occurred in the endoneurial fluid, extracellular virus was not seen in either the nerve or ganglion. Both naked and enveloped virus particles were seen in the axoplasm of nerve fibres throughout the peripheral nerve and ganglion following replication in ganglionic neurons but virus particles were not identified during the incubation period. The passage of one or two infecting particles during the latter period, however, would be very difficult to detect by all the techniques employed. Evidence is presented for the axoplasm as the pathway of the virus to the spinal ganglion and central nervous system.

Steady-state chemostat cultures of mouse LS-cells grown in a chemically defined, protein-free medium were induced to produce interferon by a mycophage double-stranded RNA in a system potentiated by DEAE-dextran. The induction was terminated by the addition of heparin. The kinetics of interferon production in glucose-limited cultures were similar to those produced by the same induction system in batch culture. A maximum titre of approximately 2.8 log units/ml was reached 7 to 8 h postinduction. Interferon production was unaffected by changes in the cell growth rate within the range of dilution rates 0.25 to 0.35 day−1. Glucose-limited chemostat cultures (0.5 mg glucose/ml) yielded higher titres of interferon than cultures with excess glucose. Repeated induction of interferon was obtained in the chemostat without the development of a refractory state, although the repeated treatment was associated with inhibition of cell growth.

Examination of chloroplasts from chinese cabbage leaves infected with turnip yellow mosaic virus (TYMV), using freeze-fracture techniques revealed, that the small peripheral vesicles induced by infection were present in clusters near the surface of the chloroplast, often being arranged in arrays that approximate to hexagonal. The small peripheral vesicles were flask shaped with their necks pointing to the chloroplast surface. The structure of the necks, which were only about 15 to 22 nm in diam., suggested but did not prove that they were open to the cytoplasmic space.

The inner membrane of the peripheral vesicles is distinguished from other chloroplast membranes in showing no particles on either the A or the B fracture faces.

In the regions where the peripheral vesicles were clustered, the number and kind of particles seen on the chloroplast membrane faces were changed compared with healthy chloroplasts or the non-vesicle bearing areas of chloroplasts from infected cells. In particular, the B face of the outer chloroplast membrane in the vesicle-bearing regions had a reduced number of particles 10 nm in diam., but in addition to these contained large numbers of particles about 5 nm in diam.

In the chloroplasts of both healthy and infected tissue we frequently observed protuberances of the stroma corresponding to the mobile phase of chloroplasts postulated from light microscopic observations.

Ethidium bromide inhibits multiplication of encephalomyocarditis virus and synthesis of infectious single-stranded virus RNA. Infectious double-stranded virus RNA and cellular RNA synthesis are inhibited to a lesser degree. It is suggested that the effect is due mainly to binding of the drug to double-stranded replicative virus RNA.

The brucella phages A422, M51 and S708 were examined and compared with the Tbilisi reference phage. A422 was found to be identical in host range, adsorption pattern, physical and chemical stability and serological specificity with Tbilisi phage. M51 and S708, although, like A422 and Tbilisi, lytic for all smooth strains of Brucella abortus, were also lytic for Br. suis and Br. neotomae. All of the phages were morphologically identical with Tbilisi phage. M51 and S708 could be distinguished from each other and from A422 and Tbilisi by their serological properties. None of the phages were lytic for Br. melitensis, Br. ovis or Br. canis.

Mycobacteriophages B01, B02a, and B02h were studied with respect to host range, plaque morphology, electron microscopic morphology, nucleic acid composition, one-step growth characteristics, heat inactivation, adsorption, and cross-neutralization.

Phage B01 can be propagated only on Mycobacterium smegmatis. It forms plaques which are clear, circular and consist of several concentric rings. It has an elongated hexagonal head and long non-contractile tail. Its nucleic acid is double-stranded DNA with a 71% GC ratio. In heart infusion broth without Tween-80, phage B01 shows a latent period of 150 min, a rise period of 230 min, and burst size of 61.

Phage B02a is polyvalent, i.e. can be propagated on a number of different mycobacterial hosts. It forms plaques which are hazy and circular. It has a hexagonal head and long non-contractile tail. Its nucleic acid is double-stranded DNA with a 72% GC ratio. In heart infusion broth without Tween-80, phage B02a shows a latent period of 260 min, a rise period of 180 min, and a burst size of 11.

Phage B02h is also polyvalent and forms plaques that are clear and circular. This phage has a hexagonal head and a non-contractile tail. Its nucleic acid is similarly double-stranded DNA with a GC ratio of 72%. It is characterized by a latent period of 320 min, a rise period of 120 min, and a burst size of 20 in heart infusion broth without Tween-80.

The addition of 0.03% Tween-80 as recommended by Bowman (1958) decreases the burst size of all the phages studied. Tween shortens the latent period of phage B01 and B02h and the rise period of phage B01, but lengthens the latent period of phage B02a as well as the rise period of phages B02a and B02h. Heat inactivation of phage B02a is more rapid than that of B01 or B02h at 56 °C and 60 °C. Phage B01 is only slightly more heat sensitive than phage B02h.

All three phages adsorb poorly to their host bacteria. Phage B02h is strongly neutralized by anti-B02a serum, whereas neutralization of phage B02a by anti-B02h serum is considerably weaker. All three phages show some degree of cross neutralization.

BHK 21 cells infected with high multiplicities of polyoma virus show transiently various characteristics of transformed cells, including the appearance of the polyoma specific surface antigen (S antigen), as detected by immunofluorescence. Various inhibitors were tested for their effect on S antigen. Drugs interfering with DNA synthesis were found to block the appearance of this antigen, but did not affect its disappearance. On the other hand, actinomycin D did not block S antigen formation, but blocked its disappearance, and appeared to favour the maintenance of the BHK21 cell in the abortively transformed state. Inhibitors of protein synthesis prevented the appearance of S antigen. These results are discussed in relation to other results on the effect of inhibitors on virus-induced antigens or on virus-induced ‘unmasking’ of some lectin sites.

Human leucocyte interferon was rapidly cleared from the circulation of rabbits receiving 3 million units intravenously, but the clearance rate was greatly decreased after 1 h. The early half-time was 13 min and the late half-time 73 min. No circulating interferon was detected beyond 6 h. Repeated injections did not affect the clearance rate.

Intramuscular injection of 3 million units of human or rabbit interferon maintained a relatively stable interferon level in the serum for 12 h. Higher doses raised the level and prolonged the persistence of circulating interferon. A single intramuscular injection of 30 million units of human interferon maintained a detectable interferon level in the serum for 48 h. Subcutaneous injections resulted in even longer persistence of measurable interferon in the blood.

No interferon was detected in the serum after oral administration of 6 million units of human interferon.

A carrier state of Sindbis virus with cycling pattern was induced in the mouse L cells and in the established mouse embryo cell line MEC/c. Chick-adapted virus readily induced the persistent infection whereas the mouse-adapted virus killed cultures during the first passage. In the MEC/c line small (sp) and large (lp) plaque mutants of Sindbis virus produced more stable carrier state than the giant (gp) plaque mutant. The persistently infected cultures periodically produced small amounts of interferon. Highly specific rabbit anti-interferon globulin, which can neutralize mouse interferon, incorporated into the growth medium of carrier L line caused a 100- to 1000-fold stimulation of the synthesis of Sindbis virus after incubation for 5 days at 37 °C. The activated virus destroyed the carrier culture.

The effect of interferon pre-treatment on early virus protein and RNA synthesis has been examined in chicken cells infected with vaccinia virus. Protein synthesis was inhibited while RNA synthesis was stimulated.

Cytoplasmic inclusions, made up of a dense, finely granular matrix up to 3 to 4 µm in diam. were consistently found in ultrathin sections of leaf tissues from Fragaria vesca infected with strawberry vein-banding virus (SVBV). Isometric particles, usually with ring-like profiles, 40 to 45 nm in diam. were seen either embedded in the dense matrix or scattered in some electron-lucent areas of the inclusion. The particle morphology and the type of the inclusion found associated with SVBV places it in the cauliflower mosaic virus group.

Rinderpest, canine distemper, and measles viruses were all grown in Vero cell tissue cultures. Each virus was treated with ferritin-tagged antibodies to each of the viruses. The cross-reactions and electron micrographs showed that they all are antigenically and morphologically similar.