- Volume 28, Issue 3, 1975

N-methyl and N-ethyl isatin beta-thiosemicarbazones inactivate cell-free Parana and Pichinde viruses as well as three strains of lymphocytic choriomeningitis virus. This antiviral activity is abolished in the presence of the chelating agent EDTA. The rate of virus inactivation by N-methyl isatin beta-thiosemicarbazone is greatly enhanced and controlled by the addition of cupric sulphate. Divalent cations of other first transition series metals are less effective. A difference exists in the copper requirement for fast inactivation of the prototype arenavirus (lymphocytic choriomeningitis) and the Tacaribe Complex of viruses (Parana and Pichinde). In the presence of 20 µm-N-methyl isatin beta-thiosemicarbazone, LCM and Pichinde viruses can be inactivated at about the same rate if 20 µm-CuSO4 is added to the former and 160 µm-CuSO4 is added to the latter. Using 20 µm-N-methyl isatin beta-thiosemicarbazone and CuSO4 the inactivation of LCM is reduced, but not eliminated, in the presence of an equal amount of infectious Pichinde virus. Crude and highly purified Pichinde virus are inactivated at the same rate when exposed to identical concentrations of N-methyl isatin beta-thiosemicarbazone and cupric sulphate. There is little detectable change in inactivation rates when Pichinde or LCM viruses are grown in a variety of different cell lines.

The SV40 DNA that was generated by undiluted passaging of the virus was analysed by agarose gel electrophoresis. Nine bands of virus DNA were distinguished and each band contained a specific size class of DNA, all shorter than the complete SV40 genome as was determined by electron microscopy measurements. A difference of 2% in length, about 100 base pairs, resulted in a clear band splitting. Two sets of undiluted passaging were established and the defective DNA in the two sets had both different and similar size classes varying in length from 96% to 73% of the unit length SV40 DNA.

The pattern of polyadenylated messenger RNA (mRNA) synthesis in BHK cell monolayers, infected under defined conditions with herpes simplex type 1 virus has been investigated by polyacrylamide gel electrophoresis of pulse-labelled RNA isolated by oligo dT-cellulose chromatography. Two classes of mRNA molecules were synthesized in infected cells; these were not detected in uninfected cells. The rate of synthesis of the larger, 18 to 30S RNA class reached a maximum soon after infection and then declined, whereas the rate of synthesis of the 7 to 11S RNA class did not reach a maximum until much later and did not decline. In the presence of cytosine arabinoside, the rate of mRNA synthesis in infected cells was reduced but the electrophoretic pattern remained the same.

The effect of concanavalin A (Con A) on the course of early infection of HeLa cells with purified radioactive human rhinovirus type 2 (HRV-2) or poliovirus type 2 (P-2) has been examined. Several early steps in infection were inhibited before the uncoating of parental virus. Con A, at 100 µg/ml, reduces attachment of virus when added to cells before infection. Con A also detectably slows the normal progression of adsorbed virus to tightly bound forms characterized, in the case of HRV-2, by resistance to elution by EDTA, or in the case of P-2, by isolation of a characteristic virus-membrane complex. When Con A is added together with either virus, it also inhibits cell-mediated eclipse of infectivity and the formation of non-infective subviral particles.

Temperature sensitive mutants of bacteriophage Qβ have been isolated which fail in the synthesis of their virus RNA at the non-permissive temperature (42 °C). Nine mutants have been studied in some detail. Cells infected with these mutants at 37 °C and incubated long enough to produce substantial amounts of Qβ RNA cease Qβ RNA replication when shifted to 42 °C. The mutants can be classified into 3 groups according to the amount of Qβ RNA replicase activity exhibited in extracts from infected cells isolated at various times after shift to 42 °C: in group 1 mutants, enzyme activity is the same, regardless of the time of isolation after shift; in group 2 mutants enzyme activity increases with time of isolation after shift; in group 3 mutants, enzyme activity decreases with time of isolation after shift. Synthesis of all virus proteins is suppressed at 42 °C in cells infected with group 1 or group 3 mutants. In cells infected with group 2 mutants, synthesis of Qβ RNA replicase subunit β is increased, but synthesis of other virus proteins is depressed at 42 °C. The inhibition of virus RNA and protein synthesis is reversible. A detailed analysis of these experiments suggests that a defective Qβ RNA replicase is involved in the inhibition of both virus RNA and protein synthesis.

Nineteen recent isolates and three laboratory strains of herpes simplex virus types 1 and 2 were tested for their ability to produce clinical signs in mice following intradermal inoculation in the ear. All viruses produced erythema at the inoculation site; this was the most sensitive clinical sign of infection. Virus multiplication in the ear tissue was similar for both types 1 and 2 up to the fifth day after inoculation but type 2 viruses persisted for longer. Latent infection was demonstrated in cervical dorsal root ganglia. Type 1 viruses required a much higher dose than type 2 to produce neurological signs and death after intradermal inoculation but the difference was less after intracerebral inoculation.

Erythema of the inoculated ear recurred sporadically during several months observation in about half the mice that survived intradermal infection with a selected type 1 isolate. The presence of virus in the ear tissue during such recurrences was confirmed by electron microscopy and isolation of infectious virus. The system of ear infection in the mouse is presented as a new model for studying neurovirulence, and latent and recurrent infection with herpes simplex virus.

Replication of human cytomegalovirus (CMV) was inhibited by 50 µg/ml of rifampin. Nevertheless, a number of functions of CMV were still expressed in the presence of rifampin, including early cell rounding, and the development of immuno-fluorescent antigen, haemadsorption antigen, complement-fixing antigen and precipitin antigens. If rifampin was kept in the culture medium for longer than 48 h, infectious virus was not synthesized, but removal of rifampin resulted in restoration of virus titre within 24 h. In parallel with the restoration of infectivity, removal of the drug resulted in a sharp increase in macromolecular synthesis, first RNA and then virus DNA. The results suggest that rifampin blocks a stage in the production of m-RNA species.

Treatment of L cells with 3 to 10 mm 3′:5′-cyclic adenosine monophosphate (cAMP) in the presence of interferon was found to potentiate the development of antiviral activity. The dose response of interferon activity at various time periods in the presence and absence of cAMP indicated that potentiation of interferon activity by cAMP occurred at an early stage in the development of antiviral activity. Among the analogues of cAMP tested for interferon-potentiating activity, only the acylated derivatives were found to be active. Combined l-epinephrine and theophylline treatment of cells elevated cellular cAMP levels and also potentiated interferon-mediated antiviral activity.

Interferon was also found to elevate cAMP levels in L cells. This activity was limited to biologically active interferon and antagonized the depression of cAMP associated with vesicular stomatitis virus (VSV) infection of L cells. These observations suggest that some aspects of interferon’s biological activity is associated with an alteration in cellular levels of cAMP.

Jack bean (Canavalia ensiformis) and French bean (Phaseolus vulgaris) cv. The Prince were useful diagnostic hosts for broad bean stain virus and Echtes Ackerbohnenmosaik-Virus. Bottom and middle component of each virus complemented one another to give greatly increased infectivity but there was no complementation between components from the different viruses. Bottom and middle components of each virus were indistinguishable serologically.

The haemagglutinating ability of three strains of IBV was investigated. It was shown that whereas strain Beaudette had no detectable haemagglutinin, both Connecticut and Massachusetts agglutinated red cells of various species. The haemagglutinin of Connecticut was detectable after sucrose gradient purification whereas that of Massachusetts required both the purification step and incubation with the enzyme phospholipase C to reveal it. The agglutination could be inhibited by specific antisera. Some studies on the nature of the red cell receptor, and the possible presence of a receptor destroying enzyme, are reported.

An examination of BHK, CEF, and FHM cells chronically infected with frog virus 3 has been made by scanning and transmission (thin section, freeze fracture, and surface replica) electron microscopy. With minor differences the pattern of virus development is similar in all three cell lines. Virus particles were detected in cell nuclei which subsequently became degenerate very late in infection. Three inclusions were associated with frog virus 3 cytoplasmic foci of infection; lamella structures, extensive microtubule formation (in BHK and FHM cells), and linear crystalline structures. The last two structures may play a role in creating or maintaining the cell rounding c.p.e. revealed by scanning electron microscopy. Very late in infection most BHK and FHM, but not CEF, cells are stripped of the plasma membrane. Replicas of frozen fractured BHK cells featured cytoplasmic foci of infection, budding at the plasma membrane, and showed that at early times when virus is detected in the nucleus, the nuclear membranes are intact and morphologically unaltered. Budding at the plasma membrane was better resolved by scanning and as surface replicas. This demonstrated that sparse to profuse localized budding occurred. Frequently virus particles were located singly, or as multiples, at the end of, or along, cytoplasmic protrusions which occur both on the body of the cells and at the cytoplasmic/coverslip ‘interface’.

A synthetic nucleoside analogue 1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin or RTCA) inhibits the replication in tissue culture of influenza B virus and also a wide range of influenza A viruses of human, animal and avian origin. The synthesis of influenza virus-induced antigens and also structural and non-structural polypeptides is inhibited by RTCA as detected by immunofluorescence and by pulse labelling experiments with [35S]-methionine. The inhibitory effects of RTCA on influenza A virus replication in tissue culture is reversed by a molar excess of guanosine or xanthosine which suggests that the compound acts at an early stage of virus RNA synthesis prior to the utilisation of the latter nucleosides. A possible inhibitory effect of RTCA on cellular DNA replication is not excluded.

The P2 phage mutation vir56, like the previously studied vir22, is the result of an unequal replacement of a chromosome segment with non-homologous DNA. The end positions of the replacements are essentially the same in the two mutants, whereas the lengths of the replacements are quite different. A third chromosomal aberration, del3, has similar structure and position. These results strengthen the suggestion that the left ends of these three aberrations coincide with the point of exchange in integrative recombination.