Ehud Katz

Kinetics of formation of neutralizing antibodies against vaccinia virus following re-vaccination.

Administration of vaccinia immune globulin (VIG), derived from vaccinated healthy adult volunteers, is the treatment-of-choice for patients suffering from severe complications following smallpox vaccination. The present study was aimed to determine the time interval after vaccination, at which the highest titer of neutralizing antibodies is obtained. Ninety-nine 18-year-old soldiers, immunized with vaccinia virus at birth, participated in the study, 87 of whom had detectable antibodies against vaccinia virus prior to re-vaccination. Their initial average neutralizing antibodies titer (NT50) was 27. Fourteen days after re-vaccination the titer reached 152 and then dropped to 136, 119, 110 and 87 at 21, 30, 45 and 60 d, respectively. The titers of vaccinia antibodies induced in vaccinees without detectable antibodies at the start of the study, were significantly lower and the titers observed after re-vaccination were: 62, 56, 66, 38 and 34, at 14, 21, 30, 45 and 60 d, respectively. In an additional study, 65 volunteers vaccinated at birth and again at the age of 8 years old were re-vaccinated. Fourteen days later their NT50 was higher than those vaccinated only at birth. It can be concluded that bleeding of vaccinees 14 d following re-vaccination is the preferable time for the preparation of VIG.

Inhibition of vaccinia virus late protein synthesis by isatin-β-thiosemicarbazone: Characterization and in vitro translation of viral mRNA

Abstract Thespecific effect of istin-βthiosemicarbozone (IBT) was manifested after vaccinia virus late protein synthesis had commenced. At 6 hr after infection, viral protein synthesis was inhibited by about 9596. We confirmed that λ portion of the virus-specific RNA appears to be degraded ( B. Woodson and W. K. Joklik, 1965, Proc. Nat. Acad. Sci. USA 54,946–953 ). Nevertheless, the amount of viral RNA that was capped, properly methylated, and polyadenylylated, was reduced by only about 50%. Moreover, RNA from IBT-treated cells stimulated cell-free protein synthesis to one-half the level obtained with RNA from control cells. Polyacrylamide gel electrophoretic analysis further demonstrated that RNA from IBT-treated cells was translated into late viral proteins in vitro. Thus, it seems possible that the inhibition of protein synthesis in IBT-treated cells does not result entirely or directly from either an inhibition of mRNA synthesis or from λ depletion of mRNA caused by accelerated degradation. An alternative possibility, that accelerated degradation is secondary to λ more immediate effect of the drug on protein synthesis, was considered.

Characterization and Mixed Infections of Three Strains of Vaccinia Virus: Wild Type, IBT-resistant and IBT-dependent Mutants

Summary The characteristics of IBT-dependent (IBT d ) and IBT-resistant (IBT r ) mutants of vaccinia virus were compared with those of the wild type (wt) strain. The mutants did not differ from the wild type strain by their sedimentation in sucrose gradients. Minor differences in the polypeptide composition of the virus particles and in the neutralization of the three virus strains by anti-vaccinia human immunoglobulin were observed. Mixed infections of the viruses in HeLa cells enabled the growth of the strains under their unfavourable conditions (wt in the presence of IBT and IBT d in the absence of IBT).

Inhibition of Vaccinia Virus Growth by the Nucleoside Analogue 1-beta-D-Ribofuranosyl-1,2,4-Triazole-3-Carboxamide (Virazole, Ribavirin)

Virazole or Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) inhibits the growth of vaccinia virus at a concentration ode to a certain extent in the presence of Virazole, the DNA fails to acquire resistance to deoxyribonuclease and virus particles are not formed. Reversibility of the antiviral effect occurs when the drug is washed out from the infected cultures or when guanosine at an equimolar concentration is added.

Antiviral activity of tunicamycin on herpes simplex virus.

Tunicamycin (0.5 microgram/ml) significantly lowers (2 to 3 log10) the infectious yield of herpes simplex virus type 1 grown in chicken embryo fibroblasts and in BSC1 cells. Although virus particles are formed and the synthesis of the viral deoxyribonucleic acid is only partially affected by the antibiotic, the glycosylation of herpesvirus glycopeptides is amost completely inhibited. The morphology of virus particles made in the presence of tunicamycin is similar to that of intact virus particles, as demonstrated by electron microscopy. This suggests that the absence of the carbohydrate side-chain from the viral glycopeptides does not affect the overall integrity of the virion but decreases very significantly the infectivity of these particles. Images

Location of vaccinia virus structural polypeptides on the surface of the virus particle.

Vaccinia virus has a complex structure. Thin sections of virus particles show three structural components: the outer envelope, the lateral bodies and a central structure, known as the ‘core’, which contains the virus DNA (Dales, 1963). Easterbrook (1966) showed that treatment with the nonionic detergent NP40, in the presence of 2-mercaptoethanol (2 ME) leads to the dissociation of the outer membrane and release of the core. By polyacrylamide gel electrophoresis, Holowczak & Joklik (1967) identified at least 17 structural polypeptides, three of which were associated with the core. Katz & Moss (1970) showed that some of these polypeptide peaks could be further separated into two to three polypeptides, thus increasing the total number of resolved structural polypeptides to 22. Our knowledge concerning the arrangement of vaccinia virus polypeptides on the particle surface is very limited. The present paper attempts to determine which of the virus polypeptides is situated on the surface.

Synthesis of Vaccinia Virus Polypeptides in the Presence of Isatin-β-Thiosemicarbazone

Isatin-β-thiosemicarbazone (IBT) at a concentration of 14 μM inhibited the multiplication of vaccinia virus in HeLa cells. For the first 3 h after infection, viral deoxyribonucleic acid (DNA) was synthesized in the presence of IBT at the same rate as in the control culture; the replication rate declined at a later stage. The DNA failed to be coated with proteins and to become resistant to deoxyribonuclease unless IBT was removed. “Early” and “late” viral polypeptides were formed in the presence of IBT, as revealed by polyacrylamide gel electrophoresis. The formation from precursor of a core polypeptide, a reaction blocked by rifampin, was not affected by IBT. Therefore, it is suggested that a maturation step later than the one blocked by rifampin is involved in the inhibition of vaccinia virus by IBT. Images

Isolation and Characterization of an IBT-Dependent Mutant of Vaccinia Virus

The antiviral activity of thiosemicarbazones against poxviruses was reported by Hamre, Bernstein & Donovick (1950). Thiosemicarbazone derivatives are clinically used in India and Africa. Appearance of drug-resistant and drug-dependent mutants in the course of viral therapy is a potential danger. Appleyard & Way (1966) and Ghendon & Chernos (1972) observed the selection of thiosemicarbazone-derivatives resistant mutants, during growth of the wild-type strain in the presence of these drugs. We shall describe here the isolation and characterization of an isatin-β-thiosemicarbazone (IBT)-dependent mutant (IBTD) of vaccinia virus, which needs IBT for its growth. The characteristics of this mutant are compared with these of the wild-type (wt) strain and an IBT-resistant mutant (IBTR), isolated in our laboratory. The two vaccinia mutants IBTD and IBTR were isolated after treatment of cells infected with wt virus with the mutagenic agent iododeoxyuridine in the presence of IBT.

An isatin beta-thiosemicarbazone (IBT)-dependent mutant of vaccinia virus: the nature of the IBT-dependent step.

Summary The IBT-dependent mutant of vaccinia virus needs IBT for its growth. IBT is not essential for the synthesis of virus DNA, but the formed DNA does not become resistant to deoxyribonuclease in its absence. Both ‘early’ and ‘late’ virus proteins are synthesized in cells infected with the mutant, but in the absence of IBT one of the virus core structural polypeptides, which is normally formed from a higher mol. wt. precursor, is not made. These findings suggest that the IBT-dependent step of the mutant occurs earlier in the maturation process of the virus than does the block produced by IBT with the wild-type strain.

Promotion of Globularia sarcophylla flowering by Uniconazol, an inhibitor of gibberellin biosynthesis

Abstract Globularia sarcophylla, originating from the Canary Islands, was recently introduced as a new cut flower in Israel. Two major problems have prevented its commercialization: the late-summer blooming and the low quality of its flowering shoots. In the present work we studied the factors affecting G. sarcophylla flowering. We found that long-day (LD) conditions slightly promote flowering but artificial photoperiodic illumination does not enable the manipulation of flowering time. On the other hand, treatment with the gibberellin (GA)-biosynthesis inhibitor, Uniconazol, had a dramatic promotive effect on flowering time. Application of Uniconazol in autumn or winter induced flowering in winter or early spring, respectively. The inhibitor did not advance flowering during the summer or in the phytotron under high temperatures. When plants were grown in the spring/summer under heavy shading, they did not flower unless they were treated with Uniconazol. Uniconazol treatment also improved flower quality by reducing the length of inflorescence pedicles. This effect was found in all seasons. Based on our results, we raise the hypothesis that Uniconazol treatment induces flowering by diverting assimilates to the apex. Inhibition of GA biosynthesis under conditions limiting photosynthetic activity reduces vegetative growth and increases the availability of assimilates to the apex, leading to flower initiation. However, under high irradiance and/or high temperatures, when the levels of assimilates are sufficient to induce natural flowering, the inhibitor has no further effect.