- Volume 32, Issue 1, 1976

The rate of virus DNA synthesis and the production of infectious virus are impaired in stationary monkey kidney CV-1 cells irradiated with u.v. before infection with herpes simplex virus (HSV). The inhibition of HSV multiplication is due to u.v.-induced damage in cell DNA.

CV-1 cells recover their capacity to support HSV growth during the 40 to 48 h after irradiation, and the final virus yield is enhanced by a factor of 10. The time course of the recovery is similar to that of the excision repair process occurring in u.v.-irradiated mammalian cells. Caffeine, hydroxyurea and cycloheximide inhibit the recovery. Fluorodeoxyuridine is without effect.

A small but significant amount of labelled dThd coming from irradiated cell DNA is incorporated into virus DNA.

HSV specified thymidine kinase seems to be more effective for virus DNA synthesis in irradiated than in control cells.

The surface nature of the proteins of a bovine enterovirus have been determined by using 125I and pyridoxal phosphate-sodium borohydride labelling techniques. As found previously, 125I labels only VP1 in intact capsid particles, whereas reaction with pyridoxal phosphate followed by reduction with tritiated sodium borohydride labels VP1, VP2 and VP3. Only VP4 is found to have no surface tyrosine, histidine or lysine available for reaction. After neutralization with homologous antisera, however, VP4 becomes exposed and is then available for labelling with 125I. This must reflect a substantial conformational change in the virus particle after neutralization.

Protection of mice against EMC virus infection by poly C and poly I has already been distinguished from interferon mediated protection in several ways. Transfer of serum from EMC virus infected and poly C or poly I treated mice to donor mice that were then infected shows that the anti-viral effect of the single-stranded poly-nucleotides is not due to boosting interferon produced by infection itself in the way that interferon can be ‘primed’ in vitro. Mice surviving infections of more than 1 × LD100 as a result of poly C or poly I treatment show no protection against re-infection 15 days after the first infection, indicating no long-term stimulation of immune responses to the virus. Mice treated with an immunosuppressive regime of cytosine arabinoside can be protected against EMC virus infection with poly C and poly I treatment and athymic ‘nude’ mice can also be protected. The possibility of IgM stimulation by poly C and poly I seems unlikely from experiments in which serum was transferred from mice treated with the polynucleotides and an inactivated EMC ‘vaccine’ to recipient mice which were then challenged with infectious virus.

Protection of mice against EMC virus by the single-stranded polynucleotides is abolished by administration of silica to the mice, implying an involvement of macrophages in the protective effects of poly C and poly I. The possibility that the polynucleotides stimulate clearance of virus particles, at least from immunologically responsive regions of the mouse, has been discounted by the inability of polynucleotide treatment to suppress ‘vaccine’ mediated protection of mice. These results indicate that macrophages are involved in the anti-viral effects of poly C and poly I either because they inhibit replication of the virus in macrophages or because direct anti-viral properties of macrophages are activated by the polynucleotides.

The first reported phages for Ancalomicrobium adetum were isolated from three pulp mill waste aeration lagoons. In situ enumeration studies indicated that the concentration of these phage can fluctuate by two to three orders of magnitude in the natural habitat. The three phages contain DNA and are morphologically similar, having a hexagonal head and long flexible tail with no base plate or tail fibres. Compared to other phage the latent phase of the phages was long, ranging from 8 to 10 h. However, the doubling time of the host strains was 5.3 to 7.0 h so that the ratio of latent phase to generation time was consistent with previous studies of phage having shorter latent periods.

Increasing the host lawn density above 4 × 106 cells/ml or incubation temperature from 21 to 30 °C decreased both the plating efficiency and maximum plaque size for the phages. These results suggest that lawn density and incubation temperature are important factors in the initial isolation of phages from natural habitats.

A new phage-host system is described in which phage α 3a grows on stationary phase Achromobacter mutant strains. Characteristic clear plaques are formed, at an e.o.p. 10-1 to 10-2, on already confluent bacterial lawns of the mutant strains. Phage growth is sensitive to aeration and growth only occurs under micro-aerophilic conditions. Lysates prepared on the mutant strains cannot transduce in contrast to transducing lysates prepared from wild type Achromobacter strains.

Examination of P3HR-1 cells (Epstein-Barr virus [EBV] producer) persistently infected with the MAL strain of herpes simplex virus type 1 (HSV-1) suggested that only a few cells were actively producing a virus indistinguishable from HSV-1 (MAL) despite the presence of immunofluorescent HSV-1 antigens associated with the majority of cells. EBV-specific immunofluorescence was not altered in HSV-1 persistently infected P3HR-1 cells. HSV-1 persistently infected cells, labelled for 72 h with 14C-thymidine, incorporated approx. 8% of the label into cell associated HSV-1 DNA as resolved by caesium chloride gradients. Values greater than 8% of the total were suggested by hybridization of gradient fractions with 3H-HSV-1 DNA.

To determine whether the establishment of HSV persistent infections in Burkitt lymphoma derived cells was a general phenomenon, six strains of HSV-1 (MAL, KOS, Patton, Syn R, BF and Syn V) and two strains of type 2 (333 and MS) were used to infect the P3HR-1 and Raji (EBV non-producer) cell lines derived from Burkitt lymphomas. In P3HR-1 cells, persistent infections were established with all strains of HSV-1 but not with HSV-2. In Raji cells, persistent infections were established with all strains of HSV-1, except Syn V, and with both strains of HSV-2. No external support was required to maintain these infections.

The relationship of vaccinia haemagglutinin (HA) to extracellular enveloped virus (EEV) was examined. EEV banded in caesium chloride gradients at a density of 1.23 to 1.24 g/ml coincident with a peak of HA activity. EEV of an HA+ vaccinia strain showed greater than 90% adsorption to rooster red blood cells (RBCs) as detected by infectivity and 3H-thymidine labelling whereas intracellular naked virus (INV) of the HA+ strain and EEV of an HA- strain failed to show significant adsorption. The adsorption was specifically inhibited by antiserum to vaccinia. Adsorption kinetic experiments demonstrated a lack of temperature dependence on the total amount of EEV adsorbed. No elution of EEV from RBCs could be detected. The capacity of EEV to adsorb to RBCs was found to be stable at 56 °C for 30 min.

The infectious virus (HVJ-pi) obtained from BHK cells persistently infected with haemagglutinating virus of Japan was found to be temperature-sensitive as well as causing little or no cytopathic effect (c.p.e.) and leading to establishment of carrier cultures in several cell lines at both permissive (32 °C) and non-permissive (38 °C) temperature.

In order to obtain information about the role of HVJ-pi in the establishment of persistent infection, comparative studies were made of some phenotypic properties of HVJ-pi and HVJ-38 which was obtained by passing wild-type HVJ in eggs at 38 °C and was proved to be highly cytopathic. HVJ-pi differed from HVJ-38 in (1) temperature sensitivity in its ability to produce virus progeny, (2) infectivity for embryonated eggs, (3) neuraminidase activity, (4) the thermal stability of HA and neuraminidase activity, and (5) the polypeptide composition of BHK-grown viruses. BHK cells infected with HVJ-pi release haemagglutinin more efficiently, and less HA was accumulated on the cell membrane.

In considering these results, it was concluded that the difference of envelope proteins might be involved in the striking difference in c.p.e. between HJV-pi and HVJ-38.

Infection of mouse L cells with VSV leads to the formation of polykaryocytes about 4 to 12 h p.i. When anti-VSV immune serum was added during the course of infection, progression of cell fusion was soon suppressed. Cycloheximide completely suppressed the cell fusion when the drug was added within 1 h p.i., while the cell fusion was not suppressed at all when the drug was added at and after 3 h. Early polykaryocyte formation, ‘fusion from without’, was observed only at a low level in cells infected at very high multiplicities.

The development of cell fusion induced by VSV was found to be different in several cell types, although all these cells produced a rather high yield of virus: L and C-243-3 mouse cell lines showed a high level of polykaryocytosis (80 to 100%), BHK and RK-13 cells responded at low level, and PS and Vero cells showed no cell fusion in response to VSV infection. In PS cells, however, cell fusion occurred when VSV-infected L cells were co-cultivated. From these observations, the mechanism of cell fusion induced by VSV was discussed.

The enhanced glucose uptake by chick embryo cells as early as 72 h after infection with Rous sarcoma virus (RSV) was confirmed in this study to be an early indicator of cellular transformation. The glucose uptake of C/E BrL cells infected by various doses of BS-RSV of subgroup A showed that the relationship between the log-dose of virus and log-uptake was linear (slope, b 1 = 1.30 ± 0.14) when the ratio of the number of infectious virus particles to the number of cells in the culture was above 1:200. But infection of cultures with a relatively high dose of virus, for instance 103.5 focus forming units (f.f.u.) was ineffective for the measurement of cellular transformation using the criterion of glucose uptake, whereas a much lower dose such as 101.7 f.f.u. was sufficient to induce foci of transformed cells. We concluded therefore that the statistic of glucose uptake assay (GUA) measured at 72 p.i. is less sensitive than that of the focus count assay (FCA) measured after 10 days as a measure of assessing cellular transformation by RSV.

Nevertheless, when the cultures were infected with a higher dose of virus (104.3 f.f.u. or more), the GUA could discriminate between the transformed (T) and non-transformed (NT) cultures. This was demonstrated in the two genetic crosses, line 7–2 × WC(F1) and line 7–2 × C line. Embryo cultures of these two test-crosses were infected with viruses of subgroups A, B, C and D, and the T and NT phenotypes were ascertained. Also, on the basis of focus counts in the cultures the genetically resistant (R) and susceptibile (S) phenotypes in response to various infections were determined. The T and NT phenotypes based on the GUA were compared with the S and R phenotypes, respectively, based on the FCA. It was found that in 47 of the 51 cultures, the phenotypic agreement was perfect, suggesting that glucose uptake by cells of embryo cultures exposed to RSV is a biochemical indicator of genetic susceptibility. The discordant results in 4 cultures are discussed in the light of present knowledge of cellular transformation by RSV.

Focus formation by Rous sarcoma virus (RSV) was significantly enhanced when virus was incubated with the saline fraction of chloroform extracted chicken egg yolk, prior to infecting chicken embryo cells. The enhancement was restricted to members of RSV subgroups B, C and D and was proportional to yolk dilution. Subgroup A virus was never affected. In all, 108 yolk samples from specific pathogen free chickens were investigated. Of these 78 to 97% stimulated focus formation. RSV(RAV-50) of subgroup D which was stimulated up to tenfold, was the most strongly affected strain. The enhancing principle was shown to be a specific yolk factor, and its effect remained constant even after several years' storage. Crude yolk specimens showed essentially the same enhancing property. The chemical nature of the yolk factor is still unknown. It must, however, be taken into account when assaying for avian leukosis virus antibodies.

Selective removal of small plaque (SP) Western equine encephalitis (WEE) virus from a population heterogeneous with respect to virulence and plaque morphology permitted direct detection of a small sub-population of virulent large plaque (LP) WEE virus. Selective removal of SP-WEE virus was achieved by intracardiac (i.c.a.) inoculation of hamsters; plasma obtained 60 min after inoculation was proportionately enriched for LP-WEE virus since only the SP-WEE virus was cleared. By this method, the proportion of LP- to SP-WEE virus, in a population of SP-WEE virus which appeared to be homogeneous by conventional plaquing methods, was calculated to be 1 LP- to 250000 SP-WEE virions. The presence of a virulent LP-WEE virus sub-population explains why a single passage of a high but not low dose of SP-WEE virus in hamsters resulted in the emergence of an LP-WEE virus population with enhanced virulence.

A bacteriophage specific for Bacillus anthracis was isolated and designated as AP50. The nucleic acid of phage AP50 is RNA and the virion contains five different phospholipids. Some physical and biological characteristics of the phage, including morphology, were examined. To the best of our knowledge, this RNA bacteriophage containing phospholipids is the first to be isolated for a Gram-positive host.

The sedimentation rate of human leukocyte interferon (HLIF) reactivated from sodium dodecyl sulphate (SDS) solution was studied by glycerol gradient centrifugation and compared to that of native HLIF. Reactivated HLIF consistently sedimented faster than native HLIF, indicating that full recovery of antiviral activity does not require renaturation of the entire interferon molecule.

Vesicular stomatitis virus (VSV) was found to multiply efficiently in whole Peregrinus maidis (Ashm.), the leafhopper vector of maize mosaic virus (MMV), a plant rhabdovirus. Insects were inoculated with VSV by means of a microsyringe, collected at 1-day intervals and tested individually for the presence of virus. Exponential virus multiplication occurred within the first 4 days, reaching titres of 106 p.f.u. per insect in days 5 to 10 after inoculation. These observations show that a common host is available to study the multiplication of a plantand an animal rhabdovirus.

Vitamin A (retinoic acid) suppressed interferon production by L cells infected with Newcastle disease virus (NDV). This suppression was maximal when cells were treated with retinoic acid for 2 h after NDV adsorption, indicating that the inhibitory step was an early event. It was not due to inhibition of total RNA synthesis. Retinoic acid treatment caused both a delay in appearance of interferon and a reduced rate of synthesis thereafter.

Athymic (homozygous nude) mice of a non-inbred stock had relatively little antiviral activity in their serum compared with normal control mice at 4, 6 and 6.5 h after the intraperitoneal injection of Newcastle disease virus. The antiviral activity in the serum had the characteristics of interferon. At 10 h after injection and thereafter, the serum titres were comparable in nude and normal control mice. Exceptional nude mice with thymus-like tissue sometimes produced interferon more or less normally. Transfer of spleen cells from normal donor mice, but not from nude donors, led to increased serum interferon levels in nude recipient mice at 4 h after virus injection.

A dosage effect of chromosomal translocation was used to locate the gene(s) which codes for the human interferon induced antiviral state on the long arm of chromosome 21.