- Volume 16, Issue 3, 1972

A hamster cell line (HDC-22) developed in our laboratory was non-permissive for herpes simplex virus type 2 (HSV-2) but not for herpes simplex virus type 1 (HSV-1). The mechanism of resistance to productive infection by HSV-2 was investigated. Adsorption studies revealed that HSV-1 and HSV-2 adsorbed at similar rates to the HDC-22 cells. The HDC-22 cells, when infected with HSV-2, produced minimal amounts of virus-specific antigens which increased slightly after several days.

The amount of HSV-2 DNA produced in the HDC-22 cells was always less than when these cells were infected with HSV-1. Cellular DNA synthesis was severely depressed by infection with HSV-1 at a multiplicity of 1 while HSV-2 had no effect at this multiplicity. At a higher multiplicity (of 5), this effect was increased with HSV-1 and noticeable with HSV-2. These studies indicate that the block for infection of the HDC-22 cell line with HSV-2 is after virus adsorption and may involve synthesis of virus DNA.

Thin sections of Datura stramonium L. leaf tissues infected either by the Rothamsted culture (HMV-r) or an Italian isolate (‘alkekengi’ strain = HMV-a) of henbane mosaic virus, were examined in the electron microscope.

The cytoplasm of most cells contained lamellar inclusions, which appeared as parallel lines, pinwheels, rings or arcs in the sections, typical of infection with viruses of the potato virus Y group, and long crystalline structures, apparently made up of elongated elements 8 to 10 nm. in diameter, arranged in a lattice. Flexuous threads, about 10 nm. wide, of moderate electron density, probably representing HMV particles in situ, were seen scattered in the cytoplasm or lying parallel to the tonoplast or the lamellar inclusions.

The most remarkable observation, however, was the presence of aggregates of numerous (up to 40) long, cylindrical mitochondria, tightly packed in certain areas of the cytoplasm. Between adjacent mitochondria there was a layer of elongated, virus-like particles, running parallel and longitudinally to the mitochondrial surface.

The fine structure of maize rough dwarf virus (MRDV) has been studied by examining particles in ultrathin sections, crude extracts, and in purified preparations from diseased plants. The virus has a diameter of 65 to 70 nm. and contains a dense core of about 40 to 50 nm. diameter that is enveloped by a thin inner capsid 3 nm across. The outer capsid is about 10 nm. wide and 18 holes are seen on the perimeter. Smaller particles, 40 to 50 nm. in diameter are found in all types of preparations and some have incomplete outer capsids consisting of 12 isolated projecting structures that adhere to the inner capsids. Purified preparations contained only the incomplete particles. The structure of MRDV particles is compared to that of reoviruses.

When centrifuged in caesium chloride solutions, purified preparations of uncloned cultures of the cam and prn strains of tobacco rattle virus contained more than one component but single lesion isolates of each strain contained only one component with buoyant densities of 1.306 and 1.324 g./ml. respectively. The buoyant density of the sp5 strain of pea early-browning virus was 1.310. As determined by electron microscopy the diameters of the particle and the core were respectively 23 and 5 nm. for prn and about 21.5 and 4 nm. for both cam and sp5. The mol. wt of the single virus protein, obtained from each freshly purified virus and estimated by polyacrylamide gel electrophoresis, were 28,500 (cam and prn) and 24,000 (sp5). Each virus produced nucleoprotein particles of two predominant lengths and these yielded two RNA species whose mol. wt were proportional to those lengths. The mol. wt (× 10-6) estimated by polyacrylamide gel electrophoresis were 0.7 and 2.5 (cam), 1.0 and 2.5 (prn) and 1.3 and 2.5 (sp5).

At pH 8.6 the electrophoretic mobility of the long particle of cam was 16% greater than that of the short particles. However, the long particles of cam have few if any antigenic determinants not possessed by short particles.

The small-plaque effect induced by exposure of herpes simplex virus to u.v. light was investigated. New results are presented showing that the effect was due to a delay of multiplication of virus in the cells initiating plaques. The delay occurred after entry of virus into the cell and before the replication of virus. The ultimate cause of the delay was probably the formation of thymine dimers, since the small-plaque effect was reversed by photoreactivation in avian cells. Evidence was obtained for co-operative reactivation and host-cell reactivation in infected BHK cells but neither of these phenomena could be related to the small-plaque effect.

Extracts of polyoma virus infected hamster cells made 8 to 10 hr after infection stimulated polyoma virus production in permissive mouse cells, whereas extracts made from 12 hr onwards inhibited virus production.

No such inhibitor was found in mouse embryo cells within 22 hr of infection under the same conditions. A genetic study of a mouse-hamster-hybrid cell line showed that non-permissiveness was not a dominant trait and seemed to depend on the inhibitor which could be expressed only in non-permissive cell lines. A specific inhibitor was found also in clones of hamster cells transformed by polyoma virus in vitro and in a clone of transformed hamster cells derived from a tumour induced by virus in vivo. This inhibitor did not reduce DNA synthesis or replication of non-infected permissive mouse cells. It reduced the number of infective centres and the yield of infective virus, infective DNA and of the polyoma DNA component I.

Particles of raspberry ringspot virus were homogeneous in immunoelectrophoresis tests but sedimented as three components, T, M and B, with sedimentation coefficients of 52, 92 and 130s respectively, and containing 0, 30 and about 44% RNA. The three components were serologically indistinguishable. In CsCl gradients, M and B were each homogeneous, with buoyant densities of 1.43 and 1.52 g./cm.3 respectively. Infectivity was associated with B. RNA extracted from the virus preparations was single-stranded and showed about 20% hyperchromicity in 0.1 m-sodium phosphate buffer at pH 7.0, indicating about 40% base-pairing. Analysis of RNA preparations by polyacrylamide gel electrophoresis revealed two predominant RNA species with mol. wt of 2.4 × 106 (RNA-1) or 1.4 × 106 (RNA-2); RNA-2 was indistinguishable in mol. wt from the RNA-2 of tobacco ringspot virus but RNA-1 migrated slightly more slowly than the RNA-1 of tobacco ringspot virus. M particles of raspberry ringspot virus contain one molecule of RNA-2; B particles contain either one molecule of RNA-1 or, probably, two molecules of RNA-2. Whereas B particles of raspberry ringspot virus were apparently homogeneous in CsCl gradients, B particles of tobacco ringspot virus formed two buoyant density classes.

Preparations of top (T), middle (M) and bottom (B) component particles were made by centrifuging purified raspberry ringspot virus in sucrose density gradients. Only B particles were infective alone, but infectivity was increased by adding excess M particles to B particles. RNA obtained from purified virus was fractionated by centrifuging in density gradients and by polyacrylamide gel electrophoresis. Preparations of the larger RNA species (RNA-1, mol. wt 2.4 × 106) were infective but those of the smaller species (RNA-2, mol. wt 1.4 × 106) were not; infectivity of RNA-1 was greatly increased by addition of RNA-2. RNA-2 from M and B particles behaved similarly. The effect of RNA-2 was not mimicked by yeast RNA and was abolished by u.v. irradiation. The RNA-1 and RNA-2 of tobacco ringspot virus behaved like those of raspberry ringspot virus in infectivity tests, but no interaction was detected between RNA species from the two viruses. It is suggested that RNA-1 and RNA-2 carry different pieces of genetic information.

Purified preparations of the golden elderberry strain of cherry leaf roll virus contain two spherical nucleoprotein components with sedimentation coefficients of 115s and 128s. Purification of the components by centrifuging several times through sucrose density gradients decreased their specific infectivity. The original specific infectivity of unfractionated virus was restored by mixing the separated components, indicating that both components are necessary for maximum infectivity. The components were indistinguishable serologically, in their extinction spectra and in their appearance in the electron microscope. Polyacrylamide gel electrophoresis of preparations from each component indicated that both contained a single protein species of 54,000 mol. wt, but that whereas the 115s component contained a RNA molecule of 2.1 × 106 mol. wt the 128s component contained one of 2.4 × 106. These and other properties suggest that cherry leaf roll virus can be considered a member of the nepovirus group. The present cryptogram of the golden elderberry strain is R/1 : 2.1/40 + 2.4/43 : S/S : S/*.

In these studies in mice, guinea pigs and rabbits infected by intraperitoneal, intracerebral or respiratory routes, the expression of virulence by a virulent/avirulent mixture of known proportion depended on the administered dose and was not a simple marker for the virus population, or for the heterogeneous wild strain which it simulated. This dependence of the virulence of a virus sample upon its dose and heterogeneity is presented quantitatively for each host by a dose-response diagram which is the necessary extension of the simple dose-response curve. The latter may be used to express single response characteristics (death only, protection only) but is inappropriate to the expression of the present dual response-dose characteristics in which protection at low dose gives place to death at high dose, or vice versa. At some proportions of virulent/avirulent sub-populations in the virus inoculum even more complex dual response-dose characteristics may be generated. Thus the specification of virulence requires the presentation of a dose-response diagram for each relevant host and route of administration of virus.

Notwithstanding these seeming complications, basic types of virulent/avirulent interaction have been demonstrated and arranged in sequence according to the susceptibility or responsiveness of the host-route systems investigated. With closer definition of population heterogeneity and dose-response relationships, other virus-host-route systems will probably fit within similar sequences.

These results are interpreted in terms of a dynamic interaction between distinct lethal and protective responses and are relevant to problems involved in the design and testing of live vaccines.

The biological activity of protein-free transfectious DNA isolated from phage 3NT of Bacillus subtilis was not altered after treatment with diethyl pyrocarbonate (DEP), and the DEP-sodium dodecyl sulphate (SDS) method could be used to extract transfectious DNA from phage 3NT. No transfectious DNA could be isolated from phage T4 of Escherichia coli by the conventional phenol-SDS or by the DEP-SDS method. The DNA extracted from T4 phage either by the phenol-SDS or by the DEP-SDS method had about the same transforming activity, and the transforming activity of T4 DNA extracted by the phenol-SDS method was not reduced by DEP treatment. On the other hand, protein-containing infectious particles obtained from T4 phage of E. coli were inactivated by DEP. It is concluded that DEP does not react with double-stranded phage DNA and can be used in the extraction of biologically active phage DNA, if no protein is needed for biological activity.

Incubation of bacteriophage φX174 with isolated bacterial cell walls in tris HCl. MgCl2 solution results in disruption of the phage with partial release of virus DNA. After incubation for 5 min., particles which sedimented heterogeneously at about 50s were formed. These particles were infectious for spheroplasts, but not for whole bacteria. Incubation for 40 min. produced 70s particles which contained less DNA than intact phage. Similar results were obtained when φX174 virus particles were incubated with starved bacteria.

The five vesicular stomatitis viruses Indiana, New Jersey, Cocal, Argentina and Brazil have been examined for cross-relationships by complement fixing and neutralization tests. With the exception of the Cocal and Argentina strains, intact virus particles showed little cross-reaction in either test. However, the infective skeleton-like structure produced by uncoating the virus with Tween-ether or Nonidet, and the infective ribonucleoprotein (RNP) produced by deoxycholate disruption of the virus, were neutralized by either homo- or heterotypic antisera and showed considerable cross-reactivity in the complement fixation test. Experiments with the isolated sub-units of the virus indicated that the major part of the cross-reactivity was associated with the RNP. The morphologically similar Piry and Chandipura viruses appeared to be unrelated to the vesicular stomatitis group in both complement fixation and neutralization tests.

Double-stranded RNA has been widely regarded as the inducer of interferon formation, both in virus-infected cells and in cells treated with synthetic polynucleotides (Colby, 1971). It is not difficult to see how infection with RNA viruses may lead to the formation of double-stranded RNA, but how do DNA viruses induce interferon formation? Colby & Duesberg (1969) have isolated a double-stranded RNA from chick cells infected with vaccinia virus, and shown that this double-stranded RNA induced interference against a virus challenge. However, they did not measure interferon production, either by virus infected cells or by the cells treated with double-stranded RNA, and since were found that chick cells infected by vaccinia virus did not produce interferon, there was some doubt about their explanation.

The genus Coulobacter has been characterized in detail by Poindexter (1964). Because of the simple yet distinct morphological and physiological changes which occur during the life-cycle of this stalked pseudomonad, it may provide a bacterial example for the study of cellular differentiation among lower life forms. The stalked, non-motile Caulobacter cell gives rise to two different cell types upon division, a motile swarmer cell with flagellum and a cell with the original stalk. The stalked cell divides again following the same pattern. However, the motile cell must lose motility and develop a stalk before cell division. It then gives rise to a motile, non-stalked cell and a stalked cell as did its parent. A key to the study of such a system with two distinct growth stages is the effective separation of the two cell types or the elimination of one or the other so that each may be studied separately.

Human lymphoid cell lines have so far only been established from cells exposed to Epstein-Barr virus (EBV) (Henle et al. 1967). Such cell lines contain virus DNA in addition to cellular DNA (zur Hausen & Schulte-Holthausen, 1970), but the virus genome is present in a repressed state (Hampar et al. 1972). Some cell lines of this type can be superinfected with EBV. This results in an abortive infection, with the expression of several early virus functions (Henle et al. 1970; Gergely, Klein & Ernberg 1971) but no production of virus particles. In the present work, biologically active EBV, labelled with [3H]-thymidine, was partly purified from the culture medium of the cell line P3HR-1 which produces virus (Hinuma et al. 1967). The fate of the radioactive virus DNA was then investigated after abortive infection of two different human lymphoid cell lines.

Several viruses antigenically indistinguishable from measles virus have been isolated from the brains of patients with subacute sclerosing panencephalitis (SSPE), a progressive neuropathy of children, by co-cultivation of brain cells with other cell types (Horta-Barbosa et al. 1969a; Payne, Baublis & Itabashi, 1969). The SSPE-1 virus isolated by Horta-Barbosa et al. (1969b) was obtained as second passage fluids from Dr Wolfgang Zeman, Indiana University Medical Center. In our laboratory, SSPE-1 virus (mantooth strain) was adapted to the brains of newborn hamsters by four pairs of alternate passages in hamster brain and monkey cell tissue culture (Byington, Castro & Burnstein, 1970). This communication presents some of the characteristics of the hamster-brain-adapted (HBS) virus in cell culture.

Stock virus was the 10th passage from the original brain isolate. It was prepared by intracerebral inoculation of neonatal hamsters with 0.02 ml. of HBS virus; affected brains were harvested at 96 hr, when pronounced neurological signs were evident.

Host cell RNA synthesis and initiation of DNA synthesis are inhibited rapidly after infection with vesicular stomatitis virus (VSV) (Huang & Wagner, 1965; Yaoi, Mitsui & Amano, 1970b; Yaoi & Amano, 1970). Since this shut-off was unaffected by extensive u.v. irradiation of the virus (Huang & Wagner, 1965; Yaoi et al. 1970b), it was concluded that proteins or lipids of the infecting virus were responsible for the shut-off of the cellular synthesis of nucleic acids and that the virus genome was not involved. It was of interest, therefore, to determine whether virus components entered cell nuclei, or whether replication of VSV was restricted to the cytoplasm (Yaoi, Amano & Mitsui, 1970a). The present paper reports the fate of radioactively labelled VSV, (New Jersey serotype) after infection of cultured chick embryo cells.

Avian infectious bronchitis virus (IBV), a coronavirus, requires initial isolation in, and adaptation to, chicken embryos (CE) before transfer to primary avian cell and chicken tracheal organ cultures. These are the only presently known cell cultures in which IBV replicates and produces cytopathic effects (c.p.e.) in serial passage (Estola, 1966; Cunningham, 1970). Monkey kidney cells have been reported (Fahey & Crawley, 1956; Steele & Luginbuhl, 1964) to support relication of IBV without c.p.e. when first inoculated with virus propagated in CE. Attempts apparently were not made to passage the virus in these cells.

Direct haemagglutination (HA) is not a normal property of IBV (Biswal, Nazerian & Cunningham, 1966) or of the human coronaviruses (Kapikian, 1969). However, human coronaviruses OC 38 and OC 43 adapted to suckling mouse brain (McIntosh et al. 1969) cause direct HA (Kaye & Dowdle, 1969) and produce syncytia and plaques in African green monkey kidney and BSC-1 cells (Bruckova, cited by Bradburne & Tyrrell, 1971).

Recent studies on adenovirus adsorption have shown (Neurath, Hartzell & Rubin, 1969, 1970) that the erythocyte receptors for adenovirus differ from the receptors for other viruses hitherto described, e.g. influenza virus (Springer, Nagai & Tegtmeyer, 1966), in that arginine residues in the virus capsid and carboxylic acid groups of aspartic and glutamic acid residues of cellular proteinaceous components are involved in the interaction of adenovirus with cell membrane. Experiments of adenovirus attachment on to permissive cells suggest that the critical arginine residues are located in the vertex projections (Philipson, Lonberg-Holm & Petterson, 1968).

Although carbohydrates do not seem to participate in adenovirus-cell interaction, it is interesting to investigate the possible role of a carbohydrate moiety (especially of neuraminic acid residues) of plasma membrane glycoproteins on the adsorption of adenovirus type 2 to intact cells under conditions of a normal virus infection.