- Volume 39, Issue 1, 1978

Replication of herpes simplex virus type 1 (HSV-1) was studied in various cell lines of rat nervous system origin. Infection of neonatal rat glial primary cells with HSV-1, strain KOS, produced normal yields of progeny virus. Glioma lines B9 and B15 were permissive, the neuronal line B50 was partially restricted (10 to 100-fold reduction) and the neuronal line B103 was non-permissive (> 1000-fold reduction) for HSV-1 (KOS) replication. Synthesis of virus DNA in infected B103 cells was not detected. However, at least some virus macromolecular synthesis was induced, including production of thymidine kinase, DNA polymerase and virus structural proteins.

Mild trauma was induced in the skin of mice latently infected with herpes simplex virus type 1 by stripping the originally infected ear with cellophane tape. Recurrent herpes simplex developed at this site 2 to 5 days later. It was detected clinically by the development of erythema and vesicles and by the appearance of virus in the skin. On any one occasion about 30% of mice showed reactivated disease and increasing the severity of trauma did not increase this proportion. However the majority of animals developed reactivated disease on some occasions when stripping was repeated at monthly intervals. The results are discussed in relation to the skin trigger theory of reactivation of herpes simplex.

Human coronavirus RNA, prepared by extraction of purified virions with phenol-chloroform, consists of a major 15 to 55S class and a minor 4S class of RNA fragments. Polyadenylic acid [poly (A)] sequences are present in 15 to 55S but not in 4S RNA, suggesting different functions for each class. A stretch of poly (A) of approximately 19 adenosine monophosphate residues was obtained in sizing experiments after digesting OC-43 RNA with pancreatic and T1 ribonucleases. An OC-43 virion RNA transcriptase could not be detected with systems optimal for detecting the transcriptases of influenza and Newcastle disease virus.

Two new virus isolates, M432 and M832, obtained from the Southeast Asian mouse have been characterized morphologically with respect to their composition and intracellular assembly. The mature virions resemble in certain respects the type B murine retroviruses. The new isolates, however, have an intracellular precursor, a type A particle, closely associated with the mitotic apparatus. The intracellular transport of the type A particles to the cell surface, where they are released by budding, is closely associated with the microtubule system of the cell.

A temperature-sensitive (ts) strain, Ls1, was isolated from a culture of L, a tomato strain of tobacco mosaic virus (TMV). Ls1 caused smaller necrotic local lesions than L in leaves of hypersensitive tobacco plants. A temperature shift treatment (22 °C for 3 days, 32 °C for 2 days and 22 °C for 1 day) allowed L to produce necrotic lesions surrounded by a collapsed area, whereas Ls1 caused necrotic lesions without a collapsed surrounding area, suggesting that Ls1 did not spread outside the lesions at 32 °C. In tomato leaf discs at 22 °C, infectivity of Ls1 increased in parallel with that of L, but at 32 °C the increase in infectivity of Ls1 was negligible and contrasted with the large increase of infectivity of L. Few mesophyll cells from Ls1-inoculated discs incubated at 32 °C were stained by fluorescent antibody but many of those incubated at 22 °C were stained. In protoplasts at 32 °C, however, Ls1 and L infected and multiplied similarly. When leaves inoculated with Ls1 were kept at 20 to 25 °C for 24 h, and discs were then prepared and cultured for 12 h at 32 or 22 °C, the proportion of mesophyll cells containing virus antigen did not increase at 32 °C but infectivity increased greatly. This suggests that Ls1 multiplied readily at 32 °C in already infected cells. At 22 °C the proportion of infected cells and the infectivity of leaf extracts increased rapidly. The growth curve of Ls1 in leaf discs at 32 °C resembled the so-called ‘one-step growth curve’ of Ls1 or L in protoplasts, and not the growth curve of L in discs, in which the virus could spread from cell to cell. These results suggest that Ls1 is a ts strain that multiplies normally at the non-permissive temperature but has a malfunction in cell-to-cell movement.

Filamentous particles of lilac chlorotic leafspot virus (LCLV), a newly recognized virus resembling closteroviruses, were found only within mesophyll and phloem parenchyma cells of infected Chenopodium quinoa and Phaseolus vulgaris leaves. In immature mesophyll cells the particles were associated with endoplasmic reticulum in the cytoplasm; in mature mesophyll cells the particles, and extensively proliferated and dilated endoplasmic reticulum, were found in ovoid cytoplasmic inclusions up to 8 µm in diam. In phloem parenchyma cells each inclusion was about 3 to 4 µm in diam. and typically occluded the cell lumen. In differentiated cells chloroplasts adjacent to the inclusions often had enlarged and sinuous thylakoids.

The intracellular inclusions induced by LCLV are similar in structure and location to those of five closteroviruses, observations supporting the inclusion of LCLV in this group.

Electron microscopic examination of haemopoietic liver tissue from mice infected in utero or when newborn showed inclusions of Colorado tick fever virus within erythroblasts, reticulocytes and erythrocytes. Inclusions were also seen within erythroblastoid cells undergoing mitosis. Other evidence of virus replication within erythropoietic cells was the presence of intracytoplasmic and intranuclear fibres, which have been shown to be associated with Colorado tick fever virus replication.

The findings reported here support the hypothesis that virus replication within infected erythropoietic cells occurs concurrently with differentiation of the infected cell, resulting in the presence of virions within erythrocytes.

A temperature-sensitive mutant of Bacillus subtilis is described which produces PBSX phage at a non-permissive temperature (47 °C). The mutant is, in the properties tested, phenotypically identical to the mutant tsi23 reported by Siegel & Marmur (1969). Its mutation (tsi85) maps in the same chromosomal region (dal-purB) in which tsi23 is located; the two mutations are shown to be distinct but probably affect the same function. Double mutants carrying the mutations tsi and xin (which blocks the induction of PBSX by mitomycin C) do not produce PBSX at a non-permissive temperature but retain their thermosensitivity. Tsi mutants display a reduced rate of RNA synthesis at the non-permissive temperature. Such a phenotype is lost in tsi-xin double mutants, revealing that it is associated with PBSX induction. The nature of the primary lesion that leads to PBSX induction in tsi mutants remains to be ascertained.

The nucleocapsid of Dane particles (= hepatitis B core antigen; HBcAg) was isolated from human sera either positive or negative for e-antigen (HBeAg) — an apparent marker for the level of infectious hepatitis B virus in serum. HBcAg from the HBeAg-positive serum pool consisted of two distinct populations of particles, one with a buoyant density (d) of 1.358 g/ml and a sedimentation coefficient (s 20, w) of ≈ 110, and another with d = 1.28 to 1.30 g/ml and s 20, w ≈ 70. Only the latter type of particles was isolated from an HBeAg-negative serum pool. HBcAg was labelled with 125I-p-hydroxyphenylpropionic acid N-hydroxysuccinimide ester, dissociated and analysed by polyacrylamide gel electrophoresis. One major and one minor polypeptide with apparent mol. wt. of 16000 ± 500 and 68000, respectively, were detected. Another component having the properties of a glycolipid with a mol. wt. in the order of 103 was observed. After isoelectric focusing, HBcAg was recovered in fractions with a pH between 4.0 and 5.8, suggesting heterogeneity in isoelectric points.

We have observed that purified polyoma virus is able to take up an amount of calf thymus histone equivalent to 10 to 50% of its normal histone content under conditions allowing the binding of considerably lesser amounts of several other proteins. Some of the bound histone could not be released by procedures routinely used for virus purification. We have also found that some of the histone present in purified polyoma virus could be selectively released without major breakdown of virus particles. Possible models for virus structure are discussed in the light of the present and other recent data.

Infection of BGM cells with the Halle isolate of subacute sclerosing panencephalitis (SSPE) gave rise to a persistent infection (BGM/Halle), whereas infection of another African green monkey kidney cell line (Vero) under identical conditions led to a lytic infection. The BGM/Halle cells multiplied more slowly than the non-infected cells (even when the medium was changed daily). Under such conditions 107 to 108 p.f.u./ml/24 h of measles virus was released into the medium.

It was established that the persistent infection was not due to the accumulation of thermosensitive mutants and that the virus was not modified as measured by several biological parameters. The virus released from BGM/Halle cells had, however, acquired an ability to give rise to a persistent infection in Vero cells. The quantity of virus released from persistently infected Vero cells was very low (102 to 103 p.f.u./ml). It was concluded that a host-cell factor plays a role in the restriction of virus replication.

Human leukocyte interferon (HuLeIF) preparations were separated into populations of molecules with different sizes, by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and with different charges, by isoelectric focusing. These populations with different sizes and charges were analysed for their antiviral activity on homologous cells and on heterologous (bovine) cells.

The distribution of interferon activity into two broad peaks by SDS-PAGE was similar whether assayed on human or bovine cells. However, within these peaks, the relative ratio of the activity in human cells and bovine cells varied significantly: while most of the size components had similar human/bovine cell activities, the fastest migrating component (apparent mol. wt. ∼ 13500) was more than 100 times more active on bovine cells than on human cells.

The peaks of activity in isoelectric focusing were distributed from pH 5.5 to 7.0. There was generally correspondence between human and bovine cell activities, but while the more neutral pH range peaks were consistently slightly more active on human cells than on bovine cells, the more acid range peaks were always slightly more active on bovine cells than on human cells. However, with the most acidic peak, there was more than 100 times greater activity on bovine cells than on human cells.

These data show that the heterogeneity of HuLeIFs is greater than merely two size populations, and data confirm that different forms of human leukocyte interferon can vary markedly in biological activity.

Immediately after purification of cowpea chlorotic mottle virus (CCMV) in situ degradation of RNA-2 into two distinct RNA fragments begins. Upon storage of purified virus, even at 4 °C, all four virus RNAs degraded into small heterogeneous pieces. Addition of thiols accelerated degradation, as did higher temperatures. Addition of chelating agents in low concentrations prevented the in situ RNA degradation.

Isolated CCMV-RNA was not degraded in the presence of thiol compounds. If the RNA was, however, reassembled with isolated coat protein into virus-like particles, degradation of the RNA occurred. A mixture of empty protein capsids and RNA also caused degradation of RNA. In a mixture of isolated RNA and purified virus, both degradation of added and in situ RNA occurred, but not to the extent of the degradation of the same amount of RNA within virions.

Addition of radical scavengers to purified virus partially prevented the in situ degradation of CCMV-RNA, suggesting a radical mediated mechanism for the nucleic acid degradation.

The three major structural proteins of vesicular stomatitis virus, M (mol. wt. 29000), N (mol. wt. 50000) and G (mol. wt. 69000) are synthesized at non-equimolar rates. The possibility of translational control of VSV protein synthesis by examining the size distributions of polysomes involved in the synthesis of these three proteins has been tested. The strategy was to label the nascent peptide chains in vivo with radioactive amino acids and to analyse separately membrane-bound polysomes, involved predominantly in synthesis of the G protein, and soluble polysomes, involved predominantly in the synthesis of the M and N proteins. Analysis by sucrose gradient centrifugation indicated that the G protein was synthesized on membrane-bound polysomes having a mean size of 8 ribosomes per polysome. The M and N proteins were synthesized on soluble polysomes having a mean size of 5 ribosomes per polysome. Both membrane-bound and soluble polysomes had a maximal size of approximately 15 ribosomes per poly-some. The distribution of M and N nascent chains within the soluble polysome gradient was determined by tryptic peptide analysis. The M nascent chains were found to be associated predominantly with the smaller polysomes. Thus, the data indicate that the average size of the polysomes involved in the synthesis of the M, N and G proteins reflects the mol. wt. of these proteins. This suggests that the relative rates of synthesis of the M, N and G proteins are not the outcome of controls at the translational level.

The freeze-fracturing of the lipid-containing bacteriophage φ6 shows the virus membrane to have structural asymmetry with smooth convex and rough concave fracture faces. The host, Pseudomonas phaseolicola, shows the four typical fracture faces of the Gram-negative cell envelope. During the infection of P. phaseolicola by φ6 the virus membrane and the bacterial outer membrane fuse to form a bulge. The nucleocapsid obviously also penetrates the cytoplasmic membrane and is seen inside the cell shortly after infection. The outer membrane of the host undulates and after infection small membrane vesicles are formed apparently from the outer leaflet of the outer membrane.

Purified preparations of isolate C of andean potato mottle virus (APMV) contained isometric particles c. 28 nm in diam. with sedimentation coefficients (s o 20, w ) of about 53, and 93 and 112S. The two nucleoprotein components, middle (M) and bottom (B), were serologically indistinguishable, and each contained similar relative amounts of two polypeptide species, of mol. wt. 22100 and 41800. M particles had a density in CsCl of 1.41 g/ml, contained a single RNA species of mol. wt. 1.4 × 106 and stained differently from B particles with uranyl formate; B particles had a density of 1.46 g/ml and contained a single RNA species of mol. wt. 2.0 × 106. Preparations of M and B particles were much less infective when inoculated separately than in mixtures. In these properties APMV resembles other comoviruses. The present cryptogram of APMV is R/1:1.4/(27) + 2.0/(34):S/S:S/*, comovirus group.

The two isolates studied, C and H, infected Gomphrena globosa systemically, and therefore differ from the type strain of APMV. Isolate C was antigenically indistinguishable from the type strain and produced similar symptoms in several solanaceous species. Isolate H differed antigenically from C and the type strain, and also differed somewhat in symptomatology and host range.

JJ/50 and four other strains of influenza C virus grew in an established line of canine kidney (MDCK) cells. Multicycle virus growth was markedly enhanced by the addition of trypsin to the culture medium and these viruses could be passaged serially in this system. The addition of appropriate concentrations of trypsin to the agar overlay medium enabled plaquing of influenza C/JJ/50 virus. Titration by plaque assay on MDCK cells was more sensitive than that by intra-amniotic inoculation of fertile hens’ eggs.

A new bacteriophage containing double-stranded DNA, Al-K-1 was isolated and characterized. The phage grows optimally at pH 10.5, is stable within the range pH 9 to 11 and quite unstable at neutral or acidic pH. The isolated DNA of phage Al-K-1 was damaged by alkaline treatment (pH 11), indicating that it was protected from the alkaline condition by the phage capsid.

The infection of barley protoplasts with brome mosaic virus (BMV) was greatly influenced by osmotic shock produced in protoplasts. Increase of osmotic pressure of the medium immediately before or during inoculation enhanced infection. The efficiency of infection increased with increasing change in mannitol concentration. In contrast, when the osmotic pressure of the medium was decreased before inoculation, few protoplasts were infected even though the other conditions were optimal for infection with BMV. When applied after inoculation, however, decrease of osmotic pressure of the medium had little effect on infection.