- Volume 36, Issue 3, 1977

Replication of the strain J 670/71 of enterovirus 70 (EV70) in non-primate cell cultures at 33 °C was studied using strain L (mouse), BHK21 (hamster), RK13 (rabbit), RK17 (rabbit), PK15 (porcine), IB-RS-2 (porcine), ESK (porcine), MDBK (bovine), and BK1 (primary bovine) together with that of the LSc, 2ab strain of poliovirus type 1 (PV1) as a control. All the cells tested adsorbed from 54 to 90% of EV70. The replication with complete c.p.e. was evident in RK13, RK17 and BK1 cells; replication without c.p.e. was shown in L, BHK21, IB-RS-2 and ESK; but PK15 and MDBK were non-permissive despite a high virus adsorption rate. On the contrary, none of these non-primate cells allowed the adsorption and growth of PV1. One-step growth of EV70 in RK13 was almost identical with that in the primate cells. Two other strains of EV70 were found having similar host range in vitro. Therefore, it is concluded that EV70 has a wider host range in vitro than ordinary human enteroviruses, and its implication is discussed.

We have examined the matrix proteins of A/Okuda/57, A/Finland/4/74 and A/New Jersey/8/76 viruses and several recombinant strains by radioiodination of the purified polypeptides followed by tryptic peptide mapping. The method is rapid and requires only small amounts of material. Reproducible differences were detected between the matrix proteins of the above parents and allowed the origin of the matrix proteins of the recombinant viruses to be determined. The possible use of matrix protein identity as a marker in recombination work is discussed.

The presence of an RNA-dependent RNA polymerase is demonstrated in purified rotavirus particles. Optimum polymerase activity was found between 45 to 50 °C, at pH 8, and in the presence of 10 mm-magnesium ions. The polymerase product was highly sensitive to pancreatic RNase (97%) in low or high salt concentration. The enzyme was activated by EDTA treatment of intact particles or heat shock. The similarities between reovirus, blue-tongue virus and rotavirus polymerases are discussed.

The reduced yields of rhinovirus type 2 at temperatures above 37 °C were shown to result from the degradation of virus-induced RNA, leaving little RNA available for inclusion into mature infectious virions. The degradation occurred about 6 h p.i., and appeared to be selectively effecting the single-stranded species. Lysosomal nucleases do not appear to have a role in this supra-optimal degradation.

Factors that control focus morphology in cell cultures infected with Bryan strain Rous sarcoma virus (B-RSV) were examined. Cloning experiments with the roundc-cell (morphr) and fusiform cell (morphf) variants of RSV (Temin, 1960), confirmed that the two mutant viruses breed true and that these two distinct focus types were under the control of the virus genome.

Three subvariants of the morphr focus type were described which failed to breed true in cloning experiments. It was concluded that this type of focus variation was not due to virus mutation.

Variation in focus morphology was observed when RSV was plated on chick embryo fibroblast cell lines from different sources, as reflected in the relative frequency of expression of the three morphr focus types. These variations were exhibited between fibroblast cell lines of different genetic background, indicating that the cell genome had an influence on focus expression. Focus expression also varied in fibroblast cell lines from chickens of different ages. The strongest host influence on focus expression was observed with fibroblast cell lines derived from selected individual tissue of single embryos. The latter two factors (age and tissue origin) suggested that the differentiated state of the cells also influences RSV focus morphology.

In an attempt to define the molecular events involved in induction of interferon, various parameters of chick cells infected with human adenovirus type 5 were analysed. It was shown by digestion with various proteolytic enzymes and by disruption of the purified virus that induction of interferon requires the interaction of infectious virus with the chick cells. Analysis of adenovirus-infected chick cells by immunological and biochemical techniques indicated that most of the cells produce some virus-specific components, and that temperature sensitive mutants which fail to induce interferon at the restrictive temperature fail to synthesize late components at that temperature. However, since it has been shown that interferon can be induced in the absence of DNA synthesis, these studies conclude that interferon induction results from an early interaction between virus (or virus product) and chick cells and moreover that this interaction is also necessary for the synthesis of virus DNA in this system.

An isolate (Mira) of cytomegalovirus is shown to replicate in human embryonic lung fibroblasts at supra-optimal temperature (40 °C). The ability of the Mira isolate to grow at 40 °C decreased as a function of age of cells in which the virus was grown. The unusual morphology of the lesions in late passage cells infected and maintained at 40 °C is illustrated.

Mean survival time following intracerebral inoculation of dengue virus was reduced and the titre of the virus in the brain of immunosuppressed mice was markedly increased. A single dose of cyclophosphamide given 24 h after dengue virus i.c. or i.p. substantially reduced the number of antibody forming cells in the spleen. Three doses of dengue virus, each followed by cyclophosphamide 24 h later, produced specific hyporesponsiveness to the dengue virus but not to a heterologous virus (Coxsackie B4), with a reduction in antibody forming cells in the spleen of such animals against dengue virus but not against Coxsackie B4 virus. Adoptive immunity by antiserum was abolished along with increased titres of the virus in the brain of immunosuppressed mice but the protection could be restored by a second dose of antiserum. Pre-treatment of mice with immune or normal spleen cells i.v. or reconstitution of immunosuppressed mice by such cells had no effect. Thus, humoral antibodies play a crucially important role in host defence mechanism in recovery of mice from primary dengue virus infection.

The purified 12000 dalton (p12) phosphoprotein of Rauscher (R) and wild mouse (WM) strains of murine leukaemia virus (MuLV) was analysed for the distribution patterns of its variously charged molecular species by urea-poly-acrylamide gradient gel electrophoresis. The distribution patterns of the p12 of two different field isolates of WM viruses, 292 and 1504, and the mouse-tropic and amphotropic clonal sub-populations of 15-4 field isolate were very similar but different from that of MuLV-R. A unique characteristic of the p12 of the WM isolates is the presence of two major apparently non-phosphorylated species in approximately constant proportions relative to the phosphorylated species. Similar studies on the p12 of the same virus (MuLV-R or WM viruses) grown in different host cells showed that the patterns of phosphorylated and non-phosphorylated species are virus-specific and independent of the cell lines of propagation. These analyses and their comparison with urea-gel patterns of the phosphoproteins of other mammalian type C viruses indicated that the number and relative proportion of the variously phosphorylated and non-phosphorylated species are predetermined for a virus. Therefore, the virus must have the genetic information for the phosphoprotein as well as other necessary genetic information which functions, perhaps in conjunction with appropriate cellular factors, in regulating the specific proportions of these multiple species. Possible biological significance of the variously charged molecular species in the phosphoprotein of RNA tumour viruses is discussed.

Four intertypic recombinants of herpes simplex virus have been shown to possess genetic information for functions characteristic of each of the two parental types. The functions were identified by (a) polyacrylamide gel electrophoresis of purified virus particles and of polypeptides synthesized in cells infected with the recombinants and (b) analysis of antigenic sites interacting with type specific neutralizing antibody. The analysis shows that each recombinant possesses a different combination of these type specific markers. Finally we have been unable to detect recombination between herpes simplex type 1 and pseudorabies viruses.

The morphology of a rhabdovirus-like particles, encountered in the gastric mill muscles of the blue crab is reported. The particles occurred in the Schwann cells, presumptive fibroblasts, haemocytes, within the cells forming walls of blood vessels and also in the extracellular spaces. Motor axons were free of such particles. The entities appeared rod-shaped with two rounded ends, measured about 30 nm in diameter and, on average, about 115 nm in length. In cross-sectional profile, they showed an outer granular layer surrounding an electron-dense ‘ring-like’ structure with a central core of low electron density. Stacked particles with regular orientation were seen within the interlamellar space of the nuclear envelope. Also, membrane bound clusters of these particles formed inclusion bodies within the cytoplasm of the ‘infected’ cells.

When avian infectious bronchitis virus (IBV) is fixed in formaldehyde, negative stain is able to penetrate the particle and an internal component is visualized. This component is seen as a tongue or flask shaped structure attached at one point to the outer virus membrane. A model yielding transmission patterns similar to the virus has been made. Gradient centrifugation studies on IBV reveal that the RNP is associated with the internal sac.

Methods are described for the preparation in vivo of 35S-methionine-labelled influenza viruses, the purification of the nucleoprotein (NP) and matrix (M) proteins and the separation of peptides obtained by protease digestion by two-dimensional thin-layer chromatography. The maps of the M proteins of A/Okuda/57 (H2N2) and A/Finland/4/74 (H3N2) were very similar overall but differed in three peptides. Hence they could be clearly distinguished. Maps of the NP proteins of the same strains showed a greater number of differences.

A recombinant strain having the haemagglutinin and neuraminidase of the A/Finland/4/74 parent and the virulence of the A/Okuda/57 parent was shown to have the M and P proteins of A/Okuda/57.

Nuclear extracts of varicella-zoster virus (VZV)-infected human embryo lung (HEL) cells were found to contain DNA polymerase activity not present in un-infected HEL cells. This enzyme was designated the VZV-induced DNA polymerase. The VZV-induced polymerase was partially separated from the cellular α- and β-polymerases by fractionation of the cells and by phosphocellulose chromatography. The separated enzymes were examined for the effect of added (NH4)2SO4, activity with synthetic templates, optimal pH, and the effect of phosphonoacetic acid. The VZV-induced DNA polymerase was distinct from cellular enzymes and had the properties of a typical herpesvirus-induced DNA polymerase.

Two nuclear polyhedrosis baculoviruses were observed to acquire an envelope by budding through the plasma membrane. Budding (synhymenosis) occurred in a variety of tissues. The mechanism involved in the budding process appears similar to that described for other viruses and results in a characteristic surface structure similar to peplomers on one end of the virus.

RNA with a sedimentation coefficient of 64S was isolated from infectious bronchitis virus, an avian coronavirus. The RNA contained a polyadenylic acid tract and was found to be infectious.

Electron microscopy of ultrathin sections indicated that nucleoprotein particles of raspberry ringspot (RRV) and tobacco rattle (strain CAM; TRV-CAM) viruses occurred in mixed aggregates in cells of doubly infected Nicotiana benthamiana leaves, and in protoplasts doubly infected by inoculation. RRV particles were attached to the sides and ends of TRV-CAM particles that were mostly bound to mitochondria. RRV particles attached to TRV-CAM particles linking mitochondria in clusters are considered to be the structures represented by the aggregates of RRV particle antigen found previously by fluorescent antibody staining in doubly infected protoplasts but not in those infected with RRV alone.

The nucleocapsid structures of Tacaribe virus, a member of the Arenaviridae, were purified from detergent-treated virus particles by equilibrium density gradient centrifugation. Negative-contrast electron microscopy indicated that they were coiled, circular filaments. They had a mean diam. of 5 to 10 nm and two predominant length classes of 640 nm and 1300 nm were found.

A bacteriophage specific for Bacillus megaterium QM B1551 was isolated. This phage (MP-7) was a lytic phage of typical morphology and distinct stability properties. The DNA was double-stranded, with a mole % G + C of 38.9 ± 0.7 and a mol. wt. of 42 to 44 × 106.