- Volume 37, Issue 1, 1977

Introduction. In this review we consider some attributes which may be characteristic of herpesviruses and some properties which distinguish between them. As we shall show, the systematic interpretation of information we now have on a few herpesviruses presents us with some difficulties. For many members of the group we are spared this embarrassment only by the virtual absence of information beyond the initial recognition of the existence of the virus and the allocation of an arbitrary label. Thus, although more than 70 viruses may be listed as presumptive members of the group, criteria allowing their inclusion vary from a brief description of particle morphology (e.g. viruses observed in snake venom, oysters and fungi) to the extensive antigenic, structural, biochemical and genetic characterization of the virus [e.g. herpes simplex virus types 1 and 2 (HSV-1, HSV-2)]. Moreover, the number of separately identifiable viruses which are currently attributed to particular hosts probably reflects the degree of interest in the host species and its afflictions rather than any preferential distribution of viruses.

The different stages of frog virus 3 (FV 3) morphogenesis have been investigated in chick embryo fibroblasts infected at an optimal temperature for virus growth (29 °C). The metabolic requirements for morphogenesis were determined by adding inhibitors of macromolecular synthesis at different periods in the virus growth cycle. The effect of a non-permissive temperature for FV 3 replication (37 °C) was also studied in shift up experiments. The following results were obtained: (1) when DNA replication was inhibited, neither immature nor mature virus particles appeared; (2) continuous protein synthesis was required for every stage of virus morphogenesis. However, the assembly of virions into paracrystalline arrays seemed to be a passive phenomenon. (3) Continuous mRNA transcription was not necessary for assembly of capsid constituents, although most of these capsids appeared empty; there was also a striking increase in the number of aberrant virus structures. (4) If infected cells were shifted to a non-permissive temperature, virus maturation was completely inhibited.

Cells infected with respiratory syncytial (RS) virus eventually die but there appears to be no specific mechanism for shutting off cellular synthesis of macromolecules. DNA and RNA synthesis, as measured by the incorporation of labelled thymidine or uridine, do not begin to shut down until some time between 11 and 18 h after infection. By 18 h their rates of synthesis are reduced to approx. 50% for DNA and 35% for RNA.

Protein synthesis continues throughout the course of infection at approximately the same rate. Synthesis of most of the cellular polypeptides also continues, but the distribution of polypeptides of high and low mol. wt. shifts. The increase in the proportion of those of high mol. wt. includes a peak that represents one of the seven previously identified virion polypeptides.

Another consequence of RS virus infection is an increase in glucosamine incorporation, beginning near the end of the virus eclipse period (12 h after infection), which may be associated with virion glycoprotein synthesis. Polyacrylamide-gel electrophoresis of glucosamine-labelled cells reveals that at 18 h after infection two of the three previously identified virion glycoproteins are present.

The rate of virus and cell DNA synthesis was studied in human embryonic lung cells pre-treated with 5-iodo-2′-deoxyuridine (IdUrd) and exposed to cytomegalovirus (CMV) or medium. Analysis of DNA in CMV-infected cells following sequential 4 h pulses with 3H-thymidine indicated that a temporal relationship existed in the pattern of virus and cell DNA synthesis. The pattern of DNA replication in infected cells resembled that of a typical cell cycle, whereas the rate of cell DNA synthesis in uninfected cells remained low throughout the study. Increased rates of cell and virus DNA synthesis began concomitantly at 16 h post-infection and reached a maximum at 36 h post-infection. The rate of DNA synthesis then declined and remained at lower levels until 48 h post-infection. This was subsequently followed by a second increase in the rate of cell and virus DNA synthesis. The rates of cell and virus DNA replication were similar throughout the study in that increased and decreased rates of synthesis occurred simultaneously. It was of interest to note that CMV induced cell DNA replication in IDUrd arrested cells; in contrast, addition of fresh serum did not induce a similar increase in the rate of DNA synthesis in IdUrd arrested, but uninfected, cells.

The polypeptides of polyoma and SV40 virions are phosphorylated. An estimate of the amount of phosphorylation of the major virus capsid protein (VP1) has been made using two-dimensional gel electrophoresis to resolve phosphorylated from non-phosphorylated forms. The results suggest that in both polyoma and SV40 virions about 12% of VP1 molecules are phosphorylated. In unassembled VP1 molecules immunoprecipitated from extracts of infected cells the proportion is greater, about 33%. The possibility that phosphorylated VP1 may form the penton proteins of the virus capsid is discussed.

A plaque reduction method for measuring lymphocytic choriomeningitis virus-sensitizing antibody in human serum is described. One volume of virus and one volume of serially diluted human serum were mixed and incubated for 2 h at 37 °C. One volume of suitably diluted anti-human immune globulin antiserum was added and incubation continued for 0.5 h. Residual infectivity was then determined by means of a plaque assay employing L cell monolayer cultures and a methyl cellulose containing overlay medium. Of 75 sera from as many persons, 20 were positive with titres ranging from 640 to 10240. All positive sera were from verified cases or from persons who had had occupational contact with the virus. Close correlation of results was found between this method and a neutralization test employing mice, the one exception indicating that the in vitro assay for sensitizing antibody is more sensitive than the mouse assay for neutralizing antibody.

Factors that influence the virulence of Semliki Forest virus for mice have been studied. In vivo experiments showed that maximum brain infectivities following i.p. inoculation of adult mice with a virus strain of low virulence were less than those found in mice showing clinical signs in virulent infections. The virulent strains generally caused death before antibody could be detected in brain tissue; the less virulent strain caused an infection that was neuroinvasive, but infectivity increased less rapidly in the brain and allowed antibody to intervene before clinical signs were apparent. IgG3 antibody was first detected in brain tissue coincident with the beginning of the decline of brain infectivities in avirulent infections; other sub-classes of IgG antibody were not detected until later.

In vitro experiments showed differences in abilities of SFV strains to replicate in tissue culture and adult mouse brain tissue, and differences in susceptibility to infection of brain tissue from mice of various ages. These in vitro results could provide a basis for differences of virulence.

Studies of the resistance patterns to infection with a murine cytomegalovirus in inbred strains of mice revealed the existence of resistant and susceptible strains. Resistance was found to be associated with possession of the H-2k allele at the major histocompatibility locus of the mouse. The F1 hybrid between a resistant strain (C3H/HeJ) and a susceptible strain (BALB/c) was found to have a resistance intermediate between that of both parents, indicating that the gene(s) controlling resistance is partly dominant. Susceptible BALB/c mice could be made resistant to lethal infection by pre-treatment with thioglycollate broth but not by pre-treatment with endotoxin or BCG. Resistant C3H/HeJ mice could be made susceptible to lethal infection by pre-treatment with cyclophosphamide.

Encephalomyocarditis (EMC) virus replication was investigated in permissive mouse MKS cells, semi-permissive monkey CV1 cells, and in somatic monkey-mouse MKCVIII hybrid cells whose permissiveness is under the negative control of the simian genome. We found that in CV1 cells the synthesis of both single- and double-stranded virus RNAs was restricted. In contrast, in semi-permissive hybrid Cl4/3 cells only the single-stranded virus RNA was synthesized in small amounts, whereas the double-stranded virus RNA accumulated late after infection. The synthesis of virus polyribosomes and virus polypeptides was lowered in semi-permissive conditions. In the presence of quaternary ammonium ions, the synthesis of EMC virus was partially relieved in CV1 cells. Thus, it can be postulated that a defective function in the replication complex is involved in the restrictive event.

Electron microscopy of poliovirus particles and empty capsids under various conditions of specimen deposition and staining visualizes the dissociation products of these particles. The dissociation proceeds in steps; it begins with the expansion of particles and leads to the final product of the dissociation — a cluster of several sub-particles of equal size (approx. 100 Å in diam.). A scheme of the dissociation is proposed on the basis of the observed intermediates.

The mol. wt. of the DNA from four nuclear polyhedrosis viruses isolated from Spodoptera littoralis, S. exempta, S. exigua and S. frugiperda were determined to be 84, 80, 68 and 74 × 106, respectively, by electron microscopy. The molecules were demonstrated to exist as double-stranded relaxed circular or supercoiled DNA, though linear forms of DNA were also observed.

The matrix (M) protein of the H2N2 virus A/Ann Arbor/6/60 may be distinguished from M protein of several H3N2 viruses and A/New Jersey/76 (Hsw1N1) by SDS acrylamide gel electrophoresis using a discontinuous buffer system. The smallest RNA (RNA 8) of the A/Ann Arbor/6/60 virus may be distinguished from RNA 8 of several H3N2 viruses by acrylamide gel electrophoresis in 3% or 3.6% gels in the absence of urea, if electrophoresis is done at 30 to 35 °C or 20 °C respectively. Ten clones of conditionally-lethal temperature-sensitive (ts) mutants were studied, which derived their cold-adaption and ts genes from mutant A/Ann Arbor/6/60, and their haemagglutinin from the H3N2 virus A/Scotland/840/74. Each clone was found to derive its M protein from A/Ann Arbor/6/60 mutant, and its RNA 8 from A/Scotland/840/74. The only assignment of genes 7 and 8 consistent with these findings for the recombinants is that in each parent virus (and in the recombinants) gene 7 codes for M protein, and gene 8 for NS protein. Furthermore, it may be concluded from the results that the biologically important ts lesions in the A/Ann Arbor/6/60 mutant parent are not present in the NS gene. In addition to the recombinants of A/Ann Arbor/6/60 and A/Scotland/840/74, five independent ts/cold-adapted recombinants of A/Ann Arbor/6/60 mutant with H3N2 and Hsw1N1 wild-type viruses were examined, and all were found to contain the M protein of the A/Ann Arbor/6/60 mutant parent. This is suggestive that M protein may be at least partially responsible for the cold-adaptation and/or ts properties of the A/Ann Arbor/6/60 mutant and the recombinants.

Cells were cultured from the breast muscle of 11- to 12-day-old chick embryos and were grown under conditions optimal for the development of the cells into terminally differentiated, fused myotubes. Myotubes were infected with influenza virus A/Ann Arbor/6/60(H2N2) at high multiplicity, and synthesis of virus-specific proteins and RNAs was detected by haemadsorption, fluorescence microscopy and/or isotope labelling and electrophoresis techniques. Provided that myotubes were maintained at temperatures below 39 °C after infection, production of virus components and yield of infectious virus in these cells was similar to those observed in infected chick kidney cells. However, if cells were maintained at temperatures of 39 ° to 40 °C after infection, virus nucleoprotein was prominent in the nuclei, and synthesis of virus-specific polypeptides and of plus-strand RNA was reduced about fourfold to 20-fold compared to that detected at lower temperatures. Moreover, infectious virus was not produced when temperatures of 39 to 40 °C were used during virus replication. The results demonstrate that under suitable conditions avian myotubes formed in culture resemble epithelioid cells in their ability to support the productive replication of influenza virus.

Black queen-cell virus was isolated from dead prepupae and pupae of queens and workers of Apis mellifera found locally in the field. Kashmir bee virus was isolated from individuals of Apis mellifera that had died in the laboratory after they had been inoculated with some preparations from Apis cerana. Both viruses have isometric particles about 30 nm in diameter, contain RNA, and are unrelated to each other or to any known bee virus. Black queen-cell virus particles sediment at 151S and have a buoyant density in CsCl of 1.345 g/ml; Kashmir bee virus particles sediment at 171 to 173S and have a buoyant density in CsCl of 1.371 g/ml. Serological evidence indicates that black queen-cell virus is common in Britain and occurs in the U.S.A. The studies involved acute bee-paralysis and sacbrood viruses and led to re-determination of the buoyant densities of these as 1.380 and 1.358 g/ml respectively.

Kethoxal bis (thiosemicarbazone) (KTS) inhibited replication of, and plaque formation by, vesicular stomatitis virus (VSV) in chick embryo cells. No other thiosemicarbazones tested were effective. Virus-specific m-RNA and protein synthesis were inhibited by KTS. However, virion RNA-dependent RNA synthesis was not inhibited by the drug. Treatment of VSV virions directly with KTS produced enhancement, rather than inactivation, of plaque formation.

KTS inhibited cellular DNA and RNA synthesis by 67 and 25% respectively. Since cellular DNA and RNA synthesis are not required for VSV replication, the inhibition of these processes is probably unrelated to the antiviral activity of KTS. Cellular protein synthesis was inhibited 24% by KTS. Unexpectedly, synthesis of four proteins was induced in KTS-treated uninfected cells.

Two morphological types of human rotavirus particles were separated in caesium chloride density gradients. The particles of higher density (‘incomplete’ particles without an outer capsid layer) banded at a density of 1.38 g/ml, while the less dense (‘complete’ particles with an outer as well as an inner capsid layer) banded at a density of 1.36 g/ml. Some particles were found with an incomplete outer layer of capsomeres. The particle/infectivity ratio for tissue cultures of the fraction containing complete particles was more than 3 log10 higher than that of the fraction containing incomplete particles. But, as there was a small number of complete particles in the fraction containing mostly incomplete particles, it was impossible to determine whether the incomplete particles had a low infectivity or whether they had none at all.

Human leucocyte interferon has antiviral activity and anticellular effects on the transformed human cell lines, RSa and RSb. Treatment of the cells with cycloheximide or puromycin at 0.5 to 5.0 µg/ml suppressed the antiviral action of interferon but increased its anticellular effects. Interferon also has antiviral activity in IFr cells, but this is relatively resistant to its anticellular action. Nevertheless, both drugs suppressed the antiviral activity of interferon and increased its anticellular action.

Sendai and measles viruses were tested for their interferon-inducing capacity in human lymphoblast cells. Sendai virus reproducibly induced considerable amounts of interferon (1 research reference interferon unit/103 cells), but no increase in infectious virus titre was observed. Two Edmonston–Enders strains of measles virus grew very well. The attenuated (A) strain was a good interferon inducer (4 units/103 cells), while the virulent (V) strain induced only minimal amounts at a high multiplicity of infection. Pre-treatment of the cells with 5-iododeoxyuridine (IdUrd) had no effect on the growth of Sendai or EE measles virus and on interferon yields induced by Sendai virus. It slightly potentiated interferon induction by EE measles virus. Partial u.v.-light-inactivation of Sendai virus infectivity resulted in a parallel loss in interferon-inducing capacity.