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'When we give a definition it is for the purpose of using it'. HENRI POINCARE in Science and Method A. Objectives The first version of this paper was written to introduce new students and fellows of my laboratory to the mysteries of herpesviruses. Consonant with this design sections dealing with well documented data were trimmed to the bone whereas many obscure phenomena, controversial data and seemingly trivial observations were discussed generously and at length. There is some doubt as to whether it was meant to be published, but it was not a review. The objective of reviews is frequently to bring order. But alas, even the most fluent summation of credible data frequently makes dull reading and too much plausible order, like very little entropy in chemical reactions, is not the most suitable environment on which to nurture the urge to discover. This version is more charitable but not less inbalanced. The bibliography reflects the intent of the paper and was updated last in December of 1968. It should be obvious without saying that no single account such as this can do justice or injustice, as the case may be, to the several hundred papers published on herpesviruses each year or to the many thousand papers published on herpesviruses since the first of the members of the family was experimentally transmitted to a heterologous host more than half a century ago (GRUTER, 1924). B. Definition 1.

Ever since arbovirus infections became known and their relative importance assessed, experiments were designed to elucidate the mode of transmission and the most important natural hosts responsible for perpetuating the infection in nature. Human infections and the disease in wild rodents, birds, and domestic animals were studied in relation to viremia and distribution of the infectious agent in the organism. With increasing epidemiological studies it became apparent that the neural manifestations of the disease are very uncommon, confined only to a small percentage of individuals of the most susceptible species. Various factors have been proposed to explain why in certain instances the virus becomes establish­ ed in the central nervous system and causes a serious or lethal disease. For example, differences in the virulence of the virus strains, varying susceptibility of individuals of one species, or intercurrent circumstances facilitating access of the virus to the central nervous system were alleged. Also, various possible routes of entry of the virus into the brain and spinal cord have been considered.

Several discoveries are noteworthy for allowing us to probe the recesses of the virus­ infected cell and to search for cryptic viral genomes which might provide clues in our studies of cancer etiology or developmental biology. One of the most notable was the dis­ covery of reverse transcriptase. This marked a momentous occasion in the history of molecular biology. Not only did it provide insight into the mechanism of persistence of retroviruses but it also provided us with an enzyme that could synthesize a DNA copy of any RNA. This DNA copy could then be used as a hybridization reagent to search for both complementary DNA and viral-specific RNA. Thus one could follow the course of any viral infection or probe in tumor cells for hidden viral genomes. Second, a great deal of credit must be given to the geneticists who isolated the various deletion mutants in the'avian retrovirus system and thus provided us with the frrst means of isolating gene-spe­ cific probes. Finally, the laboratories which have mapped the genome have provided us with the framework in which to ask very specific questions with our gene-specific probes. Recently, numerous excellent reviews concerning various aspects of the retroviruses have appeared. In this review I shall not even attempt to present a comprehensive review of retroviruses.

Prominent progress in molecular biology was only made when it became possible to separate functionally distinct molecules by taking advantage of their biophysical properties. Likewise, the analysis of the functions of hetero­ geneous populations of immunocompetent cells, as to the functional properties of their various subpopulations, can not be done until these can be isolated in reasonably pure form by selective fractionation. During the last few years significant advances have been made in this field, and cells have been separated according to size, density or charge (MILLER et aI., 1969; SHORTMAN, 1968; ANDERSSON, 1973 c), or by taking advantage of more specific surface markers to allow selective depletion or enrichment of a given subpopulation of cells (WIGZELL and ANDERSSON, 1971). Although separation techniques have been used in a variety of cellular systems, they have been particularly useful in the study of reticuloendothelial cells and primarily in the study of cells partici­ pating in the immune responses. Quite extensive reviews have been written which well cover the methods used for separation of cells and the results obtained with the various approaches (WIGZELL and ANDERSSON, 1971; SHORTMAN, 1972). To review this work is becoming a more and more voluminous task. As data rapidly accumulate, we will not try to make such a complete review.

At the end of the last century and the beginning of this century, the prob­ lems of immunity in lower vertebrates and the influence of environmental temperature attracted attention for the first time (ERNST, 1890; WIDAL and SICARD, 1897; METCHNIKOFF, 1901). However, relatively little work has been done on this subject until recently. The early investigators were chiefly in­ terested in the immuno-pathological problems. They immunized various species of lower vertebrates essentially with bacterial vaccines; agglutinating, neutralizing and protective antibodies were detected in their blood. The in­ fluence of environmental temperature on the immune response was investigated, since this subject represented great economical and theoretical importance. Epizootic diseases were observed to occur in relation to the cold season of the year, when the decrease or spontaneous increase of water temperature occurred (SCHAPERCLAUS, 1965; BESSE et al., 1965; KLONTZ et al., 1965 WOOD,1966). The immunological deficiency of fish, caused by their natural or experimental stay in cold water, is now evident for both humoral and cellular immunity. In this review we will focus on two points: We shall attempt (1) to explain the mechanism by which the environmental temperature influences the immune resistance of fish to pathogens, (2) to determine the chronological location of this temperature-sensitive stage in the process of antibody formation, and to make some approaches to the general antibody formation mechanism.

This volume is dedicated to the memory of the late Professor WERNER BRAUN, one of the most devoted and active members of the Editorial Board of the Current Topics in Microbiology and Immunology, who passed away, after suffering a heart attack, in November 1972. Dr. WERNER BRAUN was born in Berlin, Germany, on November 16,1914. During his highschool days in Berlin he did research work on problems of genetics as a young guest in the Kaiser-Wilhelm-Institut fur Biologie, in the department of Prof. R. GOLDSCHMIDT. I remember his colourful description of his discussions during this period, while still a teen-ager, with OTTO WAR­ BURG. He studied biology and medicine at the University of G6ttingen and received a Ph.D. degree in biology in 1936. In the same year he left Nazi Germany and came to the United States first as a Guest Investigator in Genetics at the University of Michigan at Ann Arbor, and then in Berkeley, where he carried out his work in the Depart­ ments of Zoology and of Veterinary Science until 1948. He was engaged during this period in the study of problems concerned with physiological genetics, bacterial variation, immunology and biochemistry.

Influenza continues to be one of the major epidemic diseases of man and is, in fact, his only remaining pandemic disease (BEVERIDGE, 1969). This is largely because influenza virus undergoes extreme antigenic variation, the mechanism of which is still poorly understood. Two kinds of antigenic variation occur in influenza viruses, antigenic drift and major antigenic shifts; both involve chan­ ges in the hemagglutinin and neuraminidase antigens on the surface of the virus. Antigenic drift, which involves gradual changes in the surface antigens of influenza virus, is thought to result from the selection by an immune popula­ tion of mutant virus particles with altered antigenic determinants. These mutants therefore possess a growth advantage in the presence of antibody (FRAN­ CIS and MAASSAB, 1965; ARCHETTI and HORSFALL, 1950; HAMRE et aI., 1958). It has been shown that antigenic mutants isolated in vitro by selection with antibody have changes in amino acid sequence in the polypeptides of the hem­ agglutinin subunits (LAVER and WEBSTER, 1968) and it is likely that antigenic drift in the neuraminidase occurs by the same mechanism.

General aspects of nucleic acid uptake by mammalian cells have been the subject of several reviews during the last few years (PAGANO, 1970; BHARGAVA and SHANMUGAM, 1971; DUBES, 1971; RYSER, 1967). These reviews covered methods used for the infection of cells by viral nucleic acids as well as interaction of mammalian cells with non-viral nucleic acids. This article is restricted to a discussion of experiments with poliovirus RNA and focuses special attention on the steps following the uptake of RNA into a cell, aspects that were not discussed in earlier review articles. The fate of input RNA once inside the cell is determined by the host cell but experimental conditions can be chosen to favor the survival of input RNA and the induction of a virus growth cycle by interfering with host-cell meta­ bolism through events that, in the case of infection with intact virus, might be controlled by viral proteins.

The expression of many bacterial genes adapts itself in an almost in­ stantaneous and reversible way to specific environmental changes. More specifically, the concentration of a number of metabolites, a function of the amounts of enzymes involved in their synthesis or degradation, in turn retroacts on the rate of synthesis of these enzymes. The genetic bases for this regulation were established by JACOB and MONOD (1961). These authors also showed how the known elements of these regulatory mechanisms could be connected into a wide variety of circuits endowed with any desired degree of stability, in order to account for essentially irreversible processes like differentiation (MONOD and JACOB, 1961). The general principles used by JACOB and MONOD in their study of negative regulation were extended to positive regulation by ENGLESBERG et al. (1965). An independent approach permitted the discovery of positive controls in temperate bacteriophages (see below, III). Each control operation is mediated by a pair of complementary genetic elements (hereafter called'control cell'): a control gene which produces a l control (or regulator) protein and a control site which is the target for the regulator protein. Negative control means that the control protein (repressor) prevents gene expression. One deals with positive control when the control protein (activator) is necessary for this expression. It has become apparent that, as initially postulated by JACOB and MONOD, control of gene expression operates, at least to a large extent, at the transcriptional level.

Phenomena as diverse as tuberculin sensitivity, delayed sensitivity to soluble proteins other than tuberculin, contact allergy, homograft rejection, experimental autoallergies, and the response to many microorganisms, have been classified as members of the class of immune reactions known as delayed or cellular hypersensitivity. Similarities in time course, histology, and absence of detectable circulating immunoglobulins characterize these cell-mediated immune reactions in vivo. The state of delayed or cellular hypersensitivity can be transferred from one animal to another by means of sensitized living lymphoid cells (CHASE, 1945; LANDSTEINER and CHASE, 1942; MITCHISON, 1954). The responsible cell has been described by GOWANS (1965) as a small lymphocyte. Passive transfer has also been achieved in the human with extracts of sensitized cells (LAWRENCE, 1959). The in vivo characteristic of delayed hypersensitivity from which the class derives its name is the delayed skin reaction. When an antigen is injected intradermally into a previously immunized animal, the typical delayed reaction begins to appear after 4 hours, reaches a peak at 24 hours, and fades after 48 hours. It is grossly characterized by induration, erythyma, and occasionally necrosis. The histology of the delayed reaction has been studied by numerous investigators (COHEN et al., 1967; GELL and HINDE, 1951; KOSUNEN, 1966; KOSUNEN et al., 1963; MCCLUSKEY et al., 1963; WAKSMAN, 1960; WAKSMAN, 1962). Initially dilatation of the capillaries with exudation of fluid and cells occurs.

Binding of various ligands (hormones, neurotransmitters, immunological stimuli) to membrane receptors induces the following changes: 1. Receptor redistribution (clustering,'capping') 2. Conformational changes that can be detected by fluorescent probes 3. Alteration in membrane fluidity (spin label and fluorescence polarization probes) 4. Changes in fluxes of ions and metabolites 5. Increased phospholipid turnover (especially of phosphatidyl inositol) 6. Activation of membrane-bound enzymes (adenyl cyclase, ATPase, transmethylases). Some of the early changes resulting from or associated with the binding (adsorption) of virions to the host cell membrane are of the same type. Adsorption of animal viruses to cells is the ftrst step in a chain of events resulting in the production of progeny virus on the one hand and in damage to cells and tissues on the other. In the classical studies of viral infection, cells are adsorbed with virus, usually for 60 min, and the changes induced by the virus in the host cell are recorded thereafter. In the past decade, more and more studies have been aimed at the events occurring in these ftrst 60 min of the so-called adsorption period. These studies deal with the nature of adsorption, e. g., the ligand-receptor type of interaction between the virus and the cell membrane. Many receptors for viruses were identifted and so were the viral proteins which take part in adsorption.

The study of the genetic regulation of immune response to natural multidetermi­ nant immunogens was undertaken by the method of bidirectional selective breed­ ing of High or Low antibody responder lines of mice. Five Selections are described: Selection I, carried out for agglutinin responsiveness to sheep erythrocytes and pigeon erythrocytes alternated in each generation. Selection II, carried out for agglutinin responsiveness to sheep erythrocytes repeated in each generation. Selection III and Selection IV performed respectively for agglutinin response to flagellar or somatic antigens of Salmonella typhimurium and Salmonella oranienburg alternated in each generation. Selection V, performed for passive agglutinin response to bovine serum albumin and rabbit gamma globulin alternated in each generation. In each Selection the character investigated is polygenic. High and Low responder lines diverge progressively during the selective breeding. The maximal interline separation (selection limit) is reached in the 7th-16th generations. High and Low responder lines at selection limit are considered homozygous for the character submitted to se~ection. Their variance is therefore only due to environ­ mental effects. The difference in agglutinin titre between High and Low lines is 220-fold in Selection I, 103-fold in Selection II, 90-fold in Selection III, 85-fold in Selection IV and 275-fold in Selection V. The partition of genetic and environmental variances in the foundation popu­ lations of the five Selections is established. The proportion of genetic variance is 60% in Selection I; 49% in Selection II; 51% in Selection III; 47% in Selection IV and 76% in Selection V.

Expression of an immune response is the net result of complex synergis­ tic and antagonistic activities performed by a variety of cell types. It includes macrophages, T and B populations which may interact in performance of a response, and suppressor cells interfering with it. Accordingly, a lack of res­ ponse may not necessarily indicate absence of immunocompetent cells, but rather nonexpression of competence. Thus, one should consider two possible situations, which are by no means mutually exclusive, to account for immuno­ logic unresponsiveness: (a) one or more of the cell populations composing the synergistic unit is absent or immature, and (b) an antagonistic unit which interferes with the response is dominating. In view of this, an approach to development of immune reactivity necessitates parallel surveys of development of cells with the potential to perform, as well as of cells which can suppress the response. Classification of the various cell types has been based so far on their phenotypic properties (e. g., membrane antigen markers, cell receptors, pro­ duction and secretion of immunoglobulins, etc.). Genotypically, T and B cells may represent either separate, independent cell lines, or different stages of development within the same cell lineage.

The processes involved in herpesvirus replication, latency, and oncogenic transformation, have, in general, been rather poorly defined. A primary reason for this is the size and complexity of the herpesvirus genome. Undoubtedly, a better understanding of the functions of the viral genome in infected and transformed cells will be achieved through studies with temperature-sensitive (ts) mutants of herpesviruses since, theoretically, any essential gene function can be affected by mutants of this type. A. The Herpesviruses A consideration of the genetic analysis of members of the herpesvirus group necessitates a description, albeit brief, of the properties of the group and, most importantly, of their genetic material. The herpesviruses comprise a group of relatively large (100-150 nm), enveloped viruses. The envelope surrounds an icosahedral capsid enclosing a core which contains double­ stranded DNA (ROIZMAN, 1969). The group is thus defined on the basis of a common virion morphology. In addition to a common structure, members of the group share a number of biological properties such as a similar replicative cycle, the ability to cause latent and chronic infections, and the ability to induce antigenic modifications of infected cell membranes. Several herpes­ viruses have been associated recently with malignancies in man and animals (KLEIN, 1972). Herpesviruses are ubiquitous and have been described in over 30 different species (HUNT and MELENDEZ, 1969; WILDY, 1971; FARLEY et aI., 1972; KAZAMA and SCHORNSTEIN, 1972; NAHMIAS et aI., 1972; ROlZMAN et aI., 1973). Their widespread occurrence in nature suggests a common ancestor.