Bluetongue may be described as an acute insect borne disease of ruminants, manifested clinically in sheep by a catarrhal inflammation of the mucous mem­ branes of the digestive and respiratory systems and associated with degenerative changes in the skeletal musculature. The profound emaciation and weakness which follow the acute disease are responsible for a protracted convalescence and for serious economic losses due to diminished productivity. II. Historical During the early colonisation of Africa, susceptible Merino and other European breeds of sheep were introduced into the Cape, at first by the Dutch East India Company between 1652 and 1785 and again later in 1870. A report of the Cattle and Sheep Diseases Commission (1876) records the appearance of a serious febrile disease amongst these imported sheep in which both morbidity and mortality was high (cited by HENNING, 1949). HuTCHEON (1881) gave this disease the name of "Fever" or "Epizootic Catarrh", in order to distinguish it from other clinical conditions of a similar nature encountered amongst sheep. In the first com­ prehensive description of this clinical syndrome and its epizootiology HuTCHEON (1902) referred to it as "Malarial Catarrhal Fever of Sheep", a designation which was obviously influenced by the mistaken belief that an intracorpuscular parasite was the primary cause of the disease. More systematic studies were conducted by SPREULL (1902; 1905), who endeavoured to immunize sheep by the simultaneous inoculation of immune serum and infective blood.

Reoviruses are ether-resistant icosahedral viruses 60 to 75 mp in diameter which contain ribonucleic acid. They have been recovered from man and lower animals and are ubiquitous in their geographic distribution. At present, the importance of these viruses as a cause of human or animal disease is still largely unknown. As a result of having a number of unusual characteristics, reoviruses have attracted the attention of many workers in the relatively short time since they were first recognized. For example, investigators interested in the molecular aspects of virology have been attracted by the unusual double-helical ribonucleic acid of high molecular weight which reoviruses possess, while those interested in epidemiology have been attracted by the occurrence of apparently identical viruses in both man and an unusually wide variety of lower animals. This com­ pilation is based on information available to the author as of October 31, 1966. II. History The term "reqvirus" was proposed in 1959 (SABIN, 1959) as a group name for a number of viruses then classified (SABIN, 1956; RAMOS-ALvAREz and SABIN, 1958) as being identical with, or related to, ECHO type lO virus.

The herpes group consists of viruses which have been placed together on the basis of a number of distinguishing features that they share in common (ANDRE­ WES, 1962). All these viruses are relatively large, possess identical morphological characteristics, contain DNA, and are extremely sensitive to inactivation by ether; these viruses are also assembled within the nucleus of the host cell and induce the formation of eosinophilic intranuclear inclusions. The epidemiology of some of the best known viruses in this group (herpes simplex, pseudorabies, and B-virus) is also similar (BURNET et aI. , 1939). Herpes simplex virus exists in the latent state in man, the natural host for this virus, and becomes overt in individuals subject to some form of stress; this condition appears to be paralleled by pseudorabies virus in its natural host, swine and by B-virus in monkeys. In each instance, transmission of the virus to a susceptible host other than the natural one results usually in marked symptoms and death. This chapter is confined to a description of herpes simplex and pseudorabies viruses; B-virus is described separately elsewhere in the Hand book. Since the clinical aspects of the diseases caused by herpes simplex virus and pseudorabies virus have been well described, greater emphasis will be placed, therefore, on the basic biological and biochemical properties of these viruses; their clinical features will be discussed only briefly.

A. Definitions of Transformation in vitro When normal tissues or organs are explanted to conditions favoring the growth of cells as individual units ("cell culture"), the original cell population undergoes a large variety of modifications. Only a minority of the cells will thrive and multiply and within a rather short period of time, the complex composition of the original explant is replaced by a much simplified one of only a few recogniz­ ably different cell types. With most organs fibroblast-like cells survive longest and outgrow other types. This is then a stable state of affairs for many gener­ ations. This treatise will not discuss whether this simplification and stabilization represents selection of certain pre-existing cell types or a modification of cells into only a few recognizably different categories; for an excellent review see HARRIS. (1964). Table 1. Terminology Employed to Describe Transformations in vitro Type of transformation Essential features Irregular growth Lack of contact inhibition of cell membrane movement ("ruffled membranes") between juxtaposed cells Unrestrained growth Deficient inhibition of the cell cycle (mitosis) in a crowded culture Infinite growth Capacity of cells to undergo an infinite number of di­ visions (formation of established cell lines) Cells may depart from this typical behavior in numerous ways involving for instance cellular morphology, immunology, chromosomes or metabolism. Such changes have, sometimes rather vaguely, been called "transformations". This is unprecise and the term "transformation" will here be used exclusively to indicate disturbances in cell growth related to neoplasia.

I. Introduction Of the ever increasing number of viruses known to affect man and higher animals, the virus of lymphocytic choriomeningitis (LCM) was one of the first to be discovered. Indeed, this virus has been known and maintained in the laboratory by passages in a relatively simple host, the mouse, for 35 years. Yet our knowl­ edge of its properties is still scanty when compared with the wealth of informa­ tion available for other viruses, some of which have come to our attention much more recently. There are at least four reasons which may help to explain this seeming paradox. (1) The early belief that the LCM virus was the frequent cause of human diseases had soon to be abandoned; infections of man with this virus are rare. (2) By way of contrast, laboratory infections are not uncommon and they frequently run severe and even fatal courses. (3) Until recently, the only means of titrating the virus was by mouse inoculation, a method in which accuracy and economy are poorly correlated. (4) The virus is of unusual lability, being quickly inactivated under conditions which leave other viruses intact. Thus, when balancing medical and theoretical importance against personal hazard and tech­ nical difficulties, the result was quite unfavorable, and lack of interest was really not surprising. In the last few years, however, the situation has gradually changed and an increasing number of workers have turned their attention to this virus.

2 D. Immunological Response . . . . . . . . . . . . . . . . . . . . 78 1. Thymectomy in LDV Infection . . . . . . . . . . . . . . . . 81 2. Effect of LDV Infection on Immune Response to Various Antigenic Stimuli. . . . . 82 E. Tumour Growth . . 87 F. Histological Changes 91 VII. Ecology . . . . . . . 97 VIII. Laboratory Methods. . 100 A. Blood Samples from Mice for LDH Estimation . 100 B. Estimation of Plasma LDH Activity . . 101 1. Quantitative Methods. . . . . . . . 101 a) Determination of Plasma LDH by Spectrophotometric Method (Backward Reaction) . . . . . . . . . . . . . . . 101 b) Determination of Plasma LDH by Spectrophotometric Method (Forward Reaction) . . . . . . . . . . . . . . . . 102 c) Determination of Plasma LDH by Colorimetric Method 103 2. Qualitative Method. . . . . . . 103 3. Units of LDH Activity . . . . . 104 C. Diagnosis of LDV Infection in Miee. 105 D. Virus Titration. 105 References. . . . . . . . . . . . . . . . 106 We wish to express our thanks to our many colleagues who generously provided us with preprints of their work, and unpublished observations. We are particularly indebted to those who donated prints of their electron micrographs of the virus. I. Introduction Inapparent virus infections of experimental animals and tissue culture systems present to the investigator a problem which it is impossible to overcome completely. Although all recognised viruses can be excluded from an experimental system, previously unsuspected viruses causing no obvious effects ('silent' viruses) will continue to be discovered. A truly silent virus would replicate, causing no change in its host cell, damage to infected tissue or immune response and would pre­ sumably be of no consequence.

I. Introduction In his biography "Arrow in the Blue" the author Arthur Koestler suggests ironically that the fate of an individual may be predicted by examining the content of the newspapers at birth. Adenoviruses were discovered in 1953 (ROWE et al. , 1953; HILLEMAN and WERNER, 1954). At this time the Salk poliomyelitis vaccine was developed (SALK et al. , 1954) and in the same year the discovery of the double helical structure of DNA (WATSON and CRICK, 1953) and the plaque assay for one animal virus (DULBECCO and VOGT, 1953) was announced. Thus, this new group of viruses was born with great hopes for progress in molecular biology and for the control of animal virus infections. In the short interval be­ tween 1953 and 1956 the adenoviruses were discovered, methods for laboratory diagnosis and serotyping were established, the epidemiology was clarified and a highly effective vaccine was developed and approved (for a review see HILLE­ MAN, 1966). Succeeding years showed, however, that the vaccines were contami­ nated with the oncogenic SV 40 virus and that the adenoviruses themselves were tumorigenic. Since the discovery of adenoviruses animal virology was developed into a quantitative science offering explanation for viral functions at the molecular level. Precise biochemical tools to characterize the genome and its transcription products as well as the structural proteins of these viruses are now available.

I. Introduction Parvoviruses belong to the large group of viral agents of which virologists have become aware by chance in many biological materials due to the availabil­ ity of more sensitive isolation techniques and the extensive use of the electron microscope. In general, many of these viruses lacked the stimulating background of an infectious disease and, therefore, have fallen into oblivion already soon after discovery. In case of parvoviruses, however, interest has been maintained because of the circumstances under which most of them were isolated. A great number of parvoviruses has been recovered from tissues of tumor­ bearing animals, from cell-free filtrates of tumors, or from stable cell lines of tumor origin. These observations necessarily suggested the newly isolated viral agents of playing an important, yet unknown role in the induction and develop­ ment of cancer. On the other hand, further parvoviruses were found constantly associated with adenoviruses. It was the experimental analysis of the multiplica­ tion behaviour which then revealed that the association between parvoviruses and tumors or parvoviruses and adenoviruses originates from the basis of a cer­ tain genetic defectiveness. For some members of the group this may be overcome by cellular helper effects in rapidly growing tissues, for several others, however, by biochemical events in the simultaneously occurring replication of an adeno­ virus only. Additional points of view in favour of parvovirus research have arisen from experimental animal studies.

2. Virological Findings. 90 3. Immunity. . . . . 90 C. Secondary Dengue: Dengue Hemorrhagic Fever and the Shock Syndrome 92 1. General Remarks. . . . . . . . . . . . . . 92 2. Clinical Course and Clinical Laboratory Findings 93 3. Virological and Serological Findings. . . 95 4. Immunopathology of Secondary Dengue. 98 XI. Immunization. . . . . . . . . . . . . . . 104 A. Anamnestic Immune Responses in Sequential Infections With Dengue and Other Group B Togaviruses . . . . . . . . 104 1. Results With Members of the Dengue Subgroup 104 2. Results With Dengue and Other Flaviviruses. 107 B. Dengue Vaccines for Use in Man 108 XII. Opportunities for the Future 113 Acknowledgments. 114 References. . . . . . . . . . . 114 I. Introduction Dengue fever is a mosquito-transmitted disease of man which has afflicted untold millions of people over the past two centuries. It is caused by viruses classified as a subgroup of the group B togaviruses. Along with other members of that group as well as group A, the dengue viruses have been investigated intensively during recent years. Certain unique aspects of their structure, composition, antigenicity, replication, and antigenic relationships have established the togavirus family as quite distinct from other families of enveloped RNA viruses (see recent review of PFEFFERKORN and SHAPIRO, 1974). The basic studies leading to this conclusion have coincided with epidemiological field investigations which have resulted in a continuing increase in the number of viruses now designated as group A or B togaviruses. This, in turn, has led to a growing appreciation of their immense importance as actual or potential pathogens of man and beast.

l RNA tumor viruses have become increasingly utilized in studies of cellular transformation and gene regulation. The genes of retroviruses exist in two forms; as extrachromosomal, RNA-containing, infectious particles and as DNA pro­ 2 viruses stably associated with cell genes. Components from the extracellular form can be collected in large quantity and purified for the preparation of molec­ ular probes. These probes can be used to dissect the sequence of events required for the establishment and expression of the integrated form. Furthermore the 2 genomes of retroviruses originated from normal cell genes, genes called virogenes • The nucleic acid and protein probes isolated from these viruses are therefore useful for studying the nature and expression of this normal cell gene and in elucidating the physiological role of its products. RNA tumor viruses perhaps offer us one of the most complete sets of biochemical reagents and biological responses for examining gene regulation in vertebrates and for studying the consequences of aberrant gene regulation on cell growth in tissue culture and in animals. Further­ more, there is an increasing conviction that virogenes play an important role in normal development and/or differentiation (RISSER, STOCKERT and OLD, 1978). Consequently, there is a growing feeling that DNA proviruses are altered viro­ genes and are capable of interfering with normal development or differentiation, causing reprogrammed growth or the incapacity to specialize.

Bluetongue may be described as an acute insect borne disease of ruminants, manifested clinically in sheep by a catarrhal inflammation of the mucous mem­ branes of the digestive and respiratory systems and associated with degenerative changes in the skeletal musculature. The profound emaciation and weakness which follow the acute disease are responsible for a protracted convalescence and for serious economic losses due to diminished productivity. II. Historical During the early colonisation of Africa, susceptible Merino and other European breeds of sheep were introduced into the Cape, at first by the Dutch East India Company between 1652 and 1785 and again later in 1870. A report of the Cattle and Sheep Diseases Commission (1876) records the appearance of a serious febrile disease amongst these imported sheep in which both morbidity and mortality was high (cited by HENNING, 1949). HuTCHEON (1881) gave this disease the name of "Fever" or "Epizootic Catarrh", in order to distinguish it from other clinical conditions of a similar nature encountered amongst sheep. In the first com­ prehensive description of this clinical syndrome and its epizootiology HuTCHEON (1902) referred to it as "Malarial Catarrhal Fever of Sheep", a designation which was obviously influenced by the mistaken belief that an intracorpuscular parasite was the primary cause of the disease. More systematic studies were conducted by SPREULL (1902; 1905), who endeavoured to immunize sheep by the simultaneous inoculation of immune serum and infective blood.

Reoviruses are ether-resistant icosahedral viruses 60 to 75 mp in diameter which contain ribonucleic acid. They have been recovered from man and lower animals and are ubiquitous in their geographic distribution. At present, the importance of these viruses as a cause of human or animal disease is still largely unknown. As a result of having a number of unusual characteristics, reoviruses have attracted the attention of many workers in the relatively short time since they were first recognized. For example, investigators interested in the molecular aspects of virology have been attracted by the unusual double-helical ribonucleic acid of high molecular weight which reoviruses possess, while those interested in epidemiology have been attracted by the occurrence of apparently identical viruses in both man and an unusually wide variety of lower animals. This com­ pilation is based on information available to the author as of October 31, 1966. II. History The term "reqvirus" was proposed in 1959 (SABIN, 1959) as a group name for a number of viruses then classified (SABIN, 1956; RAMOS-ALvAREz and SABIN, 1958) as being identical with, or related to, ECHO type lO virus.

The herpes group consists of viruses which have been placed together on the basis of a number of distinguishing features that they share in common (ANDRE­ WES, 1962). All these viruses are relatively large, possess identical morphological characteristics, contain DNA, and are extremely sensitive to inactivation by ether; these viruses are also assembled within the nucleus of the host cell and induce the formation of eosinophilic intranuclear inclusions. The epidemiology of some of the best known viruses in this group (herpes simplex, pseudorabies, and B-virus) is also similar (BURNET et aI. , 1939). Herpes simplex virus exists in the latent state in man, the natural host for this virus, and becomes overt in individuals subject to some form of stress; this condition appears to be paralleled by pseudorabies virus in its natural host, swine and by B-virus in monkeys. In each instance, transmission of the virus to a susceptible host other than the natural one results usually in marked symptoms and death. This chapter is confined to a description of herpes simplex and pseudorabies viruses; B-virus is described separately elsewhere in the Hand book. Since the clinical aspects of the diseases caused by herpes simplex virus and pseudorabies virus have been well described, greater emphasis will be placed, therefore, on the basic biological and biochemical properties of these viruses; their clinical features will be discussed only briefly.

A. Definitions of Transformation in vitro When normal tissues or organs are explanted to conditions favoring the growth of cells as individual units ("cell culture"), the original cell population undergoes a large variety of modifications. Only a minority of the cells will thrive and multiply and within a rather short period of time, the complex composition of the original explant is replaced by a much simplified one of only a few recogniz­ ably different cell types. With most organs fibroblast-like cells survive longest and outgrow other types. This is then a stable state of affairs for many gener­ ations. This treatise will not discuss whether this simplification and stabilization represents selection of certain pre-existing cell types or a modification of cells into only a few recognizably different categories; for an excellent review see HARRIS. (1964). Table 1. Terminology Employed to Describe Transformations in vitro Type of transformation Essential features Irregular growth Lack of contact inhibition of cell membrane movement ("ruffled membranes") between juxtaposed cells Unrestrained growth Deficient inhibition of the cell cycle (mitosis) in a crowded culture Infinite growth Capacity of cells to undergo an infinite number of di­ visions (formation of established cell lines) Cells may depart from this typical behavior in numerous ways involving for instance cellular morphology, immunology, chromosomes or metabolism. Such changes have, sometimes rather vaguely, been called "transformations". This is unprecise and the term "transformation" will here be used exclusively to indicate disturbances in cell growth related to neoplasia.

I. Introduction Of the ever increasing number of viruses known to affect man and higher animals, the virus of lymphocytic choriomeningitis (LCM) was one of the first to be discovered. Indeed, this virus has been known and maintained in the laboratory by passages in a relatively simple host, the mouse, for 35 years. Yet our knowl­ edge of its properties is still scanty when compared with the wealth of informa­ tion available for other viruses, some of which have come to our attention much more recently. There are at least four reasons which may help to explain this seeming paradox. (1) The early belief that the LCM virus was the frequent cause of human diseases had soon to be abandoned; infections of man with this virus are rare. (2) By way of contrast, laboratory infections are not uncommon and they frequently run severe and even fatal courses. (3) Until recently, the only means of titrating the virus was by mouse inoculation, a method in which accuracy and economy are poorly correlated. (4) The virus is of unusual lability, being quickly inactivated under conditions which leave other viruses intact. Thus, when balancing medical and theoretical importance against personal hazard and tech­ nical difficulties, the result was quite unfavorable, and lack of interest was really not surprising. In the last few years, however, the situation has gradually changed and an increasing number of workers have turned their attention to this virus.

2 D. Immunological Response . . . . . . . . . . . . . . . . . . . . 78 1. Thymectomy in LDV Infection . . . . . . . . . . . . . . . . 81 2. Effect of LDV Infection on Immune Response to Various Antigenic Stimuli. . . . . 82 E. Tumour Growth . . 87 F. Histological Changes 91 VII. Ecology . . . . . . . 97 VIII. Laboratory Methods. . 100 A. Blood Samples from Mice for LDH Estimation . 100 B. Estimation of Plasma LDH Activity . . 101 1. Quantitative Methods. . . . . . . . 101 a) Determination of Plasma LDH by Spectrophotometric Method (Backward Reaction) . . . . . . . . . . . . . . . 101 b) Determination of Plasma LDH by Spectrophotometric Method (Forward Reaction) . . . . . . . . . . . . . . . . 102 c) Determination of Plasma LDH by Colorimetric Method 103 2. Qualitative Method. . . . . . . 103 3. Units of LDH Activity . . . . . 104 C. Diagnosis of LDV Infection in Miee. 105 D. Virus Titration. 105 References. . . . . . . . . . . . . . . . 106 We wish to express our thanks to our many colleagues who generously provided us with preprints of their work, and unpublished observations. We are particularly indebted to those who donated prints of their electron micrographs of the virus. I. Introduction Inapparent virus infections of experimental animals and tissue culture systems present to the investigator a problem which it is impossible to overcome completely. Although all recognised viruses can be excluded from an experimental system, previously unsuspected viruses causing no obvious effects ('silent' viruses) will continue to be discovered. A truly silent virus would replicate, causing no change in its host cell, damage to infected tissue or immune response and would pre­ sumably be of no consequence.

I. Introduction In his biography "Arrow in the Blue" the author Arthur Koestler suggests ironically that the fate of an individual may be predicted by examining the content of the newspapers at birth. Adenoviruses were discovered in 1953 (ROWE et al. , 1953; HILLEMAN and WERNER, 1954). At this time the Salk poliomyelitis vaccine was developed (SALK et al. , 1954) and in the same year the discovery of the double helical structure of DNA (WATSON and CRICK, 1953) and the plaque assay for one animal virus (DULBECCO and VOGT, 1953) was announced. Thus, this new group of viruses was born with great hopes for progress in molecular biology and for the control of animal virus infections. In the short interval be­ tween 1953 and 1956 the adenoviruses were discovered, methods for laboratory diagnosis and serotyping were established, the epidemiology was clarified and a highly effective vaccine was developed and approved (for a review see HILLE­ MAN, 1966). Succeeding years showed, however, that the vaccines were contami­ nated with the oncogenic SV 40 virus and that the adenoviruses themselves were tumorigenic. Since the discovery of adenoviruses animal virology was developed into a quantitative science offering explanation for viral functions at the molecular level. Precise biochemical tools to characterize the genome and its transcription products as well as the structural proteins of these viruses are now available.

I. Introduction Parvoviruses belong to the large group of viral agents of which virologists have become aware by chance in many biological materials due to the availabil­ ity of more sensitive isolation techniques and the extensive use of the electron microscope. In general, many of these viruses lacked the stimulating background of an infectious disease and, therefore, have fallen into oblivion already soon after discovery. In case of parvoviruses, however, interest has been maintained because of the circumstances under which most of them were isolated. A great number of parvoviruses has been recovered from tissues of tumor­ bearing animals, from cell-free filtrates of tumors, or from stable cell lines of tumor origin. These observations necessarily suggested the newly isolated viral agents of playing an important, yet unknown role in the induction and develop­ ment of cancer. On the other hand, further parvoviruses were found constantly associated with adenoviruses. It was the experimental analysis of the multiplica­ tion behaviour which then revealed that the association between parvoviruses and tumors or parvoviruses and adenoviruses originates from the basis of a cer­ tain genetic defectiveness. For some members of the group this may be overcome by cellular helper effects in rapidly growing tissues, for several others, however, by biochemical events in the simultaneously occurring replication of an adeno­ virus only. Additional points of view in favour of parvovirus research have arisen from experimental animal studies.

2. Virological Findings. 90 3. Immunity. . . . . 90 C. Secondary Dengue: Dengue Hemorrhagic Fever and the Shock Syndrome 92 1. General Remarks. . . . . . . . . . . . . . 92 2. Clinical Course and Clinical Laboratory Findings 93 3. Virological and Serological Findings. . . 95 4. Immunopathology of Secondary Dengue. 98 XI. Immunization. . . . . . . . . . . . . . . 104 A. Anamnestic Immune Responses in Sequential Infections With Dengue and Other Group B Togaviruses . . . . . . . . 104 1. Results With Members of the Dengue Subgroup 104 2. Results With Dengue and Other Flaviviruses. 107 B. Dengue Vaccines for Use in Man 108 XII. Opportunities for the Future 113 Acknowledgments. 114 References. . . . . . . . . . . 114 I. Introduction Dengue fever is a mosquito-transmitted disease of man which has afflicted untold millions of people over the past two centuries. It is caused by viruses classified as a subgroup of the group B togaviruses. Along with other members of that group as well as group A, the dengue viruses have been investigated intensively during recent years. Certain unique aspects of their structure, composition, antigenicity, replication, and antigenic relationships have established the togavirus family as quite distinct from other families of enveloped RNA viruses (see recent review of PFEFFERKORN and SHAPIRO, 1974). The basic studies leading to this conclusion have coincided with epidemiological field investigations which have resulted in a continuing increase in the number of viruses now designated as group A or B togaviruses. This, in turn, has led to a growing appreciation of their immense importance as actual or potential pathogens of man and beast.

l RNA tumor viruses have become increasingly utilized in studies of cellular transformation and gene regulation. The genes of retroviruses exist in two forms; as extrachromosomal, RNA-containing, infectious particles and as DNA pro­ 2 viruses stably associated with cell genes. Components from the extracellular form can be collected in large quantity and purified for the preparation of molec­ ular probes. These probes can be used to dissect the sequence of events required for the establishment and expression of the integrated form. Furthermore the 2 genomes of retroviruses originated from normal cell genes, genes called virogenes • The nucleic acid and protein probes isolated from these viruses are therefore useful for studying the nature and expression of this normal cell gene and in elucidating the physiological role of its products. RNA tumor viruses perhaps offer us one of the most complete sets of biochemical reagents and biological responses for examining gene regulation in vertebrates and for studying the consequences of aberrant gene regulation on cell growth in tissue culture and in animals. Further­ more, there is an increasing conviction that virogenes play an important role in normal development and/or differentiation (RISSER, STOCKERT and OLD, 1978). Consequently, there is a growing feeling that DNA proviruses are altered viro­ genes and are capable of interfering with normal development or differentiation, causing reprogrammed growth or the incapacity to specialize.