CHAPTER 2 VARIOLA VIRUS AND OTHER ORTHOPOXVIRUSES Contents Page Introduction 70 Classification and nomenclature 71 Development of knowledge of the structure of poxvirions 71 The nucleic acid of poxviruses 72 Classification of poxviruses 72 Chordopoxvirinae: the poxviruses of vertebrates 72 The genus Orthopoxvirus 73 Recognized species of Orthopoxvirus 73 Characteristics shared by all species of Orthopoxvirus 75 Morphology of the virion 75 Antigenic structure 76 Composition and structure of the viral DNA 79 Non-genetic reactivation 80 Characterization of orthopoxviruses by biological tests 81 Lesions in rabbit skin 81 Pocks on the chorioallantoic membrane 82 Ceiling temperature 82 Lethality for mice and chick embryos 83 Growth in cultured cells 83 Inclusion bodies 86 Comparison of biological characteristics of different species 86 Viral replication 86 Adsorption, penetration and uncoating 86 Assembly and maturation 86 Release 88 Cellular changes 89 Characterization of orthopoxviruses by chemical methods 90 Comparison of viral DNAs 90 Comparison of viral polypeptides 93 Summary: distinctions between orthopoxviruses 95 Variola virus 95 Isolation from natural sources 96 Variola major and variola minor 96 Laboratory investigations with variola virus 97 Pathogenicity for laboratory animals 97 Growth in cultured cells 98 Laboratory tests for virulence 99 Differences in the virulence of strains of variola major virus 101 Comparison of the DNAs of strains of variola virus 101 Differences between the DNAs of variola and monkey- pox viruses 103 69 70 SMALLPOX AND ITS ERADICATION Page Variola virus (cont.) Genetic studies 103 Species diagnosis 104 Vaccinia virus 104 Isolation from natural sources 105 The variability of strains and their pathogenicity 105 Variation within a strain 106 Genetic studies 107 Species diagnosis 108 Cowpox virus 108 Cowpox and horsepox in Europe 108 Genetic studies 108 Species diagnosis 109 Laboratory confirmation of smallpox diagnosis 109 Preparation of a guide for laboratory diagnosis 110 Methods of laboratory diagnosis 110 Comparison of different laboratory diagnostic methods 112 Tests for species-specific viral antibodies 114 Resistance to physical and chemical agents 114 Vaccinia virus 115 Variola virus 115 Speculations about the origins of variola virus 116 The diversity and specificity of viruses of man 117 Requirements for human-to-human transmission 117 Possible derivation of variola virus from another Orthopoxvirus 118 Variola virus as the descendant of an Orthopoxvirus of early man 119 Conclusions 119 INTRODUCTION diseases with which this book is less directly concerned, was delineated as early as any other Because of its distinctive clinical picture, viral genus as the “variola-vaccinia subgroup’’ described in the previous chapter, smallpox of the poxvirus group. has been recognized as a disease entity for This chapter outlines the historical devel- many centuries. Its control by deliberate opment and current state of knowledge of the intervention, at first by variolation, then by orthopoxviruses, based primarily on studies vaccination, began long before such measures with vaccinia virus. Much of the material were adopted for any other disease. Likewise, presented will be of special interest to bio- knowledge of the virus that produced the logists, but it includes topics of greater disease and those that were used to control it, complexity and of a more technical nature variola and cowpox or vaccinia viruses respec- than can be readily understood by the other- tively, is as old as the relatively new science of wise informed general reader. However, the virology. The particles that cause these two authors consider that it is important in this diseases were seen with the microscope, and book to endeavour to embrace the full scope then by electron microscopy, before any other of currently available knowledge of the ortho- viruses had been visualized, and their chemi- poxviruses. For the virologist the account will cal composition was analysed earlier than that appear unbalanced, since the intention is to of any other animal virus. The family to limit it to providing the virological back- which they belong, now called Poxviridae, ground that is necessary to understand how was correctly categorized before any other the body responds to infection with these viral family, and the genus Orthopoxvirus, viruses, how the clinical diagnosis can be whose members are the causative, agents of confirmed by laboratory studies, and what smallpox, vaccinia and the several related other related agents may pose threats to man, 2. VARIOLA VIRUS AND OTHER .ORTHOPOXVIRUSES 71 \ The Nature of Viruses in General and Poxviruses in Particular Viruses form a distinct group of agents, which differ fundamentally from cellular microorganisms. The infective particle, known as the virion, is inert; it proceeds to a dynamic phase only after it enters a susceptible cell and loses enough of its outer protective layers to allow its genetic material to be transcribed and translated. The inert poxvirion is the largest of all virions and its genetic material, a single molecule of double-stranded DNA, is among the largest of all viral genomes. Poxviruses differ from most other DNA viruses in that they replicate in the cytoplasm rather than in the nucleus of susceptible cells. To accomplish this, they have a battery of enzymes not found in other DNA viruses, including a viral DNA-dependent RNA polymerase which transcribes messenger RNA from the viral DNA. confuse the diagnosis or give rise to problems term “elementary bodies”, originally intro- in interpreting ecological data. To do this it duced by Chaveau (1868) and used until will be necessary to describe some features of recently to describe the virions. Paschen the orthopoxviruses, such as their structure, (1906) used a modified Loeffler’s flagellar the composition of their genetic material and stain and championed the belief that the their behaviour in experimental animals, in elementary bodies of vaccinia virus thus made some detail, but it is not necessary to provide a visible with the microscope were the infective detailed analysis of the complex events of the particles ;they were later also called “Paschen replication cycle, a feature which has always bodies” in recognition of Paschen’s extensive been of central interest to virologists. work in this field (Plate 2.1). Negri (1906) had shown that the infectivity of vaccine lymph would remain after the lymph had been CLASSIFICATION AND passed through a filter that held back bacteria, NOMENCLATURE but final proof that the elementary body was indeed the infectious entity was not provided The internationally accepted classification until Ledingham (1931) showed that antisera of viruses is based primarily on the morphol- produced against vaccinia or fowlpox viruses ogy of the viral particle (virion) and the would simultaneously and specifically agglu- nature and structure of the viral nucleic acid. tinate the particles and neutralize the infec- As the largest of all viruses, the virions of tivity of the homologous but not the heter- poxviruses were the first to be seen with the ologous virus. microscope. Development of Knowledge of the Structure of Poxvirions As early as 1886, Buist (see Gordon, 1937) reported that he had seen what must have been the virions of vaccinia virus in stained smears, although he regarded them as spores. Calmette & GuCrin (1901) used the rabbit to assay batches of vaccine lymph and in the course of this work they observed that the lymph contained numerous minute refractile particles which they suggested might be the “virulent elements”. These observations were Plate 2. I. “Elementary bodies” (virions) of vaccinia confirmed by Prowazek (1905), an expert virus. Bar = lpm. Imprint of a rabbit cornea infected microscopist, who found that they could be with vaccinia virus, prepared and stained by Paschen stained by Giemsa’s method, and revived the in 1906. 72 SMALLPOX AND ITS ERADICATION Further analysis of the structure of pox (caused by a herpesvirus) and “the great POX” virions has depended on the use of the (syphilis-caused by a spirochaete) in the electron microscope with virions treated in same category as smallpox. various ways. Enzymatic digestion was used By examining sections of poxvirus-infected to demonstrate the existence of a substructure tissues, pathologists came to recognize cyto- within the brick-shaped virions (Dawson & plasmic inclusion bodies as characteristic of McFarlane, 1948). In a series of classical poxvirus infection (Guarnieri, 1892), al- studies, Peters (1956), using enzymatic diges- though for many years they were regarded as tion with deoxyribonuclease and metal shad- protozoa. Gradually, however, the signifi- owing, demonstrated the major viral com- cance of the minute particles seen in stained ponents and designated them as the outer smears was appreciated, and by the 1920s membrane, the lateral bodies and the core. Aragiio (1 927) grouped together, as belong- Thin sections of infected cells have been ing to one family, the viruses of “myxoma, particularly valuable in elucidating the mor- smallpox, molluscum contagiosum, epithe- phogenesis of the virions of vaccinia virus. lioma of fowls, etc.”. Subsequently, Goodpas- Negative staining, combined with a variety of ture (1933) formally proposed that vaccinia- methods of degrading the virion, has been variola, fowlpox, horsepox, sheep-pox, goat- useful in analysing the substructure of vac- pox, swinepox and molluscum contagiosum