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--> 3 Clinical Features of Smallpox The term pathogenesis is used to describe the mechanisms involved in the production of disease, from the spread of infection through the body to the molecular and physiological responses of host cells to a pathogen. Immune response is the constellation of mechanisms by which the host limits continued multiplication of the pathogen. These mechanisms include T cell and B cell responses that are specific for the organism and are acquired during the course of the infection. Some of these mechanisms are innate, while others are adaptive. The pathogenesis of smallpox has been studied in three ways: (1) by using material from human patients; (2) by conducting experiments with variola virus infection of nonhuman primates; and (3) by conducting experiments with model infections in mice, rabbits, and monkeys using related orthopoxviruses. Investigations in human subjects prior to eradication were limited to virological and serological tests of hospitalized smallpox patients or case contacts and case histories. Much of our understanding of the pathogenesis of generalized orthopoxvirus infections is based on studies carried out with ectromelia (mousepox). Entry and Infection The usual entry of variola virus is through the respiratory tract with infection of the oropharyngeal (mouth) or respiratory (trachea and lung) mucosa. Secretions from the mouth and nose, rather than scab material, are the most important source of human-to-human transmission. The initial infection in the oropharynx or respiratory tract produces neither symptoms nor local lesions, and patients are not infectious until an oropharyngeal enanthem appears at the end of the primary incubation period. Transmission to others is generally through coughing out of virions in oropharyngeal secretions. Patients are most infectious
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--> during the first week of rash. Scab material forms as the rash dries and usually consists of large fragments of cellular debris, with virions bound within a dense, fibrous mesh containing a large amount of the antiviral substance interferon. Infectious virus is difficult to release from scabs except by mechanical grinding. Inoculation smallpox sometimes occurs when variola virus is introduced into the skin either intentionally or accidentally. A local skin lesion appears on the third or fourth day, with fever and constitutional symptoms beginning on the eighth day. The incubation period is typically 2 to 3 days shorter than in natural smallpox. The rash, which is usually less severe than in naturally acquired smallpox, appears on the tenth or eleventh day. The milder course of disease stemming from deliberate variola inoculation was the basis for variolation, which preceded Jenner's use of cowpox and the subsequent use of vaccinia inoculation as a preventive for smallpox (as discussed further below). Very rarely, vaccinia vaccination produces dissemination vaccinia, a systemic infection characterized by malaise combined with a generalized rash similar to inoculation lesions. In another condition, called progressive vaccinia, the inoculation lesion fails to heal, and secondary lesions sometimes appear elsewhere. Both conditions are problematic predominantly for individuals with deficient immune mechanisms. Variola major caused severe problems in pregnant women. Abortions and stillbirths were frequent, and a majority of the babies born to infected women in hospital died within 15 days, most within 3 days. About half of the babies acquired infection in utero or at the time of delivery. Dissemination The appearance of high fever and then lesions on the skin marks the end of the incubation period. Smallpox pathogenesis is a poorly understood series of events in which the virus first disseminates locally, then through the lymphatic system, and finally to the skin without affecting vital organs. In mousepox, the primary source of molecular studies of orthopoxvirus pathogenesis, the infection moves from the respiratory tract to the liver and spleen following breach of the macrophage barrier. The virus then replicates extensively in both organs, which produces semiconfiuent necrosis. About a day after infection of the liver and spleen, large numbers of virions are liberated into the bloodstream, leading to secondary infection of the skin, kidneys, lungs, intestines, and other organs. This phase is followed by an interval of 2 or 3 days during which the virus replicates and reaches a high titer before visible changes are apparent in the infected organs. As noted earlier, mousepox is unusual among the generalized orthopoxvirus infections in that the spleen and liver are the major target organs for viral replication. There is inadequate evidence regarding exactly where the virus replicates during a smallpox infection. The likely sites for viral replication are the lymphoid organs (spleen, bone marrow, lymph nodes), but extensive necrosis does not occur in those sites. At this stage, the virus in the blood is largely cell-associated.
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--> The Rash The primary event that triggers the production of focal lesions in orthopoxvirus infections is the localization of virus particles in the small dermal blood vessels. Subsequently, adjacent epidermal cells are infected, and skin lesions develop. The earliest change is dilation of the capillaries in the papillary layer of the dermis, followed by swelling of the endothelial cells in the walls of these vessels and subsequently perivascular cuffing with lymphocytes, plasma cells, and macrophages. Following these early changes, the cells of the Malpighian layer become swollen and vacuolated. The cells continue to increase in size, and the nucleus usually disappears or is lysed. The cell membrane then ruptures, and the vacuoles coalesce to produce the early vesicle. Because this coalescence occurs quickly, a true papule is rarely seen, and the lesions appear vesicular almost from the beginning. Except on the palms and soles, umbilication is a common feature of skin lesions in smallpox. It is due mainly to swelling of the cells around the vesicle and proliferation of the basal cells surrounding the lesion, so that the periphery of the vesicle is raised above its center. The mechanisms that allow the localization of variola virus in the skin and the characteristic "centrifugal' distribution of the rash are not known. With the development of an effective immune response (described below), healing begins. The contents of the pustule become desiccated, and reestablishment of the epithelial skin layer occurs between the cavity of the pustule and the underlying dermis. The pustule contents become a crusty scab. On the soles and palms, the horny layer of the skin is very thick, and the dried exudate often remains for a long period if not removed artificially. The face bears the heaviest crop of lesions in most cases of smallpox, and scarring is more common there than elsewhere. Although cells of other skin appendages (hair follicles and sweat glands) are relatively unaffected by variola virus, cells of the sebaceous glands are highly susceptible. Degeneration occurs simultaneously in several parts of the gland, leading to extensive necrosis. When healing occurs, the defect in the dermis fills with granulation tissue, which frequently shrinks, leaving localized facial pockmarks. Lesions of the Mucous Membranes Although the oropharynx and respiratory tract are usually regarded as the portal of entry for smallpox, primary lesions have not been observed in these areas. The mucous membranes in which enanthem lesions later develop are, in order of frequency, the pharynx and uvula, the larynx, the tongue, and the upper part of the trachea and esophagus. Lesions of the lower trachea and bronchi are much less frequent. Epithelial cells in mucous membranes are not as tightly packed as in skin, and there is no horny layer. Therefore, there is more pronounced exudation of fluid into the subepithelial tissues than occurs with dermal lesions. This exuda-
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--> tion and the subsequent separation of cells and their degeneration are the earliest changes. Instead of a vesicle, the extensive necrosis in the epithelial cells, unrestrained by a homy layer, leads to ulceration. Later, increasing vascularization takes on the appearance of granulation tissue, with numerous polymorphonuclear leukocytes in the demarcation zone beneath the necrotic epithelium. The lack of a homy, keratinized cell layer permits the lesions on the mucous membranes to ulcerate soon afar their formation, releasing large amounts of highly infectious virus into the saliva. Viral titers, or amounts of virus in throat swabs, are at their maximum on the third and fourth days of the disease. In fatal cases, virus is usually still present in throat swabs at the time of death. The onset of infectivity coincides with the development of the rash and is due to the release of the virus from the ulcerated surfaces of these skin and mucosal lesions. Effects on Other Organs Death is usually the result of disseminated intravascular coagulation, hypotension, and cardiovascular collapse; these are exacerbated by clotting defects in the rare hemorrhagic type of smallpox. The endothelial cells lining the sinusoids of the liver are often swollen and occasionally proliferating or necrotic. Reticulum cell hyperplasia occurs in the bone marrow and spleen. The spleen is usually engorged and contains many large lymphoid cells. The liver is generally considerably heavier than normal, but this does not appear to be due to engorgement or fatty infiltration. Encephalitis is an occasional complication. Immune Response The human immune response to viruses is a complicated process about which much has yet to be discovered. Furthermore, knowledge of interactions between the immune system and variola virus is limited because modem techniques for the study of immune responses were developed after smallpox was eradicated. At least three types of cells are involved: macrophages, dendritic cells, and lymphocytes. Macrophages and dendritic cells process antigens for presentation to T cells (thymus-derived lymphocytes, of which there are several subclasses). Macrophages and dendritic cells may acquire antigen at a given location from which they migrate to specialized lymphoid tissue—lymph nodes—the architecture of which allows for the cell-cell interactions required for a proper immune response. Importantly, certain antigen-presenting cells are able to be infected with different viruses, including orthopoxviruses. B cells are responsible for producing immunoglobulin, and often require help from T cells to do so. Such B lymphocytes have immunoglobulins on their cell surface that function as antigen-specific receptors. When an antigen triggers the B cell receptor and when appropriate help is delivered to the B cells by T lymphocytes or their secreted products, the B cells are stimulated to respond
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--> clonally. B cells differentiate into antibody-secreting plasma cells or long-lived memory cells, which are critically important in mounting a secondary response upon re-exposure to antigen. While immunoglobulins can recognize antigen directly, the antigen-specific receptors on T lymphocytes interact with antigens on the surface of cells that present antigen in the form of short peptides (8–15 amino acids) in a complex with products of the Major Histocompatibility Complex (MHC). The generation of the peptides requires the proteolytic conversion of protein antigens into suitably sized peptides (antigen processing). The complicated series of reactions between antigen-presenting and T cells serves to announce to the immune system the presence of intracellular pathogens such as viruses. Not surprisingly, a number of viruses have evolved mechanisms that deflect this process, presumably helping them avoid immune recognition. T cells respond specifically to MHC antigen presentation by clonal expansion. When activated, they secrete active substances called cytokines. As with B cells, a subset of activated T cells become long-lived memory cells. There are subclasses of T cells that have different functions. Some act to modulate B cells and other T cells by enhancing or suppressing their proliferation or their production of antibodies or cytokines. T cells that recognize viral proteins differentiate on contact with a specific antigen to release cytokines, such as gammainterferon. Such cells cause delayed-type hypersensitivity reactions, thereby attracting other inflammatory cells to the sites of infection. Certain T cells are cytotoxic and actively destroy cells exhibiting specific viral proteins composed of MHC products on their surface. During infection with a virus as complex as an orthopoxvirus, antibodies specific to many different viral proteins are generated. Antibodies of three types have received specific attention: (1) those that neutralize viral infectivity; (2) those that, in conjunction with other proteins, Icad to lysis of virus-infected cells; and (3) those that combine with circulating antigens to produce immune complexes. Information about T cell responses to smallpox is very limited. However, in other viral infections, T cell responses often precede the appearance of neutralizing or other antibodies. Clearance of virus in many situations correlates with activation and expansion of virus-specific T cells. The kinetics of the appearance of antibodies is often slower than the initiation of T cell responses in the generation of cytotoxic T cells and T cells that produce interferon-gamma or other cytokines. This may be the case for smallpox as well, although the molecular tools needed to evaluate this question were not available when the disease was prevalent. In nonhemorrhagic smallpox, hemagglutinin-inhibiting (HI) and neutralizing antibody titers increase from about the sixth day of illness (approximately l g days after infection), and most patients develop immunoprecipitating antibodies that can be demonstrated by Ouchterlony-like gel precipitation assay about 2 days later. Patients with the rare hemorrhagic type of smallpox have much-reduced neutralizing antibody responses. HI titers rise and are approximately the same for patients with either ordinary or hemorrhagic smallpox.
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--> Lowered numbers of both T cells and B cells have been observed in smallpox patients, but the subtypes of the T cells have not been determined. The best information on the relative importance of cell-mediated and humoral immune responses to orthopoxvims infections in humans comes from studies of human subjects in immune-deficient states who are subsequently vaccinated with vaccinia virus. In children with immunological defects in cell-mediated immunity, vaccinia virus replicates without restriction, resulting in a continually progressive primary lesion, persistent viremia, and widespread secondary viral infection of many organs. In patients with thymic dysplasia and partially or completely intact immunoglobulin-synthesizing capacity (Nezelofs syndrome), the progression is slower and less persistent, but usually results in death. Delayed-type hypersensitivity reactions are not evoked in patients with progressive vaccinia, nor can their peripheral blood lymphocytes be stimulated to undergo mitosis by exposure to inactivated vaccinia virus. Although neutralizing antibody is sometimes present in the serum, its presence does not prevent the development of progressive vaccinia if cell-mediated immunity is defective. Orthopoxvirus-specific T cell and B cell memory can be thought of as involving previously induced lymphocytes that persist as long-lived but nonactivated cells sequestered in lymphoid tissue and in the recirculating pool of lymphocytes. The effectiveness of memory T cells in providing protective immunity against orthopoxviruses decreases as the interval between primary and secondary infection increases. Immunity Against Smallpox All orthopoxviruses induce cross-protective immunity in susceptible laboratory animals. Indeed, that is one of the ways of identifying members of the genus and is the basis for currently available vaccines. Among the orthopoxviruses that infect humans, cowpox and vaccinia viruses usually produce only local lesions and minimal systemic disturbance. Variola and monkeypox viruses cause serious systemic disturbance with high case-fatality rates. The observation that recurrences of smallpox were very rare had been made in ancient times, and led to attempts to ameliorate the severity of smallpox by administering pustular fluid or dried scab material to the nostrils or skin of persons who had not yet contracted smallpox. Much later, it was observed that similar protection against smallpox could be obtained by administering cowpox or vaccinia virus. The process of inoculating smallpox material is called variolation to distinguish it from vaccination, which uses cowpox or vaceinia virus. After variolation of the skin, a primary lesion develops at the inoculation site on about the third day, and satellite pustules are common. But the rash is usually much less severe than with naturally occurring smallpox. Historically, case-fatality rates were between 0.5 and 2 percent after variolation, compared with 20 to 30 percent from natural smallpox. Since the virus material used was not attenuated, it
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--> was possible for those receiving variolation to transmit ordinary smallpox to susceptible contacts. Early in the 18th century, the variolation procedure spread through the Balkans into central Europe and from Turkey to Great Britain and the rest of Europe. Subsequently, Jenner confirmed earlier observations that a person who had suffered cowpox did not get smallpox. In 1796 he took matter from cowpox lesions on the hand of Sarah Nelmes, a young dairymaid, and used it to inoculate an 8-year-old boy. After the boy's slight fever and iow-grade lesion disappeared, Jenner attempted to variolate him by inoculating him with smallpox. The primary lesion did not develop, and protection was complete . The educated public was receptive to this discovery. Jenner's vaccine was a way of providing the advantages of variolation without the associated risks, and there was no doubt that cowpox produced a much less severe disease than variolation. To recognize Jenner's contribution, Louis Pasteur later proposed that this method of protecting against infectious diseases be called vaccination and the product used a vaccine, although the general process is now usually called immunization. At some unknown point in time, vaccinia virus became substituted for cowpox virus, probably because it produced generally milder lesions and lower fever. Modern vaccination against smallpox consists of abrading the skin with vaccinia virus, which may subsequently spread to the lymph nodes and spleen, organs heavily involved in initiating the immune response (as discussed earlier). The result of spread to these sites is the induction of cell-mediated and humoral immunity, and long-lived memory T and B cells that recognize the virus. Replication of variola virus is completely prevented for a few years, and thereafter replication is limited so that infection is subclinical, causing no symptoms. Immunity wanes over time and can decline to levels that do not protect against illness, although the severity of disease is likely to be reduced. Passive immunization, as a natural consequence of either transmission of antibodies from mother to progeny or the administration of antisera, is less effective in modifying the course of disease than active immunization involving live virus. Active immunity, whether elicited by vaccination or the disease, provokes the complete range of cell-mediated and humoral immune responses, whereas passive immunization provides only the antibodies present in the source of the sera. Three groups of complications occurred in a small number of vaccinated subjects: abnormal skin eruptions, disorders affecting the central nervous system, and a variety of other rarer or less severe complications. Although trivial compared with the problems historically associated with smallpox, these complications posed significant health risks when smallpox receded. Therefore, vaccination was discontinued once smallpox had been eradicated globally.
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