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Factors of Emergence

VARIATION AND INTERSPECIES TRANSMISSION OF INFLUENZA A VIRUSES

Robert G. Webster, Ph.D.

Department of Virology and Molecular Biology

St. Jude Children’s Research Hospital

The problem with influenza viruses is that, unlike other respiratory tract viruses, they undergo considerable antigenic variation. Antigens, which are located on the surface of the viruses, are the substances, typically glyco-proteins, that stimulate an immune response. Influenza viruses carry two types of antigens: hemagglutinin (HA) and neuraminidase (NA). Both types undergo two forms of variation. The first type, called drift, involves minor changes in the antigens. New drift strains emerge constantly, giving rise to yearly epidemics and forcing the medical community to revise the viral strains used in vaccines. The second type of antigenic variation, called shift, involves major changes in the virus’s genetic makeup. These new variants are of even greater concern: new strains to which most humans have no immunity appear suddenly, and the resulting pandemics vary from serious to catastrophic. While we currently know the complete genome sequences of many influenza viruses, we do not understand the molecular basis of pathogenesis and are unable to predict which combinations of viral genes have pandemic potential.



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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary 3 Factors of Emergence VARIATION AND INTERSPECIES TRANSMISSION OF INFLUENZA A VIRUSES Robert G. Webster, Ph.D. Department of Virology and Molecular Biology St. Jude Children’s Research Hospital The problem with influenza viruses is that, unlike other respiratory tract viruses, they undergo considerable antigenic variation. Antigens, which are located on the surface of the viruses, are the substances, typically glyco-proteins, that stimulate an immune response. Influenza viruses carry two types of antigens: hemagglutinin (HA) and neuraminidase (NA). Both types undergo two forms of variation. The first type, called drift, involves minor changes in the antigens. New drift strains emerge constantly, giving rise to yearly epidemics and forcing the medical community to revise the viral strains used in vaccines. The second type of antigenic variation, called shift, involves major changes in the virus’s genetic makeup. These new variants are of even greater concern: new strains to which most humans have no immunity appear suddenly, and the resulting pandemics vary from serious to catastrophic. While we currently know the complete genome sequences of many influenza viruses, we do not understand the molecular basis of pathogenesis and are unable to predict which combinations of viral genes have pandemic potential.

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary Studies on the ecology of influenza viruses have led to the hypothesis that all mammalian influenza viruses originate from a reservoir of viruses in aquatic birds, particularly ducks. In wild birds, the viruses are spread by fecal–oral transmission through the water supply. Initial transmission of avian influenza viruses to mammals, including pigs, horses, and humans, and to domestic birds, including chickens and turkeys, probably also occurs by fecal contamination of water. Another method of transfer is by feeding pigs untreated garbage or the carcasses of dead birds. After transmission to humans or other mammals, the method of spread of influenza is mainly respiratory. Influenza represents one of the major success stories for the World Health Organization (WHO). To cope with the variability of influenza, WHO maintains a network of more than 100 laboratories that constantly survey influenza viruses, and this information is then analyzed in four reference centers. Based on these efforts, WHO makes annual recommendations for those virus strains to be included in the current vaccine in order to stay abreast of genetic drift. The less well resolved problem of influenza is the pandemics, which occur at irregular intervals and are to date unpredictable. In the past century, three viral subtypes have caused pandemics in humans: the Spanish flu of 1918–19, which was caused by the H1N1 subtype; the Asian flu of 1957, which was caused by the H2N2 subtype; and the Hong Kong flu of 1968, which was caused by the H3N2 subtype. The H1N1 and H3N2 subtypes also have caused disease outbreaks in pigs, and the H3N8 and H7N7 subtypes have caused outbreaks in horses. The Role of Swine in the Emergence of New Influenza Viruses Generally, influenza viruses are host specific, and viruses from one host rarely establish stable lineages in another host species. Although whole viruses may rarely transmit, gene segments can cross the species barrier through the process of genetic reassortment. Pigs have been postulated to play an important role in the process of genetic reassortment by acting as the “mixing vessel” for such events. Pigs, unlike humans, seem to be readily infected by avian viruses, and most, if not all, avian HA subtypes are capable of replicating in swine. Researchers have proposed a molecular mechanism for the susceptibility of swine to avian virus infection. Viral receptors called sialyloligosaccharides, which are present on the pig tracheal cells, possess the ability to bind to both types of viruses, with human viruses preferentially binding in one location and manner and avian viruses preferentially binding in another location and manner. Thus, pig tracheal cells can be infected not only by human influenza viruses but also by avian viruses. However, the direct chicken-to-human transmission of H5N1 vi-

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary ruses, observed during the 1997 flu outbreak in Hong Kong, argues that factors in addition to receptor specificity must be involved in influenza interspecies transmission. Influenza in swine is an acute respiratory disease, the severity of which depends on many factors, including pig age, virus strain, and secondary infections. Currently, three main subtypes of influenza virus are circulating in different swine populations throughout the world: H1N1, H3N2, and H1N2. In North America, Asia, and much of Europe, viruses of the H1N1 subtype are the most commonly isolated. The circulating H1N1 viruses differ, however, in the origins of their genomic components. The H1N1 viruses in North America and Asia belong to the classical swine lineage, which is genetically related to human H1N1 viruses responsible for the 1918 Spanish influenza pandemic. In contrast, all eight genes of the H1N1 virus circulating in Europe are phylogenetically related to the avian lineage that entered pigs in about 1979. The avian-like H1N1 virus also is present in the United Kingdom, although the virus of current concern is a reassortant H1N2 virus with gene segments derived from both human and avian lineages. Viruses of the H3N2 subtype circulate in Asia and Europe but have been infrequently isolated in North America. The most recent outbreak of this subtype in North America occurred in 1998, when a severe influenza-like illness was observed in pigs on a farm in North Carolina. Additional outbreaks among swine herds soon occurred in Minnesota, Iowa, and Texas. Genetic analysis of the viruses showed that at least two different genotypes were present. The initial North Carolina isolate contained gene segments similar to those of the human (HA, NA, PB1) and classical swine (NS, NP, M, PB2, PA) lineages, whereas the isolates from the other states contained genes from the human (HA, NA, PB1), swine (NS, NP, M), and avian (PB2, PA) lineages. Serological surveillance indicates that the latter triple reassortant virus has spread throughout the pig population of United States. Examples of Recent Outbreaks in Birds The avian H5N1 influenza virus that was transmitted to poultry and humans in 1997 in Hong Kong caused high mortality in both species, killing more than 70 percent of chickens and six of the 18 infected humans. (Hong Kong’s location at the crossroads of many trade routes makes it particularly susceptible to the outbreak of new diseases.) Surveillance studies revealed that two antigenically and genetically distinguishable variants of H5N1 were circulating among avians and humans. There was no correlation between lethality in humans and one or other of the variants. The

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary slaughter of approximately 1.6 million chickens during a 2-day period stopped the further spread of the virus to humans. The failure of H5N1 to transmit from human to human, and the slaughter of poultry in a matter of days before another strain of influenza (H3N2) began circulating in humans in Hong Kong, probably prevented the generation of reassortants with pandemic potential. Also in 1997, the avian H9N2 virus struck the live poultry market in Hong Kong. In 1998–99, observers reported that the virus had transmitted to humans and pigs. The initial report of five human cases in southern China was confirmed by the isolation of H9N2 viruses from two children in March 1999 in Hong Kong. The children had typical influenza and recovered. The isolates were genetically similar to H9N2 isolates found in quail. Further characterization of the viruses revealed that the human isolate from Hong Kong and the quail isolates shared similar genetic traits with the H5N1-like viruses from chickens and humans in Hong Kong in 1997. Thus, while avian influenza viruses can transmit directly to humans and cause disease, additional mutations and/or reassortant events are probably required to permit efficient human-to-human spread. The fact that these viral strains can transmit to and cause respiratory disease in humans confirms that the surface glycoproteins can fulfill their primary functions in mammals. Indeed, several lines of genetic evidence suggest that some of these strains have a special propensity for interspecies transmission. The continued circulation of such viruses in poultry, especially in quail, alerts us to the continuing need for active surveillance for these viruses in humans and pigs in this region. Recent Advances in Understanding Influenza Viruses A major advance in the ability to manipulate the genome of influenza viruses occurred in 1990, when scientists established a “reverse genetic” system, which permits the generation of influenza viruses containing genes derived from cloned DNA, or cDNA. A further major advance occurred in 1999 when other researchers demonstrated the generation of influenza A viruses entirely from cloned cDNA with high efficiency. These advances permit complete manipulation of all genes of influenza viruses, which means that it is now possible to tailor-make future live attenuated vaccine strains and to define all of the functional domains in the viral genes and their interaction with the host. Thus, resolution of the molecular basis of pathogenesis will be possible in the near future, and the domains responsible for interspecies transmission and ability to spread in new hosts will eventually be known. With this information in hand, it may be possible to predict which influenza viruses have pandemic potential in humans. Another potential advantage is that it will be possible to resolve the

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary question of the pathogenicity of the 1918 Spanish influenza. When the total genetic sequence of the 1918 virus is obtained, scientists will be able to recreate the virus. While of great scientific value, this possibility also raises considerable concern; such a study should be done only if the benefits warrant the risk and only if all work is performed in high-level biosafety laboratories. Once researchers have made the virus, tested its ability to interact with cultured cells, and determined which host genes are turned on or turned off, it will then be necessary to study the virus in an animal model, perhaps the mouse or the minipig. Before this happens, however, the research community and society must fully consider the ethics and safety of doing these experiments. Preparing Our Defenses Along with advancing our scientific knowledge, we also must improve our ability to detect and respond to new emerging strains of influenza virus, particularly those that appear suddenly and are capable of spreading over large areas. Many countries have prepared plans to cope with the next pandemic, which is considered imminent. Such documents must be updated as new information becomes available. The cornerstone of pandemic preparedness is surveillance, both of humans and lower animals and birds, for if we develop the ability to predict which combinations of genes have pandemic potential, we must then maintain active surveillance to detect them. Viral surveillance will continue to be the key to providing time for the preparation of vaccines ahead of worldwide spread. In the interim between detection of a pandemic and vaccine availability, it will be essential to have adequate supplies of antiviral drugs, which means that urgent attention should be given to ensuring strategic stockpiles. Overall, however, the reality is that we are not well prepared to cope with a pandemic, even a moderately severe one. We have identified where our weaknesses are, but we have not brought resources to bear on their solution. ASSESSING THE THREAT AND THE OPPORTUNITIES ACROSS THE SPECTRUM OF ZOONOTIC DISEASES Paul W. Ewald, Ph.D. Professor, Department of Biology, Amherst College Attention to emerging diseases has focused largely on acute infectious diseases that have caused outbreaks after entering humans from other host

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary species. Most of these diseases have very limited potential for spreading in human populations, particularly in wealthy countries. Of far greater potential importance to public health is discovery of infectious causes of the widespread and damaging chronic diseases. The logic leading to this conclusion emphasizes that the global emergence of highly virulent, acute infectious diseases requires special sets of conditions that are rarely met. Vectorborne diseases, for example, may persist evolutionarily in a highly virulent form, but only a very small proportion of vectorborne pathogens have the characteristics necessary to be transmitted persistently from person to vector to person. None of these pathogens have demonstrated this ability under conditions found in modern wealthy countries, characterized by screened houses, air conditioning, and primarily indoor life. Similarly, the newly emergent pathogens that cause deadly acute infections and are transmitted from person to person by air would need to have characteristics that enable them to be transmitted readily from sick hosts, particularly durability in the external environment, if they are to maintain transmission cycles. The smallpox virus and tuberculosis bacterium have these characteristics. The pathogens that have attracted the most public attention in recent years, such as Ebola and hanta viruses, do not. Evolutionary principles, current evidence, and the recent track record of recognizing infectious causation indicate that many if not most of the damaging chronic diseases are caused by infection. Some of the candidate pathogens use humans as primary hosts. Others infect humans zoonotically. In contrast to the oft-mentioned examples of newly emerging acute infectious diseases, the most damaging chronic diseases are already globally distributed and prevalent. Some of these diseases are now causing damage in human populations that is comparable to the damage that is merely feared for emerging acute infectious diseases. Atherosclerosis is the most damaging chronic disease in this category, accounting for about half of the deaths in wealthy countries. Other such diseases include schizophrenia, bipolar disorder, Alzheimer’s disease, diabetes, and breast cancer. Considering the damage, prevalence, and persistence of these diseases in human populations, investment of intellectual and economic resources in the investigation of infectious causation of these illnesses may be more beneficial than investments in efforts to monitor, study, and control the spread of the acute infectious diseases that make for sensational headlines but pose a relatively small global threat to human populations. These investments may provide particularly great health benefits because the most damaging manifestations of infection tend to occur when the infection in humans is no longer transmissible. Consequently, strategic use of antibiotics may enable the causative agents to be controlled indefinitely without the evolution of antibiotic resistance. Recent evidence sug-

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary gests, for example, that schizophrenia may be caused by infection with Toxoplasma gondii, which is transmitted in its natural cycle between cats and rodents. T. gondii damages the mental health of rodents in ways that facilitate capture of the rodents by cats, and hence its transmission to cats. Because T. gondii is not transmissible from humans, evolution of resistance should be negligible if an antitoxoplasmal drug is used only for human infection. This strategy for controlling antibiotic resistance, referred to as “dead-ending,” requires that at least two and preferably more than two antibiotics be available, so that one antibiotic can be used for the human infection and another can be used for the infection in the reservoir host. This distinction is particularly apparent for T. gondii, because cats are domestic animals that receive extensive medical treatment. When the natural hosts are unlikely to be the target of antibiotic treatment, as with the Lyme disease spirochaete Borrelia burgdorferi, the principles of antibiotic dead-ending are less relevant. The principles of dead-ending apply to vaccine use as well. Although evolutionary escape from vaccines has been documented only for a few pathogens, we can expect that the continued use of vaccines and generation of new vaccines will lead to more examples, particularly when pathogens are prone to genetic variation through high rates of mutation or genetic recombination. To reduce this danger, vaccines for humans should be antigenically different from vaccines generated for reservoir hosts. If, for example, T. gondii becomes widely recognized as a cause of schizophrenia and other damaging diseases, then the demand for a vaccine against T. gondii will increase for humans and for cats (to prevent infections in humans). If the same vaccine is used for both people and cats, then the use in cats would create a cumulative selective pressure favoring vaccine escape. The use in humans, however, would not generate a cumulative selective pressure because humans are dead-end hosts. To preserve the effectiveness of the best vaccine for humans, an antigenically different vaccine needs to be generated for cats. Developing different antibiotics or vaccines for humans and reservoir hosts is not as formidable as it may seem, because the constraints are not as severe for nonhuman recipients as they are for humans. A higher frequency of adverse reactions would be more acceptable for nonhuman hosts than for humans, as would certain kinds of adverse reactions. Neuronal damage sufficient to reduce a person’s IQ by 5 percent, for example, would not be acceptable for humans but probably would be acceptable for cats. If the reservoir host is not a valued animal, then discovery of zoonotic causes of already common chronic diseases may provide other less technologically sophisticated options. For example, one of the major candidates for breast cancer is mouse mammary tumor virus (MMTV) or a closely

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary related virus. The normal host for MMTV is the house mouse, Mus domesticus, in which the virus causes mammary tumors. This virus has been found much more frequently in tissue from human breast cancer than in surrounding healthy tissue, and breast cancer is associated geographically with the distribution of M. domesticus. Details of transmission to humans are unknown. If each human infection is directly acquired from M. domesticus, then local extermination of M. domesticus may directly protect humans from breast cancer. If transmission occurs from person to person with occasional reseeding from M. domesticus (analogously to the transmission of yellow fever virus or Yersinia pestis), then extermination may indirectly and diffusely protect the human population. Lyme disease is an example of a zoonotic agent that is already recognized as a cause of chronic disease, but the spectrum of chronic illnesses caused by B. burgdorferi appears to be broadening. Evidence indicates, for example, that B. burgdorferi is responsible for some cases of chronic diseases that have been diagnosed as multiple sclerosis, motor neuron disease, arthritis, paralysis, or myocarditis. Indeed, B. burgdorferi has been referred to as “The Great New Imitator” because of its potential involvement in chronic diseases that have been previously categorized as other diseases. This use of the term “imitator,” however, illustrates how an overarching trend that has been occurring in studies of chronic diseases may be inadvertently obscured. During the past half-century, a steadily increasing number of chronic diseases have been accepted as infectious. When a portion of a disease category is so recognized, that portion is typically given a new name to distinguish it from the rest of the category (e.g., reactive arthritis and neuroborelliosis), but this experience has not been used prospectively as a model for allocating research effort. Doing so would involve searching for the agents that will permit a subdivision of each of the umbrella categories, such as multiple sclerosis, schizophrenia, motor neuron disease, chronic fatigue syndrome, obsessive compulsive disorder, atherosclerosis, stroke, and Alzheimer’s disease. Just as we now consider such diseases as hepatitis and pneumonia to be collections of different diseases with distinct infectious etiologies, we can expect a variety of infectious etiologies for each of these umbrella diseases. If we instead search for the infectious cause of an umbrella disease, then we risk being misled by studies that do not find an association with a particular agent because that agent is rare in the study area. The ongoing resolution of hepatitis and arthritis illustrates how diverse the infectious causation of a chronic disease can be. The potential applicability to other highly damaging chronic diseases is apparent from the current evidence on infectious causation of diseases for which causation is still controversial. Atherosclerosis, for example, is associated with infections by Chlamydia pneumoniae, Porphyromonas gingivalis, Actinobacillus actinomycetocomitans, Bacillus forsythus, and cytomegalovirus, with

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary each of these pathogens being found in the atherosclerotic plaques, and some being shown to cause atheromas in animal models. Sporadic Alzheimer’s disease has been similarly linked to C. pneumoniae and human herpes simplex virus type 1. Much of the controversy over infectious causation stems from discrepancies between research teams that are unable to replicate associations with their own versions of the assays. This problem may occur because research will tend to achieve consensus most readily for those infectious diseases that are detectable even when detection techniques vary greatly, leaving in their wake those infectious diseases that are detectable only with very specific versions of experimental protocols. The candidate pathogens for atherosclerosis and Alzheimer’s disease are regularly transmitted between humans. The ambiguities due to discrepancies between research teams may be even greater for diseases that are sometimes caused by zoonotic agents, because these agents are less likely to be detected in humans where the zoonotic reservoirs are absent. Thus, if breast cancers are caused in part by MMTVs that are transmitted directly to humans from M. domesticus, then studies might be confirmatory in New York, where M. domesticus is present, but not in Japan, where M. domesticus is absent. In Japan, another pathogen, such as Epstein Barr virus, might be playing a relatively more important role. Similarly, geographic variation in pathogens might help explain why studies have found the zoonotic borna disease virus to be associated with schizophrenia in Japan, but not in other areas, where T. gondii, human herpes simplex virus type 2, and an endogenous retrovirus have been associated with schizophrenia. The proposed importance of infectious causation of chronic diseases emphasizes an irony in the attention devoted to emerging infectious diseases over the past two decades. This attention was triggered largely by the AIDS experience, in which a lethal disease arose from an exotic source and spread pandemically. This experience gave credence to concerns that other exotic diseases might similarly emerge. Concern focused on the most conspicuous examples—acute infectious diseases, such as Ebola, lassa fever, and hanta disease. But AIDS is a chronic disease syndrome. The irony, therefore, is that the alarm bell was rung in response to an emerging chronic disease syndrome, yet most of the subsequent attention has been devoted to emerging acute infectious diseases. The past two decades have not generated examples of new globally spreading acute infectious diseases that have been highly damaging to human populations; nor was there such an example for the entire 20th century. Resurgences of long-recognized global threats, such as influenza, have occurred, but concern over such resurgences was present before the recent interest in emerging infectious diseases. This recent history therefore suggests that concern over the future threat

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary of emerging diseases needs redirection. In poor countries, the resurgence of long-recognized acute infectious diseases represents a grave danger. In both poor and rich countries, grave dangers are posed by the long-standing chronic diseases that are or may soon be recognized as caused by infection. Leaders of the effort to awaken concern over infectious diseases have emphasized the danger from resurgence of known acute infectious diseases, and to some extent the growing recognition of infectious causation of chronic diseases, but most of the media attention has focused on the exotic acute diseases. A broader emphasis on studies of infectious causation of chronic diseases and the distribution of current knowledge about these diseases may be needed to direct the attention of researchers, policy makers, and the public to support efforts to identify and reduce the greatest threats to human health. PRACTICES AND POLICIES TO PROTECT HUMAN HEALTH FROM ANTIBIOTIC-RESISTANT PATHOGENS Stephen F. Sundlof, D.V.M., Ph.D. Director, Center for Veterinary Medicine Food and Drug Administration U.S. Department of Health and Human Services Antimicrobial resistance is one of the highest-priority issues facing the Food and Drug Administration’s (FDA) Center for Veterinary Medicine (CVM). The evidence of harm to the public from certain uses of antimicrobials in food-producing animals continues to grow, so CVM is taking steps to address the problem. We hope to deal with this issue through regulatory changes in the way we manage and approve drugs; through improved monitoring systems; through better risk assessment, which we think is a critical area of need; and through education. The issue of antimicrobial resistance has been around for some 30 years. The National Academy of Sciences, through the National Research Council (NRC) and the Institute of Medicine (IOM), has taken up this issue and provided input to FDA and the public. The NRC’s first report, which basically was the first risk assessment, was released in 1980. The NRC was asked to address the issue of whether subtherapeutic use of antimicrobials in feed for food animals was a potential hazard to human health. The report concluded that existing data neither proved nor disproved the potential hazards. That study was followed in 1998 by an IOM report titled Human Health Risks with Subtherapeutic Use of Penicillin and Tetracycline in Animal Feeds. The report concluded that the study committee was unable

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary to define a substantial body of direct evidence that established a definite human health hazard from the subtherapeutic use of these drugs in food animal feed. However, the committee did find considerable indirect evidence of that human health hazard. Another IOM report in 1998, on the benefits and risks of using drugs in food animals, reached a stronger conclusion: “There is a link between the use of antibiotics in food animals, the development of resistant microbes, and the zoonotic spread of pathogens to humans.” Because of the mounting evidence of risk to humans, FDA/CVM believes that there are issues we must address. From a regulatory standpoint, however, an issue as complex as antimicrobial resistance presents a tremendous challenge. To begin the process, we published in November 1998 a document titled Guidance for Industry #78. This document affirmed FDA’s position that it has the authority to regulate not just the toxic effects of drug residues, which has been FDA’s traditional role, but also the microbial effects from the antibiotics and antimicrobials that FDA regulates. In the document, we asked that two types of questions be answered for approval of a drug. First, we asked for information regarding the quantity of resistance that would be created from the use of an antimicrobial in food animals. We want to know which organisms are affected, how much resistance is likely to be created, and at what rate would resistance be likely to develop. Second, we asked for information about the change in animal enteric bacteria that are human pathogens that would come from the use of the drug. From a scientific standpoint, the problem we face is that we have a very limited ability to predict the rate and extent of antimicrobial resistance that could result from the use of an animal drug. A month later, in December 1998, CVM issued a second document, Microbial Effects of Antimicrobial New Animal Drugs Intended for Use in Food-Producing Animals, also known simply as the “Framework Document.” (The document is available on CVM’s web site at www.fda.gov/cvm.) This document lays out a conceptual, risk-based approach for regulating antimicrobial drugs so that resistance is minimized. The primary public health goal is to ensure that significant human antimicrobial therapies are not lost due to antimicrobial use in food-producing animals. The framework document was not intended to be regulation. Instead, it was an attempt to lay out what we considered to be a rational approach to dealing with this issue and to do so in a way that would be informative for a variety of stakeholders, including the animal drug industry, animal producers, scientists, and the general public. What we came up with was a fairly straightforward, risk-based approach to dealing with the regulatory issues of antimicrobial resistance. First, we thought in terms of assessing the risk. We want to know how

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary example, during 1996, approximately 77 million Americans, about 40 percent of the total population 16 years of age and older, took part in some recreational activity relating to wildlife and fish. Another potential route of infection focuses on urban and suburban environments. These locations are of special concern because of their increasing role as wildlife habitat, the greater interface between humans and wildlife that takes place within those environments, the paucity of knowledge about disease in those wildlife populations, and the general lack of orderly management for wildlife within those environments. In the wild, several trends are contributing to the growing importance of zoonotic diseases. First, the spectrum of infectious diseases affecting wildlife today is greater than at any time during the previous century. Second, the occurrence of infectious diseases has changed, from sporadic, self-limiting outbreaks that generally resulted in minor losses to frequently occurring events that generally result in major losses of wildlife. Third, disease emergence has occurred on a worldwide scale in a broad spectrum of wildlife species and habitats. Given the scope of the problem, current disease surveillance efforts are inadequate. Few state wildlife agencies allocate personnel and resources to address wildlife disease, despite their statutory responsibility for managing nonmigratory wildlife. Some state agencies provide minimal support for regional programs based at universities. At the federal level, the primary surveillance effort is conducted by the National Wildlife Health Center, operated by the U.S. Geological Survey. Outside of government, some veterinary schools, agriculture diagnostic laboratories, and other programs provide additional information on animal diseases, primarily by examining carcasses of dead wildlife submitted for analysis, and individual university-based researchers carry out a variety of studies. Typically, information about the occurrence of disease in free-ranging wildlife is derived from surveys and mortality events in areas where wildlife observations by agencies and the public are frequent enough to detect their occurrence before carcasses are removed by scavengers and predatory animals. The result is that disease occurrence is grossly underreported, heavily biased toward mortality events, and biased toward species of special concern and interest, such as game and endangered species. Therefore, the available information should be viewed as the “proverbial tip of the iceberg” relative to disease activity within wildlife populations. In general, mammals are the most important source of zoonoses transmitted by wildlife. However, birds are involved in the transmission of a number of serious zoonoses, especially vectorborne diseases. This is of special concern because of the greater geographic movement of many bird species. The 1999 outbreak in New York City of West Nile fever, which afflicted 62 people and killed 7 of them, serves as an example. After exten-

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary sive study, scientists determined that the virus apparently was carried by crows and transmitted to humans by mosquitoes. (However, it is not known how the virus was initially introduced to the region or, indeed, to the United States.) Waterfowl, such as Canada geese and mallard ducks, represent a particular threat for disease transmission to humans. These species are becoming increasingly common in urban and suburban areas, both because of habitat losses elsewhere and because of the current trend in landscape planning toward creating planned communities that feature “miniestates,” natural areas, and golf courses—environments that are attractive to waterfowl. The urban/suburban environment also has become an increasingly important habitat for songbirds. Salmonella typhimerium has emerged as an important pathogen, causing large-scale epizootics, usually in association with bird feeding. The magnitude of the potential human–songbird interaction is reflected by the 1996 expenditure for bird food in the United States of approximately $2.7 billion, with nearly 39 million people participating in this activity during that year. Environmental factors are the driving force for many emerging diseases of wildlife. In general, wildlife disease prevention and control will be most effective when environmental conditions are understood and the anthropogenic actions causing those conditions are addressed. The most important considerations can be classified under the headings of landscape changes, wildlife translocations, and human values. Landscape changes include both changes to the physical environment and the introduction of exotic species. Changes in the wetlands of the Central Valley of California provide a case in point. These wetlands lie within the Pacific Flyway, one of four primary migration corridors for birds that typically breed in remote northern areas and winter in southern areas of the North American continent and beyond. Because of their location, the wetlands have always been an important stopover area for birds. In recent years, however, approximately 90 percent of the wetlands have been converted to agricultural lands and other uses. As a result, more than 60 percent of the entire Pacific Flyway waterfowl population is now channeled into about 10 percent of the former wetland habitat. Such mass concentrations of birds for prolonged periods facilitate exposure of large numbers of birds to disease agents that may be present. The frequency of outbreaks, variety of bird species involved, and numbers of birds exposed to various pathogens provide a continuum of opportunity for the development of novel host–parasite relations. Human-created environments and exotic species are other important aspects of landscape change. For example, the Salton Sea, located in the desert of Southern California, was created in the early 1900s. Artificially sustained by agricultural drainwater, this highly saline body of water has the most productive fishery in the world and is one of the crown jewels of

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary avian biodiversity. However, since the 1990s, the ecosystem’s species richness has been tarnished by an unprecedented array of disease outbreaks that have killed large numbers of birds. The Salton Sea is an often-repeated contemporary situation that involves the creation of new environments and the mixing within these environments of multiple species that do not have established ecological relations with those that do. The opportunity for disease emergence is a component of the resulting species interactions and environmental changes taking place. Wildlife translocation, in which humans move free-ranging wildlife from one geographic area to another, is a common conservation tool that has clearly facilitated disease emergence, including zoonoses. An example was the government-directed translocation of raccoons trapped within a known enzootic area for raccoon rabies in the southeastern United States. These rabies-infected animals were the source of a raccoon rabies epizootic in West Virginia that spread to numerous other mid-Atlantic states, some coastal Atlantic states, and New England. The result is that enzootic foci for raccoon rabies are now established in geographic areas where rabies in raccoons previously had either been incidental cases due to epizootics in other species or small, self-limiting events in raccoons. Rabies also has been translocated with foxes and coyotes moved for sporting purposes. Other types of wildlife movements by humans that are contributing to disease emergence are captive rearing of wildlife for release into nature, wildlife rehabilitation and releases, and translocations for commercial purposes. Bovine tuberculosis has recently spilled over from the agriculture industry into white-tailed deer. Establishment of bovine tuberculosis within free-ranging white-tailed deer populations will pose a significant human health threat because of the pursuit of this species by millions within the hunting community. Disease emergence is as much a social issue as it is a biological issue. Of particular note, the prevailing philosophical attitude among many people within the wildlife conservation community is that disease is a natural event that need not be addressed. (Exceptions are made for transient responses to high-impact mortality events and for limited diagnostic activities in response to public inquiry about cases of visible mortality.) Proponents of this view maintain that impacts on wildlife population rather than on the individual animal should primarily determine whether or not there is a need for actions to be taken. This is a fundamental difference relative to human health and companion animal considerations, where clinical disease in individuals is of prime concern. A reasonable question is: Why is more not being done to address disease within wildlife populations? One contributing factor is that while most people believe it is possible to deal with disease threats involving humans and domestic animals, similar confidence is lacking regarding our ability to

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary control disease in free-ranging populations of animals. Response to disease in humans, livestock, and companion animals is governed by clear agency mandates supported by statutory authorities, legal mandates, laws, regulations, and other directives. The response process is facilitated by reporting systems, including designated reportable diseases; formal interagency infrastructures for disease diagnosis and control; infrastructures for epidemiological investigations; and systems for clinical treatment and fiscal considerations, among other factors. In general, all of those conditions are either absent or at best rudimentary for agencies with stewardship responsibilities for free-ranging wildlife. As a result, disease outbreaks in wildlife do not have a mandated responsibility to be investigated or dealt with. Because of the differences in agency responsibilities, agriculture agencies do not become involved unless the outbreak is known to be, or has a high probability of being, a disease of major concern for domestic animals. However, even in those instances, the wildlife are under the jurisdiction of wildlife agencies. Similar considerations are involved for zoonoses transmitted by wildlife. Complicating factors include animal rights advocates that give special attention to protecting wildlife, even when serious diseases are involved, and conservation legislation such as the Endangered Species Act and other laws that can constrain actions normally implemented when domestic animals are affected by a disease of major importance. Free-ranging wildlife populations are under the legal stewardship of state and federal government agencies. Primary responsibilities are vested in different agencies depending on the types of species involved. Shared responsibilities between state and federal agencies generally exist despite one or the other having primary responsibility for a specific situation. This stewardship form of ownership leads to two results. First, there is no private ownership of free-ranging wildlife. Instead, wildlife are held in the public trust for human society. Second, the wildlife stewardship agencies are nonprofit organizations that have little economic incentive to address the costs of disease. Thus, there are no compelling reasons for government agencies to expend resources on disease prevention and little incentive for disease control. To help in minimizing the emergence of diseases in wildlife, as well as the transmission of such diseases to humans, numerous observers have suggested a variety of actions. These actions include: Interdisciplinary collaboration and cooperation. Federal agencies should develop a tripartite cooperative program to address infectious diseases in humans, in domestic animals, and in wildlife. This program should serve as a focus for regular communications through working groups to address information transfer; to improve response to disease emergencies; to establish priorities for collaborative, focused investigations; and to pur-

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary sue other areas of mutual interest. The program also should serve as a model and catalyst to stimulate the development of similar cooperative programs between state agencies that would network with the federal program. Database development. Federal agencies should take the lead in developing a common database for disease surveillance and monitoring that can be used to track infectious diseases and the emergence of new diseases. As part of this effort, a work group should be established to develop a listing of “reportable” diseases that are to be entered into the system, with standards for data entry, reporting, and utilization by collaborating agencies and institutions. The CDC has proposed developing a national electronic disease surveillance network for state and federal public health information on emerging infectious diseases, and this network should be expanded to include wildlife surveillance information on emerging zoonotic diseases. Focused collaborative investigations. In ongoing research programs, joint development and planning can help ensure that high-quality specimens, reagents, information, and assays will be provided among collaborators at minimal costs. In other instances, joint budget initiatives will be required to provide the resources needed to carry out the monitoring programs and other focused investigations to address specific diseases. Agencies should develop agreements with one another to facilitate collaborative investigations on issues of mutual interest; such agreements should cover such issues as fund transfers, personnel assignments, and sharing of facilities and technical capabilities. Biological repositories. There is a need to develop and maintain systems for archiving materials from wildlife disease investigations for retrospective and comparative studies. Isolates of infectious disease agents, serum banks, histological specimens, and other biological reference materials need to be organized in a coherent manner that provides ready access to them by qualified investigators willing to work as true collaborators within the area of emerging infectious diseases. Disease ecology. Disease prevention and control activities will be enhanced by greater understanding of the epizootiology of wildlife diseases. This is a fruitful area for interagency and interdisciplinary collaboration. Efforts should extend beyond field and laboratory investigations to include the areas of mathematical modeling and geographic information system technology. These efforts should be supported by expanded databases of information from the physical, biological, and social sciences. Urban wildlife disease studies. Given the increasing importance of urban/suburban environments as habitats for some species of wildlife, this is an important area for collaborative investigation. Such studies have ur-

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary gency for protecting the well-being of migratory birds and other wildlife, as well as for protecting human health. Public education. A coordinated, ongoing process is needed to provide the general public with timely, accurate information about emerging diseases of wildlife and the importance of such diseases to public health, domestic animals, and the wild animals themselves. One possible route would be for federal agencies to work collaboratively with an independent organization dedicated to public outreach. Emergency response. Collaborative arrangements should be developed to integrate the emergency response capabilities within the public health, domestic animal, and wildlife conservation communities. Response to emerging infectious diseases of wildlife should be augmented as needed by the combined capabilities of the different programs to minimize the potential for establishment and spread of wildlife diseases capable of infecting other species, including humans. Technical forum. Communications need to be improved between officials with responsibility for managing wildlife on public lands and researchers who study diseases of wildlife or diseases transmitted from wildlife to humans and domestic animals. It is not sufficient to rely on the diverse scientific meetings that currently incorporate wildlife diseases as agenda topics. The North American Wildlife Conference can provide an appropriate forum for bringing wildlife disease issues before those individuals who manage public lands, and organizers of the conference should be encouraged to develop a regular forum devoted to emerging diseases. The human health community should develop a reciprocal opportunity for participation by members of the wildlife community. Guidance on landscape change. The expanding human population assures continued landscape changes. Thus, the government and other organizations should become proactive in terms of developing and disseminating information that can help guide land development in a manner that gives greater consideration to disease emergence. Initial actions that should be considered include distributing authoritative publications, sponsoring public forums, and providing consultations on particular problems. To quote the comic strip character Pogo, “We have met the enemy and he is us.” This observation continues to be demonstrated for emerging diseases. Human arrogance cannot overcome biological processes. However, by replacing arrogance with some humility and by addressing these issues from a truly collaborative perspective, we will be able to improve environmental conditions substantially and impede disease in a manner that will greatly benefit humankind and our planet’s biological resources for decades to come.

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary ANIMAL HUSBANDRY PRACTICES AND RISK FACTORS, WITH PARTICULAR REFERENCE TO BOVINE SPONGIFORM ENCEPHALOPATHY Fred Brown, Ph.D. Visiting Scientist, Plum Island Animal Disease Center U.S. Department of Agriculture Bovine spongiform encephalopathy (BSE) is caused by a member of a group of agents that collectively are known as transmissible subacute spongiform encephalopathies. These agents cause fatal human and animal neurological diseases, which are characterized by a long symptom-free incubation period followed by a short acute phase. Many such diseases have been described over the years, but attention has been focused on them most recently by the massive outbreak of BSE in cows that started in the United Kingdom in 1986. This outbreak presented a new problem for the agricultural industry because it had no precedent. The disease occurred in many herds but was of low incidence in any individual herd, with one to three animals being affected. The animals became uncoordinated and irritable, fell frequently, and eventually died. The only diseases remotely connected to BSE seemed to be scrapie in sheep and goats and Creutzfeldt-Jakob disease (CJD) and kuru in humans. Histological examination of the brains of infected animals showed that all three diseases caused similar lesions. Veterinary scientists quickly traced the source of BSE infection during the United Kingdom outbreak to the food concentrates—essentially, tissues of slaughtered cows—that were being fed to cattle to enhance their productivity. The clear question to be answered was why the disease had emerged then, when the feeding of concentrates had been part of animal husbandry for several decades. Why had the disease not emerged earlier? Another critical question was how an infectious agent could survive the severe heat treatment used in the preparation of these concentrates. The only clue to these questions seemed to be the change made in the rendering procedure by which the concentrates were produced—a change in the solvent step that was made to decrease the cost of the process. This change had been made in the 1980s, leading to the suggestion that the incubation period of the disease was about 5 years. Despite the imposition in 1988 of a ban on the feeding of concentrates, the number of infected animals continued to rise dramatically. The peak in the outbreak occurred in 1992 and 1993, confirming the initial suggestion of a 5-year incubation period. Subsequent pathogenesis studies have shown that cattle fed large amounts of the BSE agent do not develop disease earlier than 36 months, nor do their tissues contain any infectious agent before

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary that time. This led to a ban on any use of meat or other animal tissues for human consumption from animals older than 30 months. (Of course, this raises a question: if an animal develops the disease when it has received the infected concentrate 36 months previously, how safe is the precursor of the infectious agent?) Early on, many observers considered it unlikely that the agent causing BSE would lead to any problems in humans, since there was no evidence that the infectious agent that causes scrapie in animals causes disease in humans. Indeed, the Southwood Committee established by the British government reached this conclusion in 1989, as did the Spongiform Encephalopathy Advisory Committee (of which I was a member) in 1990. Still, the burning question in many quarters remained: Is beef safe? Such worries had a formidable impact on the British cattle industry. Then, in March 1996, the U.K. Ministries of Agriculture and Health announced that human cases of a new form of Creutzfeldt-Jakob disease had been detected in a small number of young people. This was unexpected because CJD does not usually occur in young people, yet histological examination of the brains of these victims showed clear similarities to BSE. Subsequent evidence has confirmed that the new disease (now called new variant Creutzfeldt-Jakob disease) and BSE are caused by the same agent. These new findings had perhaps an even greater impact, on both the cattle industry and the general public, than did the initial observations. Who was going to eat beef from a potentially infected animal? In a world demanding no risk, sales of beef plummeted, and some groups demanded that all 12 million cattle in the British herd be killed. The clear problem was to ensure public health but at the same time attempt to preserve an important and lucrative industry. Balancing these demands would require a delicate balancing act on the government’s part. The outcome, however, did not go well. Many individuals and groups argue that the BSE crisis was badly handled, both by the government and by its scientific advisers. But what were the options? There are several issues that need to be taken into account before judgment is rendered: (1) BSE was a new disease of cattle, (2) the disease was caused by an “old” agent, (3) the disease had a very long incubation period, (4) there was no in vitro diagnostic test, and (5) the assay system for detecting the agent relied on the use of mice and required months to complete. One lesson seems clear, however: the problem in Britain was so large that the government should have appointed an “overlord” who would have devoted full-time attention to issues as they emerged. Advisory committees are no substitute for day-to-day involvement. (I suggested this approach to the British government in 1991, but it was rejected.) Would such an approach have made any difference? Because of the disease’s long incubation period, it may not have done so. But having such a central authority would

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary have ensured that all the scientists who knew something about these agents and the diseases they cause would have been brought into the equation at the earliest possible stage. Neither worrying about “territory” or “turf,” nor the seeking of glory, can solve such problems—and these issues should not cloud the work under way today on other emerging diseases. NATURAL HISTORY OF SIMIAN IMMUNODEFICIENCY VIRUSES: CLUES TO THE EMERGENCE AND VIRULENCE OF AIDS VIRUSES Lisa Chakrabarti, Ph.D. Visiting Scientist, Aaron Diamond AIDS Research Center Simian immunodeficiency viruses (SIVs) infect more than 20 species of Old World monkeys and apes, all of them of African origin. Those species naturally infected include chimpanzees, mangabeys, mandrills, baboons, colobus, African green monkeys, and guenons. African green monkeys (genus Chlorocebus) were the first nonhuman primates found to harbor SIV in the wild. In most cases, SIVs are species specific, meaning that viruses obtained from animals of a given species will phylogenetically cluster together. The species-specific clustering of viral sequences suggests that virus and hosts evolved in parallel and that SIV infections are ancient. However, several exceptions to the species-specific clustering rule indicate that SIVs do occasionally cross species barriers. For instance, viruses from African green monkeys (SIVagm) appear to have been acquired by a talapoin, a patas, a white-crowned mangabey, and two baboons. The frequency of SIVagm cross-species transmission may reflect the fact that African green monkeys are particularly widespread and numerous and thus represent a major SIV reservoir. SIV infection has not been shown to cause disease in its natural hosts. This issue is obviously difficult to address in the wild but has been addressed by the epidemiological studies of sooty mangabey and African green monkey populations bred in primate centers. SIV seroprevalence typically is low in young animals but rises sharply in juveniles and young adult mangabeys, suggesting that SIV is mainly transmitted through the sexual route. However, SIVs have the potential to become pathogenic when transferred to new host species, including humans. There is compelling evidence that SIV from sooty mangabeys (SIVsm) is the recent ancestor of the human AIDS viruses HIV-2 and of SIVmac, the virus that causes simian AIDS in rhesus macaques. There also is evidence that SIV from chimpanzees (SIVcpz) is the ancestor of at least some types of HIV-1. The clustering of several

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary human and simian lentivirus pairs on phylogenetic trees indicates that cross-species transmission of SIVs to humans has been a repeated occurrence. Transmission events did not always result in the emergence of pathogenic and highly transmissible AIDS viruses, as indicated by the fact that only two of the six HIV-2 subtypes identified so far were associated with AIDS. HIV-1 types N and O are pathogenic but have a limited epidemic spread as compared to HIV-1 type M, suggesting differences in adaptation to the human host. The factors responsible for the acquisition of virulence in the human host remain to be elucidated. This issue may be more easily addressed in simian models, by comparing SIV infection in species, such as the sooty mangabey and the rhesus macaque, that are resistant and susceptible to disease. Sooty mangabeys range from Sierra Leone and Liberia to the western half of Ivory Coast. Converging evidence supports the idea that cross-species transmission of SIVsm to humans is at the origin of HIV-2. Among the evidence are the following observations: (1) SIVsm and HIV-2 are genetically close and share a common genome structure; (2) all known HIV-2 subtypes occur together only within the range of the sooty mangabey; and (3) ample opportunities exist for transmission, since people in the region hunt sooty mangabeys and keep them as pets, and the prevalence of SIVsm in sooty mangabeys is relatively high. The most convincing evidence is based on the phylogenetic clustering of SIVsm and HIV-2 isolated from the same geographic locale, either in Sierra Leone or Liberia. Evidence for the origin of HIV-1 from chimpanzees can be found in the close genetic proximity between two SIVcpz from Cameroon and HIV-1 type N, which is found exclusively in the same country, indicating both phylogenetic and geographic coincidence between the two viruses. However, only seven SIVcpz have been characterized so far, and none of them cluster closely with the HIV-1 type M and HIV-1 type O. Thus, it may be premature to draw conclusions on the most recent ancestors for these two group of viruses. They are likely to belong to the SIVcpz group, but the detection of an ancestral HIV-1 type M or O in another species cannot be entirely ruled out at this stage. Chimpanzees are known to prey on other monkey species and may thus be exposed to transmission of heterologous SIVs. One crucial question is whether cross-species transmission of SIVs to humans can readily generate an AIDS virus, or whether further adaptation to the new host is required for the emergence of a virulent HIV strain. Some insight can be obtained from studies of rhesus macaques. When infected with certain SIVsm isolates, such as B670, these monkeys readily develop AIDS, while inoculation with other SIVsm isolates does not appear to cause disease. It is also relevant to note that accidental SIVsm transmission to

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The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health - Workshop Summary laboratory and animal workers has been documented in at least two instances, with no cases of AIDS reported thus far. Taken together, these observations suggest that simian immunodeficiency viruses have the potential to cause disease upon transmission to a new host but that this may actually be a rare occurrence. The most telling piece of evidence is epidemiological. Only two of the six HIV-2 subtypes described so far have spread epidemically. The four individuals infected with subtypes C to F were all healthy (or, in one case, afflicted with a disease that is not associated with AIDS). Thus, the divergent HIV-2 subtypes C to F may represent viruses poorly adapted to the human hosts. It appears likely, then, that either the epidemic HIV-2 subtypes originated from the transmission of specific variants that happened to be pathogenic for humans or that the emergence of pathogenic HIV requires further adaptation to the human host through unknown mechanisms. What mechanisms might drive the acquisition of SIV virulence in the human host? Experiments in the macaque models have repeatedly shown that serial intravenous passages increase SIV and HIV virulence in this host. Thus, it is possible to draw a parallel and speculate that serial intravenous passages could have contributed to the propagation and the adaptation of SIVsm and SIVcpz in humans. Epidemiologists and historians have documented multiple instances of reuse of nonsterile needles or even of direct arm–arm vaccination in Africa since the beginning of the 20th century. The main reason why serial intravenous passages can promote SIV adaptation is that they provide the setting for successive viral jumps from primary infection to primary infection. A poorly adapted virus would induce a very low viral load and therefore would be very unlikely to be transmitted during the chronic phase of the infection. The only window of time during which transmission could occur would be the few weeks that precede the establishment of the antiviral immune response—that is, the primary infection.