key: cord-0690109-dh4zysz9 authors: Wakeley, Philip R.; North, Sarah; Johnson, Nicholas title: Synthesis date: 2013-10-15 journal: The Role of Animals in Emerging Viral Diseases DOI: 10.1016/b978-0-12-405191-1.00012-0 sha: ac89728a2ec18058f2cb718ce1f385e9125eb68a doc_id: 690109 cord_uid: dh4zysz9 The preceding chapters have described in detail specific examples of viruses that have emerged or re-emerged from animal sources. Most, although not all, are zoonotic and have caused significant human morbidity and mortality. This final chapter will attempt to bring together a number of the common themes raised by these examples. These include the role of historical events in shaping the emergence of viruses in the present, the ability to retrospectively test samples to trace the emergence of disease and the technologies used to detect viruses. Technologies such as the polymerase chain reaction and mass sequencing have revolutionized virus detection and enabled the rapid identification and characterization of viruses. The application of these methods has led to the identification of new groups of viruses in samples obtained from a range of animal species without the need for invasive sampling or the recovery of live virus. However, with the detection of such a large range of viruses that exist asymptomatically with other species, the challenge now becomes the identification of those with the potential to become pathogenic for humans. The examples in the preceding chapters give a comprehensive overview of the role of animals in the emergence and re-emergence of viral diseases. Every species on the planet, Homo sapiens included, hosts a range of viruses that are naturally transmitted between members of that species. However, opportunities arise for one species to encounter a virus from another species. Such opportunities lead to cross species transmission events and if the virus can infect the new host it has the opportunity to cause the emergence of a new disease. If that new species is human or livestock or, now increasingly, wildlife, there is a disease outbreak. The repeated exposure of humans to lentiviruses through hunting and butchery of bushmeat is a good example of this and has been responsible for the largest zoonotic disease outbreak in recent times, namely that of human immunodeficiency virus (HIV) . If that species is a domestic or companion animal, then the likelihood of humans being infected increases. This has been seen with the emergence of Nipah virus, through the pig, and for Hendra virus infection, through the horse, in southeast Asia and Australia, respectively. It is therefore possible to characterize the role of animals into discrete groups. The first is that of the reservoir or vector. This group potentially includes all animals, as any virus infecting an animal could theoretically adapt to infect humans. This in part depends on the binding of virus proteins to cellular receptors that can be highly selective, as seen for parvoviruses, or nonselective as observed for Nipah virus. However, most viruses appear to be strongly adapted to a particular species or genus and tend not to "jump" between species and cause disease. Bats harbor a multiplicity of coronaviruses (Tang et al., 2006) that are believed to be the progenitor of SARS coronavirus. Infection appears to be asymptomatic in bats with shedding of virus in fecal material being frequently observed. At some point a cross species transmission event occurred, leading to infection of animals that entered the wet markets of China. A further transmission to humans occurred, leading to the outbreak of a respiratory disease that through air travel affected countries around the world (Hughes, 2004) . In the Americas, bats harbor rabies virus and are becoming an increasing source of infection to humans . In this case, bats are susceptible to disease but through predation by the vampire bat (Desmodus rotundus) or chance encounters with a wide variety of insectivorous bats, humans become infected. Dogs are also efficient transmitters of rabies (see Chapter 4) and it is through them that the virus has spread throughout the world. The virus is maintained within the dog population, although can be controlled through methodical management, such as animal movement restrictions, quarantine and vaccination against disease. Rodents are the source of a range of viruses that are capable of crossing the species barrier to humans (see Chapters 5 and 10). Transmission to humans occurs through exposure to excreta; control is limited to reducing the opportunities for exposure to particular rodents or exclusion of rodents from human premises. Finally, one of the largest reservoirs for emerging viruses is the bird population. Here, birds act as the host for influenza viruses that have caused some of the largest outbreaks of disease over the past hundred years. The second role of animals is that of the amplifying or bridge host. These are those species that are not the natural host of a particular virus but, through infection and shedding of virus (amplification), provide the opportunity to infect humans. Nipah virus (Chapter 11) provides an excellent example of this. In late 1998 a new disease emerged in pig farms in Malaysia, followed shortly by cases of viral encephalitis in farm workers (Mohd Nor and Ong, 2000) . The source of the virus was identified as fruit bats that roosted close to the farms and shed the virus in fecal matter. In the case of the Hendra virus outbreak in Australia (see Chapter 6), the source was also fruit bats and horses acted as the bridge host that led to the infection of veterinarians and others who handled the horses. One final consideration is to see animals as the victims of virus emergence. Rather than this being a sentimental consideration, disease emergence can have a profound effect on particular species that in extreme cases could lead to species decline or extinction and influence the ecology of a particular environment. Outbreaks of rabies in a range of canid species have led to the possibility that rare species could be driven to extinction. Two examples from Africa illustrate this. The world's rarest canid, the Ethiopian wolf (Canis simensis), is restricted to the Ethiopian Highlands with populations limited to a few thousand. Recent outbreaks of rabies have caused dramatic disappearances of well-studied populations (Johnson et al., 2010) and it is only through interventions such as parenteral vaccinations that outbreaks have been controlled and further population declines prevented (Randall et al., 2006) . Similarly, outbreaks of rabies and, more devastatingly, canine distemper virus in populations of the African wild dogs (Lycaon pictus) has led to the decline of this species across its whole range (Goller et al., 2010) . A somewhat surprising observation from these chapters has been the role of historical events, often occurring hundreds of years in the past, influencing disease emergence in the present. Clearly human activities underpin this, but it is the emergence of disease long after the trigger event that is striking. Examples of this include the emergence of vampire bat rabies in Latin America, dog rabies in Africa, and the emergence of HIV and the current distribution of Lassa fever in West Africa. For the two examples for rabies, the key events are related to colonization by Europeans. In the New World, Europeans introduced domestic animals that provided a ready supply of prey for vampire bats that, in turn, is thought to have increased their numbers with larger, more numerous colonies. Europeans initially introduced dogs and horses to assist in the conquest and subjugation of the indigenous populations. Later introductions of domestic animals to supply food completed the radical change to the ecology of the New World. It is hypothesized that this supported an increase in the vampire bat population capable of sustaining rabies virus infection, leading to spillover transmission to human and livestock. While the initial colonization took place in the 15 th and 16 th centuries, rabies in vampire bats was only recognized in the early 20 th century and continues to be a major problem for the livestock industry in Latin America and a public health hazard. In Africa, the translocation of domestic dogs by colonists arriving in the west and the south of the continent is widely suspected of introducing dog-associated strains of rabies (Smith et al., 1992) . While there are ancestral populations of rabies variants in certain wildlife species such as the yellow mongoose (Cynictis penicillata) that likely predates colonization (Van Zyl et al., 2010) , the virus strains associated with dogs are those responsible for the vast majority of human rabies cases. Further colonial events such as economic exploitation of the human population and the environment are considered a major cause for the emergence of HIV in the human population of the Congo Basin (Chapter 9). While it is likely that human population growth and environmental destruction were inevitable without the "blame King Leopold II" explanation, Leopold's policies brought humans into contact with nonhuman primates. The exploitation of the Congo Basin by the royal private army, the Force Publique, began in the 1870s. This group terrorized the local population through mutilation and murder in pursuit of increased produce yields that in turn led to population disturbance, forced urbanization and accelerated the use of bush meat as a means of nutrition. The dating of the last common ancestors of HIV and SIV provides strong evidence for cross species transmission during this period. However, it was not until a further hundred years later that the virus achieved pandemic status. The importation of European breeds of livestock is also thought to have led to the rapid spread of diseases such as foot-and-mouth disease (Chapter 2) and rinderpest. While rinderpest has been eradicated, FMD continues to affect many regions of the world and is a major threat to the food chain. For Lassa fever virus, the key historical event that could have caused the emergence of the virus in Sierra Leone is the disruption of the West African slave trade by the British in the 19 th century. The slave trade itself was responsible for the translocation of numerous diseases to the New World from Africa that remain public health problems to the present day. There is evidence that mosquito-borne diseases such as yellow fever and dengue were translocated to the New World by the slave trade (Bryant et al., 2007) . However, in the case of Lassa fever virus, the result of this intervention that freed many slaves caused a problem in relocation. While the British Empire repudiated slavery, many countries did not, maintaining the trade in humans from Africa to the Americas. Those that were freed were forced to remain in Sierra Leone because of the danger of recapture if they returned to their country of origin. If the "humans as vectors" hypothesis is correct, this population could have been responsible for the introduction of Lassa fever virus and the emergence of Lassa fever in Sierra Leone in the 20 th century. Another common feature that has played a role in the investigation of emerging disease investigation has been the ability to use retrospective analysis of biological samples or data to pinpoint to time of emergence. Analysis of the piglet mortality index, a measure of litter deaths in the index farm where Nipah virus is believed to have been introduced has identified the likely time of cross-species transmission from Pteropid bats to livestock . Critically, this precedes the first human cases by a number of months and refutes the hypothesis that a particular environmental factor, the El Niño Southern Oscillation, triggered the emergence of the virus. Another striking case is the retrospective identification of a cluster of HIV infections in Norway, a decade before the recognition of the clinical syndrome AIDS in the USA (Jonassen et al., 1997) . The index case was a Norwegian male who worked on merchant shipping who traveled to numerous locations around the world between 1961 and 1965. On some of these, he visited ports on the west coast of Africa. He later returned, presumably after being infected, married and started a family. In 1966 he developed various disease symptoms, including persistant lymphadenopathy. His wife also developed recurrent mucocutaneous candidiasis and his daughter from the age of 2 developed a series of recurrent infections. All died in 1976. However, it was the ability to demonstrate HIV genomic RNA in formalin fixed samples using highly sensitive capture PCR that confirmed that these were perhaps the earliest cases of HIV in Europe. Further serological testing of archive samples in Africa has also identified early evidence for HIV infection as far back as the 1950s (Nahmias et al., 1986) . For the emergence of canine parvovirus (Chapter 3), clinical disease alerted veterinary authorities to the presence of a new disease in dogs. However, restrospective testing of canine serum samples allowed the presence of the causative agent to be detected and allowed the emergence of the disease to be traced through the 1970s (Parrish, 1990) . Rapid and accurate detection of pathogens in clinical or environmental samples is the cornerstone of disease diagnosis. It has also been instrumental in the detection of emerging viruses. Over the past 20 years there has been a move to enhance diagnostic capability using more conventional diagnostic tests such as ELISA, serology and virus isolation, by the introduction of molecular-based testing as an adjunct to these tests rather than replacing them. Molecular diagnostics involve the detection of specific nucleic acids and frequently the amplification of these sequences in order to allow the products to be easily detected and as such are not reliant on the viability of the virus. This is both advantageous in that generic tests can be applied where no virus isolation methodology exists (or no means to identify the virus either through a cytopathic effect or immunologically once propagated in cells-i.e., lack of monoclonal antibodies) but may also identify samples as containing dangerous pathogens when in fact the virus is nonviable and therefore presents no threat. A further benefit of these approaches is the generation of DNA samples from which the sequence of the virus under investigation can be derived and used to identify the disease-causing agent. This in turn drives the design of molecular tests, which has been greatly accelerated through advances in genome sequencing technologies that have provided the base information (the specific pathogen sequences) for the design of such molecular tests. The following sections focus on particular molecular tests that have been applied to the detection of new and emerging viruses. The polymerase chain reaction (PCR) is the most commonly used and dominating technology applied to molecular testing both in the clinical and veterinary field. Its power lies in its simplicity of design and application. The specificity of the assay relies on the hybridization of short stretches of synthetic DNA (oligonucleotides) complementary to the target sequence and amplification of the sequences between these oligonucleotides using the enzymatic activity of Taq polymerase. PCR assays are easy to design as primer design software packages are freely available on the Internet. The speed of the test using PCR is limited by the processivity of Taq polymerase and also the requirement to heat and cool the reaction through cycles of denaturation of the double-stranded DNA (95 o C), annealing of primers and elongation (68-72 o C). Various enhancements have been made to the process and the machinery to generate so-called Fast PCR, but time to result still remains somewhat slow (some commercial companies quote 40 minutes as being fast) or when compared with the rapidity (but decreased sensitivity) of direct detection methods employing lateral flow devices, for instance. The addition of sequence-specific DNA probes into a PCR enables the real-time monitoring of the reaction. The traditional probe format is a "TaqMan" probe which involves labeling a DNA probe with a fluorescent reporter and a corresponding quencher. The close proximity of reporter and quencher causes quenching of the fluorescence. The probe hybridizes with its complementary DNA target and is enzymatically cleaved, causing separation of the fluorophore and quencher and results in an increase in the level of fluorescence, which can be detected after excitation with a laser. Different probe and amplification technologies have emerged and are briefly described below: l A molecular beacon is a dual labeled fluorescent probe, complementary to the target sequence, which is held in a hairpin structure by complementary stem sequences. Upon hybridization of the molecular beacon with its complementary sequence, the hairpin structure relaxes and the fluorophore and quencher separate, resulting in emission of the fluorescence. Unlike the traditional TaqMan probe, the molecular beacon is not cleaved. Real-time PCR assays for the detection of swine vesicular disease virus (SVDV) and vesicular stomatitis virus (VSV) using molecular beacons (MBs) have been developed (Belák, 2005) . l A Scorpion probe is formed by the linkage of the primer and probe via a blocker. The probe part is held in a hairpin structure, similar to that of a molecular beacon, with a fluorescent reporter dye and quencher. The 3′ end of the probe (where the quencher is located) is linked via a blocker to the 5′ end of the PCR primer. During the amplification reaction the primer anneals to its complementary target sequence and the polymerase extends the primer. This results in the newly synthesized strand being attached to the probe. During a subsequent cycle the hairpin loop of the probe is denatured and hybridizes to a part of the newly produced PCR product. This results in the separation of the fluorophore from the quencher and causes light emission. Scorpion probes are thought to provide increased specificity and have been applied to the quantitation of HIV-1 in clinical samples (Saha et al., 2001) . l LATE (Linear After The Exponential) PCR is a form of asymmetric amplification developed by researchers at Brandeis University. LATE-PCR generates single-stranded amplicons after a short period of exponential amplification through the use of a limiting primer and an excess primer. The reaction switches from the production of double-stranded to single-stranded DNA when the limiting primer is exhausted, creating an abundance of single-stranded DNA which can be detected with probes over a temperature range. This enables the detection of low numbers of target organisms and the detection of a wide range of sequences with a single probe (Sanchez, 2004) . LATE-PCR has successfully been utilized in the detection of foot and mouth virus (Pierce, 2010) . Isothermal amplification methods are now coming to prominence principally due to the ease with which these methods can be applied in that they do not require a programmable heating block being the heart of the PCR machine, but simply a constant source of heat. This has the advantage that the engineering challenge with respect to a programmable heating device is considerably simplified. Nucleic Acid Sequence Based Amplification (NASBA) was one of the first methods to gain prominence (Guatelli et al., 1990) . NASBA is an isothermal transcription based amplification system using three enzymatic processes essential in retroviral replication. These processes involve reverse transcriptase, e.g., avian myeloblastosis virus reverse transcriptase (AMV-RT), RNase H and DNA-dependent RNA polymerase (T7 RNA polymerase, most commonly). The method, using RNA as the starting material, is particularly suitable for the detection of genomic, ribosomal and messenger RNA. As for all the isothermal methods it does not require a thermocycler but just a continuous source of heat (41 o C). The specificity of the method is conferred by a primer that hybridizes to the target. This primer also encodes a T7 RNA polymerase promoter sequence which comes into play during the exponential phase of the amplification process. Following annealing of the primer to target complementary DNA (cDNA) is generated under the action of AMV-RT, forming a DNA-RNA hybrid, the RNA being subsequently digested under the action of RNase H leaving just the cDNA. A second primer complementary to the cDNA then binds and extends a second strand of DNA leading to the formation of an intact T7 RNA promoter region. Once the promoter is formed T7 RNA polymerase is able to catalyze the production of very large numbers of RNA transcripts (complementary to the original RNA), which can themselves serve as starting material for further rounds of exponential amplification. NASBA has been applied to the detection of numerous viruses including those transmitted or amplified in animals prior to insect vector transmission. Influenza virus has been detected using NASBA from both birds (Lau et al., 2004) and more recently swine (Ge et al., 2010) . NASBA has also been used to detect West Nile and St. Louis encephalitis virus (Lanciotti and Kerst, 2001) , SARS coronavirus (Keightley et al., 2005) , rabies virus (Wacharapluesadee et al., 2011) and human rhinoviruses (Sidoti et al., 2012) . An alternative isothermal amplification method that appears to offer many advantages over others available is loop mediated isothermal amplification (LAMP). First described by Notomi et al., (2000) and improved upon with respect to speed of reaction by Nagamine, Hase and Notomi (2002) , this method employs the strand displacement activity of Bst polymerase (or equivalent) and four specific primers that hybridize to six different regions of the target DNA. Despite the apparent complexity of the molecules produced (cauliflower-like with multiple loops) they are essentially composed of repeats of the initial amplification molecule and as a consequence undergo DNA melting at a consistent and reproducible temperature in much the same way PCR products do. By incorporating thermostable reverse transcriptase (RT) able to operate at 62-65 o C into the reaction mix, it is possible to generate RT-LAMP reactions that can be used to amplify viral RNA targets. The pace with which this technology is being adopted for diagnostic testing appears to be increasing, with diagnostic LAMP reactions for a number of viruses of significance to human health emerging over the past 5 years or so, e.g., Japanese encephalitis virus , hepatitis E virus (Zhang et al., 2012) , West Nile virus (Shukla et al., 2012) , rabies virus , influenza virus including pandemic H1N1 (Nakauchi et al., 2011) and Rift Valley fever virus (Le Roux et al., 2009) . However, despite the use of this technology in many detection assays a major limitation of the technology appears to be its apparent lack of tolerance to sequence variation in the primer binding sites. This is particularly important for detection of RNA viruses. Unlike PCR, which appears somewhat tolerant to a limited number of base changes in the primer/probe binding regions (particularly if these are not at the 3′ termini of the primers), LAMP primers must bind with a high efficiency or the reaction fails to work. It is possible for particular assays to circumvent this problem by the mixing together of different primer sets compensating for sequence variation in the target region with apparently no effect on the performance of the assays when performed individually. An ingenious strategy for DNA amplification has been adopted in a process known as recombinase polymerase amplification or RPA. This isothermal amplification method is similar to PCR in that only two opposing primers are employed but where heating is used to melt the template and subsequent products globally, RPA uses recombinase-primer complexes to scan the double-stranded DNA, facilitating strand exchange at cognate sites (specific site melting, in other words). RPA and assays combining reverse transcription and RPA have demonstrated the utility of this technology for the detection of biothreat agents such as Rift Valley fever virus, Ebola, Sudan virus and Marburg (Euler et al., 2013) . Previously, methods have been described to detect Rift Valley fever (Euler et al., 2012) and HIV (Rohrman and Richards-Kortum, 2012) but it is anticipated that in time this technology will also become more prominent. PCR will continue to dominate but has and will become increasingly more rapid so that it can compete with more rapid isothermal methods. Commercial manufacturers are now claiming "cycling times to result" of 25 minutes with some, such as Applied Biosystems producing specific FAST machines, recognizing that Taq polymerase is not working at a maximum rate in conventional machines. In well-equipped laboratories the rate-limiting step is shifting from the detection method used to the method of nucleic acid extraction. Another limiting step is the transport of samples from the infected animal to the laboratory. One of the most exciting prospects for molecular testing is to take the test to the infected animal. The principle of point of care or point of decision testing has been established using RT-PCR methods and combined extraction/real-time PCR machines, such as the GeneXpert produced by Cepheid who have produced a range of assays for clinical use. However, the cost of these machines and consumables where they are most needed in the world may be prohibitive and this is clearly problematic for commercial companies producing such devices. Using isothermal amplification methods may circumvent problems of cost by using very low cost instruments or no instrument whatsoever for heating, as has been demonstrated for RT-LAMP HIV testing where a constant source of heat was provided by essentially a canister that relied on the exothermal energy derived from the reaction of calcium oxide with water (Curtis et al., 2012) . Both RPA and LAMP assays can be detected in real time using intercalating dyes or more simply at the end of the reactions using tagged primers (FITC, biotin) on lateral flow devices (LFD), which may be of considerable benefit to laboratories where capital investment in such technologies is limited. Of a particular concern with respect to point of care testing is not only the consideration of what is gained (speed of testing and reduced costs of transport) but also what is lost for those collecting epidemiological data or responsible for control of disease (centralized laboratories). This is a pressing issue, particularly in light of the availability of diagnostics via the internet. Much effort has been put into the development of microarrays that can detect multiple virus families. Such arrays consist of thousands of oligonucleotides, targeting many known pathogens. Examples of these have been deployed in the detection of virus, bacterial and protozoan pathogens (Palacios et al., 2007) . The use of pan-virus family polymerase chain reaction assays has revealed the existence of thousands of viruses in a range of hosts that previously have been poorly studied. In the wake of the SARS outbreak, the search for the reservoir host led to the discovery of a wide diversity of SARS-like coronaviruses being shed by bats. This was made possible by the application of sensitive pan-coronavirus primers to RNA extracted from throat and fecal samples in Chinese bats (Chu et al., 2006) . A similar approach has led to the detection of coronaviruses in bat populations around the world (Carrington et al., 2008; Drexler et al., 2010) . The major benefit of PCR is that it generates fragments of DNA that can be sequenced and used in phylogenetic analysis. Pan-paramyxovirus primers have been applied to the detection of viruses from this family from a range of vertebrate hosts and the phylogenies being created have led to the proposal that bats form the ancestral host for many viruses (Drexler et al., 2012) . An alternative route to the use of PCR is to directly sequence a virus from nucleic acid extracted from a sample. The development of mass sequencing and the application of algorithms that will construct contiguous sequences from large data sets have enabled the recovery of complete virus genomic sequences directly from clinical samples. A recent example of this approach detected a novel Rhabdo virus in patients suffering from acute hemorrhagic fever in Africa (Grard et al., 2012) . A key benefit is that a virus can be characterized from a clinical sample or from a degraded sample. This raises a number of fundamental questions on the nature of viruses and disease. While a virus can be detected in a sample derived from a diseased individual, can it unequivocably be said to have caused that disease if "Kochs postulates" cannot be demonstrated? Also many viruses are detected that appear to have no role in disease within a particular host. The ability of these viruses to jump the species barrier and cause disease in a new host is difficult to assess. The consequences of disease emergence are clear in human morbidity and death. In addition is the economic cost, highlighted in the foreword to this book. Disease emergence can also lead to changes in human behavior and also innovation through the development of vaccines and therapeutics. Combating disease emergence, particularly raising awareness among susceptible populations and detecting the causative agent, is critical in responding to such events. Due to the nature of diseases of animal origin it is unlikely that eradication will ever be an option (see Box 12.1) as there will always remain a reservoir of pathogen (Dowdle, 1999) . Therefore, rapid detection and response will be the most effective approaches to controlling future disease emergence. One area that has received particular attention in recent years has been the development of the One Health approach to responding to disease emergence (Hayman et al., 2012) . The One Health concept is a worldwide strategy for expanding interdisciplinary collaborations and communications in all aspects BOX 12.1 Key terms for progressive reduction of disease (Dowdle, 1999) . The reduction of incidence of a disease to an arbitrary level where it is no longer a public health priority. The interruption of transmission of the pathogen when disease incidence becomes zero in a population within a defined geographic area. The interruption of transmission of a pathogen worldwide and the reduction of disease incidence to zero. The infectious agent no longer exists in nature or the laboratory. Biological criteria for disease eradication l Humans are the sole pathogen reservoir l An accurate diagnostic test exists l An effective, practical intervention is available at a reasonable cost. of health care for humans, animals and the environment (Narrod et al., 2012) . A key observation from this that will be developed below is that intervention early in areas such as wildlife and domestic livestock will yield benefits for human health. The challenge is to persuade policy makers that this is the case and that limited resources should be targeted toward achieving this. The origins of the One Health approach are found in the observations of Robert Virchow, a nineteenth-century physician who stated that "between animal and human medicine there is no dividing line, nor should there be" (Conrad et al., 2009 ). This then developed into the concept of "One Medicine," by the veterinary epidemiologist, Calvin Swabe, who emphasized the need for clinicians and veterinarians to coordinate efforts at disease control and reverse the trend of increasing specialization and division of resources. The One Medicine approach to disease control and management has evolved into One Health, which encourages all disciplines to focus efforts on the interface between humans, wildlife and domestic animals and can be applied to the control of all zoonotic pathogens including viruses, bacteria, protozoa, spongiform encephalopathies and fungi. This has been defined as "the collective efforts of multiple disciplines working locally, nationally and globally to attain optimal health for people, animals and environment." The key benefits to this approach are reduction in human and animal morbidity and mortality, reduction in costs associated with responding to disease outbreaks or persistence, and innovations in animal husbandry and human health . The obstacles to introducing a One Health approach are weak leadership, institutional resistance, lack of finance to support linkage between human and veterinary medicine and a poor understanding of ecology. Therefore, a key challenge to introducing a One Health approach to disease management is to demonstrate its efficacy and the potential benefits. For this the most obvious one is to demonstrate cost benefit. Narrod and co-workers (2012) have developed modified risk models that attempt to calculate the economic impact of zoonotic diseases and highlight the benefits of early intervention before the disease reaches the human population. A good example of this is rabies control. The majority of governments target prevention of human deaths by reactive vaccination, commonly referred to as post-exposure prophylaxis (PEP). While highly effective at preventing human deaths from rabies, it does not prevent the persistence of rabies within animal reservoirs, mainly the domestic dog (Lembo, 2012) . By maintaining PEP, but augmenting this with measures that control dog rabies, there are clear long-term financial cost savings and the possibility of eliminating rabies at the local level (Zinsstag et al., 2009) . One final consideration is what viruses will emerge in the future. In recent years the spotlight has been on bats as a source of disease and many putative viruses have been detected within bat species from around the world (Drexler et al., 2012) using the technologies described above. Whether these will be the precursors of future disease emergence is unclear. However, future diseases will probably be caused by viruses that are similar to the ones we are aware of. Reassortment of the influenza genome and subtle changes to virus coat proteins, such as have occurred for canine parvovirus, can rapidly change an apparently harmless virus to one with radically different infectious properties. Strategies that can detect such changes in virus virulence may offer the best hope of identifying emerging viruses that will impact on human populations. Bats and Lyssaviruses The molecular diagnosis of porcine viral diseases: a review Out of Africa: A molecular perspective on the introduction of yellow fever virus into the Americas Detection and phylogenetic analysis of group 1 coronaviruses in South American bats Coronaviruses in bent-winged bats (Miniopterus spp Evolution of a transdisciplinary "One Medicine -One Health" approach to global health education at the University of California Isothermal amplification using a chemical heating device for point-of-care detection of HIV-1 The principles of disease elimination and eradication Genomic characterization of severe acute respiratory syndrome-related coronaviruses in European bats and classification of coronavirues based on partial RNA-dependent RNA polymerase gene sequences Bats host major mammalian paramyxoviruses Recombinase polymerase amplification assay for rapid detection of Rift Valley fever virus Development of a panel of Recombinase Polymerase Amplification assays for detection of biothreat agents Detection of novel swine origin influenza A virus (H1N1) by real-time nucleic acid sequencebased amplification Fatal canine distemper infection in a pack of African wild dogs in the Serengeti ecosystem A novel Rhabdovirus associated with acute hemorrhagic fever in Central Africa Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modelled after retroviral replication Evolutionary history of rabies in Ghana The application of One Health approaches to Henipavirus research SARS: an emerging global microbial threat A new outbreak of rabies in rare Ethiopean Wolves Sequence analysis of HIV-1 group O from Norwegian patients infected in the 1960s Real-time NASBA detection of SARS-associated coronavirus and comparison with real-time reverse transcription-PCR Nucleic acid sequence-based amplification assays for rapid detection of West Nile and St. Louis encephalitis viruses Nucleic acid sequence-based amplification methods for detect avian influenza virus The blueprint for rabies prevention and control: A novel operational toolkit for rabies elimination Development and evaluation of a real-time reverse transcription-loop-mediated isothermal amplification assay for rapid detection of Rift Valley fever virus in clinical specimens Reverse transcription loop-mediated isothermal amplification for rapid detection of Japanese encephalitis virus in swine and mosquitoes Nipah virus infection of pigs in peninsular Malaysia Accelerated reaction by loop-mediated isothermal amplification using loop primers Evidence for human infection with an HTLV III/LAV-like virus in Central Africa Evaluation of reverse transcription loop-mediated isothermal amplification assays for rapid diagnosis of pandemic influenza A/H1N1 2009 virus A One Health framework for estimating the economic costs of zoonotic disease on society Loop-mediated isothermal amplification of DNA Panmicrobial oligonucleotide array for diagnosis of infectious diseases Emergence, natural history, and variation of canine, mink, and feline parvoviruses Design and optimization of a novel reverse transcription linear-after-the-exponential PCR for the detection of foot-and-mouth disease virus Agricultural intensification, priming for persistence and the emergence of Nipah virus: a lethal bat-borne zoonosis An integrated disease management strategy for the control of rabies in Ethiopian wolves A paper and plastic device for performing recombinase polymerase amplification of HIV DNA Quantitation of HIV-1 by real-time PCR with a unique fluorigenic probe Linear-After-The-Exponential (LATE)-PCR.: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis Molecular detection and characterization of West Nile virus associated with multifocal retinitis in patients from southern India Development of a Quantitative Real-Time Nucleic Acid Sequence-Based Amplification Assay with an Internal Control Using Molecular Beacon Probes for Selective and Sensitive Detection of Epidemiologic and historical relationships among 87 rabies virus isolates as determined by limited sequence analysis Prevalence and genetic diversity of coronaviruses in bats from Evolutionary history of African mongoose rabies Comparative detection of rabies RNA by NASBA, real-time PCR and conventional PCR Simple and rapid detection of swine hepatitis E virus by reverse transcription loop-mediated isothermal amplification Transmission dynamics and economics of rabies control in dogs and humans in an African city Mainstreaming One Health This work has been supported through the project "Anticipating the global onset of novel epidemics (ANTIGONE)" funded by the European Union project number 278976.