key: cord- -jfawhnsq authors: caron, alexandre; cappelle, julien; cumming, graeme s; de garine-wichatitsky, michel; gaidet, nicolas title: bridge hosts, a missing link for disease ecology in multi-host systems date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: jfawhnsq in ecology, the grouping of species into functional groups has played a valuable role in simplifying ecological complexity. in epidemiology, further clarifications of epidemiological functions are needed: while host roles may be defined, they are often used loosely, partly because of a lack of clarity on the relationships between a host’s function and its epidemiological role. here we focus on the definition of bridge hosts and their epidemiological consequences. bridge hosts provide a link through which pathogens can be transmitted from maintenance host populations or communities to receptive populations that people want to protect (i.e., target hosts). a bridge host should ( ) be competent for the pathogen or able to mechanically transmit it; and ( ) come into direct contact or share habitat with both maintenance and target populations. demonstration of bridging requires an operational framework that integrates ecological and epidemiological approaches. we illustrate this framework using the example of the transmission of avian influenza viruses across wild bird/poultry interfaces in africa and discuss a range of other examples that demonstrate the usefulness of our definition for other multi-host systems. bridge hosts can be particularly important for understanding and managing infectious disease dynamics in multi-host systems at wildlife/domestic/human interfaces, including emerging infections. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. ecological functional approaches classify organisms according to what they do, and/or what they eat. they offer an alternative perspective to taxonomic classifications for identifying trends within and making sense of ecological complexity. applications of functional group concepts, which date back to fundamental ideas about biomass distributions across different trophic levels [ ] , have been crucial in advancing ecological understanding. more recently, ecological functional analyses have achieved prominence as a way of linking taxonomic survey data and the provision of ecosystem services [ ] . functional analyses thus remain an important research area in ecology. in epidemiology, functional concepts have clear potential utility but are still in a relatively early stage of development. classical epidemiology relies heavily on single-species studies, particularly those of people (e.g., analyses of measles and smallpox in human populations [ ] ). in contemporary epidemiological studies, in the last fifteen years, under the influence of ecology, the scope of epidemiology is being broadened to include plant and animal communities in which multiple different species can contribute to the maintenance and spread of pathogens in host populations [ ] . in multihost systems, the role played by each host population in pathogen dynamics is determined by the species' competence for the pathogen (i.e., its receptivity to infection and its capacity to replicate and transmit the pathogen [ ] ), its exposure to the pathogen determined by the host ecology/behaviour and its interactions with other host populations (including vectors for vectorborne infections) leading to infectious contacts, and finally, the composition of the host community that will determine the range of inter-host interactions [ ] . one of the central questions in disease ecology is that of how the community composition of potential host species relates to the dynamics of pathogen transmission within the host community, as opposed to within a population of a single species. the complexity of this problem can be simplified by assigning epidemiological functions to relevant traits that define an organism's role in the epidemiology of a given pathogen. for example, animals that undertake long movements (a trait) may contribute to the epidemiological function (pathogen disperser) of spreading pathogens over large distances (a role). grouping organisms by epidemiological functions facilitates the development of eco-epidemiological models for a given pathogen in relation to an entire animal community [ ] . this approach could potentially play an important role in guiding research, as well as in the surveillance and control of animal and zoonotic diseases [ ] . although some progress has been made in the characterization of epidemiological functional groups, (e.g., clear definition of the maintenance function [ , ] ), other epidemiological functions remain incompletely defined, especially those relating to the transmission of pathogens between groups of hosts. in this paper we first define the transmission function in relation to the maintenance function. we then focus on the concept of "bridge hosts" and demonstrate their potential importance in the ecology of disease transmission in multi-host systems. though closely related concepts have been used previously [ ] [ ] [ ] , we believe that a refined definition embedded in a clear functional framework is still lacking. lastly, we present an operational framework to identify potential bridge host populations, using as a case study the ecology of avian influenza viruses at the wild/domestic bird interface in africa and also giving other multi-host systems examples. we use "host" to refer to a host population, a host species, or a host community. the smallest epidemiological unit to which we will refer is a host population, acknowledging the fact that individual variability can also substantially impact pathogen transmission (e.g. "superspreader", [ ] ). as defined by haydon et al. [ ] and more recently revised by viana et al. [ ] , a conceptual framework for the role of hosts in epidemiology requires the definition of the target host: "the population of concern to the observer" in the geographic area under study (table ) . a maintenance host will only be relevant to a target population if it can be in contact with and able to transmit the infection to it. the maintenance function represents the capacity to maintain the pathogen within the ecosystem. a maintenance host is a host population (single population) or community/host complex (several sympatric host populations) "in which the pathogen persists even in the complete absence of transmission from other hosts" [ ] . the maintenance function depends on host density, and on intraand inter-host infectious contacts (i.e., a contact leading to infection amongst other intra-host factors; [ ] ). in multihost systems, the notion of a maintenance community in which several populations from different species play a role in the maintenance of the pathogen seems more appropriate than the "reservoir" concept [ , ] for understanding pathogen dynamics. the reservoir concept is still being used in contradictory ways, as discussed by several authors [ ] [ ] [ ] . haydon et al. [ ] extended the definition of reservoir by adding "source populations" that may not be involved in the maintenance of the pathogen but rather in the transmission of the pathogen to the target population. ashford [ ] defined a "liaison host" as linking the reservoir to another host population, with no explicit reference to target populations. we agree with ashford [ ] that source population should not be included in the definition of the reservoir, as this term is strongly linked to the concept of maintenance and because control of infection in the reservoir would be different if targeted at the maintenance or source populations. for example, aiming at controlling the infection in a maintenance vs. a source population might have different outcomes, since the maintenance host could still re-infect the source population in the latter case. to add to the confusion, suzán et al. [ ] presented a new framework to understand patterns in space and time of meta-communities of hosts and parasites. in their first figure they display in red "reservoir species" and in orange "alternative hosts", together "maintaining higher infection of prevalence". clearly, their concept of "reservoir" differs from that of haydon et al. [ ] , who argued that any host involved in the maintenance of the pathogen should be part of the reservoir. the difference in definitions is identical with plowright et al. [ ] : they present domestic horses as potential source populations (defined in the article as "recipient" and "intermediate hosts") of hendra viruses for human populations without considering them as part of the reservoir (presented as the bat community). the extensive use of the "reservoir" concept under multiple definitions and the lack of consensus around the liaison host and source population concepts (revealed by the scarcity of use of these two last terms in the literature) requires a refined conceptual framework and definitions. agreeing with others [ , ] , we thus prefer to use only maintenance host or community, a term that refers better to the dynamic aspect of the functional role than the static notion of a reservoir [ , ] . although the maintenance-target host relationship and its link with the maintenance function have been properly defined, the function of pathogen transmission to the target host needs a clearer definition. interspecific pathogen transmission is of crucial importance for infectious disease management. disease control can target the maintenance host to stop pathogen maintenance and circulation in the ecosystem (i.e. targeting the maintenance function); however, as this option is often unfeasible (for practical or ethical reasons, notably concerning wildlife populations), one could also try to break the transmission pathway that brings the pathogen to the target host. we therefore define the transmission function as the capacity to transmit the pathogen to the target host. this function must be separated from the maintenance function, as the maintenance host does not always have infectious contact with the target host. when it has direct contact with the target host, then the maintenance host is implicated in the maintenance and transmission functions. when it does not, a bridge host (table ) can connect (i.e., have infectious contact with) both maintenance and target hosts, "bridging" the gap between them. using this functional definition, the concept of the reservoir as revisited by haydon et al. [ ] and more recently by viana et al. [ ] , does not refer clearly to a single epidemiological function, because it includes maintenance host(s) involved in the maintenance function and potentially in the transmission function as well as non-maintenance population(s) only involved in the transmission function. allocating hosts belonging to the reservoir to specific functional groups that surveillance and/or control can target is therefore difficult and provides an additional reason to focus solely on the maintenance-target hosts. bridge host is therefore used, since (i) the group is distinct from the source population, as bridge hosts do not belong to the maintenance host/community, and the liaison host as a bridge host is always in reference to a maintenance-target population system; and (ii) the word "bridge" is relevant to the definition proposed (e.g. [ ] ). -hosts in which the pathogen persists even in the complete absence of transmission from other hosts [ ] x (x) -brush-tailed possums for bovine tuberculosis in new zealand [ ] -population larger than the critical community size (i.e. size under which the pathogen cannot be maintained in the community) in which the pathogen persists [ ] -white-footed mouse (peromyscus leucopus) for lyme disease in the united states [ ] maintenance host community/ maintenance host complex -one or more epidemiologically connected populations or environments in which the pathogen can be permanently maintained [ ] x (x) -anatids for avian influenza viruses worldwide [ ] -any host complex in which disease persists indefinitely is a reservoir [ ] -amphibian sp. for the trematode ribeiroia ondatrae [ ] -host for which cross species transmission and inter-species transmission are high [ ] bridge host -non-maintenance host population able to transmit a pathogen from a maintenance host/complex to the target population, otherwise not or loosely connected to the maintenance complex (this manuscript) x previous related definitions: -little studied so far -source population: any population that transmits infection directly to the target population [ ] -red deer and domestic pigs for bovine tuberculosis in new zealand [ ] ? -liaison host: incidental hosts that transmit pathogens from a reservoir to another incidental host [ , ] -peri-domestic birds such as swallow sp., sparrow sp., etc. [ ] -spatial vector: host that transport the pathogen to target populations in new locations [ ] -temporal vector: host that can transmit the pathogen to target species across temporal scale [ ] crosses in brackets indicate that maintenance host can participate in the transmission function although this is not a necessary condition. bridge hosts refer therefore to a group of hosts that perform the same epidemiological function for a pathogen that can be targeted by specific surveillance and control interventions. in suzán et al. [ ] , information about whether alternative hosts function as bridge hosts would add an important layer of information to their framework and contribute to the understanding of the spatial spread of parasites. our bridge host definition is closely related to the "spatial and temporal vector" concepts presented by nugent [ ] but unifies them with previous definitions (see above) and broadens them. a bridge host can be defined at the level of a population or a community. bridge hosts may be frequent in disease ecology, but this term has not been explicitly defined and its usage is not common when referring to the transmission function without any role in maintenance function. for example, it would be incorrect to use the term "bridge species" as the role of a bridge host would refer to a specific host population in interaction with other hosts in a given ecosystem (e.g. contact with maintenance or target populations) and at a specific density [ ] ; the host density and the network of interaction between these hosts in another ecosystem would likely be different and would make it unlikely that a species can play the same epidemiological functional role across its range. a clearer conceptual framework is thus needed to guide the identification of bridge hosts and the characterisation of their roles in disease ecology. this framework must also be operationalised if it is to guide the design of hypotheses that can be tested through field protocols to characterise the role(s) of hosts in disease ecology. using the different target-maintenance systems proposed by haydon et al. [ ] , bridge hosts can be included in target-bridge-maintenance systems in several ways ( figure ). according to our definition, a bridge host is involved in the transmission function while not involved in the maintenance function. two main prerequisites must be fulfilled for a host to qualify as a bridge host. the first prerequisite is that the host must be competent for the pathogen (i.e., must be receptive to infection, permit pathogen replication, and be able to excrete it) without being able to maintain it alone, in which case the host would be considered as a maintenance host; or alternatively, the host should be able to mechanically transport the pathogen [ , ] . its competence will influence the capacity of a bridge host to achieve the transmission function: if the bridge host has a short pathogen excretion period, it will be able to transmit the pathogen to a target population only if the time lag between contact with a maintenance and then a target host is shorter than the excretion period, or if the distance between target and maintenance is shorter than the maximum distance that the bridge host can travel during its excretion phase. similarly, for mechanical transmission, the survival of the pathogen on/in the host body part (e.g. skin, hair, mouth, feathers) exposed to the external environment will determine for how long the host can play the bridge role. the second prerequisite is that infectious contacts must occur along the maintenance-bridge-target transmission chain. these will depend on direct and indirect (e.g. environmental transmission) contacts, the mode of transmission of the pathogen, and the site of infection. the basic reproductive number r for the bridge host (not considering here mechanical transmission) should be < as it cannot maintain the infection but its force of infection, dependent on the number and extent of infectious contacts with the target host, can be high. a bridge host that compensates for a lack of infectious contacts between maintenance and target hosts can operate across different dimensions: spatial, temporal, and behavioural. the spatial dimension arises when the bridge host creates a spatial link between the separate areas in which the maintenance and the target host populations occur. this dimension typically refers to the situations developed below for wild birds and avian influenza. it has been defined as a "spatial vector" by nugent [ ] when considering the role of feral pigs (sus scrofa) in the epidemiology of bovine tuberculosis (btb) in new zealand. the temporal dimension arises when the pathogen can persist (but not be maintained indefinitely) in the bridge host for a period of time longer than in the maintenance host or during a distinct season; this has been well described by nugent [ ] as a "temporal vector", for example when red deer (cervus elaphus) transmit mycobacterium bovis to possum populations that are controlled to levels that are well under the critical community size for btb maintenance. the behavioural dimension exists when the absence of contact between sympatric maintenance and target hosts is compensated for by another host that has infectious contacts with both. situations may occur in which the microhabitat preferences and behaviours of maintenance and target hosts mean that they do not come into direct contact despite using the same locations on a daily basis. bats, for example, are believed to be the maintenance host for ebola, and can be sympatric with people; but ebola transmission from bats to humans is enhanced by the great apes (whose susceptibility to ebola seems to indicate that they are not maintenance hosts) which feed with bats and are fed upon by humans [ ] . it is interesting to note that in all cases, even a r close to zero (approximating a dead-end host) could still be important for the transmission function: the capacity to excrete the pathogen for a few hours, associated with some form of dispersal, may be sufficient for a bridge host to come into contact with the target host and infect it. for pathogens like ebola, the range of hosts that is classically considered to be important in disease ecology may have to be broadened by including hosts that are able to transmit the pathogen over short time-and space-scales. these hosts are commonly considered as playing no role in pathogen ecology and are called dead-end hosts (e.g., most wild avian hosts for avian influenza virus -aiv -apart from anseriformes and charadriiformes). amongst the multitude of those dead-end hosts, the bridge host perspective can identify some that do play a role in disease ecology. with this framework in place, we next turn to the question of how bridge hosts can be identified in the multihost context of aiv epidemiology and suggest an operational framework (partially implemented in [ ] ) that can figure definition of different target-bridge-maintenance systems (adapted from haydon et al. [ ] ). a represents the simplest maintenance-bridge-target system. in a', the maintenance and target populations are less connected (frequency/intensity of infectious contacts) than between the maintenance-bridge-target populations. in b, mitigation strategies aimed at one bridge host cannot fully control pathogen transmission to the target host because of the alternative bridge host's pathway. if both maintenance populations were in contact with both bridge hosts (i.e. if dashed arrows exist), controlling contacts between the target population and bridge hosts should be simpler than other control options. in b', according to our definition, z is not considered as a bridge population as it belongs to the maintenance community. in c, stopping contacts between the maintenance population and the target population by acting on one of the two bridge hosts would not be enough to stop transmission, which can still occur through the second bridge host. d is a special case of b, understanding the complexity of the maintenance community is not necessary to control the pathogen transmission risk to the target population, which can be achieved through the control of arrows connecting the bridge host. in e, none of the host populations can sustain the infection by itself and according to our definition, u is not considered as a bridge population as it belongs to the maintenance community. in f, the bridge host connects the target population with another maintenance host creating a system with a maintenance meta-population, which could change the epidemiological dynamics of the system and the probability of success of intervention strategies (e.g. vaccination coverage to achieve control of the infection in the target population). g is a special case where two bridge hosts are necessary to achieve the transmission function. good knowledge of the ecological interactions in the ecosystem will be necessary to identify such complex interactions between bridge hosts. enhance disease ecology as well as pathogen surveillance and control. waterfowl (defined here as ducks, geese, waders, gulls, and terns) constitute the maintenance hosts for low pathogenic avian influenza viruses (lpaiv) [ ] . aiv represent major threats to poultry production when strains originating from wild birds evolve from low to high pathogenicity in the poultry (target) populations [ ] . the transmission of lpaiv between the wild bird maintenance community and domestic populations is therefore crucial to managing the sanitary and economic impacts of the disease. in this section, the risk of lpaiv spillover to poultry populations from the maintenance populations will be used as an example. when poultry are confined in farms or buildings, their direct contacts with the maintenance waterfowl community, which mainly lives in wetlands and on coastal shorelines, are believed to be limited due to spatial segregation between populations. many outbreaks of highly pathogenic aiv outbreaks have nonetheless occurred in domestic poultry production systems. it is therefore suspected that bridge hosts play a role in transmitting waterfowl-derived strains of aiv to poultry populations. the ability of wild birds to travel long distances, and their ubiquity in most habitats, facilitate the potential for wild bird species to act as bridge hosts. several constraints limit a better understanding of aiv ecology in bird communities: ) high host diversity, that can include several hundred species in a given ecosystem; ) the costs of diagnostic techniques that limit the number and type of samples (e.g. cloacal/tracheal swabs, blood) that can be analysed; and ) the impossibility of randomly sampling from bird communities because of bias in capture techniques (e.g. walk-in traps, mist-nets). as a consequence, the information available on most wild bird species is scarce and has been obtained mostly from by-catch (i.e. captured non-targeted species) of studies investigating aiv in maintenance waterfowl, resulting in small sample sizes that are inadequate to provide epidemiological understanding of the host roles in aiv ecology in africa [ ] . the following framework used in a recent study [ ] and here developed in detail, aims at first gathering/collecting available ecological and epidemiological information; second, at synthesizing this information to provide a priority list of species that act as potential bridge hosts; and finally, at undertaking targeted sampling that can determine the competence of the high priority species and revisit the framed hypotheses. the range of methods available to characterize host competence for aiv and contact patterns between maintenance, potential bridge and target host populations is drawn from the fields of epidemiology and avian ecology ( table ) . none of these methods alone is sufficient to identify a bridge host in a given ecosystem [ ] . molecular epidemiology (e.g. gene sequencing after virus isolation) could in principle be used to identify bridge species but it is very unlikely that related strains from three different host populations (i.e., maintenance, bridge and target hosts) are concurrently isolated except perhaps during a localised aiv outbreak. virological surveillance (e.g. polymerase chain reaction -pcr techniques) can provide information about host contacts between potential bridge and maintenance hosts if data are collected close to wetlands where waterfowl communities are known to occur. serological investigation (e.g. elisa tests) can be cheaper than virological testing but provide less information on the timing of the infection [ , ] . however, a combination of epidemiological and ecological methods could provide the necessary information to infer the bridge role of a given host population. taking into account these constraints, the proposed framework aims, first, to narrow the large number of species by ranking the most probable potential bridge hosts based on proxies of host competence and/or contacts between maintenance, target and potential bridge hosts. this step can be achieved using (or combining) available published field (e.g. [ , [ ] [ ] [ ] ) and experimental epidemiological studies (e.g. [ , ] ). however, most aiv experimental studies have concentrated so far on a very limited set of species (e.g. for lpai [ , ] and for hpai [ , ] ). reviewing available pcr viral data within a given area or region can provide information on the range of host species with a competence for aiv. for example, in sub-saharan africa, the available databases are poor representations of existing avian diversity (only . % of all african species have been sampled, additional files and , and only a few species were tested with a sample size that would be sufficient to detect % aiv prevalence). this exercise can help with ranking the species or groups of species based on the rate of infection, which provides an initial prioritization list for future investigation (additional file ). however, one shortcoming of pcr data is to link detection of genetic material and state of infectiousness of the sampled individual [ ] , an issue that is often overlooked but particularly important for the identification of bridge hosts. the first step of the proposed framework must also incorporate ecological data that provide information about the presence/abundance of potential bridge hosts in the ecosystem and their potential contacts with maintenance and target hosts. however, it is a challenge to provide evidence that contacts ( ) occur; and ( ) result in successful virus transmission. different types of data can be used or collected, each with its own strengths and weaknesses: life history traits (e.g., abundance, gregarism, foraging and migratory behaviour) obtained from the literature can be used as risk factors for contacts between wild and domestic birds or exposure to infection [ , ] ; contacts between wild and domestic birds can be estimated using satellite telemetry [ ] ; capture-recapture techniques indicate population size (e.g. using colour rings at a local scale) [ ] ; and observations at focal points that are at wild/ domestic bird interfaces (e.g. around poultry production building) can be used to quantify interactions [ ] . the second step is to synthesize the ecological and/or epidemiological data to rank bird species according to the likelihood that they play a bridge role in the ecosystem under study. risk analysis can provide such a tool [ , [ ] [ ] [ ] and may be particularly important when no information is available for an ecosystem, or prior to a field survey, by highlighting the populations that could be targeted preferentially. once the bridging potential of different species has been evaluated, the third step of the framework consists in testing the host competence of the most likely bridge hosts in the ecosystem through targeted sampling. for example, caron et al. [ ] applied this framework in a southern african ecosystem and identified bridge hosts by combining bird counts with selected sampling and pcr testing. targeted sampling facilitates the concentration of resources to obtain adequate sample sizes and relevant epidemiological information and comes in place of the practice of blind sampling from wild bird communities, which is usually biased by capture techniques. hypotheses can be revisited iteratively as more is learned about the potential of highly ranked species to act as bridge hosts. this approach can also lead to the detection of inconsistencies in the initial model (e.g., the definition of the maintenance community) and the necessity to revisit it [ ] . avian influenza provides a good example of a case in which paying conceptual and practical attention to bridge hosts can enhance our understanding of pathogen dynamics in multi-host systems. although the use of the bridge host concept may not be relevant for all multi-host systems, it has the potential to contribute to structuring investigations on the ecology of emerging pathogens shared at wildlife/livestock interfaces. to illustrate this point we present two additional examples of multi-host systems. in the first, ebola in west africa, understanding could be improved by the use of the conceptual framework developed here. in the second and better-known system, bovine tuberculosis in new zealand, bridge hosts have been identified and are an important component of the problem. ebola virus spilled over in early in west africa from an unknown animal to the human index case. knowledge of ebola ecology is still limited, despite the first outbreak having being reported in . current understanding points at bats (mammalia: chiroptera) as potential maintenance hosts, and contact between humans and some bat species occurs through the bushmeat industry [ ] as well as via bat droppings and occasional cases of sick bats that are handled by humans [ , ] . however, embracing the functional approach presented here makes sense to look for potential bridge hosts that could link maintenance bats and humans. a priori, scavenging pigs, dogs, other non-maintenance bat species and wild antelope can have direct or indirect (e.g. consumption/hunting) contact with humans [ ] . targeted surveillance of such species will provide information on their competence for the virus; and host interaction protocols that identify contact networks with maintenance and target populations can provide information on the potential for viral spread (e.g. [ ] ). once the multi-host system is better understood (case b, c or d in figure ), it may be simpler to try to block transmission pathways from bridge hosts to human populations (e.g. through changes in behavior related to bridge host consumption by people) than to control the pathogen in the maintenance hosts. [ ] [ ] [ ] [ ] risk analysis x x x [ ] [ ] [ ] serological investigation x x x xx [ , , ] virological investigation xx xx xx xx xx [ , , , , , , ] telemetry study xxx xxx xxx [ ] bird ringing and monitoring xx x x [ ] bird counts xx xx x [ , , , ] molecular epidemiology xx xx xx xx xx xxx [ , ] as the number of crosses increases in the first columns the methods provide better ecological or epidemiological information; in the last columns, cost increases as the number of crosses increases. a similar yet less complex example was recently developed [ ] , indicating that domestic horses could be "bridge hosts" for hendra viruses between bats (maintenance host) and humans (target host) even if it is not yet known if horses could maintain the virus or just act as a bridge between bats and humans [ ] . as a second example of the utility of the bridge host framework, nugent [ ] offers a comprehensive description of the btb multi-host system in new zealand. the cattle industry in new zealand suffers from continuous spillover of the btb mycobacterium from the maintenance host, the brush-tailed possum (trichosurus vulpecula). the control of possum populations by depopulation is mainly implemented in areas around farms that are at high-risk of transmission to cattle, leaving high densities of possums in more distant forest and providing a gradient of btb prevalence. this apparently efficient strategy is, however, thwarted by three potential bridge hosts (feral pigs sus scrofa, red deer cervus elaphus, and feral ferrets mustela furo) that are involved in transmission (case g in figure , called "link-host" in the article but lacking a more conceptual definition). infected pigs and deer with large home ranges may leave the forest to die (or be hunted) around farms, providing an opportunity for ferrets to become infected when feeding on carcasses and subsequently infecting cattle or possums. this study is particularly interesting for reasons: ( ) the complexity and low probability of the chain of events leading to infection of the target population do not prevent btb occurrence and the failure of disease control; ( ) disease control targeted at the maintenance population prevents the transmission link between the maintenance and target hosts but the transmission pathways built by bridge hosts (case a' in figure ) reduce the effectiveness of control, proving the importance of considering this epidemiological function and host role; and ( ) the plasticity of the roles of host populations in disease epidemiology, which is heavily influenced by the environmental, ecological and anthropological context. the concepts of transmission function and bridge host contribute to a better understanding of disease ecology in multi-host systems by clarifying the epidemiological processes that are relevant for disease transmission and maintenance. this perspective fits better with the way that people operationalize the complexity theory and makes it easier to develop models of these systems. when maintenance and target hosts are not in direct contact, pathogen transmission relies on successive infectious contacts along the chain of maintenance, bridge and target hosts. bridge hosts can play a pertinent and legitimate role in disease ecology and could become the targets for surveillance and control for some multi-host systems. for example, in some ecosystems, domestic bird populations are rarely in direct contact with wild waterfowl populations but phylogenetic analyses have indicated that most precursors of hpaiv in gallinaceous poultry have originated from wild waterfowl [ ] , suggesting that bridge hosts play a role in aiv transmission at the wild/domestic bird interface. more recently, evidence supporting a role for some passerines (finches, sparrows) in the transmission of the avian-origin human influenza a (h n ) to human and poultry in china [ ] suggests a potential role for passerines as bridge hosts between poultry and humans. the functional approach emphasizes the need to focus on transmission pathways between hosts (and their directionality) instead of relying solely on intrinsic host properties (e.g. density, shedding capacity) [ , ] . the presence of a target host defines directionality in the transmission processes and implies a network of interconnected hosts with different epidemiological roles. our framework thus provides a better empirical approach to some kinds of epidemiological problems, such as the risk of spread of a specific pathogen towards a target population or the potential for disease emergence in emerging disease hotspots. the maintenance and transmission function concepts can be related to the roles of vectors in vector-borne disease ecology. blood-feeding arthropod vectors that transmit a pathogen between hosts [ ] may be involved in distinct epidemiological functions, including the transmission function. the term "bridge vector" has already been used (e.g. [ , ] ) to group mosquitoes that transmit west nile virus to humans (here the target population). however, so far, the distinction between the maintenance and transmission function has not been properly defined. this distinction could be important if maintenance and bridge vectors are different species, opening different control strategy options (i.e. on the maintenance or on the bridge hosts). the identification of bridge hosts for a given pathogen in a given ecosystem has consequences for disease management, surveillance and control. once bridge hosts are known, managers can adopt mitigation strategies specifically aimed at reducing contact between the target and the bridge populations. in the case of aiv, this mitigation can be achieved through strengthening biosecurity measures or decreasing the quantity of attractors on the farm (e.g. water sources or open feedlots) [ ] . the adoption of adequate management measures targeting contacts between maintenance, bridge and target hosts is also more environmentally acceptable than controlling (wild) host populations. the distinction between maintenance and bridge hosts may under some circumstances be difficult. in the case of aiv, for example, our current level of knowledge about the maintenance hosts and the apparent lack of contact in some ecosystems between the maintenance community and the target populations suggest a role for bridge hosts. the identification of hosts that do not fit into either maintenance or target host groups, as in [ ] , raises two possibilities: either these susceptible hosts act as bridge hosts, or they may act as previously unknown maintenance hosts for aiv epidemiology. to differentiate between these two hypotheses may require focused experimental research, for example by using infection of captive animals to determine their capacity to maintain the virus. other approaches using meta-analysis of existing data sets have also been proposed [ ] . in both cases, our conceptual framework helps with framing hypotheses based on current knowledge and using empirical tests to either confirm these hypotheses or call for a revision of our understanding of the epidemiological system (e.g. this host is not a bridge host and therefore has no (or another) role in the local context). our framework does have some weaknesses. in particular, proving that a bridge host in a complex multi-host system where maintenance communities are composed of numerous interacting populations does not take part in the maintenance function (i.e. that removing the bridge host will not drive the pathogen to extinction, according to haydon et al. [ ] ) may necessitate an experimental design that would be difficult to achieve in practice [ ] . in addition, only cases in which maintenance and target populations are not in contact have been considered so far. if they are loosely in contact (case a' in figure ), the frequency and efficacy of contacts between different pairs (maintenance-target, maintenance-bridge and bridgetarget) would need to be weighted against each other. decreasing the maintenance-target contacts through management will reveal the relative importance of bridge-target contacts and could require interventions in order to efficiently stop pathogen transmission (as in the case of control of possums for btb in new zealand mentioned earlier). finally, we have assumed that a bridge host must be competent for the pathogen but in some cases simple mechanical transmission (e.g., a bird carrying the virus on its feathers [ ] ) may be possible, relaxing the prerequisite on host competence for the bridge host. the development of complex human/livestock/wildlife interfaces, due to the encroachment of human activities within natural ecosystems, triggers new epidemiological dynamics that may permit a range of wild or domestic bridge hosts to link maintenance communities with new target hosts [ ] . we would expect that domestic species and newly farmed or traded wildlife species will increasingly play bridge host roles in the emergence of new zoonoses. the epidemiology of ebola, sars, lyme disease, and h n aiv, for example, are not yet fully understood but are known to involve multiple hosts. we believe that introducing our definitions and operational framework into research and surveillance could contribute to more efficient use of resources to fill some knowledge gaps. our approach builds on that of haydon et al. [ ] and refines it to take into account potential circumstances under which an extra conceptual development is necessary. whether this extra development will be necessary in many multi-host systems or will be used only under exceptional circumstances will be answered by studies to come. the examples given here indicate that they could be used for at least a few important diseases. the recent appearance in the epidemiological literature of similar concepts [ , , ] that are not always placed soundly within a conceptual framework and/or ignore previous definitions suggests also the need for a consolidated review and refinement of these concepts and definitions. while no individual element of our proposed framework is new, it is clear from our discussion above that approaching the problem of understanding multi-host disease systems from a more integrated, functional perspective has the potential to offer a wide range of valuable insights into both epidemiology and its applications to pathogen control. our approach, which requires both epidemiological and ecological approaches (and also social science approaches when the human host is considered) fits well within current initiatives that call for more transdisciplinary integration between the health sciences and other fields of research. finally, the global fight against emerging infectious diseases is increasingly focused on identifying potential emerging pathogens from high-risk maintenance hosts (e.g., bats and rodents, [ , ] ). recent advances in genetics and genomics have increased drastically the pace at which new micro-organisms are discovered and identified [ ] . but adding new names to the list of parasites and pathogens does not provide information on which of these microorganisms might present a significant threat to animal or human health. a maintenance population hosting a large range of potentially new emerging pathogens does not constitute a threat for target populations if no transmission route exists between the maintenance and target populations. focusing on pathogen transmission pathways, including potential hosts bridging the gap between maintenance and target populations, will help to guide "pathogen hunting" approaches as functional ecology complements taxonomy. such an approach will help to guide high-throughput sequencing tools towards key hosts within a given epidemiological context, increasing the efficiency of surveillance and control efforts. epidemiology and public health unit contrasting spatial patterns of taxonomic and functional richness offer insights into potential loss of ecosystem services infectious diseases of humans: dynamics and control causal inference in disease ecology: investigating ecological drivers of disease emergence experimental infection of north american birds with the new york strain of west nile virus the ecology of infectious disease: effects of host diversity and community composition on lyme disease risk epidemiological interaction at the wildlife/livestock/human interface: can we anticipate emerging infectious diseases in their hotspots? 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understanding the ecological drivers of avian influenza virus infection in wildfowl: a continental scale study across africa use of observed wild bird activity on poultry farms and a literature review to target species as high priority for avian influenza testing in regions of canada characterizing the interface between wild ducks and poultry to evaluate the potential of transmission of avian pathogens a comparative analysis of movements of southern african waterfowl (anatidae), based on ringing recoveries estimating dynamic risk factors for pathogen transmission using community-level bird census data at the wildlife/domestic interface wild bird movements and avian influenza risk mapping in southern africa ornithological data relevant to the spread of avian influenza in europe (phase ): further identification and first field assessment of higher risk species fruit bats as reservoirs of ebola virus bats: important reservoir hosts of emerging viruses transmission of ebola virus from pigs to non-human primates understanding pathogen transmission dynamics in waterbird communities: at what scale should interactions be studied? molecular analysis of avian h influenza viruses circulating in eurasia in - : detection of multiple reassortant virus genotypes possible role of songbirds and parakeets in transmission of influenza a(h n ) virus to humans differential sources of host species heterogeneity influence the transmission and control of multihost parasites relationship between pace of life and immune responses in wild rodents ecology: from individuals to ecosystems -, th edn west nile virus risk assessment and the bridge vector paradigm culex pipiens (diptera: culicidae): a bridge vector of west nile virus to humans sampling strategies and biodiversity of influenza a subtypes in wild birds isolation of influenza a viruses from wild ducks and feathers in minnesota conservation and development interventions at the wildlife/livestock interface: implications for wildlife, livestock and human health emerging infectious diseases: threats to human health and global stability host range and emerging and reemerging pathogens discovering the phylodynamics of rna viruses persistence of low pathogenic avian influenza virus in waterfowl in a southern african ecosystem influenza a viruses in waterbirds in africa investigating avian influenza infection hotspots in old-world shorebirds isolation of a low pathogenic avian influenza virus (h n ) from a black kite (milvus migrans) in egypt in circulation of avian influenza viruses in wild birds in inner niger delta characterization of h n avian influenza virus isolated from a wild white pelican in zambia reassortant low-pathogenic avian influenza h n viruses in african wild birds additional file : virus detection in african non-maintenance wild bird populations (order and family level). this file describes the methodology that was used to gather all the relevant information on rt-pcr aiv results of non-anseriformes and non-charadriiformes species in africa and provides the summary of findings at the bird order and family level. [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] .additional file : aiv rt-pcr results for wild birds sampled in africa until . this table displays detailed results of wild bird species and families sampled for aiv (rt-pcr) in africa until following the gathering of data as described in additional file . the authors declare that they have no competing interests. ac drafted the first version of the manuscript after in-depth discussions with the various authors about the concept and framework developed in this manuscript. jc, gsc, mdgw and ng commented on various versions of the manuscript. all authors read and approved the final manuscript. this article was made possible by the involvment of authors in three projects: the gripavi project sponsored by grants from the french ministry of foreign affairs, the tcp of fao through additional grants from the government of france, and the usaid-sponsored (through the wildlife conservation society) gains project. we are grateful to the numerous epidemiologists, veterinarians and field assistants who participated in field operations. this work was conducted within the framework of the research platform "production and conservation in partnership", rp-pcp and the ahead initiative.author details key: cord- - xbu hnq authors: slingenbergh, jan title: animal virus ecology and evolution are shaped by the virus host-body infiltration and colonization pattern date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: xbu hnq the current classification of animal viruses is largely based on the virus molecular world. less attention is given to why and how virus fitness results from the success of virus transmission. virus transmission reflects the infection-shedding-transmission dynamics, and with it, the organ system involvement and other, macroscopic dimensions of the host environment. this study describes the transmission ecology of the world main livestock viruses, in total, a mix of rna, dna and retroviruses. following an iterative process, the viruses are virtually ranked in an outer- to inner-body fashion, by organ system, on ecological grounds. also portrayed are the shifts in virus host tropism and virus genome. the synthesis of the findings reveals a predictive virus evolution framework, based on the outer- to inner-body changes in the interplay of host environment-transmission modes-organ system involvement-host cell infection cycle-virus genome. outer-body viruses opportunistically respond to the variation in the external environment. for example, respiratory and enteric viruses tend to be associated with poultry and pig mass rearing. ruminant and equine viruses tend to be more deep-rooted and host-specific, and also establish themselves in the vital inner-body systems. it is concluded that the framework may assist the study of new emerging viruses and pandemic risks. animal viruses may be split into transmissible and persistent viruses. it has been proposed that transmissible viruses correlate with replication and virulence and that virus persistence instead permits a lower transmission rate [ ] . this insight builds on earlier work suggesting that virus persistence may pave the way for virus-host symbiosis [ ] . viruses are considered essential agents within the roots and stems of the tree of life [ ] . for example, rna viruses in vertebrates tend to broadly follow the evolutionary history of their hosts that began in the ocean and extended for hundreds of millions of years [ ] . the symbiotic virus-host relationships can take many forms, from antagonistic to mutualistic, and viruses, like other symbionts, lie on a continuum that can shift with environmental changes [ ] . the present study seeks to take these insights to the next level. starting point in the analysis is the link between virus propagation and transmission success. virus transmission may be considered to present the backbone of virus ecology, determining the viruses selected for. unfortunately, the current classification of animal viruses emphasizes the importance of virus genomic architecture and the host cell infection cycle [ ] . less attention is given to why and how virus fitness results from the virus transmission success. for example, an animal virus may become established in the upper respiratory tract and transmit via aerosols to the next host. an enteric virus features a fecal-oral cycle. a skin virus may transmit on the basis of touch. a virus colonizing the distal urogenital tract may transmit during sexual contact. hence, an analysis of the virus transmission success requires a consideration of the overt clinical signs of infection, the gross pathology, the matching virus shedding profile, and of the ensuing modes of transmission. the current analysis explores how the virus molecular world and the macroscopic dimensions of the host environment are intertwined, integral to one and the same virus transmission ecology. in vertebrate hosts the more vital organ systems are shielded off from external aggressors, small or large. it may be assumed that also the host immune defenses are structured to ensure that harmful viral pathogens remain confined to the outer-body environment, the epithelia. epithelial viruses interface with the external environment and respond to the variation encountered here. opposingly, infiltrative viruses establish in the inner-body environment and are expected to evolve towards a more intimate virus-host relationship. given that the virus-host relationship changes with the position of the virus in the outer-to inner-body continuum the analysis focuses on the extent of virus host-body infiltration. the virus organ system tropism is assumed to evolve in harmony with the virus cell tropism. this may be inferred from the dichotomy in the release of viruses from epithelial cells. apart from direct cell-to-cell transmission [ ] , viruses may be released from the apical cell surface and so end-up in the outer-body environment. these viruses colonize mucosae and skin. in contrast, viruses released from the basolateral cell surface infiltrate underlying tissues. these viruses end-up in the lymph drainage, enter the blood circulation and so may infect any of the internal organs. infiltration of and establishment in the inner-body environment may translate in additional, non-epithelial transmission modes. for example, virus establishment in the reproductive organs may translate in intrauterine or lactogenic transmission [ ] . in birds, virus may be shed into yolk or albumen and so transmit vertically [ ] . virus circulation in the bloodstream may enable virus transmission via needles or arthropod vectors [ ] . taking an invasion ecology perspective, the host-body is viewed as a mosaic of organ systems. viruses and organ systems are virtually portrayed in an outer-to inner-body fashion, based on the outer-to inner-body shifts in the virus infection-shedding-transmission characteristics which, in turn, result from the shift in virus organ system tropism. it is assumed that the nature of the virus-host interaction changes with the position of the virus in the outer-to inner-body continuum. the study describes the transmission ecology of the world main livestock viruses. the rationale for selecting the world main livestock viruses relates to the host damaging effects of these pathogens, the overt clinical signs and gross pathology, translating in prominent virus shedding and obvious virus transmission modes. moreover, because of the major economic impact of these diseases, the causative viruses and the corresponding infection-transmission dynamics have been well studied. placed in a wider perspective the analysis builds on the growing perception that viruses deserve to be viewed as evolving living entities [ ] [ ] [ ] . as biological replicators viruses require a propagation strategy in order to become transmitted to the next host [ ] . for this, a virus may turn host damaging or instead evolve a friendly, persisting virus-host relationship [ ] . the analysis entailed an iterative process. as a first step, the one-to-three scores allocated to the livestock viruses for the four ecological variables were examined in more detail. the scores are shown in figure s b . the variables comprise the extent of virus host-body infiltration, the length of the infection period, the infection severity level, and the virus environmental survival rate. the one-to-three infiltration score reflects the organ systems involvement in infection-transmission and concerns, respectively, virus transmission based on the involvement of the epithelia, transmission involving epithelia and internal organs, and transmission involving just internal organs. the score for the length of the infection period reflects, respectively, acute, acute plus persistent, and persistent infections. likewise, the score for the infection severity level concerns a case fatality of less than one, one to ten, and above ten percent. the score for the virus environmental survival rate refers to the number of days that the virus remains infective outside the host-body, ranging from up to three, three to ten, to over ten days. with one exception, the variables did not increase or decrease in value together. just the association between the extent of virus host-body infiltration and the length of the infection period was found to be monotonic. spearman correlation yielded an r = . and p < . . it was thus found that viruses infiltrating internal organs either cause persistent infections or a combination of acute and persistent infections. conversely, persistent viruses either colonize internal organs or a combination of internal organs and epithelia. hence, the length of the infection period appears to present a measure for the extent of virus host-body infiltration. as a next step, the eleven virus families in the study were grouped and ranked a-d on the basis of the infiltration scores allocated to the individual family viruses, see figure . the transmission of the viruses belonging to the orthomyxoviridae and the paramyxoviridae was found to strictly result from the involvement of the epithelia. the transmission of the viruses belonging to the coronaviridae, the picornaviridae and the poxviridae was in part modulated also by the internal organ systems. the transmission of the viruses belonging to the arteriviridae, the flaviviridae, the herpesviridae, plus also the single infectious bursal disease virus (ibdv), resulted from epithelial modes as well as internal organ systems involvement. finally, the transmission of the single bluetongue virus (btv) plus the viruses belonging to the retroviridae family either reflected the involvement of epithelia plus internal organ systems or of just internal organ systems. a spearman correlation of the a-d virus family specific infiltration ranking and the length of the infection period scores yielded an r = . and p = . the result indicates that the interrelationships among virus families may be defined in ecological terms and that the virus families may be neatly lined up in an outer-to inner-body fashion, virtually. one to ten, and above ten percent. the score for the virus environmental survival rate refers to the number of days that the virus remains infective outside the host-body, ranging from up to three, three to ten, to over ten days. with one exception, the variables did not increase or decrease in value together. just the association between the extent of virus host-body infiltration and the length of the infection period was found to be monotonic. spearman correlation yielded an r = . and p < . . it was thus found that viruses infiltrating internal organs either cause persistent infections or a combination of acute and persistent infections. conversely, persistent viruses either colonize internal organs or a combination of internal organs and epithelia. hence, the length of the infection period appears to present a measure for the extent of virus host-body infiltration. as a next step, the eleven virus families in the study were grouped and ranked a-d on the basis of the infiltration scores allocated to the individual family viruses, see figure . the transmission of the viruses belonging to the orthomyxoviridae and the paramyxoviridae was found to strictly result from the involvement of the epithelia. the transmission of the viruses belonging to the coronaviridae, the picornaviridae and the poxviridae was in part modulated also by the internal organ systems. the transmission of the viruses belonging to the arteriviridae, the flaviviridae, the herpesviridae, plus also the single infectious bursal disease virus (ibdv), resulted from epithelial modes as well as internal organ systems involvement. finally, the transmission of the single bluetongue virus (btv) plus the viruses belonging to the retroviridae family either reflected the involvement of epithelia plus internal organ systems or of just internal organ systems. a spearman correlation of the a-d virus family specific infiltration ranking and the length of the infection period scores yielded an r = . and p = . the result indicates that the interrelationships among virus families may be defined in ecological terms and that the virus families may be neatly lined up in an outer-to inner-body fashion, virtually. next, the organ system tropisms of the viruses belonging to each family were collectively fitted and with the naked eye aligned with the figure line-up of families. for this, the within-group, alphabetical family order was adjusted to secure an optimal visual match. as indicated in figure , from outer-to inner-body the virus organ system appears to shift from the respiratory plus the alimentary tract to the skin, the distal urogenital tract or cloaca, the peripheral nerves and ganglia, the reproductive organs system, the lungs, to the immune plus the circulatory systems. hence, there are indications that both viruses and organ systems may be lined up in an outer-to inner-body fashion, virtually. next, the organ system tropisms of the viruses belonging to each family were collectively fitted and with the naked eye aligned with the figure line-up of families. for this, the within-group, alphabetical family order was adjusted to secure an optimal visual match. as indicated in figure , from outer-to inner-body the virus organ system appears to shift from the respiratory plus the alimentary tract to the skin, the distal urogenital tract or cloaca, the peripheral nerves and ganglia, the reproductive organs system, the lungs, to the immune plus the circulatory systems. hence, there are indications that both viruses and organ systems may be lined up in an outer-to inner-body fashion, virtually. next, the a-d family ranking was converted into a one-to-four virus infiltration score applicable to individual viruses. further, these scores are shown in supplementary figure s b. to make the scoring compatible with the a-d family ranking, the one-to-four scores reflects, respectively, virus transmission strictly based on epithelial modes, primarily based on epithelial modes, involving epithelia and internal organ systems, and primarily involving internal organ systems. there are several differences with the a-d family ranking shown in figure . among the viruses of family group b, tgev, aev, fmdv, and lsdv received a score of three for transmitting on the basis of the involvement of both epithelia and internal organs. pev and svdv of group b were considered primarily epithelial and so received a two score. both these viruses are persistently shed in feces, including in the absence of clinical signs, indicating a systemic infection component. the herpesviruses of group c were split into two. bhv- , dev, ehv- , and gahv were considered primarily epithelial while ehv- , gahv- , and shv- were considered to involve epithelia and internal organs. among the d group viruses alv was considered to involve epithelia and internal organs, unlike caev, jsrv, and mvv, for which the involvement of the epithelia did not appear to contribute to the overall virus transmission success. the latter viruses were allocated a score of four. when the new, one-to-four virus infiltration scores were matched to the scores for the length of the infection period spearman r became . , and p = . when the somewhat atypical, vector borne next, the a-d family ranking was converted into a one-to-four virus infiltration score applicable to individual viruses. further, these scores are shown in supplementary figure s b. to make the scoring compatible with the a-d family ranking, the one-to-four scores reflects, respectively, virus transmission strictly based on epithelial modes, primarily based on epithelial modes, involving epithelia and internal organ systems, and primarily involving internal organ systems. there are several differences with the a-d family ranking shown in figure . among the viruses of family group b, tgev, aev, fmdv, and lsdv received a score of three for transmitting on the basis of the involvement of both epithelia and internal organs. pev and svdv of group b were considered primarily epithelial and so received a two score. both these viruses are persistently shed in feces, including in the absence of clinical signs, indicating a systemic infection component. the herpesviruses of group c were split into two. bhv- , dev, ehv- , and gahv were considered primarily epithelial while ehv- , gahv- , and shv- were considered to involve epithelia and internal organs. among the d group viruses alv was considered to involve epithelia and internal organs, unlike caev, jsrv, and mvv, for which the involvement of the epithelia did not appear to contribute to the overall virus transmission success. the latter viruses were allocated a score of four. when the new, one-to-four virus infiltration scores were matched to the scores for the length of the infection period spearman r became . , and p = . when the somewhat atypical, vector borne bluetongue virus was removed from the correlation, r remained . for the one-to-four scoring, became . for the a-d virus family specific ranking, and . , with p = × − , for the one-to-three infiltration ranking. next, all of the above findings were considered in conjunction with the literature data on the transmission ecology collated for each of the viruses in figure s a . pieced together on this basis was an outer-to inner-body line-up of viruses by organ system or combination of organ systems, guided by the one-to-four virus infiltration score, the corresponding virus organ system tropism, the matching virus transmission modes, length of the infection and shedding periods, infection severity level, and virus environmental survival rate, see figure and, also, figure s d . bluetongue virus was removed from the correlation, r remained . for the one-to-four scoring, became . for the a-d virus family specific ranking, and . , with p = × − , for the one-to-three infiltration ranking. next, all of the above findings were considered in conjunction with the literature data on the transmission ecology collated for each of the viruses in figure s a . pieced together on this basis was an outer-to inner-body line-up of viruses by organ system or combination of organ systems, guided by the one-to-four virus infiltration score, the corresponding virus organ system tropism, the matching virus transmission modes, length of the infection and shedding periods, infection severity level, and virus environmental survival rate, see figure and, also, figure s d . for the epithelial, outer-body viruses it turned out that the length of the infection and shedding periods, as well as the virus environmental survival rate generally increased from respiratory tract to alimentary tract to skin. the respiratory viruses transmitted on the basis of aerosols, direct contact or fomites. alimentary tract viruses were found to transmit on the basis of a fecal-oral cycle, through direct contact, contamination of feed and water, or involving fomites, persons and vehicles. viruses infecting both respiratory and alimentary tract featured a mix of these transmission modes. mostly, these viruses caused rather severe infections. among the skin viruses, the more infiltrative viruses affecting all layers of the skin caused slowly healing lesions. the transmission of these deep-rooted skin viruses was found to rely on abrasion or biting flies rather than on direct touch or on indirect contact, more typical for superficial skin lesions. some of the epithelial viruses are shed in feces over a prolonged time period, also in the absence of clinical signs, and these infections were considered to feature a systemic component. next, the epithelial herpesviruses establishing latently in peripheral nerves and ganglia were found to cause a recurrence or persistence of the mucosal and/or skin infection, including of the distal urogenital tract and external genitalia. virus infiltration of the inner-body environment frequently implicated the genital tract or reproductive system in general. this was found to be the case for the rna, the dna and for the retroviruses in the study. virus establishment in the reproductive system translated in seminal transmission, haphazard abortion, late term abortion, stillbirth, birth of infected, yet apparently healthy offspring or, also, lactogenic transmission. the vertical transmission modes were common among the utmost deep-rooted viruses, the viruses infiltrating also the immune and circulatory systems. some of the utmost infiltrative viruses featured an absence of epithelial transmission modes and were environmentally labile. virus infiltration of the immune system associated with immune-suppression, severe infections, neoplasia, or instead with in-apparent, persistent infection. virus infiltration of immune and circulatory systems associated with iatrogenic transmission modes. virus circulation in the bloodstream facilitated arthropod borne transmission. as indicated in figure , the transmission of the bluetongue virus, the sole arbovirus in the study, was considered somewhat atypical because the virus usually causes a transient infection in the ruminant host while in midges remains infective for life. hence, the involvement of the biological vector complicates a direct comparison with the transmission ecology of the remaining viruses. the finding that virus environmental survival in the outer-body environment increased from respiratory tract to alimentary tract to skin and decreased with the shift from the outer-to the inner-body environment prompted a re-examination of the relationship between the extent of virus host-body infiltration and virus environmental survival. virus infiltration scores two-to-four, running from primarily epithelial transmission, to transmission involving also internal organ systems, to transmission primarily involving internal organ systems, were matched to the one-to-three virus environmental survival rate scores, yielding an r = − . and p = < . . the indication that at least in broad terms the extent of virus inner-body infiltration correlated with a loss of virus robustness was applied in the virus ranking, along with the other factors. furthermore, the outer-to inner-body shifts in virus host tropism and virus genome were examined. underlined in figure are ruminant and equine viruses, contrasted to the remaining, poultry and pig viruses. excluded from the host tropism correlations was the multiple-host fmdv. the remaining viruses all formed part of either of the two virus host groupings. it was found that the rna, dna and retroviruses broadly line up in an outer-to inner-body fashion. virus host tropism and the virus genome line-up were matched. additional correlations concerned the virus host tropism and the four virus ecological variable scores, as well as the virus genome type and the four virus ecological variable scores. the extent of the host-body infiltration was found to increase from rna to dna to retrovirus, with r = . and p < . . from rna to dna to retrovirus the ruminant and equine viruses gained in prominence, with r = . and p < . , and the infection severity level decreased, with r = − . and p < . . moreover, the ruminant and equine viruses were found to cause less severe infections than the poultry and pig viruses, with spearman r = − . and p < . . hence, from outerto inner-body, the virus genome type and host tropism appear to shift in concert, along with the infection-transmission dynamics. the synthesis of the findings is presented in figure . the host environment frames the virus transmission modes and, with it, explains the organ system involvement, the specifics of the host-cell infection cycle, and the virus genome. vice versa, the virus life history explains the virus genomics, the host-cell infection cycle and, with it, the macroscopic level virus-host interactions and the host population ecology. the interplay of the host environment-transmission modes-organ system involvement-host cell infection cycle-virus genome changes from outer-to inner-body, resulting in two opposite virus evolution pathways, respectively for generalist and specialist type viruses. the extent of the host-body infiltration was found to increase from rna to dna to retrovirus, with r = . and p < . . from rna to dna to retrovirus the ruminant and equine viruses gained in prominence, with r = . and p < . , and the infection severity level decreased, with r = − . and p < . . moreover, the ruminant and equine viruses were found to cause less severe infections than the poultry and pig viruses, with spearman r = − . and p < . . hence, from outer-to inner-body, the virus genome type and host tropism appear to shift in concert, along with the infection-transmission dynamics. the synthesis of the findings is presented in figure . the host environment frames the virus transmission modes and, with it, explains the organ system involvement, the specifics of the host-cell infection cycle, and the virus genome. vice versa, the virus life history explains the virus genomics, the host-cell infection cycle and, with it, the macroscopic level virus-host interactions and the host population ecology. the interplay of the host environment-transmission modes-organ system involvement-host cell infection cycle-virus genome changes from outer-to inner-body, resulting in two opposite virus evolution pathways, respectively for generalist and specialist type viruses. implied by figure is that the crowding conditions observed in poultry and in pig husbandry tend to attract horizontally transmitting respiratory and enteric viruses. the pathogenicity level of the viruses evolves to match the dynamics in host abundance and contact rate. at the molecular level, these rna viruses become released from the apical surface of epithelial cells directly into the implied by figure is that the crowding conditions observed in poultry and in pig husbandry tend to attract horizontally transmitting respiratory and enteric viruses. the pathogenicity level of the viruses evolves to match the dynamics in host abundance and contact rate. at the molecular level, these rna viruses become released from the apical surface of epithelial cells directly into the outer-body environment. thus, proliferative virus replication, generalized infection of respiratory plus enteric mucosae, profuse virus shedding, and swift onward transmission all go hand-in-hand. a diametrically opposite scenario is given by relatively stable host environments observed in ruminant and equine husbandry, with parent stock and their young grazing together in the open, not unlike wild herbivore ecologies. the viruses attracted and selected for establish in the vital inner-body systems and transmit vertically, via needles or via bloodsucking arthropods. at the molecular level, virus establishment in the vital body systems is matched by low replication rates and minor or slowly evolving host damage. the utmost infiltrative viruses in the study are the retroviruses. in addition, some of other rna viruses are deep-rooted. the dna viruses in the study take an intermediary position. it has been established that epithelial viruses are highly evolvable, more so than inner-body viruses [ ] . epithelial viruses are responsive to the dynamics in the environment external to the host-body. this may be illustrated on the basis of the genetically related virus pairs in the study. for example, the influenza virus circulating in horses (eiv) generates a transient, dry cough supporting swift virus transmission via aerosols [ ] . in pigs, the virus (siv) causes coughing and sneezing, resulting from significant mucus production [ ] . the virus transmits on the basis of close direct contact, in line with the social behavior and body size of pigs. the rinderpest virus (rpv) in cattle and buffaloes primarily colonizes the alimentary tract and transmits on the basis of direct muzzle-to-muzzle contact [ ] . in small ruminants, the identical peste des petits ruminants virus affects also the respiratory tract and transmits also via aerosols. likewise, the lumpy skin disease virus (lsdv) in cattle causes persistent, deep, necrotic skin plugs and transmits via biting insects, mechanically. in sheep and goats, the virus (sgpv) causes transient lesions [ ] . the caprine arthritis-encephalitis virus (caev) and the maedi-visna virus (mvv) present an example of closely related lentiviruses establishing in the inner-body organ systems of sheep and goats. the viruses display overlap in host tropism and both transmit mainly vertically via colostrum and milk. the difference between the two viruses mainly concerns the differential inner-body virus organ system tropism. projected on a long evolutionary timescale, inner-body viruses tend to become locked in within the host body. this internalization may turn progressive when the epithelial transmission modes are being replaced by internal organ system-based modes. virus establishment in the reproductive system translates in vertical transmission, in turn enhancing virus-host co-evolution [ ] . virus infiltration of also immune and circulatory systems may yield in-apparent, persistent infections, indicating low levels of pathogenicity and/or enhanced host tolerance. the division between virus and host may become blurred and given enough time the two may become one [ ] . the nature of species jumps differs between generalist and specialist type viruses. for example, an opportunistic, epithelial virus of wildlife origin is likely to be found circulating in livestock before becoming first detected in humans as host. this has been the case for influenza [ ] , henipah [ ] and mers corona viruses [ ] . further, the sars corona virus infected civet cats raised as food animals before appearing in humans as host [ ] . in contrast, more infiltrative viruses establish in the vital inner-body systems. specialist viruses circulating in the bloodstream of non-human primates may directly jump to humans as host, as a result of complex ecological, socio-economic, demographic and other drivers. examples comprise hiv-aids [ ] , chikungunya [ ] , and zika viruses [ ] . hence, knowing how species jumps differ for the different host ecologies may assist the study of pandemic risks. a subtotal of livestock viruses of global animal health significance was extracted from the oie-listed diseases, infections and infestations in force in [ ] . livestock infections and diseases resulting from virus spill-over from wildlife were excluded from the analysis. the common livestock hosts, described in the colloquial oie terminology, comprise horses, donkeys, cattle, buffaloes, sheep, goats, swine, chicken, turkeys, ducks, and geese. the total of livestock viruses belong to eleven different families and form a mix of rna (n = ), dna (n = ), and retroviruses (n = ). shown in figure s a for each of the viruses are the virus family, virus genomic architecture, virus name in full, abbreviated, and the common names given to the infection or disease. also presented is a brief summary on the transmission ecology for each virus, with references to the primary livestock host, the virus organ system tropism, the length of the infection and shedding period, the infection severity level, the transmission modes, and the virus environmental survival rate. presented in figure s b are one-to-three scores allocated to the viruses for four ecological variables. the variables comprise the extent of virus host-body infiltration, the length of the infection period, the infection severity level, and the virus environmental survival rate. the one-to-three infiltration score reflects the organ system involvement in infection-transmission and concerns, respectively, virus transmission based on the involvement of the epithelia, transmission involving epithelia and internal organs, and transmission involving just internal organs. also shown is a one-to-four virus infiltration score, an outcome of the iterative analysis process and reflecting, respectively, virus transmission strictly based on epithelial modes, primarily based on epithelial modes, involvement of epithelia and internal organ systems, and of primarily internal organ systems. the score for the length of the infection period reflects, respectively, acute, acute plus persistent, and persistent infections. likewise, the score for the infection severity level concerns a case fatality of less than one, one to ten, and above ten percent. the score for the virus environmental survival rate refers to the number of days that the virus remains infective outside the host body, ranging from up to three, three to ten, to over ten days. also indicated in figure s b is the virus host range as observed in both livestock and wildlife. figure s c lists the literature sources on which figure s a ,b is based. the analysis concerned an iterative process. as a first step, the one-to-three scores allocated to the viruses for the four ecological variables were examined in more detail and the monotonic associations subjected to spearman correlation. just the scores for the virus host-body infiltration and for the length of the infection period were found to increase in value together. next, given the coarse match between virus infiltration and persistence, it was examined how this relationship played out at the virus family level. for this, the eleven virus families in the study were grouped and ranked a-d on the basis of the one-to-three infiltration scores allocated to the individual family viruses. also, this a-d infiltration ranking was held against the length of the infection period scores. next, given the indication, from the above, that the virus families may be neatly lined up in an outerto inner-body fashion, it was examined how the organ system tropisms of the family viruses aligned with it. for this, the organ system tropisms of the viruses belonging to each family were collectively fitted and with naked eye aligned with the a-d family groups. for this, the alphabetical family order within the family groups was abandoned in order to obtain an optimal visual match. the result confirms that also the organ systems may be lined up in an outer-to inner-body fashion, virtually. next, the a-d family ranking was converted into a one-to-four virus infiltration score applicable to individual viruses, as described in section . , and also these scores were matched to the length of the infection period scores. next, since the infiltration-persistence match for the individual viruses was found to be about as strong as for the virus families, the viruses were individually lined up in an outer-to inner-body fashion, irrespective the family origin, strictly on ecological grounds. for this, all of the above obtained results were considered in conjunction with the literature data on the transmission ecology collated for each of the viruses in figure s a . pieced together on this basis was an outer-to inner-body line-up of viruses by organ system or combination of organ systems, guided by the one-to-four virus infiltration score, the corresponding virus organ system tropism, the matching virus transmission modes, length of the infection and shedding periods, infection severity level, and virus environmental survival rate. the finding that virus environmental survival in the outer-body environment increased from respiratory tract to alimentary tract to skin and decreased with the shift from the outer-to the inner-body environment prompted a re-examination of the relationship between the extent of virus host-body infiltration and virus environmental survival. virus infiltration scores two-to-four, running from primarily epithelial transmission, to transmission involving also internal organ systems, to transmission primarily involving internal organ systems, were found to match with the one-to-three virus environmental survival rate scores. the indication that, at least in broad terms, the extent of virus inner-body infiltration correlated with a loss of virus robustness was applied in the virus ranking, along with the other factors. next, furthermore examined were the outer-to inner-body shifts in virus host tropism and virus genome. for this, the ruminant plus equine viruses were contrasted to the poultry plus pig viruses. excluded from the host tropism correlations was the multiple host fmdv. the remaining viruses all formed part of either of the two host groupings. it was found that rna, dna and retroviruses broadly line up in an outer-to inner-body fashion. virus host tropism and the virus genome line-up were matched. additional correlations concerned virus host tropism and the four virus ecological variable scores, as well as virus genome type, and the four virus ecological variable scores. it was found that from outer-to inner-body, the virus genome type and host tropism appear to shift in concert, along with the infection-transmission dynamics. the collective results above served the compilation of the predictive framework for animal virus evolution shown in figure , discussion section. the online rho calculator https://www.socscistatistics.com/tests/spearman/default.aspx was used for the spearman correlations. this software has been audited by established statistics packages. virus ecology: a gap between detection and prediction. emerg. microbes infect. , , e virus-host symbiosis mediated by persistence viruses are essential agents within the roots and stem of the tree of life the evolutionary history of vertebrate rna viruses move over, bacteria! viruses make their mark as mutualistic microbial symbionts virus taxonomy: the database of the international committee on taxonomy of viruses (ictv) direct cell-to-cell transmission of respiratory viruses: the fast lanes preventive and therapeutic strategies for bovine leukemia virus: lessons for htlv detection of avian leukosis virus in albumen of chicken eggs using reverse transcription polymerase chain reaction nosocomial transmission of dengue fever via needlestick. an occupational risk what ecologists can tell virologists predicting virus emergence amid evolutionary noise life history determines genetic structure and evolutionary potential of host-parasite interactions are viruses alive? the replicator paradigm sheds decisive light on an old but misguided question the good viruses: viral mutualistic symbioses cell tropism predicts long-term nucleotide substitution rates of mammalian rna viruses equine influenza (infection with equine influenza virus) in world organisation for animal health (oie) manual of diagnostic tests and vaccines for terrestrial animals, oie technical disease cards, swine influenza. oie technical disease cards, rinderpest. oie technical disease cards, sheep pox and goat pox (oie, ) transmission modes and evolution of the parasitism-mutualism continuum on the concept and elucidation of endogenous retroviruses spatiotemporal distribution and evolution of the a/h n pandemic influenza virus in pigs in france from to : identification of a potential swine-specific lineage transmission of henipaviruses mers coronavirus: diagnostics, epidemiology and transmission beyond the cut hunter: a historical epidemiology of hiv beginnings in central africa a scoping review of published literature on chikungunya virus zika virus: history, emergence, biology, and prospects for control oie-listed diseases, infections and infestations in force in world health organization in animal health yearbook fao-oie-who this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: i am grateful to epke le rütte, lenny hogerwerf, anneke engering, marjan leneman, jelle bruinsma, dorothea van ooyen and marleen slingenbergh for discussions. the author declares no conflict of interest. sponsors had no role in the design, execution, interpretation, or writing of the study. key: cord- -e oxvgar authors: webber, quinn m. r.; willis, craig k. r. title: sociality, parasites, and pathogens in bats date: - - journal: sociality in bats doi: . / - - - - _ sha: doc_id: cord_uid: e oxvgar little is known about the ecology of many of the parasites and pathogens affecting bats, but host social behavior almost certainly plays an important role in bat-parasite dynamics. understanding parasite dynamics for bats is important from a human public health perspective because of their role as natural reservoirs for recent high-profile emerging zoonotic pathogens (e.g. ebola, hendra) and from a bat conservation perspective because of the recent emergence of white-nose syndrome (wns) in north america highlighting the potential population impacts of parasites and pathogens. although some bat species are among the most gregarious of mammals, species vary widely in terms of their social behavior and this variation could influence pathogen transmission and impacts. here, we review the literature on links between bat social behavior and parasite dynamics. using standardized search terms in web of science, we identified articles that explicitly tested or discussed links between some aspect of bat sociality and parasite transmission or host population impacts. we identified social network analysis, epidemiological modeling, and interspecific comparative analyses as the most commonly used methods to quantify relationships between social behavior and parasite-risk in bats while wns, hendra virus, and arthropod ectoparasites were the most commonly studied host-parasite systems. we summarize known host-parasite relationships in these three systems and propose testable hypotheses that could improve our understanding of links between host sociality and parasite-dynamics in bats. parasitism is ubiquitous in nature. parasites affect fitness of their hosts and thus can shape host population dynamics. defined broadly, a parasite is any organism that grows, feeds, and/or is sheltered at the expense of another organism (i.e. the host). parasites can be categorized into different ways but one common approach is to differentiate microparasites, which are typically unicellular or multicellular microbes (e.g. bacteria, prions, viruses, protozoans, fungi) with short generation times and a life cycle that occurs entirely on/in the host, from macroparasites which are multicellular parasites with longer generation times and more complex life cycles that may include multiple host species (table . ; anderson and may ; hudson et al. ) . many microparasites can be pathogenic and cause identifiable disease in their hosts with recognizable physiological or behavioral signs or symptoms (table . ). for example, batrachochytrium dendrobatiidis is a fungal microparasite of amphibians that infects keratinized tissues, thickens the epidermis, and eventually causes mortality, all of which are identifiable signs of the associated disease, chytridiomycosis (voyles et al. ; rosenblum et al. ) . macroparasites tend to result in chronic infections of their definitive hosts, decrease host fecundity, and usually cause morbidity rather than mortality (hudson et al. ) . experimental infection of great tits (parus major) with the hen flea (ceratophyllus gallinae) resulted in reduced reproductive success via increased nest failure during incubation and the nestling period (fitze et al. ) . parasite dynamics and impacts are often quantified using two metrics: intensity (i.e. load) and prevalence. intensity quantifies the number of infections per individual host within a given population, while prevalence is measured as a proportion of infected individuals within a sample from a given host population (table . ). understanding variation in parasite prevalence and intensity can thus be important for making inferences about the evolution of social behavior and the potential fitness consequences associated with parasite infection. although empirical data from wild host populations are surprisingly scarce, host behavior is considered an important predictor of parasite intensity, prevalence, and impacts (moore ) . a longstanding hypothesis in parasite ecology predicts an influence of the host social system, and the duration and frequency of social contacts within host populations, on parasite prevalence or intensity (loehle ; altizer et al. ) . social systems, defined as groups of conspecifics that regularly interact more frequently with one another than with members of other groups, represent the highest level of sociality, while social organization and social structure describe the size, composition, and spatiotemporal distribution and cohesion of social systems (table . ; whitehead ) . social systems can be further subdivided into two categories: colonies and aggregations, and this distinction is important for understanding host-parasite dynamics in bats. colonies are groups of individuals that may or may not be genetically related but which exhibit non-random patterns of association, and frequent close contact with each other (kerth ) . it is often assumed that groups of bats roosting in a common structure represent a colony but often such groups may not meet the colony definition and, instead, represent aggregations. aggregations are defined as assemblages of individuals that happen to occur in a shared environment at the same time, perhaps due to an attraction to that environment rather than social bonds with other individuals (table . ). variation in social dynamics within and between colonies and aggregations can mediate host-parasite dynamics (webber et al. ). host an animal or plant on which a parasite lives. martin ( ) reservoir host (ecological definition) hosts that do not exhibit clinical disease as a result of infection. nunn and altizer ( ) reservoir host (medical definition) hosts that serve as a source of infection and potential reinfection of people and sustain parasite populations when humans are not available. martin ( ) parasite (ecological definition) any organism that lives on and draws nutrients from another living organism (the host), usually to the host's detriment. nunn and alitzer ( ) parasite (medical definition) an organism that grows, feeds, and is sheltered on (ectoparasite) or in (endoparasite) a different organism while contributing nothing to survival of its host. martin ( ) microparasite pathogens, or disease-causing microbes (viruses, bacteria, protozoa, fungi). anderson and may ( ) macroparasite multicellular parasites (helminthes, arthropods, most ectoparasites) may and anderson ( ) disease (ecological definition) pathology caused by infection, including outward physical signs and internal or behavioral changes. nunn and alitzer ( ) disease (medical definition) a disorder with a specific cause (may or may not be known) and recognizable signs and symptoms. martin ( ) pathogen disease-causing agent. nunn and alitzer ( ) virulence (ecological definition) disease-induced host mortality and/or reductions in fecundity nunn and alitzer ( ) virulence (medical definition) the disease-producing (pathogenic) ability of a microorganism. martin ( ) infection invasion of the body by harmful organisms (pathogens), such as bacteria, fungi, protozoa, or viruses. martin ( ) prevalence a measure of morbidity based on current levels of disease in a population; measured as a proportion (i.e. number of infections divided by number of individuals). martin ( ) intensity a measure of morbidity based on current levels of disease in a population; measured as an absolute number (i.e. the total number of infections). martin ( ) epidemiology the science concerned with the study of the factors determining and influencing the frequency and distribution of disease in a defined human or animal population. martin ( ) relationships between host social systems and parasite dynamics are complex, but several mechanisms are predicted to influence these patterns and are likely important for bats. for example, hosts that occur in large, high-density colonies are predicted to have more frequent interactions resulting in more opportunities for parasite transmission (stanko et al. ; tompkins et al. ) . dense aggregations may result in contacts that are fewer and shorter in duration than those occurring in colonies, but are still likely to provide more opportunities for parasite transmission than might occur for solitary bats or those in very small colonies. this variation in host density also has the potential to influence one of the most fundamental parameters of disease ecology, the basic reproduction number or basic reproductive ratio (r ). r is an important metric of parasite fitness typically defined, for microparasites, as the number of secondary infections caused by an infectious individual in an entirely susceptible population or, for macroparasites, the number of female larvae established from a single female worm (hudson et al. ) . when r > infection persists within the host population and when r < infection cannot become established (perkins et al. ) . variation in social behavior can impact r if certain individual hosts disproportionately infect a large an individual's reaction to a novel object or situation. réale et al. ( ) number of conspecifics, and thus inflate r above the persistence threshold of one (lloyd-smith et al. ) . although empirical data from numerous vertebrates (e.g. ungulates: ezenwa ) and invertebrates (e.g. bees: otterstatter and thomson ) support theoretical relationships between parasitism and sociality, there are few data for most host-pathogen systems involving wild mammals, including bats. bats are among the most ecologically diverse of mammals with an enormous range of social systems (kunz and lumsden ; kerth ; johnson et al. ) . for example, colony or aggregation sizes of bats range from completely solitary to millions of individuals, while social systems range from small, closed societies with potentially long-term social bonds (e.g. thyroptera tricolor: chaverri ) to enormous, likely passive aggregations of individuals attracted to high-quality habitats (e.g. myotis lucifugus during autumn swarming : fenton ) . social behavior in bats presumably evolved in response to the costs and benefits associated with close conspecific contact. benefits of social roosting may be numerous and include social thermoregulation, cooperative behavior, and information transfer. many temperate bats rely on social thermoregulation to decrease energy expenditure during periods of energy limitation, such as pregnancy and lactation (e.g. eptesicus fuscus: willis and brigham ) , while some tropical species also appear to exploit social thermoregulation (e.g. uroderma bilobatum: lewis ) . the evolution of cooperative behavior in bats was likely facilitated by strong female philopatry and stable group structure (emlen ) . vampire bats (desmodus rotundus) are well known for their cooperative behavior (i.e. reciprocal altruism) and females rarely transfer between groups (wilkinson ; carter and wilkinson ) . this combination suggests an evolutionary scenario, where stable group structure ultimately led to selection favoring cooperative behavior. information transfer about predation risk (e.g. kalcounis and brigham ) and high-quality foraging sites (e.g. mccracken and bradbury ) are also commonly cited as potential benefits of sociality for bats. despite these potential benefits of social behavior, risk of infection with microand macroparasites is thought to represent a potentially pronounced fitness cost of being social (côté and poulin ) . this cost is illustrated most obviously by the recently emerged infectious disease white-nose syndrome (wns), which is caused by the fungal microparasite pseudogymnoascus destructans (blehert et al. ). wns has resulted in catastrophic declines of temperate hibernating bats in north america (frick et al. ) and prompted urgent conservation and management attention fenton ) . p. destructans is an invasive pathogen that appears to have evolved with bats from the old world, where it does not cause mortality of infected hosts, and to date, is known to occur on at least hibernating bat species (puechmaille et al. ; zukal et al. ) . p. destructans grows in exposed skin of the muzzle, ears, and wing membranes of bats during hibernation (blehert et al. ; warnecke et al. ) . for north american species, infection with p. destructans causes an increase in energy expenditure (verant et al. ) and arousal frequency (boyles and willis ; reeder et al. ; warnecke et al. ) which lead to premature depletion of fat stores during hibernation. although the mechanism inducing increased energy expenditure and arousals by infected bats is still not fully understood (for review see willis ) , variation in social behavior could mediate fungal transmission and growth, especially since affected species tend to hibernate in large colonies or aggregations in caves or mines. understanding host-parasite dynamics in the context of social behavior for wns is therefore important from a conservation perspective. in addition to serious conservation threats for some species, bats also appear to be reservoir hosts for a number of emerging infectious diseases (eids) of public health concern (luis et al. ; plowright et al. ) . reservoir hosts tend not to exhibit clinical disease as a result of infection (baker et al. ) and reservoir host populations may therefore provide large pools of infected hosts that could facilitate spillover events to heterospecifics, including humans, livestock, or pets (luis et al. ; plowright et al. ) . interestingly, their apparent ability to tolerate infection with a wide diversity of viral parasites is one factor supporting the recent hypothesis that bats are 'special' with respect to their propensity to host zoonotic microparasites (luis et al. ; brook and dobson ) . the recent identification of a number of viral, protozoan, and bacterial microparasites in bats supports this hypothesis and has prompted significant analysis and discussion about bats as natural hosts to microparasites associated with eids of humans or livestock luis et al. ; olival and hayman ; veikkolainen et al. ) . for example, recent evidence suggests that bats host more zoonotic viruses per species compared to rodents, and human encroachment into bat habitats, particularly in the tropics, could facilitate spillover events (daszak et al. ; luis et al. ) . several hypotheses have been proposed to explain the apparent zoonotic potential of bats. enormous variation in body temperature (t b ) and metabolic rate (mr) between rest and sustained flight in many heterothermic bats could reduce fitness or pathogenicity for many viral parasites (o'shea et al. ) . interestingly, widespread torpor expression in bats appears to reduce the likelihood of hosting zoonotic viruses, possibly because viral replication decreases as a result of reduced t b and mr during torpor (luis et al. ; stawski et al. ). this hypothesis suggests physiological tolerance as a mechanism allowing bats to serve as reservoir hosts, but evidence linking host sociality as an additional mechanism is limited . despite the fundamental evolutionary importance, and conservation and public health significance of bat-parasite interactions, studies of the influence of bat sociality on parasite transmission and acquisition are limited, especially for microparasites. here, we review the literature on relationships between social behavior of bats and their associated parasites. first, we provide an overview of the tools, techniques, and methodologies that have been used to quantify relationships between sociality and parasitism in bats, as well as relevant techniques that have been used for other vertebrates and which could be applied to bats. second, we summarize the role of sociality in three relatively well-studied bat-parasite systems: wns, hendra virus (hev), and arthropod macroparasites. finally, we propose testable hypotheses and observational and experimental studies important for understanding the influence on parasite dynamics of two important concepts in behavioral ecology of bats: fission-fusion social organization and individual behavioral tendencies (i.e. personality). we conducted an extensive search of the literature and compiled a list of articles based on combinations of key word searches on web of science. we searched the term "chiroptera" and "bat" with every possible combination of social*, gregarious*, colony, aggregation, fission-fusion (i.e. a common type of bat social system, see below), viral, pathogen, disease, ectoparasite*, endoparasite*, parasite*, infection* and epidemiology*. our initial search yielded unique articles but we eliminated articles that did not explicitly quantify or discuss a link between some aspect of sociality and parasite risk. this left only articles that fully satisfied our search criteria. four of these used comparative analyses to examine effects of species-specific socioecological traits on parasite risk (table . ), addressed sociality in the context of microparasites (table . ), and addressed sociality in the context of macroparasites (table . ). the studies we identified employed a range of methodologies to quantify links between sociality and parasite risk (tables . , . and . ). methods included social network analysis (n = ), epidemiological modeling (n = ), and interspecific comparative analyses (n = ). details about the underlying theory and the implementation of these methods is available elsewhere (e.g. social network analysis: croft et al. ; epidemiological modeling: may ; comparative analyses: garland et al. ) , and here we focus on the progress that has been made to date in identifying knowledge gaps where future research on bat-parasite dynamics could be focused. social network analysis is based in mathematical graph theory and has been widely applied in human sociobiology, since the s (e.g. cartwright and harary ; wasserman and faust ) . for studies of wildlife, network analysis was first applied in primate sociobiology (e.g. sade and dow ) , and has recently been applied more broadly to many other taxa (e.g. fewell ; hamede et al. ; drewe ) , including bats (for review see johnson et al. ) . networks consist of nodes (individuals or locations) and edges (interactions between nodes) through which a variety of individual and group level metrics can be quantified (wey et al. ) . the roles and importance of individuals or locations can then be assessed based on these metrics (see croft et al. for review). for example, individuals with certain combinations of traits can influence network dynamics by potentially acting as intermediaries connecting smaller subgroups within larger groups (wey et al. ; krause et al. ). in addition, bipartite networks (or two-mode networks) can be constructed to assess associations between individuals and ecologically relevant locations (e.g. a population of organisms and their nesting sites). network metrics are especially useful because they can be used as predictor variables for relevant-dependent variables (e.g. infection status or parasite intensity) in standard statistical models (e.g. general linear models). network analysis has allowed disease ecologists and parasitologists to make important strides quantifying how non-random social interactions affect parasite transmission and dynamics (for review see godfrey ). network analyses have become increasingly popular for studies of bats (johnson et al. ) . for instance, network analyses have been applied in a bat-habitat management context by identifying critical roosting locations that serve as 'hubs' within a roost network and simulating the consequences of removing those key sites for stability of the social group (rhodes et al. ; silvis et al. ) . network analyses have also been used to identify and quantify fission-fusion behavior within bat colonies (fortuna et al. ; patriquin et al. ; kerth et al. ; johnson et al. ). in the context of disease ecology, so far only two studies have connected social networks with epidemiological models to make inferences about host-pathogen dynamics (e.g. fortuna et al. ; webber et al. ) . fortuna et al. ( ) showed that giant noctule bats (nyctalus lasiopterus) form highly modular, fission-fusion colonies, and used an epidemiological model, parameterized using network metrics, to show that this arrangement reduces the spread of information or disease within the population because the colony was effectively segregated into modules divided among many roost trees (n = trees for bats: fortuna et al. ). for big brown bats (eptesicus fuscus), webber et al. ( ) recently showed that network structure depends on the habitat context. although tree-roosting e. fuscus reuse tree hollows between years (willis et al. ) , they frequently switch roosts within years (on average every . days: willis and brigham ) and rarely return to the same roost within a given summer. in building roosts, however, e. fuscus switch much less frequently and commonly return to the same roost repeatedly within the same year (ellison et al. ; webber et al. ) . webber et al. ( ) applied epidemiological models to social networks constructed based on these patterns of roosting behavior in forests versus buildings and found that parasite dynamics should vary between these habitat contexts with more rapid pathogen dissemination within building roosting colonies. constructing epidemiological models from empirical data undoubtedly improves inference for predicting epidemic outcomes, but as yet there are no studies that quantify real-world relationships between the social behavior of bat hosts and their parasites to test predictions of epidemiological models (table . ). although this form of data collection can be labor-intensive and potentially expensive (craft and caillaud ) , there are numerous examples of network analysis being used to integrate information on host-contact patterns and parasitism in systems with highly gregarious hosts. for example, network analysis elucidated the role of meerkat (suricatta suricata) allogrooming as a predictor for the prevalence of myobacterium tuberculosis, the microparasite that causes tuberculosis (drewe ) . quantifying association patterns of meerkats was largely conducted via focal animal observation, a type of data collection that is nearly impossible for free-ranging bats. thus, advances in technology, such as passive transponders (pit tags), data-logging telemetry, and/or proximity data loggers may be useful for quantifying association patterns and constructing social networks for bats with implications for epidemiology and disease ecology (e.g. willis and brigham ; patriquin et al. ; kerth et al. ; johnson et al. ). although empirical data are sparse, host-parasite dynamics, and the impacts of parasites on host populations have a rich theoretical history founded on anderson and may's ( ) seminal models. their classic microparasite epidemiological model conceptualizes host population dynamics in terms of susceptible (s), exposed (e), infected (i), and resistant/recovered (r) population pools, and these pools can be combined in a range of ways depending on the nature of host-pathogen interactions in the wild (e.g. si, sis, sir, seir models). in a standard sir model, individuals transition from s to i as a function of transmission rate (β) and from i to r as a function of survival of infection (v), while birth (a), and death rates (b, a + b for infected hosts) of each pool drive overall population dynamics (fig. . ; anderson and may ) . since being developed, sir epidemiological models have been widely used to infer microparasite impacts on host populations for humans (e.g. anderson and may ) , wildlife (e.g. mccallum et al. ) and plants (e.g. gilligan et al. ) . although appropriate for modeling the impacts of many infectious diseases (e.g. measles, whooping cough: anderson and may ) , classic sir models compartmentalize individuals into broad categories which may fail to capture variation in aspects of host biology that could influence parasite dynamics and impacts (keeling and eames ) . for example, classic sir models often assume that individual hosts in the population associate at random but, particularly for highly social species, non-random association patterns are far more likely in nature. incorporating network structure into epidemiological models eliminates random-mixing assumption of classic sir models by assigning each individual a finite number of permanent or temporary contacts between which transmission can occur (keeling and eames ; may ) . implications for network epidemiological modeling include the ability to identify highly connected individuals that may be involved in a disproportionate number of transmission events (e.g. super-spreaders: lloyd-smith et al. ) and weakly connected individuals that may benefit from lower risk of infection. epidemiological models informed by variation in social connectedness can be highly effective for predicting pathogen dynamics (lloyd-smith et al. ) but, as noted above, so far only two studies have applied these methods specifically to bats (fortuna et al. ; webber et al. ) . however, to our knowledge no study has explicitly tested model predictions on a natural bat-parasite system in the wild. an alternative to network epidemiological models informed by short-term (i.e. one season) association data, is the use of long-term population data for model parameterization. these data ideally include information on changes in population or colony sizes and changes in population demographic structure and/or vital rates. most often for bats, long-term field data on colony size and population connectivity, in combination with laboratory data on immune physiology have been used (table . ; george et al. ; plowright et al. ) . for example, field and laboratory data were combined to model ecological factors driving hev spillover events occurring in australia from flying foxes (pteropus sp.) to horses and predicted that decreased migratory behavior of the host increased host density in urban areas, which contributed to the duration and intensity of hev outbreaks (plowright et al. , see below) . similarly, mark-recapture data, combined with birth and death rates of different demographics, have been used to parameterize models of rabies dynamics (george et al. ) . rabies in e. fuscus is highly seasonal, with peaks in prevalence at maternity colonies (i.e. during june and july) after parturition when pups provide a supply of immunologically naive hosts (george et al. ). thus, the chance of spillover is greatest during the post-parturition period when females roost in large colonies and the influx of juveniles in the population increases overall prevalence. these studies highlight the value of predictive models incorporating the behavior of bats to make predictions about parasite dynamics, with potential implications for human public health. however, even models which incorporate host ecology and behavior (e.g. habitat selection) do not typically account for variation in social behavior which could also play an important role (moore ) . for microparasites, the two most likely parameters influenced by variation in social behavior are transmission rate (β) and the survival and subsequent reproduction of infected individuals (fig. . ). for example, recent appreciation of individual differences in host behavior has led to the realization that homogenous mixing or so-called 'mass action' transmission may not reflect the dynamics of most host-parasite systems (mccallum et al. ; moore ; barber and dingemanse ) . despite this realization, though, for many host-pathogen systems, estimating β can be difficult and modeling studies tend to rely on some plausible range of values for β (e.g. griffin and nunn ) . for some microparasites, however, it may be possible to experimentally manipulate transmission and determine β empirically. for example, a basic pairwise experiment, where pairs of individuals (one infected, one susceptible) are housed together could help to determine the proportion of contacts that result in transmission, and narrow the range of plausible β values. incorporating empirically derived data on association patterns of individual bats into epidemic models could help to improve our predictions of potential epidemic scenarios. these analyses (i.e. informed by empirical data) are still relatively scarce for bats (table . ), but recent interest in host-parasite interactions and social network analysis provides an excellent opportunity to connect real data with model predictions. comparative analyses provide insight into potential species-specific variation in evolutionary adaption (i.e. changes in response to natural selection) within a group of organisms (garland et al. ) . from the perspective of parasites, a host is effectively a habitat, which means principles of community ecology, and biotic and abiotic characteristics of the "host ecosystem," can be used to explain patterns of parasite community composition (morand and poulin ) . thus, in contrast to intraspecific analyses which typically assess variation in prevalence or intensity of infection among individuals, comparative studies typically examine interspecific variation in parasite species richness among host species because intensity and prevalence are unlikely to be comparable in terms of their impacts, across different host and parasite species. in most cases, comparative studies of parasite species richness "correct for" host phylogeny. two closely related species are more likely to share similar functional traits compared to two more distantly related species because of their more recent evolutionary history (ives and garland ) . more parasites are also likely to have been identified for host species that have been well-studied so, in addition, most comparative studies also account for publication bias by including the total number of publications on a given host species as a covariate in statistical models, or by using residuals of a linear regression between number of publications and parasite species richness as the response variable (e.g. nunn et al. ; lindenfors et al. ; turmelle and olival ). to date, relatively few studies have used comparative analyses to test effects of social behavior on parasite species richness in bats and, so far, most have focused on viruses with one quantifying macroparasites (table . ). in contrast, studies of primates, the best-studied mammalian taxon in terms of relationships between social behavior and parasite richness, focus on a wider breadth of parasites (i.e. ecto and endoparasites as well as bacterial and viral pathogens: nunn et al. ; vitone et al. ; griffin and nunn ) . for bats, colony size appears to be one of the most important predictors of viral richness, and epidemiological and evolutionary theory predict that highly colonial bat species should harbor greater parasite diversity, because of increased opportunities for parasite reproduction and evolution . however, observed relationships have not always followed this pattern for bats (turmelle and olival ; gay et al. ) . turmelle and olival ( ) found no effect of colony size on viral richness in bats but, rather, identified species' conservation status and global f st (i.e. population genetic structure) as the most important predictors. on the other hand, among southeast asian bats, gay et al. ( ) found a negative relationship between colony size and viral richness, which contradicts epidemiological theory (loehle ) . one potential limitation of these results could be that the authors used a categorical metric of population size (i.e. small, medium or large), which likely underrepresents the complexity of social behavior (gay et al. ) . alternatively, in the case of gay et al.'s ( ) results, it could be that bats in large colonies express immune or behavioral traits that provide protection from increased parasite risk in that social context. although they have still not been widely used, in our view comparative studies have great potential to help understand how bat social behavior influences parasite diversity and co-evolves with parasites, in part because bats are so diverse and exhibit a wide range of social and mating systems. for example, promiscuous bat species should host a greater number of parasite species compared to harem-breeding and monogamous bats because, independent of colony size or density, polygynandrous species presumably come in contact with a greater number of conspecifics during mating compared to polygynous species, which likely has implications for host-parasite coevolution. however, it is important to note that mating systems vary considerably among bats (mccracken and wilkinson ) and other aspects of bat social systems (e.g. degree of fission-fusion or roost fidelity: see below) likely co-vary with mating system and are equally important as predictors of parasite richness. thus, we recommend that future studies attempt to include more detailed information on bat social systems in comparative analyses. for example, a meta-analysis of primate social structure showed that differences in social network modularity (i.e. sub-grouping within a larger group) in species led to differences in disease dynamics among species (griffin and nunn ) . although it may not be possible to include such detailed social association data for bats at present, as more social network studies accumulate, we recommend the use of network metrics as predictor variables of parasite richness in comparative analyses. species-specific data on typical contact rates among individuals and demographics, and between sympatric species, as well as data on dispersal patterns and group stability within bat species (e.g. fission-fusion) would also be valuable as predictor variables to better understand how host social traits in bats influence their parasite ecology and evolution. although very different in terms of their ecology and population impacts, based on our literature review, the relationship between bat sociality and parasite impacts and dynamics has been relatively well-studied for three sets of parasites: p. destructans, the cold-tolerant fungal pathogen of bats that causes wns; hev, which can lead to potentially fatal zoonotic disease in humans and horses; and the many species of arthropod ectoparasites which have been relatively well-studied because they can be easily sampled from bats captured in the field. we also identified four articles which addressed the relationship between rabies, or european lyssavirus, and bat social behavior (table . ), but rabies in bats has been the focus of a number of reviews (e.g. messenger et al. ; kuzmin and rupprecht ; rupprecht et al. ; o'shea et al. ) so here we focus on less well-characterized bat-parasite interactions. wns is an eid of urgent conservation concern because it is causing staggering rates of mortality among hibernating bats in eastern north america (blehert et al. ; frick et al. ; wilder et al. ; langwig et al. ; frick et al. ) . despite its recent emergence , however, wns is among the most well-studied microparasites of bats in the context of social behavior. interestingly, one of the hallmark behaviors observed for p. destructans infected bats, both in the wild and in the laboratory, is a reduction in clustering behavior as wns infection progresses over the course of hibernation (langwig et al. ; wilcox et al. ). this phenomenon could lead to increased energy expenditure or evaporative water loss which has implications for survival and potentially future reproduction (willis et al. ; boratyński et al. ) . reduced clustering could be part of a stereotyped behavioral response by animals to infection, known as "sickness behavior" (hart ) , which could either reduce the likelihood of becoming infected, or reinfected, by sick individuals in the hibernaculum or reduce the likelihood of infecting susceptible individuals, which could be important for inclusive fitness if bats cluster with kin during hibernation (wilcox et al. ; bohn et al., in review) . in the laboratory, transmission occurs via direct physical contact and there is no evidence of airborne transmission (lorch et al. ) , while environmental transmission in the wild likely contributes strongly to infection dynamics because p. destructans can survive in hibernacula in the absence of bats (verant et al. ; hoyt et al. ) . although the most adversely affected bat species tend to associate with large numbers of conspecifics throughout the year, transmission appears to occur almost exclusively within hibernacula during autumn and throughout the winter . variation in sociality within and between bat species appears to influence host-parasite dynamics in wns. in the context of host density, there are two theories relevant to the relationship between transmission and host-parasite dynamics. first, density-dependent transmission predicts that host infection scales as a function of host density so that, at low host density, pathogen transmission declines, and a given pathogen fades out (greer et al. ) . second, frequency-dependent transmission predicts that host infection is driven by the total number of interactions among infected and susceptible individuals in the population, regardless of population density, so that when host density is low, a given pathogen can persist (greer et al. ) . interestingly, the relationship between wns transmission and host density during hibernation appears to be species-specific. among relatively solitary species (i.e. perimyotis subflavus and myotis septentrionalis) that hibernate individually or in small clusters, aggregation size (i.e. the numbers of bats in the hibernaculum) best predicted the number of surviving bats within a given hibernaculum, with larger aggregations experiencing faster apparent declines (langwig et al. ). this observation is consistent with density-dependent transmission models. in contrast, for species that vary more widely in aggregation size (i.e. m. lucifugus and m. sodalis), mortality was equally severe across a range of aggregation sizes (langwig et al. ) , which is consistent with frequency-dependent transmission models. this suggests that differences in species-specific social behavior during hibernation can modulate wns transmission and infection. unlike other infectious diseases of bats (e.g. rabies: george et al. ) demographic structure in the active season does not appear to drive wns dynamics. healthy m. lucifugus emerge from hibernation over an approximately eight-week period in spring (norquay and willis ; czenze and willis ) . females tend to emerge prior to males, with the females in the best condition emerging first. this is presumably because larger fat reserves allow females to cope with potentially inclement weather, but also initiate reproduction earlier by exploiting warmer, passively heated maternity roosts, and occasional warm nights with flying insects available (norquay and willis ) . however, bats infected with p. destructans tend to emerge from hibernation much earlier than healthy bats presumably because their fat stores are depleted. moreover, bats that survive hibernation with wns often have severe wing damage in spring and individuals with the most damaged wings tend to have the smallest energy reserves (fuller et al. ). for many diseases, an influx of immunologically naive hosts (i.e. juveniles) leads to a dramatic increase in prevalence immediately following reproduction due to vertical transmission from mothers to offspring. in wns, however, seasonal dynamics of the disease are driven by dramatic seasonal changes in host physiology, specifically sustained low body temperature during hibernation (warnecke et al. ; langwig et al. langwig et al. , . for p. destructans-infected bats in spring and early summer, an increase in body temperature limits infection and transmission despite the fact that females tend to aggregate in potentially large maternity colonies . although transmission likely does not occur readily for females at maternity colonies, high mortality rates during hibernation may decrease the number of potentially reproductive females that form maternity colonies . therefore, normal benefits incurred from colonial roosting, such as social thermoregulation, may be dramatically reduced for wns-surviving bats, which could have additional negative impacts on survivors. for example, the energetic costs of wound healing may be significant for infected individuals upon emergence from hibernation (fuller et al. ) and the decreased availability of social thermoregulation and increased likelihood of roosting solitarily may further increase energetic expenditure (wilcox and willis ) . therefore, for species affected by wns, the lack of potential colony members, and thus potential for social thermoregulation, during spring and early summer could ultimately result in decreased survival and reproduction . in addition to influencing transmission of p. destructans, theory predicts that population size influences risk of extinction (de castro and bolker ) . using counts of bats during winter hibernaculum surveys as a proxy for population size, frick et al. ( ) showed that, for five of six wns-affected species, probability of local extinction from a given hibernaculum decreased as population size increased. this suggests that host aggregation and social behavior could influence risk of extinction from wns. one possible mechanism is that larger populations have greater flexibility to cope with decreases in population numbers, and are thus less likely to face declines below population thresholds where extinction becomes inevitable. interestingly, social behavior may also help to explain differences in the consequences of p. destructans infection for european versus north american bats. prior to the emergence of wns, hibernating aggregations of affected north american species were approximately tenfold higher, on average, than colony sizes of ecologically similar european species . however, after wns emergence, colonies of affected north american species have declined precipitously and appear to be stabilizing at sizes similar to those of european bats . this suggests the possibility that european colony sizes could have been larger in the past and similar to pre-wns colony sizes in north america (frick et al. ) . taken together, empirical data from north america and inferences from europe suggest that wns could select against larger colony sizes (frick et al. ) resulting in remnant colonies which are, on average, smaller and less social than pre-wns colonies. we suggest that future studies attempt to quantify social tendencies at the individual and population levels for pre-and post-wns infected populations to assess the impact of this conservation pathogen for social evolution in bats. in australia, bats from the genus pteropus are reservoir hosts for hev, a recently emerged zoonotic virus from the family paramyxoviridae. flying foxes are only briefly infectious with no clinical disease but shed hev in urine, saliva, feces, and placental fluids (halpin et al. ) . hev is lethal to horses and humans, and transmission presumably occurs when horses ingest food or water contaminated by bats (plowright et al. ) . horses appear to act as amplifier hosts (daszak et al. ) , as all human cases have been directly traced back to an infected horse (field et al. ) . although, spillover events from flying foxes to horses are complex and few data exist linking individual social tendencies of the bats to hev transmission, host socioecology likely mediates host-pathogen dynamics for this zoonotic disease. colony/aggregation sizes of pteropus species can range from as few as , individuals in p. alecto maternity colonies to millions of individuals for p. scapulatus. based on experimental data and closely related human paramyxoviruses (e.g. measles), hev may require large host population sizes to provide enough susceptible individuals for persistence because of a relatively short infectious phase and life-long host immunity (daszak et al. ) . historically, naturally occurring nectar resources in native forests supported large, seasonally-migrating flying fox populations (eby ; plowright et al. ) but human-mediated habitat alterations have resulted in patchily distributed natural food resources, which were already ephemeral in nature (i.e. seasonally produced nectar). therefore, flying foxes have begun to colonize urban and periurban areas to exploit highly abundant, consistently available anthropogenic food resources, such as fruiting trees planted in gardens or horse paddocks (eby et al. ) . in many cases, urban bats do not migrate because food resources are consistently available, which increases the likelihood of hev-positive bats coming in contact with horses (plowright et al. ) . therefore, recent emergence and spillover of hev in australia appears directly linked to increases in the size and abundance of urban bat colonies as well as reduced migratory behavior (plowright et al. ) . although urban bat aggregations appear to remain relatively large throughout the year, temporal dynamics of hev are at least partially mediated by host socioecological traits such as colony size and migration (plowright et al. ). as noted above, social group size has been identified as a potentially important predictor of parasitism, with larger groups generally hosting more parasites (côté and poulin ; rifkin et al. ; patterson and ruckstuhl ) . among temperate bats, sexual segregation and variation in social group size between sexes appears to be among the most important mediators of ectoparasitism. adult males tend to roost individually or in small groups, while females form maternity colonies (kunz and lumsden ) . for males, parasitism tends to remain low-throughout spring and early summer and as the mating season progresses ectoparasite prevalence and intensity increases (webber et al. a) . most studies investigating the link between colony size and ectoparasitism in bats have found the predicted positive relationship (e.g. luçan ; reckardt and kerth ; encarnaçao et al. ) , although this is not always the case (e.g. zhang et al. ; postawa and szubert-kruszynska ) . one explanation for lack of correlation between group size and ectoparasitism at female colonies in some studies may be linked to variation in host association patterns and roost selection/ephemerality. for instance, bamboo shoots used as roosts by flat-headed bats (tylonycteris pachypus and t. robustula) are highly ephemeral and likely contribute to frequent roost-switching (lewis ) as well as variation in group size and composition (zhang et al. ) . frequent roost switching breaks up social contacts that could favor direct host-host transmission of ectoparasites. meanwhile, because some ectoparasites rely on stable roost structures for a portion of their reproductive cycles, bats that use ephemeral roosts rarely come in contact with ectoparasites that rely on a stable roost for reproduction (lewis ) , which could supersede the theoretical relationship between colony size and parasite risk. although, social contacts among females help to explain observed patterns of ectoparasitism at maternity colonies, social organization (i.e. composition of groups) can also mediate ectoparasitism. most females present at maternity colonies are either pregnant or lactating and links between temporal variation in social organization (i.e. transition from pregnancy to lactation) and ectoparasitism are supported by co-evolution of some host-ectoparasite reproductive cycles (christe et al. ) . for example, reproductive cycles of some mite species respond to pregnancy hormones of their female hosts (lourenço and palmeirim ) . once pups are born there is also a pulse of mites, which results in significant vertical transmission from adult females to juveniles (christe et al. ; luçan ) . lactating females may also tolerate significant ectoparasite burdens. during lactation, females may have less energy available to mount behavioral (i.e. grooming) or immunological responses to infection (zuk and mckean ; christe et al. ) . thus, while females are lactating and pups are non-volant, ectoparasite prevalence, and intensity can be exceptionally high. however, once pups have been weaned and are volant, they tend to be more heavily parasitized than females until the colony disperses because they presumably have a naive immunoresponse and less-experience grooming (christie et al. ; czenze and broders ) . although ectoparasitism tends to increase with colony size, juveniles are usually the most infected hosts at maternity colonies. adult females still face significant ectoparasite burdens, while ectoparasitism for adult males generally increases throughout swarming as conspecific contact rates increase (webber et al. a) . many bat ectoparasites are also vectors for bacterial pathogens (e.g. bartonella sp., veikkolainen et al. ). transmission of bacterial or viral pathogens via ectoparasite vectors among bat hosts could be influenced by sociality, especially for highly gregarious females at maternity colonies. in theory, the same principles which regulate host-pathogen dynamics should also apply to host-vector-pathogen dynamics, where the duration and frequency of host-host contact facilitates ectoparasite transmission, which in turn facilitates vector-based pathogen transmission. for instance, recent empirical evidence suggests that wing mites (spinturnix sp.) can transmit p. destructans propagules among hibernating bats in europe, a chain of transmission which is facilitated by direct host-host contact (luçan et al. ) . we suggest that more future studies quantify links between host sociality, vector ectoparasites, and vector-borne bacterial, viral, and fungal pathogens. progress has been made in identifying relationships between some aspects of sociality and parasitism in bats, but much more empirical data from wild and captive bats of more species is certainly needed. we propose a series of testable hypotheses and possible experimental and observational studies about the role of bat sociality in parasite dynamics. we focus on two aspects of sociality which have been relatively little-studied for bats in the context of parasitism: fission-fusion dynamics and consistent individual differences in behavior or personality. fission-fusion is the temporary splitting and reforming of colonies, where lack of consensus, or even conflict, result in temporary fission, but the cost of remaining apart is greater than subsequent fusion (sueur et al. ) . within maternity colonies, females exhibit fission-fusion sociality (e.g. m. bechsteinii: kerth and könig ; nyctalus lasiopterus: popa-lisseanu et al. ) and change roosts every few days but not all members of the group will move together, resulting in variable group size and composition (e.g. patriquin et al. ) . fission-fusion sociality and frequent roost switching by forest-living bats may have evolved as a mechanism to avoid parasites (reckardt and kerth ; bartonička and gaisler ) , although a trade-off between the costs of parasitism and benefits of remaining loyal to high-quality roosts likely exists. empirical data have linked fission-fusion behavior to ectoparasite avoidance (e.g. reckardt and kerth ) and some theoretical modeling also supports this hypothesis (kashima et al. ) . however, there are currently no studies investigating microparasite transmission within fission-fusion societies. we hypothesize that the relationship between fission-fusion dynamics and parasite risk and impacts will vary depending on the mode of transmission for a given type of parasite. we expect that for most microparasites and some ectoparasites (e.g. basilia nana: reckardt and kerth ) that require direct contact between hosts for transmission, fission-fusion dynamics could serve to disrupt parasite transmission. bats that display fission-fusion behavior typically switch roosts every - days (willis and brigham ) and data from m. bechsteinii suggest that bats are able to detect roosts that have recently been occupied by other bats (reckardt and kerth ) . thus, when bats switch roosts they appear to select sites that have not been occupied recently which could help them avoid infested roosts. in contrast, we expect that for macroparasites with alternative transmission strategies, such as mobile ectoparasites (e.g. crawling or flying), fission-fusion dynamics are likely to be less effective as a parasiteavoidance strategy. in fact, theory predicts that bats would be more likely to avoid mobile ectoparasites if they remained in a single, large group because of encounter-dilution effects (côté and poulin ; rifkin et al. ) . we suggest studies employing social network analysis of wild bats, combined with estimates of micro-and macroparasite prevalence, and intensity to disentangle relationships between host social behavior, including fission-fusion dynamics, and the ecology of parasite transmission (for review see godfrey ). animal personality refers to consistent individual differences in behavior that are stable within an individual across time and situations (sih et al. ; wolf and weissing ) . recently, personality has become more widely appreciated as a possible explanatory variable for a number of ecologically relevant traits including juvenile development (stamps and groothuis ) , energy expenditure (careau et al. ) and social structure (krause et al. ; wolf and krause ) . because personality influences non-random association patterns observed within social groups (krause et al. ) , between-individual variation in personality traits are expected to influence parasite transmission patterns within and between groups (barber and dingemanse ; kortet et al. ) . in general, the most active, exploratory or sociable (see table . for definitions) individuals are expected to face greater risk of transmitting and/or acquiring parasites (barber and dingemanse ; kortet et al. ) . despite enormous recent interest in animal personality among behavioral ecologists, evolutionary physiologists, and conservationists (for review see sih et al. ; réale et al. ; careau et al. ; wolf and weissing ) , there are currently only three published studies examining effects of personality on any ecological trait in bats (menzies et al. ; kilgour and brigham ; webber et al. b ). these studies highlight that individual bats, like many other vertebrates exhibit differences in personality but there is clearly room for much more research on personality in bats, especially in the context of parasite dynamics. we propose a series of hypotheses that would help to understand relationships between personality and parasite prevalence/intensity and transmission in the context of sociality. as for the implications of fission-fusion dynamics described above, these hypotheses vary for contact-mediated micro-and macroparasites versus mobile macroparasites with host-seeking behavior. first, we hypothesize a relationship between individual sociability and parasitism for contact-mediated parasites. theory predicts a positive relationship between host sociability and parasite risk, because the most sociable individuals are the most highly connected group-members within the social network and may therefore be exposed to, and themselves transmit, a disproportionate number of infections (barber and dingemanse ) . by contrast, for parasites with active host-seeking behavior, individual activity and exploration may be most important because less active or exploratory individuals may be easier targets for mobile macroparasites. webber et al. ( b) identified the expected negative relationship between the prevalence and intensity of fleas (a relatively mobile ectoparasite) and the activity component of personality for female, but not male m. lucifugus, which highlights the potential complexity of these relationships. personality may have had a larger effect on females because females may be selective with their mating partners, so if less active females mate with fewer males they may retain a higher proportion of fleas, which would otherwise be transmitted during mating (webber et al. b ). we suggest that future studies attempt to disentangle relationships between contact versus mobile parasites and sociability, activity, and exploration components of personality in bats. studies of host-parasite dynamics in bats are important from both a conservation and human public health perspective and, as we describe above, the implications of bat social behavior for parasite risk can be dramatic. based on our review of the literature, we identified social network analysis, epidemiological modeling (often, though not always, parameterized using empirically-derived data), and phylogenetically-informed comparative analyses as the methods most commonly used for quantifying links between sociality and parasitism in bats. in general, these studies suggest complex relationships and studies aimed at fully elucidating these links may require integration of techniques and data collection from multiple disciplines (e.g. behavior, ecology, epidemiology, parasitology, and physiology/immunology). we also found that, despite its recent emergence, wns is already among the best-studied pathogens of bats in the context of sociality perhaps, in part because researchers who focus on bats have brought behavioral and ecological expertise to bear on this important conservation problem. the link between host social aggregation and hev is also well established, again likely because of perceived societal importance, in this case due to the public health implications of a zoonotic pathogen. relationships between bats and their ectoparasites have also been relatively well studied, perhaps partly because ectoparasites are relatively easy to quantify when bats are in the hand. ectoparasite studies have identified links between parasite risk, colony size, and fission-fusion dynamics which have broad implications for understanding how sociality affects host-parasite interactions in bats. a few studies have also begun to understand bats in the context of individual animal personality. in general, however, we advocate for more research on bat parasites in the context of socioecology, especially for bat and parasite species which have received little attention to date. social organization and parasite risk in mammals: integrating theory and empirical studies population biology of infectious diseases: part i coevolution of hosts and parasites infectious diseases of humans: dynamics and control parasitism and the evolutionary 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white-nose syndrome without borders: pseudogymnoascus destructans infection tolerated in europe and palearctic asia but not in acknowledgments we thank jorge ortega for inviting us to contribute to this interesting book project and an anonymous reviewer for comments on an earlier version of this chapter. qmrw was funded through a manitoba graduate scholarship (mgs). funding for ckrw's research on host-parasite dynamics in bats is provided by a discovery grant and a discovery accelerator supplement from the natural sciences and engineering research council (nserc, canada). key: cord- - jbm r authors: hurst, christon j.; adcock, noreen j. title: relationship between humans and their viruses date: - - journal: viral ecology doi: . /b - - / -x sha: doc_id: cord_uid: jbm r nan many of the viruses that can infect humans should not be considered as viruses of humans, but rather as zoonotic. zoonotic viruses are those viruses of animals that can cross boundaries such that they occasionally infect humans. some examples of diseases induced in humans by zoonotic viruses are dengue and ebola fever, the equine encephalitids (i.e., eastern, st. louis, venezuelan, and western), hantavirus pneumonia, lassa fever, marburg fever, rabies, and yellow fever. additionally, it should be noted that the zoonotic category includes most, if not all, of the human illnesses induced either by arboviruses (viruses whose transmission is vectored by arthropods) or by the hemorrhagic fever viruses. with respect to the zoonotic viruses humans are, at best, alternate hosts. humans do in fact usually represent dead-end hosts for these zoonotic viruses, meaning that subsequent transmission of those viruses either to new humans or back to the natural host is not sustained. there is a subgroup of zoonotic viruses that, although principally remaining viruses of animals, seem to have adapted themselves to use humans as natural hosts. this adaptation is indicated by the fact that these viruses have demonstrated an ability to sustain a chain of transmission among humans. examples of zoonotic viruses that have shown this ability to adapt themselves to become viruses of humans are those members of the family flaviviridae (genus flavivirus) that induce the human diseases known as dengue and yellow fever. all of the above-mentioned zoonotic viruses contrast with the viral agents that clearly are known by their nature to be viruses of humans. examples of viruses of humans are those that induce the diseases known as acquired immunodeficiency syndrome, fever blisters, measles, mumps, polio, rubella, smallpox, t-cell leukemia, t-cell lymphoma, and type a influenza. the aim of this chapter is to address those viruses that are considered to be viruses of humans. those viruses of terrestrial mammals that are considered to be zoonotic are addressed by calisher and fenner in chapter . every virus species needs to have a successful overall approach for sustaining its existence. that overall approach must enable the virus to attain its two principal goals, namely, that the virus be able to reproduce itself within a host and that the virus then be transmitted onward to a new host. those mechanisms that any given virus species employs for achieving its sustainment, of course, have been developed through a process that involved an initiation of events by random chance followed by evolutionary selection. the most successful overall approaches may be those that subsequently evolve into the types of relationships between a virus and its host species that will allow the virus to persist without eliminating the host population. this latter point is very important, because the virus may in turn become extinct if it kills off the host population. it is for this reason that excessive virulence will be detrimental to the virus, and an interesting side point may be that, if an individual host cannot successfully surmount the infection, then the death of that individual host may be seen as an altruistic defense mechanism for the host population as a whole. achieving self-reproduction is the first principal goal of the virus. the processes involved can be divided into three aspects. the first aspect is the virus's overall approach with regard to the course of the infection it establishes within the host. this involves the time course of the infection and the extent to which infectious progeny viral particles are produced during the course of the infection. the second aspect is the replication strategy employed by the virus. this involves the issues of where the virus begins its march through the host body and the physical trajectory followed until the virus exits in search of a new host. the third aspect involves which approaches, if any, the virus uses to avoid the host defensive mechanisms. as mentioned in chapter , the goal of establishing an effective course of infection is an aspect of viral reproduction that can be attained in many ways. we can summarize the strategies that viruses use as following four basic patterns. three out of the four patterns of the course of viral infection are considered to be productive. the infection will be acquired in the form of infectious viral particles, and subsequently produced progeny viral particles then serve to infect future hosts. productive, in this context, means that the number of progeny viral particles produced during the course of an infection is sufficient for the particles to transmit the infection to a new host with some reasonable probability. the productive approach involves an evolutionary decision as to whether there will be either a very short initial but highly productive course of infection (short term-initial), a course of infection that is prolonged but only intermittently productive (recurrent), or a productive though very slow course of infection whose severity progressively increases with time to reach a dramatic end-stage (increasing to end-stage). these three patterns can be described as follows. a. short term-initial. viral production only occurs during a short time course near the time of initiation of the infection, which then abruptly ends. the human host may or may not survive beyond the course of this short infection. host survival depends on the type of virus involved, the extent to which the involved virus and humans have had time to coevolve as species, and whether or not the ancestral humans of that particular subgroup of the human host population previously have had contact with the causative virus. coevolution usually will tend to make the outcome of this pattern of viral infection sufficiently mild as to be associated with a fairly low incidence of mortality in an otherwise healthy population of human hosts. some examples of this pattern would be the infections caused by the human caliciviruses (family caliciviridae), human influenza viruses (family orthomyxoviridae), human polioviruses and human rhinoviruses (family picornaviridae), and the human rotaviruses (family reoviridae). b. recurrent. viral production, often very pronounced initially, is recurrent when the virus persists in a latent state within the host body and viral particle production periodically recurs but is not life threatening. some examples of this pattern would be the infections caused by the human herpesviruses (family herpesviridae) and the human papillomaviruses (family papovaviridae). c. increasing to end-stage. viral infection normally is associated with a slow, almost innocuous, start, followed by a gradual progression, associated with an increasing level of viral production and eventual host death. death of the host may relate to destruction of host immunological defense systems, which then results in death by secondary infections. this pattern of infection may take from l to years to kill a human host. the infections caused by the human immunodeficiency viruses and the human t-lymphotropic viruses (all belonging to the family retroviridae) represent examples of this pattern. the fourth basic pattern of viral infection is considered to be nonproductive. a nonproductive infection is one in which the production of infectious virus particles is so limited that the virus must transmit itself through other means, usually by transferring a copy of only the nucleic acid genome of the virus. in these instances, the viral infection is normally acquired by direct transfer of the viral genetic material from the human parents to their developing fetuses, such transfer occurring via the egg and sperm cells. there may be no apparent health effects associated with such an infection. an example of this pattern is the infections caused by the endogenous retroviruses (family retroviridae), whose genomes are incorporated into the chromosomal material of every cell in the human body (villareal, ) . the nonproductive pattern of infection seems to suggest the highest degree of coevolution between a virus and its host, since a nonproductive virus has no means of transmitting itself to a new host without some very active, albeit perhaps unwitting, participation on the part of the present host. this section addresses the questions of where and how the virus begins its march through the host body, and how the virus then continues the course of this attack, leading ultimately to the concept of viral reproduction strategies at the host population level. discussion of the strategy of viral replication within the body of a host organism begins at the most basic level, which is attachment of the virus to a particular molecule present on the surface of host cells. such a molecule is said to be the "virus's receptor," and will be some cellular protein or lipid component naturally produced by those cells. the virus's choice of receptor is a product of viral evolution. after binding to its receptor, the virus gains entrance to the interior of the cell and viral replication begins. viruses whose genomes are composed of dna generally replicate mainly within the nucleus. in contrast, those viruses with rna genomes generally focus their center of replication in the cytoplasm. during the course of replication, the virus must decide which cellular systems and machinery it will employ. some large viruses carry the genomic coding capacity for many of their own enzymes, while others may rely almost completely on the enzymatic machinery possessed by the host cell. many viruses, including those belonging to the genus enterovirus of the family picornaviridae, are said to be highly cytopathogenic, meaning that they usually quickly kill the host cell as a result of infection. other viruses, such as those that occupy the genus rubivirus of the family togaviridae, may cause prolonged and severe crippling of the cell rather than killing it outright. a further discussion of these issues can be found in chapter . viruses vary greatly with respect to the tissues they tend to target for infection. on a larger scale, this then leads to identification of those organs that the viruses are affecting. this selective targeting is referred to as a "tropism." viral tropisms can be divided into those considered primary and those considered secondary. primary tropisms will be associated with production of those viral particles that subsequently contribute to transmission of the viral infection to a new host. as such, the primary tropisms tend to be related to those sites (termed "portals") through which the virus either enters or exits the host body. secondary tropisms may represent accidents. some of these accidents may come about as a result of the molecule that a virus uses as its receptor, existing on the cells of tissues that are unrelated to those that the virus must employ in order to achieve transmission. nevertheless, secondary tropisms may contribute greatly to the types and severity of the illnesses associated with infection of humans by any particular virus. when considered at the host population level, the strategy of viral replication includes the ease with which or likelihood that a virus is transmitted to new hosts plus the severity of infection and accompanying likelihood of death (including the age-related likelihood of death) for any given host individual. through the course of evolution, many viruses have developed mechanisms for either countering or evading the human immune and non-immune defenses as a means for enhancing the probability of viral success. the human immune system includes both humoral (antibody-mediated) and cellular components. the cellular components can include granulomatous reactions, which play a role in defense against protozoans, though their possible role in antiviral defenses has been incompletely explored. those mechanisms that viruses use to either avoid or minimize attack by the host immune system can be divided into the four following groups. the use of these types of mechanisms seems particularly critical in association with those viral infections that persist within a human host for very long periods of time, often up to decades. a. antigenic mimicry. the produced antigens are similar to those of the host, as with prions. b. rapid viral mutation. this mechanism includes both antigenic drifting and shifting. some viral types demonstrate rapid viral mutation during the course of an infection, as occurs with the human immunodeficiency viruses of the family retroviridae. other virus types, such as the influenza viruses of the family orthomyxoviridae, demonstrate rapid viral mutation between reinfections of the same host. c. low antigenicity. some viruses inherently seem to provoke little, if any, immune response. this often occurs because the virus persists in a latent state within host cells, during which time either little or no viral antigenic material is produced. examples include the endogenous retroviruses of the family retroviridae and the human herpesviruses of the family herpesviridae. d. infect the immune cells! the most direct attack may be the most effective. exceptionally notorious examples of this approach are the genus rubivirus of the family togaviridae and the genus lentivirus of retroviridae. aside from the above groupings, some viruses such as the norwalk virus of the family caliciviridae seem to be antigenic but provoke an immune response that is minimally effective. the body has non-immune defense mechanisms that help to protect against viral infections. these mechanisms are associated with the portals through which viruses can enter the body of a host. examples of non-immune defenses include the enzymes secreted as a part of pancreatic fluid, saliva, and tears. various glands associated with mucosal tissues secrete antimicrobial compounds into the mucus produced by those tissues. some mucosal tissues also possess cilia whose movement helps to expel both the mucus and any foreign materials, including pathogens, that become entrapped within the mucus. an additional example of a non-immune defense is the stomach acid produced to aid digestion of organic compounds. many gastroenteritis viruses, such as the rotaviruses of the family reoviridae and the astroviruses of astroviridae, have evolved such an effective resistance to attack by proteolytic enzymes that those viruses virtually need partial proteolysis to facilitate their infectivity. the influenza viruses of the family orthomyxoviridae are known for their ability to paralyze the activity of the mucosal cilia located within the respiratory tract. one of the defining characteristics for the enteroviruses of the family picornaviridae is their resistance to acidic exposure. the task of achieving viral transmission between hosting individuals involves two aspects. the first is the type of infectious material in which a virus will leave its present host, while the second involves the route by which the virus can encounter its proximate host. the types of bodily materials within which viruses can be released include substances that exit during the course of normal body functions. among these substances are feces and a variety of liquids, including menstrual blood, respiratory secretions of the upper and lower tracts, saliva, semen, tears, urine, and vaginal fluid. sweat is another fluid that is naturally released from the body; however, it is not known to contain viruses. viruses can also be found in blood released from wounds in the skin; from blood acquired by blood-consuming parasitic insects, among which are the fleas and several groups of flies, ticks, and mosquitoes; and blood leaked from swollen or ruptured capillaries into mucosal tissue and skin pores. those natural routes by which viruses are transferred to and between humans are the same routes associated with all surface-dwelling terrestrial vertebrates. these routes are tightly associated with the portals of entry and exit that any particular virus family uses as it tries to survive and find its way from one host to the next. viral transmission routes can be subdivided into two broad groups. the first is transmission by direct contact (also known as direct transfer) between two members of those species that host the virus. this includes both the possibility of transmission between two members of the principal host species and the possibility of transmission between a member of that principal host species and some alternate host species, and the latter may represent a vectoring species. the second group is transmission by indirect contact (also known as indirect transfer). these routes have been described in detail by hurst and murphy ( ) and are represented in figures and of chapter herein. as explained in chapter , there are some routes of viral transmission that are considered unnatural vehicular routes. such routes represent the use of unnatural vehicles as a means to evade the host defenses associated with natural portals of entry. these unnatural routes involve invasive medical devices (such as syringes, endoscopes, and other surgical instruments) and transplanted tissues, including transfused blood and blood products. the remainder of this section describes the natural routes of viral transmission between hosts. the direct contact approach offers one major advantage and one major drawback to the virus. the advantage is that those viruses transmitted by direct contact need not be stable when exposed to ambient environments. the drawback is that the number of new hosts to which they have potential access may be smaller than for viruses transmitted by indirect contact. viruses that are endogenous by their nature will survive for as long as the host survives. although endogenous viruses can only be transmitted to host progeny, these viruses neither have to adapt themselves to nor coevolve with any other hosting species. an example of this type of endogenous agent would be the endogenous retroviruses of the family retroviridae. viruses that are venereal in nature, that is, transmitted in semen and vaginal secretions during sexual activity, have a somewhat greater potential for contacting new hosts. represented among the venereal viruses of humans are some species of the genera simplexvirus (family herpesviridae) and papillomavirus (family papovaviridae). once these venereal viruses infect a host, they remain associated with the host for the rest of the host's life in the form of a permanent infection. thus, although the frequency with which endogenous and venereal viruses can find a new host is restricted, the viruses compensate for this to some degree by remaining with the host for a very long time. the next step on the scale of host access is represented by those viruses transmitted via direct contact with insect vectors. these viruses have greatly increased access to new hosts and tend not to remain with their present host for the remainder of the host's life. those viruses transmitted by biting insects are commonly referred to as "arboviruses," which is an abbreviation of "arthropod-borne viruses." included among those arboviruses that infect humans are members of the genera alphavirus (family togaviridae); bunyavirus, nairovirus, and phlebovirus (all of the family bunyaviridae); and flavivirus (family flaviviridae). viruses transmitted by way of saliva may be perceived as bridging the categories of direct and indirect contact. if any particular type of virus that is secreted into saliva has either no stability when exposed to ambient environments in oral secretions or only limited stability under those conditions, then the virus will have to be transmitted by saliva that is transferred during oral contact between hosts. conversely, if the particular virus type has good stability when exposed to ambient environments in oral secretions, then that virus can be transmitted on shared food or in association with fomites. some of the viruses transmitted in saliva do remain associated with the host as a permanent infection, and these often are the viruses that possess limited stability in ambient environments, such as members of the family herpesviridae. many of the viruses that are secreted into saliva and can be transferred to a new host in association with fomites do not remain associated with the host as a permanent infection, such as members of the family picornaviridae. in general, those viruses transmitted by indirect contact between hosting individuals tend to produce only transient infections of their individual hosts rather than remain associated with the individual host as a permanent infection. these several latter points suggest that there may be some evolutionary relationship between ease of viral transmission to a new potential host individual or the frequency of opportunities for viral transmission, and the length of time that a virus must be capable of remaining with its present host to have a reasonable chance of achieving eventual transmission. the indirect contact approach also offers one major advantage and one major drawback to a virus. viruses transmitted by indirect contact have the advantage of potential access to a far greater number of hosting individuals than is the case for viruses transmitted by direct contact. the drawback is that the viruses must have evolved stability when they are exposed to the ambient environment. the vehicles that viruses utilize to achieve transmission between hosting individuals by indirect contact are subdivided into the following four categories: food, water, air (in actuality, this refers to aerosols), and fomites. transmission by any of these four categories of vehicles will usually be associated with some specific physical activity on the part of the present host, and will always be associated with some physical activity on the part of the proximate host. of course, foods are items intentionally ingested for their caloric or nutritional value. food contamination can occur by way of the food being a virally infected animal consumed by the proximate host. in such cases, there is no specific physical activity on the part of the present host (the one being eaten) that can be identified as having caused the proximate host to be ingesting contaminated food (indeed, perhaps it is a lack of physical activity on the part of the present host that is to blame). otherwise, viral contamination of foods can result from fecal material being transferred via contact with unwashed hands or if contaminated aerosols fall into the food. a particularly notable example of a virus of humans that is transmitted via foods is the hepatitis a virus of the genus hepatovirus (family picornaviridae). water usually serves as a vehicle after it has been contaminated with fecal material. acquisition of a viral infection from water usually results from the proximate host ingesting contaminated water. physical contact of the skin or mucosa of the proximate host with contaminated water, as may occur during recreational activities or body washing, can result in acquisition of infection. notable examples of viruses transmitted by these waterborne routes are those belonging to the viral families astroviridae and caliciviridae, and the genera enterovirus and hepatovirus of the family picornaviridae. viral contamination of air can occur by two principal mechanisms. the first, and most significant, involves release of aerosols that contain droplets of respiratory secretions (i.e., nasal, oral, or pulmonary mucus). this type of transmission route is referred to as being the route of droplet aerosols. notable examples of viruses transmitted in this manner are those belonging to the viral families coronaviridae, orthomyxoviridae, and paramyxoviridae, and members of the genus viral contamination of fomites (defined as solid environmental surfaces that can serve in transmission of infections) can occur in many ways. the variety of things that represent fomites include items used to conserve warmth (e.g., blankets, clothing), items used while eating (e.g., cups, dinner plates, and utensils), tables on which diapers are changed, doorknobs, medical devices, toilet seats, and toys. the ways by which these environmental items become contaminated include projection of droplet aerosols onto environmental objects during sneezing or coughing, aerosols falling onto objects, and unintended surface contamination (including children's clothing, blankets and toys) by blood, feces, fluid from skin lesions (rashes), nasal secretions, saliva, or urine. the task of achieving viral transmission via this route is accomplished when these objects are subsequently handled or used by a potential proximate host. among the viral genera whose members can be transmitted via fomites are orthopoxvirus (family poxviridae) and rhinovirus (family picornaviridae). twenty of the viral families contain members capable of infecting humans. together, they cause a broad range of illnesses in humans. the terminology used in describing these illnesses is given in table i . the rest of this section summarizes the ecology of these viral families. figure schematizes the manner in which the different aspects of the ecology of viral infection fit together. the literature sources used for to compile this summary include hurst and murphy ( ) , ictv ( ), evans and kaslow ( ) , and white and fenner ( ) . dae) in association with a natural host. transmission of this virus between hosts occurs when an infected animal bites an uninfected animal, with the virus being transferred by saliva into the bite wound. subsequent movement of viral infection into the nervous system and salivary glands of the newly bitten host animal is considered to represent primary tropisms, as infection at these sites is directly related to movement of virus into the body of the current host and subsequent transfer of virus to the next host. infection of the adrenal cortex is considered a secondary tropism, since those viruses produced in the adrenal cortex will not be transferred to a subsequent host animal. however, infection of the adrenal cortex may play a role in the viral ecology by augmenting the aggressiveness of this newly infected host, thereby increasing the likelihood that this animal will then bite other potential host animals. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans. adenopathy (the origin of the family name), conjunctivitis, coryza, encephalitis, gastroenteritis, keratoconjunctivitis, pharyngitis, pharyngoconjunctival fever, and pneumonia. infection course ~ productive, both short term-initial and recurrent. viral replication ~ at the individual host level, the primary tissue and organ tropisms are toward the cervix, conjunctiva, pharynx, small intestine, and urethra; the secondary tissue and organ tropisms are toward the brain, kidney, lungs, and lymph nodes; at the host population level, these viruses generally are endemic and initially acquired at a very early age, with the infections very often asymptomatic in young children. evasion of host defenses ~ uncertain. and indirect (vehicle-borne) contact via fecally contaminated water, food, fomites, and fomites contaminated by respiratory secretions. genus affecting humans: arenavirus. familial nature with respect to members affecting humans" zoonotic. natural hosts: rodents, including commensal voles and mice, as well as commercial colonies of hamsters and nude mice. arthralgia, carditis, encephalomyelitis, encephalopathy, facial edema, fetal loss, focal necrosis of liver, gastritis, hemorrhagic fever, hepatitis, inhibition of platelet function (causes the fatal bleeding associated with this virus family), malaise, meningitis, myalgia, nerve deafness, and pneumonia. infection course ~ productive, short term-initial. at the individual host level, the primary tissue and organ tropisms presumably are toward the liver and lungs; the secondary tissue and organ tropisms are toward the brain, fetus, heart, joints, and nerves; at the host population level, these viruses can be extremely devastating to individual hosts but are poorly transferred between humans, which usually represent dead-end hosts. evasion of host defenses ~ uncertain. predominant routes of transmission between hosts: indirect (vehicle-borne) contact via inhalation of particulate aerosols bearing dried rodent urine or acquisition of infectious materials through skin abrasion (a form of surface contact). genus affecting humans: astrovirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans. types of illnesses induced in humans: enteritis, gastroenteritis. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, the primary tissue and organ tropisms are toward the small intestine; the secondary tissue and organ tropisms presently are unknown; at the host population level, these viruses are endemic, principally causing a mild enteritis seen in young adults. avoids host non-immune defenses by resistance to proteolytic attack (their infectivity is actually increased by proteolytic attack). via fecally contaminated water, food, and fomites. genera affecting humans: bunyavirus, hantavirus, nairovirus, phlebovirus. familial nature with respect to members affecting humans: zoonotic. natural hosts: largely rodents, but also hares and rabbits, and some ungulates. types of illnesses induced in humans: arthralgia, encephalitis, hematemesis, hematuria, hemolytic uremia, hemorrhagic fever, hemorrhagic pneumonia, hemorrhagic (petechial) skin rash, hepatitis, melena, myalgia, pneumonia, renal dysfunction, retinitis, retroocular pain. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, the primary tissue and organ tropisms are toward the kidneys, liver, and lungs; the secondary tissue and organ tropisms are toward the brain and eyes; at the host population level, these viruses are not well sustained within human populations, and humans usually represent dead-end hosts. evasion of host defenses ~ uncertain, but may include avoiding host immune defenses by infecting immune cells. direct host-to-vector contact by gnats, midges, mosquitoes, sandflies, and ticks for the genera bunyavirus, nairovirus, and phlebovirus, and indirect (vehicle-borne) contact via particulate aerosols containing dried rodent urine, or contact with rodent excreta or contaminated fomites for the genus hantavirus. genus affecting humans: calicivirus. familial nature with respect to members affecting humans: viruses of humans and zoonotic. natural or alternate hosts: fish, terrestrial as well as marine mammals (especially swine). gastroenteritis, hepatitis (nonprogressive, but extraordinarily high fatality rate- to > % --for women if contracted during the third trimester of pregnancy), myalgia. infection course ~ productive, short term-initial. ~ral replication ~ at the individual host level, primary tissue and organ tropisms are toward the small intestine; secondary tissue and organ tropisms are toward the liver; at the host population level, these tend to be epidemic within human populations; for the hepatitis e virus it seems that acquisition occurs from swine, with the result being epidemics (often very widespread) of human disease; some acquisition from animals may come from eating infected animals; subsequent transmission of all caliciviruses within human populations is by fecally contaminated waste and thus can be very widespread. evasion of host defenses ~ avoids host non-immune defenses by resistance to proteolytic attack. indirect (vehicle-borne) contact via fecally contaminated water, food, and fomites. genera affecting humans: coronavirus, torovirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: some terrestrial ungulates and carnivores. coryza, gastroenteritis. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, the primary tissue and organ tropisms are toward the intestines, lungs (possibly), nasopharynx, and sinuses; at the host population level, these viruses are very widespread and essentially nonfatal. predominant routes of transmission between hosts: indirect (vehicle-borne) contact via fecally contaminated water, food, and fomites. familial nature with respect to members affecting humans: generally zoonotic. natural hosts: unknown, but may include bats and rodents, with primates serving as intermediary hosts leading to human exposure. conjunctivitis, hemorrhagic fever (frequently fatal, death possibly resulting from extreme inflammatory response), hepatic necrosis, myalgia, pharyngitis. infection course ~ productive, short term-initial. viral replication w at the individual host level, primary tissue and organ tropisms are toward the immune cells and liver (possibly); secondary tissue and organ tropisms are toward the adrenal glands, kidneys, liver, and spleen; at the host population level, these viruses are transferred between humans but seem unable to be sustained in human populations; humans usually represent deadend hosts. predominant routes of transmission between hosts: direct contact via host-tohost transfer of contaminated bodily fluids. genera affecting humans: flavivirus, hepatitis c-like viruses. familial nature with respect to members affecting humans: viruses of humans and zoonotic. natural or alternate hosts: members of the genus flavivirus cross-infect a variety of birds and terrestrial mammals via mosquitoes or ticks (depending on the viral species) and most clearly are zoonotic, although those that cause yellow fever and the four that cause dengue may have become viruses of humans without time to coevolve; one species of the genus hepatitis c-like viruses affects humans and seems naturally limited to humans. arthritis with rash, encephalitis, hemorrhagic fever, hepatitis (chronic, which may lead to hepatocellular carcinoma). infection course ~ short term-initial for the genus flavivirus, increasing to end-stage for the hepatitis c virus. viral replication m at the individual host level, primary tissue and organ tropisms are toward the immune cells (principally monocytes and macrophages) and liver; secondary tissue and organ tropisms are toward the brain and liver; at the host population level, most of these viruses are zoonotic, with humans representing dead-end hosts; however, some can be sustained within human populations and occasionally have high lethality rates. evasion of host defenses ~ avoids host immune defenses by infecting immune cells. for flaviviruses, direct host-tovector contact; for hepatitis c virus, presumably direct contact via host-to-host transfer of contaminated bodily fluids. genera affecting humans: orthohepadnavirus. the hepatitis d virus (hdv) is a member of the floating genus deltavirus; it is a defective satellite virus which can coinfect humans, but only in association with the hepatitis b virus (hbv) because hdv encapsidates itself with proteins encoded by the genome of the coinfecting hbv. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: one species of viral family hepadnaviridae (hepatitis b virus) is known to infect humans, and it seems naturally limited to humans. type of illness induced in humans: hepatitis, which may become chronic in adults. infection course ~ productive, short term-initial, and increasing to end-stage. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the liver; secondary tissue and organ tropisms are toward the bile duct epithelium, circulating immune cells, and pancreatic acinar cells; at the host population level, when acquired by adults and older children, these viruses generally cause an acute but short-term illness that sometimes can be fulminant; when acquired by neonates or younger children, initially tends to be subclinical but becomes chronic, and the tendency to be chronic can be racially associated (chinese, possibly also black african). evasion of host defenses ~ avoids host immune defenses by infecting immune cells. direct contact via host-to-host transfer of contaminated bodily fluids and perinatally from contaminated maternal blood. genera affecting humans: cytomegalovirus, lymphocryptovirus, roseolovirus, simplexvirus, varicellovirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans, but may pass to primates. carcinoma, carditis, chronic gastrointestinal infection, encephalitis, hepatomegaly, keratoconjunctivitis, lymphoma, myelitis, neuralgia, papular rash of skin and mucosa, paralysis, retinitis, splenomegaly. infection course ~ productive, recurrent. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the genital and oral mucosa, pharynx, and salivary glands; secondary tissue and organ tropisms are toward the eyes, kidneys, liver, lymph nodes, nervous system including brain, and spleen; at the host population level, these viruses are ubiquitous, tend to be acquired in childhood or early adulthood, and seldom directly result in host death. evasion of host defenses ~ avoids host immune defenses by infecting immune cells. direct contact via host-to-host transfer of fluid from viral-induced lesions of skin or mucosa and by saliva contaminated by chronically infected salivary glands; plus transmission to offspring either transplacentally, intrapartum (during the birth process), or via breast milk. genera affecting humans: influenzavirus a, influenzavirus b, and influenzavirus c. familial nature with respect to members affecting humans: generally viruses of humans. alternate hosts: birds (possibly), swine. types of illnesses induced in humans: coryza, malaise, myalgia, nasopharyngitis, pneumonia, retroocular pain, tracheobronchitis. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the ciliated columnar epithelium of the respiratory tract (the exact tissue tropism is directly related to the virus hemagglutinin [ha] serotype); at the host population level, these viruses constantly undergo antigenic drift and antigenic shift and cause wide-scale seasonal epidemics in humans, although infection-related fatality is usually limited to humans aged or older (most notably, age or older). evasion of host defenses ~ avoids host immune defenses by antigenic mimicry and by rapid viral mutation. indirect (vehicle-borne) contact via droplet aerosols (from sneezing and coughing) and aerosol-contaminated fomites. genera affecting humans: papillomavirus, polyomavirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans. types of illnesses induced in humans: benign tumors of skin and mucosa that may progress to malignancy, progressive demyelinating encephalopathy. infection course ~ productive, recurrent. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the mucosa and skin (genus papillomavirus) and toward the upper respiratory tract (genus polyomavirus); secondary tissue and organ tropisms are toward the brain and kidneys (genus polyomavirus); at the host population level, these viruses are ubiquitous and almost never directly responsible for host death. evasion of host defenses ~ avoids host immune defenses by antigenic mimicry. host-to-host or indirect (vehicle-borne) contact by way of fomites (genus papillomavirus); indirect (vehicle-borne) contact via aerosols (genus polyomavirus). genera affecting humans: morbillivirus, paramyxovirus, pneumovirus, rubulavirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans. bronchiolitis, conjunctivitis, coryza, encephalitis, glandular enlargement (especially salivary glands), immunosuppression (morbillivirus causes an immunosuppression that is temporary, but which is arguably the most severe induced by a virus of humans, and can result in death by other coinfecting pathogens, such as enteric protozoans, that normally would not cause fatality), macular rash, nerve deafness, orchitis, pneumonitis. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the epidermis and mucosa (including conjunctival, oral and respiratory); secondary tissue and organ tropisms are toward the brain, breasts, circulating immune cells, and testicles; at the host population level, these viruses tend to be acquired at a young age and are almost never directly responsible for host death, although severe sequelae can result if acquired beyond early childhood. evasion of host defenses avoids host immune defenses by infecting immune cells. indirect (vehicle-borne) contact via aerosols. genus affecting humans: parvovirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans. anemia, arthralgia, erythema, myalgia. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the throat; secondary tissue and organ tropisms are toward the circulatory system, erythrocyte precursor cells in bone marrow, possibly reticulocytes in blood, and skin; at the host population level, these viruses usually cause a disease of childhood; parvoviral disease is either mild or self-limiting in otherwise healthy children or adults. evasion of host defenses ~ uncertain. uncertain, but potentially direct host-to-host contact, including transplacental, and indirect (vehicle-borne) contact via aerosols and fecally contaminated water, food, and fomites. genera affecting humans: enterovirus, hepatovirus, rhinovirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans, but may pass to primates and canines. diabetes, encephalitis, macular and maculopapular rashes of skin and mucosa, meningitis, myocarditis, otitis media, paralysis of skeletal muscles (occasionally including the diaphragm), pericarditis, retroocular pain, sinusitis (genus enterovirus); hepatitis (nonprogressive) (genus hepatovirus); coryza (genus rhinovirus). infection course ~ productive, short term-initial. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the nasopharynx and small intestine; secondary tissue and organ tropisms are very genus and species specific and toward the beta cells of the pancreas, conjunctiva, liver, meninges, muscles (including the heart), neurons (including those of the central nervous system), and skin; at the host population level, infections caused by members of the genus enterovirus usually are nonfatal, and both enterovirus and hepatovirus tend to result in asymptomatic infections if acquired in infancy, though the likelihood of severe symptomatology increases with age at acquisition; infections caused by members of the genus rhinovirus generally are symptomatic but essentially nonfatal regardless of host age. evasion of host defenses m members of genus enterovirus avoid host non-immune defenses by resistance to low ph (resistant to stomach acid) and to moderate alkalinity. indirect (vehicle-borne) contact via aerosols and fecally contaminated water, food, and fomites. genera affecting humans: molluscipoxvirus, orthopoxvirus, parapoxvirus. familial nature with respect to members affecting humans: viruses of humans and zoonotic. alternate hosts: one species affecting humans (smallpox) seems naturally limited to humans; monkeypox is a very notable but rare zoonotic exception and is presumably acquired from monkeys; several other species may cycle with domesticated bovines and ovines appearing as lesions on teats and udder. types of illnesses induced in humans: necrotic lesions of abdominal organs and skin, nodules and tumors in skin, papular rash. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the skin; secondary tissue and organ tropisms are toward the internal organs and lymph nodes; at the host population level, these viruses have very low transmissibility but have prolonged survivability on fomites due to extreme resistance to desiccation. evasion of host defenses ~ avoids host immune defenses by antigenic mimicry. direct contact via host-to-host contact with skin lesions and indirect (vehicle-borne) contact via lesion-contaminated fomites (very notably blankets and other bedding items). genera affecting humans: coltivirus, orthoreovirus, rotavirus. familial nature with respect to members affecting humans: viruses of humans and zoonotic. natural or alternate hosts: those species of the genus coltivirus infecting humans seem zoonotic with terrestrial mammals (notably rodents and squirrels) serving as their natural hosts; species of the genus orthoreovirus cross-infect nearly all known terrestrial mammals (especially rodents); those species of the genus rotavirus affecting humans seem naturally limited to humans. hemorrhagic fever, meningoencephalitis (genus coltivirus); upper respiratory symptoms (possibly associated with genus orthoreovirus); gastroenteritis (genus rotavirus). infection course m productive, short term-initial. viral replication-at the individual host level, primary tissue and organ tropisms are highly genus specific, and are toward the immune cells (genus coltavirus), possibly upper respiratory area (genus orthoreovirus), and the small intestine (genus rotavirus); secondary tissue and organ tropisms are toward the brain and meninges; at the host population level, these viruses have high transmissibility, especially among newborns, for whom they usually produce asymptomatic infections; in older children and adults, these viruses likewise have a tendency to produce asymptomatic infections; although rarely fatal in wellnourished children, members of the genus rotavirus are estimated to cause a million deaths every year in undernourished children. evasion of host defenses ~ avoids host immune defenses by infecting immune cells (genus coltivirus), avoids host non-immune defenses by resistance to heat, low ph, and proteolytic attack (infectivity actually increased by proteolytic attack) (members of the genera orthoreovirus and rotavirus). indirect (vehicle-borne) contact via fecally contaminated water, food, and fomites with the orthoreovirus possibly also being spread by aerosols. genera affecting humans: blv-htlv retroviruses, lentivirus, spumavirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans. carcinoma, encephalitis, leukemia (adult t-cell), lymphoma (adult t-cell), progressive chronic immunosuppression and immunodepletion (including acquired immunodeficiency syndrome), progressive myelopathy, sarcoma. infection course m productive, short term-initial, often followed by increasing to end-stage; also may seem nonproductive in the case of some endogenous retroviruses. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the immune cells (largely t-cell populations); secondary tissue and organ tropisms are toward the brain and intestines; at the host population level, those viruses considered transmissible (i.e., excluding endogenous retroviruses) have a very low transmissibility rate, produce infections whose incubation times are very long ( -- years), and may pass through breast milk; the endogenous retroviruses are permanently integrated into the human genome and are passed genetically to all offspring. evasion of host defenses ~ avoids host immune defenses by rapid viral mutation and by infecting immune cells. direct contact via host-to-host transfer of contaminated bodily fluids. genera affecting humans: lyssavirus, vesiculovirus. familial nature with respect to members affecting humans: zoonotic. natural hosts: foxes, skunks, and vampire bats (genus lyssavirus), cattle and horses (genus vesiculovirus). neuronal infections leading to encephalitis which appears invariably fatal (genus lyssavirus); myalgia (genus vesiculovirus). infection course m productive, short term-initial. viral replication m at the individual host level, primary tissue and organ tropisms are toward the neurons, including those in the spinal cord and limbic system of the brain, and the salivary glands (genus lyssavirus); and toward either the muscles or nerves (genus vesiculovirus); secondary tissue and organ tropisms are toward the adrenal cortex and pancreas (genus lyssavirus); at the host population level, these viruses are essentially nontransmissible. evasion of host defenses ~ avoids host immune defenses by limited antigenic exposure within the host because the virus largely remains within neuronal cells until near end-stage (genus lyssavirus). direct contact via host-to-host contact associated with deposition of contaminated saliva into a bite wound and possibly associated with contamination of skin or mucosal wounds by other types of bodily fluids; in the case of the genus vesiculovirus, vesicular fluids. genera affecting humans: alphavirus, rubivirus. familial nature with respect to members affecting humans: viruses of human and zoonotic. natural or alternate hosts: species of the genus alphavirus cross-infect a wide variety of terrestrial vertebrates, mostly via mosquitoes and ticks; one species of the genus rubivirus affects humans and it seems restricted to humans. arthralgia, arthritis, diabetes, encephalitis, fetal developmental abnormalities (cardiological, diabetic, and neurological including auditory, encephalitic, and visual k caused by rubivirus if contracted during the first trimester of pregnancy), macular rash of skin, myalgia, myositis. infection course m productive, short term-initial. viral replication m at the individual host level, primary tissue and organ tropisms are toward the immune cells (specifically monocytes and macrophages in bone marrow, liver, lymph nodes, and spleen) and oropharynx; secondary tissue and organ tropisms are toward the beta cells of the pancreas, muscles, neurons of the central nervous system including the brain, skin, and synovial cells of joints; at the host population level, most members of the genus alphavirus seem poorly transmitted between humans, and humans probably represent a dead-end host; infection by the genus rubivirus is seldom fatal but highly transmissible via aerosols and usually causes a trivial exanthema of childhood or mild symptoms in adults, although infection during the first trimester of pregnancy can result in extremely severe developmental abnormalities. evasion of host defenses avoids host immune defenses by infecting immune cells. to-vector (genus alphavirus), host-to-host (genus rubivirus), or indirect (vehicleborne) contact via aerosols (genus rubivirus). there are many types of viruses that afflict humans. we have managed to coevolve with some of these to lessen our misery. the struggle will continue as new viruses appear and as the existing ones reshuffle their genes or change their antigenicity by mutation. in the end, the contest is a struggle of biology versus biology, and the basic biology of the viruses is the same as ours. viral infections of humans: epidemiology and control modeling disease transmission and its prevention by disinfection virus taxonomy: sixth report of the international committee on taxonomy of viruses on viruses, sex and motherhood key: cord- -ejvv lxv authors: bowdish, d. m. e.; davidson, d. j.; hancock, r. e. w. title: immunomodulatory properties of defensins and cathelicidins date: journal: antimicrobial peptides and human disease doi: . / - - - _ sha: doc_id: cord_uid: ejvv lxv host defence peptides are a conserved component of the innate immune response in all complex life forms. in humans, the major classes of host defence peptides include the α- and β-defensins and the cathelicidin, hcap- /ll- . these peptides are expressed in the granules of neutrophils and by a wide variety of tissue types. they have many roles in the immune response including both indirect and direct antimicrobial activity, the ability to act as chemokines as well as induce chemokine production leading to recruitment of leukocytes to the site of infection, the promotion of wound healing and an ability to modulate adaptive immunity. it appears that many of these properties are mediated though direct interaction of peptides with the cells of the innate immune response including monocytes, dendritic cells, t cells and epithelial cells. the importance of these peptides in immune responses has been demonstrated since animals defective in the expression of certain host defence peptides showgreater susceptibility to bacterial infections. in the very few instances in which human patients have been demonstrated to have defective host defence peptide expression, these individuals suffer from frequent infections. although studies of the immunomodulatory properties of these peptides are in their infancy, there is a growing body of evidence suggesting that the immunomodulatory properties of these small, naturally occurring molecules might be harnessed for development as novel therapeutic agents. that many of these properties are mediated though direct interaction of peptides with the cells of the innate immune response including monocytes, dendritic cells, t cells and epithelial cells. the importance of these peptides in immune responses has been demonstrated since animals defective in the expression of certain host defence peptides show greater susceptibility to bacterial infections. in the very few instances in which human patients have been demonstrated to have defective host defence peptide expression, these individuals suffer from frequent infections. although studies of the immunomodulatory properties of these peptides are in their infancy, there is a growing body of evidence suggesting that the immunomodulatory properties of these small, naturally occurring molecules might be harnessed for development as novel therapeutic agents. human neutrophil peptide hbd human beta defensin tlr toll-like receptor lps lipopolysaccharide lta lipoteichoic acid tnf-α tumour necrosis factor alpha mhc major histocompatibility complex class bal bronchoalveolar lavage overview host defence peptides are small (generally less than amino acids), positively charged peptides that are an evolutionarily conserved component of the innate immune response. originally characterised as natural antimicrobial agents, it is becoming increasingly apparent that these peptides have a wide range of immunomodulatory properties that are either complementary to, or independent of, antimicrobial activity. interest in the immunomodulatory functions of these peptides is increasing, and indeed many peptides and proteins with similar characteristics to host defence peptides have been found to have either antimicrobial or immunomodulatory properties in addition to their primary functions. in humans the best-characterised host defence peptides are the defensins and the sole cathelicidin, hcap- /ll- . the amino acid sequences of these peptides are summarised in table . in general, the defensins are between and amino acids long (approximately . kda) and contain three conserved disulphide bridges (white et al. ) . the genes for all human defensins are clustered on chromosome (sparkes et al. ; maxwell et al. ) . they are further subdivided to include the αand β-defensins, a distinction based on the organisation of the three characteristic cystine disulphide bonds. the canonical sequence of the α-defensins is x - cxcrx - cx ex gxcx gx ccx - , where x represents any amino acid residue. these peptides are rich in cysteine, arginine, and aromatic residues . the cysteines are linked - , - , and - . initially these peptides were isolated from the neutrophils and are thus called human neutrophil peptides (hnp)- to - . the hnps are expressed at the transcriptional level in the bone marrow, spleen and thymus, where they co-localise with peripheral blood leukocytes (zhao et al. ) . the three hnps are highly homologous, differing by only one amino acid at the nh terminus. because of the high sequence similarity and difficulties in purifying the individual peptides as well as the high degree in functional similarity, the hnp - are often studied as a group, although certain studies have demonstrated differences in their antimicrobial ) and immunomodulatory activities (chertov et al. ) . hnp was identified as a hnp due to its structural homology to the hnp - (wilde et al. ). this gene differs from the other genes of this family by an extra -base pair segment that is apparently the result of a recent duplication within the coding region (palfree et al. ) . as with the other hnps, hnp is found in the neutrophils, but is also called corticostatin because it exhibits corticostatic activity and inhibits corticotrophin-stimulated corticosterone production (singh et al. ) . despite its variation from the conserved sequences of hnp - , it appears to have much more potent antimicrobial activity (wilde et al. ) . two other α-defensins, hd and hd , are found solely in the intestinal tract. hd and hd were found to be expressed at the transcriptional level solely in the small intestine and in situ hybridisation demonstrated that this expression occurs in the paneth cells bevins , ) . southern blot analysis using a nucleotide probe for the conserved signal sequence of the defensins indicated that a number of genes with high homology to hnps exist within the human genome. the β-defensins are expressed in a variety of tissue types, including epithelial cells from the trachea and lung, in the salivary and mammary glands, in a variety of organs such as in the plasma and skin (bensch et al. ; zhao et al. ; harder et al. ; reviewed in lehrer and ganz ) . the expression of certain β-defensins is inducible upon stimulation with bacterial components or pro-inflammatory cytokines and thus these peptides are presumed to be an important component of host defence to infection or inflammation. the canonical sequence for the beta defensins is x - cx - (g/a) x cx - cx - c x - ccx n . the best characterised members of the β-defensin family are hbd - ; however, the antimicrobial properties of hbd have been recently published (garcia et al. ) and over potential β-defensin homologues have been identified in the human genome based on sequence similarity to hbd - (schutte et al. ) . the cathelicidins are an evolutionarily conserved family of host defence peptides which are found in cows, sheep, guinea pigs, rabbits, mice, and primates (reviewed in zanetti ) and are characterised by having an evolutionarily conserved n-terminal domain called the cathelin domain. in addition, these peptides have a signal sequence, which is believed to target their delivery to the secondary granules of neutrophils. the c terminal domain, which is released by cleavage of proteases, has both antimicrobial and immunomodulatory properties. despite the conserved nature of the cathelin domain, its function remains unclear, although it has been proposed to block the antimicrobial activity of the cleaved product, presumably as a mechanism which allows storage of the peptide in its inactive form , and there is some evidence that it has anti-protease activity . the sole human cathelicidin ll- /hcap- is found at high concentrations in its unprocessed form in the granules of neutrophils and is processed upon degranulation and release (sorensen et al. ) . consequently, it is found at sites of neutrophil degranulation. it is also produced by epithelial cells and is found in a number of tissues and bodily fluids, including gastric juices, saliva, semen, sweat, plasma, airway surface liquid and breast milk (bals et al. c; murakami et al. b; hase et al. ; murakami et al. ) . generally epithelial cells have been shown to produce the hcap- form. although the hcap- has been shown to be cleaved by the neutrophil protease, protease , when released from neutrophils, it is not entirely clear how or when hcap- is cleaved when it is produced by epithelial cells. there are a variety of processed forms of hcap- that result from as-yet uncharacterised cleavage processes. for example, a -kda form is found in gastric juice (hase et al. ) , while numerous cleavage products are found in the sweat (murakami et al. b) . as well, hcap- from semen is cleaved to a -amino acid antimicrobial peptide all- in the vagina, thus potentially providing some antimicrobial protection after sexual intercourse ). there appears to be some overlapping, complementary and possibly even enhanced antimicrobial activity of these isoforms (murakami et al. ) ; however, to date there is no information about their immunomodulatory properties. due to the high homology of hnp - , they are often classed as a group. the hnps are found exclusively in leukocytes and at their highest concentrations in neutrophils where they are localised to azurophilic granules. the recently described hnp is also found in azurophilic granules but at much lower concentrations (wilde et al. ) . to date there has been no indication that their expression levels can vary substantially. neutrophils stimulated with il- , fmlp or phorbol -myristate -acetate causes degranulation and release of hnp - (chertov et al. ) , thus it is believed that relatively high concentrations of these peptides are present at sites of infection or inflammation. interestingly, in the course of infection hnp levels increase systemically. although the mechanism is not entirely clear, the plasma levels of defensins increase significantly in patients experiencing septicaemia or meningitis (from approximately ng/ml to as much as , ng/ml). the concentration of neutrophil defensins generally correlates with that of il- , a potent chemoattractant for neutrophils (ashitani et al. ) , as well as the presence of other neutrophil components such as elastase (zhang et al. ) . presumably this occurs due to neutrophil degranulation in response to bacterial or pro-inflammatory stimuli as concentrations decrease after antibiotic treatment (panyutich et al. ) . hd and hd are expressed throughout the gastrointestinal tract, with the highest levels of expression occurring in the jejunum and ileum (dhaliwal et al. ) . specifically, immunohistochemical studies have shown that hd is expressed by paneth cells, some villous epithelial cells and in the terminal ileal mucosa (cunliffe et al. ) . although there is great variability between individuals, levels of these defensins are increased in certain disease states such as acute coeliac sprue (frye et al. b) and are decreased in others such as hiv-related cryptosporidiosis (kelly et al. ). in patients with crohn's disease or ulcerative colitis, but not in healthy individuals, hd immunoreactive cells are present in the crypt region of a large proportion of colonic samples, indicating that expression in these disease states might be dysregulated (cunliffe et al. ) . limited studies have demonstrated that hd and hd may also be present at low levels in airway epithelial cells (frye et al. a) or the female reproductive tract (quayle et al. ). the patterns of expression of the β-defensins are markedly different. in general hbd is not up-regulated during the course of infection or inflammation or by stimulation with pro-inflammatory cytokines or bacterial components and in many cases the presence of hbd is detectable at the transcriptional level but is undetectable at the protein level (o'neil et al. ; frye et al. b) . however its constitutive presence in airway surface liquid, in intestinal and colon cell lines and in other tissues and bodily fluids implies that it may be involved in maintenance and homeostasis of these areas (salzman et al. ) . the expression levels of hbd appear to be quite low, ranging from the pg/ml to ng/ml levels in most bodily fluids. hbd is an inducible host defence peptide whose expression is altered under both infectious and inflammatory conditions. it has been found to be up-regulated by pro-inflammatory stimuli in oral epithelial cells and keratinocytes (krisanaprakornkit et al. ) , in intestinal and colonic epithelial cell lines (o'neil et al. ; ogushi et al. ; vora et al. ) and in various lung epithelial cell lines (singh et al. ; becker et al. ) . hbd is believed to be an important component of host defences, since hbd expression is depressed in patients with atopic dermatitis who often present with cases of acute and chronic colonisation by staphylococcus aureus (ong et al. ) but is increased in psoriatic skin, a disease in which patients are fairly resistant to bacterial infection. it has been demonstrated to be upregulated by both commensal and pathogenic bacteria in the oral mucosa and keratinocytes, although by different mechanisms (krisanaprakornkit et al. ; chung and dale ) . hbd is inducible during the course of inflammation and infection in the gastrointestinal system, as observed at both the mrna and protein levels. hbd expression in intestinal and colonic epithelial cell lines is increased upon stimulation with il- α, flagellin or bacteria, in an nf-κb-dependent manner (o'neil et al. ; ogushi et al. ) , and by either lipopolysaccharide (lps) or lipoteichoic acid (lta) in a tlr and tlr -dependent manner (vora et al. ) . interestingly, other inflammatory mediators such as tnf-α and lps do not induce hbd up-regulation (o'neil et al. ) . this may reflect a predominantly intracellular expression pattern of tlr in these cells, which has been suggested to be an evolutionary adaptation to the high bacterial load in the intestine (naik et al. ; hornef et al. ) . increases in hbd expression have been detected in inflamed intestinal and colonic tissues by rt-pcr and immunohistochemistry, in crohn's disease and ulcerative colitis (fahlgren et al. ) , and in the stomach of patients suffering from helicobacter pylori-induced gastritis (wehkamp et al. ) . in the lung, hbd has been found to be down-regulated in a variety of infectious and inflammatory diseases including cystic fibrosis and infections (chen et al. ) . in lung transplant patients, it has been found to be present at greater than ten times the concentration in patients suffering from bronchiolitis obliterans syndrome, a consequence of rejection of the transplant, compared to transplant patients without signs of rejection (ross et al. ) . less is known about the newly characterised hbd . hbd expression and activity is not well characterised; however, it has been demonstrated to be inducibly expressed at the transcriptional level in bronchial epithelial cell lines stimulated with tnf-α, bacteria, or live rhinovirus (harder et al. ; duits et al. ). in addition, this peptide can be induced in amnion cells in response to bacterial components and is found at high concentrations in human fetal membranes, by immunohistology, indicating that it may be involved in maintaining the sterility of the intra-amniotic environment (buhimschi et al. ) . the pro-cathelicidin hcap is found at high concentrations in the granules of neutrophils and is produced by epithelial cells. the camp-gene promoter that directs the expression of this peptide contains many transcription factor binding sites (larrick et al. ) , including a vitamin d response element (wang et al. ) . consistent with this, hcap , and/or its processed product ll- , has been shown to be up-regulated in sinus epithelial cells, at the transcriptional level, by the bacterial products lps and lta (nell et al. ) . it is also increased in bronchial airway cells by il-α (erdag and morgan ) . in other cell types, pro-inflammatory cytokines do not increase the expression of this peptide, implying that other signalling pathways may be involved (hase et al. ). its expression is increased in gastric epithelial cells upon stimulation with a wild-type strain of h. pylori, but not a type iv secretion mutant (hase et al. ) , and is found at increased levels in other forms of bacterial infection. it is not entirely clear under what circumstances neutrophils release ll- . the antimicrobial activity of all host defence peptides is highest in media of low ionic strength, and the activity of most peptides is sensitive to the presence of physiological concentrations of ions such as na + , mg + and ca + . the antimicrobial properties of the β-defensins, for example demonstrate profound salt sensitivity, and in some cases their antimicrobial activity is completely lost at concentrations of mm nacl (bals et al. a (bals et al. , b garcia et al. ) . for example, hbd is antimicrobial towards gram-negative bacteria at concentrations of - µg/ml (singh et al. ), but its antimicrobial activity is almost completely abrogated by the presence of mm sodium ions. although hbd is slightly less sensitive to the presence of sodium ions, its antimicrobial activity is reduced from . - . µg/ml in conditions of low ionic strength to . µg/ml or more in the presence of mm sodium (singh et al. ) . of the human β-defensins, hbd is the most potent antimicrobial. this peptide is more basic, has a broader spectrum and stronger bactericidal activity against gram-positive and gram-negative bacteria, as well as yeast, and is salt-insensitive at concentrations less than mm na + ions (harder et al. ) . most biological fluids, including sputum (halmerbauer et al. ) , airway surface liquid (baconnais et al. ) and serum/plasma (hoshino et al. ) contain mg + and ca + at free concentrations between and mm, and the presence of these ions is generally more detrimental to antimicrobial activity than na + alone. the α-defensins are susceptible to concentrations of ca + and mg + as low as . mm ). in the presence of mm na + ions, the antimicrobial activity of ll- is decreased two-to eightfold (turner et al. ) , and in the presence of standard tissue culture media, which contains mm nacl and - mm of mg + and ca + , ll- has no killing activity against s. aureus or salmonella typhimurium even at concentrations as high as µg/ml . in some cases, for example in the granules of neutrophils, the concentrations of host defence peptides are estimated to be as great as mg/ml, and there is no doubt that upon ingestion of bacteria these concentrations are sufficient to cause direct antimicrobial activity, despite the presence of divalent cations or other inhibitory substances. however, it is questionable whether these concentrations are reached at mucosal surfaces. for example, in patients suffering from inflammatory lung disease or infection, the concentration of hnps in the bronchoalveolar lavage (bal) has been estimated to be . - . and µg/ml in two different studies (cole et al. ; spencer et al. ). this contrasts with antimicrobial activity in low salt medium in vitro at greater than µg/ml for s. aureus and escherichia coli (nagaoka et al. ) , and reduction of the infectivity of adenoviruses in an airway epithelial model at concentrations of between - µg/ml (spencer et al. ) . interestingly, the antimicrobial activity of the host defence peptides may also be inhibited by components of serum. for example, hnp has also been demonstrated to possess antiviral activity towards enveloped viruses, but this activity is abrogated by the presence of serum or albumin (daher et al. ). it is believed but has not been conclusively shown that the high concentrations of α-defensins in neutrophils would overcome any localised serum effects (daher et al. ). moreover, the antibacterial activity of ll- has been demonstrated to be abrogated by the presence of apolipoprotein . in contrast to the antimicrobial activity of these peptides, their immunomodulatory properties are generally studied in the presence of standard tissue culture media, which contains physiologically relevant concentrations of ions and serum proteins. under these conditions, host defence peptides have been demonstrated to induce chemokine production, reduce pro-inflammatory cytokine production, alter transcription, induce proliferation and angiogenesis, induce chemotaxis and alter dendritic cell differentiation. thus the immunomodulatory properties of host defence peptides are unaffected by physiological ion concentrations and it seems possible that they may be the predominant function of these peptides in vivo. this perspective is quite controversial, and will remain so since it is extremely difficult to discriminate between direct and indirect (i.e. through stimulation of innate immunity) mechanisms of killing. one often used argument for the likely antimicrobial function of these peptides is their substantial variation over evolution (e.g. the mouse cramp and human ll- peptides share only % homology), which could have arisen from the evolutionary pressure of dealing with different pathogens. however, not all antimicrobial proteins are as divergent, and we note that a number of proteins involved in immunity and reproduction show similar "rapid evolution" to the antimicrobial peptides (emes et al. ). there has been some debate about whether host defence peptides might be directly antimicrobial in vivo. certainly, neutrophil granules and the crypts of the lumen contain sufficiently high concentrations of peptides to ensure substantial antimicrobial activity; however, it is less clear that antimicrobial activity occurs at the lower concentrations in such sites as mucosal surfaces, and it is worth noting that such sites are often heavily colonised by a rich and diverse collection of commensal bacteria. the evidence for antimicrobial activity in certain body sites is in our opinion inconclusive. on the one hand, certain bodily fluids such as sinus fluid (cole et al. ) and gastric fluids (hase et al. ) can directly kill certain micro-organisms, and this antimicrobial activity is ablated or reduced by removal of proteins or immunodepletion with a peptide-specific antibody. however, in certain animal models in which peptides and bacteria are instilled simultaneously, bacterial counts are often not significantly different from mice treated with bacteria alone, despite improved outcome or reduced pro-inflammatory responses (sawa et al. ; giacometti et al. ) . the difficulties in assessing the role of host defence peptides in vivo are profound, as it is almost impossible to account for synergistic interactions between peptides and other factors, to assess the actual concentrations at the sites of infection and to discriminate the direct antimicrobial activity of peptides from other less direct effects such as enhancement of inflammatory mechanisms (chemotaxis and recruitment of effector cells, enhancement of nonopsonic phagocytosis, etc.). nonetheless, creative experiments and animal models have begun to elucidate the roles of these peptides in vivo. in transgenic mouse model studies in which the expression of certain host defence peptides is ablated, these mice are somewhat more susceptible to infection and carry increased bacterial loads when challenged nizet et al. ) . although this was interpreted as being due to direct antimicrobial activity, other components of host defences must be considered. for example, in a mouse model of peritoneal klebsiella pneumoniae infection, small doses of hnp ( ng- µg) caused an increase in leukocyte accumulation. in this model, it was the leukocyte accumulation which was linked to hnp induced antimicrobial activity, as the reduction in bacterial counts was significantly diminished in leukocytopenic mice. similar results were observed in s. aureus thigh infections (welling et al. ) . gain of function studies have found that introducing or increasing the expression of a host defence peptide can reduce bacterial loads in certain animal models of infection. for example, adenovirus transfer of ll- /hcap- into the lungs of mice that were subsequently challenged with pseudomonas aeruginosa led to a reduction in both the bacterial load and in production of the pro-inflammatory cytokine, tnf-α (bals et al. ) , and intriguingly, similar gene therapy decreased susceptibility to sepsis induced by lps in the complete absence of bacterial infection. in other models, the simultaneous instillation into the mouse lung of p. aeruginosa and either of hbd or a ll- derivative led to reduced lung damage and pro-inflammatory cytokine production, but did not affect bacterial counts (sawa et al. ). there are very few human diseases that are characterised by defects in host defence peptide production, perhaps emphasizing their importance. however, the neutrophils of individuals with specific granule deficiency, a disease characterised by frequent and severe infections, have a reduction in the size of the peroxidase positive, defensins-containing granules (parmley et al. ) and are deficient in defensins ). however, it is difficult to assess the extent to which these infections result from the lack of defensins, as these patients are also deficient in other neutrophil components. it is believed that the constitutive production and deposition of neutrophils is of crucial importance to maintaining the immunological balance of the mouth. patients who suffer from morbus kostman, a severe congenital neutropenia, and are treated with g-csf to restore neutrophil level, do not express ll- in these cells. one of the manifestations of this disease is frequent and severe infections and periodontal disease (putsep et al. ) . it has been proposed that the absence of ll- may give a selective advantage to bacteria that at low levels are commensals but at higher levels are responsible for periodontal disease. it is unclear, however, whether ll- is directly microbicidal towards common pathogens of the mouth or marshals other defences. although a number of oral bacteria are susceptible to ll- (< µg/ml) at mm nacl in vitro, far fewer bacteria are susceptible in physiologically more relevant isotonic environments (tanaka et al. ) . although ll- has been detected in saliva, the actual concentration was not determined (murakami et al. a) . other indirect evidence for the in vivo antimicrobial activity of host defence peptides is that a decreased level of expression often correlates with frequency or severity of disease. for example, hbd and ll- expression is depressed in patients with atopic dermatitis who often present with cases of acute or chronic colonisation by s. aureus (ong et al. ) . in contrast to atopic dermatitis, hbd expression is increased in psoriatic skin, a disease in which patients are fairly resistant to bacterial infections (harder et al. ; nomura et al. ) . whether host defence peptides are directly or indirectly antimicrobial, it is apparent that it is of advantage for bacterial pathogens to subvert their expression or activity. for example, streptococcus pyogenes binds to α -microglobin and secretes a small proteinase which inhibits ll- from interacting with the bacteria and thus prevents ll- mediated killing (nyberg et al. ). ll- expression has been shown to be decreased in shigella infection, consistent with a proposed mechanism of evasion by this bacterium (islam et al. ) . however, it is not clear whether this is a direct down-regulation of expression, or a consequence of denuding the epithelium, with reduced expression in the replacement cells. early experiments with host defence peptides demonstrated that many of these peptides have mitogenic effects on a variety of cells and cell lines. since modest to high concentrations of host defence peptides are found at sites of infection and inflammation, it has been hypothesised that this proliferative effect might be involved in wound healing and re-epithelisation. consistent with this hypothesis, both the human and mouse cathelicidins are up-regulated at sites of incision or wounding, even if the wound is sterile. the appearance of cathelicidins in the skin has been ascribed to both synthesis within epidermal keratinocytes, and deposition from granulocytes that migrate to the site of injury . upon incision, hcap- (the precursor to ll- ) has been shown to be up-regulated in the epidermis bordering the wound. this increase in expression at both the rna and protein levels was clearly evident at the migrating front of the wound during re-epithelialisation. levels of hcap- decreased following wound closure and eventually returned to baseline levels when the wound was intact and reepithelisation was complete. hcap- was found to be an active component in the process of re-epithelisation since antibodies specific for the peptide decreased the rate of re-epithelisation in a concentration-dependent manner (heilborn et al. ) . consistent with this observation, low levels of ll- (as low as ng/ml) have been demonstrated to increase proliferation in an endothelial cell line (koczulla et al. ) . the importance of this peptide in re-epithelisation has been further inferred from its presence in wounds which are healing normally, but its absence in chronic ulcers (heilborn et al. ) . hnps are potent mitogens for epithelial cells, squamous cell carcinoma cell lines and fibroblasts in vitro at low concentrations (murphy et al. ; . interestingly, in one of the earliest studies of these effects it was demonstrated that the hnps acted synergistically with insulin to induce proliferation (murphy et al. ) . in general it has been hypothesised that the mitogenic properties of the neutrophil defensins on non-myeloid cells is an important component of the healing process. however, certain tumours and tumour cell lines have been demonstrated to inappropriately express neutrophil defensins, and in such cases it is believed that this expression might lead to inappropriate proliferation. for example, in a renal carcinoma cell line, the α-defensins hnp - are expressed at both the transcriptional and protein levels. at moderate levels (i.e. ≤ µg/ml), the defensins had mitogenic activity on a subset of these cell lines. by influencing tumour cell proliferation, α-defensins could potentially modulate tumour progression of renal carcinoma cells (muller et al. ) . moderate concentrations (e.g. ≤ µg/ml ) of neutrophil defensins (hnp - ) induce proliferation of a lung epithelial cell line in vitro (aarbiou et al. ) . consistent with these observations, a combination of hnp - caused a dose-and time-dependent increase in cell migration and wound closure of an airway epithelial cell line, possibly due to an ability to induce the expression of genes involved in proliferation (aarbiou et al. ). the mitogenic activity of the hnps and cathelicidins does not appear to be shared by the β-defensins. although hbd has been demonstrated to be up-regulated in chronic ulcers (butmarc et al. ) , it has not been demonstrated to be involved in re-epithelisation. in addition, the β-defensins investigated did not increase the proliferation of epithelial cells, squamous cell carcinoma cell lines or fibroblasts (m. ). an interesting phenomenon which has been observed to occur in response to two cathelicidins, human ll- and its mouse homologue cramp, is the induction of angiogenesis, which is the process of blood vessel formation and/or growth. the formation of new blood vessels results in restoration of tissues increasing the oxygen supply and the provision of blood substances and cells to these tissues. as such it is a requirement for tissue repair and wound healing as well as for the marshalling of innate immunity. thus this function is consistent with a role for host defence peptides in the maintenance and repair of tissues. in a chorioallantoic membrane assay, µg of ll- induced an increase in blood vessel growth, while in a rabbit hind limb model of angiogenesis, collateral vessel growth and blood flow were increased (koczulla et al. ) . interestingly however, despite the known chemotactic properties of this peptide, no inflammatory infiltrate was detected. the angiogenic properties of ll- appear to stem from its direct interaction with endothelial cells rather than induction of growth factors. these data are consistent with the observation that cramp knockout mice have reduced vascular structures at the wound edge at the site of injury (koczulla et al. ) . a mouse β-defensin, defbd , has been shown to be involved in vasculogenesis, which is the differentiation of endothelial cells from progenitor cells during blood vessel development, leading to the de novo formation of blood vessels and tubes. tumours expressing defbd recruit dendritic cell (dc) precursors via ccr and result in enhanced vascularisation and growth in the presence of the cytokine vegf-a (conejo- ). interestingly, these dcs differentiate to express both dc and endothelial cell markers in response to vegf, indicating that these cells undergo endothelial cell-like specialisation after or during migration to newly formed vessels. this implies that host defence peptides may play important roles in vascular development. it has been observed that there are similarities between chemokines and host defence peptides. indeed, many chemokines have modest antimicrobial activity (hieshima et al. ; yang et al. ) , while a derivative of the highly active antimicrobial peptide, horseshoe crab polyphemusin is a potent antagonist of cxcr (tamamura et al. ) . indeed it has been proposed that certain host defence peptides have evolved from duplication of chemokine genes, although this connection is controversial (durr and peschel ; yang et al. ) ; consistent with this, certain peptides have chemotactic activity. interestingly, unlike the chemokines characterised to date, many host defence peptides appear to have chemotactic activity over a wide range of species, and generally speaking these activities are often observed at concentrations fold or more higher than observed with the classical chemokines. hnp and - have been demonstrated to induce chemotaxis of t cells in vitro at concentrations of between . - ng/ml, with maximal activity occurring at less than ng/ml (chertov et al. ) . hnp is a more potent chemoattractant of monocytes that hnp , with optimal activity at concentrations of - - - m, while hnp failed to induce significant chemotaxis (territo et al. ) . conversely, these peptides were not chemotactic for neutrophils (territo et al. ) , and indeed a subsequent study demonstrated that hnp actually suppressed polymorphonuclear (pmn) migration to formyl-methionyl-leucyl-phenylalanine but not to interleukin (grutkoski et al. ) . in balb/c mice, h after subcutaneous injection, a mixture of hnp - was demonstrated to induce infiltration of pmns and mononuclear cells, while in hupbl-scid mice the defensins-induced infiltrate consisted of modest numbers of cd + cells (chertov et al. ) . interestingly, in contrast to in vitro results, in this animal model study, the infiltration of pmns was observed. however, it is unclear whether pmn infiltration was caused by direct chemotaxis or indirect effects of the peptide treatment. further studies demonstrated that these peptides specifically lead to chemotaxis of immature dendritic cells and naïve, but not memory, t cells (yang et al. a ). collectively, these data indicate that neutrophil granules contain important chemotactic factors which promote the infiltration of cells of both the innate and adaptive immune responses. the β-defensins hbd and hbd are chemoattractants for immature dendritic cells and memory t, cells with peak activities occurring at µg/ml (yang et al. ) . these activities are mediated through the chemokine receptor ccr , which also binds the chemokine larc. hbd , but not hbd , has also been demonstrated to be a chemotactic agent for tnf-α treated human neutrophils (niyonsaba et al. ), a response that is also mediated through ccr . ll- has been demonstrated to be chemotactic for rat mast cells (niyonsaba et al. b) , mouse mononuclear cells and pmns (chertov et al. ) , as well as human neutrophils, monocytes and t cells (de et al. ) . as ll- has been demonstrated to induce a number of chemokines, there has been some debate as to whether it induces chemotaxis directly or indirectly by induction of classical chemokines. in the rat mast cell model, it appears as though this chemotaxis is a direct effect: as when mast cells are cultured with ll- and the supernatants are used for the chemotaxis assay, chemotaxis can be blocked by anti-ll- antiserum (niyonsaba et al. b) . host defence peptides may also indirectly enhance chemotaxis by inducing the production of chemokines from a variety of different cell types, including epithelial cells and monocytes. the hnps, for example, have been demonstrated to induce il- from lung epithelial cells and cell lines (van wetering et al. ; sakamoto et al. ) and to induce the production of il- β and il- mrna production (sakamoto et al. ) from a lung epithelial cell line. it is unclear whether the β-defensins have similar chemokine-inducing activities. hbd , for example, does not induce il- expression in bronchial epithelial cells (sakamoto et al. ). however, in bal from patients with diffuse panbronchiolitis, the hbd concentration correlated significantly with the numbers of cells recovered from the bal fluid (total cells, neutrophils, and lymphocytes) (hiratsuka et al. ) , implying that there might be a link between this peptide and cellular infiltration to the site of infection. ll- has been demonstrated to induce mcp- and il- release in a mouse macrophage and a human bronchial epithelial cell line, respectively, and both chemokines were increased upon stimulation with ll- in whole human blood (scott et al. ) . ll- has also been demonstrated to induce chemokine transcription (il- , mcp- , mcp- ) and release (il- ) in a mitogen-activated protein kinase (mapk)-dependent manner in human peripheral blood derived monocytes . both ll- and the hnps are neutrophil-derived peptides which are released upon neutrophil degranulation. these peptides induce the transcription and release of chemokines, specifically il- , which preferentially attract neutrophils. consequently the presence of these peptides correlates well with that of il- , a potent chemoattractant for neutrophils (ashitani et al. ) , as well as the presence of other neutrophil components such as elastase (zhang et al. ). it appears that host defence peptides induce chemotaxis in two ways: first through direct chemotactic activity of pmns and mononuclear cells mediated through ccr and other as yet to be identified receptors and second through inducing chemokine production which would hypothetically increase the numbers of neutrophils and monocytes at sites of infection. this then would have the net effect of promoting or marshalling cells important in innate immunity to the sites of excessive production (through induction) or deposition (through neutrophil degranulation) of these host defence peptides. this then begs the question as to whether host defence peptides are overtly pro-inflammatory. early experiments determined that a number of host defence peptides from various sources bound to lps from diverse gram-negative bacteria and reduced lps-induced release of pro-inflammatory cytokines (e.g. tnf-α, il- , il- ) and nitric oxide from monocyte or macrophages and protected mice from lps lethality (larrick et al. (larrick et al. , vandermeer et al. ; kirikae et al. ) . initial studies focussed on the unprocessed form of cathelicidin, hcap- (kirikae et al. ); however, it was later found that the lps-binding properties of the peptide were contained within the processed -amino acid c-terminal domain, ll- (turner et al. ). it has been proposed that the anti-endotoxic properties of these peptides are the result of the inhibition of binding of lps to cd ) and lipopolysaccharidebinding protein (lbp) (scott et al. ) , and/or indirect effects on cells (scott et al. ) . ll- has been shown to block a number of lps-induced inflammatory responses, including contractility and (nitric oxide) no release in aortic rings (ciornei et al. ) , pro-inflammatory cytokine production in a macrophage cell line and in animal models (scott et al. ) (ohgami et al. ) , suppression of leukocyte infiltration in a model of endotoxin-induced uveitis (ohgami et al. ) and lethality in animal models of sepsis (scott et al. ) . these effects occur at concentrations in the physiological range for ll- ( - µg/ml) and may reflect a natural role for ll- in the body (e.g. balancing of the potential stimulus by endotoxin from commensals). this anti-endotoxin activity appears to correlate with an ability to dampen the pro-inflammatory effects of the gram-positive surface molecule lipoteichoic acid (scott et al. ) . it appears that there may be marked differences in the ability of ll- and the defensins to inhibit the pro-inflammatory effects of endotoxin. for example, hnp and hbd are not potent inhibitors of lps-lbp binding (scott et al. ) . in ex vivo whole blood experiments, hnp was approximately , -fold less potent than bpi at reducing tnf-α in response to gramnegative bacteria and is much less potent in blocking endotoxin activity, as assessed by a surrogate assay, the limulus amoebocyte lysate assay, or in priming pmn for arachidonate release or stimulating leukocyte oxidase activity (levy et al. ) . thus, the ability to bind to and neutralise endotoxininduced activity in humans may be more evident for ll- and other proteins such as bacterial permeability-inducing protein (bpi) and lbp (weiss ) . natural killer cells are cd -cd lymphocytes that are an important component of the innate immune response. they kill transformed and infected cells, but unlike t cells they are active against cells that have decreased or ablated expression of major histocompatibility complex class (mhc ) molecules. the cytolytic properties of nk cells are increased in the presence of cytokines produced by cells of the innate immune response. nk cells themselves produce cytokines, such as ifn-γ, which are involved in the enhancement of both the innate and adaptive immune responses. nk cells contain a wide variety of cytotoxic peptides of which granulysin (nk-lysin) is considered to be the most important (kumar et al. ) . recently nk cells have also been demonstrated to express the transcripts for ll- and hnp - , and these peptides were found in the supernatants of il- -treated cells consistent with an involvement in the cytotoxic properties of these cells (agerberth et al. ) , or alternatively an immunomodulatory role. consistent with these observations, it has been shown that both tlr and tlr agonists induce the release of hnp - from nk cells into the supernatant and that this release is increased synergistically in the presence of other cytokines found at the site of inflammation (chalifour et al. ). it is not entirely clear what the role of defensins may be in modulating nk-induced cytotoxicity. in one study it was found that nk mediated cytolysis of the transformed cell line kn is decreased in the presence of hnp - in a dose-dependent manner. as well, nk cells treated with hnps had a decreased expression of both cd and cd ). this study also demonstrated that there are high concentrations of hnps due to the infiltration of neutrophils in colorectal tumours, but not in surrounding healthy tissue. thus the authors hypothesised that the presence of hnps might actually protect cancerous cells from nk cytolysis. a conflicting study demonstrated that pbmcs, treated with opsonin-coated zymosan particles, induced the release of substances that enhanced nk-mediated cytotoxicity. these substances were identified as neutral serine proteases and hnps. of the peptides tested, hnp was the most potent, increasing nk-mediated cytolysis optimally at a concentration of . µg/ml (lala et al. ) . clearly, further studies are required to fully elucidate the role that host defence peptides have on nk mediated cytoxicity. monocytes and macrophages do not express high levels of defensins or cathelicidins unless stimulated by lps or pro-inflammatory mediators (agerberth et al. ; duits et al. ) . however, when thus stimulated, they secrete as yet unidentified factors that stimulate epithelial cells and keratinocytes to produce host defence peptides . monocytes and macrophages are, however, quite responsive to stimulation with these peptides and both ll- and the defensins have been demonstrated to induce chemotaxis (territo et al. ; de et al. ) . it has been noted that host defence peptides are strong inducers of chemokine activity in monocytes (chaly et al. ; . interestingly it has been demonstrated that the hnps are able to prevent hiv replication in monocytes and monocyte-derived macrophages and that this property may be due to their ability to induce chemokine production and/or receptor antagonism (guo et al. ). hnp and hnp were both demonstrated to induce production of mip-α and mip- β, the ligands for ccr in monocyte-derived macrophages and to prevent replication of a ccr tropic strain of the virus, presumably by blocking virus binding to ccr (guo et al. ). there has been some evidence that host defence peptides might work as opsonins (fleischmann et al. ; sawyer et al. ) . although this property would be predicted to generally enhance that antimicrobial activity associated with these peptides, one study demonstrated that an ll- derivative actually promoted infectivity of coxiella burnetii, an intracellular pathogen of macrophages (aragon et al. ) . generally, host defence peptides are thought to possess anti-inflammatory properties, as described above. however, in some cases, they may actually enhance some aspects of a pro-inflammatory response. ll- , for example, has been demonstrated to enhance il- β processing and release in lps-primed primary human monocytes (elssner et al. ) . this property appears to be conserved across a range of host defence peptides from a number of different species (perregaux et al. ) . mast cells are distributed throughout the body and are also found in low amounts in the blood. these cells rapidly accumulate at sites of infection, and upon encountering certain bacterial components or pro-inflammatory stimuli they promote the inflammatory response by releasing histamine, which causes vasodilation and thus assists in the recruitment of cells and substances from the blood. two host defence peptides, ll- and hbd have been demonstrated to be chemotactic for rat mast cells, although they may work by different mechanisms (niyonsaba et al. b (niyonsaba et al. , . thus mast cells may accumulate at sites of high concentrations of host defence peptides such as at sites of neutrophil degranulation or at epithelial surfaces in vivo. hbd and ll- as well as the hnp - and hnp homologues from rabbits and guinea pigs have also been demonstrated to induce histamine release (befus et al. ; niyonsaba et al. ) . this property may be especially important in the development of host defence peptides as drugs, as mast cell degranulation is a potentially detrimental side effect. host defence peptides have been demonstrated to interact with epithelial cells. neutrophil peptides have been demonstrated to induce proliferation (m. , induce chemokine production (van wetering et al. ) and stimulate cell signalling pathways . ll- has been demonstrated to bind to a lung epithelial cell line in a manner which suggests that it may have more than one receptor . it has also been demonstrated that binding and subsequent internalisation is required in order to induce il- production . host defence peptides in the adaptive immune response in addition to apparently having multiple roles in innate immunity, it is becoming clear that host defence peptides can modulate the adaptive immune response, and several studies have now demonstrated adjuvant activities of host defence peptides in vivo. the mechanisms involved remain unclear, although these activities could reflect the innate immunity modulating activity of host defence peptides and the fact that there appears to be a strong interconnection between innate and adaptive immunity. the relatively non-immunogenic model antigen ovalbumin (ova) is widely used to study adaptive immune responses. intranasal co-administration of human α-defensins hnp - with ova was shown to enhance the production of ova-specific igg antibodies and ova-specific cd + t cells, which produced significantly more ifnγ, il- , il- , and il- (lillard et al. ). this indicated the capacity of α-defensins to alter the host response to ova, acting as adjuvants to promote a mixed t helper (th) cell response. in two other recent studies, hnp , the human β-defensins hbd and hbd (brogden et al. ) , and a simple synthetic peptide klkl klk (fritz et al. ) were also demonstrated to be effective adjuvants. the observation that such effects are observed with the model peptide klkl klk suggests the possibility of a relatively non-specific mechanism, and that such activities may therefore be seen with a broad range of host defence peptides. however, the nature of the enhanced responses may depend both on the antigen and the peptide used. in contrast to the mixed th- /th- response enhanced in the hnp - -treated animals (lillard et al. ) , ova-stimulated splenic lymphoid cell cultures were found to produce significantly decreased levels of ifn-γ, when taken from hbd -treated mice (brogden et al. ). on the other hand, although klkl klk induced a strong th- type response when co-administered with ova, it enhanced a mixed response when the trivalent influenza split-vaccine fluvirin was used as antigen, with the production of both igg and igg antibodies (fritz et al. ). interestingly, this report also demonstrated that a peptide could markedly enhance antigen association with a monocytic cell line in vitro, and that co-administration in vivo could result in the formation of a transient depot of antigen at the site of injection. these observations indicate that antigen uptake by antigen-presenting cells (apcs) might be enhanced in the presence of the peptide, and thus influence responses in the presence of klkl klk in vivo. although these studies clearly showed altered humoral and th responses to antigens, the functional consequences of these alterations were not clearly demonstrated. in another study, mice were given an intraperitoneal vaccination combining a b-cell lymphoma idiotype antigen and daily µg injections of human α-defensins. this study also demonstrated adjuvant activity, whereby the defensins led to increased levels of antigen-specific igg antibodies and enhanced ifn-γ production by splenic cells (tani et al. ) . moreover, defensins showed mitogenic properties (with a significant increase in the number of splenic b cells) and led to an increase in resistance to tumour challenge. the latter observation raised the possibility that an antigen-specific cytotoxic t cell response was being generated in addition to a humoral response. these studies collectively demonstrate that co-administration of host defence peptides with antigens can enhance and perhaps alter the nature of the host's specific adaptive immune responses in vivo. this raises the question of whether host defence peptides might naturally act as endogenous adjuvants to enhance normal immunological responses, since many peptides can be up-regulated or secreted at sites of infection and inflammation. it is unclear whether the doses used in such studies to assess in vivo immunological processes are within relevant physiological ranges. the physiological significance should be addressed by examining transgenic mice with defective production of host defence peptides, although the issue of possible functional redundancy amongst the many murine defensins must be considered when examining single gene knockouts. the published characterisations of such mice have concentrated on innate responses and have generally not described defects in adaptive immune responses morrison et al. ; moser et al. ) . however, one mbd- knockout model was found to display a defect in generating antibodies to the carbohydrate capsule of pneumococci (c. moser, personal communication) . while this is consistent with an in vivo role for this constitutively expressed defensin in generating an effective humoral response, it is clearly an area requiring further study. regardless of possible physiological significance, the adjuvant effects of host defence peptides are clearly of interest from an immunotherapeutic and vaccinology perspective. in contrast to the studies that have co-administered host defence peptides and antigens, other groups have taken an alternative dna-vaccine approach. this methodology involved immunizing mice with dna plasmids encoding non-immunogenic lymphoma antigens fused to murine β-defensins (biragyn et al. ) . successfully transfected cells of an undefined nature should then express the peptide/lymphoma antigen fusion proteins. this strategy represents an attempt to target antigen to immature dendritic cells (idcs), by exploiting the affinity of the β-defensin portion of the fusion proteins for the chemokine receptor ccr , expressed on idcs. this approach also demonstrated an adjuvant capacity for host defence peptides; however, igg responses were only observed when the plasmid encoded a fusion of the antigen and peptide, and not observed after simple co-administration of peptide and antigen. interestingly, anti-tumour activity was also generated in these mice (most effectively with murine β-defensin ), but did not correlate with the amplitude of the humoral response (superior with murine β-defensin ). furthermore, this anti-tumour activity could be transferred to other mice with the delivery of splenocytes, but not serum, from vaccinated animals, indicating the generation of cytotoxic t cells in response to non-immunogenic antigens when fused to peptides. in another recent study, using a similar approach, immunisation of mice with a plasmid fusing the human cathelicidin ll- to m-csfr (acting as a tumour antigen in this model) also generated enhanced antigen-specific humoral and cytotoxic responses, and prolonged survival in a tumour model ). ll- fusion plasmids were found to be significantly more effective than the m-csfr plasmid alone, or co-administration of separately encoded m-csfr and ll- plasmids. these animal studies all demonstrate the adjuvant capacity of host defence peptides in vivo, but the mechanisms underlying these observations have not been fully elucidated. a variety of hypotheses can be proposed, including direct modulation of lymphocyte responses, mitogenic effects, chemotactic capacity, increased apc antigen uptake and consequently enhanced presentation, activity as endogenous danger signals, alterations to the apc cytokine environment, or direct modulation of apc function (fig. ) . the most obvious mechanisms might include altered antigen uptake (fritz et al. ) and direct modulation of lymphocyte activity and proliferation (tani et al. ) , boosting apc presentation, cellular and humoral responses. this could be further enhanced by direct chemotactic effects of host defence peptides, resulting in the chemotaxis of monocytes, neutrophils, macrophages, idcs, mast cells and t lymphocytes (territo et al. ; chertov et al. ; yang et al. yang et al. , a yang et al. , b niyonsaba et al. a niyonsaba et al. , b , and the enhancement of chemokine receptor expression on these cells (scott et al. ) . in addition, host defence peptides could act indirectly to stimulate the release of potent traditional chemokines (such as il- ) from epithelial cells (van wetering et al. ) , and/or cause mast cell degranulation , enhancing vascular permeability. these direct and indirect chemotactic effects could amplify the inflammatory response and bring key cells of the adaptive immune response to the location of the antigen. while recruiting memory t cells to an infection site may induce a more rapid cellular response to previously encountered antigens, the recruitment of monocytes and idc is likely to be critical to generating the initial response. dendritic cells are sentinel leukocytes that capture antigen in the peripheral tissues and then initiate and orchestrate t cell helper (th- ) responses, the nature of which determines the character of the adaptive immune response (moser and murphy ) . this process is critical to generating a successful defence against harmful microbial non-self antigens while maintaining tolerance to self. it is dependent upon the antigen-capturing capabilities of idcs, and antigen-presenting capabilities of mature dendritic cells (mdcs). idcs are derived from circulating haematopoietic precursor cells and predc populations (monocytes and plasmacytoid cells) under the influence of specific cytokines and growth factors (liu ; pulendran et al. ). in the tissues, these cells encounter and take up antigen. stimulation of idcs by conserved structures on certain microbial antigens, acting via the toll-like receptors (tlrs) of the innate immune system (medzhitov and janeway ) or by signals from host cytokines, results in dc activation. these activated cells mature to become effective antigen-processing and presenting mdcs, migrate to the secondary lymphoid organs and interact with naïve t-lymphocytes (banchereau et al. ) . the characteristics of the mdcs determine the nature and consequences of this interaction, resulting in proliferation and differentiation, or deletion of t cells, and determine the polarisation of the th response (lanzavecchia and sallusto ) . whereas steady-state trafficking of non-activated idcs carrying self-antigen is thought to help maintain tolerance, it has been proposed that sustained trafficking of large numbers of highly stimulatory mdcs to the t cell areas is necessary for the generation of an effective t cell proliferative response (lanzavecchia and sallusto ) . this would require extensive, repeated recruitment of circulating predcs to the site of infection, with rapid differentiation to replace the "first-line" resident idcs. thus, at the simplest level, it is conceivable that the in vivo effects of host defence peptides on the adaptive immune response are the result of direct and indirect chemotaxis of idcs and monocytes to the site of inflammation. thus, chemotaxis, altered antigen uptake, and mitogenic effects on lymphocytes offer potential mechanisms by which host defence peptides may enhance responses to immunogenic antigens. however, these explanations do not account for the generation of humoral and cytotoxic t lymphocyte responses to non-immunogenic antigens observed in vivo. in these examples, an increase in the number of dcs encountered and the amount of antigen taken up should make no difference to the response in the absence of an activating signal. indeed, theoretically, this might serve to increase host tolerance to these antigens. despite this, host defence peptides clearly enhance an adaptive immune response to non-immunogenic antigens in vivo. on the basis of current literature, we propose three hypotheses that might explain these observations. the first two theories propose that these peptides directly or indirectly provide an activating signal to differentiated idcs concurrent with these cells encountering antigen, while the third proposes peptide modulation of dc differentiation from precursor cells. in one intriguing report, it was demonstrated that murine β-defensin fusion proteins were capable of activating idcs directly in a tlr- dependent manner, to produce t helper (th- ) polarised responses . in the context of these dna plasmid vaccines, stimulation of the innate pattern recognition pathways through tlr would occur in close spatial and temporal conjunction to an otherwise non-immunogenic antigen. this suggests that host defence peptides might be capable of functioning as endogenous ligands of innate pattern recognition receptors. however, activation of idcs was not observed with murine β-defensin in the absence of fusion to lymphoma antigen. although this appears to make it improbable that this mechanism is responsible for most of the above-described in vivo observations, it is possible that peptide and antigen concentrations are much higher in co-administration studies, than when relying on dna plasmid expression. thus, if the temporal coordination of tlr stimulation and antigen presentation are critical, this may be achieved by high concentration co-administration and depot formation at the site of delivery but, when utilizing a dna vaccine approach, require peptide fusion. however, despite proving effective as an adjuvant for humoral responses (biragyn et al. ) , murine β-defensin fusion proteins did not have tlr- dependent idc-activating capabilities . furthermore we have seen no evidence of an ability to directly mature human monocyte-derived dc in vitro when studying a range of peptides at or above the putative physiological concentrations, (dj davidson, aj currie,, rew hancock, dp speert, unpublished data). these data indicate that direct activation of idcs may not be an inherent property of host defence peptides. thus, although direct activation of idcs is unlikely to be the basic mechanism underlying host defence peptide adjuvant activities, we cannot rule out a peptide-specific effect in which temporal coordination of tlr ligation and chemokine receptor-directed antigen uptake by the same cell are critical. an alternative mechanism to explain altered idc activation is to suggest that the effects of host defence peptides might be indirect, acting to alter the milieu in which these cells encounter antigen. defensins have been shown to increase expression of various cytokines, including il- , il- , mcp- and gm-csf , in different airway epithelial cells, while ll- can induce il- and mcp- expression in epithelial and monocytic cells (scott et al. ) . changes to the cytokine environment may induce a myriad of effects, from the chemotactic activities of mcp- and il- , and cellular differentiation effects of gm-csf, to the enhancement of b cell proliferation and blockade of the suppressive effects of regulatory t cells by il- (pasare and medzhitov ) . possibly other factors are induced that might activate idcs even in the presence of non-immunogenic antigens. following activation of monocytes with s. aureus or phorbol myristate, human α-defensins at con-centrations as low as nm, can increase the expression of tnf-α and il- β (chaly et al. ) . these cytokines have the potential to directly induce dc maturation, sharing components of activating pathways with tlr, and thus potentially enhancing the generation of highly stimulatory mdcs. however, while such mechanisms might therefore be proposed at sites of inflammation, similar activities in the presence of non-immunogenic antigens are only speculative. the third hypothesis relates to our recent discovery that the human cathelicidin ll- can modulate the differentiation of idcs from precursor cells, with consequent impact on th cell polarisation ). the stimulatory nature of dcs is subject to dynamic temporal regulation (langenkamp et al. ) and can be modified by precursor cell lineage, the specific antigen captured, the receptors engaged, and the microenvironment for both differentiation and maturation (liu ; pulendran et al. ; de jong et al. ; boonstra et al. ) . we demonstrated that ll- has the potential to act as an endogenous environmental modifier of dc differentiation ). ll- -primed dcs displayed significantly upregulated endocytic capacity, modified phagocytic receptor expression and function, up-regulated co-stimulatory molecule expression, enhanced secretion of th- -inducing cytokines, and promoted th- responses in vitro. these results suggest the potential for host defence peptides to exert effects on the adaptive immune system by priming newly differentiating dcs to enhance their antigen uptake and presentation capabilities and influence the nature of the response they will subsequently generate. according to this hypothesis, host defence peptides would not simply affect "first-line" resident idcs, but act upon the differentiating "second-line" dcs that can sustain highly stimulatory presentation of antigen to generate an effective t cell proliferative response. in the context of a physiological role, ll- -primed idcs might be generated at sites where ll- is up-regulated in response to infection or inflammation, be matured by immunogenic antigens, and promote a more robust adaptive immune response. however, ll- -primed idcs also have increased expression of the co-stimulatory molecule cd in vitro in the absence of activating stimuli and any other signs of maturation. if such cells were generated in vivo in the presence of a high concentration depot of host defence peptides at the site of vaccination, or in an area of peptide overexpression by host cells transfected with a dna vaccine, they might be capable of presenting non-immunogenic antigen in a stimulatory context. although enhanced humoral and cytotoxic t-lymphocyte (ctl) responses in dna vaccinated mice are dependent on the fusion of ll- and the antigen , this might again relate to issues of local concentration and co-presentation to the same cells. interestingly however, enhanced splenocyte ifn-γ responses were observed not only in mice vaccinated with the ll- fusion plasmid, but also in those given separately encoded ll- and antigen plasmids. this might reflect the enhanced ifn-γ responses of t cells stimulated with ll- -primed dcs in vitro. however, further research is required to establish the in vivo significance of the ll- -priming of dcs observed in vitro. furthermore, the effects of other host defence peptides on dc differentiation have not been described, raising uncertainty about this hypothesis in the context of the in vivo studies using defensins, or synthetic peptides as adjuvants. in conclusion, the potential for host defence peptides to modulate the adaptive immune response is evident, but remains largely undescribed. in addition to further exploration of the effects in vitro, innovative in vivo modelling is a priority to dissect the mechanisms underlying these observations. a clear understanding of the extent and mechanisms of the immunomodulatory effects of host defence peptides will be fundamental to their future development as novel therapeutic agents. however, these early in vivo studies demonstrate great potential for targeting tumours, recalcitrant, antibioticresistant pathogens, infections for which effective vaccines do not exist, and vaccines, which generate suboptimal responses of an inappropriate nature. mammalian host defence peptides were originally discovered as components of the non-oxidative killing mechanisms of neutrophils. in the granules of neutrophils, these peptides are found at sufficiently high concentrations to be antimicrobial. however, it is less clear that this is the case at mucosal surfaces or in other body fluids, especially at sites that already support a rich and diverse normal flora. certain body fluids, including sinus fluid and gastric juices, have innate antimicrobial activity against certain bacteria, and the components that appear to contribute to this include a variety of antimicrobial proteins (e.g. lysozyme, secretory phospholipase a ), as well as peptides (e.g. defensins) (cole et al. ) . however, the specific contributions of each of these components to overall antimicrobial activity has not been determined, and given the moderate levels of peptides often found in these fluids, synergy between individual agents working in combination may be important, as been demonstrated for some peptides, and combinations of lysozyme and peptides in vitro (singh et al. ; yan and hancock ) .this is still further complicated at sites such as the mucosa when considering the abilities of host defence peptides to modulate innate immunity, as discussed extensively in this review. one approach to trying to resolve these mechanisms is to use genetic strategies using either knockout models in animals or specific genetic deficiencies in humans nizet et al. ; putsep et al. ; salzman et al. ) . such studies have clearly demonstrated that defensins and cathelicidins are integral components of host defence in mammals, and that these peptides are required to reduce bacterial load and inhibit infection. however, they do not always permit the discrimination between the various potential mechanisms of host defence peptides, namely direct antimicrobial activity, synergistic activity with other antimicrobial components and/or the broad range of abilities to modulate immunity. indeed, these distinctions may be unimportant, as they all have the same net result, namely the control of potentially dangerous microbes. we hypothesise that all of these mechanisms operate in the body of mammals, and that any given peptide may have different roles in anti-infective immunity according to the body site it is found, its local concentration, the prevailing physiological conditions, and the other antimicrobial and cellular components of immunity at that site. a clear illustration of this complexity is provided by a study of the use of human defensin (hnp- ) to treat experimental peritoneal k. pneumoniae infections (welling et al. ). in this model, hnp injection was shown to markedly reduce bacterial numbers, but the antibacterial effect was associated with an increased influx of leukocytes into the peritoneal cavity, and this was strongly related to the antibacterial effect, as no such activity was observed in leukocytopenic mice. a further challenge to our thinking, and possibly the most profound question in innate immunity, is how mammals manage to support a complex normal flora while retaining the ability to respond to potentially dangerous pathogens. the toll-like receptors (tlrs), which represent one of the major "triggers" of innate immunity, do not really distinguish between the conserved surface molecules from pathogens and commensal organisms. thus it is of interest as to whether host defence peptides may play a role in this delicate dance between symbiosis and pathogenesis. as shown by e. nishimura et al., many commensal bacteria from the oral cavity are quite susceptible to hbd , while chung and dale indicated that both commensals and pathogenic bacteria can induce this defensin (chung and dale ; e. nishimura et al. ). conversely, putsep et al. compared germ-free and normal mice to conclude that an intestinal microflora does not have a major influence on the production or processing of defensins (putsep et al. ) . however, we consider at least one activity of these peptides might play a role for host defence peptides in homeostasis, namely anti-endotoxic activity, which in our experience is expressed at lower concentrations of ll- than other immunomodulatory activities. it seems possible that this would provide a mechanism for balancing the po-tential stimulation of tlrs by surface molecules from commensal organisms. innate immunity would then be triggered by local perturbations of peptide concentrations though mechanisms such as degranulation of phagocytes, or specific up-regulation by certain cytokines. in addition, the efficiency of these peptides might the increased by other local factors, for example phagocytes that enter the local site (welling et al. ) or local cytokines such as gm-csf that has been shown to enhance mapk signalling by ll- . the full spectrum of the immunomodulatory properties of these peptides is not yet known and each new report demonstrates that the range and importance of these immunomodulatory effects is greater than initially suspected. most likely both antimicrobial and immunomodulatory activities are to some degree involved, as this is consistent with the redundant and efficient nature of evolution, and with the concept of innate immunity as a network of overlapping mechanisms. understanding the interplay between host defence peptides and innate and adaptive immunity will expand our knowledge of immunity in general and allow us to develop anti-infective therapies adapted from 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idiotype and carrier antigens monocyte-chemotactic activity of defensins from human neutrophils activities of ll- , a cathelin-associated antimicrobial peptide of human neutrophils effect of defensins on interleukin- synthesis in airway epithelial cells neutrophil defensins stimulate the release of cytokines by airway epithelial cells: modulation by dexamethasone protective effects of a novel -amino acid c-terminal fragment of cap in endotoxemic pigs ) β-defensin- expression is regulated by tlr signaling in intestinal epithelial cells cutting edge: , -dihydroxyvitamin d is a direct inducer of antimicrobial peptide gene expression apolipoprotein a-i binds and inhibits the human antibacterial/cytotoxic peptide ll- defensin pattern in chronic gastritis: hbd- is differentially expressed with respect to helicobacter pylori status bactericidal/permeability-increasing protein (bpi) and lipopolysaccharide-binding protein (lbp): structure, function and regulation in host defence against gram-negative bacteria antibacterial activity of human neutrophil defensins in experimental infections in mice is accompanied by increased leukocyte accumulation structure, function, and membrane integration of defensins purification and characterization of human neutrophil peptide , a novel member of the defensin family regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in innate host defense synergistic interactions between mammalian antimicrobial defense peptides beta-defensins: linking innate and adaptive immunity through dendritic and t cell ccr human neutrophil defensins selectively chemoattract naive t and immature dendritic cells ll- , the neutrophil granule-and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like (fprl ) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and t cells mammalian defensins in immunity: more than just microbicidal many chemokines including ccl /mip- alpha display antimicrobial activity antimicrobial and protease inhibitory functions of the human cathelicidin (hcap /ll- ) prosequence cathelicidins, multifunctional peptides of the innate immunity conventional mechanical ventilation is associated with bronchoalveolar lavage-induced activation of polymorphonuclear leukocytes: a possible mechanism to explain the systemic consequences of ventilator-induced lung injury in patients with ards regulation of activities of nk cells and cd expression in t cells by human hnp- , - , and - widespread expression of beta-defensin hbd- in human secretory glands and epithelial cells we would like to acknowledge support for the authors' research from the applied food and materials network, the canadian bacterial diseases network and the functional pathogenomics of mucosal immunity program funded by genome prairie and genome bc, with additional funding from inimex pharmaceuticals. rewh holds a canada research chair, dmeb is supported by a cihr studentship, and djd was funded by a wellcome trust uk, international prize travelling research fellowship ( ). key: cord- -cnz qjy authors: pedersen, amy b.; davies, t. jonathan title: cross-species pathogen transmission and disease emergence in primates date: - - journal: ecohealth doi: . /s - - - sha: doc_id: cord_uid: cnz qjy many of the most virulent emerging infectious diseases in humans, e.g., aids and ebola, are zoonotic, having shifted from wildlife populations. critical questions for predicting disease emergence are: ( ) what determines when and where a disease will first cross from one species to another, and ( ) which factors facilitate emergence after a successful host shift. in wild primates, infectious diseases most often are shared between species that are closely related and inhabit the same geographic region. therefore, humans may be most vulnerable to diseases from the great apes, which include chimpanzees and gorillas, because these species represent our closest relatives. geographic overlap may provide the opportunity for cross-species transmission, but successful infection and establishment will be determined by the biology of both the host and pathogen. we extrapolate the evolutionary relationship between pathogen sharing and divergence time between primate species to generate “hotspot” maps, highlighting regions where the risk of disease transfer between wild primates and from wild primates to humans is greatest. we find that central africa and amazonia are hotspots for cross-species transmission events between wild primates, due to a high diversity of closely related primate species. hotspots of host shifts to humans will be most likely in the forests of central and west africa, where humans come into frequent contact with their wild primate relatives. these areas also are likely to sustain a novel epidemic due to their rapidly growing human populations, close proximity to apes, and population centers with high density and contact rates among individuals. emerging infectious diseases (eids) pose a serious and increasing threat to human health and welfare (daszak et al., ; king et al., ; jones et al., ) . diseases that have recently emerged in humans include sars, hiv, and swine flu. it is projected that . million people are currently infected with hiv/aids (who, ) , and in alone, it was estimated that billion dollars would be needed to prevent future hiv transmission and provide care for those already infected (who, ) . in wildlife populations, eids also have resulted in recent and dramatic declines in mammals (e.g., ebola in african apes; walsh et al., , canine distemper virus in wild dogs and lions; roelke-parker et al., ) , amphibians (e.g., chytridi-omycosis fungus in global amphibian populations; daszak et al., ) , insects (e.g., the mite varroa jacobsoni in honey bees; oldroyd, ) , and birds (e.g., conjunctivitis due to mycoplasma gallisepticum in passerines; williams et al., ) . human eids originate from multiple sources (e.g., host shifts from animal reservoirs [zoonotics] , evolution of existing organisms, and reemergence due to antimicrobial resistance), are increasing in number, and are globally distributed (jones et al., ) . although there is not sufficient data to evaluate whether there is a similar trend for increasing frequency of disease emergence in wildlife populations, if similar drivers influence the process of disease emergence we might expect this to be the case (daszak et al., ) . the ability to predict when, where, and within which species pathogens are most likely to emerge is therefore increasingly urgent. understanding the process of disease emergence will be critical for developing strategies to minimize risk and reduce the high cost of managing outbreaks once a disease has emerged. several review papers have explored the ecological and evolutionary drivers of disease emergence in human and wildlife populations and the conditions most likely to heavily impact new host species (daszak et al., ; osterhaus, ; jones et al., ; parrish et al., ) . ecological drivers that favor emergence are most frequently associated with changes in host or pathogen ecology, specifically ( ) an increase in host population density and contact rates, ( ) environmental changes that effect host quality and demography, and ( ) changes in host mobility and behavior. evolutionary drivers of emergence include high genetic variability in the pathogen population (morand et al., ; gupta et al., ) or a lack of variability in the host (daszak et al., ; altizer et al., b) . however, sustainable emergence of a pathogen on a host is only one outcome of a complex multistep process. we focus on disease emergence after host shifts, defined as the movement of a pathogen to a new host species irrespective of the long-term consequences (antonovics et al., ) . emergence after a host shift can result in rapid spread and high virulence because naïve hosts may lack appropriate immune responses (osterhaus, ; altizer et al., b) . the longer-term outcomes of a host shift can range from transient, unsustained ''spillover'' events (e.g., hantavirus; hughes et al., ) to persistent, self-sustaining epidemics (e.g., hiv; hahn et al., ; fenton and pedersen, ) . there are three key stages required for successful emergence of an infectious disease on a ''new'' target host: ( ) opportunity, ( ) infection and transmission, and ( ) establishment and sustainability ( fig. ) . close geographic proximity among hosts likely increases contact rates and, hence, opportunity for shifts (step , fig. ). although geographic overlap may be important for determining which pathogens a host species is exposed to, successful infection and then transmission of the pathogen in the new host will depend on evolutionary and ecological factors determined by the biology of both host and pathogen (step , fig. ). for example, recent comparative analyses have demonstrated that viruses are the most likely group to cross species boundaries (cleaveland et al., ; pedersen et al., ) , perhaps facilitated by high mutation rates and fast generation times providing the potential to rapidly overcome barriers to infection (parrish et al., ) . in contrast, within primates, half of the recorded helminth pathogens are specific to a single host species . pathogen taxonomy and, by implication, pathogen biology, may then serve as an indicator of potential for host . opportunity (biogeography of host & pathogen) . transmission (evolutionary & ecological barriers) (contact rate, demography, population density) figure . three key stages of disease emergence: ( ) opportunity, ( ) infection and transmission, and ( ) establishment. the biogeography of both hosts and pathogens will contribute to the opportunity for cross-species transmission. for natural populations, opportunity for new pathogens may be restricted to neighboring species; however, wildlife trade, invasive species, and domestic animals also may be a source of new pathogens. the ability for a pathogen to infect and be transmitted within the new host species is likely driven by ecological and evolutionary barriers, such as phylogenetic relatedness of the hosts and the evolutionary potential of the pathogen. lastly, multiple demographic factors will affect the ultimate sustainability and establishment of a pathogen following a host shift, including population density, contact rates, and the rate of spread of the pathogen. shifts. however, host characteristics also are important. close evolutionary relationships among host species might translate into similar immunological responses and lifehistory traits (pfennig, ; ricklefs and fallon, ; perlman and jaenike, ) , and hence increase the likelihood of successful cross-species infection. the rate of spread in the new host and the progression through the stages of host shift to sustainable disease emergence will be affected by host and pathogen demographics (step , fig. ), for example, population density, contact rates, and the rate of spread of the pathogen in the new host (r ; number of new cases per infected individual) (dobson and foufopoulos, ; fenton and pedersen, ; . mathematical models of hostpathogen interactions (e.g., traditional sir-susceptible, infected and recovered-models) indicate that for diseases transmitted in a density dependent fashion (i.e., by direct contact), pathogens will only persist and become established in populations above a critical threshold density (r > ; anderson and may, ) . disease emergence may be inhibited at any stage, providing the opportunity for alternative management strategies; unfortunately, this complexity contributes to the difficulty in generating accurate predictive models. in davies and pedersen ( ) , we demonstrated a significant relationship between the geographical distribution and evolutionary relatedness of primate host species, and the similarity of their pathogen communities. this relationship reflects both the opportunity for host-host contact (geographic overlap), and the barriers to infection (host biology) (steps and , fig. ). we expand on our previous analysis to explore the potential for future pathogen host shifts and disease emergence within and between wild primates and humans. first, we use the relationship between host relatedness, geographic overlap, and pathogen community similarity to quantify the risk of future host shifts that each species faces. second, we evaluate how past host shifts between primates might have shaped pathogen communities by analyzing patterns of host specificity. third, we explore whether the phylogenetic risk of host shifts may have shaped host geographic distributions over time. finally, we identify regions of the globe where we predict host shifts between nonhuman primates and humans may be most frequent. this analysis provides the first quantitative attempt to assess the risk of pathogens host-shifting to humans from wildlife populations, a critical step toward predicting disease emergence. pathogen species occurrences were obtained from the global mammal parasite database www.mammalparasites.org), comprising , records representing pathogen species (including viruses, bacteria, helminths, protozoa, arthropods, and fungi) across of the wild primate species recognized in the phylogenetic tree of bininda-emonds et al. ( ) . the human disease database from taylor et al. ( ) was used to measure pathogen sharing between primates and humans. we use the term ''pathogen'' broadly to include both microparasites (i.e., viruses, bacteria, and protozoans) and macroparasites (i.e., helminths, fungi, and arthropods). for all pair-wise primate-primate combinations we estimated pathogen community similarity as: a/(a + b + c), where a is the number of pathogen species found on two host species, x and y; b is the number of pathogen species on host x that are not found on host y; and c is the number of pathogen species on host y that are not found on host x (for further details see davies and pedersen, ) . critically, this metric is not biased by differences in sampling intensity or the relative sizes of the pathogen species pools of x and y. we focus on general patterns of pathogen sharing, thus our estimate of pathogen community similarity was calculated for all pathogen types combined (helminths, protozoa, and viruses). the relationship between community similarity and evolutionary divergence may differ across pathogen taxonomy; however, we currently lack comprehensive data on pathogen occurrences (and absences), which limits the scope of more taxon-specific analyses. in theory, our approach could be applied to any pathogen subset given sufficient data. following davies and pedersen ( ) , we derived the relationship between evolutionary divergence (representing time to most recent common ancestor from the dated phylogenetic tree of bininda-emonds et al., ) , and pathogen community similarity (as described above) between each primate pair using generalized linear modeling (glm) with binomial errors and a logit link function in the statistical package r (r: a programming environment for data analysis and graphics, v. . . ; http://www.r-project.org/). we used a glm approach because this enabled the shape of the rela-tionship between pathogen sharing with evolutionary relatedness to be characterized, and followed a similar protocol to that in previously published studies (gilbert and webb, ) . because we explore the combined risk of cross-species transmission between co-occurring primate hosts separately (see below), we did not include range overlap in the glm directly. for our model, we assume that pathogen community similarity (pathogen sharing) reflects the frequency of host shifts between any pair of primate species; the greater the frequency of host shifts, the more similar the pathogen communities. however, we note that pathogen community sharing will also reflect co-inheritance of similar pathogen communities from a common primate ancestor. although the relative importance of co-inheritance versus host-shifts in structuring pathogen communities remains unresolved, experimental cross-infection of fungal pathogens among tropical plant species (gilbert and webb, ) revealed a qualitatively similar relationship between host evolutionary relationships and cross-infection success, to that between host relatedness and pathogen sharing in primates (davies and pedersen, ) . we therefore suggest that, although co-inheritance will be important, the shape of the relationship may be determined by host shifts. nonetheless, disentangling co-inheritance from host shifts should be a focus of future studies but will require detailed information on pathogen phylogeny for many species. for each primate species, we calculated a metric encapsulating the summed risk of cross-species pathogen transmission (potential for host shifts) posed by all geographically co-occurring primates. this metric was calculated from the glm of pathogen community similarity against divergence times of each primate pair. we refer to this metric as the phylogenetic risk of host shifts (prhs). phylogenetic risk for species x (prhs x ) is then calculated as: where prhs x is the summation of the phylogenetic risk (prhs y ) for each s host species that overlaps in geographic range with the target species x. the phylogenetic risk from each overlapping species (y) to the target species x (prhs y ) is derived from the glm given the phylogenetic relatedness of species x and y. we hypothesize that primate species with high prhs will share a larger proportion of their pathogens with their geographic neighbors and close relatives. we therefore predict that: ( ) pathogen species richness will correlate positively with prhs, and ( ) the proportion of host-specific pathogens will decrease with increasing prhs. our metric (prhs) assumes that each overlapping species contributes additively to the total risk of a pathogen shifting to the target species. because the number of primate pathogens is finite, it is possible that the target species could accumulate all pathogens found in the local community of hosts. although we do not have complete data on the total pathogen community size of any one host or community, the large number of species-specific pathogens (> % in wild primates; pedersen et al., ) indicates that complete pathogen sharing within a community is unlikely. our model therefore predicts a positive relationship between the number of overlapping host species and within-host pathogen species richness. correlates of pathogen species richness in primates have been explored elsewhere (nunn et al., and may be confounded by variation in sampling intensities (altizer and pedersen, ) . we focused on testing our second prediction, relating to host range, which is the primary focus of this paper. if pathogen communities are shaped by host shifts, we predict that host species with high prhs would have fewer host-specific pathogens because they would be shared with other members of the host community. to test this prediction, we constructed a further glm to evaluate the relationship between prhs and the proportion of host-specific pathogens in each host's parasite community. because the number of recorded pathogens varies considerably between hosts (see above), some estimates of host specificity will be derived from very small sample sizes. to explore model sensitivity, we therefore repeated the glm weighting each datum by the logarithm of the total number of pathogens sampled for each species. previous studies found that viruses are the least likely to be specific to a single primate host . therefore, we ran a final glm of prhs against the proportion of host specific nonviral pathogens only. we hypothesize that phylogenetic risk of host shifts might limit geographic co-occurrence between closely related host species. this process would lead to the prediction that the observed prhs x should be lower than expected from a null model of drawing neighbors at random from the regional species pool. to evaluate whether prhs across wild primates varies nonrandomly or simply reflects the geographical distribution of host species within regional communities, we compared observed prhs x for each x primate host against a null distribution generated by randomly drawing ''neighbors'' from the phylogeny. for each primate host, we performed , random draws, with sample size of neighbors, s, matching the empirical number of hosts that overlaps with each x species' range. because of the large geographic distance separating the new and old worlds, new and old world primates will never have the opportunity to overlap, thus we performed our random draw of neighbors only from within the regional primate communities. to generate a geographical representation of the potential for host shifts within wild primates, we first constructed a weighted distribution map of primates, using our prhs metric as weights. primate species distributions were obtained from grenyer et al. ( ) . because the probability of pathogen sharing was influenced only by whether two species were found in sympatry across any part of their range, but not by the magnitude of range overlap (davies and pedersen, ) , we assumed prhs to be uniform across each target host species range. spatial data manipulation was performed in arcview (gis . , environmental systems research institute inc.) and arcmap (v . environmental systems research institute inc.) at a grid cell size of ° °. cell weights (cell w ) summed the phylogenetic risk of host shifts across co-occurring species and were calculated as: where k is the number of primate hosts with ranges intersecting with cell w . hotspots represent regions with high diversity of closely related primate species-where we predict host shifts will be most frequent. next, to provide an estimate of the cross-species pathogen transmission risk from wild primates to humans, we constructed a second hotspot map, weighting each primate distribution in proportion to its evolutionary distance from humans, using the nonlinear transformation determined from the glm model coefficients described above. here, cell weights (cell w ) are calculated as: where prhs h is the phylogenetic risk of host shifts from species x to homo sapiens. our hotspot maps provide a coarse estimate of the potential for host shifts, accounting for opportunity (geography) and biology (phylogeny). however, successful establishment of a pathogen in a novel host will depend on several additional factors, including those that influence contact rates among and within host species (fenton and pedersen, ; wolfe et al., ) . for example, habitat disturbance and transformation, resulting from human growth, may increase the probability of humans encountering new pathogens from wildlife. as a first approximation, we use estimates of human population growth ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ; sedac: http://sedac.ciesin.columbia.edu/gpw/) to represent human expansion into previously untransformed or isolated habitats, and hence potential for increased human-wildlife contact. we map the product of prhs to humans (cell weights cell w ) and an index of human population growth (increase in population density during years) to highlight areas of both high population growth and many closely related primates. here, cell weights (cell w ) are calculated as: last, we consider minimum threshold population densities required for sustained transmission and successful establishment of a disease after a host shift and map human population centers (cities > , ) on the weighted human risk map. population centers in close proximity to regions with high phylogenetic risk of host shifts and human population growth are likely to be foci of disease emergence. a third of the variation in pathogen communities can be explained by phylogenetic relatedness; more closely related primate hosts have more similar pathogen communities (glm of pathogen community similarity against divergence time: p value < . , z = - . , degrees of freedom (df) = , deviance explained = %; see also davies and pedersen, ) . phylogenetic risk of host shifts (prhs), our metric summarizing the likelihood of pathogens shifting between neighboring hosts, varied considerably across primates (fig. ) fig. a ), as expected, given that our metric, prhs, is a summation of all overlapping primates. the brown capuchin (cebus apella), with a geographic range extending from colombia and venezuela to paraguay and northern argentina, has the greatest prhs ( . ), and overlaps with more than other new world primate species across its large range. critically, however, phylogenetic risk of host shifts frequently differs even between primates overlapping with the same number of neighbors. for example, demidoff's bushbaby (galagoides demidoff) and the white-fronted capuchin (cebus albifrons) both have geographic distributions that overlap with other primate species, but the former has a prhs of . , and the latter . . across primates, our randomization procedure indicates a tendency for prhs to be greater than predicted from a random draw of neighbors (sign test of the number figure . a scatter plot of phylogenetic risk against the number of overlapping species for each primate host. risk correlated positively with overlap; however, there was still large variation in risk among species with the same number of neighbors. variation in phylogenetic risk is a product of both number of neighbors and phylogenetic relationships in the primate tree. b scatter plot of the proportion of host-specific pathogens against phylogenetic risk; symbol size is proportional to the total number of recorded pathogens for each host species respectively. hosts with high phylogenetic risk have significantly fewer host-specific pathogens, supporting our hypothesis that host shifts are more frequent among high-risk species. of times observed prhs is greater than predicted from random draws: p < . ), indicating frequent geographical overlap among close relatives. primates with higher phylogenetic risk had a significantly lower proportion of host specific pathogens (all pathogens combined), consistent with an increased frequency of host shifts in species with lots of closely related neighbors, but this was found only in the weighted glm (p value < . , z = - . , df = ; p = . for the unweighted glm). when excluding viruses, the relationship between prhs and the proportion of host-specific pathogens is significant with and without model weights (weighted glm: p < . , z = - . , df = ; unweighted glm: p < . , z = - . , df = ). unsurprisingly, there was large scatter in the relationship between prhs and the proportion of host-specific pathogens recorded for each primate host (fig. b) , suggesting other factors also are important in shaping pathogen communities. there are much fewer host-specific pathogens for primates with very high prhs (> ), although the sample size is relatively small. in contrast, primates with low prhs (< ) have varying proportions of host-specific and hostgeneralist pathogens (fig. b ). we show central africa and western amazonia to be hotspots of phylogenetic risk for host shifts between wild primates (fig. a) . these regions contain many closely related primate species that overlap in their geographical ranges. we interpret these hotspots to indicate regions where the frequency of pathogen transmission between wild primates is likely to be the highest, and thus we additionally predict increased pathogen community similarity, high within-primate pathogen species richness and a low frequency of host specific pathogens within these localities. centers of high phylogenetic risk of host shifts from wild primates to humans include central and western africa. areas of low risk include much of brazil, southern africa below °s, and northern india into nepal (fig. b) . therefore, despite the high frequency of predicted host shifts between wild primates within amazonia, it is not identified as a high risk region for shifts to humans. new world primates are more distantly related to humans than old world primates, and hence pose lower phylogenetic risk. the likelihood of successful establishment and sustained within-host transmission of a novel pathogen will be affected by several ecological and demographic factors. in humans, a critical factor is likely to be human-wildlife contact rates, population connectivity, and human population density. to stimulate discussion, we map one such factor-the intersection between human population growth (as a proxy for increasing human-wildlife contact; see methods), phylogenetic risk of host shifts to humans, and the distribution of human population centers of size > , (fig. c) . we do not have accurate data to model these terms directly; in particular, it is not clear how the likelihood of disease emergence decays with distance from population centers, or how human movement facilitates disease emergence. disease emergence is a complex, multistage process, but our results show that we can begin to make predictions about when and where diseases are likely to emerge. by understanding the factors contributing to each stage: opportunity, infection, and ultimately, establishment of a novel pathogen, we can identify which lineages and within which geographic areas disease emergence may be most likely. previous work has mapped where diseases have emerged in humans in the past (jones et al., ) and which pathogens likely pose the biggest risk of host shifts to humans (cleaveland et al., ) . our study is the first to make predictions about the likelihood of host shifts in the future. specifically, we use a robust evolutionary relationship between host relatedness and pathogen sharing to quantify the phylogenetic risk of host shifts within primates. . a hotspot map of the summed phylogenetic risk for host shifts across wild primates. central africa and west amazonia represent regions of high geographical overlap among many closely related primate species. we predict host shifts among primates to be frequent in these localities. b phylogenetic risk of pathogens host shifting to humans from wild primates. west central africa is a hotspot of high risk to humans, due to the overlapping ranges of many of our closest relatives. c the intersection between high phylogenetic risk and an index of human population growth (increase in density from [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , revealing regions where we expect high rates of human contact with primate species that pose the greatest risk of cross-species pathogen transmission to humans. open circles indicate human population centers (> , ) that may facilitate disease emergence following host shift events. we identify species between which host shifts are likely to be frequent, and map geographic areas in which they are distributed. in addition, we generate a hotspot map to highlight regions where humans may be most at risk of host shifts from wild primates. last, we explore recent demographic trends in human populations that might contribute to disease emergence following successful cross-species transmission. cross-species pathogen transmission events can heavily impact both human and wildlife populations (daszak et al., ; parrish et al., ) . however, limited data on disease in natural populations, in particular, historical information on when and where a pathogen first moved between host species, has made it difficult to generate predictive models (hopkins and nunn, ) . in a recent paper, we demonstrated that pathogen community similarity among primate species-pairs was correlated closely with their geographical proximity and phylogenetic relatedness (davies and pedersen, ) . close geographical proximity provides the opportunity for host-host contact and hence pathogen transfer, whereas phylogenetic distance might be indicative of the ease with which a pathogen can infect a novel host given the opportunity. in this paper, we extrapolate the observed relationship between pathogen sharing and host evolutionary relatedness to explore the likely frequency of host shifts within communities of primate hosts. we predict that host shifts will be most frequent among co-occurring and closely related hosts. therefore, the frequency of host shifts will reflect both the geographical distribution of host species across the landscape, as well as the shape of the phylogenetic tree that connects them (fig. ) . for example, the white fronted capuchin (cebus albifrons) overlaps in its distribution with many other new world primates and is nested within the recent new world radiation of capuchin monkeys (genus: cebus). we predict the risk of cross-species pathogen transmission to be high for this species. in contrast, demidoff's bushbaby (galagoides demidoff) is common across central and western africa, and also overlaps with many other primates in its distribution, but has only few close relatives. we predict intermediate risk of cross-species pathogen transmission for this species. the lowest risk of cross-species pathogen transmission is predicted for evolutionary distinct species in regions of low primate diversity (fig. ) . our model assumes that primate hosts will differ in their vulnerability to host shifts. we predict that frequent host shifts will increase pathogen species richness and de-crease the proportion of host-specific pathogens per host, as pathogens are shared with an increasing fraction of the host community. however, evaluating pathogen species richness is confounded by sampling artifacts (altizer and pedersen, ; but see nunn et al., ; lindenfors et al., ) , which may vary both taxonomically and geographically (hopkins and nunn, ) . we therefore focus on variation in host specificity. although it is possible that a systematic geographic bias in the distribution of pathogen species richness (e.g., as suggested for protozoan parasites; might additionally influence the frequency of host shifts, we do not believe that this trend would be sufficient to affect the strong geographical patterns found here. the results strongly support our hypothesis: primates with many close phylogenetic relatives and geographical neighbors have significantly fewer hostspecific pathogens. this relationship becomes stronger when we exclude viral pathogens, which tend to be less constrained by taxonomic barriers davies and pedersen, ) . to our knowledge, our study is the first to explore variation in host specificity among within-host pathogen communities across an entire mammalian order. in addition, we suggest that future studies figure . representation of the interaction between phylogeny and geography that may determine the frequency of host shifts within host communities. in the maps, warmer colors represent areas with many overlapping species ranges (geographically clumped distributions). in the phylogeny, red branches represent samples of closely related taxa (phylogenetically clumped). we predict the greatest frequency of host shifts will be observed among species which are both phylogenetically clumped (those with many close relatives) and geographically clumped (species with many overlapping neighbors) (top left). species that are neither geographically clumped nor phylogenetically clumped are predicted to have the lowest risk (bottom right). we expect intermediate risk of host shifts for species that are phylogenetically clumped but geographically dispersed, or geographically clumped but phylogenetically dispersed. investigating correlates of pathogen species richness should consider the frequency of host shifts. our results suggest that the frequency of host shifts between primates is likely highest in central africa and western amazonia-both centers of primate diversity. these findings are important because current estimates indicate that almost % of primates are considered threatened (iucn, ) , and infectious diseases, especially those that have resulted from a host shift, have been recognized recently as a significant driver of extinction risk in wild animals (pedersen et al., ; smith et al., smith et al., , . primates suffer from multiple threats, including habitat fragmentation and transformation, and direct exploitation (iucn, ) , which may increase susceptibility to disease-mediated declines (smith et al., ) . direct exploitation through hunting for bushmeat, necessitating frequent contact between humans and wild primates, is already associated with emerging infectious diseases moving to humans (i.e., siv, ebola, stlv- ; wolfe et al., ; nunn and altizer, ) . more recently, there has been increasing awareness of the risk for diseases moving from humans to wildlife populations. in tai national park, ivory coast endangered chimpanzee populations have suffered dramatic declines from pathogens shared with humans, including three outbreaks from pandemic human viruses (human respiratory syncytial virus (hrsv) and human metapneumoniovirus (hmpv); köngden et al., ) . significant declines in both chimpanzee and gorilla populations also have been documented across central africa due to infectious diseases (leroy et al., ) . ominously, it is likely that current risk scenarios are underestimates, based on limited pathogen sampling, especially in wild primates (hopkins and nunn, ) . geographic regions with high primate species richness, particularly, high hominidae diversity, such as west africa, pose a disproportionate risk of host shifts from wild primates to humans. notably, amazonia is not a hotspot for risks of shifts to humans, despite high primate diversity. new world monkeys diverged from old world monkeys and apes * mya, hence represent only distant evolutionary relatives to humans. although our model indicates host shifts from wild primates to humans to be most frequent within central and west africa, the likelihood of a host shift will be affected by additional factors influencing host-host contact rates. predicting successful host shifts will therefore require information on these additional factors. in this study, we used the rate at which humans are transforming natural habitat, indexed by human popula-tion growth, as a proxy for encounter rates between wildlife species and their associated pathogens. as human populations grow, encroach into new areas, and transform habitats, we may be at greater risk of contacting new pathogens (daszak et al., ; dobson and foufopoulos, ; weiss and mcmichael, ; wolfe et al., ) . this is of particular concern, given the positive correlation between human population growth and our modeled risk of host shifts (spearman's r = . , p = . , adjusting degrees of freedom to account for spatial autocorrelation), suggesting that in many parts of central africa, where host shifts are predicted to be frequent, human population growth also is high. global emergence of human infectious diseases frequently originate not from the point of first infection but from human population centers, where contact rates among individuals are high and where international travel and dissemination of the pathogen to other cities is facilitated (woolhouse, ; tatem et al., ; sharp and hahn, ) . in epidemiological models, directly transmitted pathogens are not likely to persist in populations below a critical size threshold (a function of both host and pathogen characteristics; anderson and may, ) . for example, measles is thought to go locally extinct in populations comprising less than , individuals (keeling and grenfell, ) . many host shift events may therefore never become established. however, large population centers in or near areas of rapid human expansion can provide the demographic component necessary to convert a host shift into a global emergence event. here, we map the distribution of the major human population centers across the globe. population centers that have the potential to facilitate the emergence of new diseases shifting from wild primates to humans are those in west and east central africa, including: kinshasa, democratic republic of congo; brazzaville, congo; and yaounde, cameroon to the west, and kampala, uganda; kigali, rwanda; and nairobi, kenya to the east. infectious disease detection and control, before emergence events, should focus on these regions. possibly the worlds most devastating pandemic, hiv- , provides a remarkable case study, closely matching our model predictions. resulting from a host shift from chimpanzees, one of our closest living relatives (korber et al., ) , hiv- , was first described in , but was introduced to human populations several times during the past years-two of which were from sivcpzptt (simian immunodeficiency virus) of chimpanzees (pan troglodytes troglodytes) from west central africa (korber et al., ) . one shift led to the global aids pandemic (hiv- group m), and the other resulted in a localized endemic in cameroon (hiv- group n) (keele et al., ) . although siv has been found in more than african primate species, chimpanzees are the only species that harbor the strain most closely linked to pandemic hiv- (sharp and hahn, ; keele et al., ) . recent analysis of hiv- group m suggests that several variants were circulating in the s, and molecular analysis now proposes that hiv- likely emerged from sivcpzptt at the turn of the th century ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) but had a period of relatively slow growth during the next years (worobey et al., ) . it is thought that hiv- group m was first transmitted to humans from wild chimpanzees in the southwest corner of cameroon (keele et al., ) , and its establishment and spread was likely facilitated by the rise of cities, specifically kinshasa, democratic republic of congo (formerly leopoldville, zaire). in , no population centers in the area of the cross-species transmission event were greater than , people; however, kinshasa grew rapidly as a trading center and had a population between , - , during the middle of the twentieth century when hiv- can be found in human samples (sharp and hahn, ; worobey et al., ) . kinshasa is km from the location of the original host shift event; however, it was a key transportation hub through river travel (sharp and hahn, ) . the successful emergence of hiv occurred in our predicted hot spot areas of high human risk. this example highlights the complex multistage process that drives disease emergence: a region with high risk of shifts to humans; a host shift from our closest relative; delayed emergence after the local growth of the human population at a transportation center. whereas human population density and encroachment into new habitats may affect the likelihood of the successful emergence of a primate pathogen into humans, the risk map of human disease emergence also highlights geographic locations where wild primates may be at the greatest risk of contracting human diseases. many pathogens from humans have been found in wild primate populations (e.g., polio, syphilis, influenza a, and measles) and pose a significant threat to their persistence (nunn and altizer, ) . almost half of wild primates face the risk of extinction (iucn, ) and pathogens have been linked to substantial population declines (formenty et al., , leroy et al., , köngden et al., ; understanding the patterns driving cross-species transmission and host shifts in wild populations will therefore be crucial for their conservation. within wildlife populations, ecological factors may be more important in determining rates of crossspecies transmission and host shifts within wild primates. host life history, ecological niche, feeding and activity patterns, sexual behavior, group size, and territoriality are strong determinants of pathogen transmission rates within a host population (altizer et al., a; nunn and altizer, ) , and therefore may be important for determining when and to whom cross-species transmission events are likely to be successful. although a comprehensive exploration of these ecological factors is beyond our current scope, knowledge of how host ecology is likely to interact with pathogen ecology, host phylogeny, and geography will be key for developing a predictive framework of wildlife disease emergence. in our analyses, we focused on primates because they are perhaps the best studied animal group and because of their close evolutionary relationship to humans. primates represent a significant disease reservoir for humans; approximately % of zoonotic human eids are shared with primate host species; however, other taxa (e.g., bats, rodents, birds, and domestic animals) also pose a threat of host shifts to humans (woolhouse and gowtage-sequeria, ) . nonetheless, we suspect that our results, specifically that phylogenetic tree shape and geographic dispersion are likely to affect the phylogenetic risk of a host shift, will likely extrapolate across taxa (see gilbert and webb, ) . last, we note that the relationship between pathogen taxonomy, host phylogeny, and host geography may be complex and vary across the different pathogen groups. for example, we might predict a greater frequency of host shifts among more distant relatives for viruses than for helminths, which suggests that the risk of viral emergence may be even greater than predicted in our framework. although data on pathogens from natural populations is incomplete, and there may be many factors that affect the likelihood of a host shift, our results suggest that a broad biogeographical approach can help predict cross-species pathogen transmission. by combining information across multiple species and communities, it is possible to detect general trends that are not apparent from single case studies. in contrast with traditional epidemiological studies, our analysis explores macroevolutionary and macroecological patterns in the distribution of pathogens across species and the 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emergence population biology of emerging and re-emerging pathogens emerging pathogens: the epidemiology and evolution of species jumps host range and emerging and re-emerging pathogens direct evidence of extensive diversity of hiv- in kinshasa by the authors thank k. smith key: cord- -s geepsc authors: vargas-garcía, cesar; lis-gutiérrez, jenny paola; gaitán-angulo, mercedes; lis-gutiérrez, melissa title: parasite-guest infection modeling: social science applications date: - - journal: advances in swarm intelligence doi: . / - - - - _ sha: doc_id: cord_uid: s geepsc in this study we argue that parasite-host infections are a major research topic because of their implications for human health, agriculture and wildlife. the evolution of infection mechanisms is a research topic in areas such as virology and ecology. mathematical modelling has been an essential tool to obtain a better systematic and quantitative understanding of the processes of parasitic infection that are difficult to discern through strictly experimental approaches. in this article we review recent attempts using mathematical models to discriminate and quantify these infection mechanisms. we also emphasize the challenges that these models could bring to new fields of study such as social sciences and economics. considering that the evolution of parasites and pathogens is important for human health, agricultural systems and wildlife [ , ] , there is a theory that focuses on how the mechanisms of infection can evolve. because viruses are the most abundant and simple entities on the planet, they are often used as models to study the evolution of parasitic infections. in particular, parameters such as replication, mortality rate of the infected host, infection rate (absorption rate), among others, have been suggested as possible control parameters used by parasites to optimally infect hosts [ ] [ ] [ ] [ ] . this paper reviewed the different mathematical models that describe the traditional and recently proposed infection mechanisms. in addition, we reviewed how these are used in the optimal dispersion of infections through susceptible host populations. in the first section, the classic theory of the evolution of the parasite is reviewed. this theory states that natural selection maximizes the number of secondary infections resulting from infection of a susceptible host through free channels that do not involve direct contact between infected and susceptible hosts [ ] . one way of doing this is by the evolution of the infection rate, which is the probability of a parasite infecting a host after direct contact. in restricted environments, the classical theory predicts that a parasite will evolve to an infinite maximum infection rate. however, experiments using bacteria as a host and viruses as parasites show the unexpected appearance of viruses with a moderate or intermediate infection rate [ , ] . how and under what conditions this intermediate rate evolves is still an open question. the proposed section reviews the classical and recent models that try to explain this phenomenon it has been suggested that infection channels between infected and susceptible hosts may provide an advantage, either by allowing parasites to evade the host's immune response [ ] , reducing antiviral drug activity [ ] , or simply having a more efficient mode of infection. in the second section, a novel model of parasite-host interactions is proposed that accounts for transmission, both through free channels (not involving contact between infected and susceptible hosts), and through infections produced by contact between hosts. the last section examines the possible social and economic science applications that could result from this modeling. first consider a basic model for parasite dynamics introduced by [ ] . let h, i and p be the number of healthy and infected hosts and parasites, respectively. host -free infection a healthy host reproduces at a rate λ and dies at a d h rate. the parasite attacks hosts at a rate of rph, where r is the rate of infection. once the infected host dies (with latency period /d i ), a set of b-size parasites is released. alternatively, the term bd i i in ( ) can be replaced by bi in situations where infected hosts release parasites throughout their life cycle rather than dying before releasing them. parasites that are free in the environment (outside the infected host) can die at a d p rate. the level of parasites in the steady state system is: equation ( ) can be seen as a way of measuring the parasite's ability to infect. note, that for the parasite to develop its maximum infective capacity, the infection rate should be infinite (the maximum population of the parasite in a stable state is λ(b − )/d p ). alternatively, the number of secondary infections can be used to represent the performance of the parasite. remember that the infection-free steady state, given by h = λ/d h , i = , y p = , is an unstable point (meaning that infection will take place) if where r is the number of secondary infections and can be interpreted as the number of newly infected hosts produced by an infection. r can be used to infer the evolutionary outcome of the system ( ). for example, from ( ) it is derived that the parasite should evolve towards infinite infection rates to obtain the maximum fit. the experiments shown in the literature challenge the theory that parasites can evolve to an infinite infection rate, suggested by the previous model. these experiments show the unexpected appearance of parasites with moderate or low infection rates [ , ] . intermediate infection rates can be explained by the presence of the spatial structure of the host [ ] . presumably, parasites with high infection rates tend to create a shielding effect in which the local availability of healthy hosts is reduced, resulting in more interactions with the parasite-infected host, leading to a rate of new parasites equal to zero [ ] . figure shows the shielding effect. this shielding effect can be incorporated into the previous model, assuming the number of parasites released by the death of an infected host as a function of the infection rate [ ] . where b is the maximum number of parasites released that can be obtained from the death of infected cells. d f represents the ability of the newly released parasite to escape from the harmful residues produced by the death of the infected host. a larger d f means that the parasite has a high probability of finding a healthy host to infect and reproduce. x represents the average amount of waste generated by the death of a single infected host. this modification produces a new level of parasites in a stable state with an optimal finite infection rate given by ( ) in addition, there is an optimal number of secondary infections given this finite rate of reproduction [ ] . the our system ( - ) was modified to include the ability of the parasite to carry out transmission by direct contact between infected and susceptible hosts. let s the number of parasites sent through the channel formed between an infected and a susceptible host. in addition to the infections produced by the traditional mechanism of infection (without direct contact) r p h, we add an additional production of infections represented by p(s)βp h. here β is the rate of interaction between infected and uninfected hosts. the p(s) function is the probability that an uninfected host will become infected by receiving s parasites through the channel formed between an infected and a susceptible host. the probability p(s) is defined as: where σ (s) is the probability that the infected and uninfected host will form a host-tohost channel. f (s) is the probability that sending pathogens through a given host-to-host channel will result in an infection, and can be any monotonously increased function in s. assuming the probability of parasites infecting a cell as a binomial distribution. if each copy of the parasite has an r probability of successful infection, then that is, f(s) is the probability that at least one of the parasites will have a successful infection given that there is an enabled host-to-host channel. there are two possible scenarios for host-to-host channel formation: channels between infected and uninfected hosts; and channels between infected hosts. the first scenario leads to an infection with probability p(s). therefore, there is a reduction in the number of s σ(s) h i parasites that cannot be used in other infections. the other scenario arises because there is no discrimination mechanism that causes the infected host to form channels with the uninfected host. channels between infected hosts produce a waste of parasites s σ(s) s σ (s) ri that does not produce additional infections, because both cells are already infected. figure shows the three possible routes of infection from host to host: transmission of parasites without involving direct contact between hosts; transmission of parasites through channels between infected and uninfected hosts; and transmission of parasites through channels between infected hosts. including the mechanism described above in the system ( - ) results in the increased. an application to the aids virus of this augmented system is available at work [ ] . host -to -host infection ( ) host -to -host infection according to fig. , a parasite has the ability to infect cells through (a) a free channel without direct interaction; (b) through contact. in the former, infected cells produce chains of rna (red lines) that use information from the parasite stored in their genome (blue and red line), encapsulate them (blue and red concentric circles) and send these out of the cell. this paper reviewed current approaches in ecology through the use of mathematical tools such as ordinary differential equations (odes). extended models were presented that address issues under debate in ecology, such as optimizing parasite-host interactions and why host infection mechanisms can be beneficial to parasites. whether these models and their projections of infection spread can be applied to fields such as economics, business administration and public policy is relevant for future research. for example, recent studies have suggested studying crime in a region in a manner similar to an epidemic. one could, for example, predict how many additional crimes occur in a given season and design public policy using these models. in the medical field, one could determine which transmission model (host-to-host or non-host) is most effective in spreading and mitigating infections, or in agronomic science [ , ] . computational modeling approaches to the dynamics of oncolytic viruses chapter -parasite-host interactions fitness of rna virus decreased by muller's ratchet experimental selection reveals a trade-off between fecundity and lifespan in the coliphage qß conditions for invasion of synapse-forming hiv variants fitness benefits of low infectivity in a spatially structured population of bacteriophages are parasites "prudent" in space? local interactions select for lower pathogen infectivity viral infection: changing sides to get in cell-to-cell hiv- spread and its implications for immune evasion cell-to-cell spread of hiv permits ongoing replication despite antiretroviral therapy population dynamics of immune responses to persistent viruses emergence of increased frequency and severity of multiple infections by viruses due to spatial clustering of hosts optimal adsorption rate: implications of the shielding effect synaptic transmission may provide an evolutionary benefit to hiv through modulation of latency developing a thief: haustoria formation in parasitic plants prediction rules in e-learning systems using genetic programming key: cord- - lmwnfda authors: ray, sumanta; lall, snehalika; mukhopadhyay, anirban; bandyopadhyay, sanghamitra; schonhuth, alexander title: predicting potential drug targets and repurposable drugs for covid- via a deep generative model for graphs date: - - journal: nan doi: nan sha: doc_id: cord_uid: lmwnfda coronavirus disease (covid- ) has been creating a worldwide pandemic situation. repurposing drugs, already shown to be free of harmful side effects, for the treatment of covid- patients is an important option in launching novel therapeutic strategies. therefore, reliable molecule interaction data are a crucial basis, where drug-/protein-protein interaction networks establish invaluable, year-long carefully curated data resources. however, these resources have not yet been systematically exploited using high-performance artificial intelligence approaches. here, we combine three networks, two of which are year-long curated, and one of which, on sars-cov- -human host-virus protein interactions, was published only most recently ( th of april ), raising a novel network that puts drugs, human and virus proteins into mutual context. we apply variational graph autoencoders (vgaes), representing most advanced deep learning based methodology for the analysis of data that are subject to network constraints. reliable simulations confirm that we operate at utmost accuracy in terms of predicting missing links. we then predict hitherto unknown links between drugs and human proteins against which virus proteins preferably bind. the corresponding therapeutic agents present splendid starting points for exploring novel host-directed therapy (hdt) options. the pandemic of covid- (coronavirus disease- ) has affected more than million people. so far, it has caused about . million deaths in over countries worldwide (https://coronavirus.jhu.edu/map.html), with numbers still increasing rapidly. covid- is an acute respiratory disease caused by a highly virulent and contagious novel coronavirus strain, sars-cov- , which is an enveloped, single-stranded rna virus . sensing the urgency, researchers have been relentlessly searching for possible therapeutic strategies in the last few weeks, so as to control the rapid spread. in their quest, drug repurposing establishes one of the most relevant options, where drugs that have been approved (at least preclinically) for fighting other diseases, are screened for their possible alternative use against the disease of interest, which is covid- here. because they were shown to lack severe side effects before, risks in the immediate application of repurposed drugs are limited. in comparison with de novo drug design, repurposing drugs offers various advantages. most importantly, the reduced time frame in development suits the urgency of the situation in general. furthermore, most recent, and most advanced artificial intelligence (ai) approaches have boosted drug repurposing in terms of throughput and accuracy enormously. finally, it is important to understand that the d structures of the majority of viral proteins have remained largely unknown, which raises the puts up the obstacles for direct approaches to work even higher. the foundation of ai based drug repurposing are molecule interaction data, optimally reflecting how drugs, viral and host proteins get into contact with each other. during the life cycle of a virus, the viral proteins interact with various human proteins in the infected cells. through these interactions, the virus hijacks the host cell machinery for replication, thereby affecting the normal function of the proteins it interacts with. to develop suitable therapeutic strategies and design antiviral drugs, a comprehensive understanding of the interactions between viral and human proteins is essential . when watching out for drugs that can be repurposed to fight the virus, one has to realize that targeting single virus proteins easily leads to the viruses escaping the (rather simpleminded) attack by raising resistance-inducing mutations. therefore, host-( ) we link existing high-quality, long-term curated and refined, large scale drug/protein -protein interaction data with ( ) molecular interaction data on sars-cov- itself, raised only a handful of weeks ago, ( ) exploit the resulting overarching network using most advanced, ai boosted techniques ( ) for repurposing drugs in the fight against sars-cov- ( ) in the frame of hdt based strategies. as for ( )-( ), we will highlight interactions between sars-cov- -host protein and human proteins important for the virus to persist using most advanced deep learning techniques that cater to exploiting network data. we are convinced that many of the fairly broad spectrum of drugs we raise will be amenable to developing successful hdt's against covid- . in the following, we will first describe the workflow of our analysis pipeline and the basic ideas that support it. we proceed by carrying out a simulation study that proves that our pipeline accurately predicts missing links in the encompassing drug -human protein -sars-cov- -protein network that we raise and analyze. namely we demonstrate that our (high-performance, ai supported) prediction pipeline accurately re-establishes links that had been explicitly removed before. this provides sound evidence that the interactions that we predict in the full network most likely reflect true interactions between molecular interfaces. subsequently, we continue with the core experiments. we predict links to be missing in the full (without artificially having removed links), encompassing drug -human protein -sars-cov- -protein network, raised by combining links from year-long curated resources on the one hand and most recently published covid- resources on the other hand. as per our simulation study, a large fraction, if not the vast majority of the predictions establish true, hence actionable interactions between drugs on the one hand and sars-cov- associated human proteins (hence of use in hdt) on the other hand. a b c d figure . overall workflow of the proposed method: the three networks sars-cov- -host ppi, human ppi, and drug-target network (panel-a) are mapped by their common interactors to form an integrated representation (panel-b). the neighborhood sampling strategy node vec converts the network into fixed-size low dimensional representations that perverse the properties of the nodes belonging to the three major components of the integrated network (panel-c). the resulting feature matrix (f) from the node embeddings and adjacency matrix (a) from the integrated network are used to train a vgae model, which is then used for prediction (panel-d). for the purposes of high-confidence validation, we carry out a literature study on the overall drugs we put forward. for this, we inspect the postulated mechanism-of-action of the drugs in the frame of several diseases, including sars-cov and mers-cov driven diseases in particular. see figure for the workflow of our analysis pipeline and the basic ideas that support it. we will describe all important steps in the paragraphs of this subsection. this reduces the training time compared to the general graph autoencoder model. we tested the model performance for a different number of sampled nodes, keeping track of the area under the roc curve (auc), average precision (ap) score, and model training time in the frame of a train-validation-test split at proportions : : . table shows the performance of the model for sampled sugraph sizes n s = , , , and . for sampled nodes, the model's performance is sufficiently good enough concerning its training time and validation-auc and -ap score. the average test roc-auc and ap score of the model for n s = are . ± . and . ± . . to know the efficacy of the model in discovering the existing edges between only cov-host and drug nodes, we train the model (with n s = ) on an incomplete version of the graph where the links between cov-host and drugs have been removed. we further compute the feature matrix f based on the incomplete graph, and use it. the test set consists of all the previously removed edges. the model performance is no doubt better for discovering those edges between cov-host and drug nodes (roc-auc: . ± . ap: . ± . for runs). the fastgae model is learned with the feature matrix (f) and adjacency matrix (a). the node feature matrix (f) is obtained from a using the node vec neighborhood sampling strategy. the model performance is evaluated with and without using f as feature matrix. figure shows the average performance of the model on validation sets with and without f as input for the different number of sampling nodes. we calculate average auc, and ap scores for complete runs of the model. from figure , it is evident that including f as feature matrix enhances the model's performance markedly. we use the node vec framework to learn low dimensional embeddings of each node in the compiled network. it uses the skipgram algorithm of the word vec model to learn the embeddings, which eventually groups nodes with a similar 'role' or having a similar 'connection pattern' within the graph. similar 'role' ensures that nodes within the sets/groups are structurally similar/equivalent than the other nodes outside the groups. two nodes are said to be structurally equivalent if they have identical connection patterns to the rest of the network . to explore this, we have analyzed the embedding results in two steps. first, we explore structurally equivalent nodes to identify 'roles' and similar connection patterns to the rest of the networks, and later use lovain clustering to examine the same within the groups/clusters. the most_similar function of the node vec inspects the structurally equivalent nodes within the network. we find out all the cov-host nodes which are most similar to the drug nodes. while it is expected to observe nodes of the same types within the neighborhood of a particular node, in some cases, we found some drugs are neighbors of cov-host proteins with high probability (pobs > . ). sars-cov- cl protease . some other drugs such as 'clenbuterol' and 'fenbendazole', the probable neighbor of ppp cb and eef a respectively, are used as bronchodilators in asthma. to explore the closely connected groups, we have constructed a neighborhood graph using the k-th nearest neighbor algorithm from the node embeddings and apply louvain clustering ( figure -panel-c). although there is a clear separation between host proteins (including cov-host) cluster and drug cluster, some of the louvain clusters contain both types of nodes. for example, louvain cluster- and - contain four and two drugs along with the other cov-host proteins, respectively. figure panel-d represents a network consisting of these six drugs and their most similar cov-host nodes. for drug-cov-host interaction prediction, we exploit variational graph autoencoder (vgae), an unsupervised graph neural network model, first introduced in to leverage the concept of variational autoencoder in graph-structured data. to make learning faster, we utilized the fastgae model to take advantage of the fast decoding phase. we have used two data matrices in the fastgae model for learning: one is the adjacency matrix, which represents the interaction information over all the nodes, and the other one is the feature matrix representing the low-dimensional embeddings of all the nodes in the network. we create a test set of 'non-edges' by removing all existing links between drugs and cov-host proteins from all possible combinations ( cov-host × drugs) of edges. the model is trained on the whole network with the adjacency matrix a and feature matrix f. the trained model is then applied to the test 'non-edges' to know the most probable links. we identified a total of most probable links with drugs and cov-host proteins with a probability threshold of . . the predicted cov-host proteins are involved in different crucial pathways of viral infection (table ). the p-values for pathway and go enrichment are calculated by using the hypergeometric test with . fdr corrections. figure , panel-a shows the heatmap of probability scores between predicted drugs and cov-host proteins. to get more details of the predicted bipartite graph, we figure . drug-cov-host predicted interaction: panel-a shows heatmap of probability scores between drugs and cov-host proteins. the four predicted bipartite modules are annotated as b , b , b and b within the heatmap. the drugs are colored based on their clinical phase (red-launched, preclinical-blue, phase /phase -green and phase- / phase- -black ). panel-b, c, d and e represents networks corresponding to b , b , b and b modules.the drugs are annotated using the disease area found in cmap database a b c d e figure . predicted interactions for probability threshold: . . panel-a shows the interaction graph between drugs and cov-host. drugs are annotated with their usage. panel-b, c, d and e represents quasi-bicliques for one, two, three and more than three drugs molecules respectively. use a weighted bipartite clustering algorithm proposed by j. beckett . this results in bipartite modules (panel-a figure ): b ( drugs, cov-host), b ( drugs, cov-host), b ( rugs and cov-host), and b ( drugs and cov-host). the other panels of the figure show the network diagram of four bipartite modules. b contains drugs, including some antibiotics (anisomycin, midecamycin), and anti-cancer drugs (doxorubicin, camptothecin). b also has some antibiotics such as puromycin, demeclocycline, dirithromycin, geldanamycin, and chlortetracycline, among them, the first three are widely used for bronchitis, pneumonia, and respiratory tract infections . some other drugs such as lobeline and ambroxol included in the b module have a variety of therapeutic uses, including respiratory disorders and bronchitis. the high confidence predicted interactions (with threshold . ) is shown in figure panel-a. to highlight some repurposable drug combination and their predicted cov-host target, we perform a weighted clustering (clusterone) on this network and found some quasy-bicluques (shown in panel-b-e) we matched our predicted drugs with the drug list recently published by zhou et al. and found six common drugs: mesalazine, vinblastine, menadione, medrysone, fulvestrant, and apigenin. among them, apigenin has a known effect in the antiviral activity together with quercetin, rutin, and other flavonoids . mesalazine is also proven to be extremely effective in the treatment of other viral diseases like influenza a/h n virus. . baclofen, a benzodiazepine receptor (gabaa-receptor) agonist, has a potential role in antiviral associated treatment . antiinflammatory antecedents fisetin is also tested for antiviral activity, such as for inhibition of dengue (denv) virus infection . it down-regulates the production of proinflammatory cytokines induced by a denv infection. both of the drugs are listed in the high confidence interaction set with the three cov-hosts: tapt (interacted with sars-cov- protein: orf c), slc a (interacted with sars-cov- protein: orf c), and trim (interacted with sars-cov- protein: orf a) ( figure -panel-c). topoisomerase inhibitors play an active role as antiviral agents by inhibiting the viral dna replication , . some topoisomerase inhibitors such as camptothecin, daunorubicin, doxorubicin, irinotecan and mitoxantrone are predicted to interact with several cov-host proteins. it has been demonstrated that the anticancer drug camptothecin (cpt) and its derivative irinotecan have a potential role in antiviral activity , . it inhibits host cell enzyme topoisomerase-i which is required for the initiation as well as completion of viral functions in host cell . daunorubicin (dnr) has also been demonstrated as an inhibitor of hiv- virus replication in human host cells . the conventional anticancer antibiotic doxorubicin was identified as a selective inhibitor of in vitro dengue and yellow fever virus replication . it is also reported that doxorubicin coupling with monoclonal antibody can create an immunoconjugate that can eliminate hiv- infection in mice cell . mitoxantrone shows antiviral activity against the human herpes simplex virus (hsv ) by reducing the transcription of viral genes in many human cells that are essential for dna synthesis . histone deacetylases inhibitors (hdaci) are generally used as latency-reversing agents for purging hiv- from the latent reservoir like cd memory cell . our predicted drug list (table ) contains two hdaci: scriptaid and vorinostat. vorinostrate can be used to achieve latency reversal in the hiv- virus safely and repeatedly . asymptomatic patients infected with sars-cov- are of significant concern as they are more vulnerable to infect large number of people than symptomatic patients. moreover, in most cases ( percentile), patients develop symptoms after an average of - days, which is longer than the incubation period of sars, mers, or other viruses . to this end, hdaci may serve as good candidates for recognizing and clearing the cells in which sars-cov- latency has been reversed. heat shock protein (hsp) is described as a crucial host factor in the life cycle of several viruses that includes an entry in the cell, nuclear import, transcription, and replication , . hsp is also shown to be an essential factor for sars-cov- envelop (e) protein . in , hsp is described as a promising target for antiviral drugs. the list of predicted drugs contains three hsp inhibitors: tanespimycin, geldanamycin, and its derivative alvespimycin. the first two have a substantial effect in inhibiting the replication of herpes simplex virus and human enterovirus (ev ), respectively. recently in , geldanamycin and its derivatives are proposed to be an effective drug in the treatment of covid- . inhibiting dna synthesis during viral replication is one of the critical steps in disrupting the viral infection. the list of predicted drugs contains six such small molecules/drugs, viz., niclosamide, azacitidine, anisomycin, novobiocin, primaquine, menadione, and metronidazole. dna synthesis inhibitor niclosamide has a great potential to treat a variety of viral infections, including sars-cov, mers-cov, and hcv virus and has recently been described as a potential candidate to fight the / sars-cov- virus . novobiocin, an aminocoumarin antibiotic, is also used in the treatment of zika virus (zikv) infections due to its protease inhibitory activity. in , chloroquine (cq) had been demonstrated as an effective drug against the spread of severe acute respiratory syndrome (sars) coronavirus (sars-cov). recently hydroxychloroquine (hcq) sulfate, a derivative of cq, has been evaluated to efficiently inhibit sars-cov- infection in vitro . therefore, another anti-malarial aminoquinolin drug primaquine may also contribute to the attenuation of the inflammatory response of covid- patients. primaquine is also established to be effective in the treatment of pneumocystis pneumonia (pcp) . cardiac glycosides have been shown to play a crucial role in antiviral drugs. these drugs target cell host proteins, which help reduce the resistance to antiviral treatments. the antiviral effects of cardiac glycosides have been described by inhibiting the pump function of na, k-atpase. this makes them essential drugs against human viral infections. the predicted list of drugs contains three cardiac glycosides atpase inhibitors: digoxin, digitoxigenin, and ouabain. these drugs have been reported to be effective against different viruses such as herpes simplex, influenza, chikungunya, coronavirus, and respiratory syncytial virus . mg , proteasomal inhibitor is established to be a strong inhibitor of sars-cov replication in early steps of the viral life cycle . mg inhibits the cysteine protease m-calpain, which results in a pronounced inhibition of sars-cov- replication in the host cell. in , resveratrol has been demonstrated to be a significant inhibitor mers-cov infection. resveratrol treatment decreases the expression of nucleocapsid (n) protein of mers-cov, which is essential for viral replication. as mg and resveratrol play a vital role in inhibiting the replication of other coronaviruses sars-cov and mers-cov, so they may be potential candidates for the prevention and treatment of sars-cov- . another drug captopril is known as angiotensin ii receptor blockers (arb), which directly inhibits the production of angiotensin ii. in , angiotensin-converting enzyme (ace ) is demonstrated as the binding site for sars-cov- . so angiotensin ii receptor blockers (arb) may be good candidates to use in the tentative treatment for sars-cov- infections . in summary, our proposed method predicts several drug targets and multiple repurposable drugs that have prominent literature evidence of uses as antiviral drugs, especially for two other coronavirus species sars-cov and mers-cov. some drugs are also directly associated with the treatment of sars-cov- identified by recent literature. however, further clinical trials and several preclinical experiments are required to validate the clinical benefits of these potential drugs and drug targets. in this work, we have successfully generated a list of high-confidence candidate drugs that can be repurposed to counteract sars-cov- infections. the novelties have been to integrate most recently published sars-cov- protein interaction data on the one hand, and to use most recent, most advanced ai (deep learning) based high-performance prediction machinery on the other hand, as the two major points. in experiments, we have validated that our prediction pipeline operates at utmost accuracy, confirming the quality of the predictions we have raised. the recent publication (april , ) of two novel sars-cov- -human protein interaction resources , has unlocked enormous possibilities in studying virulence and pathogenicity of sars-cov- , and the driving mechanisms behind it. only now, various experimental and computational approaches in the design of drugs against covid- have become conceivable, and only now such approaches can be exploited truly systematically, at both sufficiently high throughput and accuracy. here, to the best of our knowledge, we have done this for the first time. we have integrated the new sars-cov- protein interaction data with well established, long-term curated human protein and drug interaction data. these data capture hundreds of thousands approved interfaces between encompassing sets of molecules, either reflecting drugs or human proteins. as a result, we have obtained a comprehensive drug-human-virus interaction network that reflects the latest state of the art in terms of our knowledge about how sars-cov- and interacts with human proteins and repurposable drugs. for exploiting the new network-already establishing a new resource in its own right-we have opted for most recent and advanced deep learning based technology. a generic reason for this choice is the surge in advances and the resulting boost in operative prediction performance of related methods over the last - years. a particular reason is to make use of most advanced graph neural network based techniques, namely variational graph autoencoders as a deep generative model of utmost accuracy, the practical implementation of which was presented only a few months ago (just like the relevant network data). note that only this recent implementation enables to process networks of sizes in the range of common molecular interaction data. in essence, graph neural networks "learn" the structure of links in networks, and infer rules that underlie the interplay of links. based on the knowledge gained, they enable to predict links and output the corresponding links together with probabilities for them to indeed be missing. simulation experiments, reflecting scenarios where links known to exist in our network were re-established by prediction upon their removal, pointed out that our pipeline does indeed predict missing links at utmost accuracy. encouraged by these simulations, we proceeded by performing the core experiments, and predicted links to be missing without prior removal of links in our encompassing network. these core experiments revealed high confidence interactions relating to drugs. in our experiments, we focused on predicting links between drugs and human proteins that in turn are known to interact with sars-cov- proteins (sars-cov- associated host proteins). we have decidedly put the focus not on drug -sars-cov- -protein interactions, which would have reflected more direct therapy strategies against the virus. instead, we have focused on predicting drugs that serve the purposes of host-directed therapy (hdt) options, because hdt strategies have proven to be more sustainable with respect to mutations by which the virus escapes a response to the therapy applied. note that hdt strategies particularly cater to drug repurposing attempts, because repurposed drugs have already proven to lack severe side effects, because they are either already in use, or have successfully passed the preclinical trial stages. we further systematically categorized the repurposable drugs into categories based on their domains of application and molecular mechanism. according to this, we identified and highlighted several drugs that target host proteins that the virus needs to enter (and subsequently hijack) human cells. one such example is captopril, which directly inhibits the production of angiotensin-converting enzyme- (ace- ), in turn already known to be a crucial host factor for sars-cov- . further, we identified primaquine, as an antimalaria drug used to prevent the malaria and also pneumocystis pneumonia (pcp) relapses, because it interacts with the tim complex timm and alg . moreover, we have highlighted drugs that act as dna replication inhibitor (niclosamide, anisomycin), glucocorticoid receptor agonists (medrysone), atpase inhibitors (digitoxigenin, digoxin), topoisomerase inhibitors (camptothecin, irinotecan), and proteosomal inhibitors (mg- ). note that some drugs are known to have rather severe side effects from their original use (doxorubicin, vinblastine), but the disrupting effects of their short-term usage in severe covid- infections may mean sufficient compensation. in summary, we have compiled a list of drugs, which when repurposed are of great potential in the fight against the covid- pandemic, where therapy options are urgently needed. our list of predicted drugs suggests both options that had been identified and thoroughly discussed before and new opportunities that had not been pointed out earlier. the latter class of drugs may offer valuable chances for pursuing new therapy strategies against covid- . we have utilized three categories of interaction datasets: human protein-protein interactome data, sars-cov- -host protein interaction data, and drug-host interaction data. we have taken sars-cov- -host interaction information from two recent studies by gordon et al and dick et al , . in , high confidence interactions between sars-cov- and human proteins are predicted using using affinity-purification mass spectrometry (ap-ms). in , high confidence interactions are identified using sequence-based ppi predictors (pipe & sprint). the drug-target interaction information has been collected from five databases, viz., drugbank database (v . ) , chembl database, therapeutic target database (ttd) , pharmgkb database, and iuphar/bps guide to pharmacology . total number of drugs and drug-host interactions used in this study are and , respectively. we have built a comprehensive list of human ppis from two datasets: ( ) ccsb human interactome database consisting of , genes, and high-quality binary interactions - , ( ) the human protein reference database which consists of proteins and ppis. the summary of all the datasets is provided in table . cmap database is used to annotate the drugs with their usage different disease areas. we have utilized node vec , an algorithmic framework for learning continuous feature representations for nodes in networks. it maps the nodes to a low-dimensional feature space that maximizes the likelihood of preserving network neighborhoods. the principle of feature learning framework in a graph can be described as follows: let g = (v, e) be a given graph, where v represents a set of nodes, and e represents the set of edges. the feature representation of nodes (|v |) is given by a mapping function: f : v → r d , where d specify the feature dimension. the f may also be represented as a node feature matrix of dimension of |v | × d. for each node, v ∈ v , nn s (v) ⊂ v defines a network neighborhood of node v which is generated using a neighbourhood sampling strategy s. the sampling strategy can be described as an interpolation between breadth-first search and depth-first search technique . the objective function can be described as: this maximizes the likelihood of observing a network neighborhood nn s (v) for a node v given on its feature representation f . now the probability of observing a neighborhood node n i ∈ nn s (v) given the feature representation of the source node v is given as : where, n i is the i th neighbor of node v in neighborhood set nn s (v). the conditional likelihood of each source (v) and neighborhood node (n i ∈ nn s (v )) pair is represented as softmax of dot product of their features f (v) and f (n i ) as follows: variational graph autoencoder (vgae) is a framework for unsupervised learning on graph-structured data . this model uses latent variables and is effective in learning interpretable latent representations for undirected graphs. the graph autoencoder consists of two stacked models: ) encoder and ) decoder. first, an encoder based on graph convolution networks (gcn) maps the nodes into a low-dimensional embedding space. subsequently, a decoder attempts to reconstruct the original graph structure from the encoder representations. both models are jointly trained to optimize the quality of the reconstruction from the embedding space, in an unsupervised way. the functions of these two model can be described as follows: encoder: it uses graph convolution network (gcn) on adjacency matrix a and the feature representation matrix f. encoder generates a d -dimensional latent variable z i for each node i ∈ v , with |v | = n, that corresponds to each embedding node, with d ≤ n. the inference model of the encoder is given below: where, r(z i |a, f) corresponds to normal distribution, n ( z i µ i , σ i ), µ i and σ i are the gaussian mean and variance parameters. the actual embedding vectors z i are samples drawn from these distributions. decoder: it is a generative model that decodes the latent variables z i to reconstruct the matrix a using inner products with sigmoid activation from embedding vector, (z). where, a is the decoded adjacency matrix. the objective function of the variational graph autoencoder (vgae) can be written as: the objective function c v gae maximizes the likelihood of decoding the adjacency matrix w.r.t graph autoencoder weights using stochastic gradient decent. here, d kl (.||.) represents kullback-leibler divergence and p(z) is the prior distribution of latent variable. drug-sars-cov- link prediction . adjacency matrix preparation in this work, we consider an undirected graph g = (v, e) with |v | = n nodes and |e| = m edges. we denote a as the binary adjacency matrix of g. here v consists of sars-cov- proteins, cov-host proteins, drug-target proteins and drugs. the matrix (a) contains a total of n = nodes given as: where, n nc is the number of sars-cov- proteins. n dt is the number of drug targets, whereas n nt and n d represent the number of cov-host and drugs nodes, respectively. total number of edges is given by: where, e represents interactions between sars-cov- and human host proteins, e is the number of interactions among human proteins, and e represents the number of interactions between drugs and human host proteins. the neighborhood sampling strategy is used here to prepare a feature representation of all nodes. a flexible biased random walk procedure is employed to explore the neighborhood of each node. a random walk in a graph g can be described as the probability: where, π(v, x) is the transition probability between nodes v and x, where (v, x) ∈ e and a i is the i th node in the walk of length l. the transition probability is given by π(v, x) = c pq (t, x) * w vx , where t is the previous node of v n the walk, w vx is the static edge weights and p, q are the two parameters which guides the walk. the coefficient c pq (t, x) is given by where, distance(t, x) represents the shortest path distance between nodes t and node x. the process of feature matrix f n×d generation is governed by the node vec algorithm. it starts from every nodes and simulates r random walks of fixed length l. in every step of walk transition probability π(v, x) govern the sampling. the generated walk of each iteration is included to a walk-list. finally, the stochastic gradient descent is applied to optimize the list of walks and result is returned. . link prediction: scalable and fast variational graph autoencoder (fastvgae) is utilized in our proposed work to reduce the computational time of vgae in large network. the adjacency matrix a and the feature matrix f are given into the encoder of fastvgae. the encoder uses graph convolution neural network (gcn) on the entire graph to create the latent representation (z). the encoder works on full adjacency matrix a. after encoding, sampling is done and decoder works on the sampled sub graph. the mechanism of decoder of fastvgae is slightly different from traditional vgae. it regenerate the adjacency matrix a based on a subsample of graph nodes, v s . it uses a graph node sampling technique to randomly sample the reconstructed nodes at each iteration. each node is assigned with a probability p i and the selection of noes is based on the high score of p i . the probability p i is given by the following equation: where, f (i) is the degree of node i, and α is the sharpening parameter. we take α = in our study. the node selection process is repeated until |v s | = n s , where n s is the number of sampling nodes. the decoder reconstructs the smaller matrix, a s of dimension n s × n s instead of decoding the main adjacency matrix a. the decoder function follows the following equation: a s (i, j) = sigmoid(z t i .z j ), ∀(i, j) ∈ v s ×v s . at each training iteration different subgraph (g s ) is drawn using the sampling method. after the model is trained the drug-cov-host links are predicted using the following equation: where a i j represents the possible links between all combination of sars-cov- nodes and drug nodes. for each combination of nodes the model gives probability based on the logistic sigmoid function. a new coronavirus associated with human respiratory disease in china host-pathogen systems biology host-directed therapies for bacterial and viral infections network-based drug repositioning: approaches, resources, and research directions new horizons for antiviral drug discovery from virus-host protein interaction networks drug target prediction and repositioning using an integrated network-based approach mapping protein interactions between dengue virus and its human and insect hosts a review of in silico approaches for analysis and prediction of hiv- -human protein-protein interactions network-based study reveals potential infection pathways of hepatitis-c leading to various diseases prediction of the ebola virus infection related human genes using protein-protein interaction network a genome-wide positioning systems network algorithm for in silico drug repurposing deepdr: a network-based deep learning approach to in silico drug repositioning network-based drug repurposing for novel coronavirus -ncov/sars-cov- network bioinformatics analysis provides insight into drug repurposing for covid- a sars-cov- protein interaction map reveals targets for drug repurposing comprehensive prediction of the sars-cov- vs. human interactome using pipe , sprint, and pipe-sites scalable feature learning for networks fastgae: fast, scalable and effective graph autoencoders with stochastic subgraph decoding from community to role-based graph embeddings specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus a next generation connectivity map: l platform and the first , , profiles improved community detection in weighted bipartite networks drugbank: a comprehensive resource for in silico drug discovery and exploration detecting overlapping protein complexes in protein-protein interaction networks the therapeutic potential of apigenin delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/h n virus baclofen promotes alcohol abstinence in alcohol dependent cirrhotic patients with hepatitis c virus (hcv) infection antiviral and immunomodulatory effects of polyphenols on macrophages infected with dengue virus serotypes and enhanced or not with antibodies evaluation of topoisomerase inhibitors as potential antiviral agents potent antiviral activity of topoisomerase i and ii inhibitors against kaposi's sarcoma-associated herpesvirus antiviral action of camptothecin an analog of camptothecin inactive against topoisomerase i is broadly neutralizing of hiv- through inhibition of vif-dependent apobec g degradation water-insoluble camptothecin analogues as potential antiviral drugs inhibition of hiv- replication by daunorubicin a derivate of the antibiotic doxorubicin is a selective inhibitor of dengue and yellow fever virus replication in vitro elimination of hiv- infection by treatment with a doxorubicin-conjugated anti-envelope antibody antiviral activity of mitoxantrone dihydrochloride against human herpes simplex virus mediated by suppression of the viral immediate early genes histone deacetylase inhibitors for purging hiv- from the latent reservoir interval dosing with the hdac inhibitor vorinostat effectively reverses hiv latency the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application synthesis and in vitro anti-hsv- activity of a novel hsp inhibitor bj-b heat shock protein facilitates formation of the hbv capsid via interacting with the hbv core protein dimers severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis hsp : a promising broad-spectrum antiviral drug target drug repositioning suggests a role for the heat shock protein inhibitor geldanamycin in treating covid- infection broad spectrum antiviral agent niclosamide and its therapeutic potential hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro pharmacokinetic optimisation in the treatment of pneumocystis carinii pneumonia the antiviral effects of na, k-atpase inhibition: a minireview severe acute respiratory syndrome coronavirus replication is severely impaired by mg due to proteasome-independent inhibition of m-calpain effective inhibition of mers-cov infection by resveratrol structural basis of receptor recognition by sars-cov- angiotensin receptor blockers as tentative sars-cov- therapeutics next-generation sequencing to generate interactome datasets development of human protein reference database as an initial platform for approaching systems biology in humans drugbank . : shedding new light on drug metabolism chembl: a large-scale bioactivity database for drug discovery therapeutic target database update : enriched resource for bench to clinical drug target and targeted pathway information the iuphar/bps guide to pharmacology: an expert-driven knowledgebase of drug targets and their ligands towards a proteome-scale map of the human protein-protein interaction network a proteome-scale map of the human interactome network a reference map of the human binary protein interactome stochastic backpropagation and approximate inference in deep generative models on information and sufficiency. the annals mathematical statistics key: cord- - x juiw authors: christe, philippe; morand, serge; michaux, johan title: biological conservation and parasitism date: journal: micromammals and macroparasites doi: . / - - - - _ sha: doc_id: cord_uid: x juiw nan pandas, tigers, right whales and gorillas are emblematic and charismatic species worldwide and a consensus exists for the need of their conservation. the same is true for economically important species such as salmonids and sturgeons. but do we really need the conservation of parasitic species? not really! during our childhood, we all have favourite stuffed animals (representing one of these emblematic species) we take to sleep. however, do you ever see a child squeezing a stuffed worm, flea or tick? the lack of affect for cryptic species and the perception of parasites as disgusting creatures among the public certainly leads to a disinterest among governmental and conservation agencies to preserve them. fortunately, in , donald a. windsor expressed his concern for this matter in the famous slogan: "equal rights for parasites!" (windsor ). five years later he pleaded once more for parasite conservation in an editorial that appeared in "conservation biology" (windsor ) . during the same time, the ominous term "co-extinction" was proposed to characterize the dual extinction of a host and its specialized parasite (stork and lyal ) . despite the passing of years since these passionate declarations and the exponential increase of an interest in conservation biology, we can point out that parasitic species are far from being in a leading position among current conservation priorities. very few parasites are listed on the iucn red list of threatened species (iucn, ; whiteman and parker ) . some parasite extinctions have been even intentionally provoked as revealed by the will to remove parasites from hosts in captive breeding programs (stork and lyal ) . to convince resource managers that parasites are an important component of all ecosystems, the following arguments, which mainly rely on their potential utilitarian effects, are advocated by the parasites' defenders. first, parasites are living organisms and are de facto part of biodiversity. they shape community structure by reducing competitive abilities and vulnerability to predation of their hosts and have strong impact on ecosystem functioning (hudson ) . moreover, parasites could maintain biodiversity by mediating competitive interactions between different members of an ecosystem. because the rate of molecular evolution is usually faster in parasite dna than that within the homologous loci of their hosts (moran et al. ; nieberding et al. ) , the study of the evolution of parasite dna sequences could provide valuable information on past population dynamics, evolutionary history and current demographic processes of endangered hosts (whiteman and parker ) . parasites could thus be used as a biological "magnifying glass" (nieberding et al. ) . another utilitarian effect of parasites is their potential use as indicators of environ-mental quality and ecosystem health (marcogliese ) . indeed, parasites may be used as accumulation indicators of heavy metal contamination, particularly in aquatic ecosystems (sures et al. ). in addition, parasite species and composition revealed perturbations in ecosystem structure and function (marcogliese ) . furthermore, the use of parasite in human medicine is a new promising field of investigation, as illustrated by the use of helminths as therapeutic agents for inflammatory disease (hunter and mckay ). wild animals in their natural habitat have to cope not only with predictable environmental changes such as the cycles of seasons and their associated modifications in resource availability and temperature but also with unpredictable events such as catastrophes, spread of new diseases and human disturbances. whereas animals react adaptively by behavioural and physiological modifications to predictable changes, unpredictable disturbances may have negative effects on population dynamics of living organisms. increasingly rapid disappearing and fragmentation of habitats, which may be considered as unpredictable environmental changes, translates in a cascade of negative effects and can result in physiological stress on animals (suorsa et al. (suorsa et al. , . the first physiological responses of an animal to stressful stimuli include cardiovascular effects and a hormonal response involving synthesis and secretion of glucocorticosteroids (romero ) . consequently, a corticosteroid response might be a good indicator of a stress response (hofer and east ) . it is, therefore, not surprising that corticosteroid level is measured in many studies in ecology and conservation biology that have evaluated the effect of different environmental and human perturbations on the stress level of wild animals (creel et al. ; creel et al. ; mostl and palme ; romero ; palme et al. ) . the consequences of a stress response on parasite resistance are complex and alter host immunocompetence in different ways (apanius ). the immune system appears to be down regulated under stressful environmental conditions (von holst ), particularly under severe chronic stress with prolonged periods of high cortisol concentrations (mostl and palme ) . stress stimuli may arise due to different factors in a perturbed environment. habitat fragmentation may be related to chronic food shortage (zanette et al. ) . thus degradation of environmental conditions may decrease resource availability that in turn affects body condition and immune defences (chandra and newberne ; klasing ; christe et al. ) . as body condition is usually positively correlated with immune defences , individuals with poor body condition will be especially vulnerable to attacks of parasites christe et al. ) . edge effect due to fragmentation may also be a source of stress because predators may have easy access to dense forest patches which were previously inaccessible. it has been experimentally demonstrated that exposure to predators reduced the ability of hosts to cope with parasitism mediated through effects on immune function (navarro et al. ) . consequently parasitism may be favoured in fragmented habitat through the effect of predators. thus, parasites, which can also be considered as an environmental stressor, may reinforce the effect of habitat degradation and participate in the reduction of a population. in addition to habitat degradation and fragmentation, anthropogenic factors such as environmental pollution, hunting, tourism and leisure activities exert a negative pressure on wildlife and are thought to cause stress (fowler ; mullner et al. ) . clearly, more studies are needed to investigate the relationship between anthropogenic factors, level of stress and parasitism in endangered populations. conservation biology deals with two major paradigms: population invasion and population decline. both are related to each other (i.e. decline may be a result of an invasion) and both emphasize the potential roles of parasites and/or pathogens (prenter et al. ) . theoretical, experimental and empirical studies have established clearly that parasites play important roles in regulating population dynamics (scott ; scott and dobson ; albon et al. ; rosa et al. in this volume) and structuring free-living communities (minchella and scott ; morand and arias gonzalez ; hudson and greenman ; tompkins et al. ) . parasites then have a large impact on biological conservation (dobson and may ; mccallum and dobson ; sasal et al. ) , as parasites and pathogens may compromise reintroduction or translocation programs (viggers et al. ) . they may have a higher impact on threatened species generally characterized by a lower level of genetic variability, particularly on genes associated to immune system (hedrick, ) . small-sized host populations may be prone to extinction due to stochastic events. several processes, including the allee effect ; see below), may increase the probability of extinction of small populations. these processes operate when host population size decreases to under a critical or threshold level, below which populations are almost doomed. population viability analysis is one approach that has been developed for management purposes of small-sized endangered populations. threshold size has been also extensively studied in the case of hostparasite dynamics (dobson ) . the basic reproductive number, r , is the major concept in host-parasite population dynamics. this quantity is defined as the number of new infections occurring after introduction of one parasite, or one infected host, into a naïve and susceptible host population. r is positively linked to host density in the case of direct-transmitted parasites (see rosa in this book). parasites, or infection, can spread in the population when r > and as r depends on host density, the condition of parasite invasion corresponds to a case when the density of host population exceeds a threshold density. obviously, host-parasite dynamics are viewed in terms of parasite invasion or parasite invisibility. the task of disease management is then to decrease r below one, i.e., below the threshold density. the interplay between host and parasite thresholds has not been considered adequately. deredec and courchamp ( ) emphasized the importance of the relative position of the host and parasite thresholds: when the parasite threshold is higher than that of the host, the parasite is driven to extinction and the host population is relieved of its parasite; when the host population threshold is higher than that of the parasite, the host is driven to extinction while the parasite continues to exert strong pressure on the host until it reaches its own threshold. hence, mathematical epidemiology and population dynamics are important tools for investigating thresholds and persistence of both hosts and parasites. they may help in determining the conditions to maintain a high level of parasite threshold in comparison to the host threshold. microparasites are generally considered as an important threat in conservation biology (daszak et al. ; cleaveland et al. ). all conservation textbooks refer to the canine distemper virus, rinderpest and the avian malaria as examples of pathogen-driven extinction. introduced diseases have been implicated in the local extinction of a number of species (mccallum and dobson ; vitousek et al. ) and the global species extinction of hawaiian birds (vanriper et al. ) and the thylacine (guiler ) among others. daszak et al ( ) , in their review, mentioned microparasites and no macroparasites as important threats for conservation and as zoonotic threats for human health through spill-over. the lack of reference to macroparasites may suggest that they are indeed less important, and that their survey is not of major interest, with the notable exception of ectoparasites (ticks, fleas) because of their roles as vectors of numerous virus, bacteria and protozoans. moreover, results based on comparative analyses in carnivores show that host species that live in low density within a restricted geographic area experience low parasitic pressure in terms of parasite species diversity, suggesting that parasites may not represent a particularly important risk for these host species (torres et al. ) . in contrast, widespread host species that live in high density are exposed to a wide range of parasite species that may affect drastically the population dynamics of these carnivores, suggesting that macroparasites may regulate them at least locally. these results lead to the paradox that parasite infection, and particularly that of macroparasites, is less crucial for small and isolated populations than for large populations. this paradox is apparent and resolved by considering the investment in immune defences, which is directly related to the prevalence and/or diversity of parasites as a mean to control infection (martin et al. ) . evidence comes again from comparative studies, which suggest that hosts allocate their investment in immune function as a function of their probability of exposure to parasites (møller and legendre ; møller et al. ) . large populations are composed of highly immunocompetent individuals and small populations of low immunocompetent ones. hence, parasites and pathogens are threats to small and isolated populations because of poor performance of their immune system against pathogen introduction, but parasites (and parasite diversity) are probably necessary to maintain high levels of immune defence, which helps against new pathogens. the allee effect may be defined as "a positive relationship between any component of individual fitness and either numbers or density of conspecifics" . the beneficial effects of conspecifics not only include antipredator vigilance, predator dilution, social thermoregulation, reduction of inbreeding but also social facilitation of reproduction through helpers courchamp et al. ) . when population size reaches a low density, animal species that are subject to an allee effect will suffer from a reduction in some aspects of their fitness that in turn will affect negatively growth rate of populations. because of their potential role in extinctions of declining species, the allee effects have thus become much studied in conservation biology lafferty and gerber ) . interestingly, allee effects and parasitism have several features in common that are of interest when studying population dynamics in conservation biology (deredec ) . for example, theoretical models demonstrated the importance of host density in the probability for a parasite to become established in a host population (see above) and empirical studies have shown a positive relationship between host sociality or density and parasite prevalence and intensities (anderson and may ; brown and brown ; møller et al. ; stanko et al. ; altizer et al. ) . thus, animal species that aggregated as a behavioural response to the strong allee effects, would be more prone to suffer the negative effects of parasites. parasite species may also be subject to the allee effects that influence the occurrence and the severity of epidemics as illustrated by patchy distributions of worms in hosts as a result of the necessity for female worms to find a mate in order to reproduce (cornell et al. ). it has been suggested that sexual selection, in particular female mate preferences, could lead to an allee effect (møller and legendre ) . if only males of poor quality are available for mating in a small population, females may refrain from reproduction or reproduce at a low rate. as a consequence of mating with a male of a non-preferred phenotype, females could decrease their parental investment resulting in poor reproductive success (møller and legendre ) . parasite-mediated sexual selection has been the focus of numerous studies since the influential hypothesis of hamilton and zuk ( ) . a meta-analysis of the available studies related to this topic has revealed a negative relationship between parasite load, immunocompetence and the expression of male secondary sexual characters (møller et al. ) . thus, parasites, by decreasing the expression of male secondary sexual characters, may contribute and reinforce the potential allee effects created by sexual selection. mediation of competition by parasites is one mechanism of parasite interference (anderson ; hudson and greenman ; poulin ) . parasite-mediated competition is inferred when two different host species have different susceptibilities to the same non-specific parasite species. the presence of a given host species may decrease the fitness of the other host species simply by transmitting a pathogen to the more vulnerable host species (hudson and greenman ) . the abundance of the more vulnerable host to the parasite is then depleted, potentially under the host threshold. moreover, as the parasite infects two host species, the parasite threshold is obviously low. this "apparent" competition, mediated via a shared pathogen, differs from the classical competition for limited resources. strong evidence of this competition was obtained not only from experiments but also from the field (tompkins et al. ) , e.g. red and grey squirrels in england (tompkins et al. b ) and pheasant and grey partridge in england (tompkins et al. a) . parasite-mediated competition may operate for introduced host species, as they can be best competitors simply by introducing and transmitting a new parasite to the naïve native species. this can lead to a non-fit combination that can be more pathogenic (hudson and greenman ; prenter et al. ) . in this case, the invader uses parasites as biological weapons. immune-naïve residents will be weakened or even killed by the new pathogens. the most famous example of such a process comes from the history of the expansion of european humans through america where million of native people were killed by the influenza and other pathogens that accompanied conquistadors. parasite mediated competition is not the only way by which parasites may interfere in competition processes. recently, it was shown that many introduced species lost most of their parasites from their native habitats when introduced to new ones (torchin et al. ; torchin et al. ). this could be responsible for the demographic explosion of some introduced species, formulated as the "parasite release hypothesis". the parasite release hypothesis was proposed as an ecological mechanism to explain the success of introduced species. as the introduced species lose their parasites when invading new habitats, they have a competitive advantage over local species. mitchell and power ( ) and torchin et al. ( ) found that parasitism is significantly reduced in organisms in their introduced range, supporting the "parasite release hypothesis". one cause to explain that invaders may leave behind their parasites is that many parasites have complex life cycle stages with more than one host. if one of those hosts is absent in the new colonized area, the life-cycle of the parasite would be disrupted. in the invasion process, invasive host species harbouring a high diversity of parasites in their native habitat have advantages as they lose a large number of parasites and pathogens (see above). invasive host species have another advantage if they have invested in strong immune defences in their natural range, which may then subsequently confer a better capacity to control parasites that they may acquire in the introduced habitat. hosts having evolved strong immune defences are prime candidates for successful invasion (and also more resistant towards invaders). this hypothesis was proposed in the case of introduced plants and recently for the case of introduced animals (lee and klasing ; møller and cassey ) . in contrast, hosts that are exposed to a low diversity of parasites may invest less in immune defences. maintaining a strong immune system for threatened host species, or for individual hosts maintained in captivity in the view of reintroduction, is a new task for conservation biologists. habitat fragmentation and its degradation is probably one of the main factors leading to the disappearance of many species. indeed, it often leads to a decrease in population sizes as well as to the appearance of barriers to gene flow between isolated populations. the small populations that result from this fragmentation often suffer from reduction of genetic diversity associated with genetic drift and inbreeding effects. this loss of genetic variation can result in a rapid reduction of fitness (lower possibility to adapt to long term changes in environment, poor reproductive ability associated with a lower sperm quality, higher juvenile mortality, lower general survival, etc) (o'brien ) . several recent studies (cassinello et al. ; keller and waller ; spielman et al. ) also showed that populations with a low genetic variability are generally more susceptible to infectious viruses, bacteria and other pathogens. the case of the cheetah (acinonyx jubatus) is probably one of the best known concerning this phenomenon., the two major subspecies of cheetah (a. jubatus jubatus from southern africa and a. jubatus raineyi from eastern africa) display markedly reduced levels of genetic variability compared to other mammal species (o'brien ) . this would result in intensive inbreeding. when a breeding colony of this species was contaminated by feline infectious peritonitis (fip) in oregon state (usa), % of the captive animals showed morbidity symptoms and % of them died (o'brien ) . in contrast, in domestic cats, the mortality incidence of this virus is very rare (around %). according to o'brien ( ) , the high sensitivity of this cheetah colony to the fip would be directly linked to the very low (almost monomorphic) level of variation of the major histocompatibility (mhc) genes characterising the cheetah. a wide variety of gene classes (where the mhc is the most notable but see also the eosinophil-associated rnase (ears) genes, the tumor necrosis factor gene promoter, the interleukine receptor or the -interferon receptors ; hill ; zhang et al. ) are normally variable in natural populations and could contribute to disease resistance. mhc genes encode cellsurface glycoproteins, binding antigens derived from pathogens and parasites and constitute the most polymorphic genes in vertebrates (parham ; charbonnel et al. in this volume) . they present antigens to tlymphocytes which develop the appropriate immune responses. two major groups of mhc genes are recognised: the mhc class i genes are specific to the immune defence against intracellular pathogens by binding peptides mainly derived from viral proteins or cancer infected cells. the mhc class ii genes present with t-lymphocytes, peptides essentially derived from extra-cellular parasites (bacteria, nematodes, cestodes, etc.). the variability of mhc genes is correlated with the diversity of the t-lymphocyte receptors, which, in turn, determine the resistance of an organism to pathogens and parasites (parham ) . therefore, the cheetah, with its very low variability of mhc genes, is not well protected against the fip and probably against many other pathogens and therefore is at a high risk of extinction. however, according to several recent studies, several processes would help to maintain high levels of mhc genes diversity. indeed, these studies demonstrated that the anti-gen binding sites (abs) display more non-synonymous than synonymous substitutions compared to what would be observed under neutral theory (in this condition, the rate of synonymous substitution is predicted to be larger than the rate of non-synonymous substitution as the latter change the amino acid composition and would be likely deleterious) (sommer ) . this phenomenon cannot be explained by higher mutation rates in this region (hughes and yeager ) and the hypothesis accepted at present is that this particular nucleotide diversity in mhc genes would be the result of balancing selection. this would allow the maintenance of large numbers of alleles in populations and also the persistence of allelic diversity over long periods of time. following this strategy, the binding of a large set of antigens would be possible. two main types of balancing selection have been proposed to explain high levels of genetic diversity in mhc genes of vertebrates: -"overdominance" strategy (hedrick ; richman ) , where the heterozygotes are expected to have higher fitness than parental homozygotes as the latter will carry less divergent allelic sequences and, therefore, will have less chance to resist a large panel of antigens and/or multiple types of pathogens and parasites. -"frequency dependent selection" strategy (hedrick ) . this occurs when an allele or genotype is favoured at one frequency, but disadvantaged at another frequency. this hypothesis is based on the fact that hostparasite dynamics is considered as a co-evolutionary race. pathogens adapt to infect the most common genotype, leaving rare genotypes least infected. if alleles are favoured when they are rare, but selected against when they are common, this will result in a balanced polymorphism (sommer ) different studies confirmed the effect of balancing selection on the high mhc diversity. one of the best examples concerns the nicolas island fox (urocyon littoralis dickeyi) (aguilar et al. ). on the basis of different neutral markers (microsatellites, minisatellites and allozymes), this species is considered as one of the most monomorphic among sexually reproducing species. regarding the low variability of these markers, this species would have many problems of fitness as well as low resistance to pathogens. however, it is characterised by a surprising high level of mhc diversity which makes it much more resistant to what could be expected. this observation is interpreted as being the result of intense periodic balancing selection at the mhc which may have allowed the persistence of variation within this species despite strong genetic drift. under some circumstances (for example, particular historical events such as bottlenecks or founder effects), strength of selection acting on mhc genes can be insufficient to maintain variation in small or fragmented populations over a long period of time (sommer ) . in these cases, the power of genetic drifts can be stronger than the power of selection. this can lead to a loss of genetic diversity not only on the neutral markers but also on the mhc genes. this would explain the very low genetic variability in highly threatened species such as the cheetah (acinonyx jubatus) (see above), the asian lion (pantera leo persica) (o'brien, ) , the common hamster (cricetus cricetus) in the netherlands (smulders et al. ) , the scandinavian beaver (castor fiber) (ellegren et al. ) , the northern elephant seal (mirounga angustirostris) (hoelzel et al. ) and the scandinavian moose (alces alces) (ellegren et al. ) . under these circumstances, threatened species present a high risk of extinction as they can be very sensitive to new diseases and changes in environment. however, other studies demonstrated that endangered species such as the przewalski's horse (equus przewalski) (hedrick et al. ) , the arabian oryx (oryx leucoryx) (hedrick et al. ) and the malagasy giant jumping rat (hypogeomys antimena) (sommer ) are characterised by a low number of mhc alleles but which are separated by a high level of nucleotide and amino acid divergence. analysis at the abs showed that non-synonymous substitutions were higher than synonymous ones, suggesting selection leading to an increase of amino acids changes in the abs region and thus to higher divergences between mhc alleles (sommer ) . these studies indicated that other selection processes are able to maintain some mhc polymorphism (not on the number of alleles but rather on the genetic difference between the existing alleles) even in species surviving bottlenecks. this would be sufficient to prevent immediate pathogen-induced declines. however, such kind of adaptive processes to changing conditions is probably limited and does not predict the outcome effects of introduced pathogens, which differ from commonly encountered diseases. probably, the maintenance or even renewal of variation in functional important regions of the mhc, either from mutation, recombination or immigration from other populations, would be an important genetic component to allow an appropriate immune response (sommer ) . however, too strong genetic bottlenecks, leading to important inbreeding depressions, do not permit such kind of processes to operate and this explains why some species like the cheetah or the asian lion are so sensitive nowadays to diseases. as mentioned by mccallum and dobson ( ) , "diseases and parasites pose particularly severe problems in captive populations, in which animals are held at high density, may be stressed and may be exposed to crossspecies transmission". during the last years, a great amount of zoos worldwide have participated in the management of endangered species., many threatened species have captive populations that act as insurance against extinction in the wild and, indeed, captive breeding programs have saved some endangered species from extinction (e.g. père david's deer, european bison, etc) (frankham et al. ) . because parasites may have negative effects on their host, veterinarians in zoos take great care to reduce or even to remove entirely parasite loads on captive animals. as the ultimate goal of breeding programs in zoos is to increase threatened populations or to reintroduce individuals into the wild, parasites play an important role. what could be the consequences of maintaining hosts during many generations in a parasite-free environment? the potential risk is to release into the wild individuals that have lost their defences against pathogens and diseases. once in the wild, they will be in contact with a vast array of parasite species and may be unable to resist to their detrimental effects. maintaining some parasites on individual hosts kept in captivity could be a way to solve part of this problem. macroparasites, because of chronic infections, have evolved several kinds of immune evasion strategies (charbonnel et al. in this volume) . some strategies of immunomodulation displayed by many macroparasites may have some beneficial effects on their hosts by regulating th /th cytokine responses (weinstock et al. ). th responses induce inflammatory cell activity to control intracellular infections while th responses drive humoral immune responses to control extra-cellular parasites (see weil et al. in this volume) . mice with helminths have blunted th responses while helminths promote th responses associated with production of interleukin (il- ), which helps impede th cell differentiation. thus, induction of il- could underlie the alterations seen in host immunity (i.e. high inflammatory activities). helminths also appear to protect the host from aberrant th diseases such as asthma and food allergy (weinstock et al. ) , and there is now an immunological basis for protection by helminths. human epidemiological data and several animal studies support the notion that helminths protect the host from immunological disease (elliott et al. ) , particularly those caused by the activation of the th response by microparasites. for example, helminths protect mice and rats from experimental autoimmune encephalomyelitis, as welll as other diseases of immunity. thus, natural exposure to helminths may guard animals from developing severe immunological diseases, suggesting that helminths should be useful in conserving both endangered and captive species. gompper and williams ( ) proposed a series of measures to maintain endangered parasite species originating from threatened hosts in captive breeding program. however, they pointed out that because most of the public disapprove of protecting parasite species, attempts to conserve unique species of parasites could result in a hostile public response against efforts to preserve hosts. therefore they proposed a series of measures aimed to save parasite species without damaging attempts to conserve hosts. one of those measures was to find alternative hosts to maintain parasite populations for potential reintroduction once the host population was restored. however, the problem of parasite conservation concerns mainly highly host-specific parasites. to find alternative hosts on which specialist parasite populations would be viable may be a difficult task because experiments on cross-species infection have shown a strong decrease on both parasite survival and reproductive success on the foreign host, even if this new host species belongs to the same host genus (giorgi et al. ) . the consequence of human population growth is closer contact between human and reservoir hosts of numerous diseases. the spread of disease to endangered wildlife species due to contact with humans and domestic animals, and vice versa, increases as humans and their domestic animals get in more contact with these species due to habitat fragmentation. emergence of new diseases and particularly those from small mammals such as rodents or bats are of great public health concern (leroy et al. ) . conservation medicine, a new theme within the field of conservation biology, has been viewed as the application of medicine to improve the conserva-tion of wildlife and ecosystems . conservation medicine, according to otsfeld et al. ( ) is "devoted to understanding the interactions among human-induced and natural changes in ( ) climate, habitat and land use; ( ) pathogens, parasites, and pollutants; ( ) biodiversity and health within animal communities; ( ) health of humans" . the "anus horribilis" for bats worldwide illustrates the importance of this new field of investigations. while it was discovered in china that bats are the reservoir of sars virus (lau et al. ; li et al. ) , it was found in africa that they are probably the reservoir for ebola virus (leroy et al. ) . we strongly hope that this chapter will convince ecologists and conservation biologists that pathogens and parasites, mostly investigated by veterinarians and physicians, should not be ignored or eradicated because of their crucial importance to wild and domestic animals and humans. high mhc diversity maintained by balancing selection in an otherwise genetically monomorphic mammal the roles of parasites in the dynamics of a reindeer population social organization and parasite risk in mammals: integrating theory and empirical studies the ecological relationships of meningeal worm and native cervids in north america regulation and stability of host-parasite population interactions. . regulatory processes stress and immune defense ectoparasitism as a cost of coloniality in cliff swallows (hirundo pyrrhonota) relationship between coefficient of inbreeding and parasite burden in endangered gazelles nutrition, immunity, and infection immunocompetence and nestling survival in the house martin -the tasty chick hypothesis variation in intensity of a parasitic mite (spinturnix myoti) in relation to the reproductive cycle and immunocompetence of its bat host (myotis myotis) differential species-specific ectoparasitic mite intensities in two intimately coexisting sibling bat species: resource-mediated host attractiveness or parasite specialization? the role of pathogens in biological conservation stochastic and spatial dynamics of nematode parasites in farmed ruminants multipack dynamics and the allee effect in the african wild dog, lycaon pictus radiocollaring and stress hormones in african wild dogs snowmobile activity and glucocorticoid stress responses in wolves and elk wildlife ecology -emerging infectious diseases of wildlife -threats to biodiversity and human health de la menace à l'outil: modélisation de l'impact du parasitisme en biologie de la conservation the population biology of parasitic helminths in animal populations restoring island ecosystems: the potential of parasites to control introduced mammals major histocompatibility complex monomorphism and low levels of dna fingerprinting variability in a reintroduced and rapidly expanding population of beavers limited polymorphism at major histocompatibility complex (mhc) loci in the swedish moose a. alces helminths modulate mucosal immunity and inflammation behavioral and hormonal responses of magellanic penguins (spheniscus magellanicus) to tourism and nest site visitation introduction to conservation genetics causal mechanisms underlying host specificity in bat ectoparasites parasite conservation and the black-footed ferret recovery program the former distribution and decline of the thylacine heritable true fitness and bright birds: a role for parasites? balancing selection and mhc major histocompatibility complex variation in the endangered przewalski's horse major histocompatibility complex variation in the arabian oryx the immunogenetics of human infectious diseases molecular genetic diversity and evolution at the mhc dqb locus in four species of pinnipeds biological conservation and stress introduction -parasites, diversity, and the ecosystem competition mediated by parasites: biological and theoretical progress natural selection at major histocompatibility complex loci of vertebrates review article: helminths as therapeutic agents for inflammatory bowel disease inbreeding effects in wild populations nutritional modulation of resistance to infectious diseases good medicine for conservation biology: the intersection of epidemiology and conservation theory severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats a role for immunology in invasion biology fruit bats as reservoirs of ebola virus bats are natural reservoirs of sars-like coronaviruses parasites of the superorganism: are they indicators of ecosystem health? does clutch size evolve in response to parasites and immunocompetence? detecting disease and parasite threats to endangered species and ecosystems parasitism -a cryptic determinant of animal community structure release of invasive plants from fungal and viral pathogens on the relationship between t-cell mediated immunity in bird species and the establishment success of introduced populations allee effect, sexual selection and demographic stochasticity parasites and the evolution of host social behavior condition, disease and immune defence parasitism, host immune function, and sexual selection coevolutionary arms races: increased host immune defense promotes specialization by avian fleas faster evolutionary rates in endosymbiotic bacteria than in cospeciating insect hosts is parasitism a missing ingredient in model ecosystems? hormones as indicators of stress exposure to ecotourism reduces survival and affects stress response in hoatzin chicks (opisthocomus hoazin) predation risk, host immune response, and parasitism a parasite reveals cryptic phylogeographic history of its host a role for molecular genetics in biological conservation conservation medicine. the birth of another crisis discipline stress hormones in mammals and birds. comparative aspects regarding metabolism, excretion, and noninvasive measurement in fecal samples virtual reality in the mhc the functional importance of parasites in animal communities: many roles at many levels? roles of parasites in animal invasions evolution of balanced genetic polymorphism physiological stress in ecology: lessons from biomedical research experimental approach to the importance of parasitism in biological conservation regulation of mouse colony abundance by heligmosomoides polygyrus (nematoda) the role of parasites in regulating host abundance complete loss of mhc genetic diversity in the common hamster (cricetus cricetus) population in the netherlands. consequences of conservation strategies effects of habitat fragmentation and changes of dispersal behaviour after a recent population decline on the genetic variability of noncoding and coding dna of a monogamous malagasy rodent the importance of immune gene variability (mhc) in evolutionary ecology and conservation does inbreeding and loss of genetic diversity decrease disease resistance? mammal density and patterns of ectoparasite species richness and abundance consequences of the allee effect for behaviour, ecology and conservation what is the allee effect? extinction or co-extinction rates forest management is associated with physiological stress in an oldgrowth forest passerine effects of forest patch size on physiological stress and immunocompetence in an area-sensitive passerine, the eurasian treecreeper (certhia familiaris): an experiment parasites as accumulation indicators of heavy metal pollution field evidence for apparent competition mediated via the shared parasites of two gamebird species differential impact of a shared nematode parasite on two gamebird hosts: implications for apparent competition parasite-mediated competition among red-legged partridges and other lowland gamebirds parapoxvirus causes a deleterious disease in red squirrels associated with uk population declines kuris am ( ) parasites and marine invasions introduced species and their missing parasites parasite species richness of iberian carnivores: influences of host density and range distribution the epizootiology and ecological significance of malaria in hawaiian land birds the importance of diseases in reintroduction program introduced species: a significant component of human-caused global change the concept of stress and its relevance for animal behavior using parasites to infer host population history: a new rationale for parasite conservation heavenly hosts equal rights for parasites food shortage in small fragments: evidence from an area-sensitive passerine evolution of the rodent eosinophilassociated rnase gene family by rapid gene sorting and positive selection key: cord- - b ycv authors: gavora, js title: resistance of livestock to viruses: mechanisms and strategies for genetic engineering date: - - journal: genet sel evol doi: . / - - - - sha: doc_id: cord_uid: b ycv nan maximum survival of livestock, with good health and well being are conditions for efficient animal production. many of the current livestock disease problems that prevent the realization of this optimal production goal are caused by viruses, described by peter medawar as &dquo;pieces of bad news wrapped in protein coat&dquo;. this review deals with possible new, genetic engineering strategies for the improvement of resistance to viruses in livestock. since work on genetic engineering of disease resistance is more advanced in plants than in livestock, information on research in plants is also reviewed. the use of livestock for food, fibre and draft over hundreds of years has led to a significant influence by humans on the evolution of domesticated animal species. some of the changes induced by artificial selection parallel in their significance speciation. a modern meat-type chicken can be viewed as a species different from a modern egg-type chicken. similar differences exist between breeds of dairy and beef cattle. this 'genetic engineering' of livestock was achieved through the long-term use of conventional genetic improvement methods. it can be argued that gene transfer represents just another phase in the development of genetic engineering of livestock and that it would be foolish not to take advantage of the new technologies. thus introduction of new mechanisms of disease resistance in livestock by gene transfer may be viewed as a logical continuation of the creative influence of humans on the evolution of farm animals and birds that could benefit mankind by improvements in food safety and production efficiency. increased disease resistance will also improve the welfare of livestock. the latter consequence may make this type of genetic engineering more acceptable to the general public than other types of gene transfer. if there is one attribute that is common to viruses, it is the lack of uniformity in all aspects of their existence. nevertheless, this review attempts to find general elements and common patterns in the subject discussed. as background for the discussion of the subject, the article deals briefly with coevolution of hosts and parasites and principal elements of virus-host interactions, and reviews past improvement of disease resistance in plants and livestock by conventional breeding and genetic engineering, as well as the potential 'biological cost' of genetic manipulation. it includes prerequisites for and principles of the design of new resistance mechanisms, and proposes possible strategies for the introduction of disease resistance mechanisms by gene transfer. the main goal of this review is to inform readers from both research and industry about this area of long-term interest to animal agriculture and outline the potential use of the concept of new resistance mechanisms for the benefit of mankind and improvement of animal welfare. basic understanding of the parallel evolution of viruses and their hosts provides a useful starting point for the consideration of strategies for genetic engineering of new mechanisms of resistance. therefore, principal elements of the coevolution of viruses and hosts are briefly reviewed. viruses are obligatory, intracellular parasistes with limited genome sizes that code for functions the virus cannot adopt from host cells (strauss et al, ) . viruses have their own evolutionary histories, independent of those of their hosts. it is not clear whether viruses had a single or multiple origin. the origin of a virus is defined as that time when its replication and evolution became independent of the macromolecules from which it was derived (strauss et al, ) . viruses may have arisen ( ) by selection from an organelle; ( ) from cellular dna or rna components that donate macromolecules which gain the ability to replicate and evolve independently; or ( ) from self-replicating molecules. polymers of ribonucleotides can contain both the information required and the functional capacity to form a self-replicating system (watson et al, ) . the main mechanisms of viral evolution are mutation, recombination, and gene duplication. viruses have a very short generation interval and high mutation rate. for example, the mutation rate of a chicken retrovirus is - nucleotide/replication cycles -approximately eight orders higher than that of the host cell genome (dougherty and temin, ) . nevertheless, the virus always retains its origin of replication. recombination has also a large role in viral evolution because it allowed viruses to 'try out new gene combinations'. an example of an unusual acquisition of genes by a virus are three trna genes in bacteriophage t a type of gene only observed in eukaryotes (gott et al, ) . although it is possible that the genes evolved within t , the phage may also have acquired the genes from an eukaryotic host (michel and dujon, ) . similarly some retroviruses such as rous sarcoma virus acquired oncogenes for their genome. in general, dna viruses are more stable than rna viruses and do not cause rapidly moving pandemics as is the rule for rna viruses; in contrast, dna viruses tend to establish persistent or latent infections which may lead to malignant transformations (strauss et al, ) . exceptions to the general rule include the herpesvirus of marek's disease, a dna virus that can cause rapidly moving disease outbreaks in chickens, and the avian leukosis viruses, rna viruses that exhibit a period of latency and seldom cause high mortality. a disease of the host is not an evolutionary goal of the parasite. compatibility is preferable to incompatibility. subclinical infections are common; they are the rule -diseases the exception. there is no selective advantage to the virus in making the host ill, unless the disease aids in the transmission of the virus to new hosts, such as in the case of diarrhea. in some instances, disease may also result from an overzealous immune system. hence the interplay between microbes and hosts should not necessarily be seen as an ongoing battle but as a coevolution of species (pincus et al, ) . general considerations susceptibility (in the narrow sense) is the capacity of cells to become infected. for a virus to survive and reproduce, essential viral genes have to ensure: ( ) replication of viral genomes in which the involvement of viral genes varies from assisting host enzymes, to actually replicating the viral genome, although even the most selfdependent viruses use some host cell function in the process; ( ) packaging of the genome into virus particle -viral proteins do the packaging, although host proteins may complex with viral ones in the process; and ( ) alteration of the structure or function of the infected cell -the effects may range from cell destruction to subtle, but significant changes in function and antigenic specificity of infected cells. in general, once it enters, no virus leaves a cell unchanged. during their replication, viruses exploit host cell molecules at the expense of the cells. there are three types of viral infection (knipe, ) . ( ) ( ) productive infection is when the cell produces the virus but, as a consequence, dies and lyses. ( ) productive infection is when the cell survives and continues to produce the virus. the levels of injury to the cells resulting from viral infection range from no visible effects to cell death and include inclusion body or syncytium formation and cell lysis. in most instances cell injury is a consequence of processes necessary for virus replication but at least in one known instance, the penton protein of the adenovirus, which has no known purpose in the viral cycle, causes cytopathic effects in monolayer cells (valentine and pereira, ) . genetic engineering strategies that prevent entry of viruses into host cells would be effective against all three types of viral infection. other strategies discussed below can deal with various stages of viral life cycles and would accordingly affect the outcome of viral infection. to provide a basis for the examination of the opportunities to devise and genetically engineer new resistance mechanisms, the viral life cycle that consists of three fundamental steps, attachment, penetration, and replication (roizman, ) will be examined in sequence. attachment of virus to the host cell attachment of the virus to the host cell is, in most instances, through a specific binding of a virion protein, the antireceptor, to a constituent of the cell surface, the receptor. complex viruses, such as vaccinia, may have more than one species of antireceptor or antireceptors may have several domains, each reacting with a different receptor. mutations of receptors may cause a loss of the capacity of a receptor and antireceptor to interact and thus lead to resistance to viral infection. it seems likely that mutations in antireceptors preventing viral attachment will be automatically eliminated from viral evolution, unless they are able to interact with a substitute host. the number of receptors for which information is accumulating is rapidly increasing. examples in table i show that receptors are mostly glycoproteins. not all cells in a susceptible organism express viral receptors, a phenomenon that may limit susceptibility. even though our understanding of receptors is still at an early stage, it is obvious that viral receptors are molecules that have a normal physiological function in the host. while there is a great deal of variability in the types of molecule in viral receptors, some cell surface molecules are used by multiple, often unrelated viruses (table i) . when viewed across host species, for example, histocompatibility molecules are receptors for both semliki-forest togavirus and human coronavirus; sialic acid residues serve as receptors for both the influenza myxovirus and reoviruses, although there are rotaviruses that do not require their presence (mendez et al, ) and low density lipoproteins (ldl) are receptors for both the human minor cold picorna virus and avian leukosis viruses. viruses compete with molecules that require receptors for a physiological function of the host. for example, ldl and the human minor rhinovirus compete for ldl receptors (table i) , and cells with down-regulated ldl receptor expression yield much less virus than up-regulated cells (hofer et al, ) . viruses tend to use abundant molecules as receptors, so that reduction in availability of the molecules for the physiological function is not lethal, or molecules whose function can be substituted by other molecules. there are alternative viral strategies to deal with the receptor problem. the part of the sodium-independent transporter of cationic amino acids, used as the receptor for ecotropic bovine leukemia virus (table i), is different from the part of the protein directly involved in the amino-acid transport function. thus the physiological function of the receptor can continue, despite binding of virus to the receptor (wang et al, ) . another example confirming this possibility is the sodium-dependent transporter of inorganic phosphate that serves as the receptor for the gibbon ape leukemia virus (table i). productive infection of cells expressing this receptor results in complete blockage of the uptake of inorganic phosphate mediated by the receptor. nevertheless, the infection is not cytotoxic. hence, there is likely more than one phosphate transport mechanism in these cells (olah et al, ) . this aspect of viral strategies may open up possibilities to block the receptor sites, thus preventing entry of a virus without serious impairment of physiological function of the receptor. the receptor for herpes simplex virus exemplifies a situation of special interest from the point of view of future engineering of disease resistance. the viral receptor heparan sulfate is present on cell surfaces but body fluids also contain heparin and heparin-binding proteins, either of which can prevent binding of herpes simplex virus to cells (spear et al, ) . hence spread of the virus is likely influenced by both immune response and the probability that the virus will be entrapped and inhibited from binding to cells by extracellular forms of the receptor (heparin or heparan sulfate). similarly, soluble molecules of the cd receptor for human immunodeficiency virus, as well as fragments of the critical cd domains can inhibit infection . it has been suggested that a secreted receptor for avian leukosis virus might similarly be able to neutralize the virus (bates et al, ) . penetration of a virus into the cells is usually an energy-dependent process that occurs almost instantly after attachment. as summarized by roizman ( ) , penetration can occur as ( ) translocation of the entire virus particle across the cell membrane; ( ) endocytosis resulting in accumulation of virus particles in-side cytoplasmatic vacuoles; or ( ) fusion of the cell membrane with the virion envelope. non-enveloped viruses penetrate host cells by the first two processes. uncoating of the virus particle takes place after penetration. for some viruses, such as orthomyxoviruses and picorna viruses, divestiture of the protective envelope or capsid takes place upon their entry into cells. for others, such as herpes viruses, the capsid is transported along the cytoplasmic cytoskeleton into nuclear pores. with reoviruses, only a portion of the capsid is removed and the viral genome expresses all its functions even though it is never fully released from the capsid. while several genetic engineering strategies to prevent attachment of viruses to host cells can be devised and are proposed below, strategies to prevent penetration of viruses attached to cells are much less obvious. viruses use many strategies for replication leading to ( ) encoding and organization of viral genomes, ( ) expression of viral genes, ( ) replication of viral genes, and ( ) assembly and maturation of viral progeny. the key event in these processes is the synthesis of viral proteins. regardless of its size, organization, or composition, a virus must present to the cell's protein synthesizing mechanisms an mrna that the cell recognizes and translates. the interaction between the viral cell attachment protein and host-cell receptors is the principal determinant of tropism, but there are other factors involved. for retroviruses and papovaviruses, cis-acting elements of the viral genome, gene enhancers, which are usually - bp in size and often repeated in tandem, stimulate transcription (serfling et al, ) . they may serve as an entry point for rna polymerase ii. enhancers may be both cell-type-specific and cell-differentiationspecific, in that they function mainly in certain cell types (tyler and fields, ) . for avian retroviruses, enhancer regions within the long terminal repeat (ltr) are an element of the viral genome that determines cell tropism of disease expression ). the cell imposes three constraints on the virus at the point of virus multiplication. ( ) the cell may lack enzymes to synthesize mrna off the viral rna genome, or may lack enzymes to transcribe viral dna. ( ) eukaryotic host cell protein-synthesis machinery translates only monocistronic messages and does not recognize internal initiation sites within mrna. as a consequence the virus must synthesize either a separate mrna for each gene or an mrna encompassing a 'polyprotein' to be later cleaved. ( ) the expression of viral proteins is in competition with cellular genes. viruses evolved strategies that either confer competitive advantage to viral mrna or abolish translation of cellular mrnas. the host range of a virus defines both the kinds of tissue or cells and animal species in which a virus can enter and multiply (roizman, ) . receptors may be species specific. for example, the poliovirus receptor is only found on primate mammalian cells (mclaren et al, ) . a tissue-specific receptor is exemplified by the cd receptor for the hiv virus, which is present only on t-lymphocytes (table i) . species-specifity of receptors is one of the components of non-host resistance that will be discussed in more detail below. infection with some viruses leads to inhibition of transcription of cellular proteincoding genes by host polymerase ii, possibly through competition for transcription between cellular and viral genes. herpes simplex virions contain a transcriptional activator complex (post et al, ) , while adenovirus provides a trans-acting eia gene product responsible for increased polymerase activity after adenovirus infection (nevins, ) . viruses can also induce or express new dna-binding proteins. thus a retrovirus encodes a homolog to cellular transcription factor ap- (bohmann et al, ) . splicing of viral mrna precursors is accomplished by cellular enzymes. influenza and retroviruses can regulate the extent of the splicing, adenovirus inhibits maturation of cellular mrna, and influenza virus transcription complexes intervene in the host mrna maturation (knipe, ) . many viral mrnas are capped, in that they contain a single major initiation site near their ' end, and their translation is similar to that of host mrna. however, inhibition of host mrna translation provides the virus with increased availability of ribosomal units. thus herpes simplex and poxvirus degrade cellular mrna to decrease its translation (inglis, ; fenwick and mcmenamin, ) . other mechanisms include competition for the host translational apparatus by production of large amounts of viral mrna, or viral mrna with higher affinity to ribosomes than cellular mrna (knipe, ) and changes in the specificity of host translational apparatus; for example, extracts from poliovirus-infected cells translate poliovirus but not host mrna (rose et al, ) . both rna and dna viruses cause inhibition of host-cell dna synthesis (knipe, ) . eukaryotic cell proteins contain signals that target them to a specific cell compartment or organelle. viral proteins may also contain similar signals for their localization within the cell. viral proteins make use of cellular chaperone proteins to secure their proper folding. similarly, many post-translational modifications of viral proteins are performed by cellular enzymes. for example, tissue-specific proteases cleave specific proteins on the virion surface thus facilitating virion infectivity (scheid and choppin, ) . maintenance of viral dna in the host cell and release of progeny virus there are two types of mechanism for maintaining viral dna in the host cell: ( ) virus dna is integrated into the cellular genome, eg, in retroviruses; or ( ) viral dna is maintained as extrachromosomal circular molecule in the infected cell, eg, epstein-barr virus, or bovine papilloma virus. viruses that persist in the body may cause damage, and prevention of persistence may be the next best defence if prevention of virus entry is impossible. persistence is usually in differentiated cells that remain morphologically unchanged but may lose their differentiated or 'luxury' function, as well as their homeostasis. persistent viruses can negatively influence host cells in two ways: ( ) virus presence and replication causes damage resulting in a selective disadvantage; and ( ) in such a way that the virus will gain an evolutionary advantage for which there will be selection pressure to maintain. alternatively, some viruses undergo a latency stage in their life cycle that seems to cause little damage. enveloped viruses move from infected cells either by budding through the plasma membrane or by secretion vesicles containing virus particles within the plasma membrane (knipe, ) . non-enveloped viruses are mostly released by lysis of the cells but they can also leave without cell lysis as in simian virus (norkin and ouelette, ). to facilitate their survival and spread throughout the body, some viruses have evolved strategies to modulate the immune response of their host to their favor, a phenomenon recently reviewed by fujinami ( ) . virus infection can lead to development of immune responses against the host's own tissues and viruses can also code for proteins, homologous to cellular proteins, that modify the host's immune response. for example, epstein-barr virus produces a bcrf protein similar to the interleukin il- protein (a cytokine-inhibiting factor) that inhibits the production of il- and il- , tumor necrosis factor, gamma interferon, and macrophage-granulocyte colony-stimulating factor. the herpes simplex virus- (hsv- ) but not hsv- can interfere with the complement system by producing a protein that acts as a receptor for the component of the complement cascade. virus infections can also interfere directly with the major histocompatibility system (mhc). cytomegalovirus encodes an mhc class i heavy-chain homolog that limits expression of the cellular class i molecules on cell surfaces and this may reduce killing of infected cells by host defences. most animal and plant species are resistant to the great majority of viruses. nonhost resistance is the rule, susceptibility the exception. however, the nature of nonhost resistance is not sufficiently understood to fully explore the incompatibility between viruses and non-hosts (wilson, ) . nevertheless, it is certain that we, as well as all animals, are &dquo;continuously bathed in a sea of microbes, yet harmed by a relatively few&dquo; (oldstone, ) . to coexist, viruses and their hosts have established, to a greater or lesser degree, an equilibrium. in general, normal coevolution of parasites and their hosts is from disoperation, through exploitation, to toleration and from facultative to obligatory mutualism, but genetic changes may also bring reversals to this process (dobzhansky, ) . none of the strategies for the creation of new, genetically engineered viral resistance mechanisms proposed in this article are derived from non-host resistance. nevertheless, a brief discussion of the subject is included to stimulate further exploration of this widespread phenomenon as the possible basis for protection of livestock against viruses. some knowledge of non-host resistance mechanisms is emerging from experimentation with plant viruses that infect permissible but normally resistant cells by bypassing the resistance barrier (dawson and hilf, ) . viral host range is determined by interactions between existing viral gene products and corresponding host components. because of the obligately parasitic nature of viruses, viral host range is not determined by a particular gene product that enables the virus to overcome host defences but by a 'fit' between viral gene product and certain gene products of the host. there are two general prerequisites for successful infection: ( ) presence of all conditions necessary for viral infection. absence of the conditions results in 'passive resistance mechanisms' in plants, that tend to be recessive or incompletely dominant. ( ) absence of successful host defences. adaptation mechanisms of viruses that enable them to infect potential hosts protected by non-host mechanisms may include an ability to overcome a host block by a mutation or recombination with another virus, or acquisition by the virus of capabilities formerly provided by the hosts that are not available in resistant plants. a virus can capture such genetic information from the host. there are many mechanisms of resistance to viral diseases. for our purposes, emphasis will be placed on non-immune mechanisms. of particular interest in this review are those mechanisms that prevent the entry of viruses into host cells. viral receptors can be variable so that some alleles of the receptor may make the potential host resistant to viral infection. however, it is only rarely that resistance to infection is observed in otherwise susceptible host species. this indicates that during virushost coevolution, viruses tend to utilize evolutionarily stable molecules as receptors. resistance to infection by parvovirus b in some humans is due to lack of a specific virus receptor. people who do not have the erythrocyte p antigen parvovirus receptor (brown et al, ) are naturally resistant to the virus (brown et al, ) . another example is resistance to coronaviruses in mice. a monomeric protein has been identified as a receptor for mouse hepatitis virus on intestinal and liver cells. the presence of this receptor appears to be the principal determinant of susceptibility to infection (boyle et al, ) . similar variation in viral receptors is observed in genetic resistance to avian leukosis virus (alv) infection in chickens (payne, ) . the alv receptors, which belong to the family of receptors for ldl (bates et al, ) , include recessive alleles that do not allow viral entry into potential host cells and render some chickens resistant to the virus. the receptor for subgroup a alv was shown to map to tva * s known as the dominant gene for susceptibility to subgroup a virus (bates et al, ) . susceptibility of cells to infection needs to be distinguished from permissiveness, which can be defined as the ability of a cell to support viral replication. for example, chick cells are not susceptible to poliovirus but are permissive to its replication following their transfection with poliovirus rna (roizman, ) . such cells are potential hosts for a virus, providing a mutation provides means for the virus to enter the cells. in laboratory mice, alleles at the fv- locus determine susceptibility to infection with ecotropic murine leukemia viruses and the resistance is dominant in heterozygous mice (ikeda and odaka, ) . a viral protein gp normally interacts with the viral receptors on cells. however, in resistant mice, the specific receptor on cell membranes seems already bound by the gp whose production is controlled by the mouse fv- '' resistant allele. this system is similar to that in chickens, where the endogenous retroviral gene ev- , expressing the subgroup e endogenous viral envelope also controls resistance to infection by subgroup e virus (robinson et al, ) . resistance of mice to certain strains of influenza virus is a dominant trait associated with the allele mx on chromosome (staehli et al, ). the resistance is mediated by action of alpha-and beta-interferons that induce mx protein expression which inhibits synthesis of viral mrna (krug et al, ) . a recent review of natural, 'preimmune' resistance loci in mice (malo and skamene, ) includes genes controlling resistance to influenza virus, cytomegalovirus, ecromelia, friend leukemia virus, mink cell focus-forming virus, moloney leukemia, radiation leukemia, and rous sarcoma virus. the resistance genes represent a variety of mechanisms that do not involve viral receptors. for example, the cmvl gene, associated with resistance to cytomegalovirus, appears to control host responses mediated by natural killer and inflammatory response cells. similarly, the resistance loci in friend leukemia control the susceptibility of target cells to viral replication. it is not the purpose of this review to provide a detailed account of immune mechanisms that protect against virus infection. the brief text below will give only a general outline of immune responses and examples of how the system may be influenced by viruses. acquired immune responses involve phagocytic, humoral and cell-mediated systems. only the cell-mediated immune response that is especially effective against cells containing actively replicating virus and, as a rule, is the most important defence against viral infections will be discussed briefly. the cellular immune system becomes sensitized to viral infection only after viral proteins are degraded to short linear peptide epitopes that become complexed with class i or ii major histocompatibility complex proteins. the resulting complexes are transported to cell surface, where they are presented as 'non-self' entities to t-lymphocytes. if the viral antigen has not previously encountered the t-cell repertoire of the host, the initial antigen-specific activation event requires appearance of mhc-peptide complexes on antigen-presenting cells. but if activated t-cells, previously sensitized to the viral epitopes are available, then a broader class of antigen-presenting cells can be targeted for clearance by cytotoxic t cells. in both events, the ability to discriminate self molecules from the viral epitopes depends on the presentation of the non-self peptide to t-cells in specific peptide-binding grooves of the mhc molecules on antigen-presenting cells. mcfadden and kane ( ) summarized how dna viruses perturb the mhc and alter immune recognition. a number of gene products of dna viruses have been identified as directly affecting mhc expression or antigen presentation, whereas rna viruses interact with mhc by indirect mechanisms. most dna viruses are able to modulate cellular immunity. it seems that many viral gene products remain to be identified among the open reading frames of as yet unknown function that exists in these viruses. besides a trivial strategy of hiding dna molecules in cells, such as neurons that lack mhc surface molecules, viruses can modify mhc expression directly within cells or indirectly at the level of cytokine regulation. there is now evidence that viruses can combat antiviral effector t cells directly by blocking their antiviral activity (bertoletti et al, ) . in humans infected with hiv- and hepatitis b viruses, naturally occurring variants of epitopes recognized by cytotoxic t lymphocytes may act as antagonists in vivo because the corresponding peptides prevent a cytotoxic t cell response. although exactly how the antagonists function is not known, it is evident that the presence of these antagonists prevents the t cell from performing its function. endogenous viruses represent a separate phenomenon with regards to the immune system. as a rule, the host is completely immunologically tolerant to endogenous viruses. however, antibodies against endogenous retroviruses were found in mice (miyazawa et al, ) . how the immune system makes antibodies against endogenous retroviral gene products is unknown but this ability may relate to the expression of such genes after the establishment of immunological tolerance to endogenous retroviral antigens expressed earlier in life (miyazawa and fujisawa, ) . a similar delay in expression of the endogenous viral gene ev- has been described in chickens (crittenden, ) and may serve as a model for construction of similar 'self-vaccinating' transgenes in the future. given the potential benefits that can be derived from the use by the host of parts of a pathogen's genome to induce resistance, the paucity of pathogen-mediated resistance mechanisms in nature is surprising. the situation begs the question whether evolution exhausted all such possibilities in the development of host defences. why did certain mechanisms develop and others not? a reason for the absence or rare occurrence of pathogen-mediated defence mechanisms may be that they encompass some disadvantage for the host. one example in which a viral genome has become an integral part of the host are endogenous proviruses found in germ cells of all vertebrates. for example, in the laboratory mouse endogenous proviruses occupy more than . % of the cellular dna (pincus et al, ) . in the genomes of chickens, there are several families of retrovirus-related permanent insertions. in the most thoroughly studied family of endogenous viral genes, there are more than endogenous proviruses in various parts of the genome (crittenden, ) . the presence of some of these proviruses may interfere in the spread of the generally non-pathogenic endogenous virus produced by other such proviruses. however, the endogenous proviruses do not protect the host against infection with similar but more harmful, pathogenic exogenous viruses. on the contrary, the antigenic similarity between the products of the endogenous proviruses and the exogenous viral antigens reduces the ability of birds with certain types of these proviruses to mount an immune response against the exogenous virus (crittenden et al, ; gavora et al, b) . a possible reason why other endogenous proviral sequences did not evolve as resistance mechanisms is that their expression may adversely affect important physiological processes of the host (gavora et al, a,b) and reduce the ability of the host to resist the exogenous analogues of the proviruses. genetic variation is a prime prerequisite for genetic change by selection. as a general rule, genetic variation exists in the ability of livestock to tolerate infectious diseases. and it was this variation that allowed populations of domestic animals and birds to survive under continuous exposure to rapidly evolving disease agents. before domestication, disease resistance of today's livestock species was influenced by natural selection and the current status of variable resistance to multiple disease agents can be considered to be the result of a response to the selection pressure of multiple pathogens. as a consequence of domestication, a significant new element that entered this evolutionary system was artificial selection for characters that benefit humans as users of livestock. simultaneously, housing conditions evolved towards increased concentration of animals and birds and thus provided opportunities for spread of pathogens. improved disease prevention and control measures now provide some compensation for the larger population sizes used in current production systems. selection for disease resistance plays a relatively minor but increasingly important role in livestock improvement. the choice of selection criteria and the emphasis they receive in the context of total selection pressure available to a practical breeder are decided by market demands and economic considerations. disease resistance traits receive attention from the breeders mainly when a specific disease is a major cause of economic loss. although in most instances existing genetic variation provides an adequate basis for resistance selection, selection may not always be practised. such selection is expensive because the expression of resistance traits requires exposure of selection candidates or their relatives to the disease agent. this is why industries prefer to look for indirect selection techniques that do not require pathogen challenge. recent developments in gene mapping provide good prospects for progress in this direction. indirect selection for resistance to the herpesvirus of marek's disease in chickens, by increasing the frequency of the 'resistant' major histocompatibility haplotypes, is one example of such a technique. it has been practised by most of the world's poultry breeding companies over the past two decades (gavora, ). conventional procedures for direct and indirect selection for disease resistance will in the foreseeable future be the main route for genetic improvement of disease resistance. one disadvantage of their application is the general absence, with rare exceptions mentioned above, of genetic variation in resistance to infection. thus genetic improvements in disease resistance by conventional means lead mostly to better resistance of livestock to disease developmenta situation where the organism becomes infected but tolerates the pathogen and reduces its ill effects. hence development of new genetic mechanisms that prevent entry of a pathogen into the host, or otherwise substantially improve the position of the host in the pathogen-host interaction is justified. while conventional selection leads to quantitative improvement of resistance, the new mechanisms would represent a qualitative change that, at least in some instances, will justify the large effort and cost. the expenses will be further justified if the new, engineered mechanism proves to be stable and remains effective despite evolution of the pathogen and functions without harmful effects on the animal's production capacity. improvement in the welfare of the modified livestock will be an automatic, additional benefit. in crops despite large differences between animals and plants, sufficient similarities exist in their resistance mechanisms to justify examination of the situation in plants with regards to genetic engineering of viral resistance. for example, normal virus replication requires a subtle balance of virus and host coded proteins, present in critical relative concentrations at specific times and locations. therefore, wilson ( ) suggests that any unregulated superimposition of protein or nucleic acid species interacting with the virus can result in plants in an apparently virusresistant phenotype. the results from experimentation with animal cells into which a viral gene was inserted indicate that a similar situation may also exist in animals (gavora et al, ) . the idea that viral components contained in plants might interfere with virus infection was first proposed well before gene transfer techniques became available (hamilton, ) and the concept of pathogen-derived resistance was first put forward in a formal statement by sanford and johnston ( ) . there are several approaches to the introduction of disease resistance by gene transfer in plants (fitchen and beachy, ) . they include transfers of segments of viral genome encoding capsid or coat proteins, viral sequences encoding proteins that may be subunits of viral replicase, sequences incapable of encoding proteins, entire genomes of defective, interfering viruses, and complete genomes of mild virus strains. the transgenes may act on initiation of infection, replication of virus, spread of infection throughout the plant, and symptom development. the level of protection derived from the transgene ranges from low to high and its breadth of host range from broad to narrow. the available data are not sufficient to firmly establish the molecular mechanisms of the protection. in general, although a viral sequence may confer resistance in one virus-host system, an analogous sequence from a different virus in another virus-host system may not be effective. the conceptual simplicity of the approach and availability of virus coat gene sequences facilitated broad implementation of this strategy. fichten and beachy ( ) list published examples of this approach. it is unlikely that a single mechanism accounts for the observed resistance of the transgenic plants but regardless of the mode of the transgene action, resistance results from a block in an early event in the infection process (fichten and beachy, ) . in resistance to some viruses other than tobacco mosaic, it seems that accumulation of the coat protein transgene rna, rather than the virus coat protein itself is responsible for resistance. resistance has been observed even in plants that transcribed a translation-incompetent coat protein mrna (kawchuk et al, ; de haan et al, ) . it seems that even in the absence of understanding of its mechanism, the strategy can be extended to other plant species and viruses. protection by sequences encoding replicase-related proteins replicase-mediated resistance was first demonstrated against tobacco mosaic virus (golemboski et al, ). the number of initially infected cells in transgenic and non-transgenic plants was the same but virus replication was markedly reduced in cells of the transgenic plants. replication of the virus was severely impeded and little or no systemic spread of the virus occurred (carr and zaitlin, ) . protection by the accumulation of rna plants were protected by rna-mediated resistance to a degree comparable to protein-mediated resistance. transgenic tobacco plants, carrying a translationally defective tomato spotted wilt virus nucleocapsid gene exhibited resistance similar to that in experiments with translationally competent gene constructs (de haan et al, ) . other examples include potato plants with constructs producing sense and antisense transcripts of potato leafroll virus (kawchuk et al, ) and tobacco plants and similar transcripts of tobacco mosaic virus (powell et al, ) . protection by transgene copies of mild strains, satellites and satellite rnas, and defective interfering viruses transgenic tobacco plants carrying cdna of a mild strain of tobacco mosaic virus developed only mild symptoms when challenged with severe strains of the virus (yamaya et al, (gerlach et al, ) . nevertheless, this approach does not seem desirable because the transgenes may produce active pathogens by recombination or a pathogenic mixture. also, transgene components may recombine with another virus, thus extending its host range or virulence (fitchen and beachy, ) . the identification of a variety of disease resistance (r) genes is expected to facilitate identification and introgression of new resistance from wild species into new plant varieties. it is well known that a new resistant plant variety developed over a long time and with great effort is often overcome by a new pathogenic race an immensely wasteful situation. such breakdown of resistance is much less likely in varietal mixtures that carry an array of different r genes. once different r genes are cloned, varieties can be produced that consist of mixtures of lines differing only in the r gene allele they carry (staskawitz et al, ) . for genetically engineered resistance, pathogen-inducible promoters, such as the prpl-i promoter in potato (martini et al, ) may be the most advantageous as they induce the 'resistance' peptide(s) only in cells that are being challenged by a compatible pathogen (de wit, ) . the extent of the research effort to genetically engineer new resistance mechanisms in animals is much smaller than that in plants and available data on the subject are reviewed below. the first successful introduction of pathogen-mediated resistance to disease in animals was reported by salter and crittenden ( ) . they produced several lines of chickens, each with an insert of a recombinant avian leukosis retroviral genome at a different locus within the host genome. the transgenic birds that expressed only the viral envelope coding region of the recombinant genome were shown to be resistant to the corresponding subgroup of the avian leukosis virus (salter and crittenden, ; gavora et al, a) , due to a blockage of virus receptors by the viral envelope proteins. another introduction of a new virus resistance mechanism into a livestock host was attempted by clements et al ( (owens et al, ) . expression under the control of metallothionein of a single glycoprotein d gene from herpes simplex virus (hsv- ) rendered cells resistant to infection by hsv but not by other viruses (johnson and spear, ) . the mechanism of this resistance is not known but it seems likely that d interacts with a cell surface component required for viral penetration. in an attempt to introduce resistance to bovine rotavirus that causes calf diarrhea and results in large economic losses, two genes that code for rotavirus capsid proteins, implicated in early virus-host cell interactions, were transferred into the genomes of susceptible cells in culture and, one of the genes, also into genomes of laboratory mice (gavora et al, ) . the transgenes produced mrna of the relevant viral genes but no corresponding protein was detected either in the cells or in the mice. nevertheless, several of the transformed cell lines showed significantly increased resistance to bovine rotavirus (gavora et al, ) , while no increase in the resistance of four similarly transformed lines of mice was detected following challenge of pups shortly after birth with the virus (js gavora, unpublished results). antisense rna although not yet tested in vivo, the use of antisense rna to combat viruses has received attention by researchers and presents another possible avenue for the construction of new resistance mechanisms. the possibilities of inhibiting retroviral replication by antisense molecules before its integration into a host chromosome has been demonstrated (to and neiman, ) . to block viral integration, antisense sequences can be designed to target regions essential in the synthesis of viral dna intermediates or viral integration. replication of a recombinant avian retrovirus, carrying a neomycin resistance gene neo' in the antisense orientation was blocked when cells expressed high levels of neo' rna molecules in the sense orientation, suggesting that antisense rna inhibition may be a useful strategy for inhibition of retroviral infections . it was hypothesized that when sequences immediately upstream of the polypurine tract are hybridized to antisense molecules, rnase h failed to process the rna sequences in the polypurine tract into a functional primer for the synthesis of plus-strand dna (to and neiman, ) . they suggested that an antisense segment in that region can be defined for use in a large number of pathogenic retroviruses. these experiments also showed that constructs expressing the antisense rnas can be delivered by replication-competent retroviral vectors to host cells in culture, thereby immunizing the host cells against superinfection with different retroviruses. the advantage of the antisense rna approach may be that only about basepairs are needed to bind the antisense rna with absolute precision to a unique mrna and intensive research is now under way to develop antisense therapeutics (bradley et al, ) . even though the mechanism will not prevent viral entry into host cells, it may prevent integration of the viral genome in the host chromosome. catalytic rnas, known as ribozymes, are not rare in nature and it is possible to engineer an intron that can repeatedly perform the first chemical step in the splicing process (parker et al, ) . ribozymes have been shown to cleave target rna and to inhibit mrna transcript activity (edington and nelson, ) . the principal advantage of ribozymes is their ability to cleave and thus inactivate multiple targets. even though ribozyme-mediated gene inhibition involves a mechanism (target cleavage) different from that of bacterial antisense rnas, many of the essential steps of the two mechanisms are identical. ribozymes were shown to successfully inhibit gene expression in xenop!s oocytes in tissue culture (cotten and birnstiel, ) and may be another possible approach to the engineering of new disease resistance mechanisms for livestock. transfer of resistance genes from another species as was mentioned above, the murine mxl is a protein with activity against influenza virus. garber et al ( ) inserted cdna encoding this protein into chicken embryo fibroblasts through the use of a replication-competent avian retroviral vector. cells infected with the vector were resistant to infection with avian, as well as human influenza viruses but susceptible to enveloped rna viruses. biological costs of and risks associated with genetic engineering conventional methods of genetic improvement are rather forgiving in the sense that they induce gradual changes and provide time for the breeder to correct disturbances in biological equilibria that might be harmful to the animals. gene transfer, on the other hand, may induce dramatic, undesirable changes that will disturb development or physiological functions that are difficult to correct. however, new technological developments, such as homologous recombination and use of embryonic stem cells for gene transfer will likely reduce the risks. given the extent of work on transfer of disease resistance-inducing genes in both plants and animals, surprisingly little research has been done on the possible physiological consequences of adding such new genes to cells. consequences of transgenes have been demonstrated in plants by hilder and gatehouse ( ) . they studied lines of transgenic tobacco containing a cowpea trypsin inhibitor gene construct which expressed the transgene at various levels and plants that possessed, but did not express, the gene. small, but in some instances, significant differences between the transgenic and non-transformed plants were found in various parameters but there was no additional difference between plants that expressed the transgene and those that did not. they concluded that although the transformation may have some small effects on non-targeted phenotypic characteristics, the expression of the transgene at high levels imposed no additional yield penalty on the plants. negative genetic correlations between disease resistance and production traits have been reported (eg, gavora, ) but their basis as to linkage or pleiotropy is not clearly established. design of genetically engineered resistance mechanisms may have to take possibility of such negative correlations with production traits into consideration. as mentioned above, a transgene that successfully induced resistance of chickens to avian leukosis retrovirus subgroup a in chickens (salter and crittenden, ) was shown to result in a sizeable reduction of egg production rate (gavora et al, a) . it was suggested that the reduced ovulation rate was due to interference of the viral envelope protein produced by the transgene with the attachment of the virus to host cells and also with transport of lipids into the developing egg yolk, since the virus uses an ldl receptor for entry into host cells (bates et al, ) . on the other hand, a transgene containing a gene for a capsid protein of bovine rotavirus in laboratory mice (gavora et al, ) was not associated with any significant effects on their growth and reproductive performance (j nagai and js gavora, unpublished results). hence, significant 'biological costs' may not always accompany insertion of transgenes but they need to be considered in strategies for genetic engineering of new resistance mechanisms. reports on work on assessment of risks involved in the production of varieties with new, genetically engineered resistance are only available for plants. transgenic plants expressing viral pathogen-derived dna sequences have been considered sites for hyperevolution of viruses through recombination of a mild or defective viral genome with the transgene (de zoetten, ) . however, there is no experimental evidence to confirm this supposition. on the contrary, evidence against this type of event exists through one to up to eight viral passages, even though heteroincapsidation of viral rna by transgenically expressed viral coat proteins has been observed (wilson, ) . the danger that transgenic crops may generate new viruses and diseases has been assessed by falk and bruening ( ) . they provide evidence that genomic recombination was observed when transgenic tobacco plants expressing a segment of cowpea chlorotic mottle virus genomic rna were inoculated with a mutant of the same virus that contained a deletion (greene and allison, ) . the important question is whether such recombination can produce dangerous new viruses. rna-rna recombination has indeed been demonstrated for four groups of rna plant viruses. the recombination occurs between closely related rna molecules, possibly at sites of similar rna structure. under usual crop production circumstances, opportunities exist for genetic interaction between plant viruses in mixed virus infections. since both crop plants and weeds may be present in a field, recombinations between a virus that cannot infect a plant and one that can, do not have a zero probability. nevertheless, mixed infections rarely result in new plant pathogenic viruses. instead, new viral diseases are usually due to minor variants of already known viruses. generally, however, existing viruses are stable, having to fit hosts that evolve only slowly. falk and bruening ( ) believe it is unlikely that recombinations between transgene rna and viral genomic rna will occur at greater frequencies than the recombinations already occurring between virus genomic rnas in natural infections. in the past, development of resistant plants by traditional breeding fostered the emergence of virulent virus strains (dawson and hilf, ) but the cost of this phenomenon is much less than the cost of abandoning plant breeding. similarly, the benefits of engineered plant resistance genes far outweigh the vanishingly small risk of creating harmful new viruses in significant excess over those being created by natural processes (falk and bruening, ) . in mice, endogenous proviruses are known to recombine with exogenous viral sequences to give rise to novel viruses with unique properties (pincus et al, ) . similar recombinants between exogenous and endogenous avian retroviruses had been produced in vitro and used as transgenes to induce resistance to the exogenous retrovirus in chickens (salter and crittenden, ) . endogenous viral genes may be regarded as prototypes of transgenes in animals. early evidence that rous sarcoma virus recombined with envelope protein of endogenous avian virus was provided by hanafusa et al ( ) . recently, an env gene related to endogenous viral gene was found on the exogenous avian leukosis virus subgroup j (bai et al, ) . there is also evidence that the alv transgene that expresses the avian leukosis virus subgroup a envelope can recombine with endogenous virus from gene ev to produce subgroup a infectious virus (lb crittenden, personal communication). until more results become available in animals, we could assume that a situation similar to that described above for plants will also exist in livestock. however, it is imperative to keep the possible risks in mind in designing strategies for induction of resistance by genetic engineering and to experimentally assess the recombinations, if any, between transgenes and existing viruses in farm animals and birds. an example of an increase in the virulence of an animal virus that may be associated with improved resistance of the host by vaccination and genetic means is the emergence of highly virulent marek's disease herpesviruses in chickens (witter, ) . the viruses may have emerged as a consequence of vaccination and conventional selection for resistance that included efforts to increase the frequency of major histocompatibility haplotypes associated with such resistance. genetically engineered resistance may provide a more stable solution to the marek's disease problem. conventional breeding and vaccination improved survival of chickens infected by marek's disease virus. however, the virus continues to be present in vaccinated birds so there are ample opportunities for its mutations towards higher virulence. a genetically engineered mechanism that would prevent the entry of the virus into the host cells would reduce the size of the viral population and thus reduce the possibility of such viral evolution. unfortunately emergence of viral mutations to overcome the genetically engineered barrier to virus entry would be difficult to eliminate. it seems that the arguments used by plant breeders in favor of continuing research toward new, engineered resistance genes should also be valid for livestock. a necessary prerequisite for this development has to be an adequate system of controls and thorough testing of the engineered livestock. as mentioned above, any introduction of new genetic material into a cell carries with it a risk of disrupting cell functions. this risk has to be kept in mind in the design of new resistance mechanisms. it may be possible to minimize such risks on the basis of a thorough understanding of the physiology of virus-infected animals and interactions between the virus and the host. another, no less important aspect of the design of new resistance mechanisms is their long-term stability. the new mechanism may become ineffective through evolution of the virus which will overcome the resistance provided by the transgene. evolution of pathogen virulence genes that overcame resistance induced by conventional breeding is well known and documented in plants (flor, ; wilson, ) , and a possible instance of a similar phenomenon observed with marek's disease herpesvirus in chickens was mentioned above. the design of new mechanisms and strategies of disease resistance to be introduced into livestock by genetic engineering techniques is a search for mechanisms that did not, for whatever reason, develop by evolution. unlike most of the mechanisms of defence of the hosts against viruses that resulted in virus tolerance by the host, the ideal goal of the new, engineered mechanisms should be prevention of viral entry into host cells. it may be easier to develop new resistance strategies for viruses which depend for most of their functions on the host cell than for those that provide for the functions in their genome. new techniques of molecular and cell biology allow transfers of genes between species, taxonomic genera and even kingdoms so that we are no longer limited by the constraints of sexual compatibility. recent progress in the development of techniques of homologous recombination, together with the use of embryonic stem cells for gene transfer provide good prospects for progress in this area of research (first et al, ) . while the use of both of these techniques is now routine in laboratory mice, their application in animal agriculture is hampered by the unavailability of a reliable technique for the production of embryonic stem cells in any of the livestock species. nevertheless, given the high level of interest and scientific activity in this area in several countries, it is likely only a matter of time before embryonic stem cells will become available for introduction of new genetic information into the genomes of farm animals and birds. homologous recombination and use of embryonic stem cells will allow insertion of a transgene in a predetermined location in the genome. in the case of gene constructs designed to induce new resistance mechanisms, the insertion will likely be targeted into a 'neutral' region of the genome, to minimize the potential disruption of important genomic functions. after successful insertion, it will be possible to test the transformed embryonic stem cells in culture for the expression of the transgene, its stability and, as much as possible, its undesirable effects on the cells. preliminary testing in cell culture for resistance to the pathogen in question will be also possible. only the embryonic cell lines that will meet criteria of acceptability in the above tests will be used for the introduction into developing embryos with the goal of producing disease resistant transgenic individuals. it is anticipated that the protocol will make the introduction of new disease resistance mechanisms into livestock less expensive. the approach will also be less risky as the dangers of disruption of important genetic mechanisms by the transgene insertion will be reduced by gene targeting. moreover, the reduction of such risks will make the research more acceptable for both livestock producers and the general public. unfortunately, the use of advanced techniques of gene transfer will likely be limited to developed countries. because of their relative simplicity and small size, the genomes of viruses are generally better understood than those of host cells. many viral genomes have been sequenced and it is generally easy to obtain the necessary sequence information for viral genes that are candidates for inclusion into potential resistance-inducing transgene constructs. the general principles for the design of new resistance mechanisms and the new defence strategies can be summarized as follows. the most useful would be mechanisms based on an element common to the life cycle of multiple viruses thus inducing resistance simultaneously to more than one virus. the new mechanisms should be designed to minimize their biological and financial costs. targeting of transgenes into 'neutral' regions of the genome may be one such strategy. the 'neutrality' of such regions can be tested by inserts of non-functional genes. the regions proven to be 'neutral' would be subsequently used for inserts of resistance genes. ideally the functioning of the new mechanisms should be triggered by the presence of the inducing virus, otherwise the mechanism should remain 'silent'. this type of mechanism would minimize its biological cost to the host. despite preliminary testing of transformed cells in culture, it will be essential to subject livestock carrying the resistance transgenes to a series of rigorous tests gama et al, ) . the tests need to prove the genetic potential of the new stock for economically important production traits, general viability, as well as resistance against the disease for which the transgene was designed. in instances of slight impairment of the production capacity of the transgenic, compared to the original stock, decisions on the practical usefulness of the modified animals will depend on comparison of the economic benefit derived from the transgene against the cost of the animals' reduced production performance. in this context, the prevalence of the pathogen in question and the damage it causes in the production areas for which the resistant animals are intended will be, no doubt, important considerations. based on considerations of the viral life cycle, and natural and genetically engineered resistance mechanisms that were already tested, several possible strategies can be proposed and are listed below according to stages of viral life cycle. the strategies are identified in a general manner, without reference to specific viruses. therefore, no description of details of their design and implementation is attempted. the aim of this list is to stimulate further activity in this area by outlining the opportunities that exist. without a doubt, a new resistance mechanism that would prevent viral attachment and penetration into host cells represents the most desirable approach. those acting on subsequent phases of viral life cycle are less desirable and should be considered if prevention of viral attachment and penetration is impossible. viral attachment and penetration into host cell transgenes that produce viral antireceptor (virion surface) proteins to block cellular receptors; -produce soluble receptors or their components to block virion surface proteins and prevent their interaction with cellular receptors; -replace host receptor genes by a modified form that is able to perform the receptor's physiological function but does not allow the attachment of the virus; -produce substances that interfere with viral penetration into host cells. transgenes that induce antisense rna to a part of the viral genome crucial for virus multiplication; -cause multiplication and accumulation of viral or modified viral rna in host cells; -disturb viral replicase or its function; -produce ribozymes attacking viral rna; -produce a defective viral protein that competes with the normal one to produce a high proportion of non-infectious virions. transgenes that induce and maintain a latent state of the virus; -do not allow activation of a virus from its natural latent state. transgenes that protect against perturbances of the host's immune system; -produce the vaccinating antigen only after the immune system is fully developed (self vaccinating transgenes). conclusions enormous variability of viral types in their strategies for life and survival will likely make it difficult to engineer generalized resistance to viruses. in their evolution, some viruses have developed strategies that do not harm the host sufficiently to cause extinction of the host -and the virus. nevertheless, in some instances virus-host coevolution has resulted in disease-producing relationships that cause economic losses and suffering of the animals and birds. conventional breeding methods will remain the principal approach to the improvement of disease resistance in livestock but in some instances, introduction of new genetically engineered resistance mechanisms may be justified. prerequisites for the design of new resistance mechanisms include good knowledge of the viral genome and life cycle (keeping to a minimum the biological cost of the new strategies to the host) and of the probability that the strategies will be overcome by viral evolution. a combination of gene targeting techniques with embryonic stem cells, when such cells become available for livestock, will greatly facilitate the introduction of new, genetically engineered virus resistance. all livestock with new resistance mechanisms will have to be subjected to thorough testing. there are several possible strategies for the development of new resistance mechanisms in livestock. the transgenes to be designed for such strategies can act at various phases in the viral life cycle. ideally, expression of the transgenes should be triggered by the presence of the inducing virus, otherwise the resistance mechanism should remain 'silent'. strategies that prevent viral entry to the host are expected to be most valuable as they could eliminate all damage to the host caused by the virus. hprs- (exogenous avian leukosis virus, subgroup j) has an env gene related to those of endogenous elements eav-o and e and an e element found previously only in sarcoma viruses isolation and characterization of a . kilobase-pair cdna fragment encoding the binding domain of the bovine leukemia virus cell receptor attachment of sa- rotavirus to erythrocyte receptors varmus he ( ) a receptor for subgroup a rous sarcoma virus is related to the low density lipoprotein receptor the cloned receptor for subgroup a avian leukosis virus maps to tva * s, the dominant gene for susceptibility to subgroup a virus natural variants of cytotoxic epitopes are t-cell receptor antagonists for antiviral cytotoxic t-cells vaccinia virus -kilodalton protein: relationship to several mammalian proteins, including two growth factors human proto-oncogene c-jun encodes a dna binding protein with structural and functional properties of transcription factor ap- genetic resistance to mouse hepatitis virus correlates with absence of virus-binding activity on target tissues antisense therapeutics vaccinia virus encodes a polypeptide homologous to epidermal growth factor and transforming growth factor long terminal repeat (ltr) sequences, env, and a region near the ' ltr influence the pathogenic potential of recombinants between rous-associated virus types and erythrocyte p antigen: cellular receptor for b parvovirus resistance to parvovirus b infection due to lack of virus receptor (erythrocyte p antigen) a sialoglycopeptide from human erythrocytes with receptor-like properties for encephalomyocarditis and influenza viruses resistance in transgenic tobacco plants expressing a nonstructural gene sequence of tobacco mosaic virus is a consequence of markedly reduced virus replication does the beta-andrenergic factor receptor function as reovirus receptor? development of transgenic sheep that express the visna virus envelope gene human coronavirus oc interacts with major histocompatibility complex class i molecules at the cell surface to establish infection ribozyme-mediated destruction of rna in vivo influence of endogenous viral (ev) gene expression and strain of exogenous avian leukosis virus (alv) on mortality and alv infection and shedding in chickens retroviral elements in the genome of the chicken: implication for poultry genetics and breeding host-range determinants of plant viruses characterization of rna-mediated resistance to tomato spotted wilt virus in transgenic tobacco plants determinants essential for the transmissible gastroenteritis virus receptor interaction reside within a domain of aminopeptidase n that is distinct from the enzymatic site molecular characterization of gene-for-gene systems in plant fungus interactions and the application of avirulence genes in control of plant pathogens risk assessment: do we let history repeat itself? determination of the rate of base pair substitution and insertion mutation in retrovirus replication utilization of ribozymes in plants. plant viral resistance will transgenic crops generate new viruses and new diseases? early virion-associated suppression of cellular protein synthesis by herpes simplex virus is accompanied by inactivation of mrna epstein-barr virus receptor of human b lymphocytes is the cd receptor cr systems for production of calves from cultured bovine embryonic cells genetically engineered protection against viruses in transgenic plants the complementary genetic systems in flax and flax rust molecular mimicry: virus modulation of the immune response transgene effects, introgression strategies and testing schemes in pigs avian cells expressing the murine mxl protein are resistant to influenza virus infection disease genetics introduction of resistance to bovine rotavirus by gene transfer influence of the alv recombinant avian leukosis virus transgene on production traits and infection with avian tumor viruses in chickens endogenous viral genes influence infection with avian leukosis virus construction of a plant disease resistance gene from the satellite rna of tobacco ringspot virus plants transformed with tobacco mosaic virus nonstructural gene sequence are resistant to the virus multiple self-splicing introns in bacteriophage t , evidence from autocatalytic gtp labelling of rna in vitro recombination between viral rna and transgenic plant transcripts the major human rhinovirus receptor is icam- defences triggered by previous invaders: viruses recovery of a new virus from apparently normal chick cells by infection with avian tumor viruses human (hla-a and hla-b) and murine (h k and h d) histocompatibility antigens are cell surface receptors for semliki forest virus phenotypic cost to plants of an extra gene members of the low density lipoprotein receptor family mediate cell entry of a minor group common cold virus trans-dominant inhibition of human immunodefficiency virus type rev occurs through formation of inactive protein complexes cellular expression of murine leukemia virus gp -related antigen on thymocytes of uninfected mice correlates with fv- gene-controlled resistance to friend leukemia virus infection inhibition of host protein synthesis and degradation of cellular mrnas during infection by influenza and herpes simplex virus herpes simplex virus glycoprotein d mediates interference with herpes simplex virus infection sense and antisense rna-mediated resistance to potato leafroll virus in russet burbank potato plants t-lymphocyte t molecule behaves as the receptor for human retrovirus lav virus-host-cell interactions haller ( ) inhibition of influenzal viral mrna synthesis in cells expressing the interferon-induced mx gene proct genetic control of host resistance to infection promoter sequences of a potato pathogenesis-related gene mediate transcriptional activation selectively upon fungal infection how dna viruses perturb functional mhc expression to alter immune recognition the mammalian cell-virus relationship attachment of poliovirus to cultivated cells of primate and nonprimate origin cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily binding to sialic acids is not an essential step for entry of animal rotaviruses to epithelial cells in culture genetic exchange between bacteriophage t and filamentous fungi? a family of retroviruses that utilize related phosphate transporters for cell entry physiology and pathology of host immune responses to exogenous and endogenous murine retroviruses -from gene fragments to epitopes pathogenesis of arteritis of sl/ni mice. possible lytic effect of anti-gp antibodies on vascular smooth muscle cells identification and chemical synthesis of a host cell receptor binding site on hepatitis b virus control of cellular and viral transcription during adenovirus infection cell killing by simian virus : variation in the pattern of lysozomal enzyme release, cellular enzyme release, and cell death during productive infection of normal and simian virus -transformed simian cell lines the cellular receptor for gibbon ape leukemia virus is a novel high affinity sodium dependent phosphate transporter rous-whipple award lecture: viruses and diseases of the twentieth century adeno-associated virus rep proteins produced in insect and mammalian expression systems: wild-type and dominantnegative mutant proteins bind to the viral replication origin ribozymes: principles and designs for their use as antisense and therapeutic agents genetics of cell receptors for avian leukosis viruses the interplay of microbes and their hosts regulation of alpha genes of herpes simplex virus: expression of chimeric genes produced by fusion of thimidine kinase with alpha gene promoters protection against tobacco mosaic virus in transgenic plants that express tobacco mosaic virus antisense rna host susceptibility to endogenous virus: defective glycoprotein-expressing proviruses interfere to infections multiplication of viruses inhibition of translation of poliovirus: inactivation of a specific initiation factor artificial insertion of a dominant gene for resistance to avian leukosis virus into the germ line of the chicken the concept of parasite-derived resistance-deriving resistance genes from parasite's own genome protease activation mutants of sendai virus. activation of biological properties by specific proteases enhancers and eukaryotic gene transcription blocking of hiv- infectivity by a soluble, secreted form of the cd antigen on the use of transgenes in livestock improvement heparan sulfate glycosaminoglycans as primary cell surface receptors for herpes simplex virus interferon-regulated influenza resistance gene m! is localized on mouse chromosome molecular genetics of plant disease resistance virus evolution inhibition of retroviral replication by antisense rna the potential for effective antisense inhibition of retroviral replication mediated by retroviral vectors constitutive expression of the nef gene suppresses human immunodeficiency virus type (hiv- ) replication in monocyte cell lines pathogenesis of viral infections antigens and structure of the adenovirus a critical site in the cell surface receptor for ecotropic murine retroviruses required for amino acid transport but not for viral reception herpes simplex genes: the blueprint of a successful human pathogen the molecular biology of the gene structure of the influenza virus hemagglutinin complexed with its receptor, sialic acid strategies to protect crop plants against viruses: pathogen-derived resistance blossoms very virulent marek's disease viruses: importance and control cross protection of transgenic tobacco plants expressing a mild strain of tobacco mosaic virus mouse hepatitis virus receptors: more than a single carcinoembryonic antigen the author wishes to express his gratitude to the institut national de la recherche agronomique, for the provision of a pleasant, friendly, and stimulating working environment at the laboratoire de g n tique factorielle at jouy-en-josas, france, where he compiled this review during a six month stay in . helpful comments and suggestions were provided during the preparation of the manuscript by lb crittenden, ri hamilton, and jl spencer. key: cord- -jtv jmkn authors: wang, lin-fa; walker, peter j.; poon, leo l.m. title: mass extinctions, biodiversity and mitochondrial function: are bats ‘special’ as reservoirs for emerging viruses? date: - - journal: curr opin virol doi: . /j.coviro. . . sha: doc_id: cord_uid: jtv jmkn for the past – years, bats have attracted growing attention as reservoirs of emerging zoonotic viruses. this has been due to a combination of factors including the emergence of highly virulent zoonotic pathogens, such as hendra, nipah, sars and ebola viruses, and the high rate of detection of a large number of previously unknown viral sequences in bat specimens. as bats have ancient evolutionary origins and are the only flying mammals, it has been hypothesized that some of their unique biological features may have made them especially suitable hosts for different viruses. so the question ‘are bats different, special or exceptional?’ has become a focal point in the field of virology, bat biology and virus-host co-evolution. in this brief review, we examine the topic in a relatively unconventional way, that is, our discussion will be based on both scientific discoveries and theoretical predictions. this approach was chosen partially because the data in this field are so limited that it is impossible to conduct a useful review based on published results only and also because we believe it is important to provoke original, speculative or even controversial ideas or theories in this important field of research. lin-fa wang , peter j walker and leo l m poon for the past - years, bats have attracted growing attention as reservoirs of emerging zoonotic viruses. this has been due to a combination of factors including the emergence of highly virulent zoonotic pathogens, such as hendra, nipah, sars and ebola viruses, and the high rate of detection of a large number of previously unknown viral sequences in bat specimens. as bats have ancient evolutionary origins and are the only flying mammals, it has been hypothesized that some of their unique biological features may have made them especially suitable hosts for different viruses. so the question 'are bats different, special or exceptional?' has become a focal point in the field of virology, bat biology and virus-host co-evolution. in this brief review, we examine the topic in a relatively unconventional way, that is, our discussion will be based on both scientific discoveries and theoretical predictions. this approach was chosen partially because the data in this field are so limited that it is impossible to conduct a useful review based on published results only and also because we believe it is important to provoke original, speculative or even controversial ideas or theories in this important field of research. bats (order chiroptera), one of the most abundant, diverse and geographically dispersed vertebrates on earth, have recently been shown to be reservoir hosts of a number of emerging viruses responsible for severe disease outbreaks in humans and livestock [ , , ]. the first recognition that bats are involved in the ecology of human disease came during the s when rabies virus was identified in bats in south and central america [ ] . however, the discovery of henipaviruses in the mid- s and the subsequent recognition that bats may be a natural host of sars-like coronaviruses and filoviruses marked a new era of fresh research into the role of bats as an important reservoir host of viruses which have the potential to cause disease in humans and livestock [ , [ ] [ ] [ ] [ ] [ ] . the recent surge of interest in bats as a reservoir of viruses was driven by two factors. first, in less than years, several high profile viral pathogens have been proven or hypothesized to have a bat origin. since hendra virus was first discovered in , there have been at least known spillover events in australia with a mortality rate in humans of approximately % [ ] . the closely related nipah virus has been responsible for devastating disease outbreaks in malaysia, bangladesh and india with mortality rates ranging from % to %, resulting in the deaths of approximately humans [ ] . filoviruses (ebola and marburg viruses) have caused outbreaks in africa with associated human mortality rates as high as %, and have been linked to mass gorilla die-offs, making them both a public health and conservation concern [ ] [ ] [ ] . the outbreak of severe acute respiratory syndrome (sars) in - , due to a previously unknown coronavirus, resulted in more than human infections with a mortality rate close to % and an estimated cost of $ billion in lost tourism and trade [ ] [ ] [ ] . the association of these high profile pathogens and disease outbreaks with bats has led to an increase in public interest, funding and research activities on these and many other bat-borne viruses. however, it should be emphasized that, although closely related sars-like coronaviruses have been detected in horseshoe bats, the exact natural reservoir of the coronavirus responsible for the sars outbreaks is still unknown [ , ] . the true natural reservoir of ebola virus is also still being debated as rodents, insectivores and bats have all been identified as potential sources of infection in primates [ , ] . the second driver for the recent surge in bat virus research has been advances in modern molecular techniques which have presented opportunities for discovery of novel bat viruses, that were considered impossible or nonpractical just a decade ago. using pan-virus-specific primers and next-generation sequencing, it is now possible to detect and characterize novel viral sequences without the need for virus isolation by cell culture or the identification of virions by electron microscopy. numerous publications in the past few years have reinforced the observation, first made by sulkin and allen in [ ], that bats carry a wide range of novel rna and dna viruses. these results also provide support to the notion, as first observed during the investigations of bat coronaviruses (see below), that bats within a geographic location and/or taxonomic group have an unusual ability to harbor a large number of genetically diverse viruses. more recently, two metagenomic studies on bat fecal samples have revealed a great number of novel bat viruses, some of which have moderate sequence identity to previously known mammalian viruses, including members of the parvoviridae, circoviridae, picornaviridae, adenoviridae, poxviridae, astroviridae, herpesviridae and coronaviridae [ , ] . further systematic surveillance will be required to determine whether bats are the natural hosts of these novel viruses, but these results clearly indicate that there are many bat viruses yet to be identified. interestingly, similar to other metagenomic analyses of human or other animal fecal samples, these studies have also identified numerous sequences derived from viruses infecting insects, plants and bacteria. although these viruses are unlikely to infect bats, one might hypothesize that they could play an important role in facilitating the dispersal of these viruses to different geographical locations and different hosts. the high detection rate and great genetic diversity of viral sequences from bats have not only propelled further scientific and public interest in this field, but also led to debate on the importance of bats as reservoirs of zoonotic viruses. the question 'are bats different?' has been raised at many international conferences and has been the topic of several previous reviews [ , , , ]. while the currently available data are too limited to provide a conclusive answer, this review aims to examine different hypotheses which may eventually allow us to resolve this intriguing and fundamentally important question. it should be noted, however, that some of the discussions presented in this review are largely speculative or even controversial. this has been done intentionally by the authors to invigorate discussion and further research on this topic. bats have several features that might help to explain the seemingly high rate of virus detection. bats constitute the second largest order of mammals. there are about bat species worldwide, which represents more than % of all mammalian species [ ] . bats are classified in the order chiroptera in which there are two suborders: the yinpterochiroptera (also known as megachiroptera), which contains the megabats, and the yangochiroptera (microchiroptera), which includes the majority of microbat families [ ] . the wide range of bat species could provide a large 'breeding ground' for viruses. the earliest known bat fossil dates to . million years ago (mya) [ , ] . extrapolation of fossil records and genetic data has suggested that the basal split from other placental mammals in the superorder laurasiatheria occurred during the late cretaceous period approximately - mya, with extensive diversification of extant bat families commencing approximately mya [ ] [ ] [ ] . bat viruses may therefore have co-evolved with or adapted to bats over many millions of years. besides, bats are the only mammalian species that can fly and some bat species can migrate hundreds of miles to their overwintering or hibernation sites [ ]. thus, bats have more opportunities than terrestrial mammals to have direct or indirect contact with other animal species at different geographical locations, thereby enhancing the opportunity for interspecies virus transmission. in addition, some insectivorous bats exhibit exceptionally long life-spans of - years and live in panmictic populations comprising of millions of individuals. the long life-span of bats may facilitate the transmission of chronic persistent infections, whereas the unusually large and complex structure of bat populations may ensure a sufficient number of immunologically naive juveniles for bat viruses to persist in bat colonies. some bat species also have a capacity for hibernation over winter or to enter into daily torpor to conserve energy. the reduced body temperature and metabolic rate may suppress robust immune responses and reduce the rate of virus replication, thereby delaying virus clearance from bat populations [ , ]. purely a numbers game: more bat species = more viruses? in one of the most comprehensive reviews on bat viruses, calisher et al. [ ] listed different bat viruses that have either been isolated or detected. since then, many more novel bat viruses, as well as variants of previously known bat viruses, have been reported. in total, virus families - families of rna viruses and five families of dna virus -are known to infect bat genera [ ] . the detection rate of novel viruses or viral sequences appears to have been higher in bats than that in any other mammalian species for the past two decades or more. it could be argued that, as bats represent the second largest group of mammals (comprising % of all mammalian species), it is not entirely surprising that there are many bat viruses. however, some of our recent indirect evidence suggests that bats may be atypical hosts of at least some viruses. firstly, the genetic diversity and prevalence of infection of some rna viruses in bats is unusually high. we previously reported the detection of genetically highly diverse astroviruses and coronaviruses in bat fecal samples, with the prevalence of infection of these novel bat viruses in the range of - % [ , ] . however, similar surveillance studies for astrovirus and coronavirus in rodents sampled at the same geographic location indicated that none of the samples (n = ) were positive for coronavirus, whereas only . % of the tested brown rat (rattus norvegicus) samples (n = ) were positive for astroviruses [ ] . at least at this location, bats appear to harbor many more coronaviruses and astroviruses than rodents. secondly, phylogenetic analysis of viral sequences has revealed that a large number of coronaviruses recognized in other mammalian species share a common ancestor with various other bat coronaviruses ( figure ). these findings suggest that bats are likely to be the natural reservoir from which all presently known mammalian coronavirus lineages have evolved [ ] . the high prevalence of viral infection in bats, together with some of the unusual characteristics of bats discussed above, may have facilitated the transmission of bat viruses to other mammals. as surveillance data for viruses in wildlife are currently scarce, it may be premature to conclude that bats host a greater diversity of viruses than other animals. for example, more than hantaviruses have been identified in rodents -the largest group of mammals on earth ( % of all mammalian species) -and each hantavirus appears to have co-evolved with a specific rodent [ , ] . rodents are also considered to be the natural reservoir of arenaviruses with which they appear to have co-evolved [ ] and waterfowls are known to be the natural reservoir of influenza viruses [ ] . it is possible that bats, rodents, birds and other wildlife may be ancient reservoirs of different sets of virus taxa. further systematic surveillance for viruses in different wildlife populations using metagenomics or other molecular approaches is required to determine if the large number of viruses identified in bats is just simply numbers game. nevertheless, the prevalence of infection of certain bat virus families appears to be much higher than has been reported for the viral families co-evolved with rodent and avian species, suggesting that bats may have some intrinsic properties which make them more suited as a reservoir host. the five great mass extinctions that have punctuated the history of life on earth have played a major role in shaping the modern biosphere [ ] and it is reasonable to assume that mass extinctions will also have impacted profoundly on the evolutionary history of viruses. the most recent mass extinction, the k-t extinction, occurred million years ago. it followed the earth impact of the large bolide that created the - -km-wide chicxulub crater in northern yucatan, mexico [ , ] and resulted in - % reduction in marine diversity at the species level, % at the genus level, and the loss of % of all species worldwide [ , ] . the k-t extinction will also have impacted on viral diversity. indeed, as the survival of virus populations is inextricably linked to the survival of their host species, the rate of virus extinction during precipitous mass extinctions is likely to have been far greater than that of their hosts. virus extinction will have occurred not only as a consequence of host extinction but also through decreases in host population size and host isolation to a level that could not sustain ongoing virus transmission (table ) . even temporary host species decline or isolation, followed by recovery and survival, will have had potential for virus extinction. survival will have favored those viruses that could persist either in the environment or in the host, those that caused no disease or mortalities, those that were transmitted vertically, those with a broad host range, and those for which the host survived with little impact on population size. surviving host species that were largely unaffected by such a devastating mass extinction event are likely therefore to have been important sources of extant viral biodiversity. because of the exceptional paucity of the fossil record, the evolutionary history of bats is not as well documented as many other vertebrate lineages. however, as discussed above, bats are known to have origins in the late cretaceous period and appear to have diversified rapidly during the period immediately after the k-t extinction [ , , ] . it has been argued that the short intense heat pulse caused by the ballistic atmospheric re-entry of ejecta following the bolide impact created a catastrophe that set the stage for later evolutionary events [ ] . indeed, ancestral bats, rodents, insectivores and some birds are likely to have had the characteristics of animals mass extinctions, biodiversity and mitochondrial function: wang, walker and poon table effects of the k-t bolide impact on host populations and likely consequences on the contemporary virosphere. characteristics required for survival of the nuclear winter and food chain collapse that is predicted to have followed the initial impact [ ] . bats are now second only to rodents as the most ecologically and morphologically diverse mammalian clades, adapting to almost every terrestrial environment and accounting for over % of extant mammalian diversity [ ] . it follows that bats may also be one of the most important sources of extant mammalian virus diversity and supports the view that bat viruses may have ancient origins and a long history of co-evolution with their hosts. as described above, bats do appear to host a strikingly wide range of viruses and are likely the natural reservoir from which all presently known mammalian coronavirus lineages have evolved [ ] . it has also been suggested that the ubiquity, wide genetic diversity and deeply rooted phylogeny of bat lyssaviruses and paramyxoviruses indicate that bats may be their natural ancestral hosts [ , ] and bats, rodents and shrew have been found to contain integrated filovirus-like genome elements that suggest a very ancient relationship [ ] . although there may not be a direct link between host diversity and virus diversity, the long evolutionary history and the ecological diversity of bats will also have presented a myriad of opportunities for cross-species transmission of viruses to and from many other host species, further enhancing their role as amplifiers of viral biodiversity. the discovery of endogenous viral elements (eves) integrated into animal genomes appears to provide the long-sought opportunity to trace the deep evolution of viruses and the role bats may have played in shaping the modern virosphere [ ] . as discussed above, persistence in the absence of pathology or disease appears to be a common characteristic of bat viruses in their natural host population and this is also indicative of a highly evolved relationship [ , , , ] . the ecological balance that maintains infection and transmission in the absence of disease favors both pathogen and host and it can be argued that each may have contributed to its evolution [ ]. the host aims to detect and contain or eliminate the pathogen through an effective immune response to avoid disease or mortality. the virus needs only to maintain replication and transmission beyond the extinction threshold (ro > ) and the long-term survival of the virus may be improved if this can be achieved in the absence of disease or mortality [ ] . however, the high replication rate, mutation frequency and potential for recombination of viruses, particularly rna viruses, provide a potential for continual adaptation and refinement that far exceeds that of their hosts [ ] . it could be argued, therefore, that the most significant characteristic of viral infections in bats may not be the effectiveness of a highly evolved host immune response, but rather the absence of pathology as the result of an ancient and highly evolved viral survival strategy. for many rna viruses such as those commonly infecting bats, accessory proteins and evolved secondary functions of other viral proteins play a key role in infection by blocking host innate immune defences, modulating cellular signaling pathways and re-directing normal cellular functions [ ] [ ] [ ] . the refinement of these functions during a long evolutionary history in bats may well have defined a successful strategy for long-term survival, even through the periods of catastrophic environmental disruption and diminished biodiversity. conversely, the severe pathology and disease that often occurs as a result of spill-over of bat viruses into other vertebrate hosts may result not from an inherently less effective immune response but from the disturbance of this finely tuned interaction of viral proteins with their targets in host cells. it can also be argued that there are several ways in which the harboring of well-adapted viruses might also bring a biological advantage to bats. one possibility is through symbiotic enhancement of innate immunity. although innate immunity has long been considered a broad, nonspecific and nonanamnestic first line of host defence, recent studies have demonstrated that persistent infection with one pathogen may prime host innate immunity to provide cross-protection from others. this has been best illustrated by a study in mice demonstrating that herpesvirus latency confers protection from bacterial in-mass extinctions, biodiversity and mitochondrial function: wang, walker and poon table favorable characteristics for survival and proliferation following the k-t mass extinction of bats and other potential sources of extant viral biodiversity. survival fection [ ] . in a paper by roossinck, examples of 'good viruses' and virus-host symbiosis have also been reviewed for viruses infecting human, wasps, plants, fungi, aphids and bacteria [ ] . highly adapted viruses persistently infecting bat populations might also serve to protect bats at the species or population level from predators (e.g., tree roosting animals such as raccoons and opossums, owls and hawks, and primates) in a sense acting as defensive 'biological weapons'. the best defensive weapons are those that do no harm to the host species and are released only when there is an imminent threat of danger and the emerging bat viruses (e.g., henipaviruses and filoviruses) satisfy these requirements. henipaviruses are believed to persist in bat populations at a very low viral load and are totally harmless to their natural host. however, under stress, the viral load increases, facilitating transmission to other animals [ , ] . they have a very broad range of susceptible hosts and are highly lethal in many different vertebrate species [ ] . such a mechanism might not be able to protect every individual animal in a population, but it would be an effective way to preserve the species. in principle, such a symbiotic relationship with viruses would benefit any animal species and there is evidence that such relationships do exist in very different hosts including humans, mice to fungi and bacteria [ ] . it is perhaps the long period of co-evolution and some unique selective pressures that have driven its emergence and dominance in bats. bats have a relatively low reproductive rate (usually one birth with one pup per year) compared to other animals such as rodents and, as discussed above, bats tend to live in very large and dense populations. these biological and behavioral characteristics may demand far more robust mechanisms to fight infection and predation in order to avoid extinction. flight capability, longevity and innate immunity -are they linked? as discussed above, some of the 'unique' biological characteristics of bats are believed to contribute to the observation that they appear to harbor a large number of viruses without clinical signs of disease. while the scientific data are not sufficient to make any conclusive link, it is tempting to speculate on the interplay for some of these factors. in table , three key aspects of the biology of bats are analyzed in the context of their impact on cellular metabolism and infectious agents. flight ability is the most distinguishing feature of bats amongst mammals. flight consumes a large amount of energy, demanding a much higher rate of metabolism. in general, it is believed that a high metabolic rate, such as that in bats, is likely to generate more metabolic byproducts, which, in turn, will increase the rate of oxidative damage to mitochondrial dna and other cellular structures [ ] . according to the 'rate of living' theories, animals with a high metabolic rate are likely to be short-lived [ , ] . although the combination of small body size, high metabolic rate and long lifespan in bats does not seem to be compatible with this view, recent studies on mitochondrial dna and cellular processes have indicated that multiple mechanisms exist in bats (and other long lifespan animals such as birds) to allow them to be more efficient in resisting oxidative damages than short lifespan animals [ ] . oxidative damage to dna is also an important mechanism of tumorigenesis [ ] . it is therefore interesting that unpublished anecdotal observations suggest that bats have a lower rate of tumorigenesis than most other animals. an extensive literature search revealed only a few recent papers describing tumors in egyptian fruit bats [ ] [ ] [ ] . in one case, a sarcomatoid carcinoma was diagnosed in the lung of a -year-old male captive bat, and in the other case a gastrointestinal leiomyosarcoma was found in a -yearold female bat. during our own study to establish bat cell lines, a wide international collaborative effort examining bats from australia, asia and africa failed to identify any tumors from a large number of individual bats representing more than ten different bat species [ ] (g. crameri, l.-f. wang, unpublished observations). although the jury is still out, it is not impossible that efficient mechanisms for countering oxidative damage in bats result in a lower rate of tumorigenesis. on the other hand, it is also possible that the low reporting rate of bat tumors results from a lack of appropriate detection/diagnostic methods for bat tumors or general interest in this area of research. mitochondria are key organelles in controlling cellular metabolism. for bats, the efficient function of mitochondria emerging viruses table potential association of unique bat biological features with a symbiotic relationship with viruses. unique biological feature impact on metabolism impact on infectious agents true ability to fly requiring more energy efficient metabolism greater chance of inter-species and long distance transmission rapid change of body temperature highly efficient sensing and regulation of temperature effect on immune system favoring persistence long lifespan relative to body size more efficient mechanism to prevent oxidative damage to dna is likely to be essential for key biological characteristics such as flight, body temperature changes and lifespan, all of which could impact on the ecology of viral infection (table ) . until very recently, it was not recognized that mitochondria also act as a center of signaling pathways for apoptosis, inflammation and innate immune responses [ , , ] . this is a very new and rapidly evolving field of research but it is clear that mitochondria are involved in signaling for antiviral and antibacterial immunity [ ] . all published studies to date have been conducted in human or mouse cell lines so it will be extremely interesting and important to conduct parallel studies in bat cells to determine whether mitochondria have similar functions in controlling innate immune responses in bats. in summary, we speculate that the key unique biological features of bats, that is, ability to fly, high metabolic rate and longevity, are functionally interconnected and mitochondria are the key cellular organelles that link all of these processes. these features, in turn, all have an impact on the bat's ability to control tumors and infection. this fundamental and common innate ability of bats may help explain their seemingly super anti-ageing, antitumor and anti-infection characteristics. multiple hypotheses are presented in this review in an attempt to address the question as to whether bats are special as reservoir hosts of viruses. while we are not able to provide a definitive answer to the question, we hope that the range of new ideas and angles presented here will stimulate those who work in the field to explore further in the future. it is possible that all of the aspects discussed here, although some of them seem to be mutually exclusive, may play a part in the overall picture of high-rate detection of viruses and infection with no diseases in bats. if bat's innate ability to counter biological imbalance proves to be different from or more robust than other mammals in whatever way or shape, it will provide a tremendous opportunity for us to 'learn from bats' and apply some of these principles to human and animal health, either via therapeutic intervention in humans or transgenic modification in livestock animals. however, one must recognize that despite the great interest in bat viruses in recent years, bat biology research is in its infancy compared with existing knowledge of infection in humans and other animals such as rodents. there is a total lack of research tools and reagents to address any of the hypotheses in depth. thus, there is an urgent need to advance the basic study of bat biology and bat immunology to help remove the road blocks. taylor dj, leach rw, bruenn j: filoviruses are ancient and integrated into mammalian genomes. bmc evol biol , : . this is the first report of endogenization in the mammalian genome of nonretroviral rna viruses with extranuclear replication. the endogenous viral elements were most commonly detected in bats, rodents and insectivores. phylogenetic analysis suggested an ancient association between filoviruses and mammals that was dated to tens of millions of years ago. olival kj, epstein jh, wang l-f, field he, daszak p: are bats unique virus reservoirs? in conservation medicine, edn . edited by aquirre aa, ostfeld rs, daszak p. oxford university press (in press). a recent review on the same topic as this review, but focusing on different aspects. it is worth to read this book chapter in conjunction with the current review for a more complete appreciation of the subject. bats and viruses: a brief review sur une grande epizootie de rage swanepoel r: fruit bats as reservoirs of ebola virus a morbillivirus that caused fatal disease in horses and humans nipah virus: a recently emergent deadly paramyxovirus bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats henipaviruses: emerging paramyxoviruses associated with fruit bats henipavirus vaccine development. j bioterrorism biodefense ebola outbreak killed gorillas the ecology of ebola virus bats, clocks, and rocks: diversification patterns in chiroptera order chiroptera early eocene bat from wyoming linking the wasatchian/bridgerian boundary to the cenzoic global climate optimum: new magnetostratiographic and isotopic results from south pass a highresolution genetic signature of demographic and spatial expansion in epizootic rabies virus placental mammal diversification and the cretaceous-tertiary boundary a molecular phylogeny for bats illuminates biogeography and the fossil record host and viral ecology determine bat rabies seasonality and maintenance an example of using mathematical modeling to identify factors that are important for viral infection dynamics in bat populations novel astroviruses in insectivorous bats identification of a novel coronavirus in bats detection of novel astroviruses in urban brown rats and previously known astroviruses in humans the most recent paper out of a series studies on astroviruses conducted by the hong kong group, which demonstrated that the genetic diversity of astroviruses in bats is greater than those in other mammals evolutionary insights into the ecology of coronaviruses emergence and persistence of hantaviruses virus evolution and genetic diversity of hantaviruses and their rodent hosts evolution of the old world arenaviridae and their rodent hosts: generalised host-transfer or association by descent? influenza: emergence and control mass extinctions in the marine fossil record new links between the chicxulub impact structure and the cretaceous/tertiary boundary extraterrestrial cause for the cretaceous-tertiary extinction. experimental results and theoretical interpretation extinctions in the fossil record selectivity of end-cretaceous marine bivalve extinctions the delayed rise of present-day mammals a phylogenetic supertree of the bats (mammalia: chiroptera) survival in the first hours of the cenozoic evolutionary biology -a first for bats primitive early eocene bat from wyoming and the evolution of flight and echolocation a bony connection signals laryngeal echolocation in bats energy, volatile production, and climatic effects of the chicxulub cretaceous/ tertiary impact reassessing conflicting evolutionary histories of the paramyxoviridae and the origins of respiroviruses with bayesian multigene phylogenies genomic diversity and evolution of the lyssaviruses endogenous viral elements in animal genomes this paper reports the use of a systematic screening in-silico to detect the common occurrence of endogenous elements derived from a diverse array of dna and rna viruses in animal genomes. analysis of the sequences of the endogenous viral elements (eves) with respect to extant viruses indicated ancient origins infectious disease modeling and the dynamics of transmission the evolution and emergence of rna viruses mechanisms of severe acute respiratory syndrome pathogenesis and innate immunomodulation understanding the accessory viral proteins unique to the severe acute respiratory syndrome (sars) coronavirus inhibition of interferon induction and signaling by paramyxoviruses virgin hwt: herpesvirus latency confers symbiotic protection from bacterial infection the good viruses: viral mutualistic symbioses an updated review on virus-host symbiosis. it is an excellent paper to read for those wishing to learn a bit more about the importance and progress in this area of research anthropogenic deforestation, el nino and the emergence of nipah virus in malaysia nipah virus outbreak in malaysia hendra and nipah viruses: different and dangerous oxidative damage to dna: relation to species metabolic rate and life span bats and birds. exceptional longevity despite high metabolic rates an excellent review for anyone who is interested in the subjects of aging, longevity and flying ability of animals in the rate of living viral oncogene-induced dna damage response is activated in kaposi sarcoma tumorigenesis sarcomatoid carcinoma in the lung of an egyptian fruit bat (rousettus aegyptiacus) gastrointestinal leiomyosarcoma in an egyptian fruit bat (rousettus aegyptiacus) microchip-associated leiomyosarcoma in an egyptian fruit bat (rousettus aegyptiacus) establishment, immortalisation and characterisation of pteropid bat cell lines emerging role of damageassociated molecular patterns derived from mitochondria in inflammation this paper describes the establishment of bat primary and immortalized cell lines which are becoming increasingly important for isolation of bat viruses and for basic study on virus-bat interaction recent advances in apoptosis, mitochondria and drug resistance in cancer cells mitochondria in innate immune responses mass survival of birds across the cretaceous-tertiary boundary: molecular evidence the role of mitochondria in innate immunity is a relative new topic in immunology and this updated review provides an excellent starting point to review the recent discoveries and future research directions bat mating systems impact winter and the cretaceous-tertiary extinctions -results of a chicxulub asteroid impact model a theoretical exercise in the modeling of ground-level ozone resulting from the k-t asteroid impact: its possible link with the extinction selectivity of terrestrial vertebrates evidence for echolocation in the oldest known bats impact of the terminal cretaceous event on plant-insect associations we thank e.c. holmes for useful discussions on the evolution of bat viruses. key: cord- -x f hn f authors: tzelepis, ilias; kapsetaki, stefania-elisavet; panayidou, stavria; apidianakis, yiorgos title: drosophila melanogaster: a first step and a stepping-stone to anti-infectives date: - - journal: curr opin pharmacol doi: . /j.coph. . . sha: doc_id: cord_uid: x f hn f following an expansion in the antibiotic drug discovery in the previous century, we now face a bottleneck in the production of new anti-infective drugs. traditionally, chemical libraries are screened either using in vitro culture systems or in silico to identify and chemically modify small molecules with antimicrobial properties. nevertheless, almost all compounds passing through in vitro screening fail to pass preclinical trials. drug screening in drosophila offers to fill the gap between in vitro and mammalian model host testing by eliminating compounds that are toxic or have reduced bioavailability and by identifying others that may boost innate host defence or selectively reduce microbial virulence in a whole-organism setting. such alternative screening methods in drosophila, while low-throughput, may reduce the cost and increase the success rate of preclinical trials. drosophila melanogaster: a first step and a stepping-stone to anti-infectives ilias tzelepis , stefania-elisavet kapsetaki , stavria panayidou and yiorgos apidianakis following an expansion in the antibiotic drug discovery in the previous century, we now face a bottleneck in the production of new anti-infective drugs. traditionally, chemical libraries are screened either using in vitro culture systems or in silico to identify and chemically modify small molecules with antimicrobial properties. nevertheless, almost all compounds passing through in vitro screening fail to pass preclinical trials. drug screening in drosophila offers to fill the gap between in vitro and mammalian model host testing by eliminating compounds that are toxic or have reduced bioavailability and by identifying others that may boost innate host defence or selectively reduce microbial virulence in a whole-organism setting. such alternative screening methods in drosophila, while low-throughput, may reduce the cost and increase the success rate of preclinical trials. a common countermeasure to the ever-growing antibiotic drug resistance is the production of new effective drugs. nevertheless, the rate of production of new antibiotics is steadily declining [ ] . one reason for this might be the chemical screening methods that rely solely on in vitro culture systems. traditionally, drug research is moving from in vitro small molecule screens to preclinical assessment in mammalian hosts. there are two problems with this approach: first, tests in mammals are costly and can usually be restricted to a few compounds at a time, and second, in vitro assays are inappropriate to capture the complexity of an infected host [ ] . live hosts are preferable because they enable drug toxicity and bioavailability assessment at the organismal level [ ] and [ ] . in addition, drugs that might interfere with the host microenvironment or microbial virulence per se can only be assessed upon the interaction of microbes with a host. thus, quality antiinfective drug assessment in simple model hosts might be a more effective way to identify new drug leads. in this review, we aim to examine the suitability of drosophila melanogaster as a model organism for anti-infective drug assessment due to its high degree of molecular, cellular and physiological conservation with humans, which allows the modelling of infections that recapitulate aspects of human disease [ , ] . in this respect, drosophila might fill the gap between in vitro screens and preclinical trials or be used directly, instead of in vitro screens. drosophila has a short life cycle of days from egg to sexually mature adult as compared to the . months of mice (table ) . large numbers of flies can be propagated quickly, since tens of females can produce hundreds of offspring within two weeks. the offspring become sexually mature very early in their adult life, enabling the life cycle to continue [ ] . due to its small size of mm in length thousands of flies can be contained in a space that would normally fit less than mice. in addition, fly food is usually made of grocery store ingredients such as cornmeal, yeast and sucrose, thus the cost of maintenance is quite low. moreover, there are no ethical concerns or regulated protocols for its use in biomedical research. as an advantage over caenorhabditis elegans, a popular invertebrate model host, drugs can not only be mixed in the fly food but also administered by injection (table ) . precise doses of - nl of drug solutions can customarily be injected in each fly [ ] and less than ml on a paper disc suffice to feed flies for hours [ ] . hence, only small quantities of drugs are required during experiments; yet another reason why drug tests in flies are not expensive. in addition, drosophila can be used for toxicological studies because the relative toxicity of chemicals in flies correlates well with that in mammals [ ] . finally, drosophila infection and inflammation can easily be studied in relation to aging overcoming the barrier of long experimental time [ ] . this is because drosophila maximum life span ranges between and days, with day of the fly roughly corresponding to year of humans. that is, flies exhibit aging effects as early as days post the onset of adulthood. drosophila has a long history as a model organism for genetics and a significant similarity with humans in terms of gene homologs. it has functional homologs for % of human disease related genes [ ] , more than any other invertebrate model host studied today (table and [ ] ). its genome is fully sequenced and is one of the bestannotated among eukaryotes. thus, many technologies have been developed and techniques are easily and commonly used, such as transgenesis, rna interference (rnai) technology and gene microarrays. doublestranded rnas have been synthesized for almost all genes and the tools are commercially available for the conditional inactivation of essentially any gene of interest in vivo or in cell culture [ ] . for example, drosophila cells have been used in genome-wide rnai screens to rapidly identify genes required for replication of influenza and dengue viruses [ , ] . furthermore, there are large collections of mutants and transgenic drosophila stocks maintained at bloomington and other stock centers around the world (http://flybase.org). moreover, the drosophila genome contains fewer genes than humans, and consequently, presents less overall genetic redundancy. this allows for an easier target identification, although multiple or modified drugs might be needed in mammals to affect the multiple gene variants. finally, a variety of genetic tools and markers are available today in order to study the role of microbial pathogenicity tissue-specifically using the gal /uas system [ ] . this is an advantage over other model hosts, because expression of any drosophila gene can be controlled time and tissuespecifically (table ) . for example, tissue-specific and temporal rnai allowed the identification of the jak/ stat signalling pathway as a regulator of the intestinal immune response and regeneration in the fruit fly [ ] . in addition, intestinal damage and regeneration can be studied by flip-out clones of cells emanating from intestinal stem cells [ , ] , as well as mitotic clones using either the b-galactosidase marker or the ''mosaic analysis with a repressible cell marker'' method [ ] . drosophila physiology and the immune system -conservation and significance for mammalian research several organs and specific cells fundamental to the immune response are highly conserved between flies and mammals. this is the most significant advantage over all other invertebrate model hosts studied today (table ) . flies have a defined brain that interacts with other organs, for example, the fat body and the intestine via cytokines and insulin peptides, respectively [ , ] . the fat body is the equivalent of the mammalian liver, an innate immunity and a metabolic organ [ , ] . the fly intestine bears many similarities with that of mammals in terms of cellular and molecular biology and epithelial architecture [ ] . plasmatocytes are the macrophage-like cells of drosophila that detect and phagocytose microbes and secrete cytokines and antimicrobial peptides [ ] . the muscle cells of the drosophila flight muscle, heart and intestine are stratified or smooth similarly to those of humans and share a role in host response to infection [ , ] . the drosophila trachea is an air-transporting organ with similarities to the human vasculature [ ] . finally, the nephrocytes and the malpighian tubules are kidney-like cells with a role in host defence [ , ] . the drosophila epithelia that are attached to the cuticle, as well as those of the intestine and the trachea are physical barriers to pathogen invasion and the first to respond to external microbes. should microbes invade anti-infectives these epithelia other local tissues, such as the drosophila flight muscle, respond to wound infection eliciting a localized host defence response orchestrated by the highly conserved jnk pathway [ ] . importantly, muscle responses to wound infection appear to be conserved in mice and in humans [ , ] . on the other hand, when bacteria enter and damage the intestine, they induce enterocyte regeneration, which serves as a defence response to protect the host [ ] . numerous conserved signalling pathways are involved in intestinal regeneration upon infection, including the wnt/wg, notch, hippo, jnk, insr/inr, k-ras/ras , jak-stat and the nf-kb pathways [ ] . in case microbes pass through intestinal or other barrier epithelia, additional mechanisms of protection take place. these include phagocytosis by the plasmatocytes, which are analogous to the mammalian macrophages, and the production of antimicrobial peptides by the fat body [ ] . many bacteria and fungi induce the toll and/or the immune deficiency (imd) pathways, which are the two highly conserved nf-kb pathways of the systemic drosophila immune response [ ] . viral infections elicit systemic immune responses via the universally conserved rnai mechanism. the drosophila small interfering rna pathway is activated by doublestranded viral rna or dna [ ] . moreover, dexd/h box helicases, cell autophagy as well as the conserved jak/stat, imd/tnf and/or the toll/tlr innate immune cascades play a crucial role in responding to viral rna in flies and mammals [ ] . many human bacterial pathogens have been studied in drosophila including the gram-positive bacteria enterococcus faecalis, staphylococcus aureus, steptococcus pneumoniae, bacillus cereus and listeria monocytogenes, and the gram-negative bacteria vibrio cholerae, serratia marcescens, pseudomonas aeruginosa, salmonella typhimurium, chlamydia spp., burkholderia cepacia, yersinia pseudotuberculosis, francisella tularensis, legionella pneumophila and mycobacterium marinum [ ] . of those, p. aeruginosa and m. marinum may suppress the innate immune response as part of their virulence repertoire [ , ] . interestingly, the antibiotics rifampicin, dinitrobenzamide, amikacin and isoniazid show good bioavailability, because when fed to the flies they alleviate systemic m. marinum infection. of special note, the success of the antituberculosis drugs isoniazid and pyrazinamide against the tuberculosis model microbe m. marinum is facilitated by a boost in host cell autophagy in flies and mammals [ ] . these data suggest that not only direct antibacterial efficacy but also innate immune induction share similarities between flies and mammals and can be exploited for pharmacological assessments in flies. intestinal p. aeruginosa induces damage and apoptosis of midgut enterocytes in drosophila, which in turn induces intestinal stem cell proliferation, a process that is however reversible upon bacteria clearance by the common food preservatives methyl paraben and propionic acid [ ] . strikingly, k-ras/ras oncogene expressing drosophila hindgut cells induce tumors and delaminate through the basal side of the epithelium upon p. aeruginosa infection, which is an additional process that can be inhibited by eliminating infection using food preservatives [ , ] . furthermore, -aminoacetophenone, a small chemical produced by p. aeruginosa, has been shown to reduce p. aeruginosa virulence in drosophila and mice [ ] . finally, researchers have exploited phages as anti-infectives against p. aeruginosa using drosophila. fruit flies infected with p. aeruginosa can be treated with bacteriophages mpk , mpk by feeding [ , ] . such findings encourage future assessment of food preservatives and natural or biological products, including bacterial metabolites and bacteriophages, as anti-infectives. apart from bacteria, human fungal pathogens can also inflict disease in flies. candida albicans, aspergillus fumigatus, aspergillus hyphae, cryptococcus neoformans, cunninghamella berthollethiae, scedosporium spp. and fusarium spp. have been studied in flies [ , ] . of those, the zygomycete c. berthollethiae has been meticulously studied in combination with chemical modifiers of iron in drosophila. enhancers of zygomycetes virulence traditionally used in humans, such as corticosteroids, increase iron supply, and iron availability through treatment with deferoxamine dramatically increases pathogenicity by zygomycetes. accordingly, iron starvation induced by treatment with the iron chelator deferasirox significantly protects infected flies [ ] . another common antifungal, voriconazole is potent against f. moniliforme and s. apiopermum infection in flies [ ] . moreover, combinatorial drug assessment assays in drosophila reveal a synergism between voriconazole and terbinafine against aspergillus fumigatus, similar to that seen in mammals [ ] . recently, another synergy was shown between tarcolimus and posaconazone in flies and mice against ryzopus oryzae [ ] . because all of the aforementioned treatments were administered by feeding in flies, while infections were either superficial or systemic, many antifungal drugs appropriate for humans may have good bioavailability and efficacy in flies. [ ] . the last three of those have also been studied in adult flies, thus allowing the assessment of treatments against them in a whole organism setting. pertinent to the identification of gene target against these viruses, drosophila nramp and its human homologue nramp have been identified as necessary for the entry of sindbis virus into the host cells [ ] . in addition, drosophila toll- has been identified similar to its mammalian ortholog tlr- as important for host defence to infection against vesicular stomatitis virus via the induction of cell autophagy [ ] . finally, west nile virus 'untranslated region-derived rna molecule, known as subgenomic flavivirus rna, suppresses the sirnainduced and mirna-induced rnai pathways in both mammalian and insect cells [ ] , indicating that rnaibased therapies might be a goal for the near future against insect-borne flaviviruses. despite the numerous advantages of d. melanogaster as a model organism for the study of anti-infectives, there are also several shortcomings. that flies are infected and maintained at a temperature of - c can be a problem for the study of pathogens and virulence factors that require the mammalian body temperature, that is, c [ ] . also, its inability to simulate human intestinal anaerobic microflora can be a disadvantage. while microaerophilic and aerotolerant bacteria might be used to infect flies, the presence of oxygen in the fly intestine prohibits fly infections with strict anaerobes, which are plentiful in the human gut [ ] . nevertheless, as with any microbe that is difficult to establish an infection with, specific virulence factors can be expressed or administered to flies to study their virulence. moreover, pharmacokinetic analyses are still problematic in insects as there is not a precise method to measure the levels of administered drug tissue-specifically and insect xenobiotic metabolism might be very different from that of mammals. furthermore, as opposed to mammals, drosophila lacks an adaptive immune system and specialized immune response cells, such as dendritic cells (dc), b and t lymphocytes, which are responsible for immunological specificity and memory [ ] . in addition, despite the significant conservation of the core of drosophila signalling pathways, some of them might be activated differently between flies and mammals. for example, the mammalian toll/tlr pathway that is directly activated by microbially associated molecular patterns, while the drosophila toll is activated indirectly through a cascade of proteases [ ] and the mammalian tlr- that is localized in intracellular membranes versus the plasma membrane-localized drosophila toll- [ ]. finally, high-throughput screening for anti-infectives has not been developed in drosophila and this is its major drawback as compared to other invertebrate hosts (table ) . in recent years, the conventional methods used in most pharmacological studies for the discovery of new therapeutic drugs are based either on screening of small molecule libraries for the capacity to induce a specific phenotype in vitro or in silico [ , ] . however, the efficacy of these methods is very low, because they lack the complex and dynamic host-pathogen interactions, which occur in vivo. consequently, the use of mammalian hosts in such studies is needed and seems to be very widespread and prevalent nowadays. even so, using a conventional animal model for this purpose can be time-consuming, laborious and expensive, not to mention the ethical concerns. exploiting alternative strategies, d. melanogaster is a very promising and useful host, which may cover this gap between the computational or cellular testing studies and the tests in mammals (figure ). while low-throughput drug assessment in drosophila has been proven meaningful, large-scale assessments might also be possible on the basis of protocols used for the identification of molecules that modify disease progression in fragile x syndrome though a screen of compounds in fmr -mutant flies [ ] and a screen of small molecules that identified reserpine as a sleep regulator [ ] . in addition, the fly can be used to assess drugs already approved for human use ( figure ). indeed, the efficacy of a number of licensed anti-infective agents has been evaluated in drosophila, demonstrating a significant correlation in drug efficacy between flies and mammals. therefore, the use of drosophila for anti-infective drug discovery may be a promising auxiliary tool for preclinical research. drosophila can be used either to validate candidate drugs or in combinatorial drug assessment assays to identify synergistic drug combinations. flies have significant similarities with humans enabling a facile and cost effective assessment of anti-infective drugs during the interaction of microbes with a host. hits selected from in vitro or in silico chemical screens can be further screened in drosophila survival or microbial colonization assays to select drug candidates that will have a higher success rate in preclinical trials. in addition, natural products, for example, microbial secondary metabolites and drugs approved in humans can be tested for the fist time combinatorially in flies to identify synergistic effects between two or more chemicals. antibiotics in the clinical pipeline in drosophila melanogaster as a model for human intestinal infection and pathology recombinagenic and mutagenic activities of fluoroquinolones in drosophila melanogaster chemotherapyinduced toxicity is highly heritable in drosophila melanogaster human disease models in drosophila melanogaster and the role of the fly in therapeutic drug discovery drosophila as a genetic model for studying pathogenic human viruses drosophila melanogaster as a model host for studying pseudomonas aeruginosa infection a quorum sensing regulated small volatile molecule reduces acute virulence and promotes chronic infection phenotypes feeding or injecting a p. aeruginosa metabolite to flies and mice reduces virulence of its own species. an example of how bacterial metabolites from pathogens might help us fight infection with the same pathogens drosophila melanogaster as a model to characterize fungal volatile organic compounds intestinal barrier dysfunction links metabolic and inflammatory markers of aging to death in drosophila a systematic analysis of human disease-associated gene sequences in drosophila melanogaster glö ckner g, vilcinskas a: a comprehensive transcriptome and immune-gene repertoire of the lepidopteran model host galleria mellonella new tools for the study of fungal virulence, pharmacology and immunology drosophila rnai screen identifies host genes important for influenza virus replication discovery of insect and human dengue virus host factors genome-wide rnai screen identifies genes involved in intestinal pathogenic bacterial infection synergy between bacterial infection and genetic predisposition in intestinal dysplasia cytokine/jak/stat signaling mediates regeneration and homeostasis in the drosophila midgut drosophila cytokine unpaired regulates physiological homeostasis by remotely controlling insulin secretion infection-induced intestinal oxidative stress triggers organ-to-organ immunological communication in drosophila rä met m: drosophila phagocytosis -still many unknowns under the surface involvement of skeletal muscle gene regulatory network in susceptibility to wound infection following trauma molecular architecture of the fruit fly's airway epithelial immune system bringing together components of the fly renal system immune response and anti-microbial peptides expression in malpighian tubules of drosophila melanogaster is under developmental regulation down-regulation of glutatione s-transferase a (hgsta ) in the muscle of thermally injured patients is indicative of susceptibility to bacterial infection regenerative inflammation: lessons from drosophila intestinal epithelium in health and disease the dna virus invertebrate iridescent virus is a target of the drosophila rnai machinery mycobacterium marinum infection in drosophila melanogaster for antimycobacterial activity assessment host cell autophagy activated by antibiotics is required for their effective antimycobacterial drug action an elegant demonstration of how two antimycobacterial drugs, apart from their direct antibiotic role, induce a conserved in flies and mammals autophagy-dependent host defence response immune response to bacteria induces dissemination of ras-activated drosophila hindgut cells bacterial clearance or pharmacological inhibition of jnk eliminates innate immune-induced enterocyte invasion ras-oncogenic drosophila hindgut but not midgut cells use an inflammation-like program to disseminate to distant sites antibacterial efficacy of phages against pseudomonas aeruginosa infections in mice and drosophila melanogaster drosophila models of anti-infective drug discovery tzelepis et al. www.sciencedirect.com current opinion in pharmacology antibacterial efficacy of temperate phage-mediated inhibition of bacterial group motilities bacteriophage treatments are simple to perform in flies. their specificity and effectiveness in both flies and mice is a promising way to develop biological anti-infective therapies drosophila melanogaster as a model organism for invasive aspergillosis drosophila melanogaster as a model host to dissect the immunopathogenesis of zygomycosis virulence studies of scedosporium and fusarium species in drosophila melanogaster toll-deficient drosophila flies as a fast, high-throughput model for the study of antifungal drug efficacy against invasive aspergillosis and aspergillus virulence kontoyiannis dp: tacrolimus enhances the potency of posaconazole against rhizopus oryzae in vitro and in an experimental model of mucormycosis in vivo combinatorial drug assays reveal that antifungals may work synergistically and in a similar fashion between flies and mice natural resistance-associated macrophage protein is a cellular receptor for sindbis virus in both insect and mammalian hosts this put drosophila in an excellent position for screening for pharmacological inhibitors of sindbis virus entry into the host cells noncoding flavivirus rna displays rna interference suppressor activity in insect and mammalian cells a human gut microbial gene catalogue established by metagenomic sequencing drosophila immune priming against pseudomonas aeruginosa is short-lasting and depends on cellular and humoral immunity evolutionary relationships, but functional differences, between the drosophila and human toll-like receptor families pharmacophore selection and redesign of non-nucleotide inhibitors of anthrax edema factor screening for small molecule inhibitors of toxoplasma gondii identification of small molecules rescuing fragile x syndrome phenotypes in drosophila small-molecule screen in adult drosophila identifies vmat as a regulator of sleep funding from marie curie (gig-infectioncancer) and fontation sante (yasante ) to ya. key: cord- - vjqrnwh authors: hraber, peter; o’maille, paul e.; silberfarb, andrew; davis-anderson, katie; generous, nicholas; mcmahon, benjamin h.; fair, jeanne m. title: resources to discover and use short linear motifs in viral proteins date: - - journal: trends biotechnol doi: . /j.tibtech. . . sha: doc_id: cord_uid: vjqrnwh viral proteins evade host immune function by molecular mimicry, often achieved by short linear motifs (slims) of three to ten consecutive amino acids (aas). motif mimicry tolerates mutations, evolves quickly to modify interactions with the host, and enables modular interactions with protein complexes. host cells cannot easily coordinate changes to conserved motif recognition and binding interfaces under selective pressure to maintain critical signaling pathways. slims offer potential for use in synthetic biology, such as better immunogens and therapies, but may also present biosecurity challenges. we survey viral uses of slims to mimic host proteins, and information resources available for motif discovery. as the number of examples continues to grow, knowledge management tools are essential to help organize and compare new findings. viruses exploit host cellular processes to replicate, and have developed myriad ways to subvert host immune defenses. molecular mimicry (see glossary) is a common and effective strategy, enabling a pathogen to usurp host protein function by resemblance [ , ] . molecular mimicry varies over a continuum, from one extreme that includes sequence and structural similarity (i.e., orthologs) of entire proteins, to another extreme of chemical similarity at only a few localized sites, as is the case for short linear motifs (slims). the growing body of literature on slims indicates that some important virushost interactions can be attributed to a few well-chosen aas [ ] [ ] [ ] [ ] [ ] . rather than devote entire proteins to one function, slims enable multifunctional viral proteins. interactions between globular virus and host proteins have picomolar affinities, while slims have micromolar binding affinities with globular host proteins [ ] . moderate binding affinity of slims facilitates disruption of signaling interactions, rather than competing for stable formation of persistent protein complexes. synthetic biology practitioners can benefit from an introduction to how slims enable viral interference with host cell functions and computational resources available for slim analysis. viral slims are potentially useful in synthetic biology, to provide a toolkit for new functions, for example, to modulate immune responses or to complement and interact with newly developed adjuvants in a synergistic manner [ ] . research efforts to develop broad-spectrum antiviral compounds or design broadly cross-protective vaccine immunogens benefit directly from knowledge of gene products, protein functions, and motifs involved with viral immune interference. n-linked glycosylation of the png sequon is a well-known example used by viral glycoproteins as camouflage against immune recognition [ , ] . the distribution of n-linked glycosylation sites has recently been recognized as essential for the design of immunogens to induce broadly cross-reactive immune protection against such challenging viruses as hiv- [ , ] . motifs associated with cellular trafficking (localization, transport, secretion, and sequestration) are readily edited to modify where expression products go, and change interaction profiles with other proteins [ ] . in addition to motifs that stabilize the structure of immunogens, such as trimerization ('foldon') [ ] [ ] [ ] [ ] and dimerization [ , ] domains, motifs that interact with cellular processes for innate antiviral pathways could be used to enhance immunogenicity. while slims in eukaryotic proteins have been discussed extensively, slim involvement in viral immunomodulation remains less thoroughly explored, and suggests new opportunities for use in engineered biotechnology applications. the ability to transfer genetic components across species, or to introduce such components de novo, enables new functions. while such functions are generally well intended, some risk also exists for harmful effects. subject to the technical advances of synthetic biology, such effects are not short linear motifs (slims) are patterns of three to ten consecutive aas used by eukaryotic cells for tasks that include: signaling, localization, degradation, and proteolytic cleavage. viruses use slims to their advantage, including interference with antiviral innate immune pathways. viral slims can tolerate mutations, evolve quickly to modify host interactions, and co-occur in a modular manner or involve multiprotein complexes. slims are useful in synthetic biology, where minor edits can alter target specificity, modulate persistence, reprogram interactions with cell-signaling domains, and alter protein function in myriad other ways. aside from possible beneficial uses, for example, to produce better immunogens and develop therapeutic interventions against infectious disease, slims may help characterize new and emerging threats to global health. necessarily a taxonomically relevant property. it may be necessary to evaluate risks of new functions by other means than taxonomy or even protein functional evaluations. instead, new methods are needed that assess functions at a finer resolution than the gene, whether by computational analysis or functional phenotypic assessments [ ] [ ] [ ] . slim analysis might help with such assessments. viral proteins can modulate immunity in several ways, which include: shutdown of host macromolecular synthesis, inhibiting antigen production or apoptosis, and interference with such processes as antigen presentation by mhc, natural killer (nk) cell function, antiviral cytokines, or interferon responses. each of these processes involves coordination among multiple components in host cells. viral interference with these functions is frequently attributed to entire proteins, but in some important cases has been localized to slims. because of their compact size, slims are modular, rapidly evolvable sequence elements. different instances of a given slim can vary in sequence while maintaining the overall functional profile, that is, the regular expression for the sequence motif, where a few positions are invariant while other positions tolerate numerous substitutions. thus, partial sequence matches are sufficient for transient binding interactions with target domains, for example, signal transduction proteins. this observation led to the proposal of ex nihilo slim evolution -the evolution of a novel slim 'from nothing' -the appearance of a new functional module from a previously nonfunctional region of protein sequence [ ] . because hosts' interaction networks are often conserved, slims represent a significant vulnerability for opportunistic exploitation. these properties enable pathogens to acquire host-like slims rapidly through ex nihilo convergent evolution, to rewire host interaction networks, and to acquire tropism and virulence traits needed for successful adaptation and propagation [ ] . over motifs are known, with , validated instances, and many more motifs may await discovery [ , ] . focus on viral motifs may reveal practical utility, to broaden the repertoire of tools available to reprogram molecular function in synthetic biology. one example of how viral proteins use slims to subvert host cell function is illustrated by epstein-barr virus (ebv), which persists in resting memory b cells of nearly all (> %) individuals throughout their adult lives [ ] . latent membrane protein (lmp ) is central to ebv persistence. the cytoplasmic tail of this membrane-bound protein includes pxxpxp and pxqxt motifs that recruit signaling proteins (jak , that is, janus kinase , and several trafs, tumor necrosis factor receptor-associated factors, respectively) [ ] . together, the motifs mimic the cytoplasmic domain of cd to activate nuclear factor-kb via intermediates, including a third motif yyd$ (where $ denotes the c terminus), the tradd (tumor necrosis factor receptor-associated death domain) binding domain. the overall result is that lmp inhibits apoptosis and infected b cell proliferation, to confer viral persistence [ ] . other examples of viral slim contributions to motif mimicry involved with immune function include: protein degradation, transcription, translation, and transport into and out of the nucleus [ ] . given the continued growth of this field [ ] , established frameworks can manage and exploit this knowledge beyond catalogues of currently known motifs [ , , ] or details on contributions of one viral protein (e.g., [ , ] ). work to use slims in bioengineering can benefit from understanding viral protein function. this information is organized in viral knowledge bases, such as viralzone (box ). ontologies describe systematically the many different functional roles of viral proteins, including immune evasion. by promoting use of standard terms for relationships between concepts, an ontology arranges concepts into a framework that can be updated as knowledge grows. protein function is captured broadly in such a framework, though the nuanced details of interactions with other molecules are not localized to domains or motifs. go is an authoritative resource for annotating functions of gene sequences [ , ] . an example of interest is 'evasion or tolerance by virus of host immune response' [ ] (www.ebi.ac.uk/quickgo/ glossary adjuvant: an additive to a vaccine that promotes nonspecific immune responses. when administered together with an antigen, it induces more potent responses than the antigen alone. autophagy: an evolutionarily conserved degradation system for maintaining cellular homeostasis and innate immunity to clear pathogens from cells. domain: structure-based modular subunit of a protein, often with a specific function. domains are generally larger and associated with structural subunits, while motifs are shorter and associated with intrinsically disordered protein regions. elm: the eukaryotic linear motif resource (elm.eu.org), a repository of known slims; includes annotation from primary literature and information about experimental assays used. glycosylation: post-translational modification by host transferases linking sugar molecules to side chains of either nitrogen (nlinked) in asparagine or oxygen (o-linked) in serine or threonine. viral proteins like hiv- envelope can be heavily glycosylated, providing camouflage against immune recognition, shifting as the png sequon mutates. go: gene ontology, an ontology for genetic products of any and all organisms, providing a framework to annotate gene and protein functions in genetic sequence databases and bioinformatics analysis procedures. go includes multiple dimensions to capture biological complexity in adequate depth: molecular function, cellular component, and biological process. go development is conducted by a consortium of research communities and databases, which regularly solicits input and feedback from the broader research community. immune modulation: interference with an immune-related process by a pathogen. intrinsically disordered protein/ domain: regions of a protein sequence that are predicted or experimentally shown not to form consistent structure, e.g., a helices or b sheets. such regions tend to be more accessible for interactions with other proteins. term/go: , figure ). concepts are hierarchically organized, and include a definition, synonyms, and lists of parents and children. functional annotation in go reflects the diverse effects of viral proteins on immune interference. modulating autophagy is an example of recent advances in this research area [ , ] . a growing number of reports describe how virus proteins and slims therein modulate autophagy to promote various aspects of their life cycle [ , ] . both go and viralzone have developed concepts to detail molecular mimicry: structural similarity that enables repurposing or hijacking of molecular function by pathogens, such as viruses. molecular mimicry varies in extent from entire globular proteins, to localized domains, down to short linear motifs. motif class: a regular expression that summarizes known variants to define a sequence motif. motif instance: a particular motif as found in a protein or translated genetic sequence from a specific organism, strain, or isolate. ontology: a representation of domain-specific knowledge organized as concepts, their properties, and their relationships. png sequon: three aa motif n [^p] [st] , that is, asparagine, then any aa except proline, then serine or threonine, recognized by glycosyltransferase as a potential nlinked glycosylation site. regular expression: a string of characters that concisely represents many alternative sequence variants; may include wildcards to represent any character, groupings of possible characters, repetition, negation, start and end of sequence, etc. short linear motif (slim): also known as minimotifs or morfs (molecular recognition features). frequently represented as regular expressions, typically three to te-naas long. viralzone: a knowledge base (https://viralzone.expasy.org) that documents viral families, genome architectures, proteins, hostprotein interactions, and an ontology for the functions of viral proteins. a bridge that links literature reports to go term annotation, viralzone is an online knowledge base that contains 'textbook' information about viral taxonomy, replication, genome organization, and virion structure, and provides links to viral sequence data [ , ] . importantly, viralzone staff collaborate with the go consortium to define entries for virus-specific molecular functions [ ] [ ] [ ] [ ] . viralzone cross-references its keywords with go consortium terms and uniprot [ ] identifiers. this makes it possible to search for viral proteins by their functional role. viralzone staff have developed go concepts specifically for viruses, to represent the diversity of viral replication and processes involved with viral entry, replication, and egress [ ] . viralzone staff have also developed a detailed listing of virus-host interactions, with entries for functions and go terms [ , ] . each entry ('keyword') has a unique identifier. unlike enzyme commission (ec) numbers [ ] , viralzone ids are arbitrary numbers and do not indicate position in the concept hierarchy. instead, organization of the keyword hierarchy is provided online. the web address https://viralzone.expasy.org/ is an entry point into the viralzone concept space ( figure i) . blue text indicates a link to more detail. shown is the vertebrate host-virus interactions page [ ] . also available are summaries for invertebrate, plant, and bacterial host-virus interactions. adapted from www.ebi.ac.uk/quickgo/term/go: [ ] . most arrows indicate 'is a' relations, where the hierarchy is refined by specialization. blue arrows indicate 'part of' relations, which relate to the symbiont process as parts to a whole. autophagy processes, including positive and negative regulation of xenophagy, the selective autophagy of pathogens [ ] [ ] [ ] . understanding the functional roles of slims can help identify related mechanisms or processes, or possibly identify knowledge gaps where slims may be posited but not yet identified. an overview of slim functions may also help to prioritize which are of greatest potential for use or abuse when artificially added to modify protein function. databases and discovery tools are also useful to identify known and new slims. identification of shared structural features across divergent protein families led to analysis and identification of modular protein domains. protein domains are used to categorize protein function, and the interpro database [ ] (www.ebi.ac.uk/interpro) aggregates information at this within-protein level. the identification of protein domains led to recognition of slims as compact, small-scale functional modules [ ] . elm is a database of eukaryotic motifs (box ), though its representation of viral-host interactions is not fully developed. at present, elm entries map to go terms. most motifs map to multiple go terms; the median is seven go terms per motif and the maximum is (mod_plk_ ). immuneassociated function of the lig_irf _lxis_ motif is involved in signal transduction responses to pathogen-associated molecular patterns; this motif maps to go terms, but none occur in the viralzone vocabulary. in total, only three elm entries utilize go terms from viralzone: lig_bh_bh _ , lig_hcf- _hbm_ , and lig_rb_pabgroove_ . these three motifs all map to the most general (ii) deg, degradation sites, part of polyubiquitination; (iii) doc, docking sites, involved in protein recruitment but not directly targeted by an active site; (iv) lig, ligand binding sites, primarily for protein-protein interactions; (v) mod, post-translational modification sites; and (vi) trg, targeting sites for subcellular localization. elm has also spun-off several specialized databases: phospho.elm for phosphorylation sites with experimental evidence [ ] , switches.elm for conditional molecular switches, such as requiring that a site be modified [ ] , and ielm, with an emphasis on protein-protein interactions [ ] . a detailed tutorial provides orientation for elm use [ ] . elm documents each motif class with a concise description of its function. for example, one type of nuclear localization signal (nls), trg_nls_bipartite_ the 'classic bipartite nls', which binds to importin-a for nuclear pore transfer and is utilized by the pb protein of influenza a, is documented here: elm.eu.org/elms/ trg_nls_bipartite_ . the abstract and functional site descriptions summarize what is known about the motif. go term, go: ('modulation by virus of host morphology or physiology', a synonym of 'virushost interaction'). this underscores the prevalent mode of elm motif discovery and annotation does not emphasize host-virus interactions, but rather systems-level interactions within eukaryotic cells. thus, better integration of viralzone-go-term vocabulary with elm or another domain-level representation of viral slims is needed to promote potential utility for biotechnology. despite not using the viralzone ontology, elm documents other motif classes with go terms that refer to 'viral', 'virus', 'immune', or 'immunity' (table ) . because the focus is motif function in the host cell context, elm does not directly indicate how viral immune interference results. further, the relative lack of viral motifs in elm does not indicate their absence in vivo, but rather the evidence-based requirement for elm inclusion. related to the earlier observation, a review of how viruses use slims to interfere with host cells [ ] lists examples that represent viral mimicry of host slims (table ). only % of these have corresponding elm entries, though the slims are known. the remaining % indicate that elm does not fully capture all known viral motifs. this strong requirement by elm for evidence-based motif classes and instances is not strictly a drawback. indeed, the elm creators are very aware that computational analysis alone is error prone and can yield misleading outcomes. in [ ] , they discuss this issue in depth, and recommend a workflow for slim discovery that culminates in experimental validation, whether in vivo or in vitro. working with viral-host systems adds layers of difficulty to experimental motif validation, so it should not be surprising or to the detriment of available information resources that viral slims are less thoroughly documented. searching arbitrary sequences for motif instances is computationally straightforward. box provides an example of motif searching with elm, which might facilitate comparative analysis of two related proteins from different species of human herpesvirus (hsv). resources such as interpro and uniprot are able to perform similar assessments, but give broader, domain-level representations with less functional detail than the slim searches enabled by elm. reports in the primary literature take a different approach, by marking slims in a protein alignment, which includes orthologues to mark conservation (e.g., figure in [ ] ). the elm-generated report combines predicted slims with information from annotated domains and local disorder predictions, for a perspective that complements the other approaches. clearly, false positives are inevitable among slim search results. this makes it necessary to filter for the most significant and informative outcomes. this leads to consideration of in silico (computational) methods for slim evaluation. a highly recommended, authoritative review of slim discovery techniques, from an author of the slimsuite software package, discusses motif identification techniques in depth [ ] . methods for slim discovery can be divided into two broad classes: (i) de novo discovery of new slims, and (ii) instance prediction to find new occurrences of known slims. there are currently at least eight software packages available to discover new slims and packages ( stand-alone programs or servers and two software suites that consist of multiple tools: slimsuite [ ] , which includes ten utilities, and meme [ ] , which consists of five tools for slim instance detection). though meme was developed for discovery of dna sequence motifs, it generates ungapped, profile-based motifs using the expectation-maximization (em) algorithm. no single method is inherently better than the rest, but the choice of which to use depends on several factors, such as the input sequence data, whether one sequence, an alignment, or a collection of nonhomologous sequences [ ] . to illustrate the diversity of motif discovery methods, this section mentions only a handful of the software tools available (table ) . readers seeking to learn more about the full set of alternatives are strongly encouraged to consult [ ] , particularly tables , , and therein. another helpful resource ( table in [ ] ) lists online motif discovery bioinformatics services. several alternative approaches for discovery of new motifs have been advanced. edwards and palopoli [ ] review the alternatives in depth, discussing their merits and drawbacks. briefly, they can be divided into alignment-based and alignment-free methods. an alignment-based approach looks for conserved sites among homologous sequences, but can be misled by high sequence conservation in globular domains. a program called slimprints works around this with a specialized approach to model substitutions [ ] . slimprints uses a statistical model of relative local conservation, which looks for clusters of overly constrained sites in a window of about aas, using iupred scores (intrinsically unordered prediction; see later) to weigh sites in intrinsically disordered protein regions more heavily than sites in globular (ordered) regimes [ ] . in contrast, alignment-free methods look for enrichment of amino acid patterns in proteins that are expected by other means to perform similar motif-related roles, for example, by go category annotations or protein-protein interaction (ppi) data, that is, via databases that capture experimental evidence for protein colocalization and functional interactions. an important caveat is that to assume such sequences are independent could yield spurious enrichment of shared patterns, so alignment-free methods need to compensate for evolutionary constraints at the domain level, rather than for full-length homologous proteins. the development of such corrections and their relative advantages are detailed in [ ] . some programs (e.g., slimdisc [ ] , slimfinder [ , ] , and dilimot [ , ] ) produce regular expressions that compensate for phylogenetic relatedness, while others (meme suite, glam [ ] , and nestedmica [ , ] ) produce probabilistic profiles. for more discussion of these and issues of concern for computational motif discovery, see [ ] . filtering methods control high false positive rates from slim instance detection. structural information, whether known or predicted, can be used for filtering. box illustrates how elm filters results to hsv- and hsv- virulence factor icp . assists in viral immune evasion by molecular mimicry. hsv- neurovirulence protein icp . , encoded by the g . gene, initiates immune interference by binding and sequestering cellular proteins that would stimulate autophagy, translational arrest, and type i interferon responses. hsv- icp . binds tank-binding kinase (tbk ) to prevent type i interferon induction [ ] , beclin- to prevent autophagy [ ] , and both pp a and eif a to overcome translational arrest [ ] . hsv- g . contains an intron not present in hsv- , and up to four isoforms of hsv- icp . are known [ ] . full-length hsv- icp . has conserved pp a and eif a-binding domains, but lacks tbk and beclin- binding domains [ ] . additional hsv- motifs influence intracellular localization [ ] , virion maturation, and egress [ ] , not yet characterized in hsv- . hsv- is recognized as more virulent than hsv- , but both can cause neuropathology, including viral encephalitis and meningitis [ ] . to attenuate virulence, icp . is routinely deleted or inactivated when making hsv- constructs for oncolytic therapy [ ] . both are the same length and share domain structures, and partially share slim compositions ( figure i ). identifying differences in slims from each could provide clues for more detailed experimental investigations to understand icp . virulence determinants and host protein targets. exclude a region predicted to fold as globular protein. these predictions were made by smart (simple modular architecture research tool) [ ] and pfam [ ] domain matches, corroborated by globplot [ ] . another widely used approach is to identify regions of local disorder, where protein structure is not clearly defined, making that region accessible to interact with other proteins. iupred [ ] is commonly used for this task, though the choice of parameter settings and how to interpret results varies. elm results include an iupred disorder score and a simple cutoff of . to define the disorder transition. above this value, local protein structure is considered accessible for interaction with other proteins. scoring schemes filter for statistical enrichment of motif instances. an approach of filtering by homology [ ] seems inappropriate for use to detect virus interactions with host proteins, as it may exclude nonhomologous regions with motifs that do interact, yielding false negatives. regardless, failure to consider evolutionary relatedness among sequences being searched could introduce bias due to common ancestry, rather than independence, among sequences. a simple approach to instance prediction is a stand-alone program called shettimotif [ ] . it was used to scan protein sequences from poxviridae genomes (an average of proteins per poxvirus) for low-complexity regions and regular expressions defined by prosite. the approach compared numbers of proteins per genome that carry each motif, and doubtlessly includes many motif instances that are not functional as slims. also, shorter motifs occur more frequently than longer motifs [ , ] , partly due to chance alone. regardless, systematic error may be considered a source of background noise across the large number of proteins in viral proteomes, each having different host specificities, to enable somewhat meaningful comparisons, in such a 'statistical genomics' approach [ ] . the comparisons could be more meaningful if false positive motif instances were reduced. becerra et al. developed another approach to instance counting [ ] , which involves comparison with a null distribution from permuting primary sequence and testing for presence of the motif in the permuted sequences. a motif is considered rare and therefore significantly unlikely to occur by chance if it is present at or below some cutoff frequency. restricting the sequence region that is used for permutation testing, such as by use of structural considerations, can further focus the search. indeed, such a hybrid filtering approach was described recently and evaluated on the hiv- proteome [ ] . following methods described in an earlier study [ ] , becerra et al. used iupred with a modified, window-based scoring procedure to identify intrinsically disordered protein regions, and tested for statistical rarity below % of shuffled variants. the approach further considered conservation above % in a set of aligned sequences, though combining three filtering criteria was too stringent and excluded all motif candidates [ ] . while algorithmic approaches seek to identify a broad range of slim types, more specialized resources have emerged to track the distribution of a particular slim in viral proteins. for example, ilir@viral is a web resource dedicated to detecting lir motif-containing proteins in viruses [ ] . lc interacting regions (lir motifs) are slims that mediate protein-protein interactions involved in autophagy, as used by influenza a virus m protein to subvert autophagy and maintain virion stability [ ] . using curated text mining analysis and position-specific scoring matrices, ilir@viral analyzed reviewed viral sequences available from uniprot across individual viral species and found that viral sequences contain lir motifs. while many predicted instances may represent false positives, the enrichment of lir motifs in viral sequences is consistent with viral adaptation to host xenophagy [ ] . curiously, elm currently lists the lir motif as a candidate, rather than an accepted motif class. embedding slims into engineered constructs may enable specific effects on cellular immune processes, for applications that include targeted drug delivery, pathogen-specific adjuvants, potent and broadly effective immunogens, transformational medical countermeasures, and improved design of vectors for gene therapy. slim modularity may enable easy ways to reprogram protein function with a few localized modifications. to realize the potential utility of slims in synthetic biology, more research is needed to expand and integrate our collection of knowledge on viral slims (see outstanding questions). detecting slims in variant sequences may help to identify functional innovation or changes in virulence, in a manner that does not rely strictly on functional assessment at the whole-gene level, to identify how sequence-specific variation may interact with host responses. this may be particularly useful and important to understand new variants and assess the risk that they may spread and cause harmful effects on human health or agricultural interests. such knowledge is needed in an era where synthetic biology may introduce new risks for biological error and biological terror. detecting and understanding slim variants can help to reduce such risks and identify newly emerging threats to global health and security because watch lists for harmful organisms to ensure public safety by preventing access to select known risks may be inadequate [ ] [ ] [ ] . slims in viral proteins can interact in many different ways with host proteins to modulate immune responses. a motif may be necessary but not sufficient for any inferred function. the simplest case is where a viral slim interacts directly with a host protein to yield an immunomodulated phenotype. more elaborate cases are known, such as the multifunctional proteins e a (ebv), nef (hiv- ), and icp . (hsv). computational prediction of slim classes and new instances is a process, which involves experimental confirmation and validation. high-throughput methods for experimental assessment of protein interactions are useful to validate computational predictions [ , ] , and more assays are needed to evaluate functional and phenotypic effects of adding or deleting slims. resolved map of human-virus protein-protein interaction networks. plos pathog. , e what specific constraints limit slim evolvability? what strategies are most effective to advance knowledge of viral immunomodulatory slims in the design of vaccines and therapies to promote global health? for example, can some viral peptides be useful as adjuvants? signatures of pleiotropy, economy and convergent evolution in a domain pathogen mimicry of host protein-protein interfaces modulates immunity use of host-like peptide motifs in viral proteins is a prevalent strategy in host-virus interactions slimsearch . : biological context 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functionality of the eukaryotic linear motif resource elm: a database of phosphorylation sites -update the switches.elm resource: a compendium of conditional regulatory interaction interfaces ielm -a web server to explore short linear motif-mediated interactions exploring short linear motifs using the elm database and tools control of tank-binding kinase -mediated signaling by the g . protein of herpes simplex virus hsv- icp . confers neurovirulence by targeting the beclin autophagy protein a conserved domain of herpes simplex virus icp . regulates protein phosphatase complex in mammalian cells up to four distinct polypeptides are produced from the g . open reading frame of herpes simplex virus herpes simplex virus icp . confers neurovirulence by regulating the type i interferon response an n-terminal arginine-rich cluster and a proline-alanine-threonine repeat region determine the cellular localization of the herpes simplex virus type icp . protein and its ligand, protein phosphatase replication of herpes simplex virus depends on the g . functions that facilitate virus response to interferon and egress in the different stages of productive infection the herpes simplex virus neurovirulence factor g . : revealing virus-host interactions elm: the status of the eukaryotic linear motif resource key: cord- -q tk tq authors: baker, kate s.; murcia, pablo r. title: poxviruses in bats … so what? date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: q tk tq poxviruses are important pathogens of man and numerous domestic and wild animal species. cross species (including zoonotic) poxvirus infections can have drastic consequences for the recipient host. bats are a diverse order of mammals known to carry lethal viral zoonoses such as rabies, hendra, nipah, and sars. consequent targeted research is revealing bats to be infected with a rich diversity of novel viruses. poxviruses were recently identified in bats and the settings in which they were found were dramatically different. here, we review the natural history of poxviruses in bats and highlight the relationship of the viruses to each other and their context in the poxviridae family. in addition to considering the zoonotic potential of these viruses, we reflect on the broader implications of these findings. specifically, the potential to explore and exploit this newfound relationship to study coevolution and cross species transmission together with fundamental aspects of poxvirus host tropism as well as bat virology and immunology. been identified in bat populations for which the zoonotic potential is unknown, including novel influenza types and hepadnaviruses [ , ] . as a result, there has been well-grounded speculation that owing perhaps to physiological, ecological, evolutionary, and/or immunological reasons, bats may have a "special" relationship with viruses [ , , ] and be particularly good viral reservoirs with exaggerated viral richness [ ] . indeed, a recent intensive study found that a single bat species likely carries ≥ different viral species from only nine viral families [ ] . as well as the obvious first step of considering the zoonotic potential of newly identified bat viruses, further exploring the impacts of these findings and the opportunities they present for multiple research fields is necessary to capitalize on these discoveries. poxvirus infections have recently been identified in bats, comprising part of the increase in viral families newly identified in this taxonomic order. here, we review the current evidence of poxvirus infections in bats, present the phylogenetic context of the viruses within the poxviridae, and consider their zoonotic potential. finally, we speculate on the possible consequences and potential research avenues opened following this marrying of a pathogen of great historical and contemporary importance with an ancient host that has an apparently peculiar relationship with viruses; a fascinating and likely fruitful meeting whose study will be facilitated by recent technological advances and a heightened interest in bat virology. there are three documented detections of poxviruses in bat populations under distinct circumstances (summarized in table ). the viruses were detected in animals from both bat suborders on three different continents. they had varied clinical impacts on their hosts and were phylogenetically dissimilar. genetic sequence of one bat poxvirus was detected at high prevalence during active surveillance on apparently-healthy african straw-colored fruit bats (eidolon helvum) [ ] . metagenomic analysis of pooled throat swabs collected from e. helvum in ghana in contained poxvirus sequences most closely related with molluscum contagiosum (mocv) a human-only pathogen ( figure ). detected sequences were distributed across the mocv genome and reconstructed sequences relating to viral genes were deposited in genbank as being derived from eidolon helvum poxvirus [ ] . retrospective analysis of throat swabs from individual bats revealed a high prevalence of this virus in the apparently healthy study population with % (n = / ) of swabs containing poxviral dna. notably, the detection of true poxvirus sequences in this metagenomic study, in which sequences related to multiple genes distributed throughout the genome were found and reconfirmed in individual throat swab samples, is distinct from the detection of poxvirus-like sequences described in other metagenomic studies performed on pooled bat feces, whose presence was ultimately attributed to the presence of other (non-pox) viruses or viral elements integrated into host genomes [ , ] . between and , a poxvirus associated with pathology (tenosynovitis and osteoarthritis) was detected in six adult big brown bats (eptescicus fuscus, a microbat) sampled at a wildlife center in the north western united states [ ] . the clinical illness of the bats was progressive and ultimately led to their euthanasia. histopathological examination of the joint lesions was indicative of poxvirus infection, which was confirmed by electron microscopy. the virus was successfully isolated on an african green monkey cell line (bsc ) and the genome was partially characterized (seven full protein coding sequences). phylogenetic analysis revealed that the novel eptesipox virus was most closely related with cotia virus, a virus detected in sentinel suckling mice in sao paulo, brazil in ( figure ) [ , ] . finally, a bat poxvirus was again detected in a clinical setting, in south australia in . the virus was identified as an incidental infection during investigation of an outbreak of parasitic skin disease in a population of southern bentwing bats (miniopterus schreibersii bassanii, a critically-endangered microbat species) [ ] . bats presented with white nodular skin lesions that contained encysted nematodes. however, in one of the twenty-one bats examined, an independent (non-nematode associated) lesion contained intracytoplasmic inclusion bodies indicative of poxvirus infection, which was confirmed with electron microscopy [ ] . no further confirmation or characterization of the virus was reported, and both the epidemiology and consequent conservation implications of poxviral disease for this species remain unknown. the three detections of poxviruses in bat populations are distinct and inherently incomplete stories with very few common threads; high-prevalence detection in throat swabs from apparently healthy african megabats, severe joint disease in several north american microbats and, negligible though comorbid skin disease in an endangered australasian microbat. further to their varied clinical impact, the partial genetic characterization of the former two viruses shows that these viruses are genetically diverse. the two viruses are most closely related with the very distinct poxviruses, molluscum contagiosum virus and cotia virus respectively (figure ), and although only partially genetically characterized, a small ( amino acids) region of overlap in their rap proteins has only % amino acid identity (please see table s in the supplementary files). that this is as far as these new viruses can be contrasted demonstrates the dearth of information currently available for further investigation of poxviruses in bats. the finding of poxviruses in bats is not unique among wildlife taxa (in fact it would have been more surprising had they not been found to carry poxviruses) and there is no reason to believe they would have greater zoonotic potential than other animal poxviruses. poxviruses with varying zoonotic potentials have been found in a broad range of wildlife taxa including hundreds of bird species, reptiles, marine mammals, macropods, marsupials, monotremes, ungulates, equids, and primates [ , , , [ ] [ ] [ ] [ ] and there is currently insufficient evidence available to determine what the zoonotic potential of bat poxviruses might be on this spectrum. for example, although eidolon helvum poxvirus is closely related to mocv, a human-only contagion, poxvirus-associated lesions mirroring mocv-disease have also been found in horses, donkeys and a red kangaroo [ ] [ ] [ ] . similarly, the discovery of eptesipox virus in north american brown bats is analogous to the discovery of the other north american poxviruses found in voles, skunks, raccoons and squirrels, which are also detected at high prevalence in their reservoir hosts [ , ] . notably however, in the initial eptesipox virus report, the authors comment that poxvirus infection manifesting as musculoskeletal disease (osteomyelitis) has also been reported in human varv and vaccinia virus (vacv) infections [ ] . however, given that no bat poxviruses identified to date are orthopoxviruses, and the little information available, it is clear that much more detail is needed before the potential threat of bat poxviruses to man can be commented on. notably however, the two hosts in which poxviruses have been identified are widely distributed across their respective continents (africa and north america) and both habit urban areas, so have ample opportunities for contact with potential spillover hosts (i.e., humans and domestic animal species). to determine the zoonotic risk posed by bat poxviruses there are, as for other novel viruses, a number of obvious and relatively straightforward investigations that can be done. full genomic characterization of these viruses to identify known and putative poxvirus host range genes (discussed further below) would be an obvious step. similarly, testing the in vitro host range of isolated viruses such as eptesipox virus would help inform whether human and further animal cell lines are permissive for infection (i.e., that they contain the necessary host factors to support infection and do not contain antiviral components that restrict infection). serological and clinical surveillance of human populations for poxvirus infections in geographical regions near detection sites, and/or overlapping with bat home ranges would be a direct approach that would provide samples useful for evaluating multiple candidate zoonoses. whether bat poxviruses pose a zoonotic threat will likely comprise part of the future research agenda as these investigations are prudent for the discovery of all novel viruses. however, our current knowledge on bat poxviruses does not allow us to make firm predictions about their ability to infect humans. irrespective of their potential role as zoonotic agents however, the study of poxviruses in bats opens unique avenues of highly relevant research for multiple research fields beyond the individual host-pathogen relationships. further field (in situ), in vitro and in silico studies could elucidate the possible coevolution, cross species infections and mechanisms of host range restriction of bat poxviruses, the implications of which are relevant for bat ecologists, virologists and emerging infectious disease specialists (including those with a specific interest in bats) alike. it is likely that comparative phylogenetics of bats and poxviruses would inform and deepen our understanding of origins and evolution of both elements. bats and poxviruses are diverse host and pathogen taxa respectively and given their . million years of likely co-existence [ ] , there is surely a vast amount of knowledge to be gained by studying the phylogenetic relationships between bats and poxviruses. further sampling of bat populations for poxviruses would undoubtedly dramatically expand the poxvirus phylogeny, as has occurred subsequent to the study of other viral taxa in bat populations [ ] [ ] [ ] [ ] [ ] [ ] [ ] . comparative phylogenetics of bats and their poxviruses could differentiate between ancient co-speciation, or a more recent introduction and dissemination, of poxviruses among bat species. the two thus far partially characterized bat poxviruses are quite distinct from each other and are both relatively basal (i.e., have older most recent common ancestors with other extant viruses) in the poxvirus phylogeny when compared with other mammalian-infecting poxviruses. it is possible that if evidence of coevolution between bats and poxviruses were present, as has been suggested for the north american poxviruses [ ] , this could inform the phylogenies of both bats and poxviruses which are complicated by convergent evolution and horizontal gene transfer respectively [ ] [ ] [ ] . in addition to allowing the study of co-evolution, such studies provide the context for the identification of cross-species infections. with concerted research effort to identify reservoir species of bat poxviruses and cross species infections of poxviruses in bats could be identified and would have important implications for both bat and zoonotic-disease specialists. continued serological and molecular studies of naturally infected bat populations would allow the clinical effect and ecological impact of cross species poxvirus infections in bats to be assessed. we already noted that poxvirus infections across species barriers can devastate wildlife populations (e.g., squirrelpox, see introduction), an effect so severe that it was used to control introduced rabbit species in australia in the s [ ] . white nose syndrome, a fungal pathogen causing massive die offs in north american bat populations, is an unfortunate contemporary example of the severe ecological impacts that emerging pathogens can have on bat populations [ , ] . hence, from an ecological perspective if a bat poxvirus, e.g., eptesipox virus with its severe disease manifestations, were an emerging cross-species infection it would be useful to identify this rapidly, especially in already endangered species as is the case of the southern bentwing bat in which a poxvirus was reported. further to the conservation implications of such research, combining data regarding cross species infection and ecological aspects of host taxa (e.g., behavior, habitat, range overlap, host relatedness) will likely inform key concepts of virus sharing among bat species, as has been done with lyssaviruses [ , ] . given the heightened interest in bat virology, further analysis of bat poxviral isolates from both within-and cross-species infections will allow for a deeper understanding of the extent and mechanisms of poxvirus host restriction. many bat cell lines have now been developed [ ] [ ] [ ] [ ] [ ] , and at least one of these allows productive poxvirus infection [ ] . such tools will allow the in vitro refinement of host range definitions beyond detection in the field. furthermore, full genome sequencing information of poxviruses (now a comparatively easy and cost effective task) would facilitate the in silico identification of poxvirus host range gene orthologues, as recently done by bratke and colleagues who performed a systematic survey for the presence of known poxviral host range genes on among chordopoxviruses [ ] . furthermore, applying new bioinformatics tools to genomic sequence information and host range data could facilitate the identification of novel host-range determinants, perhaps even unique to bat poxviruses [ , ] . in addition, with the aforementioned in vitro tools in place, hypothetical host range genes can be validated, advancing our fundamental knowledge of poxvirus host range restriction. finally, and most speculatively, the identification of genes involved in poxvirus host range restriction in bats may represent a unique opportunity to study bat immunology, which may have broader implications for their confirmed roles as zoonotic reservoirs. since genes that interplay with the host innate immune system, not those involved with cell entry, are typically responsible for host range determination in poxviruses [ , ] , the identification of bat-unique poxvirus host range genes could facilitate the cognate identification of (possibly novel) host immune factors. this is particularly important for bats as they potentially have antiviral immunity distinct from our own, which seemingly allows them to harbor numerous human pathogens viruses asymptomatically [ ] . some preliminary evidence of this distinction existing for poxviruses is that in the single described report of infection of bat cell lines with poxviruses, bat cells were found to behave very differently from other mammalian cell lines, being susceptible to a highly attenuated strain of vaccinia virus [ ] . with several bat genomes recently sequenced [ ] and the capabilities of newer proteomic approaches, it is realistic that novel non-orthologous innate immune factors of bats (if they exist) could be identified. that these novel immune factors might then be candidate therapeutics against a range of viral zoonoses for which bats are the natural reservoir is an exciting, if not fantastical, point to ponder. recent advances in the study of bats and their viruses as well as the current biotechnological revolution leave us in a position to explore questions of virology as never before. the recent detection of poxviruses in some bat species has occurred consequent to a heightened interest in bats" role as viral reservoirs. these new findings enable us to ask many exciting and important questions about both bats and poxviruses independently as well as their ecological and evolutionary relationships. integrating the new and exciting tools of the "omics revolution with traditional laboratory and field studies allow us to interrogate these questions as never before. poxvirus tropism pathology and preliminary characterization of a parapoxvirus isolated from a california sea lion (zalophus californianus) a survey of host range genes in poxvirus genomes zoonotic poxvirus infections in humans human monkeypox: an emerging zoonosis epidemics of squirrelpox virus disease in red squirrels (sciurus vulgaris): temporal and serological findings an increasing danger of zoonotic orthopoxvirus infections poxviruses and the evolution of host range 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characterisation of pteropid bat cell lines authentication of the r e fruit bat cell line establishment of cell line from embryonic tissue of pipistrellus ceylonicus bat species from india & its susceptibility to different viruses type i interferon reaction to viral infection in interferon-competent, immortalized cell lines from the african fruit bat eidolon helvum genome-wide association study identifies vitamin b biosynthesis as a host specificity factor in campylobacter comparative analysis of bat genomes provides insight into the evolution of flight and immunity the authors thank daniel streicker and gustavo delhon for their helpful comments on the manuscript. both authors reviewed the literature, wrote, and edited the manuscript. the authors declare no conflict of interest. key: cord- -wy zk p authors: blinov, v. m.; zverev, v. v.; krasnov, g. s.; filatov, f. p.; shargunov, a. v. title: viral component of the human genome date: - - journal: mol biol doi: . /s sha: doc_id: cord_uid: wy zk p relationships between viruses and their human host are traditionally described from the point of view taking into consideration hosts as victims of viral aggression, which results in infectious diseases. however, these relations are in fact two-sided and involve modifications of both the virus and host genomes. mutations that accumulate in the populations of viruses and hosts may provide them advantages such as the ability to overcome defense barriers of host cells or to create more efficient barriers to deal with the attack of the viral agent. one of the most common ways of reinforcing anti-viral barriers is the horizontal transfer of viral genes into the host genome. within the host genome, these genes may be modified and extensively expressed to compete with viral copies and inhibit the synthesis of their products or modulate their functions in other ways. this review summarizes the available data on the horizontal gene transfer between viral and human genomes and discusses related problems. the relationships between viruses and their hosts are in fact more complex and diverse than is generally perceived. if their interactions are transient and limited to the single virus host paradigm, it largely determines the diagnosis of an infection, and the virus is considered an absolute parasite; this is the approach currently practiced by clinicians. however, on a larger time scale, transient interactions of this kind represent only a specific case of a more general role of viruses, since they are the basis of the evolutionary progression of the whole biological system where viruses and their hosts are constantly adapting to each other, either gaining certain advantages or suffering considerable losses. this is a continuous, ongoing process with a varying rate. both sides act on the population level and exhibit different extents of aggression and plasticity. for this reason, it can be extremely difficult to determine the causes and the nature of a pathology (especially chronic) in a given individual because they are often modified by virological events that occurred many generations ago. therefore, it seems fairly reasonable to consider a potential viral origin (in a broad sense) for almost any human disease with unclear etiology, even though it appears noninfectious, especially taking into account that, in particular, this origin implies the possibility of horizontal gene transfer, a phenomenon that is most efficiently mediated by viruses and largely determines the evolutionary progress as the consequence of the total sum of numerous elementary interspecies interactions [ ] . the initiation of a viral infection depends on the presence of specific receptors on host cells, i.e., on the host sensitivity to a particular virus. the host's ability to resist infection or to develop a mutual relationship with the virus determines a favorable outcome, while weaker elements can be eliminated from the host population by the lethal course of infection. the distribution of genes that encode these receptors in the human genome is shown in fig. [ ] . the parallel evolution of viruses and their host's sensitivity on the immune response level can lead to a decrease in the virus's pathogenicity [ ] . for instance, this process underlaid the spreading of the low-virulence poxvirus variant, alastrim, in the years that preceded the eradication of smallpox [ ] [ ] [ ] . viruses recognize specific receptors on the surface of host cells; otherwise, if the expression of these receptors is inhibited, they become noninfectious. for instance, this happens if the human gene that encodes the ccr receptor to hiv is damaged by a -bp deletion [ ] . in some cases, a virus receptor can be blocked by a protein, the gene of which was previously acquired by the host from the same virus via horizontal transfer. on the molecular level, a mutualistic solution has the form of a latent infection; a viral agent can per-reviews udc . . sist for a long time in a host's cells, which protect it from external factors, while the host organism can make use of the reactivation of the virus, the expression of certain viral genes, or the production of latent rnas for its own benefit; at the same time, sporadic reactivation and release from the host body enable the virus to maintain the level of genomic variation sufficient for its evolutionary promotion. horizontal transfer involves fragments of genetic information that vary strongly in size, in particular depending on the buffer genome capacity of each participant. in the human genome, this capacity is determined by the portion of chromosomal dna, which does not contain species-specific protein-encoding sequences and, thus, can basically make a place for novel information that will be modified to reach a new balance. if we consider full-size genes, the essential sequences occupy ~ % of the human genome, while only . % of the genome are gene exons. the reverse process, i.e., the acquisition of host genes or shorter sequences by viruses, is also possible, although viral genomes obviously have a lower abso-lute capacity for storing the acquired material. however, there still are certain provisions; for instance, it was shown that up to % of genes in the herpesvirus [ ] [ ] [ ] [ ] and adenovirus [ ] [ ] [ ] [ ] genomes can be removed and substituted with foreign dna without losing virus viability. in some cases, it is difficult to identify the direction of the initial horizontal transfer (i.e., to determine whether a gene was transferred from the virus to the host or the other way around), because these genes start to perform important functions in both the virus and the host. considerable interest is drawn to viral trna-like (clover leaf, or l-form) structures present in the human genome and in some viral genomes, such as alphavirues and endogenous retroviruses. these structures can participate in the stabilization of viral rna, as well as in viral replication and translation; all of these functions are determined by the folding of these structures [ , ] . in human dna, viral insertions can be present as full-size genome sequences, but also as smaller i ii iii iv v vi vii viii ix x (b) genome segments, individual viral genes or their clusters, and short sequence fragments. the genetic material of all known virus types using all possible replication strategies can reach animal germ cells and be transferred to subsequent generations, which determines the evolutionary role of the gene flow from viruses to animals [ ] . retroviruses have certainly left the most extensive and frequent evolutionary ancient viral traces in host dna: in the human genome, sequences of human endogenous retroviruses (herv) amount to ~ % of its total size [ ] [ ] [ ] and are derived from at least phylogenetically different sources [ ] . this fact is related to specific characteristics of the retrovirus replication machinery, which transcribes the genetic information carried by the viral dna into dna that is subsequently incorporated into the host genome. these incorporated viral sequences can be maintained in the host genome for a long time, either in the initial form, or with some modifications, and can be inherited. modifications are determined by the activity of a number of different factors, including mobile elements of the host genome and transposons, which makes it usually very difficult to identify the source of a given insertion. further on, viral genes integrated into host chromosomes can act as alleles that modify the host phenotype and sometimes provide a considerable selective advantage. for example, it is believed that this phenomenon contributed to the evolution of viviparous placental mammals; their genomes carry retroviral insertions that encode syncytins, proteins that serve to form the syncytiotrophoblast layer of the placenta and to ensure the immunological tolerance of the mother towards the embryo. importantly, not all of the mammals (and not only mammals) possess a well-developed placenta, but those who do also have syncytins derived from surface glycoproteins of different retroviruses, the insertion of which occurred at different moments of mammalian evolution. in retroviruses, these surface glycoproteins contain immunosuppressing domains [ , [ ] [ ] [ ] [ ] [ ] [ ] , and it is these domains that are used by the novel hosts. relic retroviral sequences (herv-k) can be found in the genomes of human ancestors, old world primates, nearly to the moment of their separation from the new world primates [ ] . human chromosomal dna contains - herv-k copies, some of which contain genes that exhibit low levels of expression in normal testicular and placental tissue [ ] . at the same time, endogenous retroviruses and retrotransposons can induce carcinogenesis in somatic cells [ , ] . molecular other rna viruses. in fact, the scope of the described phenomena is not limited to retroviruses as such, since the ubiquity of retroviral elements in animal genomes, their activity in germline cells [ ] , along with the fact that viral replication depends significantly on rna expression, allow retroviruses to contribute in different ways to the insertion of nonretroviral genes into animal germline cells. the genomic integration of nonretroviral genes can be mediated by nonhomologous recombination with chromosomal dna [ ] or by interaction with retroelements of the host cells [ , ] . it has been shown that retrotransposons can help the host borrow sequences from different rna and dna viruses. for example, recombination between the rna of the lymphocytic choriomeningitis virus and a murine iap retrotransposon results in reverse transcription of the rna and in its integration into the host genome [ ] . in some species, such insertions were shown to provide advantages; for example, bees that have acquired the gene that encode structural proteins of dicistrovirus become resistant to this agent, which causes acute paralysis in wild-type individuals [ ] . this phenomenon is very common in the kingdoms of plants and fungi [ , ] . in human, genomic incorporation of nonretroviral sequences has been described for such rna viruses as ebola [ , , , ] and marburg viruses (family filoviridae), agents of the born disease (family bornaviridae) [ , , , ] , and polioviruses (order picornavirales) [ ] . the first two families belong to the order mononegavirales, which also includes paramyxoviruses and rabdoviruses. these viruses have different virion structures, but share a common trait: their genome is composed of a single-stranded negative-sense rna molecule up to kb long. filoviruses cause extremely severe acute infections in humans (with - % lethality), and bornaviruses causes an equally severe disease in horses (with up to % lethality). bornaviruses have also been detected in humans; in patients with severe mental disorders, such as schizophrenia [ ] , as well as in individuals without any pronounced clinical presentation in the cells of which these viruses persisted over long periods of time [ ] . fragments of filo-and bornavirus genomes are inserted into the host genome via interaction with lines [ ] , the most common mobile elements in higher eukaryotes, which bear a reverse transcriptase gene. the most frequent findings in the host genomes are inserted fragments containing bornavirus genes n and l, which encode nucleocapsid protein n and rna-dependent rna polymerase (p ), respectively. it is difficult to say when these fragments were inserted into the host genomes; presumably, this event occurred about ma ago. its initiation and rate varied among different hosts and for different bornaviruses species. at present, in many animal species, bornavirus-derived genes have evolved into homologous own genes, ebln and ebln [ ] . these acquisitions provided an important selective advantage, enabling the host to resist devastating bornavius epizootics. it was shown that animals that possess ebln genes are resistant to species-specific bornavirus infections, or the course of the disease is less severe in them [ ] . the molecular basis of this resistance is the excessive synthesis of a protein n analog, which inhibits p polymerase and, thus, decreases the virus yield. the initial functions of inserted genes can undergo gradual modification with time; this phenomenon is referred to as exaptation. filovirus genes appropriated by vertebrates are those that encode np nucleoprotein and structural vp protein, which inhibits interferon production in the host. cellular analogs of these genes have been found in bats, bandicoot, wallaby, and other animals of the area. the borrowed viral genes can be partially transcribed, and the resulting truncated n-terminal np fragments (the full-size sequence of the host gene apparently is not expressed [ ] ) compete with the corresponding viral component, inhibiting the replication of the virus [ ] . retroviruses are not the only group for which nucleotide sequences can be fixed in eukaryotic genomes. in the s, v.m. zhdanov, an outstanding soviet virologist, hypothesized that it should be possible for other rna viruses [ ] . in a later work, l.yu. frolova et al. showed that trna-encoding sequences in the human genome homologous to trna-like elements encoded by ltrs of endogenous retroviruses, can act as targets for alphaviruses [ ] . presumably, virus-specific revertase is not at all necessary for a viral nucleic acid to insert into a host genome [ ] . some features of this process are similar in very different viruses and are worth a detailed analysis. for example, the stable incorporation of viral genes into the host genome typical for retroviruses was also described in filoviruses (although it is less frequent), while in many other viruses, this feature is less prominent or unknown at all. the ebola virus genome has an interesting feature, which probably does not explain its ability to integrate into the host genome (most probably occurring by homologous recombination), but is nevertheless worth mentioning. it is a short ( - amino acids) immunosuppressing fragment p e, which exhibits a high level of homology in ebola and retroviruses [ ] . importantly, the function of this domain is activated as a result of the incorporation of an additional adenine, which results in a reading frame shift. this adenine insertion in p e serves as a marker of pathogenicity of both retroviruses and the ebola virus when they infect a new host. the human genome contains a number of immunosuppressing fragments: they are expressed within syncytin genes in plancetal cells [ ] . insertions of nonretrovirus genes can basically have occurred as result of interactions between the gene source and a retrovirus (most commonly, a line retrotransposon). among the proteins, the genes of which were borrowed from a virus, those that perform a primarily protective function are the most likely to be fixed in their new environment. for example, fv is a protein similar to ca protein of the murine leukemia virus and competing with it; it binds the viral capsid and blocks reverse transcription, providing insensitivity to infection. in human and other animals, there are also protective proteins encoded by genetic elements of viral origin incorporated in the host genome. in particular, trim (and its analogs present in some primates) can inhibit the proliferation of some retroviruses in largely the same way as fv and, at the same time, affects the proinflammatory transcription factors nf-κb and ap- , which control the expression of genes involved in immune response, apoptosis, and cell cycle [ ] . dna retroviruses. elements derived from genomes of dna retroviruses (e.g., the duck hepatitis b virus) are found significantly less frequently in host dna than those originating from rna retroviruses [ , , ] ), even though they possess revertase, which could be expected to enable efficient incorporation of viral sequences into the host genome. other dna viruses. host genomes also bear traces of encounters with dna viruses. most commonly, these are members of the large parvovirus family (parvoviridae): dependoparvoviruses (adenoassociated agents that can replicate only in the presence of a helper adenovirus or herpesvirus), nonpathogenic in humans. dependoparvovirus genes have been found in the dna of pigs, cattle, rats, mice, and other animals [ , ] . probably (although it has not been proven), these insertions serve to protect against parvovirus infections. papillomaviruses were also shown to integrate into human genome [ ] . apart from the above agents, it was also found that the pig genome contains relic copies of the circovirus genome (noninfectious for humans). genes of nanoand geminiviruses were found in plant genomes [ , ] . human dna was also found to contain genes and larger genome elements of herpesviruses, including the epstein-barr virus (human herpesvirus , hhv ), human herpesvirus (hhv ), and other members of this superfamily [ ] . finally, at least % of the human genome is composed of fairly large virus-like sequences: the so-called selfish dna, the origin of which is unclear, while the only observed type of activity is autoreplication. the most active group are transposons of the line class (long interspersed elements); in human, they harbor approximately one in thousand genetic mutations [ ] [ ] [ ] . on the whole, virus-like components of the human genome account for nearly a half of the chromosomal dna, and some of them play an important role in the host organism, but hardly anything is known about the origin and functions of the others. antiviral host response can transform an acute infection into the chronic or even the latent form (as it happens with herpesviruses), and a reservoir of viruses is thus conserved in their natural host, who will remain their target in case of reactivation. it should be underlined that, for many important reasons, the body of relevant data available from the existing publications is far from being complete [ ] . first of all, not all of the known viruses have been studied as potential sequence donors for the host genome, and not all the potential host species have their genomes sequenced. secondly, an insertion of a viral sequence may represent a temporary outcome of a single infection event and will not be maintained in subsequent host generations. moreover, the host species may also be eliminated from the evolution (as a result of extinction), which means that the acquired insertions will only be conserved if they are no longer limited to the extinct species and have become specific for a more general taxonomic branch, such as genus, order, or higher. next, the relatively recent insertions may be insufficiently widespread to be identified and thus evade observation. finally, the ability to incorporate parts of the viral genome into the chromosomal dna of host germline cells can vary strongly among different taxonomic groups of viruses, i.e., orders, families, genera, and even species if insertions of viral sequences remain functionally active in the host cell genome, they can give rise to either proteins that function in a new environment or untranslated rnas of different sizes. if these insertions are inactive, they can merely witness a history of close and evolutionary long-term interactions between the virus and the host. the characteristic trait of human herpesviruses is that, in their typical latent state, they can persist and replicate in the form of an episome in the direct vicinity of the host genomic dna. these viruses have long coexisted with their hosts and the hosts' phylogenetic ancestors, and their genomes carry full-size genes that in turn were captured at some moment from the host and can often be expressed in their new environment [ , ] . the nucleotide sequences of these genes, the encoded amino acids, and even the functions of the resulting proteins do not correspond strictly to their cell counterparts, but the range of their functions in the virus certainly suggests a relationship between the viral and the host genes. some herpesviruses (such as hhv associated with kaposi's sarcoma) have acquired genes of serpentines (g-protein-coupled receptors; gpcr), which they employ at the early stage of lytic infection of lymphoid cells and of sarcoma itself; moreover, the inhibition of viral dna synthesis does not affect the functioning of these molecular biology vol. no. genes. their function is not quite clear, since they act mainly at the early stages of infection: gpcr transcripts, which have mainly bicistronic structure, protect the '-region of the coding sequence of another hhv gene, К ; while monocistronic transcripts analyzed in model experiments did not exhibit such properties. this probably indicates that the translation of gpcr transcripts may be reinitiated and suggests the need for further analysis of all the functions of gpcr itself in the pathogenesis of kaposi's sarcoma. the product of the gpcr-encoding gene bilf captured by hhv acts as a specific inhibitor of class i major histocompatibility complex. the reverse process can also be imagined easily, as well as subsequent gpcr modifications as a result of the repeated gene capture with the acquisition of new functions. in fact, no such data are currently available, but the research has only begun very recently. a recent study detected the first endogenous herpesvirus (genus roseolovirus) in the genome of the philippine tarsier (order primates), while insertions of nearly full-size hhv -and hhv -like genomes were found in the dna of other primates: aye-aye, lemur, and chimpanzee [ ] . in this context, it is also worth mentioning that more data exist on the integration of herpesviruses into host genomes [ ] , as well as on the incorporation of herpesvirus dna (hhv ; also of the genus roseolovirus) into the telomeric zones of human chromosomes. the significance of these insertions (which can be transmitted vertically if occur in germline cells) for human diseases or for the functioning of the immune system is currently absolutely unclear [ , , ] . similar data were obtained for viruses of other taxonomic groups. a systematic analysis showed that sequences derived from a wide range of animal viruses other than retroviruses are present as endogenous elements in mammalian, avian, and insect genomes. these elements of animal genomes represent the full spectrum of viral replication strategies [ ] ; moreover, the larger the sample of animal genomes, the wider the diversity of endogenous viral elements. obviously, the more ancient these elements are, the smaller the number of host species needed to detect them. to identify more recent viral insertions, a much wider sample on the order and genus level is required. however, as we have pointed out above, the currently available data are far from complete. the diversity of the known virus isolated as represented in virus gene/protein banks is but a small portion of the total virus diversity. in view of their likely ancient origin, members of the many virus families may be much more widely distributed among their mammalian hosts than we currently imagine, both as separate entities and as genome fragments in the host dna. this is also reflected in the virus phylogeny, which was constructed using endogenous virus insertions along with exogenous viruses; close exogenous relations frequently are either not identified or were only described in the past decade [ ] [ ] [ ] . the recently discovered relationship between filoviruses and marsupials has suddenly specified this infraclass of mammals as a potential filovirus reservoir. the presence of viral insertions may become an important factors for evaluation of findings obtained using metagenomic approaches [ , , ] . all of these data have been discussed in several comprehensive reviews [ , , ] . the capture of certain fragments of a viral genome by the host may be a random event; however, should the captured genes reach germline cells and prove useful to the host, they can become fixed in subsequent generations. it may prove interesting to consider information that concerns shorter insertions of viral origin. they can be expected to exist in much higher numbers, since the probabilities of their incorporation, as well as of transposition or multiplication, seem higher than for larger insertions. fragments of - nt long would have the same size as the biologically active rnas, such as those involved in rna interference. numerous studies, e.g., those reviewed in [ ] , indicate that such molecules can directly participate in the regulation of mammalian viruses. for instance, liverspecific mir- suppresses the replication of hepatitis c virus [ ] , while several human micrornas, such as mir- a- p, mir- , and mir- a- p, inhibit hepatitis b virus [ ] . other human micrornas are targeted against the influenza virus (mir- , mir- , and mir- ), the vesicular stomatitis virus (mir- and mir- ), and against hiv (mir- , mir a, mir- b, mir- , mir- , and mir- ) [ ] . there are mirnas against hhv and hhv herpesviruses (mir- / and mir- b/ ) [ ] [ ] [ ] , coxsackie virus (mir- - p) [ ] , and human papilloma virus [ ] . it is also known that some viral mirnas can circulate in human. for instance, mirna-ul - is encoded by the human cytomegalovirus (hhv ; genus cytomegalovirus, family herpesviridae), and its target is mrna of the pre-early viral protein ie [ ] ; mirna of hhv is targeted against bart lmp mrna [ , ] , while mirnas of herpes simplex viruses (hhv and hhv ) , mir-h , mir-h , and mir-h , are directed against mrna of viral proteins icp and icp . [ ] . similar mirna-mrna pairs were detected in experimental models of a retroviral infection [ ] . it was shown that endogenous small interfering rnas (sirnas) can regulate the activity of some endogenous retroviruses [ ] . however, it is currently hardly possible to identify the targets of micrornas based on their sequences only, since they do not need to be strictly complementary. in addition to the clip-seq and par-clip techniques that employ immunoprecipitation of rna-protein complexes with subsequent sequencing [ ] , it may prove helpful to analyze the data on the coexpression of mirnas and their putative gene tar-gets [ ] . two other groups of molecules participating in rna interference are sirnas and so-called piwirnas (or pirnas). the latter are - nt long; their activity is mediated by a specific mechanism involving enzymes of the piwi family, and their targets are usually transposons, retrotransposons, and endogenous retroviruses [ ] . the - -nt long fragments that form sirnas and pirnas must be almost fully complementary to their target sequences (with mismatches of no more than nt). these rnas are mainly targeted at exons, which facilitates the in silico search for putative small insertions in the recipient dna. the functional activity of sirnas and pirnas should be determined experimentally. the detection of nucleotide fragments of the specific size in the escherichia coli genome invited the hypothesis that bacteria possess an antiviral immune defense system [ ] , which later became the basis for the development of the revolutionary gene editing tools, crispr cas /cpf [ , ] . we attempted to identify -to -nt-long homology stretches (which we provisionally refer to as hits) in the human genome and in the genomes of human adenoviruses and herpesviruses and showed that their number was significantly higher than in extended samples that included either viruses that do not directly infect human, such as bacteriophages, or artificially generated sequences of the same as the herpesvirus genome [ ] . later, we showed that, even in a given group of viruses (hhvs), the portion of the viral exome that corresponds to hits, i.e., short - -nt long sequences homologous between the virus and the host, is specific to each virus type and, as the first approximation, can be related to the destructive effect of the viral infection [ ] . we have also proposed a hypothesis that reactivated hhv could exhibit oncolytic properties in vivo [ ] , because the body of rl (which encodes icp . protein) contains a large number of virus/human homologous sequences (hits); in artificial hhv -based oncolytic constructs, this gene is switched off first. obviously, our hypothesis requires experimental verification. the fig. schematically shows the load of human dna with viral relics. we define the known viral insertions and their derivatives, including transposons, as relics, since we are currently able to identify only those that were incorporated into the host genome long time ago. human translated genes constitute approximately % of the total information volume of genomic dna, whereas as virus relics and virus-like structures (with some reservations, in addition to endogenous retrovi-ruses, these may be assumed to include retrotransposons and dna transposons) amount to nearly half of the human genome. this information suggests that there might be a need to reconsider the notion of a virus. from the traditional medical point of view, a virus is a parasite that infects a sensitive cell and ultimately causes its destruction. the virus (its genome) can also infiltrate the host dna, occupying the free space or adding to its size. by replicating in the host genome, the virus ensures its genetic diversity and at the same time acquires a safe depot protecting it from external factors. this viral activity can be reasonably classified as parasitic. however, from this viewpoint, we overlook the potential evolutionary advantages that the host population may gain as result of a viral infection: viruses are the most efficient vectors, which might transfer novel genetic information. for this reason, we propose to define a virus as an information carrier indistinguishable from a specific one and possessing an autonomous autoreplication program that employs the addressee's reading, synthesizing, and metabolic machinery for its own realization. this definition underlines the major evolutionary function of viruses as carriers of genetic information and is free from moral judgment, which is foreign to nature. moreover, this definition has a more general character than the one specifying viruses a parasites. certainly, these considerations do not imply that homo sapiens does not need to struggle with destructive viral infections. moreover, the fact that, in nature, portions of the human genome occupied by protein-coding genes ( %) and relic viral sequences: endogenous retroviruses ( %), dna transposons ( %), sines ( %), and lines ( %). dark area of the human genome ( %) also contains other viral sequences (see text), genes whose activity is limited to transcription, and structures whose function is currently unknown. [ ] [ ] [ ] . as for the short regions of homology to herpesvirus or adenovirus dna (which can basically be viral insertions), their distribution in the human genome is specific for every virus species, and on the whole, as we showed previously [ ] , it can hardly be random. it was not the main objective of our brief review to list all of the available data on the question. the body of these data is rapidly growing, which, however, does not seem to change the existing consensus view on the problem. obviously, mutual insertions of genomic fragments in virus-host pairs occur regularly, if not very frequently. their exact frequency remains to be determined both on the population level and on the level of single individuals. the objective is not as much to determine the number of already incorporated foreign genes but rather to find out how this frequency depends on the type of the virus and its host. it is also important to determine the consequences of each insertion, either transient or fixed, can have for the human host within the lifetime of the hosts or of their ancestors. it should be evaluated how often these insertions reach the host's germline cells and what key factors govern these events. there are currently no answers to these questions, but these issues are addressed in different areas of study, which sometimes produces unexpected results. for instance, the haldane and waddington problem about the number of generations were required to obtain recombinant inbred strains was initially only solved by the authors for the cases of two and three genes [ ] . recently, for the general case samal and martin proposed an approach based on a statistical formalism rarely used in areas other than physics [ ] ; surprisingly, it provided an exact solution to the problem involving any given number of genes. although the above example lies in the area of population genetics, while the number of studies on our topic of interest is insufficient to allow population-level generalizations, it nevertheless confirms the need to continue the efforts and emphasizes the productivity of breaking traditional thinking patterns, even in such conservative fields as medical diagnostics and therapy. the exchange of genetic information between living organisms is a complex albeit an infrequent phenomenon and generates a considerable uncertainty when we attempt to provide a comprehensive explanation of the causes and nature of a given pathological condition. the dynamic equilibrium between the human genome sensu stricto and the integrated viral sequences that perform protective and regulatory functions in the host organism represents a much deeper relationship than the organism's interaction with the internal microbiota. currently, these relationships constitute the subject of separate omicstype research. they are the consequence of a multisided (not just a two-sided) encounter, where one side is represented by a viral population, which is heterogeneous in each individual case and consists of different proportions that are both fully featured and defective, as well as mutant particles, while the other side is a multicellular organism that developed as a result of macro-and microevolution. viruses have enriched the host genome in functional virus relics, the amounts and diversity of which greatly exceed the total of the host's own genes. on the other hand, the multicellular organism has developed a system of antiviral defense and continues to develop it while employing the full range of available molecular mechanisms. predicting the consequences of this encounter and the results of their targeted modification for the benefit of the infected macroorganism is a problem that can only be solved after considerable advances in the techniques of analyzing and modeling the underlying mechanisms. vol. no. endogenous viral elements in animal genomes cellular analogs of viral proteins genes in the terminal regions of orthopoxvirus genomes experience adaptive molecular evolution a probable molecular factor responsible for generalization of variola virus infection entire coding sequence of the variola virus comparison of the genetic maps of variola and vaccinia viruses resistance to hiv- infection in caucasian individuals bearing mutant alleles of the ccr- chemokine receptor gene halliburton i.w. . molecular genetics of herpes simplex virus: demonstration of regions of obligatory and nonobligatory identity within diploid regions of the genome by sequence replacement and insertion clustering of genes dispensable for growth in culture in the s component of the hsv- genome dna of herpes group viruses the open reading frames ul , ul , ul , and ul are dispensable for the replication of herpes simplex virus in cell culture adenoviral vectors for gene transfer and therapy production of first generation adenovirus vectors: a review adenovirus: from foe to friend adenovirus: the first effective in vivo gene delivery vector comparison and functional implications of the d architectures of viral trna-like structures an rna tertiary switch by modifying how helices are tethered long-term reinfection of the human genome by endogenous retroviruses initial sequencing and analysis of the human genome new bioinformatic tool for quick identification of functionally relevant endogenous retroviral inserts in human genome the envelope glycoprotein of ebola virus contains an immunosuppressive-like domain similar to oncogenic retroviruses dormant" immunosuppressive domain in filoviruses the gp-protein of marburg virus contains the region similar to the 'immunosuppressive domain' of oncogenic retrovirus p e proteins when genes go walkabout transposon-mediated rewiring of gene regulatory networks contributed to the evolution of pregnancy in mammals can viruses make us human? retroviruses and primate evolution herv-k: the biologically most active human endogenous retrovirus family quantitative studies of naturally occurring murine leukemia virus infection of akr mice molecular functions of human endogenous retroviruses in health and disease reverse transcriptase activity in mature spermatozoa of mouse nonreplicative rna recombination in poliovirus effects of retroviruses on host genome function recombination of retrotransposon and exogenous rna virus results in nonretroviral cdna integration epigenetic regulation of an iap retrotransposon in the aging mouse: progressive demethylation and de-silencing of the element by its repetitive induction isolation and characterization of israeli acute paralysis virus, a dicistrovirus affecting honeybees in israel: evidence for diversity due to intra-and inter-species recombination unexpected inheritance: multiple integrations of ancient bornavirus and ebolavirus/marburgvirus sequences in vertebrate genomes endogenous non-retroviral rna virus elements in mammalian genomes ebola-associated genes in the human genome: implications for novel targets ebola virus vp protein binds 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cells identification of new herpesvirus gene homologs in the human genome mammalian retroelements transposable elements and the evolution of gene expression transposable elements as sources of variation in animals and plants the latent human herpesvirus- a genome specifically integrates in telomeres of human chromosomes in vivo and in vitro the first endogenous herpesvirus, identified in the tarsier genome, and novel sequences from primate rhadinoviruses and lymphocryptoviruses herpesviruses and chromosomal integration chromosomally integrated human herpesvirus : questions and answers mapping the telomere integrated genome of human herpesvirus a and b quaranfil, johnston atoll, and lake chad viruses are novel members of the family orthomyxoviridae liao ning virus, a new chinese seadornavirus that replicates in transformed and embryonic mammalian cells multiple diverse circoviruses infect farm animals and are commonly found in human and chimpanzee feces rapid identification of known and new rna viruses from animal tissues bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses a technique for genome-wide identification of differences in the interspersed repeats integrations between closely related genomes and its application to detection of human-specific integrations of herv-k ltrs antiviral effects of human micrornas and conservation of their target sites modulation of hepatitis c virus rna abundance by a liver-specific microrna role of micrornas in hepatitis b virus replication and pathogenesis micrornas and human retroviruses cullen b.r. . the viral and cellular microrna targetome in lymphoblastoid cell lines ebv and human micrornas co-target oncogenic and apoptotic viral and human genes during latency kaposi's sarcoma-associated herpesviral il- and human il- open reading frames contain mirna binding sites and are subject to cellular mirna regulation regulation of cellular mirna expression by human papillomaviruses suppression of immediate-early viral gene expression by herpesvirus-coded micrornas: implications for latency modulation of lmp protein expression by ebv-encoded micrornas modulation of lmp a expression by a newly identified epstein-barr virus-encoded microrna mir-bart mammalian alphaherpesvirus mirnas replication competent hiv- viruses that express intragenomic microrna reveal discrete rna-interference mechanisms that affect viral replication an inside job for sirnas genome-wide identification of mirna targets by par-clip crosshub: a tool for multi-way analysis of the cancer genome atlas (tcga) in the context of gene expression regulation mechanisms rna interference against viruses: strike and counterstrike unusual nucleotide arrangement with repeated sequences in the escherichia coli k- chromosome multiplex genome engineering using crispr/ cas systems cpf is a single rna-guided endonuclease of a class crispr-cas system how many antiviral small interfering rnas may be encoded by the mammalian genomes? short nucleotide sequences in herpesviral genomes identical to the human dna rna interference: the next genetics revolution? in: horizon symposia; understanding the rnaissance rna interference and its role in cancer therapy the second coming of rnai inbreeding and linkage statistical physics methods provide the exact solution to a long-standing problem of genetics key: cord- -x r bz authors: chai, qiyao; wang, lin; liu, cui hua; ge, baoxue title: new insights into the evasion of host innate immunity by mycobacterium tuberculosis date: - - journal: cell mol immunol doi: . /s - - -z sha: doc_id: cord_uid: x r bz mycobacterium tuberculosis (mtb) is an extremely successful intracellular pathogen that causes tuberculosis (tb), which remains the leading infectious cause of human death. the early interactions between mtb and the host innate immune system largely determine the establishment of tb infection and disease development. upon infection, host cells detect mtb through a set of innate immune receptors and launch a range of cellular innate immune events. however, these innate defense mechanisms are extensively modulated by mtb to avoid host immune clearance. in this review, we describe the emerging role of cytosolic nucleic acid-sensing pathways at the host–mtb interface and summarize recently revealed mechanisms by which mtb circumvents host cellular innate immune strategies such as membrane trafficking and integrity, cell death and autophagy. in addition, we discuss the newly elucidated strategies by which mtb manipulates the host molecular regulatory machinery of innate immunity, including the intranuclear regulatory machinery, the ubiquitin system, and cellular intrinsic immune components. a better understanding of innate immune evasion mechanisms adopted by mtb will provide new insights into tb pathogenesis and contribute to the development of more effective tb vaccines and therapies. introduction tuberculosis (tb) remains a serious global public health threat, accounting for over . million deaths per year. mycobacterium tuberculosis (mtb), the etiological agent of tb, is estimated to have infected . billion people worldwide. despite the availability of anti-tb medications, cure rates are low (~ % globally) for continuously emerging drug-resistant tb cases, which necessitate the use of more complex and toxic regimens and even pose risks of transmitted resistance. , therefore, rational design of novel tb vaccines and therapeutics based on an in-depth understanding of the intimate interplay between mtb and host immunity is required. innate immunity plays a dominant role in protecting the host from early infection with mtb, as indicated by the majority of mtb-exposed individuals being able spontaneously control the infection despite a conspicuous delay of acquired immunity; however, an intact adaptive immune system is insufficient to restrict mtb growth within a host deficient in innate immune responses. , as first-line defensive patrols that quickly respond to mtb infection, innate immune cells perform the duty of immune surveillance via a range of pattern recognition receptors (prrs). activation of these immune receptors leads to a range of cellular events that contribute to host anti-mtb immunity, such as phagocytosis and apoptosis. however, long-standing coevolution with the human host protects mtb from the effects these antibacterial mechanisms, leading to its persistent infection. furthermore, in recently emerging pathogenic strategies, mtb can directly target and modify various aspects of the molecular regulatory machinery of host innate immunity, such as the intranuclear regulatory machinery, the ubiquitin system and cellular intrinsic immune components, to evade host clearance. in this review, we summarize recently emerging aspects of innate immune evasion mechanisms adopted by mtb to benefit its own intracellular survival, including the role of cytosolic nucleic acidsensing pathways at the host-mtb interface; novel mechanisms adopted by mtb to circumvent host cellular innate immune events, such as membrane trafficking and integrity, cell death, and autophagy; and newly elucidated mtb strategies to manipulate the host molecular regulatory machinery of innate immunity. a better understanding of the intricate interplay between mtb and the host innate immune system may provide new insights into tb pathogenesis and contribute to the development of valid vaccines and therapies. emerging roles of cytosolic nucleic acid-sensing pathways in host-mtb interactions the core duty of the mammalian innate immune system to recognize infective pathogens is evolutionarily designed to rapidly sense and eliminate foreign threats. to prevent the successful establishment of mtb infection in the lungs, host immune cells, and various nonclassical immune cells in the airway are equipped with a set of cell-surface and intracellular prrs to recognize the invading mycobacteria, such as toll-like receptors, c-type lectin receptors, nod-like receptors (nlrs), complement receptors, and scavenger receptors (srs). these innate immune sensors play critical roles at the interface of host mucosal immunity and mtb pathogenesis and have been extensively reviewed elsewhere. [ ] [ ] [ ] in this section, we focus on the recently emerging role of cytosolic nucleic acid-sensing pathways in host-mtb interactions (fig. ). cytosolic dna-sensing pathways although the immunostimulatory effects of mycobacterial dna on mammalian hosts were receiving attention decades ago, hostresponsive dna-dependent cytosolic surveillance pathways were not elucidated until recently. initially, mtb was thought to be able to translocate from phagosomes into the cytosol by virtue of its esat- secretion system- (esx- ) system during infection of host cells, , and this process provides a potential opportunity for host cytosolic receptors to sense mycobacterial extracellular dna. in addition, the blood of patients with active tb is characterized by a distinct transcriptional signature related to type i interferon (ifn) signaling, and this hallmark was proposed to be associated with the activation of the host cytosolic surveillance pathway, which can result in the robust production of type i ifns. based on these observations, manzanillo et al. first tested the role of two putative cytosolic dna sensors, z-dna binding protein (zbp ) and ifnactivable protein (ifi ; the mouse ortholog of human ifi ), in host cytosolic surveillance of mtb and found that only ifi contributes to the type i ifn response to mtb infection via the stimulator of ifn genes (sting)/tank binding kinase /ifn regulatory factor (irf ) axis in macrophages. interestingly, the deletion of irf to subvert this signaling pathway in mice decreased the host expression of type i ifns and enhanced host resistance to long-term mtb infection. these results indicate a negative regulation of type i ifns in host anti-mtb immunity and suggest a potential strategy by which mtb hijacks the cytosolic surveillance pathway to facilitate its own infection. cyclic gmp-amp synthase (cgas) is a recently characterized dna sensor. upon direct binding with cytosolic dna, cgas is activated to catalyze the production of cyclic gmp-amp (cgamp), leading to the activation of the downstream sensor sting. , according to pioneering studies, cgas functions in the cytosol, where it cooperates with sting to activate both nuclear factor-κb (nf-κb) and irf signaling pathways to induce the transcription of type i ifns and various pro-inflammatory t helper type (th ) cytokines with action against viral infections. [ ] [ ] [ ] [ ] nevertheless, our recent findings and those of others suggest that cgas can change its subcellular location and enter into the nucleus or reside on the plasma membrane, which is a possible strategy adopted by the host to distinguish self-and nonself dna through the exertion of distinct cgas-dependent functions. [ ] [ ] [ ] the involvement of the cgas-mediated dna-sensing pathway in host anti-mtb immunity is indicated by the findings that cgas expression is upregulated and that cgas is colocalized with mycobacteria in human tb lesions, and its deficiency impairs the induction of type i ifn responses and autophagy in mtb-infected macrophages. [ ] [ ] [ ] recent studies also suggest that the cgas/sting immunesensing pathway is necessary for host dendritic cell (dc) activation because it increases the expression of type i ifns upon mycobacterial infection. , interestingly, despite confirmation of cgas/sting-dependent bacterial control in macrophages, cgas −/− and sting −/− mice show comparable lung bacterial burden and inflammation levels to those of wild-type control mice after mtb exposure, , , suggesting that additional host dna sensors or other immune receptors may compensate for cgas/ sting-dependent antimycobacterial immune responses in vivo. apart from type i ifn stimulation, the detection of intracellular dna may also lead to inflammasome activation with the production of mature pro-inflammatory cytokines, including interleukin- β (il− β) and il- , via the absence of melanoma (aim ). , in macrophages, aim responds to mtb genomic dna and results in increased caspase- cleavage and il- β and il- release, a finding consistent with the observation that aim deficient mice show an increased susceptibility to mtb infection with impaired pro-inflammatory responses. similarly, infection with virulent mycobacterium bovis can also activate the aim inflammasome in macrophages. notably, compared with nonvirulent mycobacteria containing a compromised esx- secretion system, such as mycobacterium smegmatis, mycobacterium fortuitum, mycobacterium kansasii, and attenuated mtb h ra strains, virulent mtb h rv has a significant inhibitory effect on aim dependent innate cytokine responses. this finding seemingly contradicts the accepted idea that esx- is essential for activating host cytosolic surveillance pathways. most likely, esx- is required for mtb to deliver a number of effectors into the host to remodel the intracellular environment to improve its chance for survival, despite its role in inducing immune recognition. in addition, it should be noted that individual effectors delivered by the mtb esx- secretion system may play independent immunoregulatory roles with different host targets, and thus, the mechanisms underlying esx- -dependent stimulation or evasion of the host cytosolic surveillance pathway both function during host-mtb interactions. this notion is supported by the finding that blocking the secretion of esxa, a major substrate of esx- , significantly reduced cgas/sting-mediated ifn production while leaving the inflammasome-mediated il- β response virtually intact. therefore, specifically targeting mycobacterial esx- products or host regulatory factors might enable the selective regulation of inflammasome and cgas/sting pathway activation and, hence, contribute to the recovery of the equilibrium between th -type cytokine and type i ifn responses in tb patients to improve their anti-mtb immunity. cytosolic rna-sensing pathways the immunomodulatory activity of mycobacterial rna in mammalian hosts received attention as early as the s and s. recently, it was reported that mtb-infected macrophages can deliver extracellular vesicles (exosomes) containing abundant mycobacterial rna to recipient cells, suggesting that mtb rna is probably released into host cells to trigger the rna-dependent cytosolic surveillance pathway. the cytosolic rna-sensing pathway was initially identified as a key part of host immune surveillance against rna virus infection. in mammalian cells, retinoid acid-inducible gene i (rig-i)-like receptors (rlrs) are wellconserved cytosolic prrs that recognize cytosolic viral rnas and activate downstream immune pathways to promote the production of type i ifns and other pro-inflammatory cytokines. rig-i and melanoma differentiation-associated protein (mda ) are the best characterized rlrs, which preferentially recognize short polyphosphorylated double-stranded rna (dsrna) and long dsrna, respectively. after sensing foreign rnas, rig-i, and mda transmit signals via a common adapter, mitochondrial antiviral signaling (mavs), which forms large prion-like polymers and recruits tumor necrosis factor (tnf) receptor-associated factors (trafs) to further activate nf-κb and irf immune signaling pathways. [ ] [ ] [ ] recently, the rlr-mediated cytosolic surveillance pathway was also shown to participate in the host immune response to various bacterial pathogens, such as mtb, legionella pneumophila, helicobacter pylori, and listeria monocytogenes. [ ] [ ] [ ] the involvement of the rlr-dependent rnasensing pathway during host-mtb interactions is implied by the increased expression of rig-i and mda mrnas in mtb-infected macrophages. further investigation of recombinant mtb strains demonstrated that mtb seca and esx- secretion systems are critical for the delivery of mtb rna into the host cell cytosol, resulting in ifn-β production through the host rig-i/mavsmediated rna-sensing pathway. the role of the mda mediated rna-sensing pathway in detecting mtb infection was also confirmed by a recent study, which showed that deletion of mda impaired ifn-β production and bacterial control in human macrophages, results similar to those obtained by the deletion of rig-i or mavs. nonetheless, rig-i, not mda , appears to interact with the mtb-specific mrnas pola and ppe (ref. ), suggesting that these rlrs probably play nonredundant roles in detecting different types of mycobacterial rnas. furthermore, mavsdeficient mice showed obviously increased resistance to mtb infection with attenuated bacterial growth in their lungs, as was also observed in irf -deficient mice, supporting a potentially negative role of type i ifns in host anti-mtb immunity in vivo. in addition to the rig-i/mda /mavs axis, protein kinase r (pkr) has been identified as another host sensor of cytosolic dsrna, which can interact with the natural rna derived from diverse viruses or bacteria, leading to the activation of irf , nf-κb, and other various innate immune signaling pathways. according to an infection model based on the interaction of m. bovis bacillus calmette-guérin (bcg) and primary human blood monocytes, the mycobacteria-induced production of inflammatory cytokines is regulated by the phosphorylation and activation of pkr. a recent study also demonstrated that mtb infection results in +increased expression of pkr and increased phosphorylation of its substrate, eukaryotic translation initiation factor a, in human cells, and pkr deficiency leads to enhanced intracellular growth of mycobacteria. however, the in vivo role of pkr in host immunity challenged by mtb infection remains unclear. although a research group has reported that mice lacking pkr show reduced mycobacterial burden with less severe pulmonary pathology than shown by wild-type mice, they recently attributed this observation to different genetic backgrounds of the mice rather than to a direct role of pkr. aside from the rig-i/mda -and pkr-mediated cytosolic rnasensing pathways, intracellular nlr family members, including nlrp and nod , can also recognize foreign dsrna and singlestranded rna (ssrna), respectively. , mtb infection activates both of these nlrs in an esx- -dependent manner to trigger various host downstream innate immune responses, such as nlrp inflammasome formation, autophagy initiation and nf-κb and irf pathway activation, which have been extensively reviewed elsewhere. , however, it is still unclear whether nlrp and nod , which respond to a range of pathogen-derived stimuli, , can be activated by direct binding to mycobacterial extracellular rnas, although a recent study reported that dsrna from mtb cultures is able to induce caspase- activation in retinal pigment epithelium. in summary, host cytosolic dna-and rna-sensing pathways are newly emerging innate immune recognition mechanisms of host-mtb interactions. growing evidence indicates that there is intimate cross talk among the components of different cytosolic nuclear acid-sensing pathways, , and these immune surveillance pathways probably play nonredundant roles in host anti-mtb immunity. however, the in vivo data from animal infection models show that activation of cytosolic cgas-or rlr-mediated sensing pathways can induce a strong type i ifn response that appears to impair host resistance to mycobacterial infection, , suggesting that mtb may exploit the host cytosolic surveillance pathways to facilitate its own growth. in contrast, activation of other cytosolic pathways during mtb infection, such as that mediated by aim , nod , and nlrp , can promote the production of protective inflammatory cytokines. hence, further investigation may be focused on how to spatiotemporally and selectively regulate these cytosolic surveillance pathways to optimize host anti-mtb immunity. furthermore, a recent study demonstrated that drug treatment targeting cytosolic rna sensors benefited the host by controlling mycobacterial intracellular growth, highlighting the potential value of targeting the cytosolic immune surveillance pathway for novel host-directed anti-tb therapy. new aspects of mtb-modulated cellular innate immune events the activation of host innate immune-sensing pathways by mtb infection leads to a range of subsequent cellular antimicrobial events, such as phagocytosis and apoptosis; however, these effects can be modulated by mtb to benefit its long-term intracellular survival. in this section, we focus on recently emerging aspects of regulatory strategies adopted by mtb to interfere with host membrane trafficking and integrity, cell death, and autophagy processes (fig. ). the leveraging of host membrane trafficking in infected cells is a key strategy for the notorious success of mtb as a highly adapted intracellular pathogen. upon infection, mtb is engulfed by host phagocytic cells such as macrophages, neutrophils, and dcs and internalized in a phagosome, the organelle responsible for routine clearance of pathogens. notably, while phagosomes in macrophages and neutrophils are generally designated to rapidly eliminate pathogen-associated cargo, dc phagosomes tend to moderately degrade their internalized substrates to preserve antigenic peptides for priming adaptive immune responses. however, it has been well documented that mtb recruits the gtpase rab , but not rab , away from the phagosome to inhibit phagolysosome maturation. , the prevention of the biogenesis of phagolysosomes plays a vital role in mtb infection, transmission, latency, and immune evasion. , multiple routes and numerous effectors are employed by mtb for the suppression of phagosome maturation and acidification, which have been extensively summarized elsewhere. , notably, the ability of mtb to manipulate host membrane trafficking may also contribute to the targeting of the host endosomal sorting pathway by human immunodeficiency virus during viral budding, thus favoring synergism of these two pathogens during coinfection. recently, the spatiotemporal dynamics of mtb phagosomal morphology and composition have received growing attention. during maturation, phagosomes associate with early and late endosomes, as well as other intracellular organelles such as golgiderived vesicles, the endoplasmic reticulum (er) and mitochondria, and these interactions are very dynamic and can promptly change both the phagosomal membrane and luminal components with the principal aim of restraining the growth of internalized pathogens. upon infection, mtb alternatively localizes to two morphologically different types of phagosomes, tight and spacious phagosomes, which are consistently observed in both tb patients and other animal hosts. [ ] [ ] [ ] a recent study revealed that ifn-γ can facilitate endosomal interactions with mtb phagosomes via the regulation of the rab -dependent vesicular trafficking pathway, which promotes membrane influx into tight phagosomes and shifts them into spacious and proteolytic compartments that restrict mtb growth. however, mtb can avoid being directed to rab -positive spacious phagosomes via its esx- system. another study has demonstrated that patient-derived mtb strains can produce large amounts of -tuberculosinyladenosine ( -tbad), which acts as a bacterial antacid and selectively accumulates in host cellular acidic compartments, resulting in phagosomal swelling and the obliteration of the lysosomal multilamellar structure. the phagosomal components also appear to be fine-tuned by mycobacteria, given that the mtbspecific phagosome proteome shows distinct characteristics from that of latex bead-or other bacterial pathogen-containing phagosomes. it is conceivable that mtb must remodel the intravacuolar microenvironment to establish a pathogen-friendly niche. for example, mtb can encode various effectors, such as ptpa, -tbad, and marp, to elude, neutralize or tolerate the acidic environment of phagosomes. , , mycobacteria also avoid being trafficked with bactericidal molecules, such as lipocalin , an innate immune protein that disrupts bacterial iron acquisition, to their compartments while retaining access to transferrin for iron uptake through the rab + endocytic recycling pathway. the change in mtb phagosomal content is also a hallmark of accumulated lipid droplets, which probably depends on rab , according to a recent study. although it was proposed that mtb can disrupt mitochondrial fatty acid oxidation to promote lipid body deposition in macrophages for utilization, another study demonstrated that increased formation of lipid droplets in mtbinfected cells actually facilitates host biosynthesis of eicosanoids and restricts bacterial growth. therefore, the multifaceted role of lipid bodies in mtb phagosomes requires further elucidation. membrane rupture, which depends on the mycobacterial esx- system, is another typical characteristic of mtb phagosomes. , , this phenomenon has long been considered a pathogen-driven event utilized by mtb to escape from a bactericidal phagosome and enter the host cell cytosol, where it can obtain abundant nutrients. however, a recent study demonstrated that the inhibition of phagosomal maturation and acidification is a precondition for mtb phagosomal damage. furthermore, as identified in other successful intracellular pathogens, such as l. pneumophila and brucella abortus, the establishment of a sheltered niche within a vacuolar compartment mimics a normal cellular organelle and enables the pathogen to avoid host immune surveillance and clearance. , this finding one to wonder why a mycobacteria departs from a cozy niche to enter the cytosol where it must confront a series of cytosolic immune sensors? to date, no direct evidence indicates an obvious advantage of mycobacterial extra-phagosomal survival. one possible explanation is that the success of persistent mtb infection requires the esx- secretion system to damage the phagosomal membrane and deliver numerous secretory effectors into the cytosol to target and regulate cellular immune components. this assumption is supported by accumulating evidence that indicates an indispensable role for the esx- system in mtb pathogenesis, as it has been linked to host cytosolic surveillance evasion, , phagosome maturation arrest, , cell death reprogramming, , autophagy inhibition, , etc. alternatively, escape from phagosomes facilitates mtb esx- -dependent plasma membrane damage, facilitating efficient mtb replication and spread to neighboring cells and, eventually, to new hosts. by using time-lapse microscopy at the single-cell level, ruptured host cell plasma membranes were observed at the contact points of mtb with the plasma membrane, which provides direct evidence for this assumption. however, this evidence does not exclude the other possibility: the host may actively promote mtb phagosome rupture at the early stage of infection to eliminate the pathogen. as described above, host cytosolic immune sensors, [ ] [ ] [ ] , , , , , as well as other diverse defense molecules, , , can recognize and target either damaged mtb phagosomes or cytosolic mycobacteria for immune clearance. accordingly, a recent work revealed that a host deficient in endosomal sorting complex required for transport, machinery thought to be important for repairing esx- -dependent damage of mycobacteria-containing vacuoles, shows restricted intracellular bacterial growth. in addition, several independent studies using different experimental methods consistently found that the majority of intracellular mycobacteria are not localized in the host cytosol until a very late stage of infection, , , suggesting that mtb may prepensely escape from phagosomes for rapid replication and preparation for further transmission, which occurs only after the host cells are compromised by immune responses that are attenuated after prolonged interaction with the mycobacteria. aside from membrane changes related to phagosomelysosome fusion (and autophagosome formation, which is discussed below), recent studies have indicated that mtb is also involved in the modulation of other cellular membranes. for example, the translocation of the golgi apparatus and lysosome-derived vesicles to the plasma membrane is required for the repair of mycobacteria-induced disruptions of the macrophage plasma membrane, whereas virulent mtb strains are able to prevent this process and induce necrosis of infected cells. in addition, mtb infection has also been associated with the induction of mitochondrial membrane permeability transition (mpt), which causes host cell necrosis. [ ] [ ] [ ] interestingly, pathogenic mycobacteria may also coopt the host autophagic machinery to break through the plasma membrane and depart from their host cells through an f-actin-based vacuolar compartment termed an "ejectosome", which is proposed to be a nonlytic cell-to-cell bacterial transmission mechanism. , furthermore, mtb can alter the protein composition of exosomes secreted by infected human macrophages. these actions indicate that mtb is involved in the host exosomerelated vesicular trafficking pathway, but its significance for tb pathogenesis remains largely unexplored. in conclusion, the success of the intracellular lifestyle of mtb largely depends on the establishment of an easeful niche within a nonfusogenic phagosome. in fact, growing evidence suggests that the phagosome is more likely serving as a signaling platform than as clearance machinery, and mtb is likely to promptly interact with the cellular membrane trafficking system to sense and change the host immune and metabolic conditions. these assumptions, as well as the potential interplay between mtb and other host cellular organelle membranes, warrant further indepth investigations. the development of central necrosis in granulomatous lesions, which induces lung cavity formation and promotes mtb transmission to another human host, is a hallmark characteristic of severe tb cases. hence, mtb-induced host cell death during infection likely plays a crucial role in tb pathogenesis. initially, virulent mtb strains were thought to induce host cell apoptosis in an esx- -dependent manner, as indicated by an in vitro infection model using immortalized murine macrophage cell lines. [ ] [ ] [ ] however, several studies using human macrophage cell lines have indicated that virulent mtb leads to a lower apoptosis rate than attenuated strains [ ] [ ] [ ] and even inhibits apoptosis by employing a wide variety of effector proteins (which are effectively summarized in ref. ) to evade host downstream immune responses. most likely, the integrity of cell deathassociated molecular pathways in certain cell lines accounts for these discrepancies. further investigations suggested that virulent mtb strains can switch the induction of host cell apoptosis to necrosis via manipulation of eicosanoid metabolism pathways. , in contrast to apoptosis, which is proposed to result in the containment of mycobacteria, the propensity of mtb for inducing necrotic death likely benefits the release of bacteria into the permissive extracellular microenvironment they have modulated for better growth. however, a recent study using time-lapse imaging suggested that mtb-induced necrosis predominantly benefits the growth of the bacteria within dead cells, as indicated by the observation of the accelerated intracellular replication of mtb after host macrophage death, which was much faster than it was in either live cells or in the extracellular milieu. in addition, the phagocytosis of dead infected cells containing aggregated mycobacteria by bystander macrophages would cause further necrosis. regardless of the debate on the benefit of necrosis on intra-or extracellular mycobacterial growth, these studies have established the currently accepted concept suggesting that mtb can reprogram host cell death and that it preferentially induces necrosis rather than apoptosis to facilitate its survival and dissemination. more recent studies have pointed out that mycobacteriainduced host cell necrosis is a programmed cell death process, termed "necroptosis", which is stimulated by host tnf via tnf receptor (tnfr ) and is dependent on receptor-interacting serine-threonine kinases (ripk )/ripk . , mtb infection markedly increases mixed-lineage kinase domain-like protein (mlkl), the effector protein in the ripk /ripk -mediated necroptosis pathway, and other pronecroptotic molecules such as tnfr and zbp (ref. , ). however, deletion of mlkl or inhibition of ripk in macrophages does not completely rescue mtb-infected cells from death, , suggesting that, although the deficiency of mlkl or ripk can abrogate the canonical necroptosis pathway, upstream tnf/tnfr -mediated signaling may stimulate the induction of other cell death pathways during mtb infection. alternatively, mtb may bypass the tnf/tnfr /ripk cascade to cause necroptosis, a notion supported by a recent study showing that mtb can secrete a nicotinamide adenine dinucleotide (nad + ) glycohydrolase to induce host cell necroptosis independent of ripk and tnf. furthermore, mlkl-deficient or ripk -inhibited humanized mice exhibited bacterial burdens and lung histopathology indistinguishable from those of the control mice in response to mtb infection. these results imply that, although tnf/tnfr /ripk -dependent necroptosis is activated by mtb, this type of cell death seems to play a restricted role in tb pathogenesis. hence, additional mechanisms underlying mtbinduced host cell death and their association with tb pathogenesis should be taken into account. in addition to those identifying necroptosis, a number of studies have identified multiple other types of programmed necrosis in mammalian host cells in response to mtb infection, such as inflammasome-mediated pyroptosis and neutrophil extracellular trap (net)-associated netosis, which have recently been extensively reviewed. notably, it was reported that mtb inhibited macrophage inflammasome activation and pyroptosis via its secreted effectors zmp and rv c, thus limiting host pro-inflammatory immune responses. , furthermore, netosis seemingly facilitates the interactions between neutrophils and other immune cells rather than killing mtb directly. , more recently, amaral et al. found that mtb-induced macrophage necrosis was characterized by elevated levels of intracellular iron and mitochondrial superoxide. increased lipid peroxidation, and downregulated glutathione and glutathione peroxidase- , findings that are in line with the hallmark characteristics of a typical and regulated necrosis process termed "ferroptosis". using a mouse model of acute mtb infection, the same group confirmed the association between lung necrosis and mtb-induced ferroptosis, which indicated that ferroptosis probably contributes to tb pathology and allows mtb to thrive and spread. , more importantly, treatment with the ferroptosis inhibitor ferrostatin- reduced the bacterial burdens and attenuated pulmonary necrosis in acutely mtb-infected mice, suggesting that the targeting of the host ferroptotic pathway may be a potential strategy to control tb infection and reduce lung damage. in summary, diverse host cell death pathways are involved in mtb infection, acting either as host protective mechanisms or as bacterial survival strategies. notably, the preference for these different cell death modalities likely depends on both the mycobacterial strains and molecular integrity of cell death pathways in a certain host cell type. therefore, identification of and interference with mycobacterial effectors or potential host molecular switches that can control the death modes of infected cells might be a new approach to control tb infection and diminish mtb-caused tissue damage. exploitation of the autophagy process by mtb our knowledge of the physiological and immunological roles of autophagy has recently expanded greatly. autophagy is a cellular mechanism evolutionarily conserved from yeast to mammals that involves the degradation of cellular materials such as damaged organelles, unwanted proteins or foreign pathogens by capturing them in a double-membrane structure termed the "phagophore", which can subsequently develop into a mature autophagosome and fuse with lysosomes. , the protective role of autophagy in host defense against mtb was first investigated by gutierrez et al., who noted that a portion of mycobacteria are sequestered into autophagosome-like compartments during infection in macrophages and that exogenous stimulation to enhance autophagy restricted mtb intracellular survival. a subsequent study confirmed this observation and revealed that while the th cytokine ifn-γ can induce host macrophage autophagy to control mtb infection, the th cytokines il- and il- abrogate such autophagy-mediated killing of intracellular mycobacteria. furthermore, it has been reported that autophagy is involved in regulating other multiple anti-mtb mechanisms, such as the mycobactericidal capacity of the lysosomal soluble fraction, the expression of srs on macrophages, and mycobacterial antigen presentation. taken together, these findings indicate an essential role of autophagy in both host innate and adaptive immunity in mtb infection. more recently, researchers noted that eukaryotic cells could allocate specific cellular materials to the autophagy pathway, which is considered a selective process. host selective autophagy of foreign pathogens is termed "xenophagy". deletion of xenophagy-associated genes leads to significantly enhanced mycobacterial survival in macrophages and in mice, , , , [ ] [ ] [ ] [ ] [ ] further supporting a protective role of autophagy in host anti-mtb immunity. during mtb infection, ubiquitin-ligating (e ) enzymemediated ubiquitin attachment to bacteria is a key step for host initiation of xenophagy, through which various autophagy receptors, such as p (sqstm ), nbr , ndp , and optineurin, are recruited and subsequently engage with autophagosomal membrane-associated protein lc to capture bacteria into autophagosomes. , [ ] [ ] [ ] [ ] [ ] to date, only two e ubiquitin ligases, parkin and smurf , have been found to control ubiquitin targeting of mtb for xenophagy initiation, which was realized through the mediation of k -and k -linked ubiquitination of mtb-associated substrates, respectively. , in addition, a recent study demonstrated that human makorin ring finger protein (mkrn ) is an mtb-specific e ubiquitin ligase that can mediate the ubiquitination of mtb in vitro in conjunction with ubiquitin-activating enzyme e (ube ) and ubiquitin conjugating enzyme e d (ube d ), although its intracellular role during mtb infection has not been illustrated. however, the protein substrates on mtbcontaining phagosomes or mycobacterial surfaces that can be ubiquitinated by these e ligases remain unidentified. parkin −/− mice fail to restrict mtb replication during acute infection, and smurf −/− mice display an attenuated capacity to control mtb infection during the chronic phase, , suggesting that they have different roles in host anti-mtb immunity. apart from e ligase-mediated xenophagy, we recently identified an mtb surface protein, rv c, which can directly bind host cytosolic ubiquitin chains via a eukaryotic-like ubiquitin-associated (uba) domain to recruit autophagy components and trigger a xenophagic response. therefore, both e ligase-dependent and e ligaseindependent mechanisms are involved in host ubiquitin targeting of intracellular mtb for xenophagy initiation. furthermore, it is notable that the host can also drive ubiquitin-independent xenophagy. in salmonella typhimurium-infected cells, host galectin- detects invading bacteria by binding glycans on damaged bacteria-containing vacuoles and further interacts with the autophagy receptor ndp to recruit lc and activate antibacterial autophagy. given that galectins also participate in the cytosolic recognition of mtb-damaged phagosomes, , ubiquitin-independent xenophagy may also occur during mtb infection. in addition, in view of growing eukaryotic-like effectors identified in mtb, , it is not surprising that mtb might retain certain surface proteins that can be directly recognized by new insights into the evasion of host innate immunity by mycobacterium. . . q chai et al. autophagy receptors or lc family proteins via protein-protein interaction motifs to trigger host xenophagy. in response, mtb adopts multiple strategies to avoid autophagyrelated immune clearance during infection, and an effective mechanism involves directly or indirectly targeting autophagy machinery by delivering effector proteins into host cells. for example, mtb-secreted acid phosphatase (sapm) has been found to target host rab to prevent autophagosome-lysosome fusion. another mtb effector, enhanced intracellular survival (eis), which is an n-acetyltransferase that has been reported to increase the acetylation level of histone h to upregulate il- , results in autophagy suppression via the activation of the akt/ mtor/p s k pathway. recently, a host noncanonical autophagy pathway, named lc -associated phagocytosis (lap), was identified in the context of a fungal infection and involved in the recruitment of lc and other components of the canonical autophagy machinery on pathogen-containing phagosomes for lysosomal degradation. notably, lap does not rely the preinitiation complex in ulk signaling, instead requiring rubicon and nadph oxidase (nox ), molecules, which are not involved in the canonical autophagy pathway. interestingly, mtb is insensitive to nadph oxidase and lap trafficking, and nox deficient mice show few differences compared with the control mice in controlling mtb infection. the mtb protein cpsa has been proven to cause autophagy resistance, but its direct target in the host lap pathway remains unclear. interference with host micrornas (mirnas) is another efficient strategy by which mtb disturbs the host autophagy pathway, as shown by mirna often simultaneously targeting multiple interrelated genes, thereby leading to a potent cumulative effect on a certain molecular pathway. mycobacteria can modulate the expression of diverse host mirnas, such as mir- and its passenger strands mir- *, mir- a, mir- , mir- , and mir *, which results in autophagy inhibition through the direct repression of a wide range of key autophagy effectors. , [ ] [ ] [ ] [ ] in addition, we recently found that mtb infection induces the expression of mir- a, the mirna that targets the er-located ca + transporter cacna d to inhibit the downstream calcium-associated xenophagy pathway in the host. taken together, these findings support a prevailing view that autophagy is a host mechanism of intrinsic defense against intracellular bacteria, and under certain circumstances, mtb attempts to adopt it for its own benefit. several recent studies have raised questions about the exact role of autophagy in host-mtb interactions. on the one hand, growing studies support an autophagy-independent role of the autophagy machinery during infection. for example, a study showed that mice lacking atg , atg , atg , atg , or atg l in myeloid cells displayed few differences in bacterial loads compared with those of the control mice during acute mtb infection and argued that host atg -dependent resistance to mtb predominantly depends on its regulatory functions in neutrophilrelated immunopathology rather its function in the autophagy pathway. hence, the multifaceted protective role of autophagyrelated genes in host anti-mtb immunity should be taken into consideration and need to be further characterized. on the other hand, it has been shown that the mycobacterial esx- secretory system is required for activation of the host xenophagy pathway, , which might support the supposition that mtb prevents autophagosome-lysosome fusion at the late stage of infection. , furthermore, by monitoring autophagosome formation and subsequent degradation of autophagic cargo (a process termed autophagy flux) in infected cells, a research group found that virulent mtb strains selectively prevented autophagosomes from fusing with lysosomes, while the autophagosomes that did not contain mtb developed normally. , these findings imply that mtb has probably adapted to persist in autophagosomal vacuoles by inhibiting their degradation, which means it creates a sheltered environment for prolonged intracellular survival. moreover, mtb appears to selectively prevent xenophagic flux rather than the entirety of autophagic flux in host cells, which would likely result in hyperinflammatory responses and cell death. these hypotheses are supported by our finding that cytosolic mtb can induce autophagy recognition and activation via a highly conserved ubiquitin-binding associated (uba) domain on its surface to avoid excessive host inflammatory responses. consistently, it has also been reported that, in a certain case, xenophagy can be beneficial for mtb replication. notably, the host autophagy pathway has been proposed as a potential target for host-directed anti-tb therapy, and based on these new concepts, a promising candidate of drugs or agents is expected to selectively target mtb-containing autophagosomal vacuoles rather than cause nonselective overall interference in host autophagic flux. in addition, these drugs should not only enhance autophagy activation but also overcome the mtbinduced blockade of autophagosome-lysosome fusion. novel mechanisms by which mtb targets innate immune regulatory machinery the increase in the number of studies has tremendously expanded our understanding of multifaceted molecular mechanisms by which mtb modulates the host immune regulatory network for its own advantage. in this section, we discuss the newly elucidated strategies adopted by mtb to manipulate the host regulatory machinery of cellular innate and intrinsic immune responses via direct host-pathogen molecular interactions (fig. ) . mtb targeting of intranuclear immune regulatory machinery nucleus targeting has been emerging as a new aspect of the regulatory mechanism adopted by bacterial pathogens to manipulate host cell physiology and subvert immune defenses. in particular, an increasing number of bacterial effectors have been found to enter the infected cell nucleus to hijack host nuclear processes, and these nuclear attackers are named "nucleomodulins". bacterial nucleomodulins may mimic eukaryotic transforming factors, transcription factors, chromatinregulatory factors or posttranslational modifiers, intervening in host gene transcription, chromatin reorganization, rna processing or dna replication and repair. recent studies have identified several mycobacterial nucleomodulins that exert a range of intranuclear regulatory functions, which are described below. first, some mtb nucleomodulins function as histone-modifying enzymes to engage in epigenetic regulation of host immune responses. histone modification probably plays an essential role in the regulation of host anti-mtb immunity, since inhibition of histone deacetylases (hdacs) in human monocytes leads to attenuated host immune clearance of mtb. , in addition, suppression of hdacs decreases matrix metalloproteinase- and - in mtb-infected macrophages, whose proteins drive tb lung immunopathology. furthermore, histone methylation and acetylation are closely associated with bcg-induced host-trained immunity against mtb. , pulmonary tb patients undergo obvious changes in histone modification in blood leukocytes; similarly, individuals with clinical resistance to mtb infection (known as tb resisters) display an altered expression pattern of genes related to histone modification in blood monocytes. to date, three mtb effectors that target and modify host histones have been identified: eis, rv , and rv . . as previously described, mtb eis increases the acetylation level of histone h to regulate host autophagy activation during infection. mtb rv localizes to the host chromatin during infection, serving as a functional methyltransferase that dimethylates an arginine residue at h r to repress a range of host genes involved in reactive oxygen species (ros) production, such as nox , nox , and noxa and nitric oxide synthase (nos ). although the significance of rv on mtb pathogenesis has not been identified, the expression of rv in nonvirulent m. smegmatis new insights into the evasion of host innate immunity by mycobacterium. . . q chai et al. markedly enhanced bacterial survival in infected mice. mtb rv . was isolated from the chromatin of mtb-infected human macrophages where it displayed histone acetyltransferase activity and targeted host h k and h k (ref. ). similarly, recombinant m. smegmatis rv . exhibited advanced intracellular survival in macrophages. second, mtb nucleomodulin rv c was identified as a methylcytosine-specific dna methyltransferase that participates in the methylation of host genomic dna primarily at non-cpg cytosines upon infection. however, the immunomodulatory role of rv c in host-mtb interactions has not been clarified. despite limited knowledge of the pathogenic contribution of mtbinduced host dna methylation changes, in mtb-infected macrophages, hypermethylation was predominantly observed on genes related to host immune responses, such as nlrp inflammasome activation and pro-inflammatory cytokine production. [ ] [ ] [ ] these characteristics have been consistently observed in blood monocytes isolated from tb patients. in addition, blood monocytes from bcg-vaccinated individuals also displayed a different dna methylation pattern and advanced capacity for mycobacterial control, indicating the involvement of dna methylation in hosttrained immunity against mtb. third, some mtb protein effectors exhibit dual regulatory functions that not only target host cytosolic components but also mimic eukaryotic transcription factors involved in host intranuclear processes. for instance, the mtb secretory protein ppe was found to directly interact with the host cytosolic subunit of nadph oxidase, p phox , via an sh -like domain to inhibit ros production and favor intracellular survival of mtb in macrophages. intriguingly, ppe also contains a eukaryotic-like nuclear localization signal (nls), by which it can be translocated into the host nucleus via the classical importin α/β pathway. thereafter, ppe binds to the nos promoter and limits host ros production. in another example, early studies have demonstrated that mtb ptpa is delivered into the host cytosol, where it directly targets the vacuolar-h + -atpase machinery to inhibit phagosome acidification and the nf-κb pathway to suppress host inflammatory immune responses. , moreover, we recently found that mtb ptpa can also enter the nucleus of infected cells, where it binds to and modulates the expression of diverse host genes, such as gadd a, to affect cell proliferation and migration. the host nucleus plays a central role in governing the all cellular activity, through which both genetic and epigenetic regulation of host immune responses to mtb are driven. however, our understanding of the mechanistic and pathological implications of mtb-hijacked intranuclear processes in the host remains limited. for example, it remains unclear how mtb spatiotemporally regulates the intra-and extranuclear functions of these nucleustranslocated effectors. furthermore, the majority of the identified nucleomodulins do not contain a classic nls or nuclear export signal; what is the mechanism by which they shuttle between the mtb targets the host ubiquitin system the ubiquitin system refers to a network of proteins comprising enzymes that engage in ubiquitination and deubiquitination of cellular targets and ubiquitin receptors that decipher the ubiquitin code and translate it into cellular responses. this elaborate system regulates a wide range of cellular immune responses and plays a vital role in host-pathogen interactions. furthermore, it was recently reported that another mtb-secreted virulence factor, lpqn, directly interacts with the human e ubiquitin ligase cbl, which plays a regulatory role in cell-intrinsic responses to infection. intriguingly, mycobacteria possess pupylation, the covalent modification of protein lysine residues with a ubiquitin-like protein called pup, but not ubiquitination as in eukaryotic cells. our recent study noted that to efficiently interfere with host immunity, mtb not only simply inhibits ubiquitin ligase-mediated immunomodulatory functions but also subtly exploits the host ubiquitin system for its own advantage. we found that, by direct interaction with ubiquitin via a unique ubiquitin-interacting motif-like region, mtb ptpa is activated to dephosphorylate host p-jnk, p-p , and p-vps b, leading to suppression of innate immune responses. similarly, we verified that another mtb ubiquitin-binding protein, rv c, resides on the bacterial surface, as mentioned above, and it directly recruited host cytosolic ubiquitin to trigger xenophagy to restrict host inflammatory responses. more recently, we identified a mtb-secreted protein effector, rv , as a key suppressor of host nf-κb activation, showing that it undergoes k -linked ubiquitination mediated by the host e ubiquitin ligase anaphase promoting complex (apc) subunit (anapc ). interestingly, rather than inducing the apcmediated canonical ubiquitin-proteasome degradation pathway, k -linked ubiquitination of rv facilitates the interaction between src homology region domain-containing phosphatase- and its adapter protein traf , which blocks the k -linked ubiquitination and activation of traf , leading to inhibition of the nf-κb signaling pathway. in conclusion, targeting the host ubiquitin system is a recently emerging aspect of the tactics mtb uses for immune evasion, which has received growing attention. curiously, growing evidence suggests that the ubiquitin system is often coopted by invading pathogens and then plays an altered regulatory role in host immune responses. future research will continuously expand our understanding of the ubiquitin system at the interface of host-mtb interactions, particularly the undefined roles of host-originated and mtbmimicking e ubiquitin ligases, deubiquitinases, and ubiquitin receptors. mtb targets intrinsic cellular immune components mtb has evolved to secrete a wide range of protein effectors via its sophisticated esx secretion systems to counter host immunity. , in particular, growing numbers of mycobacterial effectors have been linked to direct protein-protein interactions with the host to target and modify key cellular intrinsic antibacterial machinery. for example, it has been found that mtb encodes eleven eukaryote-like serine-threonine protein kinases, including pkna to pknl (but not pknc), and two tyrosine phosphatases, ptpa and ptpb. among these proteins, pkng is likely to selectively downregulate host pkc-α to inhibit the biogenesis of phagolysosomes. ptpa dephosphorylates host p-vps b, p-jnk, and p-p as described above, inhibiting phagosome acidification and the production of tnf and il- β in macrophages , ; ptpb decreases the phosphorylation of host p , ikkα, erk / , and p , suppressing macrophage apoptosis and the secretion of inflammatory cytokines. , both ptpa and ptpb are indispensable for mtb intracellular survival. , although the host substrates of these mtb eukaryotic-like kinases/phosphatases remain largely unknown, their essential roles in mtb virulence have been well documented. , apart from phosphorylation-associated regulation of host factors, it was found that mtb eis can target and acetylate mitogen-activated protein kinase phosphatase- to prevent host jnk-dependent immune responses. in addition, in our recent work, we revealed an mtb disulfide-bond-forming-like protein, mpt , that can directly oxidize thiols on tak to facilitate tak -mediated host hyperinflammatory immune responses. in contrast to the abovementioned cellular factors that control pathogen infection indirectly through the activation of signaling cascades followed by innate immune responses, some other host proteins are constitutively expressed in certain cell types and directly act to restrict pathogen growth, and they are termed "restriction factors". cellular restriction factors provide a frontline defense against invading microorganisms in a system known as host "intrinsic immunity"-a form of innate immunity initially elucidated in hosts as a mechanism to control viral infections. as discussed before, a range of antiviral immune mechanisms, such as cytosolic immune surveillance and the type i ifn response, are also involved in the host control of mtb infection, indicating that the host might adopt certain shared cellular immune machinery, which may include the similar restriction factors, upon infection by viruses and bacteria. for example, ifn-induced transmembrane (ifitm) family proteins are well-characterized host antiviral restriction factors critical for controlling the entry and intracellular replication of viral pathogens, which has recently been associated with host anti-mtb defense mechanisms as well. specifically, iftm , iftm , and iftm are required for the host restriction of mtb intracellular growth in both human macrophages and lung alveolar cells, among which iftm was shown to colocalize with mtb phagosomes and contribute to phagosomal acidification. another group of host intrinsic antiviral restriction factors, tripartite motif proteins (trims), have also been demonstrated to engage in the host control of mtb infection. , , in turn, these host restriction factors may be targeted by mtb for immune evasion. as discussed above, mtb lpqn is able to interact with host cbl, which is a restriction factor that regulates the balance between cellular intrinsic antibacterial and antiviral responses. similarly, mtb ptpa can directly bind host trim to antagonize its intrinsic immune functions. furthermore, it was reported that trim is recruited to mtb phagosomes in macrophages to act as a negative regulator of host cytosolic dnasensing pathway-dependent mycobacterial restriction. together, these findings suggest potential strategies utilized by mtb to avoid host intrinsic immunity. despite the compelling findings supporting an essential role for various cellular intrinsic protein factors in host anti-mtb immunity, the determinant molecules of host resistance to tb infection new insights into the evasion of host innate immunity by mycobacterium. . . q chai et al. remain largely unexplored. the application of recently developed research methods, such as genome-wide association analysis of human tb patients, may help to reveal the genetic etiology of tb and to identify key anti-mtb intrinsic immune components. furthermore, there is still a limited understanding of the direct interactions between mtb-secreted proteins and host proteins, which play central roles in tb pathogenesis. thus, more studies based on valid screening systems, such as the affinity tag purification mass spectrometry system, the mycomart transposon system, and the crispr-cas screening system, , , are warranted to further improve our understanding of the mtb-host network of molecular interactions. our understanding of the interplay between mtb and the host innate immune system has extensively expanded in recent years. as summarized in this review, upon mtb infection, various cellular antimicrobial components respond to the activation of host innate immune surveillance pathways, which might be modulated by mtb for its benefit. moreover, an increasing number of studies have revealed emerging mtb strategies to exploit the host molecular regulatory machinery of the innate immune system, including mtb-mediated disruption of the host intranuclear immune regulatory machinery, the ubiquitin system and intrinsic cellular immune components. thus, recent research on host-mtb interactions has changed the traditional view that the pathogen is incompatible, and in conflict with its host until one is overwhelmed. as a particularly successful intracellular pathogen, mtb has evolved much more moderate and nuanced strategies for immune modulation and evasion, with the principal aim of adapting to an intracellular niche for prolonged survival, rather than simply destroying the host. therefore, it is not surprising that some mycobacterial factors have an inhibitory effect on host cellular antibacterial mechanisms (e.g., interfering with protective th -type cytokine production, vacuolar membrane trafficking, or autophagy activation), while others appear to play an opposite regulatory role. in fact, host immune responses are spatiotemporally regulated and dynamically changed throughout the course of tb. , therefore, mtb probably tends to employ distinct effectors at different stages to bilaterally modulate the host immune machinery to establish a successful long-term infection. this concept is supported by compelling evidence indicating that, while an early protective th -type response favors a host-controlled infection, the machinery is often suppressed or exploited by mtb, for example, to induce hyperinflammation at the late stage of infection, which causes lung cavitation and thus benefits bacterial transmission. therefore, more in-depth studies are warranted to 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kappab/beclin pathway trims and galectins globally cooperate and trim and galectin- co-direct autophagy in endomembrane damage homeostasis trim is a key regulator of the type i interferon response during mycobacterium tuberculosis infection genome-wide association study identifies two risk loci for tuberculosis in han chinese comprehensive identification of conditionally essential genes in mycobacteria improved vectors and genome-wide libraries for crispr screening inflammatory signaling in human tuberculosis granulomas is spatially organized spatial and temporal localization of immune transcripts defines hallmarks and diversity in the tuberculosis granuloma ecology and evolution of mycobacterium tuberculosis competing interests: the authors declare no competing interests.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- -gsvswr v authors: hedblom, grant a.; reiland, holly a.; sylte, matthew j.; johnson, timothy j.; baumler, david j. title: segmented filamentous bacteria – metabolism meets immunity date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: gsvswr v segmented filamentous bacteria (sfb) are a group of host-adapted, commensal organisms that attach to the ileal epithelium of vertebrate and invertebrate hosts. a genetic relative of the genus clostridium, these morphologically unique bacteria display a replication and differentiation lifecycle initiated by epithelial tissue binding and filamentation. sfb intimately bind to the surface of absorptive intestinal epithelium without inducing an inflammatory response. rather, their presence impacts the generation of innate and differentiation of acquired immunity, which impact the clearance of extracellular bacterial or fungal pathogens in the gastrointestinal and respiratory tracts. sfb have recently garnered attention due to their role in promoting adaptive and innate immunity in mice and rats through the differentiation and maturation of th cells in the intestinal tract and production of immunoglobulin a (iga). sfb are the first commensal bacteria identified that impact the maturation and development of th cells in mice. recently, microbiome studies have revealed the presence of candidatus arthromitus (occasionally designated as candidatus savagella), a proposed candidate species of sfb, in higher proportions in higher-performing flocks as compared to matched lower-performing flocks, suggesting that sfb may serve to establish a healthy gut and protect commercial turkeys from pathogens resulting in morbidity and decreased performance. in this review we seek to describe the life cycle, host specificity, and genetic capabilities of sfb, such as bacterial metabolism, and how these factors influence the host immunity and microbiome. although the role of sfb to induce antigen-specific th cells in poultry is unknown, they may play an important role in modulating the immune response in the intestinal tract to promote resistance against some infectious diseases and promote food-safety. this review demonstrates the importance of studying and further characterizing commensal, host-specific bacteria in food-producing animals and their importance to animal health. the distal gastrointestinal tract of all animals is colonized by a diverse array of bacterial, fungal, and protozoan species. an animal host maintains a mutualistic relationship with its microbial inhabitants in which the microbes provide protection from pathogenic bacteria through competing for ecological niches and fostering a stable environment for the development of host immunity (bäckhed et al., ) . many vertebrate intestines (such as mice, rats, chickens, humans, and turkeys) harbor commensal organisms named segmented filamentous bacteria (sfb) that bind specifically to the host intestinal epithelium. these organisms are closely related to the genus clostridium, and appear as long, segmented filaments that bind tightly to the host epithelium via a specialized structure (chase and erlandsen, ) . these bacteria were initially detected through microscopic examination of the gastrointestinal epithelium of mice (davis and savage, ) . sfb drew the attention of researchers due to their unique morphology, life cycle and binding location (schnupf et al., ) . since their discovery, a large body of research has been generated to characterize these bacteria and understand their role in the host-microbiome relationship. through examining mouse and rat models, it has been revealed that these bacteria play an important role in adaptive and innate immunity of the host. segmented filamentous bacteria are gram-positive, sporeforming bacteria that possess the capability to develop into long filaments, which are divided via the production of transverse septa (ericsson et al., ) . these bacteria exist in a developmental or vegetative form, which are characterized by the presence of intrasegmental bodies and spores, respectively (figure ) . sfb produce intrasegmental bodies, which can appear as either a spore or a holdfast. the distinction between these morphologies indicates that the holdfast form serves as the vegetative form, while the spore serves as the dormant form (chase and erlandsen, ) . segmented filamentous bacteria selectively attach to the ileal epithelium of the host species via the production of rounded, nipple-like projections called holdfasts. the holdfast serves as an anchoring mechanism and point of filament elongation, attaching to host enterocytes in the mucous membrane of the figure | gram stain of candidatus arthromitus from turkey ileum containing intrasegmental bodies ( , × magnification) (reiland, ) . epithelium, without penetrating the host cell wall (sanford, ) . this structure leads to the displacement and destruction of the intestinal microvilli surrounding the point of attachment (snellen and savage, ) , and leads to alterations in the electron density of both the host cell plasma membrane and apical cytoplasm (ericsson et al., ) . once attached, actin polymerization occurs directly underneath the holdfast structure and creates a pedestal-like formation similar to the adherence mechanism of salmonella enterica serovar typhimurium (jepson et al., ; ericsson et al., ) . though this attachment mechanism resembles that of pathogenic bacteria, it does not damage the host cell and causes no inflammatory response in the lamina propria (caselli et al., ) . once attached to the host epithelium, the filament begins to extend from the distal end, releasing additional holdfasts and spores from the maturing filament (chase and erlandsen, ) . the life cycle of an sfb filament is assumed to be about - days based on the rapid shedding of the intestinal epithelium of rodents, and longitudinal studies have shown that sfb appear in juvenile mice that are around days in age (davis and savage, ) . at this stage of development, sfb proliferate to become a dominant gut microbe and then recede in mature vertebrates to lower levels. during the early stages of colonization, sfb are transiently colonized with rod-shaped bacteria (schnupf et al., ) . it is believed that the spread of sfb then occurs via vertical transmission of spores from parents to offspring, as these bacteria are widely considered to be obligate anaerobes and have also appeared in the intestinal tissue of weaning mice (schnupf et al., ) . colonization occurs in mouse and rat hosts at the onset of the weaning process and has been found to be the same in arbitrary human microbiome studies (ericsson et al., ) . studies performed on human subjects ages months to years old revealed that % of individuals carry sfb in their gut from ages - months, % carry sfb from ages - months, and only . % carry sfb from ages - (yin et al., ) . the age-related drop in sfb intestinal carriage may be pharmaceutically reversed. for example, transient feeding mice rapamycin enhanced their lifespan, while dramatically increasing the prevalence of sfb in the small intestine (bitto et al., ) . in chickens, sfb colonization peaked at approximately weeks of age, and decreased as they aged to weeks of age. the decrease was inversely proportional to the amount of intestinal iga present (liao et al., ) . it is unknown whether increasing the prevalence of sfb in the intestinal tracts of adults is beneficial, or may result in autoimmunity (e.g., rheumatoid arthritis) (wu et al., ) . segmented filamentous bacteria spores germinate in the host's gut to produce teardrop-shaped, single-celled bacteria referred to as intracellular offspring (schnupf et al., ) . amongst intestinal commensals and symbionts, sfb are unique because they penetrate the intestinal mucus layer and intimately associate with host cells without invading the host (chase and erlandsen, ; sanford, ) . it is assumed that the intracellular offspring use flagella to reach the apical surface of polarized epithelium (kuwahara et al., ) . though cellular flagella have not been observed microscopically, an analysis of a sfb genome revealed a full set of chemotaxis and flagellin biosynthesis genes, strongly suggesting the presence of flagella in the early stages of spore maturation (kuwahara et al., ) . intracellular offspring attach to absorptive epithelial cells via their holdfast and induce condensed actin rearrangements underneath the point of attachment while displacing some of the neighboring microvilli structures (chase and erlandsen, ) . before luminal attachment, the nucleoid region of the intracellular offspring appears condensed, suggesting reduced amounts of transcription. however, when the intracellular offspring attaches to the host, the nucleoid region decondenses and allows for genomic transcription (chase and erlandsen, ) . holdfast attachment to enterocytes in the terminal ilea causes the intracellular offspring to increase in size, reaching up to µm in length before bacterial division commences through transverse septum formation (chase and erlandsen, ) . filaments continue to grow and divide from their distal end, reaching their maximum length of around - µm (martin et al., ; schnupf et al., ) . once the filament reaches its maximum length, a second round of symmetric division begins from the distal end to divide each original segment in half into secondary undifferentiated cells. these secondary cells range in length from to . µm, forming segments containing - cells (chase and erlandsen, ) . after elongation, the filament will often separate from the holdfast segment and enter into the ileum. these secondary segments then undergo differentiation form a mother cell and a daughter cell (klaasen et al., ) . differentiation of these filaments appears to be more pronounced in the presence of slightly aerobic conditions, when oxygen concentrations range from to . % environmental oxygen (schupf et al., ) . once differentiated, the mother cell engulfs and houses the daughter cell, where the daughter cell undergoes division into two intracellular offspring (klaasen et al., ) . the intracellular offspring contained within the mother cell are then subject to two fates, dispersal from the filament or sporulation (davis and savage, ; klaasen et al., ) . if favorable growth conditions are present, the septa that separate individual mother cells are degraded to form a tube in which the intracellular offspring are dispersed into the host's intestinal tract. these released offspring are then allowed to colonize additional host tissue and undergo filamentation and differentiation, thus completing the sfb lifecycle (martin et al., ; schnupf et al., ) . when an unfavorable or hostile environment is presented, the two intracellular offspring produce a single spore coat that covers both of the cells. once coated with the layer of peptidoglycan, the spore matures into a complete endospore inside of the mother cell, and is released from the filament (martin et al., ; schnupf et al., ) . these spores lack the ability to colonize the host until favorable environmental conditions, such as appropriate concentrations of oxygen are presented, and shed in the host's feces. once shed, the spores can be transmitted to another host via horizontal transmission (davis and savage, ) . though it was previously proposed that the entire sfb lifecycle occurred while attached to the host tissue (chase and erlandsen, ) , filaments containing intracellular offspring have not been observed in published tem and sem images of filaments attached to enterocytes (caselli et al., ) . alternatively, filaments containing differentiated intracellular offspring only appeared as detached and free-floating, indicating that maturation and differentiation of segments occur independently from host tissues. these filaments are separated from the holdfast segment, which then penetrates the epithelium until it undergoes endocytosis, phagocytosis, or transcytosis (caselli et al., ) . the ingestion of the holdfast segment presents a great number of bacterial antigens to antigen presenting cells and lymphocytes contained within the ileal epithelium (caselli et al., ) . in rare cases, a small number of segments may remain attached to the holdfast, but these segments often exhibit irregular morphologies and present enlarged intrasegmental junctions (caselli et al., ) . the genome of a rat-isolated sfb and a number of mice sfb isolates have recently been sequenced and published (kuwahara et al., ; prakash et al., ; sczesnak et al., ; pamp et al., ) . these sfb genomes are highly similar but do contain several species-specific genes of unknown function that may be involved in the species-specificity of sfb colonization (prakash et al., ) . all sequenced sfb have displayed a small genome size of . - . mbp, low g+c content ( . %), and around , - , protein encoding genes (prakash et al., ) . sfb possess a highly reduced genome similar to their genetic relatives within the genus clostridium (ericsson et al., ) . the biosynthetic pathways of most amino acids, vitamins and cofactors (such as b , b , and b , pyridoxine, nicotinamide, pantothenate, and biotin) are incomplete or absent altogether in sfb genomes (kuwahara et al., ; sczesnak et al., ; pamp et al., ) . sfb are also unable to synthesize nucleotides independently; instead they utilize alternative pathways that rely on the uptake of nucleotide bases (prakash et al., ) . to obtain nucleotides, amino acids, and peptides from the environment, sfb genomes contains genes encoding two extracellular nucleases as well as a list of proteases and peptidases, of which are membrane associated and to that are thought to be secreted (kuwahara et al., ; sczesnak et al., ) . in addition, sfb genomes contain numerous open reading frames (orfs) thought to encode a large number of transporters and permeases for small molecules and ions (such as amino acids, oligopeptide, dipeptides, manganese, zinc, iron, and phosphate) compared to other organisms with small genomes (sczesnak et al., ) . a particularly strong requirement for iron uptake was noted by sczesnak et al., since six different orfs for iron transporters are found in the mouse genome as well as three orfs for ferric iron regulator family proteins (sczesnak et al., ) . sfb also have several orfs for phosphotransferase systems predicted for uptake of sugars such as mannose, cellobiose, mannitol, and fructose (prakash et al., ; sczesnak et al., ) . finally, the sfb genomes contain genes for the non-oxidative pentose phosphate pathway and a complete glycolysis pathway to convert glucose to pyruvate, but are deficient for genes encoding almost all components of the krebs cycle, which is required for aerobic respiration (prakash et al., ; sczesnak et al., ) . however, sfb can tolerate small concentrations of oxygen and counteract oxidative stress, as sfb genomes contain genes predicted for two catalases, a peroxidase (rubrerythrin), and an arginase, which might limit nitric oxide production through catabolism of arginine (kuwahara et al., ; pamp et al., ) . these protective mechanisms are likely essential, given the replicative niche of sfb at the surface of the small intestinal epithelium where the oxygen tension is estimated to be around . % (he et al., ) . there are several factors that have been discovered about sfb that explain their auxotrophic nature. the genome of sfb isolated from a rat host (rat-yit) contains putative genes predicted to encode for proteases and for peptidases along with many other genes through to be involved with sporulation and germination (prakash et al., ) . peroxidase and catalase genes were also found, which explains the potential for sfb to exist in microaerophilic environments (prakash et al., ) . the genomes of sfb sequenced from mice and rat hosts revealed several clustered regularly interspaced palindromic repeat (crispr) loci, which serve as a prokaryotic defense mechanism, indicating that sfb genomes may have had exposure to invading dna throughout their evolutionary history (prakash et al., ) . flagellar, pilus, and chemotactic genes have been found in sfb genomes that suggest motility, which explains the organism's ability to penetrate the mucus layer lining of intestinal epithelial cells (prakash et al., ; schnupf et al., ) we have generated a representation of the metabolic and physiological predicted capabilities inferred from the genome contents of publicly sequenced genomes of sfbs (figure ). an extensive body of work has evidenced the presence of sfb in a large number of animal species, such as horses, cattle, pigs, turkeys, chickens and even humans (ericsson et al., ) . in nearly all of the mammalian species studied for the presence of sfb, the bacteria selectively colonize the ileum of the host, with the exception of fish species that lack a welldefined ileum (ericsson et al., ) . in poultry, sfb colonizes ileum (figure ) , the cecal tonsil or cecum (figure ; rahimi et al., ; bohorquez et al., ; liao et al., ) . attempts to colonize an animal species with sfb from another species have been unsuccessful. when germ-free mice and rats were inoculated with ileal homogenates containing sfb from both species, animals became colonized with sfb from their own species, indicating that sfb are host-specific and host selective (tannock et al., ) . host-specificity of sfb may be due to differences in the sequences of flagella genes flic and flicc , which show greater variability than flic and flic (chen et al., ) . it is not known with which host proteins the sfb flagella proteins interact to become adherent, however, sfb flagellar proteins may induce th cells by signaling through toll-like receptor (tlr ) in a subset of cd c hi cd b hi intestinal dendritic cells (uematsu and akira, ). for most animal species, holdfast cells have the capability to attach to goblet cells, m-cells, absorptive enterocytes, and cellular junctions of the ileal epithelium (meyerholtz et al., ) , whereas less is known for poultry. species to species variance exists in the preferred cell of attachment. for example, sfb in rats and figure | metabolic features of candidatus arthromitus through inference of genome contents from annotated genomes through use of rapid annotation using subsystem technology (reiland, ) . frontiers in microbiology | www.frontiersin.org pigs attach to follicle-associated epithelial cells (over peyer's patches) and absorptive ileal villi (tannock et al., ) . in mice and horse, attachment occurs primarily to follicle-associated epithelial cells and mainly to absorptive ileal villi for rabbits, cattle and canines . sfb are unique amongst intestinal commensals and symbionts because they penetrate the intestinal mucus layer and intimately associate with host cells, but do not invade the host (sanford, ) . the exact mechanism of host specificity remains unclear, but it is believed that initial binding of the holdfast segment to the host epithelium serves as a ligand-receptor interaction, triggering a response from the host (kuwahara et al., ) . sfb binding elicits actin polymerization and condensation at the point of attachment (chase and erlandsen, ) , suggesting a specific host response. host specificity of sfb suggests that these bacteria have coevolved with their hosts to promote the commensal relationship that exists between the two species. though initially considered to be common commensal members of the host microbiota, recent research suggests that sfb serve an important role in modulating host microbiome and immunity. sfb are unique amongst intestinal commensals and symbionts because they penetrate the intestinal mucus layer and intimately associate with host cells without invading the host (chase and erlandsen, ; lee et al., ; mortha et al., ; atarashi et al., ; bunker et al., ; farkas et al., ; furusawa et al., ; sadler et al., ) . the holdfast binding of sfb to the ileal mucosa does not elicit a strong inflammatory response (klaasen et al., ) . several interactions between the host and sfb have been investigated, indicating almost entirely positive associations between the sfb and the host, with rare exceptions. intestinal colonization of sfb in rainbow trout may produce a fatal disease called rainbow trout gastroenteritis (rtge), where sfb expand to large numbers in the intestinal tract and cause enterocytes to detach (del-pozo et al., ) . sfb were not always present near to all intestinal rtge lesions, suggesting that sfb affects intestinal barrier function and other bacteria may not be crucial for producing rtge lesions. experimental infection of susceptible rainbow trout stock with feces from rtge animals induced colonization and characteristic histopathological lesions containing sfb and an unidentified gram-negative coccus, suggesting that sfb, in part, may be the etiological cause of rtge (mccarthy et al., ) . recent advances have identified different methods to culture sfb in vitro, accelerating research to study the interaction of sfb with different members of the intestinal microbiota (ericsson et al., ; schupf et al., ) . a large amount of research has demonstrated the indispensable role that sfb play in the maturation of the host gut immune barrier, inducing both innate and adaptive immune responses (ivanov et al., ; sonnenberg et al., ; mortha et al., ; atarashi et al., ; bunker et al., ; farkas et al., ; furusawa et al., ; edelblum et al., ; schnupf et al., ) . immune modulation of sfb may extend beyond the intestinal tract in the blood, or to other mucosal barriers (mcaleer et al., ) . mice colonized with sfb were more resistant to sepsis secondary to experimental cecal ligation and puncture injury (cabrera-perez et al., ) . oral vancomycin treatment to mice diminished the number of intestinal grampositive bacteria (including sfb), which negatively impacted anti-fungal th immunity in the respiratory tract (mcaleer et al., ) . these data suggest that the composition of intestinal microbiota, especially sfb, is vital for impacting immunity to bacterial and fungal pathogens beyond the intestinal tract. segmented filamentous bacteria are best known for their ability to induce the differentiation of naïve cd + t cells to form antigen-specific th cd + cells (schnupf et al., ) in the terminal ileum of mice . ivanov et al. first demonstrated that conventionally raised b mice purchased form taconic farms were highly colonized with sfb; these bacteria were absent from conventional raised mice purchased from the jackson laboratory (ivanov et al., ) . introduction of sfb to b mice from the jackson laboratory induced il- a and il- production from intestinal cd + t cells, which became refractory to colitis induced by the intestinal pathogen citrobacter rodentium (ivanov et al., ) . il- is a cytokine that enhances production of antimicrobial peptides from intestinal epithelial cell and prevent bacterial pathogens from inducing attaching and effacing lesions (schupf et al., ) . sfb are not the only bacteria capable of inducing th cd + t cells in the intestinal tract of animals. virulent shiga-toxin producing e. coli (o ) and citrobacter rodentium induced a th response in the murine intestinal tract, and was dependent on bacterial adherence to host cells (atarashi et al., ) , as well as the commensal bifidobacterium adolescentis (tan et al., ) . th cells are a subset of cd + t cells that are distinguished by the expression of t cell receptor cd , nuclear transcription factor rar-related orphan receptor gamma t (rorγt) and production of interleukins il- a, il- f, il- , and il- (schnupf et al., ) . other immune cells present in the intestinal lamina propria are capable of secreting il- a and il- or express rorγt [e.g., innate lymphoid cells type (ilc ) and lymphoid tissue inducer-like cells (lti)], but these cells lack expression of cd (sonnenberg et al., ; mortha et al., ) . however, ilc and lti are not dependent on sfb for their induction. in the intestinal epithelium, il- a and il- f help to modulate neutrophil chemotaxis through producing cxcl chemokines via binding interactions with il- receptors il- ra and il- rc. il- a and il- f additionally aid in regulating the activation and differentiation of host neutrophils, and stimulate the production of host-defense peptides (schnupf et al., ) . systemic depletion of neutrophils in mice caused increased production of il- a and ileal sfb colonization (flannigan et al., ) . thus, neutrophil recruitment may lessen il- a and chemokine production, and serve as a negative feedback loop to limit sfb colonization. the regulation of these immunestimulatory compounds and cell types is essential in combatting intestinal colonization and infection from microorganisms. th cells provide colonization resistance to other pathogenic bacteria present at mucosal barriers, such as escherichia coli in the intestinal tract (zheng et al., ; edelblum et al., ) and respiratory fungi (mcaleer et al., ) . in newborn or germ-free mice, the presence of th cells in the lamina propria is rare, appearing only after colonization by microbes (gaboriau-routhiau et al., ) . the role of sfb in th cell production was initially demonstrated when mice were inoculated with mouse, rat, and human microbiota containing bacterial spores similar to that of the genus clostridium. only the experimental mice inoculated with a mouse-derived bacteria were shown to produce th cells in response to colonization. mice colonized with rat-and human-derived bacteria produced much less of a th response when compared to the mousederived microbiome treatment, indicating host-specific bacteria (such as sfb) as the causative agent of the immune response (gaboriau-routhiau et al., ; chung et al., ) . this association was also confirmed when s rrna sequencing was performed on the gut microbiome of mice presenting ileal th cells, revealing the presence of sfb (ivanov et al., ) . in experiments testing the reactivity of mouse lamina propria against a sfb expression library, two proteins of unknown function elicited a th cell response (yang et al., ) . it was predicted that these unknown proteins may serve as cell surface proteins, potentially elucidating the role that sfb attachment may serve in stimulating host immunity (yang et al., ) . proteins from sfb, secreted or bacterial-associated, are believed to interact with host cells and modulate immunity include adpribosyltransferases and a myosin-cross reactive antigen . the exact antigen presenting cell responsible for immune modulation by sbf is controversial, but it appears that sfb antigens presented to both intestinal macrophages or cd + intestinal dendritic cells (goto et al., ) are involved. because of the intimal relationship of sfb with intestinal epithelium, it is possible that metabolites from sfb may also impact the differentiation of th cells. intestinal macrophages, and not intestinal dendritic cells, appear to be vital for generating sfb-specific th responses in the murine ileum . analysis of the t cell receptor repertoire of th cells recognize peptide antigens produced by sfb (yang et al., ) . the addition of the th -indcing bacterial pathogen listeria monocytogenes failed to impact induction of th cells in sfb colonized mice (yang et al., ) , suggesting that the match of t-cell effector function with antigen specificity is driven by the type of bacteria that produce the antigen. th cell differentiation is additionally mediated through the production of serum amyloid a (saa) and reactive oxygen species (ros) produced in response to sfb binding. production of saa in the host epithelium is initiated by sfb binding and subsequent actin rearrangements, leading to a signal amplification via il- and ilc , both of which aid in th cell differentiation (schnupf et al., ) . saa also stimulates intestinal antigen presenting cells to secrete il- , which assists in th activation and survival (schnupf et al., ) , but il- also has as an antagonistic effect on development of th immunity (shih et al., ) . ros produced as a consequence of sfb binding to enterocytes helps to create a chemical environment that promotes th differentiation, as demonstrated in mice treated with ros scavenging compounds having lower amounts of th cells in vivo (atarashi et al., ) . sfb flagellar proteins may induce th cells by signaling through tlr in a subset of cd c hi cd b hi intestinal dendritic cells (uematsu and akira, ). the flagellar binding motifs that are targeted by tlr appear to be highly conserved in sfb and is nearly absent from other similar clostridium species, suggesting a specific role for sfb to modulate th immunity (prakash et al., ) . although sfb-induced th immunity may benefit the animal host, there are long-term consequences. sfb-induced th immunity is linked to the development of autoimmunity in susceptible breeds of mice (lee et al., ; yang et al., ; teng et al., ) by inducing differentiation and egress of t follicular cells from peyer's patches . it is unknown whether natural colonization by sfb in poultry is capable of promoting autoimmunity, but it must be considered if sfb, or its antigens are utilized as immunomodulators for food-producing animals. segmented filamentous bacteria mono-associated mice display rapid growth and development of peyer's patches, and sfb can also stimulate the formation of lymphoid follicles and tertiary lymphoid tissues in the host (lecuyer et al., ) . this activation of the host's intestinal immunity causes a drastic increase in fecal concentrations of secretory immunoglobulin a (siga), as the number and activity of iga secreting b-cells rises (klaasen et al., ) . germ-free mice monoassociated with sfb triggers the production of iga serum levels equivalent to that of specific pathogen free, sfb-negative mice (klaasen et al., ) . the expansion and stimulation of germinal centers present in peyer's patches is not entirely unique to sfb and has been seen to occur in other commensal bacteria such as morganella morganii and bacteroides distasonis, however, the response from sfb is much greater than these other organisms (schnupf et al., ) . both t-cell dependent (b- cell) and t-cell independent (b- cell) production of siga occurs in sfb mono-associated mice (schupf et al., ) . the amount of sfb-specific iga produced by the host in response to bacterial colonization is as high as . % of the total iga of the organism (talham et al., ) . iga transmitted by nursing mice to suckling pups has been shown to inhibit sfb colonization, and only after weaning do sfb populations begin to increase, coinciding with the time in which sfb colonization is typically recognized in mice (jiang et al., ) . the induction of siga by sfb may serve as a negative feedback mechanism to prevent overcolonization by sfb and dysbiosis in older animals (ohashi et al., ; liao et al., ) . light turkey syndrome (lts) is growing problem facing commercial turkey production in the united states. lts is a condition in which turkey flocks fail to meet their genetic potential weight, yielding birds that are - pounds below the industry standard for average flock weight (danzeisen et al., ) . birds affected by lts display symptoms similar to poult enteritis complex (pec), a disease in which birds experience weight loss, diarrhea, lethargy, and depression (mor et al., ) . however, lts is dissimilar to pec in that birds do not experience watery and pale intestinal contents or distended ceca, indicating differences between the syndromes (morishita et al., ) . the causative agent of pec is suspected to be microbial in nature, as inoculation of healthy birds with fecal homogenates derived from birds experiencing pec produced light weight poults when compared to un-inoculated birds, but a single responsible microbe has yet to be determined (mor et al., ) . similarly, inoculating healthy birds with fecal homogenates derived from turkeys with lts produced birds that were lighter than the control groups (mor et al., ) . the two conditions are not dependent on each other, as lts can occur in the absence of pec (danzeisen et al., ) . there exist a number of potential factors that may lead to the development of lts, such as colonization by pathogenic bacteria, viral infection, stunting of immune system development, inhibited nutrient absorption, and alterations to gut microbiome (danzeisen et al., ) . typically, lts/pes affects birds less than weeks of age (morishita et al., ) . a higher number of different pathogenic organisms are found in these younger birds than in birds aged - weeks. virus strains such as astrovirus, reovirus, and rotavirus types were detected in the host, and are not associated with poult mortality. coronavirus, which is commonly associated with mortality, was not detected in lts/pes poults (morishita et al., ) . in an attempt to understand the microbial basis of lts, danzeisen et al. performed s rrna microbiome analysis of low-performing and high-performing (based upon flock weights) flocks to determine the role of microbial succession in promoting digestive health samples were sequenced to discern the presence and abundance of dominant otus present in higher-performing flocks as compared to lowerperforming flocks (danzeisen et al., ) . after analysis it was determined that at the age of - weeks, higher-performing turkey flocks harbored significantly higher proportions of clostridium bartlettii and candidatus division arthromitus, a sfb (danzeisen et al., ) . previous studies regarding sfb colonization of poultry have identified sfb as a causative agent in intestinal disease (goodwin et al., ) , but sfb was later ruled out as a causative agent (sell et al., ) . also, sfb belong to several microbial taxa and are not considered a homogeneous group (thompson et al., ) . as demonstrated in mice and rat models, sfb have been proven to be potent stimulators of host immunity and ileal health. though the role of sfb (in particular candidatus arthromitus) in the digestive health of turkeys is not quite understood, the evidence provided by danzeisen et al. suggests that epithelial binding of these bacteria may promote early digestive health (danzeisen et al., ) . the potential for candidatus arthromitus to serve as an immunostimulatory probiotic makes it an organism of great interest to poultry researchers, as the turkey production industry is in need of alternatives to promote animal health in this age of restricted use of antibiotics in food-producing animals and increasing antimicrobial resistance. since their initial characterization in the s, sfb have transitioned from being considered an interesting and unique member of the gut microbiome with a unique morphology, to serving as a model organism to study immunomodulatory symbiotic bacteria and their effects on the host. the hostspecific binding mechanism employed by these bacteria to attach to ileal epithelium is similar to that of enteric pathogens. unlike enteric pathogens, sfb do not harm the host epithelium and instead live in a commensal, if not mutualistic manner. intimate binding to the host mucosal epithelium allows sfb to receive nutrients from the host, satisfying their auxotrophic requirements, while delivering antigens to the host. epithelial binding also initiates several immune responses from the host. as demonstrated in mice and rat models, sfb have been shown to stimulate the maturation of the host's th and iga responses, improving the ability of the host to protect against invading pathogens. additionally, sfb compete with other members of the intestinal microbiota by modulating access to nutrients and occupying available ecological niches. the fitness-bolstering effects produced by sfb in mouse models are well-understood, but little is known about the roles these bacteria play in the other vertebrate animals. it has been suggested through microbiome analyses of turkeys that sfb, specifically candidatus arthromitus, may provide a protective role in preventing the onset of the enteric condition lts, the cause of which is not well understood. the role of sfb in turkeys must be better elucidated to determine the beneficial effects these bacteria 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cells in enterobacterial infection gut-residing segmented filamentous bacteria drive autoimmune arthritis via t helper cells focused specificity of intestinal th cells towards commensal bacterial antigens comparative analysis of the distribution of segmented filamentous bacteria in humans, mice and chickens interleukin mediates early host defense against attaching and effacing bacterial pathogens we thank judi stasko (usda ars national animal disease center) for technical assistance with scanning electron microscopy. key: cord- -c skxte authors: méthot, pierre-olivier; alizon, samuel title: emerging disease and the evolution of virulence: the case of the – influenza pandemic date: - - journal: classification, disease and evidence doi: . / - - - - _ sha: doc_id: cord_uid: c skxte “why do parasites harm their host?” is a recurrent question in evolutionary biology and ecology, and has several implications for the biomedical sciences, particularly public health and epidemiology. contrasting the meaning(s) of the concept of “virulence” in molecular pathology and evolutionary ecology, we review different explanations proposed as to why, and under what conditions, parasites cause harm to their host: whereas the former uses molecular techniques and concepts to explain changes and the nature of virulence seen as a categorical trait, the latter conceptualizes virulence as a phenotypic quantitative trait (usually related to a reduction in the host’s fitness). after describing the biology of emerging influenza viruses we illustrate how the ecological and the molecular approaches provide distinct (but incomplete) explanations of the – influenza pandemic. we suggest that an evolutionary approach is necessary to understand the dynamics of disease transmission but that a broader understanding of virulence will ultimately benefit from articulating and integrating the ecological dynamics with cellular mechanisms of virulence. both ecological and functional perspectives on host-pathogens’ interactions are required to answer the opening question but also to devise appropriate health-care measures in order to prevent (and predict?) future influenza pandemics and other emerging threats. finally, the difficult co-existence of distinct explanatory frameworks reflects the fact that scientists can work on a same problem using various methodologies but it also highlights the enduring tension between two scientific styles of practice in biomedicine. the question "why do parasites harm their hosts?" is recurrent in evolutionary biology and ecology, and has several implications for the medical sciences, particularly public health and epidemiology. the question is perplexing because of its paradoxical aspect. indeed, one wonders why natural selection favours high virulence if this inevitably results in both the host and the pathogen's deaths. shouldn't host and pathogens peacefully coevolve, and thus maximize both their chances of survival, instead of engaging in a near-infi nite arms race? very much along these lines, the lives of a cell ( ) by american physician lewis thomas refl ected the conviction that "there is nothing to be gained, in an evolutionary sense, by the capacity to cause illness or death" (thomas , ) . thomas' views on the nature of disease were once widely accepted among medical scientists during the past century. the possibility of eradicating diseases like smallpox, combined with the belief that evolution was going to naturally wipe out infections, worked together in supporting the idea of the end of infectious diseases (levins ) . physician and epidemiologist aidan cockburn, for instance, stated confi dently: "it seems reasonable to anticipate that within some measurable time, such as years, all the major infections will have disappeared" (cockburn , ) . following the improved control over infections provided by vaccines, antibiotics, and chemotherapy, biomedical authorities in the s and s, particularly in the u.s., ceased to regard infectious diseases as one of the major causes of death and morbidity, and argued, furthermore, that fundamental research on microorganisms could be halted altogether (burnet in fantini . this perspective was also refl ected at the political and economic levels. after the "war" on cancer and cardio-vascular diseases was declared in the early s, for instance, the budget of the national institute of health (nih) doubled in years, while the funding for the national institute of allergy and infectious diseases (niaid) grew by only % (krause , ) . the belief in the power of medical technology to conquer infectious diseases with newly developed drugs resulted in the idea that given suffi cient time most of these diseases would naturally decline as a result of the evolutionary dynamics that govern host and pathogens' relation and lead to lower levels of virulence over time (méthot a ; snowden ) . the return of infectious diseases from the early s onwards turned this perspective on its head, however, as the responses of modern medicine seemed no longer adequate in the face of the steep rise of nosocomial infections and the evolution of drug resistance worldwide. particularly, the acute sense of control over infectious disease felt by many was thrown into disarray with the onset of the hiv pandemic and other emerging infections such as ebola fever, sars, and more recently with the return of h n infl uenza. partly because "many people fi nd it diffi cult to accommodate the reality that nature is far from benign" (lederberg , ) , the rationale of the "conventional wisdom" (as named by may and anderson ) -namely that hosts and pathogens should coevolve towards a state of harmlessness -was promoted far into the second half of the twentieth century (see ewald for a review). an additional reason for the success of this avirulence hypothesis, besides its intuitive soundness, was the fact that no serious alternatives to it were introduced before the late s (alizon et al. ), even though some like zoologist gordon ball ( ) did raise important objections to the conventional wisdom. the thesis of a natural decline in the virulence of infectious disease postulated by earlier evolution-led models has been challenged on both theoretical and experimental grounds in the last years. empirical evidence and advances in modelling in evolutionary ecology (e.g. the trade-off model) have shown, for instance, that the evolution of hosts and parasites into a commensal state is not the vanishing, obligate point it was once held to be, but is rather only one of the possible evolutionary outcomes (anderson and may ; levin and pimentel ; ewald ; reviewed in alizon et al. ). as biologist carl bergstrom recently stressed: "we cannot count on evolution to do our work for us" (bergstrom , ) . selective pressures, on the contrary, can drive the emergence of new diseases (antia et al. ) . and as some have argued, humans may well be the "world's greatest evolutionary force" (palumbi ) behind the increased virulence of pathogens. through new social and cultural practices we open-up new routes for "viral traffi c" (e.g. blood transfusions, organ transplants), foster behavioural changes facilitating pathogens' transmission (e.g. air travel, migrations, sexual practices, use of drugs, etc.), and introduce "new" pathogens from different parts of the world into immunologically naive populations (morse (morse , (morse , . this of course adds up to the continuing emergence of human pathogens through zoonotic reservoirs (wolfe et al. ) . infectious diseases continue to be a serious threat to human health, and some diseases once believed to be eradicated might return. between and , diseases have emerged in human populations, the majority of them appearing during the s after rapid increase in drug resistance was detected (jones et al. ) . despite the recent steep rise in chronic and degenerative illnesses, emerging infections are still a global challenge for twenty-fi rst century biomedicine and they continue to claim million lives annually (morens et al. ; fauci ) . following the resurgence of infectious diseases as a leading cause of death and morbidity, and the detection of previously unknown diseasecausing entities, the idea that newly emerged pathogens have thrown the natural world "out of balance" (garrett ) has garnered a signifi cant amount of scientifi c attention and has led to the adoption of new international health regulations in order to monitor, limit, and control the spread of communicable diseases (castillo-salgado ) . here, we explore how, and in what contexts (molecular, ecological, and evolutionary) , knowledge claims about disease emergence and changes in virulence are made and justifi ed in the case one specifi c example: the - infl uenza pandemic. emerging diseases are usually defi ned as diseases whose incidence has signifi cantly increased within a population over a defi nite period of time (morse ) . as weir and mykhalovski ( ) recently observed, two of the most infl uential books on emerging diseases in the early s (lederberg et al. ; morse ) have stressed the need to investigate factors driving disease emergence from both an ecological and a molecular-genetic point of view. both books argued that the biology of the host and the pathogen, in addition to their complex interactions in changing ecological and evolutionary contexts, must be carefully considered in order to devise appropriate public health measures. in practice, though, it remains a challenging task to integrate those perspectives. indeed, our starting point is the current gap -and lack of integration -in the literature between studies of virulence as applied to emerging disease in the biomedical sciences broadly understood and in molecular pathology and evolutionary ecology in particular. integration is a multi-faceted concept that is often promoted as a promising goal of scientifi c practice. as discussed by philosophers of science, integration in science is a complex process that encompasses several activities such as methodological integration, data integration, and explanatory integration (o'malley and soyer ; see also mitchell ), among others. more rarely is the possibility that integration will fail discussed, however (see o'malley ( ) for an example of such). as this chapter exemplifi es, ecological and molecular methodologies have yet to come together to provide a broader picture of changes in virulence in emerging diseases. here, we focus particularly on experimental and modelling practices in molecular biomedicine and evolutionary ecology and on their respective explanatory limitations. very often, explanations of the virulence of a pandemics are constructed as an alternative between knowing the biological nature of the pathogen or that of the environmental conditions that facilitate its transmission. while both consider the nature of the host as part of the disease process, most of the time one branch of the alternative alone is considered as the right (or at least suffi cient) explanation while attention to other explanatory schemes is scant. using the - infl uenza pandemic as a case study of a particularly virulent emerging disease, we illustrate the enduring persistence of two distinct scientifi c styles of practice in the recent history of virulence studies. beginning with a discussion of the evolution of virulence as seen through the lens of ecological and molecular perspectives in biology, we show how each of them conceptualizes both the nature of virulence and emergence in quite different ways. next, we describe the biology of infl uenza viruses with a focus on the - pandemics and we move on to the ecological-evolutionary explanations of its exceptional virulence, paying attention to the trade-off model, before turning to molecular on the history, epistemology, and social aspects of the concept of emerging disease see grmek ( ); farmer ( ) , king ( ) ; and weir and mykhalovski ( ) . see the recent special issue on integration in studies in history and philosophy of the biological and biomedical sciences ( ). pathology. we argue that an evolutionary approach is necessary to understand the dynamics of disease transmission and evolution but that a broader understanding of virulence will ultimately benefi t from articulating the ecological dynamics with cellular mechanisms of virulence. in sum, both ecological and functional perspectives on host-pathogens' interactions are required to answer the opening question of this essay but also to devise appropriate health-care measures in order to prevent (and predict?) future infl uenza pandemics and other emerging threats. the diffi cult co-existence of distinct explanatory frameworks refl ects the fact that scientists can work on a same problem using distinct methodologies (godfrey-smith ) , but it also highlights the enduring tension between two scientifi c styles of practice in biomedicine. evolutionary biologist ernst mayr has long suggested that functional (proximate) and evolutionary (ultimate) perspectives in biology lack unifi cation (mayr ; see morange ) . more recently, evolutionary ecologists have argued in the direction of a better integration of those perspectives ( frank and schmid-hempel ) . while mayr's point that proximate and ultimate explanations are not alternatives is sound, developmental biology advocates, among others, have persuasively argued that evolutionary questions are relevant to understanding developmental processes, and vice-versa (see laland et al. for a review). today, another, and perhaps equally signifi cant divide, seems to be that between ecological and functional (or proximate) approaches to biological systems and their evolution. as we show, what we call exogenous and endogenous approaches to virulence both make knowledge claims based (sometimes loosely) on evolutionary theory, although each of them invokes one particular aspect of the theory. whereas the ecological (or exogenous) style focuses on processes (e.g. selective pressures, population density, within and between host competition, and so on) acting on the hosts and the pathogen, the molecular (or endogenous) style traces the evolutionary pathway, or patterns, of the infl uenza virus from animal(s) to man, and, by constructing molecular phylogenies, identifi es particular genes for pathogenesis and mutation sites within lineages. in other words, the former analyses one of the main mechanisms of evolution (i.e. natural selection) and the latter describe the path of evolution (i.e. they construct phylogenies) (ruse ) . the construction of molecular phylogenetic trees by the use of the concept of "exogenous" and "endogenous" styles is inspired by the work of historian of science ton van helvoort ( ) . in turn, this approach is indebted to polish immunologist and epistemologist ludwik fleck ( ). patterns derive from processes. the former can be described as the "study of order in nature" while the second refers to "mechanisms generating and maintaining this order" (chapleau, johansen, and williamson , ) . molecular pathologists refl ects the recent "data-driven" trend itself supported by genomics, molecular biology, and the development of high throughput technologies. the use of "evolution" by molecular pathologists is, however, secondary to fi nding molecular mechanisms for pathogenesis and thus explaining changes in virulence mechanistically. each perspective also provides a different way of thinking about disease emergence. briefl y, the endogenous view describes how bacteria and viruses can be transformed into pathogenic, emerging diseases by gaining intracellular and genetic material such as, for instance, a polysaccharide capsule, a large plasmid, a set of virulence genes, or pathogenicity islands (friesen et al. ). these and similar fi ndings have led some to claim that pathogens can evolve in "quantum leap" (groisman and ochman ) . point mutations allowing the virus to bind to a host receptor also belong to this category. while the capacity to cause disease due to new sets of genes is a crucial aspect of how organisms become pathogenic, this capacity can also occasionally result from genomic deletion and gene loss (maurelli ) . in sum, acquisition of novel "virulence factors" (or deletion of other genetic elements) can rapidly lead to the emergence of new diseases or enhanced virulence in some pathogens. for molecular pathologists the concept of virulence is similar to the traditional defi nition of plant pathologists, i.e. the infectivity: a strain is virulent if it is able to infect a host. this defi nition could be traced back to the work of pasteur, for whom "virulent cultures killed, attenuated ones did not" (mendelsohn , - , p. ) . a more classical defi nition is the ability to generate symptoms. in both cases, virulence is an all or nothing trait; it is qualitative and not quantitative. note that these defi nitions have the advantage that they can be translated at different levels, for instance at the cellular level, where virulence can be the ability to infect cells. the ecological or exogenous style adopts another approach to disease emergence, virulence, and evolution. often described as a two-step process, disease emergence requires the introduction of a pathogen within a population followed by its successful dissemination (morse , - ) . the "rules of viral traffi c" (morse ) dictate that both steps usually result from one or several changes in the environment, not from a modifi cation in the biological characteristics of the pathogen. for instance, in a change in the air conditioning system in a hotel in philadelphia facilitated the spread of legionellosis, a bacterium usually commensal to humans, which caused an outbreak of fever and pneumonia now known as legionnaire's disease. however, there are cases supporting a biological explanation of emergence, for instance when a maladapted strain mutates into a well-adapted strain before going extinct (antia et al. ) . the trade-off model developed by robert may and roy anderson, and independently by paul ewald, in the early note that, for historical reasons, in the phytopathology literature the virulence used to refer to the ability of the pathogen to infect a plant (i.e. a qualitative trait). since the american phytopathological society has decided to use the term virulence to refer to the damage done to the host and the term pathogenicity for the ability to infect the plant but few researchers have adopted it. in a way, the debate between two fi elds (evolutionary ecology and molecular biology) has already happened within one of the fi elds (see shapiro-ilan et al. ; thomas and elkinton ; shaner et al. ). s currently underpins the bulk of the theoretical research on host-pathogen's interactions in evolutionary ecology. put simply, the model postulates the existence of ecological trade-offs between a number of epidemiological variables. as a consequence, the evolution of virulence becomes linked to several factors: host resistance and recovery rate, pathogen transmission rate, the timing of infection lifehistory events and population density, among others. the trade-off model permits the investigation of the role of environmental changes broadly conceived (including within and between hosts selection) and selective pressures acting on pathogen transmission, and thus on the level of virulence (alizon ) . while molecular geneticists quickly adopted the concepts of virulence genes and pathogenicity islands, evolutionary ecologists working with the trade-off model continued to regard them with suspicion (see poulin and combes ) . we think this suspicion is probably due to the way virulence is defi ned. for evolutionary biologists, virulence typically is a quantitative trait that can be measured. therefore, genes that are suffi cient to render a pathogen virulent and essentially act as a qualitative trait are diffi cult to fi t into the picture. furthermore, there is no such thing as pathogen virulence alone in ecology. virulence, typically, is a "shared trait" that results from the interaction between a host genotype, a parasite genotype and their environment. in other words, some parasite genotypes might only cause virulence when they infect some host genotypes or some parasites may only be virulent to hosts in certain contexts (e.g. starvation). for evolutionary biologists and ecologists, virulence is the harm a pathogen does to its host, i.e. the reduction in host fi tness due to the infection (read ). fitness is notoriously diffi cult to evaluate but arguably the two most common measures are lifespan and fecundity. one problem is that a pathogen strain described as being very virulent in vitro could turn out to be mild in vivo (and vice-versa) . furthermore, recent work shows that levels of virulence can actually be the result of the immune system's over-response itself (see graham et al. for a review). in the end, evolutionary ecologists focus on a combination of within-host processes when they refer to virulence. importantly, this does not mean that they disregard the molecular processes that lead to virulence. for instance, studies have shown that immune-pathology contributions to virulence lead to a different evolutionary outcome than "virulence factors" produced by pathogens ( alizon and van baalen ) . recent explanations advanced to account for the rapid changes in virulence during the - infl uenza pandemic refl ect the polarity between ecological and molecular explicative strategies. applying the trade-off model to the - pandemic, paul ewald has argued that the proximity of soldiers in the trenches, the hospitals, the transport, and the military camps during world war i greatly facilitated transmission of the virus from host to host. high viral replication rate by natural selection was therefore favoured, which resulted in exceptionally high virulence and the high level of mortality of the pandemic (ewald (ewald , (ewald , . but since the late s, molecular pathology has provided an alternative viewpoint on the evolution of virulence in the pandemic. the identifi cation of the viral rna from frozen bodies and wax blocks in the u.s. and its further sequencing has led to a renewed emphasis on genetic and molecular determinants of the virus as being the most important cause of this dramatic event (see holmes ) . according to molecular pathologist jeffrey taubenberger, one of the leading scientists involved in reviving the infl uenza strain, "it is possible that a mutation or reassortment occurred in the late summer of , resulting in signifi cantly enhanced virulence" ( , ) . taubenberger believes that this "unique feature" of the virus -its extreme virulence -"could be revealed in its [genetic] sequence" ( , ) . both approaches -the exogenous and the endogenous -evolved along parallel lines during most of the twentieth century, and though the concept of emerging infectious diseases brought them closer to one another in the s, we show how they remain in tension (méthot b ) . before describing in more details the potentials and limits of these two perspectives we fi rst describe significant aspects of the biology of infl uenza viruses. the natural history and ecology of infl uenza a virus has been extensively studied (webster (webster , webster et al. ; webster and rott ) . the virus' natural reservoir is the wild waterfowl, as supported by the fact that species of wild duck are not affected by the virus and remain "healthy". the virus replicates inside the host, mostly in the intestinal tract, and is then washed into the ponds where ducks live and breed (webster ) . the relative harmlessness of this relationship is similar to the way myxoma virus is adapted to its natural host, the south american rabbit (see fenner and fantini ) . the family tree of infl uenza viruses contains two genera: one that includes infl uenza a and b viruses and the other infl uenza c viruses. the two genera are distinct in terms of host range and virulence factors. type a is the most common of all, and can infect a wide range of hosts, including, pigs, horses, seals, whales and birds. this type of virus is also the most redoubtable as it has the potential to cause pandemics. type b is believed to infect only humans (especially young children) and type c (another genus) can infect both humans and swine. in this sense infl uenza can hardly be regarded as a "single disease" (johnson , ) . infl uenza viruses are enveloped negative strand rna viruses and belong to the genus orthomyxoviridae (taubenberger ) . the virus of the spanish fl u pandemic belongs to the type a infl uenza, known as h n . infl uenza a and b viruses contain eight discrete gene segments, coding for at least one protein. the surface of infl uenza a viruses is covered by three types of proteins hemmagglutinin (ha), neuraminidases (na) and matrix (m ). the structural confi guration of ha proteins is that of a triangular spike. these spikes allow the virus to bind to red blood cells by causing the latter to agglutinate (i.e. hemmagglutinin). they facilitate entrance into the host and they trigger the infective processes. once the infection is over, antibodies responding to hemmagglutinin spikes are formed, allowing the immune system to recognize the signature of the viral strain in case of another infection episode. in contrast, neuraminidases (na) also form spikes on the surface coat of the virus but their function is to cleave glycoproteins into two and to facilitate the propagation of the virus from cell to cell. na proteins open-up cells for infection, so to speak. antiviral drugs target na in order to block their exit, and antibodies to na are also produced after the infection. infl uenza a viruses are further subdivided into serological types, which is the genetic characterization of the surface glycoproteins ha and na. ha and na proteins have been described to date. these surface glycoproteins defi ne the virus' identity in terms of what the immune system detects and attacks. the different major families of fl u are combinations of the two, hence the designation "h n " for the recent threat. the virus was h n . the genes coding for these glycoproteins can reassort (i.e. reshuffl e) due to two processes known as antigenic drift and antigenic shift. the former consists in the accumulation of point mutations in the genome of the virus, modifying both the shape and the electric charge of viral surface antigens and preventing their recognition by the antibodies of the host that were developed in reaction to previous exposures to the virus. the need to update the infl uenza vaccines every year illustrates the evolutionary success of antigenic drift. in contrast, antigenic shifts refer to the introduction of whole or part of infl uenza genes into viruses that circulate among human populations. this form of genetic reassortment or reshuffl ing occurs especially in swine that act as "mixing vessels" for the viral strains and are considered the intermediate host between birds and humans (webster and kawaoka ) . the introduction of a new hemmagglutinin gene (ha) is often hailed as the responsible factor for increased virulence (bush ). the fast reassortment of nucleotides and the high rate of mutation in infl uenza viruses result in infl uenza posing a continual threat for human and animal health. as a result, infl uenza is regarded as being a continually "re-emerging" disease (webby and webster ; webster and kawaoka ) , and international efforts are made to understand why the - pandemic was so exceptionally virulent. the motivation behind these global efforts in gaining a better understanding of this pandemic is to draw lessons from the past in order to be better prepared for the rise of future infl uenza and other viral pandemics. recorded history suggests that the fi rst infl uenza pandemic occurred in . beginning in asia, it rapidly spread to africa, america and to europe. between the eighteenth and the nineteenth century, medical historians identifi ed (at least) pandemics out of epidemics of infl uenza a virus (beveridge ) . the most devastating pandemic, however, occurred in - ( fig. ) . the emergence of on the history of the infl uenza pandemic, see barry ( a ) , johnson and mueller ( ) , van helvoort ( ), crosby ( ) , and burnet and clark ( ) . for a short but informative "chronicle" of infl uenza pandemics, see beveridge ( ) , and for a detailed scientifi c account of the biology of infl uenza see stuart-harris ( ) . the (misnamed) "spanish" infl uenza pandemic of - is the fi rst of the three major infl uenza pandemics that occurred during the past century -and is regarded as one of the most devastating episodes in medical history (mcneill ) . once described as "the biggest unsolved problem of theoretical epidemiology and public health practice" (burnet and clark ) , its consequences rendered many wary about the emergence of respiratory disease pandemics in a near future (webby and webster ) . in addition to the - pandemic, two other major infl uenza pandemics occurred in - ("asian" infl uenza, h n ) and in ("hong kong" infl uenza, h n ). the death of david lewis, a soldier at the military camp of fort dix in the u.s., and the infection of a few hundreds of others in led public health authorities to believe they were facing a new infl uenza epidemic. amidst some scepticism, vaccines against h n fl u were quickly stockpiled as president ford gave the green light to mass vaccination. however, no epidemic occurred while a number of vaccinated individuals came down with guillain-barré syndrome, an autoimmune disease, a few weeks later (see krause ) . one year later, in , the h n virus, which had disappeared in , reappeared (the socalled "russian" infl uenza) in the soviet union and spread to taiwan, the philippines, singapore, and within months had reached south america and new zealand. the virus was similar to a virus isolated in the u.s. in at fort warren and had perhaps been accidentally released from a laboratory located in the former the reference to spain is due to the fact the publication of medical reports on infl uenza was authorized in spain during the war, in contrast to other countries at war. as a consequence, the disease became associated with spain that was subsequently blamed for it and considered responsible. one of the fi rst papers to appear in london times (june ) was titled "the spanish infl uenza -a sufferer's symptoms" (in johnson , ) . like syphilis, the spanish infl uenza received other names in other countries, however. for instance, it was called the "swiss fl u" in france. soviet union (berche , ) . affecting mostly individuals born after , this virus coexisted with the h n virus until , when a new variant of h n emerged (the "swine" fl u, which is the latest pandemic) that replaced the variant. in comparison to the spanish fl u pandemic of , the hong kong and the asian pandemics were more "benign", the former causing between . and million deaths, and the latter million. the recent h n pandemic caused a few deaths only between and (taubenberger , ) . despite the (crucial) facts that antibiotics were available during the second two pandemics, and that medical care had signifi cantly improved and was more effi cient after , this raises the question: why was the - pandemic so deadly to humans? a fi rst important aspect of the - infl uenza pandemic is its likely western origin. in part because of its extensive pig-duck farming industry, china was previously singled out as the possible origin of most infl uenza pandemics. however, whereas most pandemics to have befallen man have come from china (morse , ) the "spanish" fl u originated (likely) from france as early as causing acute respiratory symptoms closely resembling the phenotype of the disease during the - fl u pandemic (oxford et al. (oxford et al. , . some have recently argued that there was an early wave of infl uenza in new york between february and april (olson et al. ). the precise geographical origins of the pandemic are still a matter of debate, however. the world's deadliest fl u pandemic kicked off in october and in just a few months, the virus killed between and million people (philips and killingray ; johnson and mueller ; crosby ; some estimate deaths to number about million, see mcneil ) . according to the "three waves theory", infl uenza swept through all fi ve continents in three recurrences. the fi rst wave (or the "spring wave") of the fl u started in march in the u.s. (mid west) before moving to europe, then to asia and north africa before reaching australia in july . while morbidity was high, mortality was not higher than the habitual norm (reid et al. , ) . the second wave (or "fall wave"), however, was highly devastating and rapidly went extinct after causing millions of deaths worldwide, with peaks in october and november. it started in late august and within week reports of the virus came from distant cities, including boston (u.s.), freetown (africa), and brest (france). on many accounts, this second wave lasted until november. the speed at which the virus circulated makes it diffi cult to pinpoint one specifi c location as being "the" source of the pandemic but a western origin appears to be the most plausible hypothesis according to the available evidence. reports indicated a further third wave that hit in the fi rst months of but was much less severe (burnet and clark ; barry a ). however, the three waves pattern of the pandemic is not uniformly applicable to all countries; for instance australia experienced a single occurrence of the fl u pandemic (johnson and mueller ; ). in , during an attack of infl uenza most victims died of secondary infections as death often resulted from bacteria invading the lungs of immunocompromised individuals (burnet and clark ) . symptoms lasted generally between and days and could, more rarely, be extended up to weeks. the respiratory disease was characterized by fever, body pain, and severe headaches. without the possibility of treating patients with antibiotics, bacteria turned "those vital organs [lungs] into sacks of fl uids […] effectively drowning the patient" (philips and killingray , ). people therefore died within just a few days of hemorrhagic pulmonary oedema and other lung affl ictions (bush ; see also taubenberger et al. ) . related to this, the second striking aspect of the infl uenza pandemic is the young age of the victims, which was qualitatively distinctive: most of them were men, supposedly healthy, of between - years old (some say - ), irrespective of whether the country was involved in the war or not. instead of forming a u shaped mortality curve, the shape of the pandemic was w shaped. an additional peak (the central peak in the w) represents the male victims of the fl u. figure (above) shows the u shaped curve of and the w shaped one of . the distribution of deaths on this curve refl ects the virulence of the pandemic and underlines the pattern of mortality of a group usually not affected by seasonal fl u. finally, the third and most signifi cant feature of the pandemic was its lethality: the disease was of exceptional virulence and estimates suggest that the pandemic claimed more victims than the first world war (mcneil ) . this central aspect was almost universally recognized as being somewhat unusual and very specifi c to it although the estimates of fatalities during the twentieth century vary between and million deaths (johnson and mueller ) . infl uenza type a viruses are not at all uncommon, and strains had circulated in human populations for a few centuries before since a few centuries when the pandemic broke out. in the united states alone only, annual death tolls related to seasonal infl uenza are estimated to be about , individuals (thompson et al. ) . seasonal outbreaks of infl uenza normally last a few weeks and then disappear abruptly; they result from infl uenza viruses present in human populations that are able to infect individuals due to antigenic drift. on occasions, however, the virus can infect up to % of the world population. during these pandemic years, in contrast, the number of deaths rises way above the average, claiming millions of victims all around the globe. in the course of seasonal epidemics strains of infl uenza type a and b can sometimes coexist, if at different frequencies among populations. so why was the spanish infl uenza so devastating? recent work in molecular biology argues that the waves pattern, the group mortality, and the clinical course of the disease "may fi nd their explanation in genetic features of the virus" (reid et al. , ) . others, however, defend the view that the changes in virulence result from signifi cant changes in the wider ecological context in which the outbreak occurred (ewald ) . in the next sections, we review both ecologicalenvironmental and molecular-led approaches to this problem, we indicate some of the limitations of each and we suggest that a better integration of those perspectives would lead to positive outcomes regarding prediction, prevention, and preparedness in the face of other similar infl uenza and other bacterial or viral pandemics. from an ecological point of view, for a disease to emerge in a population the basic reproductive rate of the pathogen ( r ) must be higher than , where r is the average number of secondary infections that follow from one infected individual in a wholly susceptible population (anderson and may ) . in other words, a pathogen must cause at least one subsequent infection to persist in the host population. the classical formula used to capture the trade-off model is as follows: where r serves as a measure of darwinian fi tness of the pathogen at the epidemiological level. in the denominator are α , the host mortality due to the infection (i.e. the virulence), μ , the rate of microparasite independent-mortality and γ is the rate of recovery of the host. the inverse of μ + α + γ is the average duration of the infection. in the numerator, we have β , the transmission rate, and s , the host population size. importantly, one should not confuse β , which is a rate (number of infections per unit of time and per susceptible host in the population) and r , which is roughly the number of new hosts infected over the whole duration of the infection. overall, this expression indicates that parasite fi tness is the product between the number of secondary infections generated per unit of time and the duration of the infection. any animal that produces less than one offspring over its lifetime infections generating less than secondary infection will eventually become extinct and die out. this r > threshold is of course a simplifi cation. for instance, in the early stages of an outbreak, emerging pathogens infect very few hosts which means that they are particularly prone to extinction going extinct due to stochastic effects. in fact, it can be shown that in an ideal situation where all the hosts would be identical, the probability of emergence of a pathogen with an r strictly greater than unity is only of - / r , due to these stochastic effects (diekmann and heesterbeek ) . conversely, pathogens with r < can nevertheless be dangerous because they can persist in the population for a while stochastically, which leaves time for a variant with an r > to evolve (antia et al. ) . in other words, the transmission between hosts (in these cases humans) must be effective for disease emergence to occur. historically, the fi rst example of a trade-off came from the analysis of myxoma virus infecting rabbits (anderson and may ; fenner and fantini ) . however, since then, clearer examples have been worked out. fraser et al. ( ) , for instance, combined data on virulence from an amsterdam cohort and data on transmission rate in discordant hiv-infected couples from a rakai cohort to show that individuals with a higher set-point viral load (i.e. the viral load during the asymptomatic stage, which has the property to often remain constant over several years) have a shorter lifespan and a higher transmission rate. when they combined host lifespan and virulence together to obtain a measure of parasite fi tness (i.e. r ), they found that viruses with an intermediate virus load achieved the highest fi tness. they also show the observed abundance of virus loads in a human population follows the distribution virus fi tnesses. the classic trade-off model focuses primarily on pathogen populations and their evolution. it often ignores host evolution because generation times for hosts (here, humans) tend to be much longer and so evolution in the host population is likely to be slow. from a trade-off model perspective, pathogens' rate of replication within a host, which usually increases the probability of its being transmitted to a new host, is balanced with its negative effect on the duration of the infectious period (may and anderson ) . if the pathogen is not virulent, it is unlikely to reach a high transmission rate. conversely, a pathogen that replicates intensively in the host will have a higher transmission rate but over a shorter time because rapid host exploitation also means shorter host lifespan. similarly to achilles who, according to homer, had to choose between a short and glorious life or a long but dull one, the pathogen has to evolve a strategy between causing long and mild infections or short and virulent infections (alizon et al. ). if such a trade-off is at work, external factors can affect virulence evolution in a predictable way. for instance, the lower the baseline mortality of the host (independently of the disease), then the higher the pathogen virulence should be. this is so because the infectious period is reduced and the pathogen has to use up the host resources in a shorter time. there is also a growing interest in the host reaction to an infection, which can broadly be split into resistance (i.e. fi ghting the disease, which decreases both virulence and transmission) or tolerance phenomena (decreasing only the virulence, not the transmission rate) that affect parasite evolution (raberg et al. ; boots et al. ). the density of susceptible hosts can also affect short-term evolutionary dynamics of virulence, as clearly shown by a recent evolutionary epidemiology framework that combines epidemiology and population genetics (day and proulx ) . indeed, early on during the course of an epidemic, most of the hosts are susceptible to infection so natural selection acts to favour strains with a high transmission rate (which happen to be more virulent according to the trade-off hypothesis). once the disease has reached an endemic stage, the pool of susceptible hosts is smaller (hosts are either already infected, dead, or immunised) and natural selection then acts to favour strains that cause longer infections. in conclusion, virulence can thus be expected to vary over the course of an epidemic for rapidly evolving pathogens. another dimension of the model is that it does not concern itself with morbidity (at least not explicitly). thus, symptoms like pain or injuries are not taken into account by the trade-off model and are implicitly integrated with other variables like host recovery and parasite transmission (levin ) . this assumption impacts on the ways in which virulence will be measured and operationalized. whereas for doctors morbidity (illness) is a key feature of virulence, for evolutionary biologists or population biologists host's pathological factors do not need to be taken into account when measuring virulence; what matters are effects that modify the pathogen's fi tness (i.e. that appear in the expression of r ). in sum, the model rests on the idea that the pathogen transmission rate cannot increase beyond a certain point without at the same time infl icting damage to the host which would, in turn, be harmful to the pathogen by decreasing the duration of the infection. what matters for an evolutionary biologist is the fi tness of an individual where the fi tness of a parasite strain typically is given by the r , i.e. the number of secondary infections. in other words, for a given parasite species, natural selection favours strains with the highest r . this can explain why the highest possible level of virulence is not always the evolutionary stable ("optimal") strategy to increase parasite's fi tness: increased transmission will indeed increase one component of parasite fi tness r (the transmission rate) but it will also decrease another component of r that is the duration of the infection (through increased virulence) as the host is likely to die more rapidly from the infection. the balance between the two selective pressures (transmission favouring higher virulence and duration of infection favouring lower virulence) determines the evolutionary stable level of virulence. ewald's early work on pathogen's virulence and transmission developed a verbal theory for the trade-off model by comparing diseases with different transmission routes (ewald ) . his work was based on the concept of "cultural vectors" and on the assumption that parasites that do not rely on host mobility for transmission should evolve towards higher levels of virulence. in ewald's terminology, a cultural vector is "a set of characteristics that allow transmission from immobilized hosts to susceptible when at least one of the characteristics is some aspects of human cultures" ( ; ; see also ewald ) . in the case of waterborne transmission, cultural vectors include contaminated bed sheets in hospitals, sewage systems carrying the pathogens, medical staff disposing of the contaminated water to water supplies, and so on. waterborne diseases can become more virulent because they do not rely on host mobility for transmission (see ewald , ) , that is, the host can be isolated and still be highly contagious; a "healthy" host is not needed for transmission (in contrast with what was postulated by the conventional wisdom). note that implicit in his reasoning is the idea that more virulent pathogens have a higher transmission because they produce more spores. applying the trade-off model to the case of the pandemic, ewald argued that host proximity and population density were key elements in enhancing virulence. more precisely, he argued that the exceptionally high virulence resulted from rapid passages of the virus in soldiers, recruits and wounded people in hospitals during the war. though a similar explanation had already been heralded in the s- s, it had to be supplemented with an essential "evolutionary mechanism": the classical explanation is based on the analogy with rapid passages of a viral strain through a series of animals (i.e. guinea pigs) in a laboratory that can enhance virulence (as pasteur et al. ( pasteur et al. ( [ ) had experimentally demonstrated, see mendelsohn ( ) ). ewald's argument is that, just like biological vectors, cultural vectors enhance virulence by facilitating transmission. the central point about the serial passages is that it removes the "requirement that hosts be mobile to transmit their infections" (ewald , ) . once this obstacle is lifted nothing (a priori) stands in the way of a steep increase in virulence. in a laboratory context, experimenters inoculate different animals with artifi cially selected viral strains; in the fi eld, this selection process results from another cultural vector, namely the warfare conditions. in the trenches, during the great war, conditions were such that transmission was maximized and with it, the observed level of virulence. as postulated by the trade-off model, the density of the population ( s ) infl uences the level of observed virulence in a biological system (at least for short-term evolutionary dynamics). in this case, the high density resulted from the proximity of the soldiers in the trenches, in hospitals, on trains bringing soldiers to the front, and in military camps. in turn, this resulted in the unusual situation that immobilized individuals who normally should not be able to infect new people (because they would be isolated in a hospital) were now easily able to transmit the infections. similarly, removing wounded soldiers from the trenches and bringing them to war hospitals facilitated transmission. the constant arrival of new susceptible individuals into the population through transport networks resulted in maintaining a high density of infected people; and as a consequence, an equally high level of virulence. related to this is the idea that spatial structure in the host population affects virulence evolution. if hosts tend to have few contacts among them, e.g. because they live in isolation (the technical term to describe such a population is "viscous"), then a parasite has to keep its host alive suffi ciently long enough to be transmitted. on the other hand, if the population is "well mixed", host encounter rate is not an issue -as in the - example -and parasites can afford to be more virulent (boots and sasaki ) . despite its theoretical appeal, some detected a number of problems in the explanation advanced by ewald and with the trade-off model in general. for other evolutionary ecologists, ewald's cost-benefi t argument is too adaptationist -i.e. virulence is depicted as being always adaptive for the parasite. as a consequence, "alternatives such as virulence being non-adaptive, or virulence being a consequence of short-sighted, within-host evolution of the parasite are ignored" (bull and levin , ) . evolutionary theory states that virulence can be directly selected but it can also be coincidental with other infection or biological processes (levin and edén ) , and in some cases it can be potentially maladaptive. this point connects to one of the usual critiques levered against the trade-off hypothesis, namely that it is very verbal and lacks empirical support (levin and bull ; lipsitch and moxon ) . however, the lack of support largely comes from the diffi culty of fi nding an appropriate biological system; arguably, when people have looked for a trade-off in a host parasite system that satisfi es the assumptions of the theory they have found it (alizon et al. ). there is actually a tendency to challenge the trade-off hypothesis using host-parasite systems that do not fi t the underlying model (see alizon and michalakis ( ) for an illustration). a second problem stems from the low level of transmissibility in infl uenza viruses and rate of pathogens' reproduction. when taken into account, this concern weakens the claim that high transmission in the case of the pandemic has favoured high virulence because transmission was lower than with most infectious diseases. r are typically variable but given ewald's argument it would be expected to fi nd a high transmissibility rate between the virus and its hosts. in turn, this would support the claim that natural selection acted on transmission in ways that increased the overall level of virulence. moreover, the trade-off assumes a homogeneous population and was developed for diseases transmitted by contact like infl uenza. however, a comparison of r between the - pandemic with other major disease outbreaks in recent history, or with infl uenza pandemics in general, does not reveal a signifi cantly higher transmissibility in the case of the spanish infl uenza. calculations suggest that the basic reproductive rate of viruses during infl uenza pandemics ranges from and (mills et al. ) . in comparison, the reproductive rate during an outbreak of measles in england in - was between and secondary infections; a pertussis outbreak in maryland (u.s.) in yielded a reproductive rate between and ; and a mumps outbreak in the netherlands during the s produced between and secondary infections in a wholly susceptible populations (anderson and may , ) . in the case of the pandemic, more recent calculations suggest that r was perhaps equal to (morse , ) . finally, a recent article on the transmission of infl uenza in households during the pandemic (fraser et al. ) used historical data and mathematical models to study the rate of transmission. the authors found a relatively low level of transmission between individuals and suggest that prior immunity to the virus should be considered. though transmissibility may, theoretically, have been fostered so that the virus reached unprecedented virulence, the trade-off model alone does not fully explain why it was so deadly. a third issue is the lack of empirical details in ewald's explanation of the steep increase in virulence circa - . to make his argument more compelling, ewald needs additional data that accurately and empirically describe the environmental conditions in the trenches. for instance, how close were the troops? how many soldiers were there? and more importantly, what was the rate of transmission between hosts? if a similar study to fraser et al. ( ) could be conducted on viral transmission in the trenches it would perhaps yield interesting insights into the changes of virulence. to date, no epidemiological data exists that could serve as a basis to model the dynamic patterns, however. though ewald's account seems to suffer from a number of theoretical and empirical problems, it nevertheless supports the argument that properties other than those of the virus need to be taken into account and that without them we would are not able to fully understand the changes in virulence that occurred. as he remarked, progress towards the evolution of virulence "has largely been limited to improve understanding of the genetic mechanisms of antigenic changes and the infl uences of these changes and host immunity on the occurrence of epidemics" (ewald , ) . the recent work of microbiologist john oxford on what we could call the "war hypothesis" reinforces ewald's conclusion by feeding in some of the missing empirical and historical data. while many would agree that the great war is a variable that must be included, in one way or another, in the broader explanation of the steep evolution of virulence of the - pandemic, ewald is convinced that the infl uenza pandemic was "caused evolutionarily by the war rather than being just coincidental with the war" (ewald , ) . the "war hypothesis", as we may call it, received new support from oxford ( ; reid et al. ; oxford et al. ) who does not claim that the great war caused the disease, evolutionarily or otherwise, but instead that the war created the right environment for the virus to become extremely deadly. when the pandemic broke out air travel was minimal and this suggests, according to oxford, that "earlier 'seeding' has occurred" ( , ) . taking an environmentally oriented approach to the evolution of virulence, oxford and his colleagues argued that the - pandemic originated in france in before going global years later. they did not postulate the evolutionary emergence of a mutant strain but rather that the ecological conditions facilitated the spread of a pre-existing infl uenza strain. studying several epidemiological and medical reports of sporadic outbreaks of respiratory infections at the british base camp in the town of etaples in northern france in , oxford argued that the disease's clinical picture maps very precisely onto the description of the - infl uenza: not only were the respiratory diseases extremely deadly, but post-mortem examination revealed in most cases clear evidence of bronchopneumonia and histological analyses of lung tissues indicated "acute purulent bronchitis" (oxford (oxford , . in an article published a few years later (oxford et al. ), oxford and his colleagues took their examination of the situation one step further. rejecting the possibility that "a particular virulence gene of infl uenza" could help to identify future pandemics, they argued that surveillance and detection of emerging infl uenza pandemics would be best served by understanding the contexts that give rise to pandemics, rather than by an analysis of genetic factors alone. in particular, concerning the pandemic, they noted that so far "there is no clear genetic indication of why this virus [the strain] was so virulent". they also remarked that what is needed is a closer examination of the environmental and social conditions of the time such as population upheavals to explain the exceptional virulence. the authors asked specifi cally whether "the special circumstances engendered in the war itself have allowed or caused the emergence, evolution and spread of a pandemic virus" (oxford et al. , ) . for them, the "unprecedented circumstances" of the war in europe were critical. back in , the front was a landscape that was contaminated with respiratory irritants such as chlorine and phosgene, and characterized by stress and overcrowding, the partial starvation of its civilians, and the opportunity for rapid "passages" of infl uenza in young soldiers would have provided the opportunity for small mutational charges throughout the viral genome […] could have been important factors in the evolution of the virus into a particularly virulent form (oxford et al. , ) . the military camp of etaples in france was subject to high traffi c in - . in addition to soldiers moving up to the front and back, , sick and injured individuals were in the hospitals "at any given time", making them overcrowded and allowing the virus plenty of opportunities for "rapid passages". overall, it is estimated that the region of etaples hosted two million soldiers who camped there during the war, in addition to the six million others who occupied and fought in the trenches system that connected the english channel with switzerland (oxford et al. , ) . secondly, as the camp had an "extensive piggery", villagers could buy geese, ducks, and chickens, providing ideal conditions for the infl uenza virus to undergo antigenic shift. thirdly, the extensive use of gases during the war (estimated at one hundred tons), some of which were mutagenic rendered the soldiers immunocompromised and more susceptible to infl uenza infections. finally, demobilisation after the war sent the soldiers back home by boat or by train, and contributed to the spread of the disease by person-to-person contact all over the world (oxford et al. ) . taken together, all these factors (overcrowding, being immunocompromised, pig-duck farming, demobilisation) created exceptional conditions for the virus to go pandemic. ewald had noted that in the absence of a recreation of those circumstances it is unlikely that such a severe pandemic will happen again. what oxford and his colleagues emphasized in their turn is that the appropriate response to a future pandemic cannot rest of putative virulence genes alone; one has also to consider the context that will allow the virus to spread in a pandemic fashion. at the same time that this ecological perspective was developed, another view on the sources of virulence was well underway in the united states. in the mid-twentieth century, leading british bacteriologist wilson smith, co-discoverer of the viral nature of infl uenza in humans in , doubted that the exceptional virulence could be linked to a particular genetic or molecular structure of the virus alone: "if we had the chance of getting a - strain of the infl uenza virus now", he said, "it is at least conceivable that, on comparing with the asian strain, we might fi nd no difference in intrinsic virulence at all, but the conditions in the human population during the two epidemic periods might have affected the degree of heterogeneity displayed by viruses possessed of the same intrinsic virulence" ( , ) . his comments were intended to provide support to a paper delivered earlier by edwin kilbourne, an american virologist who specialised in infl uenza, who had argued that the greater virulence of the pandemic was due to a combination of "the emergence of a new antigenic type in a population with little specifi c immunity" and "the dislocation of and crowding of wartime which favoured not only dissemination and high dosage of virus but spread of bacterial pathogens to an unusual degree" (kilbourne , ) . kilbourne argued that the "study of the host and his environment are more crucial to the interpretation of virulence than laboratory study of the virus itself" ( , ) . attempts to locate the cause of virulence inside specifi c genes or to relate them to other mobile, structural elements (i.e. plasmids) were met with scepticism by people like wilson, kilbourne, until the late s and early s, and the work of olitsky and gates and richard shope, most bacteriologists believed that the causative organism of infl uenza was the gram negative pfeiffer bacillus ( haemophilius infl uenza ) isolated by german scientist richard pfeiffer in . however, the causal role of this organism in infl uenza aetiology was also disputed, particularly during the - pandemic (witte ) . the discover of the viral nature of infl uenza was made by smith, andrewes, and laidlaw in (smith et al. ). and burnet who were interested in large-scale ecological processes and in the formation of evolutionary equilibriums between hosts and parasites. ecologically minded biologists were also reacting against the growing place of molecular biology since the s and its reductionist vision of the life process and the life sciences. this is how we can interpret kilbourne who scornfully remarked that "ironically in this era of molecular biology, the control of no infectious disease has yet depended on understanding its molecular mechanisms" ( , ) . in the early s, scientifi c expeditions were organized to discover the remains of victims of the spanish fl u in the hope of fi nding traces of the virus. one of the expeditors was john hultin ( -), a pathologist from sweden who immigrated to iowa in to study medicine. as part of a project funded by the university of iowa, he travelled in to a small inuit village whose population was decimated by the pandemic, which had killed people in a week, a loss amounting to % of the inhabitants. hoping to fi nd preserved corpses buried in the permafrost hosting traces of the infectious organism, hultin travelled to the seward peninsular of alaska in a village known as teller mission (taubenberger ) . he extracted lung tissue from several bodies he exhumed from the village cemetery, but all attempts to culture remaining traces of the virus of infl uenza from these samples failed to give any result. forty years later, in the context of the human genome project, the idea of resurrecting the infl uenza virus surfaced again, this time powered by genomic technology. since the late s, a renewed emphasis has been placed on the molecular, internal constituents of virulence. newly developed technology and the availability of pathogenic viral and bacterial material have facilitated the development of this approach towards explaining infectiousness. this led, in , to the publication of the complete infl uenza virus' genomic map in both nature and science . though all samples of the strain were thought to be long extinct and lost, bits of rna of the virus were discovered and processed in order to generate the complete map of its genetic structure. after the discovery of frozen individuals killed by the - pandemic and preserved in permafrost, scientists worked on the pathogenic mechanisms that possibly enabled the infl uenza virus to achieve unprecedented levels of virulence. microbiologist jeffrey taubenberger of the national institute of allergies and infectious diseases in washington led this work together with terrence tumpey from the center for disease control in atlanta. we now turn to this recent technological success and the diffi culties of pinpointing any particular molecular feature of the fl u virus of that could account for its exceptional virulence. in a u.s. lab-group based at the armed forces institute of pathology in washington (d.c.), and led by molecular pathologist jeffrey taubenberger, published a piece in science titled "initial genetic characterization of the - ʻspanish' infl uenza virus" (taubenberger et al. ) . the article provided a fi rst and partial genetic map of the virus from "archival formalin-fi xed, paraffi nembedded autopsy tissues of fl u victims" (taubenberger , ) . the examined samples were kept at, and provided by, the national tissue repository of the armed forces institute of pathology. as several mutations in hemmagglutinin, especially on cleavage-sites, often contribute to the virulence (e.g. on infl uenza subtypes h and h ) by increasing the tissue tropism, it was hoped that the genetic make-up of the virus would provide insights into the virulence of the spanish infl uenza pandemic. the goal of the project was "fi rst, to discover where the infl uenza came from, and how it got into people, and second, whether there were any genetic features of the sequence that would give insight into the exceptional virulence of the strain" (taubenberger , ) . this fi rst publication of the team describes the technique used to obtain, amplify (pcr), and sequence the genetic material. the main fi nding of the paper, based on molecular phylogenetic analyses of gene segments, was that the pandemic was caused by a strain of h n infl uenza virus, and that it was of avian origin ( , ) . in their fi rst article, taubenberger et al. randomly selected cases of paraffi n-embedded tissues collected from army servicemen who died during the pandemic for pathological review, searching for symptoms indicative of death by infl uenza. most of the individuals examined died of secondary pulmonary infection, which was a common feature of victims of the pandemic. in effect, bacterial infection very often works together with the infl uenza virus in delineating the clinical picture of the disease. one case, indeed ( case ), could be linked to viral pneumonia and exhibited symptoms of acute pneumonia in the left lung combined with an acute form of bronchiolitis in the right lung, a pathological characteristic typical of a "primary viral pneumonia". focusing on case , researchers performed control amplifi cation of reversetranscribed genetics of the nine gene fragments of the virus using the technique of polymerase chain reaction (pcr). they then carried out phylogenetic analyses based on the gene sequences to reconstruct the genealogical relationships between these elements. it was concluded that the genetic sequence of this strain was different from every other infl uenza strain, and that it was more closely related to strains found in birds than in mammals (taubenberger et al. ) . this partial analysis of the genetic map of human infl uenza was soon followed by a complete sequencing of the hemmagglutinin gene (ha) -a gene long believed to be "pivotal" in the pathogenicity of infl uenza a viruses (webster and rott ; see also cox and bender ) . this gene codes for a protein located on the surface of the virus that plays a crucial role in allowing the virus to bind to host cells. if the virus is able to spread to another species this means it has somehow (through antigenic drift) acquired a new protein that enables it to bind on a different receptor. however, the team did not identify a mutation of the cleave site of the hemmagglutinin gene taubenberger et al. ) . two years later, the team published another article on the "origin and evolution of the 'spanish' infl uenza virus hemmagglutinin gene" (reid, fanning, hultin, and taubenberger ) . johan hultin, the pathologist who attempted to fi nd traces of the infl uenza virus in alaska in the early s, was among the authors of the study. after reading the science paper, hultin wrote a letter to taubenberger offering to return to brevig mission to look for samples of people who had died of the fl u (taubenberger , ) . against all odds, hultin was successful. after he received the approval of taubenberger he set out to alaska for a second time and in august he found in situ frozen lung biopsies. once in the village, he was granted permission from the council to dig the graveyard again; with the help of a few villagers and after days work, he unearthed the body of a year-old woman whom he called "lucy". opening up her chest he found two frozen lungs that he immediately sent to taubenberger's laboratory in washington, along with some tissues taken from three other frozen corpses (berche ) . reid et al. ( ) reported on the full sequence of the hemmagglutinin gene using rna fragments from case discussed in the fi rst article. they investigated three case histories to fi nd evidence of infl uenza rna. the fi rst one was a year-old man who died at fort jackson in south carolina. pathological records indicate he had pneumonia and infl uenza symptoms; he was admitted to the camp hospital on september th and died within days. the autopsy records also show that his left lung suffered from an acute and fatal attack of pneumonia, whereas his right one showed acute bronchiolitis and alveolitis -a clear sign of infl uenza infection. no rna was found in the left lung. however, the team performed a minute microscopic analysis on the paraffi n-embedded tissue of the right lung and tissues tested positive for infl uenza rna. the fragments of fi ve genes were sequenced, amplifi ed through pcr technique and then determined. the second case was also a male soldier, this one years old and based at camp upton in the state of new york. he was admitted to hospital with pneumonia and died within days on the rd of september . microscopic examination of his lungs by taubenberger and his team revealed acute pulmonary oedema and acute bronchopneumonia. formalin-fi xed, paraffi n-embedded samples of lung tissues tested positive for infl uenza rna, the sequence of which was no longer than nucleotides. the third case history was the one found by hultin in brevig mission, alaska. using the sequences of these three case histories, the washington-based team worked out the genealogical relationships between them. their analysis reasserted that the virus that caused the pandemic was avian in nature and that it entered human populations between and , following the modifi cation of the binding site on the ha protein. in , taubenberger and tumpey published two separate articles in nature and science : the fi rst provided the complete genomic sequence of the infl uenza virus and the second revealed the methods used to artifi cially reconstruct it. yet, even before the complete genomic map of the virus was made available, it became unclear whether the genes of the infl uenza virus had indeed disclosed the causes of its exceptional virulence (see taubenberger et al. ). moreover, their argument of a likely avian origin of the virus was criticized. efforts to sequence the virus that caused the - infl uenza pandemic were motivated by the possibility of understanding the genetic origin and virulence of such an organism. while this work allowed for a more precise characterization of the hemagglutinin, neuraminidase, matrix, and nucleoprotein gene segments from a functional point of view, it is less clear, however, whether the fi rst goal was achieved. in effect, the washington team reported that a cleavage-site mutation on the hemmagglutinin gene that played a crucial role in the virulence of the hong kong pandemic in was not found in the strain obtained from the south carolina case. sequencing the specifi c cleavage site in the rna of the virus obtained from the brevig mission case and new york case also confi rmed that this mutation was absent. inquiring into this mutation site (hemmagglutinin) -understood as a key determinant of virulence -was a central motivation of taubenberger's work as it would have "offered an appealing explanation of the 's fl u virulence" (taubenberger , ). yet taubenberger was forced to recognize that "the strain (as confi rmed by all three cases) does not possess a mutation at this site" (ibid.; see also reid et al. ; stevens et al. ). in the light of this conclusion, virologist and infl uenza expert robert webster wrote that the secret of the spanish infl uenza will "remain elusive". webster commented that such "biological properties" [i.e. virulence] may "not be resolved" and suggested that the results of the sequencing project could only provide a partial explanation of this phenomenon. indeed, for him "the entire gene sequence is unlikely to reveal the secret of the high pathogenicity of the spanish virus" (webster (webster , . while taubenberger's paper ends with some remarks about the complex, likely polygenic, nature of virulence determinants in a particular strain, it also concludes -contra webster -with the hope that more sequencing would "shed additional light on the nature of the infl uenza virus" (reid et al. (reid et al. , . another molecular explanation of the - fl u pandemic emerged in from another research team. virologists hideo goto and yoshihiro kawaoka published a paper in the proceedings of the national academy of science on a novel mechanism for the acquisition of virulence by human infl uenza a viruses. there, they argued that a change in another major protein -neuraminidase -able to increase the cleavage of ha could bring about higher levels of virulence. in fact, goto and kawaoka even suggested that a change in a single amino-acid sequestering plasminogen might facilitate the cleavage of na. the authors were cautious, however, stating they "do not conclude that single mutation will convert nonplasminogen-binding nas to effi cient plasminogen binders, thus rendering the virus highly virulent" ( , ). yet, they acknowledged at the same time that it is "tempting to speculate that the pandemic strain […] may have acquired its unprecedented virulence from the mechanism we describe" (ibid). but such a change in amino-acid was also absent (or at least not observed) in the neuraminidase sequence (taubenberger , ; ; see also reid et al. ; kawaoka and watanabe ) . also, similar to taubenberger, goto and kawaoka concluded with a plea for "further sequencing", in order "to address the issue of its [the - pandemic] unprecedented virulence" (goto and kawaoka , ) . in , both taubenberger and tumpey acknowledged the lack of evidence provided by the molecular structure of the virus to explain its virulence: sequence analysis of the infl uenza virus from fi xed and frozen lung tissue has provided molecular characterization and phylogenetic analysis of this strain. the complete coding sequence of the nonstructural (ns), hemagglutinin (ha), neuraminidase (na), and matrix (m) genes have been determined; however, the sequences of these genes did not reveal features that could account for its high virulence" (tumpey et al. ; emphasis added) . and yet, despite evidence for an absence, there seems to be something particular about the structure of the ha protein that contributes to an enhanced level of virulence (morange ) . indeed, using a mouse model, another team of molecular pathologists (kobasa et al. ) showed that when the ha protein taken from the viral strain is inserted into mice it confers high pathogenicity and facilitates lung infections. for instance, infected mice show , times more virus particles after infection with the strain than with other viral strains like the texas virus, and infected mice died after days following infection with the strain, while all survived when infected with the texas virus (von bubnoff , ) . the particular structure of the protein responsible for such pathological effect remains to be found, however, and it is unclear whether similar effects could hold true in humans as well. as we have described, taubenberger's team provided the fi rst molecular characterisation of the spanish infl uenza organism based on the construction of phylogenetic trees of of the rna-polymerase genes of the - virus (taubenberger ) . the authors of this research project that spanned several years concluded that the virus did not originate from gene reshuffl ing (or reassortment) but rather that it jumped from birds to humans shortly before the onset of the pandemic. the virus was thus of avian origin. however, their interpretation of the similarity by descent, and thus of the genealogical relationships between the virus and today's avian viruses was disputed (gibbs and gibbs ; antonovics et al. ) . as the current head of the viral pathogenesis and evolution section at the national institute for allergy and infectious diseases, taubenberger's work is underpinned by evolutionary considerations. but what aspects of his work exactly are evolutionary or darwinian? philosopher michael ruse has long pointed out that the term "darwinism" carries two broad meanings. it can be used fi rstly in a metaphysical sense to characterize change, development and transformation in the natural world. in this sense, the concept of darwinism is older than darwin himself. another sense of darwinism is important to acknowledge. in this second sense, darwinism is a scientifi c notion that emerges in the work of naturalist charles darwin and refers to the fact of evolution, the paths (phylogenies) of evolution, and the mechanism (natural selection) of evolution (ruse , ) . this distinction between path and mechanism maps on the more traditional distinction between patterns and processes in evolutionary biology mentioned above. the research of taubenberger and ewald -and more generally molecular pathology and evolutionary ecology -displays these two aspects of darwinian theory. arguably, both accept evolution as a "fact". however, the former is more interested in the "patterns" of evolution and uses evolutionary thinking to unravel the biological (including genetic) and adaptive processes that led to an increase in virulence. in contrast, ewald focuses on the "process" of evolution -natural selection -as it occurred in various environments and populations of hosts and pathogens. as described above, taubenberger's research focuses on precise and minute description of the small steps that allow viruses to infect more than one species; this work painstakingly tracks changes in nucleotides and charts the genealogical relationships between several strains of infl uenza. ewald and oxford, in contrast, take a broader view and ask why those mutations were selected, what were the selective pressures that drove them to be passed on and conserved in the gene pool, and especially, what is the role of the milieu, largely understood, in shaping virulence. though the centrality of the concept of natural selection is not really in dispute here, the ways in which taubenberger and ewald (and other evolutionary ecologists) understand these processes differs signifi cantly on one important point: whereas the former describes the small incremental steps leading to the high, observable level of virulence, the latter looks for a plausible, eventually testable evolutionary scenario leading to the accumulation and conservation of these small, gradual changes. in other words, the second approach, the ecological one, seeks not only to describe organic changes leading to the formation of new viral strains, for example, but also attempts to give an account of the adaptive value of these transformations in the particular milieu in which the microorganisms lived, reproduced and eventually died. these two components of evolutionary theory -patterns and processes -are well known in the history of biology. evolutionary ecologists nowadays might want to argue that taubenberger is primarily interested in constructing and comparing distinct phylogenetic trees, no matter what the signifi cance of their (evolutionary) relationship may be. we think that the two aspects of evolutionary theory discussed here, however, refl ect more broadly the existence of two distinct styles of scientifi c practices in biomedicine. the diffi culty in addressing both aspects of the theory at the same time is indicative of a genuine tension between distinct explanatory strategies where knowledge claims are made according to different assumptions as to what counts as explanatory. confronted with the lack of evidence supporting a molecular explanation, and in the light of the limitations of the environmental-ecological account, one could have expected researchers to seek support in each other's work in order to complement their researches, and to move beyond the limitations of their own methodologies and research paradigms. yet it is striking to note that reid, taubenberger et al. ( ) , on the one hand, and goto and kawaoka ( ) , on the other, reached a conclusion diametrically opposed to that of webster and also ewald: for the former, in order to explain better the infl uenza pandemic, more genomic sequencing is needed. instead of considering other possible explanations of the exceptional virulence (i.e. ecological explanations) they persist in their attempt to provide a complete and satisfying explanation within a single explanatory framework. at this point, a few remarks are in order. firstly, and from a broad sociological point of view, this may just be a sign of our times: sequencing genetic material is an effective, and now rather inexpensive, way of obtaining prestigious research grants. proposals in genomics, synthetic biology, and other cognate fi elds with a strong engineering approach to biology can highlight potential fi ndings and even future applications, some of which are likely to be patentable and thus rapidly rentable from a fi nancial point of view. in brief, promoting more sequencing is likely to provide additional research money. while this may be a reason why taubenberger's team value more genetic sequencing other reasons of a more epistemological and historical nature must also be envisaged. a second reason to consider has to do with what historians and philosophers of science have called a scientifi c "style of practice" (keating and cambrosio ) . derivative of ian hacking's concept of "styles of reasoning" -itself inspired by alistair crombie's "style of scientifi c thinking in the european tradition"-(hacking ; crombie ), the notion of "style" typically refers to the historical formation of distinctive practices and methodologies in science. styles frame what counts as evidence, relevant questions to ask, truth-value, and sound explanation in distinct research and/or cultural contexts. alongside the development of individual styles of practice one fi nds the emergence of new standards for measurements, objectivity, proof, and so on (hacking ). though styles are fl exible they are not loose or relativist categories; they admit rules, systems of norms, stabilization techniques, and methods of justifi cation. as they progressively become stabilised over time and entrenched within scientifi c activities, however, the very existence of styles of reasoning and their historical development become taken for granted. while the notion of style is often employed to analyse scientifi c controversies (amsterdamska ; fujimura and chou ) , it is interesting to note here that the two styles at play in the present case study have grown in relative ignorance of each other. going back to the missing mutations, we can see that even though taubenberger's programme did not provide the answers it sought it could not be halted hastily, especially after gathering immense publicity and funding. on the contrary, it is expected that these scientists, working within their style of practice, continue to do so until all possibilities of fi nding the key to the exceptional virulence have been looked at and examined in detail. from this point of view, their persistence in seeking a complete molecular explanation makes sense -even if, from a public health and biosecurity point of view, their research raises ethical concerns about the development of dual-use technologies (rappert ) . moreover, the results obtained on the biology of infl uenza a viruses and the methods developed by taubenberger and his team now enable worldwide researchers to better understand the molecular differences between various infl uenza strains. what appears as a sign of determination in pursuing a research objective can also refl ect a lack of communication between distinct scientifi c communities, the problems of interdisciplinary work, the self-containment of styles of scientifi c practice, and/or the resistance offered by epistemological obstacles. the current gap between ecological and molecular explanations, as it emerged in the present case, may be due to the fact that functional explanations such as those constructed in molecular biology tend to appear "self-suffi cient", as historian and biologist michel morange recently put it ( ). this sort of epistemological obstacle means that, for many, there is no (obvious) need to complement molecular explanations with ecological considerations. to say that integration between mathematical modelling and molecular microbiological approaches has failed in this case would be going too far, however. indeed, integration of ecological and molecular approaches of virulence evolution has not even been seriously attempted so far. also, it would be misleading to suggest that molecular pathologists wholly ignore the environmental perspectives on virulence evolution and emerging diseases. yet when they do take them into account, the result does not necessarily amount to a better integration of data, theories, or methods but reveals, instead, the heights of disciplinary boundaries and the valuing of one style of practice over another. for example, in one of the last publications of taubenberger and his colleagues at the national institute of health, the authors concluded that the diminution of severity of infl uenza pandemics over time "is surely due in part to advances in medicine and public health, but it may also refl ect viral evolutionary choices that favor optimal transmissibility with minimal pathogenicitya virus that kills its host too fast or sends them to bed is not optimally transmissible " (morens et al. , ; emphasis added) . in other words, the biological interests of the virus will best be served by evolving lower virulence over time in order to facilitate transmission to new hosts, an explanation that rests on the conventional wisdom rejected by most evolutionary ecologists who advocate the theoretical trade-off model but that is still defended by some microbiologists. this may come as a surprise given that anthony fauci, on of the authors of the paper, and current head of the national institute for allergy and infectious diseases, has long criticized this view (see fauci ) . it shows, however, that branches of sciences in which the same problem is addressed, through distinct methodologies, can be surprisingly disconnected and separated by epistemic gaps, professional or institutional barriers. in other words, integration is no easy goal to achieve. problems within the molecular style of practice, however, are not only epistemological but also ethical and social. the publication of the whole sequence of the - strain in sparked lively debates among scientists and the public as it raised concerns as to whether it was safe to publish the methodology used to resurrect the pathogen (rappert ; selgelid ) . what if someone with nefarious intentions reconstructs the virus? how likely is it that this genetic information be used for harmful purposes? what if, by accident or not, the virus escapes into the environment? for some, like biologist richard h. ebright from rutgers university, "there is a risk, verging on inevitability, of accidental release of the virus" but "there is also a risk of deliberate release of the virus". yet others argued that the work of taubenberger and tumpey was entirely legitimate and could be applied to other areas and problems in virology such as the h n pandemic and "could have an immediate impact by helping scientists focus on detecting changes in the evolving h n virus that might make widespread transmission among humans more likely". the case of the spanish infl uenza pandemic is today a classical example of a technology that has the potential for "dual-use" research (i.e. it could help to understand the disease and fi ght it, but it also could be used to disseminate it further in a population). a recent case of potential dual-use consequences in infl uenza research involving a group of researchers led by ron fouchier in the netherlands and by yoshihiro kawaoka, from madison, in the u.s, led in january to a day suspension of research on infl uenza and virulent diseases, following consensus to delay publication. both teams had submitted a paper, to science and nature respectively, describing the methodology employed to artifi cially render an h n infl uenza strain transmissible between ferrets (which is, arguably, a reliable indicator of possible transmission to humans) due to a mutation on the hemmagglutinin protein. both studies have now been published (imai et al. ; herfst et al. ) . as an aside, it is interesting to see that these two studies, although undoubtedly driven by molecular biology questions, were based on classical approaches in evolutionary ecology known as serial passage experiments (ebert ). in the early s, the institute of medicine's report on microbial threats (lederberg, shope, and oaks ) and stephen morse's emerging viruses (morse ) have emphasized how emerging infectious diseases are posing a renewed threat to public health that needs to be addressed on a global scale, from the combined perspective of ecological and molecular approaches. the concept of emerging diseases has helped focus international efforts to contain infectious diseases within well-defi ned geographical and temporal limitations. with the (re)creation of the - infl uenza strain and others (e.g. yersinia pestis , polio virus, h n ), a different form of biological threat arises and it requires different political, institutional, and legal response mechanisms. indeed, while the threat of emerging infections was mostly perceived as coming from outside northern-hemisphere countries, it now appears to be growing from within the heartland of western countries itself. instead of stressing possible disease invasions in previously unexposed countries (or with only low incidence of a particular disease), recently developed technologies in synthetic biology and genomics have opened-up the possibility to artifi cially create new diseases, or to resurrect old ones such as the plague, the infl uenza strain responsible for the pandemic, and the polio virus (bos et al. ; taubenberger ; tumpey et al. ; celo et al. ; rosengard et al. ) . on the other hand, it might be argued that new variants appear constantly and the risk of a laboratory accident might be comparable to what happens naturally in the fi eld. moreover, while dual-use is a characteristic of most life sciences nowadays (atlas ), only a small number of experiments and experimental practices are, overall, seen as posing real threats to public health and global security (for a recent analysis see aucouturier ; morens et al. ) . finally, it is worth noting that dual-use technologies -like scientifi c research more generally -are often characterized by unexpected fi ndings such as, for instance, the accidental discovery that a modifi ed virus injected into mice was lethal to otherwise vaccinated animals (jackson et al. ) . as is often the case in science, the experimental system designed to answer certain questions opens-up theoretical and practical possibilities that could sometimes not be envisaged at the outset (rheinberger ) . if unpredictability and unforeseen results are truly the essence of scientifi c research, dual-use technologies are then an unavoidable tradeoff to deal with, a point that reinforces the need to develop appropriate governance responses to biomedical research programmes on pathogens and potentially pathogenic organisms (méthot ) . more generally, those new research avenues underline the need for the development of a "culture of responsibility" (nsabb ) in the life sciences, that is, a new ethos to address and balance questions of biosecurity and risk with scientifi c autonomy and progress, among others. the two most important glycoproteins allowing infl uenza viruses to invade host tissues -hemmagglutinin (ha) and neuraminidase (na) -were signifi cant molecular determinants of the virulence of infl uenza pandemics in and , and can yield potentially pathogenic effects when inserted into some animal models. considerable explanatory power was placed on these special proteins that seemed to provide a fi rst-hand, adequate, simple and certainly elegant mechanism to account for the exceptional virulence of the pandemic. indeed, the "most popular theory" was that the virus had "unique pathogenic properties, most likely encoded within the hemagglutinin protein" (holmes ) . identifying a molecular and genetic basis of virulence could not only provide a window into the most devastating epidemic of modern times, but could also help to prevent and predict those to come. overall, the remarkable technological success -i.e. the retrieving and sequencing of the avian virus -promised nothing less than to unlock one of the oldest and well-kept secrets in the whole of medical history. however, after sequencing the genome of the viral strain that killed perhaps up to million people according to the who estimates, both factors were found to be lacking in the killer strain. one might wonder about the extent to which it is possible to generalize from the example of the spanish infl uenza pandemic to other cases. researchers on ancient pathogens using high throughput technologies have recently claimed to identify the causal organism of the black death ( yersinia pestis ) in the fourteenth century and the sources of its virulence in the form of a single plasmid (schuenemann et al. ). however, determining why an organism is pathogenic or what makes it a pathogen is not straightforward and is rarely based on a specifi c structural characteristic alone (méthot c ) . as microbiologist charles nicolle once said ( ) , virulence is the expression of a "mosaic of powers" resulting from a constellation of factors that are irreducible to any particular structure and must be understood against a broad biological and even historical background. it is interesting to note, therefore, that the same team went on to revise its position in a subsequent article by pointing out the inherent limitations of molecular-oriented explanation and, furthermore, emphasized the need to widen the explanation and integrate ecological factors as well. they write: regardless, although no extant y. pestis strain possesses the same genetic profi le as our ancient organism, our data suggest that few changes in known virulence-associated genes have accrued in the organism's years of evolution as a human pathogen, further suggesting that its perceived increased virulence in history may not be due to novel fi xed point mutations detectable via the analytical approach described here. at our current resolution, we posit that molecular changes in pathogens are but one component of a constellation of factors contributing to changing infectious disease prevalence and severity, where genetics of the host population, climate, vector dynamics, social conditions and synergistic interactions with concurrent diseases should be foremost in discussions of population susceptibility to infectious disease and host-pathogen relationships with reference to y. pestis infections (bos et al. ; emphasis added) . in sum, the study of bos et al. ( ) did not reveal any signifi cant genetic or evolutionary change in years that could explain the virulence of plague in the fourteenth century. as a consequence, they argue that a molecular approach only provides an incomplete picture when applied in isolation, and that a complementary ecological perspective is needed. more precisely, the more recent study emphasizes that a full understanding of the evolution of virulence requires a multi-dimensional framework that encompasses host resistance, ecological factors, and the interactions between the different diseases occuring in a well-defi ned geographical area over a specifi c time period. to go beyond the limitations of analytical approaches that investigate one disease at a time, a synthetic and global approach is necessary in order to understand more broadly the evolution of emerging diseases that compose the past, present, and future of any "pathocenosis" (grmek ) . to conclude, our analyses of the case of the spanish infl uenza 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re-emerging disease infl uenza virus a pathogenicity: the pivotal role of hemagglutinin evolution and ecology of infl uenza a viruses global public health vigilance. creating a world on alert the spanish infl uenza pandemic of - . new perspectives. . studies in the social history of medicine origins of major human infectious diseases key: cord- -m dkkbq authors: pulliam, juliet r. c. title: viral host jumps: moving toward a predictive framework date: - - journal: ecohealth doi: . /s - - - sha: doc_id: cord_uid: m dkkbq in order to predict pathogen emergence, we must distinguish between emergence phenomena that occur via different processes. focusing on the appearance of viral pathogens in new host species, i outline a framework that uses specific molecular characteristics to rank virus families by their expected a priori ability to complete each of three steps in the emergence process (encounter, infection, and propagation). i then discuss the degree to which the patterns expected, based solely on molecular-level structural characteristics, agree with observations regarding the ability of animal viruses to infect humans. this approach yields predictions consistent with empirical observations regarding the ability of specific viral families to infect novel host species but highlights the need for consideration of other factors, such as the ecology of host interactions and the determinants of cellular susceptibility and permissivity to specific virus groups, when trying to predict the frequency with which a virus will encounter a novel host species or the probability of propagation within a novel host species once infection has occurred. emerging infectious diseases present challenges to the scientist and layperson alike (morse, ; burke, ; daszak et al., ; cleaveland et al., ; dobson and foufopoulos, ; taylor et al., ) . these infections of wildlife, domestic animals, and humans seem to be increasing and more problematic every year. the cause of the recent increase in scientific and public interest in emerging infections is probably twofold. on the one hand, technological advances in detection methods and in global communications allow us to be increasingly aware of emergence events as they occur (garnett and holmes, ) . many pathogens, including west nile virus, monkeypox virus, and h n highly path-ogenic avian influenza (hpai) virus have attracted widespread media attention. online news sources provide gripping, up-to-the-minute accounts of outbreaks occurring in locations that their primary audience may not even be able to identify on a map, such as the multimedia presentations produced by bbc news in of a marburg virus outbreak in angola (phillips, ) . on the other hand, anthropogenic activities have increased the actual frequency of pathogen emergence. habitat modification brings previously separated populations into contact and disrupts natural mechanisms regulating population dynamics. for example, increases in rodent populations due to clear-cutting of forested areas for the planting of food crops (particularly corn) is thought to have been responsible for outbreaks of several hemorrhagic fever viruses in south america, including junín and machupo viruses (buchmeier et al., ) . barriers limiting pathogen transmission are altered as we change the landscape and increase global transportation, providing unprecedented opportunities for transmission to new populations and species (schrag and weiner, ; smith et al., ) . these changes have implications not only for human health but for wildlife conservation and domestic animal health. long-distance animal transport, both intentional (for agriculture or the pet trade) and unintentional, facilitates pathogen introduction (daszak et al., ) . for example, illegal bird transport is the suspected source of the recent introduction of h n hpai into a poultry farm in the uk (promed-mail, abc) , and accidental transportation of mosquitoes aboard aircraft is thought to pose the highest risk for the introduction of west nile virus into hawaii and the galapagos islands (kilpatrick et al., (kilpatrick et al., , . considering the broad public and scientific interest in infectious disease emergence, surprisingly little work has been done to quantitatively describe broad-scale patterns of emergence in light of pathogen characteristics. much work on emerging infections has focused on specific emergence events, trying to determine when, where, and how a particular pathogen entered a new host population. the emergence of human immunodeficiency virus (hiv), for example, has been well-studied on both ecological and molecular levels. strong evidence suggests that the bushmeat trade in west africa has facilitated repeated retrovirus transmission events from wild primates into humans (wolfe et al., ) , and independent introductions of hiv- subgroups (m, n, o) from chimpanzees (pan troglodytes) and hiv- subtypes (a, b) from sooty mangabeys (cercocebus atys) are believed to be responsible for the ongoing hiv/aids pandemic (sharp et al., ) . in order to predict (rather than reconstruct) pathogen emergence, however, it is necessary to extend the domain of inquiry beyond the focus on a single disease. specific ecological processes and pathogen characteristics must affect the probability of emergence, and until these factors are recognized we will have little predictive power. burke has suggested that the high ''evolvability'' of rna viruses facilitates host jumps (burke, ) . such evolvability is derived from a combination of high replication error rates and the ability to reassort and recombine. although he makes no attempt to quantitatively determine the relative frequency of emergence for different types of pathogens, burke claims that recent pandemics in humans and wildlife have mostly been caused by rna viruses, citing multiple examples (influenza a, hiv- , enteroviruses and , human t-cell lymphoma virus, three paramyxoviruses, porcine respiratory coronavirus, and a calicivirus that causes hemorrhagic disease in rabbits). he goes on to predict that future global pandemics will be caused by groups of viruses that have caused major epidemics in humans or animals and that have high intrinsic evolvability; however, this approach does not allow for identification of viral groups with high emergence potential that have not produced large epidemics to date. in particular, if undiscovered virus families exist that have a high potential for emergence-whether in humans or in animals-a priori identification of this potential may facilitate preparedness for and rapid response to emergence events. a more quantitative approach was taken by three studies published in a special issue of philosophical transactions of the royal society of london in . the first two of these studies (cleaveland et al., ; taylor et al., ) used a broad-scale literature search to construct a database of known infectious diseases of humans and domestic animals and to classify these diseases as emerging or not. the authors were then able to describe general patterns related to emergence status. they found that viruses are significantly more likely to be classified as emerging than are bacteria, fungi, helminths, or protozoa and that the ability to infect multiple host species is a significant risk factor for emergence in humans and domestic livestock. a recent update of the work on human pathogens has confirmed these findings with a larger dataset (woolhouse and gowtage-sequeria, ) . a similar approach was taken by dobson and foufopoulos ( ) , who surveyed emerging infectious diseases of wildlife based on promed reports over a -year period. emerging pathogens of wildlife were also found to be primarily viral. the results of these studies suggest that further examination of the molecular characteristics that determine viral emergence will go a long way toward being able to predict pathogen emergence. to develop a predictive framework, however, we must distinguish between the different phenomena that the term ''emergence'' commonly encompasses. these include the appearance of a pathogen in a new host species (woolhouse, ; antia et al., ; fenton and pedersen, ; , the appearance of a pathogen in a new population of an established host species (or new geographic region) (lederberg et al., ; cleaveland et al., ; taylor et al., ; smolinski et al., ; smith et al., ) , the appearance of an antimicrobial-resistant variant of a pathogen within an established host population (lederberg et al., ; smolinski et al., ) , and a change in the immunological interaction between a pathogen and an established host population (smolinski et al., ) . different mechanisms drive each of these phenomena; therefore, the ability to predict each type of emergence will require different approaches. i focus on the appearance of viral pathogens in novel host species. i first describe the process by which this type of emergence occurs and then ask the question of whether specific molecular characteristics can be expected a priori to affect the crucial steps in this process. this approach allows for the generation of testable hypotheses regarding the emergence potential of specific viral groups. i outline these hypotheses and then determine whether expectations based on the molecular characteristics discussed correspond to observed broad-scale patterns. agreement with observed patterns may suggest that the molecular characteristics explored will yield substantial predictive power and a useful framework for ranking virus groups in terms of emergence potential. inconsistencies between the patterns expected based on molecular characteristics alone and observed patterns highlight specific parts of the emergence process as highly dependent on host-host or host-virus interactions and less dependent on molecular characteristics of the viruses themselves. a glossary is provided to clarify the use of terms that may be unfamiliar to the reader or that are used inconsistently within the current emerging infectious disease literature. a virus is an obligate intracellular parasite that relies on the molecular machinery of its host for reproduction. all viruses comprise genetic material (rna or dna) and a protein coat. virus particles (virions) of many species also contain host-derived lipids and carbohydrates. to reproduce, a virus must encounter a host organism and make its way to the site of replication within a host cell that is both susceptible and permissive. the virus then makes new copies of itself, which requires transcription of the genome to produce mrna, protein synthesis, genome replication, and packaging. new virions must exit the cell to infect new host cells and, potentially, new host organisms. to persist, emerging viruses must be able to perform all of these steps within their new host species (webby et al., ) . host jumps, or cross-species transmission events that result in the successful infection of a potential host species, may encounter barriers at any step in this life-cycle. molecular characteristics of the virus itself are expected to play a large role in determining both whether a host jump occurs and the probability of transmission between individuals of a new host species. host jumps will be considered to occur as a three-step process . encounter between a virion and a potential host species, . infection, or replication within an individual of a novel host species, and . propagation, or transmission of infection from one individual of the new host species to another. the probability that, and frequency with which, a potential new host will encounter a virus circulating in another species may be determined by the nature of interaction between current and potential hosts, by the frequency, duration, and distribution of viral shedding in the current host population, and by the virion's subsequent stability in the environment. ecological interactions between current and potential host species can be divided into four categories: . those involving contact with bodily fluids, . those involving direct contact between individuals but not contact with bodily fluid, . those involving contact with a shared resource, and . those involving spatial overlap. interactions involving between-species contact with bodily fluid, such as predation, seem most likely to facilitate host jumps. slaughter and consumption of bush-meat, for example, is responsible for the transmission of multiple retroviruses from wild primates to humans (wolfe et al., ) , lending evidence to the idea that host jumps are likely to occur as the result of trophic interactions. shared vectors are also known to mediate transmission of viral infections between species, as occurs when japanese encephalitis virus is transmitted between waterfowl (particularly herons), pigs, and people (endy and nisalak, ) . because predators have direct contact with their prey, they are likely to be exposed to any infections that their prey harbor, regardless of the site of excretion or transmission route, whereas a shared vector will only facilitate cross-species transmission of pathogens with high viremia. frequent direct contact, such as commensal relationships between humans and domestic carnivores, is the second most likely type of interaction to lead to the encounter of virions shed by other host species. encounter will be high for infections transmitted via respiratory secretions, urine, feces, or skin lesions (such as an infectious rash). depending on the nature of the commensal relationship, viral shedding in saliva may also lead to frequent encounter, either directly (as a result of licking or biting) or via a fomite (such as a pet's toy). virions found exclusively in the semen, milk, and blood, on the other hand, will have a low probability of transmission between commensal species. encounter may also occur between species competing for a shared resource. while competition rarely involves direct contact between individuals of different species, dissemination of virus particles through respiratory secretions, urine, or feces may be concentrated around a shared resource. under these circumstances-as with fomite transmission between commensal species-the stability of the virion in the environment is likely to be important in determining the probability of encounter between an infectious virus particle and a potential host species. similarly, transmission between species that overlap only in their spatial distribution may occur when virus particles are resistant to degradation but may be less frequent than in the case where current and potential host species compete for a particular resource. figure summarizes the expected interaction between the ecological relationships of current and potential host species and the frequency of encounter.because direct contact with infectious fluids implies encounter, ecological considerations will dominate the encounter process for the majority of source and recipient host pairs. molecular considerations will be most important when there is no direct contact or shared vector between current and potential hosts. in such cases, the frequency with which a potential new host encounters infectious virus particles will depend on virion stability in the environment. two main properties of the virion are likely to influence the rate of inactivation: whether the virus particle requires an envelope for entry into the host cell and the arrangement of structural proteins within the virion. though the relative rates of inactivation of different virus species depend on specific environmental conditions (such as temperature and relative humidity), requiring an envelope for successful cellular infection may generally decrease the ability of a virus to survive outside a host, since the phospholipid membrane will be sensitive to desiccation, ultraviolet radiation, changes in ph, and ozone toxicity (cox, ) . thus, the ability to enter host cells without an envelope is expected to increase emergence potential. similarly, isometric and complex structural arrangements contain many contacts between proteins relative to their helical counterparts. these arrangements are therefore more stable against physical damage, reducing the rate of inactivation and increasing expected frequency of encounter. once a virion encounters an individual of a potential host species, the probability it will be able to replicate depends on the specificity of infection and the ability to generate genetic diversity. viruses preadapted for emergence are likely to enter cells via highly conserved receptors (woolhouse, ) , to have generalized immune evasion strategies that modulate the host's innate immune response (webby et al., ) , and to have the potential for rapid evolution (burke, (burke, , . since the receptors that mediate host-cell entry are not generally known, grouping viruses by the level of taxonomic conservation of the receptors required for cell entry remains an important goal for the future. similarly, while interference with host immunity has been demonstrated for several emerging viruses (webby et al., ) , the ability of other viruses to modulate the innate immune response has been largely unexplored. the site of replication within a cell may also influence infection specificity. entry into the nucleus provides an additional barrier to successful replication not present for viruses that replicate in the cytoplasm. replication in the nucleus may therefore increase specificity and decrease potential for infection of novel host species. finally, the generation of genetic diversity increases the probability of infecting a potential host species by increasing the genetic state-space covered by the virus population and therefore the chance that some virus particles will successfully infect a novel host and reproduce (burke, ; webby et al., ) . although successful invaders may make up a tiny fraction of the population within the source host species, natural selection will increase the abundance of these variants within an infected individual of the new host species. the ability to generate genetic diversity depends primarily on the replication error rate and the potential for exchange of genomic material between genetically distinct virus particles during cellular coinfection (burke, ) . while many authors (burke, (burke, , cleaveland et al., ; suggest that rna viruses are more likely to emerge than dna viruses because of their high replication error rates, it may be more useful to focus on the primary reason for the difference in error rates: the availability of proofreading mechanisms during genome replication. although host proofreading mechanisms are used when replication relies on a host-derived polymerase, most viruses that encode their own polymerase (except large dna viruses; king et al., ; huang et al., ; willer et al., ) lack error correction and therefore have relatively high replication error rates. additionally, the exchange of genetic material between closely related viruses is facilitated by two processes during cellular coinfection: . recombination (exchange of genetic information during replication, usually via template switching), and . reassortment (copackaging of genome segments from genetically distinct parent virions). packaging multiple genome copies facilitates the recombination process (lederberg et al., ; burke, ) ; however, additional mechanisms that may promote viral recombination are poorly understood. reassortment requires only that the viral genome be divided into distinct segments (analogous to eukaryotic chromosomes) and that cellular coinfection occurs in nature. the probability of transmission between individuals of a new host species is of interest in addition to the probability of infection of a novel species (may et al., ) . evolutionary potential to increase spread between individuals of the new host, however, should rely on the virus's ability to produce genetic diversity, similar to the ability to infect and reproduce in a new host species (schrag and weiner, ) . as demonstrated by antia and colleagues, even a small increase in the basic reproductive number of a pathogen within a new host species can substantially increase the probability that the pathogen will eventually evolve the ability to propagate at a sufficient level to produce an epidemic within the new host species (antia et al., ) . substantial evidence suggests that rapid adaptation of plant and animal populations to a novel environment is more likely to occur through limited gene flow between related (but significantly diverged) populations than through point mutation (lewontin and birch, ; reiseberg et al., ) . if this holds for virus populations, recombination and reassortment may be the primary evolutionary factors permitting adaptation to propagation within a new host species; however, several virus species well known for their ability to propagate within a novel host species demonstrate that figure . the expected interaction between the ecological relationships of current and potential host species and the frequency of viral encounter. the darkest boxes represent interactions where the frequency of encounter is likely to be high, the lightest boxes represent interactions where encounter is expected to occur at very low frequency, and medium boxes represent interactions where the frequency of encounter will be highly dependent on the stability of the infectious virion in the environment, as described in the text. for all ecological relationships between species, the actual frequency of encounter via different transmission routes will depend on the exact nature of the relationship. substantial genetic change is not necessary for adaptation to propagation in all cases. sars coronavirus, for example, appears to have adapted to transmission between humans through a series of point mutations in multiple genes (holmes, ) . several mutations in the receptor-binding domain of the sars coronavirus glycoprotein that increased binding affinity to human ace (the receptor for entry into the cell) are well documented and appear to have been especially important (the chinese sars molecular epidemiology consortium, ; li et al., ) . table groups mammalian virus families according to each of the molecular traits discussed above for which data are available. mammalian viruses are used for the purposes of illustration because they are particularly well studied and because mammals represent many of the source and recipient host species of primary interest (e.g., humans, domestic livestock, and domestic carnivores). a rough assessment of expected emergence potential is made by assigning scores for encounter, infection, and propagation potential, which are given and described further in table . the score indicates how many traits a virus group possesses that are expected to increase success in that step, based on two standard reference texts (van regenmortel et al., ; tidona and darai, ). thus, virus families can be ranked based on molecular characteristics alone in order of the a priori expectation that they will be able to complete a particular emergence step. complex or isometric protein arrangement and the absence of an envelope are expected to increase encounter when there is no direct contact between current and potential host species. virus families with both of these characteristics provide some examples of host-jumping viruses; however, the absence of these characteristics clearly does not prohibit cross-species virus transmission. the orthomyxoviridae, coronaviridae, filoviridae, paramyxoviridae, and rhabdoviridae all have encounter scores of / but provide a plethora of examples of host-jumping virus species: the influenzaviruses, sars coronavirus, ebola and marburg viruses, hendra and nipah viruses, and rabies virus, respectively. assuming the traits of interest are good indicators of a virion's susceptibility to environmental stresses, stability in the environment does not seem to be a necessary prerequisite for cross-species transmission, probably because many hosts interact in a way that permit direct viral transmission, as noted earlier. in order to assess how well expected patterns of cross-species infection match observed patterns, it is necessary to consider infection within a context where encounter is expected to occur. when we consider the probability that a virus population will be able to reproduce within a potential host that has close contact with a current host, the characteristics in the left-hand columns of table dominate emergence potential. reassortment or recombination, low replication fidelity (i.e., lack of proofreading), and the ability to complete replication within the cytoplasm are all expected to increase cross-species infection potential. using these three criteria to rank mammalian virus families in terms of expected infection potential indicates that three families present the highest risk: arenaviridae, which comprises multiple viral species transmitted from rodents to commensal humans, including those responsible for lassa fever and four south american hemorrhagic fevers; bunyaviridae, which includes the hantaviruses, similarly transmitted from rodents to humans; and reoviridae, of which of the species that circulate among rodents and livestock are known to infect humans (tidona and darai, ) . in addition, nearly all high-profile viral zoonoses have at least two of the three characteristics expected to promote infection of a novel host species, including hiv-like viruses (retroviridae), influenza viruses (orthomyxoviridae), rabies virus (rhabdoviridae), ebola and marburg viruses (filoviridae), hendra and nipah viruses (paramyxoviridae), and sars coronavirus (coronaviridae) (tidona and darai, ) . one exception is monkeypox virus (poxviridae), which replicates in the cytoplasm but has high replication fidelity and, according to the simple genome-packaging division used here, little recombination/reassortment potential. it is worth noting that recombination of poxviridae species has been demonstrated in laboratory experiments (see, for example, yao and evans, ) . thus, rather than implying that recombination/reassortment potential is not an important contributor to infection of novel host species, this exception may indicate that the simple molecular traits considered here are insufficient to capture the full range of recombination mechanisms available to host-jumping viruses. of the mammal viruses lacking all three traits associated with infection of a novel host (genome reassortment a recombination potential is determined by the number of copies of the genome that are packaged in a virus particle (although other, unknown factors also facilitate recombination); reassortment potential is determined by the number of genome segments (single versus multiple). high recombination or reassortment potential is expected to increase potential for both infection and propagation. b replication fidelity is determined by the origin of the polymerase (host or viral) and the associated proofreading activities. virus groups that rely on a host polymerase for replication are subject to host proofreading mechanisms and have high replication fidelity. most virus groups that encode their own polymerase have no proofreading and therefore low replication fidelity; however, polymerases encoded by members of the poxviridae, adenoviridae, and herpesviridae are known to have ' to ' exonuclease activity (king et al., ; huang et al., ; willer et al., ) , which is the primary type of error correction expected to affect viral replication fidelity. viruses in these groups are therefore expected to have high replication fidelity. low replication fidelity is expected to promote the generation of genetic diversity and therefore increase potential for infection and propagation. c the site of replication is considered to be the cytoplasm if all steps in the replication cycle take place within the cytoplasm of the infected cell; the site of replication is denoted as the nucleus if any step in the replication cycle (e.g., genome replication or transcription into mrna) requires nuclear entry. the ability to complete replication within the cytoplasm is expected to increase relative infection potential, as nuclear entry provides an additional barrier to replication. d the presence (+) or absence (-) of a lipid envelope will affect the stability of a virion in the environment. e the arrangement of structural proteins within the virion is denoted as isometric, helical, or complex. isometric and complex arrangements are more structurally stable than helical ones due to the larger number of protein contacts. f virions of some genera of the poxviridae are enveloped upon exiting the cell; however, an intact envelope is not required for the virus particle to remain infectious (tidona and darai, ) . g the lone member of the family asfarviridae, african swine fever virus, encodes a viral polymerase responsible for genome replication (fauquet et al., ) . no studies were found in web of science searches that examined whether the viral polymerase has ' to ' exonuclease activity; however, the other families of large dna viruses do encode polymerases with known proofreading activity (king et al., ; huang et al., ; willer et al., ) and it is assumed here that the polymerase encoded by african swine fever virus has undocumented exonuclease activity. or recombination, error-prone replication, and ability to replicate in the cytoplasm), none is known to infect humans, regardless of close association between humans and the animal host. though rough, these three criteria appear to yield good predictive power regarding which viruses will be able to infect and replicate within novel host species. finally, the probability that a viral epidemic will occur via transmission within the new host species is of interest. table accounts only for the role of generation of genetic diversity in propagation. drawing from examples of mammal viruses that jump to humans, it becomes clear that other factors must be important determinants of propagation. while hiv (retroviridae) and influenza (orthomyxoviridae) propagate well in human populations, as predicted, members of the arenaviridae and bunyaviridae are rarely transmitted from person to person, despite having both characteristics expected to be associated with high chances of propagation. as with encounter, molecular characteristics of the viruses themselves do not appear to provide an a priori indication of a viral group's ability to propagate within a table . encounter, infection, and propagation scores for mammalian virus families, determined from table virus family a encounter b infection c propagation d reoviridae / / / retroviridae / / / astroviridae / / / caliciviridae / / / picornaviridae / / / poxviridae / / / adenoviridae / / / circoviridae / / / papillomaviridae / / / parvoviridae / / / polyomaviridae / / / arenaviridae / / / bunyaviridae / / / arteriviridae / / / flaviviridae / / / togaviridae / / / bornaviridae / / / hepadnaviridae / / / asfarviridae / / / herpesviridae / / / orthomyxoviridae / / / coronaviridae / / / filoviridae / / / paramyxoviridae / / / rhabdoviridae / / / a families are sorted in order of decreasing encounter score, then decreasing infection score, and finally decreasing propagation score. b the encounter score denotes the number of characteristics a group has that are expected to increase exposure frequency when the current and potential hosts have little or no interaction. characteristics that increase encounter will be those that increase the stability of a virion in the environment: being infectious without a lipid envelope and having an isometric or complex virion. c the infection score denotes the number of characteristics a group has that are expected to increase the ability to infect a novel host species: high recombination/reassortment potential, low replication fidelity, and an ability to complete replication within the cytoplasm. d the propagation score denotes the number of characteristics a group has that are expected to increase propagation: high recombination/reassortment potential and low replication fidelity. novel host species. host ecology and host-virus interactions rather than general molecular characteristics appear to determine propagation potential. the approach taken so far has shown that the ability to infect a novel host species depends heavily on molecular traits of the virus, independent of host ecology and details of the host-virus interaction; however, the approach has also highlighted the need to understand the ecology of interactions between species when considering expectations of encounter and the ecology of within-species interactions when considering expectations of propagation. host-virus interactions occurring primarily at the molecular level may also to play a large role in determining encounter and propagation, and the next steps in developing a predictive framework will examine these issues more closely. stability in the environment does not appear to limit the step of pathogen encounter for most host-jumping viruses. this is not surprising in light of the expected roles of host-host and host-virus interactions in determining encounter, which are outlined above and depicted in figure . determining whether the proposed interactions work as predicted will be extremely difficult. in fact, observation of encounter independent of infection may be impossible, whether in the field, in the laboratory, or using a synthetic database approach. one useful approach may be to look within a particular category of ecological relationships and at potential source-potential recipient host pairs. the relative frequency of different sites of excretion of viruses infecting the source host could then be compared to the relative frequency of different sites of excretion of viruses transmitted from the source host to the recipient host. databases used for previous analyses of emerging infections in livestock, domestic carnivores, and humans (cleaveland et al., ; taylor et al., ; woolhouse and gowtage-sequeria, ) could be combined with data on the site of excretion as a first step in quantifying the interactions between ecological relationships, site of viral excretion, and risk of encounter. while this approach will measure infection rather than addressing encounter directly, infection implies that encounter and factors that affect infection directly, such as the molecular traits discussed here, can be controlled for in the analysis. consistent patterns of shedding in the source host across host pairs with a particular type of ecological relationship will indicate that viruses shed through particular sites of excretion are more likely to be encountered than others. as ecological relationships between species change, knowledge of these patterns will provide an indication of the risk of encounter with viral species harbored by a newly contacted host species. in the pet trade, for example, where species that are isolated in nature live in artificial, commensal-like conditions, sometimes at high densities, the expectations laid out in figure suggest that viruses shed via respiratory secretions, urine, feces, or skin lesions-or those shed in the saliva if animals are kept in close enough quarters for biting to occur-are likely to be transmitted between species. this type of change in ecological relationships was responsible for the emergence of monkeypox virus in the us in , as african rodents were brought into contact with prairie dogs, which then transmitted the virus to humans (guarner et al., ) . practical application of this type of analysis is likely to be case-specific: the approach may be useful when evaluating threats in a specific situation, such as when interested in risks to a particular endangered wildlife species (specific recipient host) or when interested in risks associated with introduction of a particular species of animal into the wildlife pet trade or wild animal markets (specific source host). the ability of some pathogens to propagate within novel host species has highlighted both our economic vulnerability to previously unknown pathogens and weaknesses in our current medical and veterinary infrastructure (and in communication between these entities; see kahn, , for a recent discussion). much epidemiological theory has been devoted to understanding the effect of within-species host interactions on the invasion and persistence of infectious diseases in novel host populations (anderson and may, ) . understanding molecular-level determinants of propagation potential is also an important goal, however difficult it may be. factors determining a virus's ability to propagate appear to depend heavily the distribution of susceptible and permissive cells within the host. malaysian nipah virus, for example, infects lung tissue of domestic pigs, causing severe respiratory symptoms that easily disseminate virus particles (hooper et al., ) . humans living and working near infected pigs can also become infected; however, very few virions are able to replicate in human lung tissue. instead, virions replicate in cerebral tissue, producing an encephalitis (hooper et al., ) . replication in the brain does not promote propagation, and transmission between humans does not occur. a variant of nipah virus found in bangladesh, on the other hand, replicates in human lung tissue, causes respiratory disease, and can propagate within the human population (hsu et al., ) . even differences in tissue tropism within a given organ system may affect propagation potential. one hypothesis as to why h n hpai has so far failed to produce large chains of humanto-human transmission is that its viral attachment is concentrated in the lower respiratory tract, whereas access to the upper respiratory tract is insufficient to produce large amounts of aerosolized virus (van riel et al., ) . some ability to generate genetic diversity may be necessary for significant transmission within a novel host species (antia et al., ) , as was seen with sars (holmes, ) ; however, a more accurate assessment of propagation potential requires a detailed understanding of the determinants of cellular susceptibility and permissivity. large-scale characterization of receptors used for cellular entry and of the distribution of these receptors within humans, domestic animals, and wildlife could provide an invaluable tool in predicting specific interactions between viruses and potential hosts. while this task may seem daunting, host-jumping viruses often gain cellular entry via membrane proteins conserved between source and recipient host groups (woolhouse, ) . thus, characterization and distribution studies should target potential receptor proteins that are conserved among and between vertebrate lineages to maximize the practical benefit of this type of basic research. knowledge of the factors that increase a pathogen's ability to jump to, and propagate in, a new host will be invaluable in the effort to prevent, prepare for, and predict viral threats to human and animal health. we currently have a good general understanding of the three steps required for a host jump to occur: encounter is heavily dependent on ecological relationships between species and often requires close contact between a recipient host and the site of viral excretion in the source host; infection depends on a viral species' evolutionary potential and the barriers to replication that it must overcome in a new host cell, which in turn are tied to specific, conserved viral molecular characteristics; and propagation may be facilitated by a viral species' evolutionary potential but also relies on conservation of underlying mechanisms of cellular entry and other determinants of tissue tropism. the next step toward being able to predict viral host jumps between species of interest is to quantify our understanding of the determinants of encounter and infection and thereby develop indicators of risk from a given viral group. virus groups for which the risk of encounter and infection is substantial, particularly those with inherently high evolvability, should be prioritized for receptor characterization, and those found to rely on broadly-conserved receptors should be considered at high risk for emergence. researchers interested in characterizing the risk to a particular target species, such as humans, domestic animals, or endangered wildlife, may then be advised to determine the distribution of receptors-and any other determinants of susceptibility and permissivity that can be characterized-within the species of interest. glossary current/established host. a species supporting active viral infection. dissemination. the distribution of infectious virus particles in the environment. emergence. the appearance (or reappearance) of an infection in a new host population or species, or a change in interaction between host and pathogen due to pathogen evolution or changes in host immunity. host jumps (see below) are the primary type of emergence discussed here. emergence potential. the probability that a given virus population will be able to infect and reproduce in a potential host species. encounter. contact between an infectious virus particle and a species other than its source host species. envelope. the host-derived lipoprotein membrane that surrounds the protein core of some virus particles. most enveloped virions require an intact membrane for receptor binding and entry into the host cell. host jump. a cross-species transmission event that results in the successful infection of the potential host species. infection. the entry and replication of a virus particle in an individual, referring here to infection of an individual of a new host species. new/novel host. a species that has acquired a viral infection as the result of a host jump. permissive cell. a cell in which a virus particle can replicate. potential host. a species that does not currently support active viral infection. propagation. transmission of an infection between individuals of a single host species, usually referring to transmission within the new host species. reassortment. the packaging of genome segments from genetically distinct parent virions in a coinfected cell. receptor. the site on the outer membrane of a cell to which a virion binds for cellular entry. recipient host. a host species, or individual of a host species, that has acquired an infection as the result of a host jump. recombination. the exchange of genetic information between closely-related virus particles during replication in a coinfected cell, usually via template switching. source host. a current host species, or individual of a current host species, that produces virus particles actually or potentially able to infect a potential host species. susceptible cell. a cell that permits viral entry. tissue tropism. the affinity of a virus particle for cells of susceptible and permissive tissues. zoonosis. an animal infection that can be transmitted to and replicate in humans. the role of evolution in the emergence of infectious diseases arenaviridae: the viruses and their replication recombination in hiv: an important evolutionary strategy evolvability of emerging viruses diseases of humans and their domestic mammals: pathogen characteristics, host range, and the risk of emergence airborne bacteria and viruses emerging infectious diseases of wildlife-threats to biodiversity and human health anthropogenic environmental change and the emergence of infectious diseases in wildlife emerging infectious pathogens of wildlife japanese encephalitis virus: ecology and epidemiology community epidemiology framework for classifying disease threats the ecology of emergent infectious disease monkeypox transmission and pathogenesis in prairie dogs adaptation of sars coronavirus to humans comparative pathology of the diseases caused by hendra and nipah viruses nipah virus encephalitis reemergence effects of exonuclease activity and nucleotide selectivity of the herpes simplex virus dna polymerase on the fidelity of dna replication in vivo confronting zoonoses, linking human and veterinary medicine predicting pathogen introduction: west nile virus spread to galapagos quantitative risk assessment of the pathways by which west nile virus could reach hawaii processive proofreading by the adenovirus dna polymerase-association with the priming protein reduces exonucleolytic degradation hybridization as a source of variation for adaptation to new environments structure of sars coronavirus spike receptor-binding domain complexed with receptor infectious disease dynamics: what characterizes a successful invader? examining the origins of emerging viruses video: angola hit by deadly marburg virus avian influenza ( ): uk-hungary virus sequence avian influenza, poultry vs migratory birds major ecological transitions in wild sunflowers facilitated by hybridization emerging infectious disease: what are the relative roles of ecology and evolution? the origins of acquired immune deficiency syndrome viruses: where and when? globalization of human infectious disease microbial threats to health: emergence, detection, and response. washington, dc: the national academies press taylor lh molecular evolution of the sars coronavirus during the course of the sars epidemic in china virus taxonomy: classification and nomenclature of viruses molecular constraints to interspecies transmission of viral pathogens in vitro concatemer formation catalyzed by vaccinia virus dna polymerase naturally acquired simian retrovirus infections in central african hunters population biology of emerging and reemerging pathogens host range and emerging and reemerging infectious diseases emerging pathogens: the epidemiology and evolution of species jumps effects of dna structure and homology length on vaccinia virus recombination the author gratefully acknowledges l. enquist for early discussion of these ideas and insightful comments on the manuscript; p. daszak key: cord- - wezrr b authors: watanabe, tokiko; kawaoka, yoshihiro title: villains or heroes? the raison d'être of viruses date: - - journal: clin transl immunology doi: . /cti . sha: doc_id: cord_uid: wezrr b the relationship between humans and viruses has a long history. since the first identification of viruses in the th century, we have considered them to be ‘pathogens’ and have studied their mechanisms of replication and pathogenicity to combat the diseases that they cause. however, the relationships between hosts and viruses are various and virus infections do not necessarily cause diseases in their hosts. rather, recent studies have shown that viral infections sometimes have beneficial effects on the biological functions and/or evolution of hosts. here, we provide some insight into the positive side of viruses. viruses, which consist of nucleic acid encased in a protein shell, are parasites of host organisms. the term 'virus' comes from the latin word 'venom', which means poison, because a virus is generally considered to be a causative agent like a poison that causes infectious diseases. these tiny, living entities have considerable import, because they can cause substantial damage to humans and non-human animals and other living organisms. the relationship between humankind and viruses has a long history. for example, the earliest evidence of smallpox was found in -year-old egyptian mummies, who had smallpox-like eruptions on their skins. the overall mortality rate of smallpox was around %, making it one of the most feared infectious diseases. in - , during world war i, influenza a virus caused the spanish flu pandemic, resulting in infection of approximately million people and more than - million death worldwide. since the initial isolation of viruses in the th century, scientists have identified and characterised a wide variety of viruses, and the field of virology has progressed remarkably since then, enabling us to combat the frequently deadly effects of these viruses. one of the greatest achievements is the complete eradication of smallpox. although smallpox was once rampant in the world, vaccination of the entire population has eradicated this disease. similarly, the poliovirus vaccine has significantly reduced the incidence of poliomyelitis. despite the progress of virology, we still have many unconquered viral diseases and we are confronted with the problem of emerging infectious diseases, which are caused by newly identified species or strains. for example, ebola virus disease and acquired immunodeficiency syndrome emerged in and , respectively, - and more recently, severe acute respiratory syndrome (sars), highly pathogenic avian influenza viruses and middle east respiratory syndrome (mers) have appeared in human society. [ ] [ ] [ ] [ ] [ ] [ ] therefore, it is important to continue studying the mechanisms of viral replication and pathogenicity. yet, these negative aspects of viruses do not tell the whole story since the relationships between hosts and viruses are multitudinous, and virus infections do not necessarily lead to disease symptoms in hosts. rather, recent studies suggest that there are viruses that are beneficial to the biological functions and/or evolution of their hosts. recently, we established a research consortium, designated as 'neo-virology', which is supported by grants-in-aid for scientific research on innovative areas from the ministry of education, culture, science, sports, and technology (mext) of japan. in this consortium, we define a virus as a component of the global ecosystem. our aim was to elucidate the roles of viruses in host organisms and the global ecosystem, in contrast to traditional virology research, which tends to focus on pathogenic viruses that cause diseases in their hosts. this research project is expected to develop into an important scientific field that examines the interactions between the global ecosystem and viruses. in this brief review, we give some insights into the positive side of viruses. in traditional virology, most viruses found in humans are considered to be pathogenic to their hosts; however, recent studies have shown that there are some viruses that have symbiotic relationships with their hosts and do not cause disease. infection with one virus may protect the host from a superinfection with another pathogen. barton et al. demonstrated that latent infection with the herpesviruses murine gammaherpesvirus or murine cytomegalovirus, which are genetically related to the human pathogens epstein-barr virus and human cytomegalovirus, respectively, led to crossprotection in mice. infection with these viruses induced prolonged production of the antiviral cytokine interferon-gamma and systemic activation of macrophages that protected the mice from subsequent bacterial infections with either listeria monocytogenes or yersinia pestis. moreover, it has been reported that superinfection with hepatitis a virus suppressed hepatitis c virus replication in patients with chronic hepatitis c in at least two cases, and infection with human cytomegalovirus (hcmv) suppressed superinfection with hiv- in vitro as a result of the downregulation of the expression of ccr , a co-receptor for hiv- , induced by the hcmv infection. some viruses also have beneficial effects with respect to non-infectious diseases. epidemiologic studies suggest that virus infections in childhood might confer protection against some cancers later in life. for example, the risk of chronic lymphoid leukaemia in subjects who had measles in childhood is relatively low, and mumps infection in childhood might protect against the development of ovarian cancer in adults. however, infection with oncoviruses is known to increase the risk of development of some cancers (e.g. cervical cancer and liver cancer induced by the human papillomavirus and hepatitis b virus/ hepatitis c virus infection, respectively). such information is important when considering strategies for cancer immunotherapy and/or vaccination campaigns. in addition, the infection of non-obese diabetic mice with lymphocytic choriomeningitis virus prevented the infected mice from developing autoimmune disease and subsequent type i diabetes (insulin-dependent diabetes mellitus). , chronic viral infection of mice with murine cytomegalovirus (cmv) increased epithelial turnover and wound repair via antiviral cytokine type i interferons (ifns), but cmv infection can promote cancer malignancy; this phenomenon is known as 'oncomodulation'. , recent metagenomic studies have revealed that virus infection sometimes confers benefits including the regulation of microbiota in the gut. bacteriophages are abundant in the gut and are thought to modulate the gut microbiota by infecting specific bacterial populations. accordingly, potential therapeutic applications of bacteriophages in humans (e.g. control of antibiotic-resistant bacteria, stabilisation of healthy gut microbes) have been considered. , therefore, the elucidation of the symbiotic effects of viruses on the physiological functions and immune responses of their hosts, as well as clarification of the functional mechanisms involved, will lead to an understanding of the essential roles of viruses in regulating the biological processes of their hosts. retroviruses are found in almost all mammals and other vertebrates, and approximately % of the human genome is composed of retroviruses in the form of endogenised proviruses. , given that only about % of genomic dna is made up of protein-coding genes, the abundance of retrovirus sequences in the human genome is remarkable. retrovirus sequences are conserved in humans and other primates, and therefore, the endogenisation of retroviruses is thought to have occurred millions of years ago. some endogenous retroviruses have been shown to play beneficial roles in their hosts, including host evolution. for example, envelope genes from endogenous retroviruses contribute to the formation of the placenta during the fusion of syncytiotrophoblast cells in mammals. , in addition, endogenous retroviral elements are known to protect host cells from infection with exogenous retroviruses in some mammals. [ ] [ ] [ ] in addition to retroviruses, recent studies have shown that non-retroviral viruses have also endogenised in many mammalian species. [ ] [ ] [ ] for example, tomonaga's group, which is one of the research groups in our neo-virology research consortium, discovered that bornaviruses, a genus of non-segmented, negative-strand rna virus, have been endogenised in the genomes of many mammals, including humans. since bornaviruses do not encode reverse transcriptase and integrase genes, integration of bornavirus segments is believed to be mediated by long interspersed nuclear element- (line- ), which is a retrotransposon widely distributed in mammalian genomes. tomonaga's group also showed that an endogenous bornavirus-like element in the ground squirrel genome blocks infection and replication of extant endogenous bornavirus. together, these findings indicate a potential role for endogenous non-retroviral elements in antiviral defence. this group also performed an evolutionary phylogenetic analysis to elucidate the functions of endogenous bornavirus in mammalian genomes. thus, it has become apparent that a large number of endogenous viral elements have accumulated in host genomes, more than previously expected. therefore, it is important that we understand the significance of endogenous viral elements to the biological function and evolution of hosts. the first virus to be identified in humans was the yellow fever virus in after the discovery of tobacco mosaic virus in in plants and footand-mouth disease virus in in animals. since then, new virus species that infect humans have been identified almost every year. woolhouse et al. reviewed human viruses that had been described in the literature and recognised by the international committee on taxonomy of viruses (ictv), and drew a discovery curve for human viruses by plotting the cumulative number of species reported to infect humans; they showed that new species of human viruses have been discovered at a rate of three or four per year. currently, there are approximately viruses from viral families that are known to be able to infect humans according to the latest ictv report. in the last a few decades, emerging infectious diseases caused by newly identified viruses, such as ebola virus, - sars and mers coronaviruses, [ ] [ ] [ ] human immunodeficiency virus (hiv), nipah virus and hendra virus, [ ] [ ] [ ] [ ] have appeared in human society. most emerging infectious diseases are zoonotic, caused by viruses that originate in wild animals, such as primates, rodents and bats [ ] [ ] [ ] ; in particular, bats have drawn attention because a recent comprehensive analysis of mammalian hostvirus relationships indicated that bats have a significantly higher proportion of zoonotic viruses than all other mammalian orders. this analysis was part of a study supported by the united states agency for international development (usaid) emerging pandemic threats predict programme (https://ohi.vetmed.ucdavis.edu/programs-projects/ predict-project), which was initiated in , working with partners in over countries on global surveillance of viruses to identify and monitor zoonotic pathogens. to date, the predict programme has found over viruses in animals and humans, including a new ebola virus and mersand sars-like coronaviruses. in , the global virome project (http://www.globalviromeproject. org) was launched, which aims to conduct viral surveillance on an even larger scale than the predict programme. those involved in this project estimate that~ . million yet-to-be-discovered viral species from key zoonotic viral families exist in mammal and avian hosts, and expect that - of these unknown viruses have zoonotic potential. in addition to viral surveillance in mammals and avian hosts, zhang and holmes's group recently conducted a screen for rna viruses in diverse host species (more than species other than mammalian and avian hosts) and identified about vertebrate-associated rna viruses in fishes, reptiles and amphibians. they also found that vertebrate-specific viral families or genera known to infect mammals and birds, including influenza viruses, filoviruses and hantaviruses, are also present in amphibians, reptiles and fish. in addition to the identification of new virus species in diverse hosts, new virus lifestyles have also been found in nature. suzuki's group, which is one of the groups in our neo-virology research consortium, recently reported a new virus lifestyle exhibited by two rna viruses: a double-stranded (ds) rna virus (yado-nushi virus , ynv ) and a positive-sense, single-stranded [(+)ss] rna virus (yado-kari virus , ykv ) in a phytopathogenic fungus, rosellinia necatrix. they found that the (+)ssrna virus (ykv ), which does not have its own capsid protein, hijacks the capsid protein of the dsrna virus (ynv ) to replicate. our world is made up of vastly different physical environments and the various organisms that have adapted to live in those environments. the complex interactions between the living and nonliving components of these environments are the basis of the global ecosystem. various schemes have been proposed to classify living organisms: the one most often used currently defines all living organisms as archaea, bacteria or eukaryotes. therefore, viruses are not considered living components of the global ecosystem. given that approximately virus particles exist on earth , and all of them are parasitic in living organisms, it is not hard to imagine how virus infection might affect the physiological functions of both hosts and the ecosystem. although 'traditional' virology research tends to focus on pathogenic viruses that cause diseases in their hosts, the recent progress in next-generation sequencing (ngs) technologies and data analyses has enabled us to discover a wide variety of new viruses, some of which do not cause diseases in their hosts. some obstacles to comprehensive virome analyses remain, such as viral dark matter, which are sequences that originate during virus metagenomics but cannot be aligned to any reference sequences of viruses. nonetheless, recent viral metagenomic studies using ngs technologies and bioinformatic analyses have identified a large number of viruses in environmental samples, including plants and oceans. , characterisation of these newly identified viruses may provide new insight into the significance of viruses and virus-mediated processes within global ecosystems. the history of smallpox and its spread around the world on the origin of smallpox: correlating variola phylogenics with historical smallpox records integrating historical, clinical and molecular genetic data in order to explain the origin and virulence of the spanish influenza virus from emergence to eradication: the epidemiology of poliomyelitis deconstructed isolation and partial characterisation of a new virus causing acute haemorrhagic fever in zaire viral haemorrhagic fever in southern sudan and northern zaire: preliminary studies on the aetiological agent isolation of marburg-like virus from a case of haemorrhagic fever in zaire commission r of an i. ebola haemorrhagic fever in zaire hiv- at isolation of a novel coronavirus from a man with pneumonia in saudi arabia coronavirus as a possible cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome a pandemic warning? clinical features and rapid viral diagnosis of human disease associated with avian influenza a h n virus characterization of an avian influenza a (h n ) virus isolated from a child with a fatal respiratory illness herpesvirus latency confers symbiotic protection from bacterial infection hepatitis a virus infection suppresses hepatitis c virus replication and may lead to clearance of hcv human cytomegalovirus modulation of ccr expression on myeloid cells affects susceptibility to human immunodeficiency virus type infection childhood infectious diseases and risk of leukaemia in an adult population mumps and ovarian cancer: modern interpretation of an historic association early-life exposures to infectious agents and later cancer development prevention of type i diabetes in nonobese diabetic mice by virus infection treatment of nonobese insulindependent diabetes mice with virus prevents insulindependent diabetes mellitus while maintaining general immune competence from their initial discovery ( - ), and to the present ( - ), viruses have been known as agent type i interferons link viral infection to enhanced epithelial turnover and repair the story of human cytomegalovirus and cancer: increasing evidence and open questions the human cytomegalovirus, from oncomodulation to oncogenesis phage therapy: a renewed approach to combat antibioticresistant bacteria the human gut microbiota and virome: potential therapeutic implications initial sequencing and analysis of the human genome effects of retroviruses on host genome function virus evolution syncytin is a captive retroviral envelope protein involved in human placental morphogenesis paleovirology and virally derived immunity studies of endogenous retroviruses reveal a continuing evolutionary saga late viral interference induced by transdominant gag of an endogenous retrovirus endogenous nonretroviral rna virus elements in mammalian genomes unexpected inheritance: multiple integrations of ancient bornavirus and ebolavirus/marburgvirus sequences in vertebrate genomes endogenous viral elements in animal genomes comprehensive analysis of endogenous bornavirus-like elements in eukaryote genomes inhibition of borna disease virus replication by an endogenous bornavirus-like element in the ground squirrel genome paleovirology of bornaviruses: what can be learned from molecular fossils of bornaviruses virology: from contagium fluidum to virome human viruses: discovery and emeraence the global virome project a morbillivirus that caused fatal disease in horses and humans the authors the natural history of hendra and nipah viruses outbreak of nipah-virus infection among abattoir workers in singapore fatal encephalitis due to nipah virus among pig-farmers in malaysia host range and emerging and reemerging pathogens global trends in emerging infectious diseases risk factors for human disease emergence host and viral traits predict zoonotic spillover from mammals the evolutionary history of vertebrate rna viruses chapter sevenviruses of the white root rot fungus, rosellinia necatrix a capsidless ssrna virus hosted by an unrelated dsrna virus the gene weavers marine viruses -major players in the global ecosystem origins and challenges of viral dark matter metagenomics of plant and fungal viruses reveals an abundance of persistent lifestyles viral macro-and micro-diversity from pole to pole we thank susan watson for editing the manuscript. this is an open access article under the terms of the creative commons attribution license, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. key: cord- - g lio l authors: keesing, felicia; belden, lisa k.; daszak, peter; dobson, andrew; harvell, c. drew; holt, robert d.; hudson, peter; jolles, anna; jones, kate e.; mitchell, charles e.; myers, samuel s.; bogich, tiffany; ostfeld, richard s. title: impacts of biodiversity on the emergence and transmission of infectious diseases date: - - journal: nature doi: . /nature sha: doc_id: cord_uid: g lio l current unprecedented declines in biodiversity reduce the ability of ecological communities to provide many fundamental ecosystem services. here we evaluate evidence that reduced biodiversity affects the transmission of infectious diseases of humans, other animals and plants. in principle, loss of biodiversity could either increase or decrease disease transmission. however, mounting evidence indicates that biodiversity loss frequently increases disease transmission. in contrast, areas of naturally high biodiversity may serve as a source pool for new pathogens. overall, despite many remaining questions, current evidence indicates that preserving intact ecosystems and their endemic biodiversity should generally reduce the prevalence of infectious diseases. supplementary information: the online version of this article (doi: . /nature ) contains supplementary material, which is available to authorized users. i n june , a new organization, the intergovernmental science-policy platform on biodiversity and ecosystem services (ipbes)patterned after the intergovernmental panel on climate change (ipcc)-was established to assess changes to the diversity of life on the earth and how these changes will affect human well-being . human well-being would be adversely affected by biodiversity losses if ecosystems with reduced biodiversity are less able to provide the ecosystem services-such as carbon sequestration, nutrient cycling and resistance to drought-on which humans rely. in recent years, a consensus has emerged that ecosystem functions decline as biodiversity is lost . here we examine how biodiversity affects the transmission and emergence of infectious diseases and evaluate the evidence that reduced disease transmission is an important ecosystem service provided by high biodiversity. biodiversity encompasses the diversity of genes, species and ecosystems. increases in human populations have resulted in an unprecedented and precipitous loss of biodiversity . current extinction rates are estimated to be at least - , times background extinction rates and future extinction rates (over the next years) are estimated to be to times present extinction rates . a large proportion of species in all assessed taxa are currently threatened with extinction ( % of birds, % of mammals, % of amphibians; % of gymnosperms; % of corals ) and the best estimate of population trends of birds, mammals, amphibians, reptiles and fish indicates that since global population sizes have declined by almost % (ref. ) . global and local extinction rates of some taxa, particularly microbes, have not been well characterized. for the many organisms that are symbionts of other organisms, extinction of their hosts can cause their extinction too . collectively, these declines and extinctions are caused by changing the earth's ecosystems to meet growing demands for food, fresh water, fibre, timber and fuel, and by climate change. changes in biodiversity have the potential to affect the risk of infectious disease exposure in plants and animals-including humansbecause infectious diseases by definition involve interactions among species. at a minimum, these species include a host and a pathogen; often many more species are involved, including additional hosts, vectors and other organisms with which these species interact. intriguingly, biodiversity may play a dual role in the emergence and transmission of infectious diseases. on the one hand, high biodiversity may provide a larger potential source of novel pathogens, but on the other hand, biodiversity can reduce further pathogen transmission for both longestablished and newly emerging diseases. we first review the effects of biodiversity on the transmission of established diseases and then turn to disease emergence. transmission of pathogens between species biodiversity loss might affect disease transmission through several mechanisms (box ). if the effect of each species on pathogen transmission were entirely idiosyncratic, one would expect that diversity declines would be equally likely to cause a decrease or an increase in disease transmission in the remaining species. however, in recent years, a consistent picture has emerged-biodiversity loss tends to increase pathogen transmission and disease incidence. this pattern occurs across ecological systems that vary in type of pathogen, host, ecosystem and transmission mode ( table ) . as an example, west nile virus is a mosquito-transmitted virus for which several species of passerine birds act as hosts. three recent studies detected strong correlations between low bird diversity and increased human risk or incidence of west nile encephalitis in the united states - . communities with low avian diversity tend to be dominated by species that amplify the virus, inducing high infection prevalence in mosquitoes and people, while communities with high avian diversity contain many species that are less competent hosts. for hantavirus pulmonary syndrome, a directly transmitted zoonotic disease, correlational and experimental studies have shown that a lower diversity of small mammals increases the prevalence of hantaviruses in their hosts, thereby increasing risk to humans (box ). diversity has a similar effect for plant diseases, with species losses increasing the transmission of two fungal rust pathogens that infect perennial rye grass and other plant species . recent attention has focused on assessing the mechanisms by which reduced biodiversity increases pathogen transmission (box ). biodiversity loss can clearly increase transmission if it reduces predation and competition on reservoir hosts, thereby increasing their density. however, controversy has centred around whether the loss of species can increase transmission in other ways . this is because field studies like those on west nile virus, hantaviruses and rye grass have typically not controlled for changes in host density that can result from changes in 'species richness' (the number of species present in a community, which is a measure of taxonomic diversity). as a consequence, it has been difficult to separate the effects of higher density from those of reduced diversity. recent experiments confirm that increases in disease transmission can occur when species richness declines even if host density stays constant. one of the best examples comes from a study of schistosoma mansoni, a trematode that causes schistosomiasis in humans. the parasite alternately infects snails and humans via free-living infectious stages. host snails were placed in tanks at a constant density either alone or with one or two other species of non-host snails and then exposed to the parasite . in single-species treatments, host snails were % more likely to be infected because parasites in multi-species treatments often ended up in dead-end hosts. increased parasite-host encounter rates caused by reduced diversity are sufficient to increase disease transmission for schistosoma. the loss of species can increase encounter rates between pathogens and hosts, as in the schistosoma example, when the lost species are not hosts for the pathogen. but if the lost species are indeed hosts capable of transmission, this declining diversity could also reduce the total number of hosts, thereby decreasing transmission if all else remains equal , . certainly reductions in the number of hosts can reduce the number of vectors and also their infection prevalence , , but empirical examples are relatively rare, in part because the issue has been neglected, and also because all else rarely remains equal. for example, the loss of hosts can cause compensatory increases in the abundances of other hosts, such that total host abundance changes little relative to total host abundance in more diverse communities. even when total host abundance does decline in less diverse systems, differences in host quality among species can alter simple correlations between host abundance and infection risk . pathogen transmission is not always a function of host density. for example, the number of infectious bites delivered by highly mobile vectors like mosquitoes can be independent of the density of the host population . transmission of directly transmitted pathogens like hantaviruses can also be independent of host density if transmission involves behavioural encounters, for example, aggressive interactions between rodents, and if the frequency of these encounters does not vary much with host density , . in systems like these, the loss of host species can actually increase transmission if the lost hosts are suboptimal for parasite development and reproduction; this is because these suboptimal hosts absorb pathogens but are poor at transmitting them. in sum, reducing biodiversity can increase disease transmission when the lost species are either not hosts for the pathogen or are suboptimal ones. for pathogens for which transmission is a function of host density, loss of diversity is most likely to increase transmission if the loss causes an increase in the density of competent hosts. the number and diversity of examples of pathogens for which species loss leads to increases in total transmission suggests that these conditions are frequently met (table ) . additional studies in other disease systems would better establish the generality of these relationships. the loss of particular species in a community clearly has the potential to increase disease transmission. but does reducing diversity itself increase transmission, or is increased transmission the consequence of the removal of particular species? the answer depends on how species composition changes as richness changes , . for example, if those host species most responsible for amplifying the pathogen tend to persist or even thrive as biodiversity is lost, then disease risk will consistently increase as biodiversity declines. on the other hand, if amplifying species tend to disappear as biodiversity declines, then biodiversity loss will tend to reduce disease risk. these hypothetical possibilities indicate the importance of understanding both the non-random sequences by which species are lost from communities, and whether the species that tend to occur only in more species-rich communities tend to amplify or buffer pathogen transmission. in several case studies, the species most likely to be lost from ecological communities as diversity declines are those most likely to reduce pathogen transmission. in the lyme disease system of eastern north america, for example, the white-footed mouse is simultaneously the most abundant host species, the most competent host for the lyme bacterium, and the highest-quality host for immature tick vectors the loss of biodiversity can affect the transmission of infectious diseases by changing: ( ) the abundance of the host or vector. for plants, seeding experimental fields with plant species that are not hosts for fungal pathogens decreased threefold the pathogen load of species that are hosts, apparently by reducing host density through competition . on the other hand, a greater diversity of host species can sometimes increase pathogen transmission by increasing the abundance of vectors . ( ) the behaviour of the host, vector or parasite. in a more diverse community, one of the parasitic worms that causes schistosomiasis (which infects million people worldwide) is more likely to end up in an unsuitable intermediate host. this can reduce the probability of subsequent infection of humans by - % (ref. ) . for hantavirus in utah, usa, rodent hosts on more diverse plots are more likely to come in contact with heterospecific mammals and less likely to come in contact with conspecifics, reducing the probability of transmission of the virus . in principle, higher diversity could influence behaviours with a resulting increase in disease transmission or could alter the evolutionary dynamics of virulence and transmission pathways. ( ) the condition of the host or vector. in experimental rice fields in china, rice plants in genetically diverse mixtures had drier leaves because the mixture changed microclimatic conditions . as a consequence, infection with rice blast fungus was less prevalent in diverse fields. genetically diverse plantings can also lead to induced resistance in host plants because they are exposed to similar pathogens that are specialists on the other cultivars . for some disease systems (for example, lyme disease), multiple mechanisms operate in concert, leading to a compounding effect of biodiversity loss on increased disease transmission (table ) . . ticks that attempt to feed on virginia opossums are likely to be groomed off and killed. green-andyellow circles show the mean number of ticks per hectare fed by mice or opossums; yellow shading shows the proportion of ticks infected after feeding. blue circles show the mean number of ticks per hectare groomed off and killed. ticks that feed on mice are highly likely to become infected with the bacterium that causes lyme disease, whereas those that feed on opossums are not. case study of hantavirus pulmonary syndrome hantaviruses are a group of negative-stranded rna viruses associated with murid rodents. they can cause severe morbidity and mortality in humans, with case-fatality rates near % (ref. ) . infected rodents shed hantavirus in saliva, urine and faeces; transmission to humans occurs through inhalation of aerosolized excreta as well as through rodent bites . the risk of human exposure increases as the density and infection prevalence of rodent reservoirs increase . in a field study in oregon, usa, the only variable significantly linked to infection prevalence in deer mouse host populations was mammalian species diversity, with the prevalence of the hantavirus sin nombre virus rising from % to % as diversity declined. deer mouse population density was not statistically associated with sin nombre virus infection prevalence, suggesting that high diversity reduced intraspecific encounters rather than host abundance . a study in utah, usa , also found a negative correlation between small-mammal diversity and sin nombre virus infection prevalence in deer mice. as in oregon, high diversity reduced infection prevalence apparently by reducing intraspecific encounters rather than by reducing host density, a result supported by experiments . the conclusions of these studies were supported by an experimental study of hantaviruses in small mammal communities of panamá . in replicated plots, small-mammal diversity was reduced by trapping and removing species that are not hosts for the virus; infection prevalence in hosts was compared on manipulated and unmanipulated plots (box figure) . experimentally reduced small-mammal diversity caused an increase in the density of host species and also in seroconversion rates and seroprevalence within hosts (box figure) . review research (fig. ) . as a consequence, this host species infects a high proportion of the ticks within forest communities. the white-footed mouse is also an ecologically resilient species, present in both species-rich and speciespoor communities . in contrast, virginia opossums are poor hosts for the pathogen, kill the vast majority of ticks that attempt to feed on them, and are absent from many low-diversity forest fragments and degraded forests where mice are abundant , . therefore, as biodiversity is lost, the host with a strong buffering effect-the opossum-disappears, while the host with a strong amplifying effect-the mouse-remains. the primary hosts for the pathogens that cause west nile encephalitis, hantavirus pulmonary syndrome, and bartonellosis also appear to be resilient species that increase in abundance as biodiversity is lost , , . whether an organism's host competence and its resilience to factors that reduce biodiversity are causally related is an unresolved but critical issue. traits that make a host resilient to biodiversity loss may also make them susceptible to pathogen infection and transmission. such a relationship would explain the frequency with which the link between diversity loss and disease transmission has been observed in nature ( table ). for plants, species that are fast-growing and nutrient-rich with relatively high metabolic rates-characteristics of 'weedy' speciescan be more competent hosts for arthropod vectors and plant pathogens than those with less weedy traits . plants with these weedy traits are also more likely to become more abundant when plant diversity declines . consequently, the very species that have traits permitting persistence in degraded and species-poor ecosystems are also more likely to carry high pathogen and vector burdens. a similar pattern may occur in vertebrates-resilience in the face of disturbances that cause biodiversity loss, such as habitat destruction and fragmentation, is facilitated by lifehistory features such as high reproductive output and intrinsic rates of increase . vertebrates with these features tend to invest minimally in some aspects of adaptive immunity [ ] [ ] [ ] ; we hypothesize that this may make them more competent hosts for pathogens and vectors. understanding the interrelationships among pathogen transmission, biodiversity loss and interspecific differences in immune function is an important area for future research. such studies would illuminate how frequently resilient species are also those that increase pathogen transmission, and might provide general rules about the impact of biodiversity loss on disease transmission. could changes in biodiversity within the bodies of organisms also alter pathogen transmission? recent improvements in the ability of researchers to detect unculturable microbial species have allowed documentation of the tremendous diversity of microbes upon and within plants and animals. in human bodies, for example, % of all cells are microbial . a number of studies have begun to show links between diseases and the diversity of an organism's 'microbiome'. changes in the composition of microbiomes are frequently associated with infection and disease. for example, corals suffering from white plague disease have microbial communities distinctly different from those in healthy corals . in humans, bacterial vaginosis results from changes in the composition of the vaginal microbial community , and this in turn increases the risk of hiv infection . although changes in microbial species composition associated with infection are welldocumented, few studies have investigated the effects of changes in diversity itself. in a recent investigation, patients with recurrent episodes of infection caused by the bacterium clostridium difficile had significantly lower diversity of intestinal microbes than did control patients . correlational studies such as these, though intriguing, make it difficult to determine whether changes in microbial communities are the cause or the consequence of infections. but some experimental studies clearly demonstrate that increasing microbial biodiversity can protect against infection. for example, children with a history of ear infections given a mixture of five strains of streptococcus were less likely to develop subsequent infections compared to a control group . similarly, reducing microbial diversity within a host can increase transmission. when mice with persistent infections of c. difficile were treated with antibiotics that reduced the diversity of intestinal microbes, they began shedding c. difficile spores at high rates . in some of these examples, a rich microbial community appears to regulate the abundance of endemic microbial species that can become pathogenic when overly abundant . in other cases, high microbial species diversity can prevent colonization by invasive pathogenic species. for example, the more diverse the microbiome surrounding the roots of wheat plants, the more protected the plants were against invasion by the pathogenic bacterium pseudomonas aeruginosa . similarly, piglets raised in natural environments supporting a high diversity of microbes were more resistant to invasion by pathogenic gut microbes than those raised in more sterile environments . the effects of microbial diversity within and upon host bodies show intriguing similarities to the effects of macroscopic species diversity on disease transmission in aquatic and terrestrial ecosystems. further exploration of these similarities, and particularly the specific mechanisms operating within hosts, is a critical research frontier because changes in microbial diversity might accompany biodiversity loss in their hosts. for pathogens already established within ecological communities, we have shown that biodiversity loss frequently increases the rate of transmission. but what role, if any, does biodiversity have in the processes by which new pathogens emerge? between and , over emerging disease events were identified in humans around the world . concomitantly, other emerging infectious diseases also appeared in wildlife, domesticated animals, and crop and wild plants. emerging infectious diseases include those in which the pathogen has evolved into a new strain within the same host species, for example, through the evolution of drug resistance (methicillin-resistant staphylococcus aureus or mrsa) or switched to new host species (for example, human immunodeficiency virus or hiv, severe acute respiratory syndrome or sars). in some cases, the switch to new host species is accompanied by a change in geographic range (for example, west nile virus in the americas). for pathogens that establish in new species, the emergence process involves multiple steps, including the initial invasion into the new host ('spillover'), the production of transmission stages within the new host, and the establishment of the pathogen in the host population as a whole , . the effect of biodiversity may vary for each of these steps. for the initial invasion, biodiversity may act as a source pool. this hypothesis is supported by surveys of emerging diseases of humans: most are zoonotic-jumping to humans from other vertebrate animals . in one recent analysis, the probability of emergence of pathogens from wildlife to humans was positively correlated with mammalian wildlife species richness when data were corrected for reporting bias . other environmental and socioeconomic factors that bring humans into closer contact with potentially new pathogens (for example, forest clearing for agriculture, wildlife hunting) may also contribute to this pattern. indeed, almost half of the zoonotic diseases that have emerged in humans since resulted from changes in land use, from changes in agricultural or other food production practices, or from wildlife hunting (fig. ) . these human activities increase rates of contact between humans and animals, which may be a critical factor underlying spillover. once spillover of the pathogen into a new host has occurred, high densities of that host species may facilitate pathogen establishment and transmission within the new host . for example, nipah virus spilled over from wild fruit bats to domestic pigs in malaysia; high densities of pigs in local farms appear to have facilitated establishment of pig-to-pig transmission, and the pathogen then spilled over from pigs to humans . such high densities of domesticated species are almost always associated with low biodiversity. in contrast to emergence through host-switching, % of emergence events between and arose through the evolution of drug resistance . for these cases, biodiversity of microbial communities within hosts may have a protective effect; human use of antibiotics is research review thought to select for resistant microbes by eliminating the great diversity of non-resistant microbial strains and species that suppress resistant strains in the absence of antibiotics. investigations using recent advances in microbial detection support this idea , . thus, reduced microbial diversity may be an important underlying cause of the emergence of drug-resistant pathogens; this too requires further investigation. the addition of particular species-for example, natural enemies or competitors-can reduce the impacts of established pathogens. for example, experimental addition of a naturally occurring bacterium, janthinobacterium lividum, to the skin of the endangered frog rana mucosa eliminated frog mortality from experimental infection with chytridiomycosis, which is devastating amphibian populations worldwide . for corals, application of phages isolated from natural communities can control the spread of bacterial infections . the growing interest in 'probiotics' for humans and harvested species provides another example of this approach . more broadly, biodiversity itself seems to protect organisms, including humans, from transmission of infectious diseases in many cases (table ) . preserving biodiversity in these cases, and perhaps generally, may reduce the incidence of established pathogens. to preserve high diversity in nature, conservation scientists have developed robust methods that reflect the key principle that larger areas sustain larger numbers of species . methods of conserving microbial diversity within and upon bodies or in the environment are less well developed, but avoiding the overuse of antimicrobial compounds is essential. critically, future research on the relationship between biodiversity and disease must avoid conflating the effects of biogeographic patterns of biodiversity (for example, higher diversity in lower latitudes) with those of anthropogenic reductions in extant biodiversity, because policy and management options can far more readily affect the latter than the former. for emerging diseases, the observation that a more diverse microbiome within a host suppresses strains that are resistant to antimicrobial compounds suggests that avoiding the over-use of these compounds in medicine and agriculture can prevent the emergence of resistant strains. for pathogens that emerge by switching host species, three management approaches are warranted. first, potential emergence 'hotspots' could be predictable on the basis of land-use change and underlying biodiversity patterns; these areas should be targeted for surveillance of endemic wildlife pathogens that have the potential to jump host species , . second, preserving and protecting intact habitats in these hotspots provides a simple, direct way of reducing human-animal contact and reduces the likelihood of emergence of new pathogens, although methods for achieving reduced contact are not always straightforward . and third, to reduce the probability that pathogens become established and transmissible within a new host population once spillover occurs, the husbandry of high-density monocultures of domestic animals, particularly in areas at high risk of spillover, should be subject both to more intensive surveillance and to measures that reduce contact between wildlife and livestock. managing potential emergence hotspots by attempting to eliminate them is likely to backfire because the species most resilient to habitat destruction and degradation may be those that amplify pathogen transmission. despite many recent advances in our understanding of biodiversity and disease, much remains to be learned. first, we must increase the number of disease systems for which we understand the effects of biodiversity loss on disease transmission across a range of spatial and temporal scales. we must also focus on how to implement specific policies informed by this science. future research, for example, should monitor changes in epidemiology in regions in which conservation measures are imposed compared to reference sites. a major challenge will be to untangle the complex ways in which other global anthropogenic trends-such as climate change, biotic exchange, nutrient pollution, armed conflict and economic collapse-interact with biodiversity loss to influence disease dynamics, and which of these trends have the greatest impacts on human well-being. despite remaining questions, connections between biodiversity and disease are now sufficiently clear to increase the urgency of local, regional, and global efforts to preserve natural ecosystems and the biodiversity they contain. globally, almost half of these diseases resulted from changes in land use, changes in agricultural and other food production practices, or through wildlife hunting, which suggests that contact rates between humans and other animals are an important underlying cause of zoonotic disease emergence. 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ecological determinants of american crow mortality due to west nile virus during its north american sweep effects of species diversity on disease risk effects of grassland plant species diversity, abundance, and composition on foliar fungal disease zooprophylaxis or zoopotentiation: the outcome of introducing animals on vector transmission is highly dependent on the mosquito mortality while searching comparison of four species of snails as potential decoys to intercept schistosome miracidia panicle blast and canopy moisture in rice cultivar mixtures use of multiline cultivars and cultivar mixtures for disease management hantavirus pulmonary syndrome in five pediatric patients-four states the ecology and evolutionary history of an emergent disease: hantavirus pulmonary syndrome author information reprints and permissions information is available at www.nature.com/reprints. the authors declare no competing financial interests. readers are welcome to comment on the online version of this article at www.nature.com/nature. correspondence and requests for materials should be addressed to f.k. (keesing@bard.edu). supplementary information is linked to the online version of the paper at www.nature.com/nature. key: cord- - w de authors: sironi, manuela; cagliani, rachele; forni, diego; clerici, mario title: evolutionary insights into host–pathogen interactions from mammalian sequence data date: - - journal: nat rev genet doi: . /nrg sha: doc_id: cord_uid: w de infections are one of the major selective pressures acting on humans, and host-pathogen interactions contribute to shaping the genetic diversity of both organisms. evolutionary genomic studies take advantage of experiments that natural selection has been performing over millennia. in particular, inter-species comparative genomic analyses can highlight the genetic determinants of infection susceptibility or severity. recent examples show how evolution-guided approaches can provide new insights into host–pathogen interactions, ultimately clarifying the basis of host range and explaining the emergence of different diseases. we describe the latest developments in comparative immunology and evolutionary genetics, showing their relevance for understanding the molecular determinants of infection susceptibility in mammals. supplementary information: the online version of this article (doi: . /nrg ) contains supplementary material, which is available to authorized users. the way in which these analyses have helped to clarify the genetic determinants of species-specific infection and disease, as well as the reasons behind pathogen emergence. although arms races involve both the host and the pathogen, in this review we only focus on genetic diversity in mammalian hosts. host-pathogen genetic conflicts are not confined to mammals (and their pathogens): they drive molecular evolution in most realms of life, including bacterial-bacteriophage systems , plants and their infectious agents , as well as invertebrates and their pests , . although we review studies and methods (boxes - ) that analyse genetic diversity at the inter-species level, the investigation of intra-species and intra-population signatures of pathogen-driven selection has also provided extremely valuable insight into infectious disease susceptibility, especially in our species. the interested reader is directed towards several recent reviews for more information [ ] [ ] [ ] [ ] [ ] . the dynamics of host-pathogen interactions a central tenet of the red queen hypothesis is that organisms must continually adapt to survive and thrive in the face of continually evolving opposing organisms. nonetheless, evolution is not all about biotic interactions. at a macroevolutionary level, mixed models of evolution are likely to operate; biotic factors mainly shape species diversity locally and over short time spans, comparisons among species take a snapshot of selective events that have been unfolding over long timescales. most of these approaches use extant genetic diversity and phylogenetic relationships among species to infer underlying evolutionary patterns. briefly, inter-species approaches rely on the alignment of orthologous coding sequences, analyse these alignments site-by-site, and at each site determine which, among all possible substitutions, would be non-synonymous (amino acid replacing) or synonymous (non-amino acid replacing) (see the figure) . the observed number of non-synonymous differences per non-synonymous site (dn) and the observed number of synonymous differences per synonymous site (ds) are then estimated. under neutral evolution, the rate at which amino acid replacements accumulate is expected to be comparable to the rate for silent changes and, therefore, dn/ds should be equal to (green codons in the figure). nonetheless, most amino acid replacements are deleterious and, as a consequence, are eliminated by selection; this results in a large preponderance of sites with dn/ds < , a situation referred to as purifying (or negative) selection (shown in blue in the figure) . conversely, the selective pressure exerted, for instance, by a pathogen, may favour amino acid replacements (for example, changes that modify the sequence and structure of a cellular receptor): in this case, dn/ds may reach values greater than , a hallmark of positive (or diversifying) selection (red in the figure) . the figure shows a hypothetical example whereby a virus uses a cellular receptor to infect the host. to prevent viral binding and infection, selection favours variants that modify the sequence and structure of the host receptors; on the other side, the virus adapts to such changes by gaining mutations that keep re-establishing receptor binding. this process fuels a genetic conflict, which is evident at the interaction surfaces. some lineages may be under stronger selective pressure than others and may display lineage-specific selected sites (episodic selection; cyan). in this case the branch of the phylogeny leading to these species may show significant evidence of positive selection . whereas shifts in the physical environment (for example, climate changes and oceanographic and tectonic events) drive evolution at a large scale, across much longer time periods . recently, a new interpretation of the red queen hypothesis was proposed ; the analysis of several phylogenies from different taxa indicated that speciation mostly occurs at a constant rate through rare stochastic events that cause reproductive isolation . this view curtails the role of biotic interactions as major determinants of species diversity . despite these observations, the red queen hypothesis has proven to be an extremely useful framework for the study of host-pathogen interactions. in this context, red queen dynamics can be divided into different types (see ref. for a recent review). frequency-dependent selection, for example, determines allele frequency fluctuations in both host and pathogen populations. in this scenario, rare alleles are favoured by selection (the pathogen, for instance, may be adapted to the most common host genotype and may fail to infect hosts carrying a rare allele), and diversity within populations is maintained. escalatory arms races are another form of selection that usually apply to quantitative or polygenic traits and proceed through recurrent selective sweeps. selection results in an escalation in the phenotypes of both the host (for example, resistance) and the pathogen (for example, virulence). finally, in chase red queen scenarios the host is under pressure to reduce the strength of the interaction through de novo evolution of novelty, whereas the pathogen evolves to tighten the interaction by reducing phenotypic distance. chase scenarios occur when host-pathogen interactions have a complex genetic basis (polygenic); they determine selective sweeps and tend to reduce genetic diversity within populations. over the years, the red queen hypothesis has been supported by the description of rapid rates of evolution in genes involved in genetic conflicts and, in a few instances, by the temporal reconstruction of host-pathogen co-evolution in natural settings . more recently, the development of experimental evolution approaches has allowed its formal testing , . although extremely valuable, laboratory-based studies often use an isogenic host population that is infected by one or a few pathogen strains, and such studies only partially recapitulate the complex nature of host-pathogen interactions that occur in real life. for instance, phenotypic plasticity (an environmentally based change in the phenotype) and multiway host-pathogen interactions are common in nature. a remarkable example of phenotypic plasticity is the vertebrate adaptive immune system: through rearrangement and somatic hypermutation, the same genetic arsenal is used to combat a wide array of pathogens and to develop lifelong resistance to some infections. despite the relevance of adaptive immunity for host defence, its action does not preclude pathogen-driven selection at several genes involved in innate immunity or, more generally, in the interaction with pathogens (these represent the focus of this review). as for multiway interactions, these represent the norm: the same host can be infected by multiple pathogens (or even by multiple strains of the same infectious agent) during its lifetime, whereas pathogens differ in their ability to infect one or more host species. thus, multiple host-pathogen interactions might drive the evolution of the same or different molecular systems, blurring the expectations of the red queen hypothesis. finally, hosts with long generation times (such as mammals, which are the focus of this review), evolve at lower rates compared with most of their pathogens and also display smaller population sizes, resulting in an asymmetry of the arms race (although parasites with life cycles involving two or more species may be constrained in their ability to adapt (reviewed in ref. ) ). even in the presence of a strong selective pressure (for example, a fatal infection), several generations may be required before the molecular signatures of the genetic conflict can be detected in mammalian host genomes . nevertheless, natural selection signatures have been described at several mammalian genes that interact with recently emerged human infectious agents (for example, hiv- ), possibly as a result of the pressure imposed by extinct pathogens or because these agents have established long-lasting interactions with non-human hosts. the 'site models' implemented in the phylogenetic analysis by maximum likelihood (paml) package are widely used to infer positive selection and to identify positively selected sites. these models allow dn/ds to vary from site to site, assuming a constant rate at synonymous sites. data (alignment and phylogenetic tree) are fitted to models that allow (selection models) or do not allow (neutral model) a class of codons to evolve with dn/ds > . likelihood ratio tests are then applied to determine whether the neutral model can be rejected in favour of the positive selection model. if so, the gene is declared to be positively selected. also, if (and only if) the null hypothesis of neutral selection is rejected, a bayes empirical bayes (beb) approach can be used to detect specific sites targeted by selection (beb calculates the posterior probability that each site belongs to the class with dn/ds > ) , . the paml approach implicitly assumes that the strength and direction of natural selection is uniform across all lineages. because this is often not the case, murrell and co-workers recently developed the mixed effects model of evolution (meme, hyphy package) . meme allows the distribution of dn/ds to vary from site to site and from branch to branch; thus, the method has greater power to detect episodic selection, especially if it is confined to a small subset of branches in the phylogeny. a major issue related to these approaches is their extreme sensitivity to errors in sequence (coverage), annotation and alignment. misalignments and incorrect sequence information may result in apparently fast evolutionary rates and thus inflate the false-positive rate [ ] [ ] [ ] . the use of specific alignment algorithms (for example, prank) and filtering procedures (for example, guidance) may partially overcome this problem . likewise, genetic variability that is generated by recombination can be mistaken for positive selection . thus, to limit false positives, alignments should be screened for recombination before running positive selection tests (and, if necessary, split on the basis of recombination breakpoints) or recombination should be incorporated into the model. the accumulation of favourable amino acid-replacing substitutions, which results in more non-synonymous changes than expected under neutrality (dn/ds > ). coronavirus (mers-cov) as a dangerous human pathogen. both ebov and mers-cov are thought to have originated in bats and spread to humans either directly or through an intermediate host. because eids are almost inevitably caused by an existing pathogen that adapts to infect a new host, comparative analyses of different species may help to unveil the genetic and immunological determinants underlying pathogen spillover and infection susceptibility. hiv- , for example, originated from the crossspecies transmission of the simian immunodeficiency virus siv cpz , which naturally infects chimpanzees . old world monkeys are resistant to hiv- infection owing to a post-entry viral block operated by cellular restriction factors. this differential susceptibility to infection was exploited to isolate tripartite motif-containing protein (trim ; also known as trim α), a major retrovirus restriction factor, from a rhesus macaque cdna library . the protein product of trim binds directly to the incoming viral capsid and targets it for disassembly. whereas macaque trim is highly efficient against hiv- , the human protein is not . most species-specific determinants of antiviral activity were mapped to a short amino acid stretch in the so-called b . (or spry) domain of trim (ref. ). in primates, this region has evolved under positive selection, and the human lineage shows some of the strongest selection signatures . why then is human trim so highly inefficient against hiv- ? possibly because the human gene evolved to fight another retrovirus. in a seminal paper, kaiser and co-workers resurrected an extinct pan troglodytes endogenous retrovirus (pterv ) and showed that the amino acid status of a single residue in the trim b . domain modulates its activity against pterv and hiv- , with the gain of restriction for one virus resulting in decreased control of the other one . human trim is very active against pterv , suggesting that our ancestors adapted to fight this virus or some related retrovirus, and this left them (us) unprepared against the hiv- epidemic. more recently, several genes identified as hiv- host factors were analysed in primates, and evidence emerged of positive selection at five of these (ankyrin repeat domain a (ankrd a), cd , microtubule-associated protein (map ), nucleoporin kda (nup ) and ran binding protein (ranbp )) . importantly, most of the positive selection targets in cd , map and nup are located in protein regions or domains that are responsible for direct interaction with the virus. the authors suggested that the selective pressure on these genes was exerted by ancient lentiviruses , . overall, a number of concepts can be taken from these studies: past infection events may leave a signature that affects the ability of extant species to fight emerging pathogens. evolution may act through trade-offs, whereby changes that are favourable in one specific environment (in this case, the presence of a specific pathogen) may be unfavourable when conditions change. protein regions at the host-pathogen interface are expected to be targeted by the strongest selective pressure. evolutionary studies based on inter-species comparisons allow the identification of molecular determinants of infection susceptibility at single amino acid resolution. mammals display different susceptibility to distinct pathogens, and infection with the same agent can have extremely different outcomes in diverse species (see ref. for a recent review). thus, domestic and wild mammalian (and non-mammalian) species represent natural reservoirs of human pathogens and/or may provide the adaptive environment for pathogen spillover. because host reservoir species and their pathogens often signatures of selection along specific branches can be detected through the so called 'branch-site' models implemented in the phylogenetic analysis by maximum likelihood (paml) package . in analogy to the site models described in box , alignment errors result in high false-positive rates when branch-site models are applied ; this issue can be partially mitigated by the use of specific aligners . branch-site models require the phylogeny to be divided into 'foreground' and 'background' branches. a likelihood ratio test is then applied to compare a model that allows positive selection on a class of codons for the foreground branches with a model that does not allow such selection . designation of the foreground branches needs a priori information, possibly based on biological evidence. if no clues are available as to which branches are more likely to have undergone selection, it is still possible to run the analysis by designating each branch of the tree as 'foreground'; this generates a multiple-hypothesis testing problem that must be appropriately corrected . two alternative methods can detect selection at specific lineages without a priori branch partition. the branch site-random effects likelihood (bs-rel) method considers three different evolutionary scenarios (purifying, neutral and diversifying selection) for all branches in a given tree, and each branch is considered independently from the others; the algorithm applies sequential likelihood ratio tests to identify branches with significant evidence of positive selection . the second method, the covarion-like codon model (fitmodel) , allows each site to switch between selective regimes at any time on the phylogeny. thus, switches are not necessarily associated with tree nodes. recently, this approach was shown to be more powerful than the branch-site tests if a priori information is available . both fitmodel and the paml branch-site methods envisage a bayesian approach to identify sites evolving under episodic positive selection. however, extensive simulations revealed that the branch-site approach is accurate but has limited power at detecting sites . this problem has been referred to as the 'selection inference uncertainty principle' -that is, it is difficult to simultaneously infer both the site and the branch that are subject to positive selection . co-evolve for millions of years, evolutionary analyses may help to explain host adaptive events associated with low susceptibility and mild disease outcomes. the most extensive body of knowledge on host-pathogen specificity focuses on viral infections, as the example of trim mentioned above testifies, but recent work has also shed new light on bacterial diseases. leptospirosis, one of the most prevalent human bacterial zoonoses worldwide, is caused by bacteria of the leptospira genus. wild rodents are considered to be the main reservoirs for human leptospirosis, but a study of malagasy small mammals indicated that several endemic species of tenrecs and bats are also infected with leptospira species that are markedly specific to their hosts, suggesting long-term adaptation of the bacterium to different hosts . a feature that pathogenic leptospira species share with other bacteria is complement evasion. indeed, these spirochetes have evolved different strategies to elude complementmediated killing; thus, leptospiral immunoglobulin-like (lig) proteins can bind complement factor h (cfh) and c b-binding protein (c bp) to mediate complement inactivation at the bacterial surface. a genome-wide analysis of positive selection in six mammalian species indicated that the complement system has been the target of extremely intense selective pressure . similar results were obtained by analysing positively selected genes in the bat myotis brandtii . thus, selection-driven speciesspecific differences at complement genes might explain differential susceptibility to infections. in line with this view, human-specific pathogens such as neisseria gonorrhoeae and neisseria meningitidis bind cfh of human origin, but not cfh from other primates, and a single amino acid change (n r) in the chimpanzee molecule restores cfh binding to sialylated gonococci and bacterial killing . several sequenced mammalian genomes are now available; it will be important to study the detailed pattern of molecular evolution at complement genes, with the aim of gaining insight into the determinants of species-specific complement evasion. yersinia pestis provides another remarkable example of differential susceptibility to a bacterial infection. again, rodents act as a natural reservoir for this human pathogen. as with other gramnegative bacteria, lipid a, the biologically active component of y. pestis lipopolysaccharide (lps), is recognized by toll-like receptor (tlr ) and its co-receptor lymphocyte antigen (ly ; also known as md ) (see below). recent data showed that, compared with mouse cells, human cells respond less efficiently to hypoacylated lipid a; this effect is almost entirely due to differences in tlr and ly sequences, as assessed by the generation of humanized mice . different responsiveness to variably acylated lps from other sources (for example, escherichia coli) had previously been described . starting from this premise, ohto and co-workers solved the crystal structure of the mouse tlr -ly -lps and tlr -ly -lipid iva (a synthetic tetra-acylated lipid a precursor) complexes and compared them to the human counterparts. structural differences were detected in the interaction of lipid iva with the two mammalian receptors, with some amino acid replacements in ly and tlr possibly being responsible for the observed differential binding . analysis of tlr in mammals revealed that the receptor has evolved adaptively . we mapped positively selected sites onto the structure of the human and mouse complexes and observed that some of these may indeed account for structural differences between humans and mice (fig. ) . rodents are the most established animal model for human disease, including for susceptibility to infection. in recent years, however, technological advances have made the sequencing of whole genomes a relatively quick and inexpensive process. the genome sequences of non-model mammals that serve as natural reservoirs of human infectious agents are now available, allowing the unprecedented opportunity to exploit these data for molecular evolution studies. bats, for example, are known to host a wide range of viruses that are highly pathogenic to humans . the genomes of six bat species have been sequenced so far, and three of these (m. brandtii, pteropus alecto and myotis davidii) were analysed in detail to unveil the evolutionary history of specific traits . results showed that different families of immune receptors -including killer cell immunoglobulin-like receptors (kirs), killer cell lectin-like receptors (klrs), sialic acid-binding immunoglobulin-like lectins (siglecs) and leukocyte immunoglobulin-like receptors (lilrs) -have expanded or contracted in distinct bat species. also, in these three bat species, as well as in the common ancestor of p. alecto and m. davidii, genes involved in immunity represented preferential targets of positive selection . this is not unexpected: immune-response genes have been shown to have evolved rapidly in most mammalian species analysed to date . thus, although these sequenced bat genomes have not yet provided an explanation as to why bats are tolerant to ebov, for instance, they pave the way for further analyses to test specific hypotheses and/or to address the molecular determinants of host-pathogen interactions. in a recent study, demogines and co-workers showed how this can be accomplished. the authors focused on angiotensinconverting enzyme (ace ), which serves as a receptor for severe acute respiratory syndrome coronavirus (sars-cov) cell entry. in particular, the receptorbinding domain of the viral spike protein is responsible for ace binding and is a major determinant of host range . although the human sars epidemic was suggested to have originated from the zoonotic transmission of sars-cov from bats to humans, possibly via an intermediate host (for example, palm civets) , , no ace -binding sars-cov-like virus had been identified in bats when demogines and collaborators started their work . the authors analysed ace genes in bat species, and results revealed that the gene evolved adaptively and that the positively selected residues of the bat genes map at the ace -sars-cov interaction surface (fig. ) . positive selection localized to a subset of sites or confined to a few species in a phylogeny. these data led to the conclusion that ace -binding coronaviruses originated in bats . this finding was confirmed in a subsequent study that isolated an ace -binding sars-like coronavirus from horseshoe bats in china , highlighting the power of evolutionary studies in predicting host range and disease emergence. similarly to sars-cov, mers-cov is thought to have originated in bats and to have spread to humans via an intermediate host, possibly dromedary camels . infection is initiated by binding of the mers-cov spike protein to human dipeptidyl peptidase (dpp ; also known as cd ) . recent data indicate that five amino acids in dpp that differ between humans (mers-cov susceptible) and hamsters (non-susceptible) are key determinants for host specificity (fig. ) . we extended a previous evolutionary analysis of mammalian dpp (ref. ): strong evidence of positive selection was found with episodic selection in the vespertilionidae bat family and the panda and ferret branches, as well as in the dog lineage (fig. ; see supplementary information s ,s (box, table)). as shown in fig. , most positively selected sites are located at the dpp -spike protein interaction surface , and one of these is among those described as binding determinants . thus, as observed for ace , mers-cov and related viruses (for example, coronavirus hku ) are likely to act as drivers of molecular evolution on mammalian dpp genes; it will be especially interesting to evaluate the contribution of positively selected sites in ferrets because these animals are resistant to mers-cov infection. immune responses in mammals are highly coordinated processes involving multiple systems that sense infection, activate antiviral and antimicrobial responses, and trigger adaptive immunity. the evolutionary history of several such systems has been analysed in detail, and below we describe the most recent findings. innate immune receptors. the mammalian immune system is endowed with a repertoire of molecular sensors called pattern-recognition receptors (prrs). these molecules detect pathogen-associated molecular patterns (pamps) and initiate a downstream signalling cascade that culminates in the production of cytokines and antimicrobial factors. the main families of prrs include tlrs, nod-like receptors (nlrs), rig-like receptors (rlrs) and aim -like receptors (alrs). in the host-pathogen arms race, these molecules represent one of the foremost detection-defence systems; consistently, several studies have reported adaptive evolution at genes encoding mammalian prrs. analyses in primates, rodents and representative mammalian species indicate that positive selection shaped nucleotide diversity at most tlrs, with the strongest pressure acting on tlr (refs , , ) . similarly to tlr (fig. ) , several positively selected sites in other tlrs are located in pamp-binding regions, raising questions as to whether species-specific host-pathogen co-evolution is occurring, and how these sequence changes translate into differential pamp recognition. in fact, as mentioned above for lps, species-specific differences in ligand binding by tlrs seem to be common and potentially affect the overall immune response to specific pathogens . integration of evolutionary, immunological and genetic studies will be instrumental in the future for medical applications, especially in light of nature reviews | genetics sites that are positively selected in mammals are mapped onto the tlr structure (red): several of these flank or correspond (orange) to residues that differ between humans and mice and that surround the phosphate groups of lipid iva (yellow) . if lys and arg are replaced with the human residues (glu and gln , respectively), the responsiveness of mouse tlr -ly to lipid iva is abolished. b | structures of human cd (white; transmembrane and juxtamembrane region) and mir (grey; encoded by kaposi sarcoma-associated herpesvirus). cd sites that are involved in the interaction and that are positively selected in mammals are shown in red. c | complex of transferrin receptor protein (tfr ) with the surface glycoprotein (gp ) of machupo virus (macv), a rodent arenavirus that can also infect humans through zoonotic transmission. tfr residues involved in the interaction with gp are in yellow, positively selected sites are in red and positively selected sites that directly interact with gp are in orange. the proposed use of tlr ligands as vaccine adjuvants, a step that may require tailoring to distinct species . compared with tlrs, mammalian alrs are much less conserved and more dynamic, with distinct species carrying different sets of functional genes (ranging from in mice to none in some bats) , . as a consequence, analysis of several mammals indicated that, with the exception of absent in melanoma (aim ), which is non-functional in several species, no unequivocal orthologues can be inferred for the remaining alr genes. this prevents the application of standard codon-based tests across the entire mammalian phylogeny, although closely related species can be analysed. thus, interferonγ-inducible protein (ifi ) and aim were shown to have evolved under positive selection in primates. positively selected sites were observed to mainly localize near to regions or domains involved in dna binding and protein-protein interaction, suggesting modulation of substrate specificity or genetic conflicts with viral inhibitors . positive selection was also described for the three mammalian rlrs (retinoic acid-inducible gene i (rigi; also known as ddx ), melanoma differentiation-associated (mda ; also known as ifih ) and lgp (also known as dhx )), the primate nlr family apoptosis inhibitory protein (naip) and rodent naip genes , . indeed, as is the case for alrs, rodents have multiple naip paralogues that show widespread evidence of inter-locus recombination. this led to the application of a dn//ds sliding window approach: the naip sites evolving with dn/ds > were found to be located in the bacterial ligand domain . studies on antiviral restriction factors have been extensive because these molecules represent obvious targets in hostpathogen arms races. specifically, genetic conflicts between host restriction factors and viral components often play out in terms of binding-seeking dynamics (the host factor adapts to bind the viral component) and binding-avoidance dynamics (the virus counter-adapts to avoid binding and restriction by the host factors). the evolutionary history of antiviral restriction factors has been comprehensively reviewed elsewhere [ ] [ ] [ ] , and we only highlight a few recent developments here. the first restriction factor to be identified was the product of the mouse gene friend virus susceptibility (fv ), a protein that protects against murine leukaemia virus (mlv) infection . the origin and evolution of fv is extremely interesting: early sequence analysis revealed that it derives from the gag gene of an ancient endogenous retrovirus that is not directly related to mlv . thus, fv exemplifies a paradoxical twist of the arms race scenario whereby a viral gene is co-opted by the host to serve an antiviral function (this is not the only instance, see ref. ) . recent results showed that the fv gene was inserted into the mouse genome between million and million years ago, long before the appearance of mlv. thus, the selective pressure exerted by other viruses must have maintained fv function and driven its evolution . indeed, analysis of fv from wild-type mice indicates that different fv products can recognize s (box, table) ). genes that evolved from a common ancestral gene through speciation. homologous genes created by a duplication event within the same genome. the observed number of non-synonymous substitutions per non-synonymous site. the observed number of synonymous substitutions per synonymous site. and block multiple genera of retroviruses (for example, equine infectious anaemia virus and feline foamy virus), and a number of positively selected sites in the carboxy-terminal region of fv are directly involved in restriction specificity . thus, in a similar way to trim , fv was identified for its ability to restrict an extant virus, but its evolution was driven by different waves of retroviral species, some of which are likely to be extinct. other restriction factors that have been the topic of recent investigation are encoded by two paralogous genes, myxovirus resistance (mx ; also known as mxa) and mx (also known as mxb). the protein products of the two genes display high sequence identity but different antiviral specificity. mx has broad activity against rna and dna viruses. recently, mitchell and collaborators showed the potential of evolutionary analyses to generate experimentally testable hypotheses on the nature of genetic changes that affect species-specific susceptibility to infection. the authors applied an evolution-guided approach and identified a cluster of positively selected residues in an unstructured surface-exposed mx loop (loop ), which confers antiviral specificity; genetic variation in loop is a major determinant of mx antiviral activity against thogoto and avian influenza a viruses, and replacements at a single positively selected site alter the ability of mx to restrict these pathogens . more recently, the selection pattern at the mx gene, which encodes an antiretroviral effector , was shown to parallel that of mx , with most selected sites located in loop (ref. ). in mx , sites selected in the primate lineage were detected outside loop , and mx also showed evidence of selection in other domains , ; these sites are promising candidates for being additional determinants of antiviral activity. antigen presentation and the ensuing t cell activation are central processes in mammalian cellmediated immune response (fig. ) . therefore, a convenient strategy for pathogens to elude immune surveillance is to hijack the molecular pathways responsible for these processes , . in line with the arms race scenario, there is evidence of positive selection at several mammalian genes involved in antigen presentation and the regulation of t cell activation , (fig. ) . the pathogen-driven mechanisms underlying evolution at these genes are likely to be manifold. one mechanism is genetic conflict with a pathogen-encoded component, evidence of which can be seen in the protein cd . positively selected sites in the transmembrane and juxtamembrane region of cd interact with mir (fig. ) , a kaposi sarcomaassociated herpesvirus (kshv) protein that downmodulates cd expression , . a second mechanism is the use of cell-surface molecules as viral receptors: some adenovirus strains, for example, have been reported to exploit cd and cd for cellular attachment , . a third mechanism is the broadening or tuning of the host's ability to process and present pathogen-derived components. for example, a positively selected site in the carbohydrate-recognition domain of cd (also known as langerin; a birbeck granule molecule) affects an amino acid position that is directly involved in the binding of pathogen-derived glycoconjugates . these mechanisms are not mutually exclusive. for example, a plethora of viral pathogens (such as herpes simplex virus , human papillomavirus, hiv- and kshv) interfere with cd d trafficking and recycling , . as a consequence, the cytoplasmic and transmembrane regions of cd d display positively selected sites, one of which is within a primate-specific trafficking signal. additional positively selected sites are located in the cd d extracellular region and flank the t cell receptor interaction surface and the lipid-binding pocket, which suggests that they exert an effect on antigen-binding specificity . finally, we draw attention to one of the few attempts at assessing the part that helminth infections have played as selective pressures for mammals and at integrating epidemiological information into molecular evolutionary approaches. machado and co-workers found evidence of positive selection at the mammalian gene fc fragment of igg, low affinity iiib, receptor (fcgr b), which is expressed by eosinophils and is involved in the binding of immunoglobulin g (igg)-coated parasites. notably, the authors also tested a specific hypothesis whereby mammalian lineages hosting a wider range of helminth species should show stronger evidence of selection compared with other species (this was accomplished by running the phylogenetic analysis by maximum likelihood (paml) branch-site models with helminth-rich lineages as foreground branches . their hypothesis was verified, providing a plausible explanation for the evolutionary pattern at fcgr b and suggesting that similar approaches may be used to detect other mammalian genes involved in genetic conflicts with helminth parasites. as exemplified by ace , host-pathogen interactions are not limited to immune system components. the reasons why genes with no specific defence function may be targeted by the selective pressure imposed by infectious agents are manifold. the best known instances probably refer to gene products that act as incidental receptors for pathogens, as is the case with ace . other host gene products that engage in genetic conflicts include those that participate in the coagulation cascade and the contact system, which are commonly hijacked by bacterial pathogens to promote tissue invasion or to elude detection by immune cells (see ref. for a review). an alternative possibility is that the host builds a line of defence based on the sequestration of essential micronutrients from the pathogen, a phenomenon known as 'nutritional immunity' . housekeeping genes. incidental receptors are often represented by the products of housekeeping genes, which are typically expressed at high levels by different cell types. among these, the transferrin receptor (tfrc) gene encodes a cell-surface molecule (transferrin receptor protein (tfr )) that is essential for iron uptake. tfr is used as a receptor by mouse mammary tumour virus, canine parvovirus and rodent new figure | genes involved in antigen processing and presentation and t cell regulation are common targets of positive selection in mammals. all pathway components are designated using official gene names (excluding the major histocompatibility complex (mhc) and t cell receptor (tcr)) and are highlighted in red if they are targets of positive selection in mammals or primates , , . the molecular components of different antigen processing and presentation pathways are shown (details from refs , ) to provide a general overview of the extent of positive selection and to highlight the function of positively selected genes, as most of their protein products directly interact with the antigen. thus, the figure is not meant to show all molecules involved in the process or to convey mechanistic insights. also, some genes may show tissue-specific expression or may be induced under specific circumstances: their products are nonetheless included for the sake of completeness. as for t cell regulatory molecules, the representation does not reflect the stoichiometry of binding (for example, cd functions as a dimer). notably, the same molecule may be expressed by different populations of t cells, although here each molecule is shown on one t cell type only (to avoid redundancy). the dashed arrows and '?' indicate steps that lack clear molecular definition or are only inferred. the orange circles, and red and blue shapes at the bottom of the figure represent proteolytic fragments. b m, β -microglobulin; blmh, bleomycin hydrolase; calr, calreticulin; cd lg, cd ligand; ctla , cytotoxic t lymphocyte protein ; cts, cathepsin; cyb, cytochrome b; erap, endoplasmic reticulum aminopeptidase; havcr , hepatitis a virus cellular receptor ; hla-dm, major histocompatibility complex, class ii, dm; icos, inducible t cell co-stimulator; icoslg, icos ligand; ifi , interferonγ-inducible protein ; inkt, invariant natural killer t; itcr, invariant tcr; lgmn, legumain; lnpep, leucyl-cystinyl aminopeptidase; ncf, neutrophil cytosol factor; npepps, puromycin-sensitive aminopeptidase (also known as psa); nrd , nardilysin; pdcd , programmed cell death ; pdcd lg , programmed cell death ligand ; pdia , protein disulfide-isomerase a ; ros, reactive oxygen species; tap, antigen peptide transporter; tapbp, tap-binding protein (also known as tapasin); thop , thimet oligopeptidase ; tpp , tripeptidyl-peptidase . the elimination of deleterious amino acid-replacing substitutions, which results in fewer non-synonymous changes than expected under neutrality (dn/ds < ). world arenaviruses. in line with the arms race scenario, tfrc evolved adaptively in rodents and caniforms, and positively selected sites are mainly located in the extracellular domain regions that interact with rodentinfecting arenaviruses (fig. ) and carnivore-infecting parvoviruses, respectively , . interestingly, positive selection at the primate transferrin (tf) gene, which encodes the tfr ligand, was also recently described ; in this case, selection is driven by bacteria, not viruses . transferrin is the major circulating iron transporter in mammals and is also thought to participate in nutritional immunity by sequestering iron from bacteria. consistently, most positively selected sites were found to have evolved to counteract binding by bacterial transferrin surface receptors that scavenge host iron . thus, different selective pressures exerted by distinct molecular mechanisms contributed to shaping the evolution of a central homeostatic process -in this case, iron transport in mammals. another housekeeping gene product that acts as a viral receptor is niemann-pick c protein (npc ), a sterol transporter located in the membrane of late endosomes and lysosomes. npc is expressed by most cell types and is used by filoviruses (such as ebov and marburg virus). evolutionary analysis of mammalian npc genes indicated that three positively selected residues are located in the amino-terminal portion of the second npc luminal loop; binding of this loop by the ebov glycoprotein (gp) is necessary and sufficient for the viral receptor activity of the sterol transporter , (fig. ) . the second luminal loop of npc is also bound with high affinity by the gp encoded by lloviu virus, a bat-derived, ebov-like filovirus . thus, npc may represent a universal receptor for filoviruses, and the constant selective pressure exerted by such viruses might have greatly contributed to shaping mammalian genetic diversity at loop . these data may have great and immediate practical values. in fact, small molecules that directly target npc and disrupt gp binding are regarded as possible therapeutic compounds against ebov [ ] [ ] [ ] (fig. ) . because mammalian npc diversity at the interaction surface is driven by selection, future efforts in this direction are likely to benefit from the incorporation of evolutionary analysis; this would be especially important when testing therapeutic molecules on model organisms and non-human mammals. in humans, mutations in npc cause niemann-pick disease type c , a progressive neurodegenerative condition. this is in line with the central role of this transporter in housekeeping functions; thus purifying selection. is expected to constrain variation in the gene. indeed, the human-mouse dn/ds calculated for the npc whole-gene region is definitely lower than , as is the case for most genes (fig. ) . in fact, mammalian npc genes show a large preponderance of codons evolving with dn/ds < , and positive selection is extremely localized in loop (fig. ) . this specific example illustrates a general concept, whereby molecules involved in central homeostatic processes may be engaged in genetic conflicts with pathogens, although in several instances the sequence space accessible for adaptive mutation without a high fitness cost is expected to be limited. the coagulation cascade and contact system. as anticipated above, several components of the coagulation cascade and contact system evolved adaptively in mammals, most likely as a result of genetic conflicts with bacterial pathogens , . for instance, staphylococcus aureus is endowed with an arsenal of proteins that target such systems, including two cysteine proteinases (scpa and sspb) that cleave plasma kininogen at each terminal side of the bradykinin domain to generate kinins, with a consequent increase of vascular leakage . these events are central for bacterial virulence and are linked to the pathogenesis of sepsis. in kininogen (kng ), positively selected sites are located in all domains, with the exception of the highly conserved bradykinin region . one of the positively selected sites defines the n-terminal cleavage site of scpa and sspb, suggesting that sites flanking the bradykinin sequence are evolving to avoid recognition and cleavage by bacterial-encoded proteases. in analogy to the strong purifying selection acting on the bradykinin region, analysis of calculation cascade genes indicated that disease-causing mutations are more likely to occur at sites targeted by purifying selection and are rarer at positively selected sites . again, these observations highlight the coexistence of distinct selective regimes at the same gene regions and exemplify the concept of evolutionary trade-offs. the advent of high-throughput sequencing technologies has allowed for the generation of an unprecedented wealth of genetic data, including the whole-genome sequences of host reservoir species for human pathogens, as well as genetic information for multiple microbial and viral species and strains. moreover, large-scale approaches such as rna interference and mass spectrometry are providing detailed pictures of host-pathogen interactomes , . finally, an increasing number of crystal structures of interacting host and pathogen proteins solved in complex are available, allowing the opportunity to determine the structural basis of these interactions to identify regions or amino acids that lie at the host-pathogen contact surface. integration of these data with evolutionary analysis will allow the testing of specific hypotheses, including which species have responded to the pressure exerted by one or more pathogens (see the sars-cov example), which molecules and residues have participated in the arms race and which host-pathogen interacting partners are expected to have co-evolved. these advances are also expected to progressively change evolutionary genetics from a hypothesis-driven to a hypothesis-generating discipline. in this respect, we note that although the arms race scenarios we have described in this review imply some form of host-pathogen co-evolution over time, the nature of the interaction and its dynamics have often been inferred from the observed pattern of variation. indeed, the fact that the same residues that affect specific host-pathogen interactions are targeted by positive selection does not necessarily imply a causal link, and in many instances the specific selective agents underlying molecular adaptations remain to be determined. as shown above, these may well be accounted for by extinct pathogens or by agents that had a major co-evolutionary role in the past but that are now fading away from the landscape of common infections. with a few exceptions , , evolutionary studies only investigate extant genetic variation and modern pathogens, with little reconstruction of past events. nevertheless, we do not necessarily need to go back in time: evolutionary analyses can be used as predictive tools to pinpoint which genes and residues are more likely to contribute to present-day host-pathogen interaction and help explain species-specific susceptibility nature reviews | genetics the values for some of the genes discussed in this review are indicated. data were derived from the ensembl biomart database (see further information). b | natural selection acting on mammalian niemann-pick c (npc ) genes. npc is shown with its predicted membrane topology and protein regions coloured in hues of blue that represent the percentage of negatively selected sites (as detected by the single-likelihood ancestor counting method using datamonkey); the darker the blue, the higher the percentage. the location of three positively selected residues (red) is indicated on the left, and an alignment of the corresponding region is shown on the protein to the right (with red and blue representing positively and negatively selected sites, respectively). the interaction with the glycoprotein (gp; green) of filoviruses (such as ebola virus, marburg virus or lloviu virus) is shown. gp binds npc after processing by cellular proteases. ace , angiotensin-converting enzyme ; darc, duffy blood group, atypical chemokine receptor; mx , myxovirus resistance ; ssd, sterol-sensing domain; tfrc, transferrin receptor; tlr , toll-like receptor . to infection. several studies mentioned above, including those investigating selection at mx (ref. ), tfrc (refs , ) , tf and other protein-coding genes , , , , used experimental analyses to show that evolutionary information can indeed be exploited to gain highresolution insight into the molecular determinants of binding affinities at host-pathogen interfaces. the studies of iron transporters hold particular value because the authors analysed the genetic variability of both the host and the pathogen and showed that both parties evolved in response to mutually exerted pressures, in line with the red queen principles. so far, few attempts have been made at integrating evolutionary analyses of host and pathogen interacting partners into a common framework. however, efforts in this direction hold the promise of improving our understanding of the strategies used by both hosts and pathogens to adapt and counter-adapt. in turn, this knowledge has possible biomedical and therapeutic implications, given the ability of different pathogens or distinct strains of the same infectious agent to elude not only natural host defences but also drugs and vaccination strategies. as a final note, we mention that we have exclusively focused on adaptive events involving coding gene regions. nevertheless, several recent studies (see ref. for a review) have highlighted the role of non-coding variants as important determinants of susceptibility to infection within species. thus, host-pathogen conflicts are more than likely to have contributed to adaptive evolution at regulatory elements during speciation. detection of these adaptive events will benefit from the availability of high-throughput techniques (for example, rna sequencing and chromatin immunoprecipitation followed by sequencing) and the development of methodological approaches for dissecting molecular evolution in non-coding regions; notably, recent data have shown the usefulness of a framework similar to dn/ds to analyse the evolutionary history of mammalian transcriptional enhancers . application of this methodology (or extensions thereof) to the study of host-pathogen interactions will provide valuable information on which non-coding sequence changes have been targeted by selection and thus modulate susceptibility to infection or related phenotypes. signatures of environmental genetic adaptation pinpoint pathogens as the main selective pressure through human evolution a new evolutionary law running with the red queen: the role of biotic conflicts in evolution the causes of evolution (longmans, green, & co, ) revenge of the phages: defeating bacterial defences genomic variability as a driver of plant-pathogen coevolution? mainstreaming caenorhabditis elegans in experimental evolution insights from natural host-parasite interactions: the drosophila model from evolutionary genetics to human immunology: how selection shapes host defence genes population genetic tools for dissecting innate immunity in humans the red queen's long race: human adaptation to pathogen pressure human genome variability, natural selection and infectious diseases natural selection and infectious disease in human populations the red queen and the court jester: species diversity and the role of biotic and abiotic factors through time phylogenies reveal new interpretation of speciation and the red queen host-parasite 'red queen' dynamics archived in pond sediment a matching-allele model explains host resistance to parasites antagonistic coevolution accelerates molecular evolution biological and biomedical implications of the co-evolution of pathogens and their hosts global trends in emerging infectious diseases origins of hiv and the aids pandemic. cold spring harb the cytoplasmic body component trim α restricts hiv- infection in old world monkeys positive selection of primate trim α identifies a critical species-specific retroviral restriction domain restriction of an extinct retrovirus by the human trim α antiviral protein positive selection of primate genes that promote hiv- replication hiv- capsid-cyclophilin interactions determine nuclear import pathway, integration targeting and replication efficiency this is an excellent review highlighting the importance of non-model organisms in understanding zoonotic infections diversification of an emerging pathogen in a biodiversity hotspot: leptospira in endemic small mammals of madagascar patterns of positive selection in six mammalian genomes genome analysis reveals insights into physiology and longevity of the brandt's bat myotis brandtii this work helps to clarify the species specificity of n. gonorrhoeae infection by analysing the binding of sialylated gonococci to human and chimpanzee cfh humanized tlr /md- mice reveal lps recognition differentially impacts susceptibility to yersinia pestis and salmonella enterica lipid a modification systems in gram-negative bacteria structural basis of species-specific endotoxin sensing by innate immune receptor tlr /md- signatures of positive selection in toll-like receptor (tlr) genes in mammals mass extinctions, biodiversity and mitochondrial function: are bats 'special' as reservoirs for emerging viruses? an extremely interesting study providing an overview of the evolutionary history of three bat genomes, with possible implications for immunity-related evidence for ace -utilizing coronaviruses (covs) related to severe acute respiratory syndrome cov in bats a good example of how evolutionary studies can provide insight into host range and disease emergence retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier isolation and characterization of viruses related to the sars coronavirus from animals in southern china severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats isolation and characterization of a bat sars-like coronavirus that uses the ace receptor full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus a central work showing that dpp of human and bat origin acts as a functional receptor for mers-cov host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase adaptive evolution of bat dipeptidyl peptidase (dpp ): implications for the origin and emergence of middle east respiratory syndrome coronavirus molecular basis of binding between novel human coronavirus mers-cov and its receptor cd adaptation and constraint at toll-like receptors in primates contrasted evolutionary histories of two toll-like receptors (tlr and tlr ) in wild rodents (murinae) variation matters: tlr structure and species-specific pathogen recognition extensive evolutionary and functional diversity among mammalian aim -like receptors ancient and recent selective pressures shaped genetic diversity at aim -like nucleic acid sensors rig-i-like receptors evolved adaptively in mammals, with parallel evolution at lgp and rig-i molecular basis for specific recognition of bacterial ligands by naip/nlrc inflammasomes rules of engagement: molecular insights from host-virus arms races evolutionary conflicts between viruses and restriction factors shape immunity a cross-species view on viruses positional cloning of the mouse retrovirus restriction gene fv paleovirology and virally derived immunity evolution of the retroviral restriction gene fv : inhibition of non-mlv retroviruses a study in wild mice showing that fv antiviral activity is broader than previously thought. it identifies positively selected residues in the c terminus that contribute to antiviral specificity evolution-guided identification of antiviral specificity determinants in the broadly acting interferon-induced innate immunity factor mxa a seminal paper that applies an evolution-guided approach to detect mx residues that confer antiviral specificity human mx is an interferon-induced post-entry inhibitor of hiv- infection evolutionary analysis identifies an mx haplotype associated with natural resistance to hiv- infection manipulation of costimulatory molecules by intracellular pathogens: veni, vidi, vici!! plos pathog mhc class i antigen presentation: learning from viral evasion strategies an evolutionary analysis of antigen processing and presentation across different timescales reveals pervasive selection a million year history of t cell regulatory molecules reveals widespread selection, with adaptive evolution of disease alleles the intertransmembrane region of kaposi's sarcoma-associated herpesvirus modulator of immune recognition contributes to b - downregulation the nef protein of hiv- induces loss of cell surface costimulatory molecules cd and cd in apcs members of adenovirus species b utilize cd and cd as cellular attachment receptors structural basis for langerin recognition of diverse pathogen and mammalian glycans through a single binding site hiding lipid presentation: viral interference with cd d-restricted invariant natural killer t (inkt) cell activation a threonine-based targeting signal in the human cd d cytoplasmic tail controls its functional expression evolutionary history of copynumber-variable locus for the low-affinity fcγ receptor: mutation rate, autoimmune disease, and the legacy of helminth infection one of the few studies of helminth-driven selective pressure in mammals that also integrates evolutionary analysis with epidemiological information thrombosis as an intravascular effector of innate immunity dual host-virus arms races shape an essential housekeeping protein an extremely interesting study extending the arms race scenario to a housekeeping protein, the transferrin receptor evolutionary reconstructions of the transferrin receptor of caniforms supports canine parvovirus being a re-emerged and not a novel pathogen in dogs escape from bacterial iron piracy through rapid evolution of transferrin mammalian npc genes may undergo positive selection and human polymorphisms associate with type diabetes niemann-pick c (npc )/ npc -like chimeras define sequences critical for npc 's function as a flovirus entry receptor cell entry by a novel european filovirus requires host endosomal cysteine proteases and niemann-pick c multiple cationic amphiphiles induce a niemann-pick c phenotype and inhibit ebola virus entry and infection inhibition of ebola virus infection: identification of niemann-pick c as the target by optimization of a chemical probe small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection evolutionary analysis of the contact system indicates that kininogen evolved adaptively in mammals and in human populations positive selection during the evolution of the blood coagulation factors in the context of their disease-causing mutations induction of vascular leakage through release of bradykinin and a novel kinin by cysteine proteinases from staphylococcus aureus viral immune modulators perturb the human molecular network by common and unique strategies genome-wide rnai screen identifies human host factors crucial for influenza virus replication a novel test for selection on cis-regulatory elements reveals positive and negative selection acting on mammalian transcriptional enhancers paml : phylogenetic analysis by maximum likelihood accuracy and power of bayes prediction of amino acid sites under positive selection bayes empirical bayes inference of amino acid sites under positive selection detecting individual sites subject to episodic diversifying selection high sensitivity to aligner and high rate of false positives in the estimates of positive selection in the drosophila genomes class of multiple sequence alignment algorithm affects genomic analysis estimates of positive darwinian selection are inflated by errors in sequencing, annotation, and alignment the effects of alignment error and alignment filtering on the sitewise detection of positive selection effect of recombination on the accuracy of the likelihood method for detecting positive selection at amino acid sites evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level the effect of insertions, deletions, and alignment errors on the branch-site test of positive selection multiple hypothesis testing to detect lineages under positive selection that affects only a few sites a random effects branchsite model for detecting episodic diversifying selection modeling the site-specific variation of selection patterns along lineages performance of standard and stochastic branch-site models for detecting positive selection among coding sequences statistical properties of the branch-site test of positive selection towards a systems understanding of mhc class i and mhc class ii antigen presentation cd antigen presentation: how it works the authors declare no competing interests. key: cord- -fb rtmx authors: joseph, maxwell b.; mihaljevic, joseph r.; arellano, ana lisette; kueneman, jordan g.; preston, daniel l.; cross, paul c.; johnson, pieter t. j. title: taming wildlife disease: bridging the gap between science and management date: - - journal: j appl ecol doi: . / - . sha: doc_id: cord_uid: fb rtmx . parasites and pathogens of wildlife can threaten biodiversity, infect humans and domestic animals, and cause significant economic losses, providing incentives to manage wildlife diseases. recent insights from disease ecology have helped transform our understanding of infectious disease dynamics and yielded new strategies to better manage wildlife diseases. simultaneously, wildlife disease management (wdm) presents opportunities for large‐scale empirical tests of disease ecology theory in diverse natural systems. . to assess whether the potential complementarity between wdm and disease ecology theory has been realized, we evaluate the extent to which specific concepts in disease ecology theory have been explicitly applied in peer‐reviewed wdm literature. . while only half of wdm articles published in the past decade incorporated disease ecology theory, theory has been incorporated with increasing frequency over the past years. contrary to expectations, articles authored by academics were no more likely to apply disease ecology theory, but articles that explain unsuccessful management often do so in terms of theory. . some theoretical concepts such as density‐dependent transmission have been commonly applied, whereas emerging concepts such as pathogen evolutionary responses to management, biodiversity–disease relationships and within‐host parasite interactions have not yet been fully integrated as management considerations. . synthesis and applications. theory‐based disease management can meet the needs of both academics and managers by testing disease ecology theory and improving disease interventions. theoretical concepts that have received limited attention to date in wildlife disease management could provide a basis for improving management and advancing disease ecology in the future. population density decreased due to disease, contact rates would become too low for transmission to continue; thus, pathogens would be extirpated before host populations (anderson & may ) . however, disease-induced declines and extinctions of wildlife resulting from small population sizes, reservoir hosts, host switching and heterogeneity in contact rates, susceptibility and transmission within and among populations have forced a re-evaluation of this perspective (de castro & bolker ) . for example, when contact rates among individuals do not depend on host density, pathogens are more likely to drive populations to extinction because transmission continues as host populations are reduced, as seen with tasmanian devil sarcophilus harrisii (boitard ) facial tumour disease where transmission appears to be related to mating behaviours (mccallum ) . white nose syndrome in bats also seems to be more likely to cause extinctions owing to social behaviours in which hosts cluster in hibernacula, reducing the correlation between contact rates and population densities (langwig et al. ) . in recent decades, wildlife disease management (wdm) has been increasingly used to conserve threatened wildlife populations (deem, karesh & weisman ) . for example, wdm has controlled outbreaks of feline leukaemia in critically endangered iberian lynxes lynx pardinus (temminck ) and rabies in endangered ethiopian wolves canis simensis (r€ uppell ), both of which are associated with domestic animal disease reservoirs (haydon et al. ; l opez et al. ). despite the wealth of empirical wdm research, management outcomes can be difficult to predict because system-specific information is lacking for novel pathogens and many theoretical concepts in disease ecology (see table for a subset) have not been widely tested in the field, leading to uncertainty in their generality. this is unlike other environmental management disciplines such as fisheries ecology, which has effectively used theoretical models to predict yields, manage harvest timing and limits and design reserves (e.g. gerber et al. ) . indeed, theoretical applications in fisheries ecology have also produced insights into density-dependent population dynamics, metapopulation theory and the evolution of life-history table . selected theoretical concepts in disease ecology: theoretical concepts in disease ecology theory that apply to wildlife disease management, some direct management implications and a theoretical reference for each concept theoretical concepts management applications selected references host population regulation by disease disease reductions may increase host abundance and/or survival anderson & may ( ) trade-offs between transmission and virulence artificial stocking may increase virulence, and culling may reduce or increase virulence depending on pathogen life-history, culling selectivity and transmission dynamics frank ( ) seasonal drivers of disease emergence and dynamics intervention timing and frequency matters; control efforts can target transmission peaks altizer et al. ( ) pathogen interactions within hosts managing one pathogen alters the transmission and virulence of other pathogens fenton ( ) multi-host species disease dynamics reservoir hosts can drive the extinction of alternate hosts; rates of interspecific transmission may be inferred by managing one host species; management may need to target multiple host species dobson & foufopoulos ( ) spread of disease in spatially structured hosts corridor vaccination can reduce disease in metapopulations; movement controls are unlikely to work for chronic infections keeling & eames ( ) transmission increases with host density host density reductions may reduce disease transmission, and density thresholds for disease persistence may exist anderson & may ( ) transmission increases with disease prevalence independent of host density transmission associated with sexual interactions is more likely to cause host extinction, and non-selective culling may not reduce transmission getz & pickering ( ) predation as a regulator of host population and disease predator conservation may reduce disease in prey populations packer et al. ( ) community composition, diversity and disease risk biodiversity loss and community disassembly may increase disease as predators and less-competent hosts are extirpated, depending on community composition and transmission dynamics keesing, holt & ostfeld ( ) environmental reservoirs and indirect transmission duration of disease control must scale with the environmental persistence; host extinction is more likely joh et al. ( ) individual-level variation and superspreading heterogeneity in individual resistance and infectiousness within a host population can lead to 'superspreaders' that account for a large portion of transmission; management can target superspreaders lloyd-smith et al. ( ) strategies (frank & leggett ) . in this review, we assess the extent to which a similar union between theory and practice has been achieved in wdm. we use a quantitative, case-based approach to provide a critical retrospective of wdm over the last four decades to: (i) quantify how frequently specific theoretical concepts from disease ecology have been applied in the literature, (ii) identify prevailing management objectives, groups and reported outcomes and (iii) assess taxonomic biases in wdm literature. we then present methodological and conceptual opportunities to facilitate the newly emerging synthesis of disease ecology and management, drawing from environmental management and biomedicine to outline steps towards more cost-effective, efficacious and informative wdm. this synthesis aims to facilitate the development of a more predictive framework for disease interventions while simultaneously building empirical support for understanding of disease processes across systems. we compiled wdm case studies using a systematic, twostep search process with specific criteria for inclusion in our review. in the first stage, we searched titles and abstracts of records included in isi web of science using specific terms [(wild*) and (disease* or infect* or pathogen* or parasit*) and (manage* or conserv*)] to capture breadth in published wdm records. additionally, we searched for case studies in grey literature using the following online resources: national wildlife health center, wildpro, national biological information infrastructure wildlife disease information node, u.s. government printing office and the u. s. fish and wildlife service. no case studies identified in the grey literature met our criteria that were independent of cases identified in the scientific literature. case studies were also identified using previous review papers and books (lafferty & gerber ; wobeser ; hudson et al. ; wobeser ; ostfeld, keesing & eviner ) . we conducted a follow-up search with isi web of knowledge to capture subject depth for each managed disease or pathogen identified in the first step, using a search string that included all pathogen and disease names along with terms related to management interventions: (e.g. (rabi* or lyssavir*) and (vaccin* or treat* or manag* or control* or preval* or incidence or cull*) and (wild* or free-ranging or free ranging). the initial web of science search returned articles dating back to , but our disease-specific search strings often returned results dating back to the s or earlier. historical accounts of wdm are probably under-represented in the literature available online, and those returned by our search strings were often less readily accessible than recently published articles. as a result, the cases reviewed here primarily represent recently published cases of wdm. the publication dates of included cases range from to , and % of the cases included in our review were published after . for each article that met our criteria, we recorded (i) pathogen and host characteristics, (ii) management motivations, strategies and outcomes and (iii) whether and how disease ecology theory was incorporated in each article that satisfied our criteria. we only included cases that provided quantitative data on disease in a population or area (number of cases, seroprevalence, prevalence, incidence, etc.). when multiple records were encountered for a single management event, we used the most recent record (as of spring ). cases that only described disease management in humans, livestock or plants were excluded. finally, we only included studies that described management of diseases in populations (operationally defined as groups of > individual) of free-ranging wildlife. incorporation of disease ecology theory was defined broadly as the explicit use or discussion of theoretical concepts relating to transmission dynamics, host population regulation by disease, pathogen evolution, host or pathogen community effects on transmission, spatial heterogeneity in disease dynamics, life stage-or age-specific disease dynamics, endemic vs. epidemic disease states and herd immunity (see table for a list of specific concepts used to define theory in the literature search). four broad management objectives were identified, including conservation of a host species, prevention of disease transmission to humans, prevention of disease transmission to livestock and basic research. studies falling into our basic research category were usually an attempt to better understand the system, determine the extent of the disease problem or provide insight into future management opportunities. to investigate differences in theory application and objectives among managing groups, we also classified author affiliations for each paper as academic, governmental, private or some combination thereof. university or university laboratory affiliations were considered academic, and we used the same criteria for governmental and private affiliation. mixed author affiliations (e.g. academic and governmental) were recorded for individual authors and for papers with multiple authors with different affiliations. we characterized management outcomes by recording whether the disease was eradicated, and if not, whether there were changes in the prevalence, incidence or intensity of disease. ideally, these changes could be quantified and compared across disease systems, but in many cases, inconsistent reporting of results and a lack of pre-management or control data complicate meaningful quantitative comparisons of effect sizes across studies. finally, we considered whether the original management objective was attained using the following categories: 'apparent success', meaning that there was no unmanaged control population or area to compare to the treated area; 'partial success', meaning that at least some of the management objectives were reported as fulfilled; and 'success,' for cases that had controls and reported fulfilment of all management objectives. while management outcomes are rarely clear-cut in this practice, this simplified classification system facilitated coarse comparisons across disease systems and among management studies with variable monitoring time-scales. in total, scientific articles among the identified from the search strings satisfied our criteria (see appendix s and table s in supporting information). many ( %) cases consisted of collaborations between government agencies and academic researchers (fig. ) . conservation motivated % of management that involved private groups, whereas basic research was only conducted when academics were involved. overall, host conservation was the most common objective ( % of cases), while reducing disease risk to humans and domestic animals were the next most common objectives ( % and % of cases, respectively; fig. ). disease ecology theory as defined above has only recently been incorporated consistently into wdm literature (fig. a) . some theoretical concepts such as density dependence in transmission were frequently applied, while others such as pathogen evolution and the role of predators and biodiversity in regulating disease were not (fig. , table ). unexpectedly, papers authored by academics were not more likely to incorporate theory (fisher's exact test, p = Á ). management outcomes were related to theory incorporation (fisher's exact test, p = Á ). the three papers that reported disease increases following intervention explained their results in terms of disease ecology theory, providing insights into transmission and optimal control strategies (e.g. cross et al. ; fig b) . however, there was no relationship between management objective attainment and theory incorporation (fisher's exact test, p = Á ). nevertheless, some counter-intuitive but successful management distribution of management outcomes according to whether disease ecology theory was incorporated. reductions and increases refer to changes in prevalence, incidence, infection intensity or diseaseinduced mortality; eradication refers to local rather than global eradication. programmes clearly benefited from theory. for example, control of classical swine fever in wild boar sus scrofa (linnaeus ), is often hampered by stage-dependent transmission dynamics. susceptible piglets are hard to target with baited vaccines and act as disease reservoirs. by allowing an epidemic to peak such that most adults are immune, then culling only piglets, swiss academics and governmental groups successfully eradicated the disease from a -km region near the italian border (schnyder et al. ) . reductions in prevalence, incidence or infection intensity were reported in % of cases, with vaccination and host treatment as the most commonly applied intervention strategies (fig. b) . ninety-four percentage of cases reported management in terrestrial systems, with % and % of cases reporting management in freshwater and marine systems, respectively. the majority ( %) of reported management efforts were directed towards mammals, with birds and fish representing % and % of cases, respectively. however, mammals are less speciose and less threatened by disease than amphibians (vi e, hilton-taylor & stuart ), for which we found no published wdm records. taxonomic bias could arise because vaccines and drugs are developed primarily to protect human, livestock or poultry health. relatively few cases ( %) reported a failure to meet management objectives, possibly due to negative publication bias. collectively, our analyses indicate that while academics and government agencies collaborate to manage wildlife diseases, collaborations do not necessarily lead to an integration of disease ecology theory with management. density-dependent transmission was often assumed to justify control efforts, but other theoretical concepts were rarely applied (fig. ). data quality issues and potential publication biases currently hinder the application of metaanalytical techniques for wdm, and there is a paucity of published records on non-mammalian management. overcoming challenges to theory-based management while collaboration alone may not necessarily lead to an integration of disease ecology theory and wdm, it should provide a starting point for such integration. academics and managers have unique needs, constraints and knowledge-seeking behaviour that challenge such collaborations. for instance, untreated control areas or pre-treatment data can be unavailable or even unethical in wdm, but are critical for experiments in disease ecology. while academics may design field experiments to test and refine theoretical models, managers need practical, effective and uncontroversial management strategies that succeed in particular systems. such strategies may not be easily identified in the literature from model systems, which managers may be unable to access. modelling wildlife disease systems requires decisions about model complexity. in our experience, theoreticians prefer simpler, more general models that may be applicable to many systems. these models are easier to parameterize and analyse, and the resulting papers are likely to have a wider academic audience. on the other hand, simple models are easily discarded by managers because they lack system-specific detail. this tension is likely to continue, but we recommend additional flexibility on both sides. in particular, managers should appreciate that the addition of modelling details that are only weakly supported by data may not lead to better predictions. meanwhile, theoreticians may develop general models that bear little resemblance to any biological system. furthermore, individuals may be most interested in a particular suite of theoretical concepts, but a narrow approach can impede management by ignoring the full range of phenomena relevant to producing desired management outcomes (driscoll & lindenmayer ) . thus, academics and managers are challenged to take a broad view that incorporates relevant theoretical concepts and an appropriate amount of biological realism, which may require collaboration among researchers with different areas of expertise (driscoll & lindenmayer ) . unfortunately, such large collaborative efforts may bring a loss of autonomy at odds with academic or governmental bureaucracy. a diverse body of literature addresses the gap between academics and environmental managers and provides examples of successful strategies for integration. for instance, international symposia have improved information transfer in invasion biology (shaw, wilson & richardson ) . social networking, joint appointments, interinstitutional sabbaticals, fellowships, concise reporting table , and their application in the literature was included in this review, showing that some concepts such as densitydependent transmission are well represented, while others were less frequently (or not at all) applied. of relevant science to managers and targeted calls for research proposals by managers can all help to foster cooperation (gibbons et al. ) . interdisciplinary working groups for particular management issues can ensure that the needs of multiple stakeholders are considered together when organizing such activities (gibbons et al. ) . groups such as the wildlife disease association and applied journals including the journal of wildlife diseases have encouraged interdisciplinary collaboration, and a broader recognition of the complementarity between disease ecology theory and wdm can provide the impetus for expanding interdisciplinary work in this important field. theory can help address unprecedented management challenges and can be refined in the process. disease outbreaks are often caused by novel pathogens or the appearance of known pathogens in new hosts. often, details of host-pathogen interactions are unknown. by combining limited information with general principles of disease ecology (table ) theory is often refined by evaluating competing hypotheses. therefore, adaptive management is one way to integrate theory and management, especially if multiple management hypotheses can be tested (holling ) . differentiation among competing hypotheses is synonymous with identifying optimal management in this framework. thus, monitoring the effects of disease interventions on prevalence, virulence and host vital rates can help to estimate model parameters including transmission and recovery rates and help in evaluating management outcomes. when agencies have limited flexibility in decisionmaking, thus precluding adaptive management, the best available theoretical and system-specific knowledge can at least produce a 'best guess' management strategy (gregory, ohlson & arvai ) . failed management is still valuable in this framework because outcomes can be compared to predictions from competing models of disease dynamics that can be selectively eliminated, as with tasmanian devil facial tumour disease (mcdonald-madden et al. ). this approach produces mechanistic insights that might be missed if management strategies are characterized simply as effective or ineffective based on management outcomes. if many groups apply adaptive management separately in similar systems without communicating, generalities that benefit management and disease ecology may remain elusive. systematic reviews, invaluable to biomedicine, can help establish which interventions are effective and explain heterogeneity in effectiveness with a standardized meta-analytic approach. guidelines for systematic reviews in environmental management exist, but have not been applied in wdm (sensu pullin & stewart ) . our metadata indicate that this may be due to a lack of data quality and quantity. simple recommendations to facilitate the production of data suitable in quality for systematic review include: (i) establishing unmanaged control areas and/or baseline data, (ii) achieving replication, (iii) reporting precision for estimates of model parameters, prevalence and effect size, (iv) publishing and mechanistically explaining failed management and (v) reporting the spatiotemporal extent of management. data quantity may be lacking because of publication biases and a lack of incentives for managers to publish when working independently. this latter issue is minor if collaborations involve academics, but even motivated scientists may have difficulty publishing if management has no effect. however, management failures are as important to report in the literature as successes for systematic reviews and meta-analyses. an evaluation of the applicability of a theoretical concept in a particular case will rely on comparisons of observed data with explicit predictions from theoretical models, which can often be derived through mathematical modelling. theoretical concepts can be explicitly built into systemspecific mathematical models to identify and evaluate management strategies, as exemplified in a modern wdm challenge: chronic wasting disease (cwd). in , the state of wisconsin began culling white-tailed deer odocoileus virginianus (zimmermann ) and lengthened the hunting season in an attempt to eradicate cwd. these efforts were mandated despite uncertainty over transmission dynamics, the environmental persistence of prions that cause cwd and the time of cwd arrival to the state (bartelt, pardee & thiede ) . five years later, prevalence was still slowly increasing (heisey et al. ) . as this epidemic has unfolded, several models have been used to describe the dynamics of cwd (gross & miller ; wasserberg et al. ; wild et al. ) . simple models of cwd do not tend to produce plausible results. purely density-dependent transmission models predict increases in prevalence that are too rapid, while frequencydependent transmission models predict rapid host extinction or epidemics that are very slow to develop (on the order of centuries). modelling indirect transmission via environmental contamination results in a wider range of outcomes and produces several patterns observed in the field including a slow disease progression with prevalences of over % and significant host population reduction without rapid extinction (almberg et al. ) . recent analyses did not provide much support for models with variable increases in transmission over models with variable starting prevalence, suggesting that host density effects may be relatively weak in this system (heisey et al. ) . taken together, these results suggest that managers would have to reduce deer densities to extremely low levels, probably for decades, at which point other stakeholders such as hunters may wonder whether it is worse to have a lower deer density due to disease impacts or disease control efforts. disease ecology theory is not a 'silver bullet' for solving management problems. indeed, some have pointed out that application of theory under certain circumstances can lead to poor management (driscoll & lindenmayer ) . misapplication of theory at an inappropriate scale, or in a system that does not meet necessary assumptions, could cause undesired consequences. for instance, an assumption of broad-scale culling as a disease management intervention is that pathogen transmissions scale positively with host population density. however, density-dependent changes in social behaviour can alter dispersal patterns that violate this assumption, increasing transmission, as seen with bovine tuberculosis (tb) in cattle and european badgers meles meles (linnaeus ) (woodroffe et al. ) . work in the badger-tb system has refined our understanding of the effects of culling on social animals. however, one could argue that if culling-induced dispersal had been discovered in another disease system, the unintended increase in tb transmission to cattle following badger culling might have been avoided. unfortunately, had this been the case, the applicability of the social perturbation-transmission increase phenomenon to the badger-tb system would have remained uncertain. this underscores the value of moving beyond a case studydominated paradigm, towards a rigorous and empirically verified contingency-based understanding of theory applicability to disease management. such a framework could test and refine theoretical concepts that have shown promise in model systems, but have been infrequently applied in the wdm literature (fig. ) . what are the future directions for wdm? recent advances in disease ecology based on co-infection provide new ways to reduce disease susceptibility and transmission. for example, in african buffalo syncerus caffer (sparrman ), gastrointestinal nematodes reduce individual resistance to mycobacterium tuberculosis, which causes bovine tb, because of cross-regulatory immune responses to micro-and macroparasites (ezenwa et al. ) . hence, deworming drugs may increase resistance to tb and improve tb vaccination efficacy, raising the possibility that tb could be managed indirectly through nematode control (elias, akuffo & britton ; ezenwa et al. ) . management involving immunological trade-offs could improve general understanding of immune-mediated parasite interactions. for instance, interventions aimed at helminth parasites, which accounted for % of cases in our review, are predicted to differentially affect microparasite transmission depending on recovery rates and virulence (ezenwa & jolles ) . these predictions could be evaluated opportunistically by monitoring non-target pathogens. similarly, management in systems with co-infecting parasites could be used to understand virulence evolution in response to changing co-infection dynamics (alizon & van baalen ) . there is increasing recognition that microbial symbiosis can play a role in host health. using mutualistic microbes to control disease, a technique known as probiotics therapy, has benefitted aquaculture, agriculture and human medicine. for example, addition of bacillus and pseudomonas bacteria controls pathogenic vibrio that infect prawns, salmon and crabs in aquaculture (irianto & austin ; panigrahi & azad ) . bifidobacterium and lactobacillus can ameliorate escherichia coli infection in pig farms (zani et al. ; shu, qu & gill ) . in humans, probiotics can treat diarrhoea caused by clostridium difficile infection and antibiotic therapy (mcfarland ; rohde, bartolini & jones ). probiotics may prove useful for wdm. frogs with certain skin bacteria may be less susceptible to population extirpation caused by chytridiomycosis, a fungal disease that implicated global amphibian declines (lam et al. ) . experimental augmentation of skin bacteria reduces mortality of susceptible amphibians in captivity, and field experiments are underway to determine whether probiotics can prevent extirpations in nature (harris et al. ; rex ) . probiotics can also reduce vector populations. for instance, laboratory-reared mosquitoes with a maternally heritable probiotic that disrupts dengue fever virus transmission can locally replace wild mosquito populations and reduce dengue fever risk (hoffmann et al. ) . the successful use of probiotics depends on an understanding of microbial ecology, especially with respect to long-term probiotic maintenance in a host or environment. risks associated with introducing non-native microbes may be ameliorated by isolating probiotic agents from native hosts. as data accumulate, it will be important to evaluate whether the risks of probiotics outweigh those associated with antibiotic treatment in terms of antibiotic or probiotic resistance and pathogen virulence evolution. finally, linking these within-host processes to among-host processes (e.g. microbial community structure and transmission) is an important frontier for wdm and disease ecology. optimal management strategies depend on the degree to which transmission is driven by host population density and the amount of individual and population heterogeneity in contact or transmission rates. host population size, aggregation patterns and contact rates can be altered through hunting, artificial feeding, predator and scavenger conservation, fertility control, culling, translocation of individuals, artificial stocking, movement barriers, etc. understanding the functional form of the relationships among host contacts, density and transmission in real systems is critical to predicting the impacts of such interventions. therefore, field manipulations will play a crucial role in refining our mechanistic understanding of disease transmission. optimal management strategies are not static; contact rates, host abundance and demography can change naturally over time, in response to disease and due to management. for example, group sizes and contact rates may remain constant for highly social species despite management-induced population reduction. reservoir hosts may increase disease risk for other species if infected individuals maintain high fitness via increased reproductive output (fecundity compensation, for example, schwanz ) . fertility control of such reservoir hosts may protect other species that are less tolerant to infection. lastly, if transmission peaks in a short time period, perhaps due to breeding or a pulsed influx of juveniles (altizer et al. ) , management may be applied optimally in a narrow time interval. brucellosis in the elk (cervus elaphus linnaeus ) populations of the greater yellowstone ecosystem of wyoming illustrates how management can capitalize on temporal transmission dynamics. every year, wildlife managers provide supplemental feed to elk at sites in the region. contrary to theoretical predictions, elk abundance at each feeding site is uncorrelated with brucellosis seroprevalence (cross et al. ), but locally, host contact rates correlate positively with elk density (creech et al. ) . these seemingly contradictory findings are explained by variation and interaction between transmission and host density over time, which suggests that brucellosis seroprevalence may be reduced by shortening the length of the feeding season in early spring when transmission is highest (maichak et al. ). this option is appealing because vaccination has had limited, if any, effect (cross et al. ) , and a testand-remove programme, although effective, is financially prohibitive to implement across a broad region. establishing contact networks for a variety of disease systems across a range of densities and over time will help to identify life-history traits, social structures and other species characteristics that predictably influence transmission. taken together, these population-level tools can advance general understanding of transmission dynamics and optimize the application of disease control strategies. community-level interactions including predation and competition can influence disease management outcomes. predation on hosts can increase or decrease disease prevalence and the likelihood of epidemics depending upon predator selectivity, as well as behavioural and demographic effects on host populations that influence transmission and disease susceptibility (packer et al. ; holt & roy ) . interspecific competition can also influence host background mortality and thus the net effect of disease in a population (bowers & turner ) . unintended consequences when managing predators or competitors may be of less concern if coupled with ongoing management such as predator restoration and invasive species control. interspecific transmission of generalist parasites is hard to quantify, but attempts to control generalist parasites in one host species can reveal the extent to which other hosts contribute to transmission. for example, tsao et al. ( ) vaccinated white-footed mice peromyscus leucopus (rafinesque ) in southern connecticut to reduce the prevalence of borrelia burgdorferi, the bacterium that causes lyme disease. based on the strains of b. burgdorferi found in ticks in vaccinated plots, and the relationships between mouse density and tick infection prevalence, the authors concluded that other host species contributed more to tick b. burgdorferi infections than previously thought. thus, vaccination would have to target multiple host species to be effective. contact prevention between wildlife and livestock also provides an opportunity to prevent disease spillover, and when linked with monitoring of both wildlife and domestic populations, can be used to estimate relative rates of within-and among-species transmission. novel management strategies may target ultimate causes of disease emergence once they have been identified. for instance, lyme disease risk in the north-eastern united states increases with habitat fragmentation, which leads to extirpations of (i) predators and competitors that limit white-footed mouse abundance and (ii) less-competent hosts for b. burgdorferi and ticks (ostfeld & logiudice ) . in this system, biodiversity conservation might be an option for proactive wdm. management interventions that recognize and target community-or ecosystem-level processes are rare, but in some cases may more directly address disease threats than focusing solely on individuals or populations. a black box? common wdm interventions have evolutionary consequences that remain largely unexplored. in contrast, a vast literature in the biomedical sciences describes the effects of vaccination on the evolution of human pathogens. generally, (i) some pathogens tend to evolve vaccine resistance, (ii) imperfect vaccines that confer partial immunity select for increased virulence, and (iii) live attenuated vaccines can revert to virulence if inadequately distributed (anderson, crombie & grenfell ; gandon & day ; mackinnon, gandon & read ) . together, these observations provide strong incentives for an 'all or nothing' approach to vaccine-laden bait distribution programmes, which may jeopardize long-term success if low-coverage field trials using vaccines of limited or unknown efficacy precede full distribution of an effective vaccine. selective culling (analogous to selective predation) whereby managers remove infected individuals from the population to prevent disease spread may also have unintended consequences. it can select for increased virulence, because there are relatively more susceptible hosts available for the pathogen, and pathogens must transmit to susceptible hosts faster to avoid being culled along with their hosts (choo, williams & day ) . in many cases, selective culls are based on serological tests that do not discriminate between recovered and infectious individuals. removal of recovered individuals may actually result in more explosive epidemics later on due to a reduction in herd immunity (ebinger et al. ) . experiments and genetic analyses of wildlife pathogens that are often treated by vaccination or culling could reveal the extent to which these concerns are realized. aside from developing new vaccines, these risks may be mitigated if management capitalizes on immune-mediated parasite interactions, employ probiotic approaches and consider population-and community-level management interventions. the use of multiple strategies (seen in % of our case studies) may provide one means with which to avoid problems such as antibiotic or vaccine resistance resulting from the overuse of any one strategy. a more complete integration of disease ecology with wdm can benefit both disciplines. management provides unique opportunities to test disease ecology theory while building system-specific understanding. by evaluating management outcomes in terms of theory, managers can better identify effective strategies even in the face of management failures. we have presented specific recommendations, methodological tools and conceptual approaches to achieve a stronger integration of theory and practice, which we hope will facilitate the development of a strong predictive framework for wdm. the generality of this framework is currently limited by the lack of theoretical and taxonomic breadth of coverage. however, these biases are beginning to be addressed, and disease ecology theory is being integrated with wdm with increasing frequency. by continuing to incorporate ecological and evolutionary ideas in the development and evaluation of management actions, disease ecology and wdm are likely to continue to advance towards a more unified body of theory and evidence. multiple infections, immune dynamics, and the evolution of virulence modeling routes of chronic wasting disease transmission: environmental prion persistence promotes deer population decline and extinction seasonality and the dynamics of infectious diseases the epidemiology of mumps 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test-and-remove as brucellosis control measures in bison helminthes could influence the outcome of vaccines against tb in the tropics from host immunity to pathogen invasion: the effects of helminth coinfection on the dynamics of microparasites hidden consequences of living in a wormy world: nematodeinduced immune suppression facilitates tuberculosis invasion in african buffalo worms and germs: the population dynamic consequences of microparasite-macroparasite co-infection models of parasite virulence fisheries ecology in the context of ecological and evolutionary theory the evolutionary epidemiology of vaccination population models for marine reserve design: a retrospective and prospective synthesis epidemic models: thresholds and population regulation some practical suggestions for improving engagement between researchers and policymakers in natural resource management deconstructing adaptive management: criteria for applications to environmental management chronic wasting disease in 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individuals with anti-batrachochytrium dendrobatidis skin bacteria is associated with population persistence in the frog rana muscosa sociality, density-dependence and microclimates determine the persistence of populations suffering from a novel fungal disease, white-nose syndrome should we expect population thresholds for wildlife disease? management measures to control a feline leukemia virus outbreak in the endangered iberian lynx virulence evolution in response to vaccination: the case of malaria effects of management, behavior, and scavenging on risk of brucellosis transmission in elk of western wyoming disease and the dynamics of extinction active adaptive conservation of threatened species in the face of uncertainty meta-analysis of probiotics for the prevention of antibiotic associated diarrhea and the treatment of clostridium difficile disease infectious disease ecology: effects of ecosystems on disease and of disease on ecosystems community disassembly, biodiversity loss, and the erosion of an ecosystem service keeping the herds healthy and alert: implications of predator control for infectious disease microbial intervention for better fish health in aquaculture: the indian scenario guidelines for systematic review in conservation and environmental management scientist at work: toiling to save a threatened frog the use of probiotics in the prevention and treatment of antibiotic-associated diarrhea with special interest in clostridium difficile-associated diarrhea epidemiology and control of an outbreak of classical swine fever in wild boar in switzerland. the veterinary record persistent effects of maternal parasitic infection on offspring fitness: implications for adaptive reproductive strategies when parasitized initiating dialogue between scientists and managers of biological invasions probiotic treatment using bifidobacterium lactis hn reduces weanling diarrhea associated with rotavirus and escherichia coli infection in a piglet model an ecological approach to preventing human infection: vaccinating wild mouse reservoirs intervenes in the lyme disease cycle wildlife in a changing world. an analysis of the iucn red list of threatened species host culling as an adaptive management tool for chronic wasting disease in white-tailed deer: a modelling study the role of predation in disease control: a comparison of selective and nonselective removal on prion disease dynamics in deer disease management strategies for wildlife. revue scientifique et technique de l'office international des epizooties disease in wild animals: investigation and management culling and cattle controls influence tuberculosis risk for badgers effect of probiotic cenbiot on the control of diarrhea and feed efficiency in pigs we thank y.p. springer, v.j. mckenzie, s.h. paull, s.a. orlofske, s. ellis, t.j. zelikova, the cu disease discussion group and the johnson lab for insightful comments. any use of trade, product or firm names is for descriptive purposes only and does not imply endorsement by the u.s. government. a.l.a., d.l.p., j.g.k., j.r.m. and m.b.j. were supported by the nsf graduate research fellowship program. p.c.c.'s work was supported by u.s.g.s. and the nsf/nih ecology of infectious disease program deb- , and some ideas stem from working groups sponsored by the nih/dhs-funded rapidd program. p.t.j.j. was supported by a fellowship from the david and lucile packard foundation and grants from the national science foundation (deb- , ) and the morris animal foundation. the authors have no conflict of interests with regard to this research or its funding. additional supporting information may be found in the online version of this article.appendix s . references for reviewed articles. table s . metadata from reviewed articles. key: cord- -t dg oj authors: parker, michael t. title: an ecological framework of the human virome provides classification of current knowledge and identifies areas of forthcoming discovery date: - - journal: yale j biol med doi: nan sha: doc_id: cord_uid: t dg oj recent advances in sequencing technologies have opened the door for the classification of the human virome. while taxonomic classification can be applied to the viruses identified in such studies, this gives no information as to the type of interaction the virus has with the host. as follow-up studies are performed to address these questions, the description of these virus-host interactions would be greatly enriched by applying a standard set of definitions that typify them. this paper describes a framework with which all members of the human virome can be classified based on principles of ecology. the scaffold not only enables categorization of the human virome, but can also inform research aimed at identifying novel virus-host interactions. the term "microbiome" was coined by whipps, lewis, and cooke in , defined as "a characteristic microbial community occupying a reasonably well defined habitat which has distinct physico-chemical properties" [ ] . many attribute the popularization of the term to nobel laureate joshua lederberg, where he (anthropocentrically) defined the microbiome as "the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space and have been all but ignored as determinants of health and disease" [ ] . the community of microorganisms referenced in these definitions includes viruses, bacteria, fungi, protozoa, and archaea. within this community there is an inherent plasticity arising from the interaction of organisms with one another and with their environment. such meta-interactions lead to complex repercussions for each level of life, and this review will focus on the consequences that have implications for human health. the viral component of the microbiome, termed the "virome" [ ] is a poorly understood facet of the microbiome, despite having the potential to significantly impact human health. from a philosophical perspective, viruses were likely integral to the very existence of man as the primordial precursor to all current life on earth [ ] . more tangibly, during the course of human evolution, the viruses in and around humans not only drove evolution via selective pressure, but also contributed novel genetic material. in fact, approximately percent of the human genome is composed of viral sequence [ ] . this role as a major agent driving horizontal (i.e., non-reproductive) gene transfer between biomes [ ] [ ] [ ] [ ] and the fact that the diversity, complexity, and abundance of viruses surpasses any other biological entity make it apparent that understanding viromes will impart unparalleled understanding of the organisms they inhabit [ ] . however, the obvious importance of viruses in the composition of all biomes has not (yet) been met with an appropriate fervor for the characterization of the viral review recent advances in sequencing technologies have opened the door for the classification of the human virome. while taxonomic classification can be applied to the viruses identified in such studies, this gives no information as to the type of interaction the virus has with the host. as follow-up studies are performed to address these questions, the description of these virus-host interactions would be greatly enriched by applying a standard set of definitions that typify them. this paper describes a framework with which all members of the human virome can be classified based on principles of ecology. the scaffold not only enables categorization of the human virome, but can also inform research aimed at identifying novel virus-host interactions. component of microbiomes. the original explosion of microbiome information was spurred by the utilization of s sequencing technology, which can give vast information about microbial communities using universal primers [ ] . however, this technique is specific to organisms with ribosomes, so early research focused on analysis of bacterial sequences rather than much more technically challenging viral sequences [ ] . thankfully, recent advances in next generation sequencing technologies are making virome characterization more technologically and financially possible and will ensure an explosion of virome description in the near future [ , ] . in an effort to facilitate such classification of the human virome, it is prudent to use basic ecological interactions both to organize current knowledge as well as identify areas where new information may be found. this review will build a framework of the human virome from an ecological perspective, identify currently underappreciated and possibly undiscovered roles of the virome, and apply these principles to analysis of the role of the virome in human health. such a systematic conception of the structure of these ecological interactions can also facilitate the application of similar conventions to the other components of the microbiome. while appreciation of the possible benefits of the human microbiome accumulated in the latter half of the th century, a concordant acknowledgement for the human virome was not so quickly realized [ ] . it is likely this was because human viruses rely on invasion of host cells to replicate and this provokes an overwhelmingly negative impression. however, all interactions of viruses with their hosts are symbioses in the classical sense [ ] and fall along a spectrum from exclusively antagonistic to exclusively mutualistic [ ] . in general, these interactions are facultative for the human and obligate for the virus, but cases like the presence of viral sequences in human genomes exemplify where this simplistic view breaks down [ ] . while the position of a particular virus along this spectrum is not fixed, it is obvious that many viruses have varied, intimate relationships with their hosts. botanists and entomologists were among the first to notice the intricacy of virus-host interactions, which challenged the purely parasitic dogma of virus-host interaction (reviewed in [ ] [ ] [ ] [ ] ). for example, parasitoid wasps harboring polydnavirus genomes utilize virally packaged host genes to prevent rejection of wasp eggs introduced into the parasitized caterpillar [ ] . in a more benign example, the white clover suppresses nodulation in conditions of sufficient nitrogen in a manner dependent on viral coat proteins of a persistently infecting cryptic virus [ ] . while the breadth of knowledge of interactions types is surely incomplete, their presence in other organisms al-ludes to the possibility of the existence of similar interactions between humans and viruses. recent reviews have addressed the issue of the current, primarily negative, view of the human virome [ , [ ] [ ] [ ] [ ] . virologists are beginning to realize that the viruses within us may be more important than previously appreciated. the discovery of intimate interactions of viruses with humans, like the role of endogenous retrovirus (erv †) syncytins in placentation [ ] , are categorically dissimilar to the classical view of viruses only as parasites and brings to issue how scientists are approaching the study of the virome. a lack of structure in this endeavor has arguably hindered the true understanding of non-pathogenic consequences of human-virome interactions. to address this issue, an ecological framework can be constructed for more informative classification of the virome based on how viruses fill niches in humans. here, i describe a conception of the human virome with three general classifications of virus types: parasitic, commensal, and mutualistic ( figure ). within these groupings are subcategories containing more specific characteristics germane to different viral groups. table illustrates how representative members of the human virome spanning a vast array of families, replication schemes, genome architectures, and tropisms can be succinctly classified with this scheme. this arrangement provides functional information about the nature and plasticity of virus-host interaction, the most critical defining characteristic of a virus in the context of human health, in a way absent in the more classical types of organization mentioned above. in the rest of this review, i will characterize each grouping and provide examples from the literature supporting this classification. the application of this scaffold will not only deepen the understanding of known virus-host interactions in the ecological context of the virome, but will also identify logical next steps and gaps in current knowledge that are tantalizing areas for future exploration. the canonical classification of viruses is as parasites. in general, parasitic viruses are transmitted horizontally from host to host, and this process is necessary for the virus' survival. their obligate life cycles necessitate manipulation of cellular processes and have been classically linked to disease manifestation. while the role of viruses in human disease was not appreciated until the th century [ ] , throughout human history, the diseases caused by particularly pathogenic viruses have been some of the most feared and deadly. currently, there are known human viral pathogens [ ] and at least viruses that can infect humans [ ] , with more being discovered every year [ , ] . the apparent bias toward pathogenic species is undeniably because phenotype (read: pathogenesis) has traditionally been a major factor in the discovery of new viruses. while parasitic viruses are a morose facet of human history, they have contributed to man's evolution as a species. the biologist's viewpoint includes the selective pressure on man in which viruses kill susceptible hosts, leading to the emergence of fitter genotypes. some viruses play a similar role by limiting an infected host's reproductive fitness [ ] . the presence of deadly viruses has also supplied pressure for behavioral and psychological adaptation via influence on practices in mate selection, food preparation, and sanitation [ ] . a modern example of such behavioral adaptation is the development of vaccination, kick-started by edward jenner's famous work with cowpox [ ] . however, not all parasitic viruses are deadly. in fact, excessive pathogenicity is an evolutionarily poor strategy for a virus' survival unless balanced with a number of other trade-offs such as transmission rate and recovery rate [ ] . most parasitic infections occur horizontally (though there are rare exceptions [ ] ) and the equilibrium between pathogenicity and other trade-offs is crucial to ensuring continuation of the viral lineage. while parasitic viruses by definition have some level of pathogenic effect on their host, it is not the intent of the virus to do so, because viruses don't have intentions. viral genomes (particularly those of rna viruses) exist as a pool of non-identical, related sequences, termed quasispecies, and are constantly adapting in response to selective pressures [ ] . in some cases, the virus' environment can select for changes that create a balance of the above-mentioned trade-offs where pathogenicity may be a successful, or at least not detrimental, strategy [ ] . in this light, i will define types of parasitic viruses according to both their degree of pathogenesis and the way in which they interact with their host. the key characteristic of pathogenic viruses is that they cause an acute disease in which the host either lives or dies, with the virus simply using it as a vessel to move to the next host. in these interactions, hosts typically mount strong immune responses and literally fight for their lives. this is not to say that all pathogenic infections are deadly or resolved immediately, since the degree of pathogenicity and time to resolution vary widely, as will be discussed in following sections. rather, the virus-host interactions classified here as "pathogenic" represent perturbations of homeostasis for which the only resolution is that one side must prevail. either the host completely clears its body of the virus, or it succumbs to infection. while this sounds like a poor transmission strategy, history has shown it can be quite effective, as long as the proper trade-offs are satisfied (see the parasitic virome, above). influenza virus is a prime example of the success of a pathogenic strategy. most humans can expect to be infected with at least one influenza virus per year, and in general they will observe a fairly robust set of symptoms including, but not limited to, fever, chills, headache, nasal congestion, and coughing [ ] . while infection can be severe and even cause death, this is typically limited to hosts in an immunocompromised state, as selective pressure typically drives virus evolution to less virulence [ ] . in general, virus is shed one to two days after infection, with symptoms usually presenting one day later, ensuring that a person shedding virus will be out and about among currently uninfected individuals [ ] . severe symptoms may be due to viral replication and destruction of tissue; however, over-reaction of the immune system is often the cause [ ] . this is exemplified by secondary bacterial pneumonia where blooms of pathogenic and/or commensal bacteria can result in severe disease or death [ ] [ ] [ ] [ ] . however, by the time a person has become ill, they have likely spread the virus by direct contact with an uninfected individual, contaminated fomites, or aerosolized respiratory droplets [ ] . the fact that many pathogenic viruses have circulated in humans for hundreds to thousands of years, despite our best efforts to eliminate them, speaks to their success. as far back as the th- th centuries b.c., there is evidence of poliomyelitis caused by poliovirus in egypt [ ] . a more recent example is measles virus, which appears to have diverged from rinderpest sometime in the th or th century b.c. [ ] . the long histories of human disease caused by these and other pathogenic viruses made them low hanging fruits for study in the new field of microbiology in the early th century [ ] . this can explain much of the bias of current knowledge toward pathogenic viruses. while it can be argued that in the majority of human history these viruses were the most formative in terms of social, cultural, and behavioral characteristics of humans, it cannot be overlooked that this point may also be similarly biased. the notion that some viruses could live in relative homeostasis with the organisms they infect is very recent, and such interactions may prove to be a renaissance in the thinking of the importance of different portions of the virome. in fact, some of the most clinically important viruses of the last century were not classically pathogenic, but rather, persist within the host in relative silence for long periods of time. the characteristic strategy of persistent viruses is one of immune evasion and long term interaction with the host. typically, these viruses exhibit mild and short-lived acute phase infections followed by establishment of a long term niche within a host. in this niche, the virus may go through stages of dormancy and reactivation, or slow replication for a long period of time [ ] . this ability to progress to persistence for months, years, or the entire life of the host clearly delineates these viruses from the pathogenic viruses [ ] . these relationships straddle the border somewhere between symbiosis and pathogenesis, traditionally making it difficult to classify them. in this context, i will typify long-term infections with well-described detriment to the host as persistent, and other longterm interactions as either commensal or mutualistic (see respective sections, below). while the virus has a safe home during persistence, the question arises that if a virus is not replicating, or is present at very low levels, how is it transmitted to the next host? the answer lies in the spread of virus via close intrahost interactions that may occur many times during the host's life. the most conventional method by which persistent viruses are transmitted is contact between mucous membranes. this includes kissing and sexual intercourse, but can also include transmission vertically from mother to child, in rare cases [ ] . the prototypical example is herpes simplex virus (hsv- ) which, after an initial acute or subclinical infection, moves into a sensory neuron and enters a period of latency [ ] . later in life the virus can break latency, move down the neuron to the skin, and begin to replicate, the prototypical presentation of which is a cold sore. these lesions release infectious virus that can be transmitted to a new host. after a short time, the virus returns to latency and the lesion will heal [ ] . the success of this strategy of latency and mild pathogenic effect is made obvious by the fact that approximately two-thirds of the world's population is infected with hsv- [ ] . non-conventional methods of inoculation have arisen concomitant with certain human tendencies and interactions. the most common examples include intravenous drug use, blood transfusion, and organ transplantation, all of which break typically impassable barriers [ ] . for viruses like hepatitis c virus (hcv) and human immunodeficiency virus (hiv) that circulate in the blood of their host, these disruptions of natural barriers created a heyday for transmission. an important development of modern medicine was the implementation of standard blood screening for these and other persistent viruses and has led to a decrease in medically acquired disease [ , ] . because of the longevity of asymptomatic interaction, those who are infected with persistent viruses may not know for years. this has consequences not only for transmission to others, but also for eventual progression to disease, as treatment is delayed until symptoms emerge when the damage is often irreparable. for hiv, the condition of acquired immune deficiency syndrome (aids) weakens the immune system drastically, allowing opportunistic microbes to cause fatal disease [ ] . hcv is the most common cause of chronic liver disease, cirrhosis, and liver cancer in the form of hepatocellular carcinoma, all caused by years of persistent viral presence [ , ] . the delayed onset and long-term homeostasis of persistent viruses within their hosts makes them a unique class within the human virome. understanding the biology of these viruses and the development of methods to control and eliminate them is an area of much importance in future medical science. additionally, further characterization of the human virome is likely to uncover more viruses that persistently infect humans [ ] , and such discoveries could pave the way for the treatment of diseases of currently unknown etiology. as will be discussed in later sections, not all viruses are pathogenic. herein, i classify viruses that impact host physiology without directly eliciting a disease state as atypically pathogenic viruses. for example, some viruses infect humans without ever causing disease while others inhabit and change the composition of the human microbiome without ever infecting human cells. since our immune system can't tell the difference between harmful or benign viral signatures, the mere presence of these viruses harbors potential to cause disease. so far, this type of interaction has not been defined as a facet of the virome, but in this section i will review examples from the scientific literature that hint at such scenarios. endogenous retroelements (eres) represent a unique class of human-colonizing viral material that is resident in the germ line and include endogenous retroviruses (ervs), retrotransposons, and retrotranscripts [ ] . while the general classification of these genetic elements will be defined in this work as mutualistic (see the mutualistic virome, below), it is evident from a number of studies that they can sometimes contribute to pathogenesis. in the autoimmune disease multiple sclerosis (ms), human endogenous retrovirus (herv) syncytin expression is upregulated in lesions and leads to an increase of cellular protein oxidation and destruction of oligodendrocytes [ ] . another possible autoimmune contribution of eres is by their engagement of nucleic acid pattern recognition and downstream immune activation during both their rna and cdna synthesis [ , ] . carcinogenesis may also occur, as exemplified by the rec protein of herv-k(hml ), which can facilitate tumorigenesis when expressed in mice [ ] . a final example of deleterious consequences conferred by eres is their ability to destabilize the genome via insertion, rearrangement, and deletion [ ] . continued exploration is necessary to expand upon these data to elucidate the mechanisms behind the negative impacts of eres on human health. bacteriophages (from here, referred to as phages), which are canonically classified as commensals (see the commensal virome, below), may also play an atypically negative role. while these viruses do not infect human cells, the estimated phages present in the human gut [ ] and those elsewhere on the body are not likely to be completely benign. while it is known that phages can be co-opted for benefit to the host [ ] , i postulate that the presence of this amount of genomic and proteinaceous material is unlikely to be immunologically inert. an example of how a phage could affect human health is as an endogenous ligand for immune activation. it is known that humans harbor antibodies to phage proteins [ ] [ ] [ ] [ ] and that phagocytic cells of the immune system can ingest phages [ ] , but whether this has any negative impact on health is unknown. toll-like receptors (tlrs) - and - play key roles in the production of autoantibodies, presumably via sensing of nucleic acid containing immune complexes (reviewed in [ ] ). these autoantibodies could be derived from any number of endogenous sources, including those of phages. interestingly, identifying the particular nucleic acids responsible for this activation in vivo has been challenging, though functional work has vetted this mechanism with immune complexes containing nucleic acids [ , ] . it is possible that b-cell receptor (bcr)-mediated endocytic or plasmacytoid dendritic cell (pdc) phagocytic internalization of phages could deliver ligands necessary for these responses. in this light, i would argue that correlating phage localization and expression with disease state is a particularly intriguing area of research moving forward. such research may also elucidate mechanisms by which specific bacteria (and their viral cargo) are linked to autoimmune, allergic, and pathogenic phenotypes. protozoan parasite-associated viruses have emerged as another source of atypically pathogenic effects in humans [ ] . such interactions are reminiscent of the uses of viruses as agents for forwarding their host's survival in parasitoid wasps and plants (see an ecological framework of the human virome, above). the recognition of the leishmania-infecting lrv virus by human macrophages can promote inflammation, subvert the immune response, and ultimately promote persistence of the parasite [ ] . trichomonas vaginalis harbors a trichomonasvirus that triggers ifn responses via tlr- , leading to inflammatory sequelae [ ] . at present, there is insufficient data as to whether these are evolutionarily adapted mechanisms meant to perturb host immunity to benefit the parasite, but the extensive literature of such interaction in insects and plants makes this a tantalizing conjecture. additional characterization should explore the nature of the interactions of protozoan viruses with humans. while all the above postulates are conjecture on the limited knowledge base regarding atypically pathogenic virus-host interactions, it must be noted that these are certainly not overlying principles. associated pathologies likely require particular genetic dispositions and/or external stressor states. it cannot be understated, however, that a better understanding of the viruses that typically inhabit humans will likely lead to a concomitant rise in the cases of viruses identified as atypically pathogenic. the term commensal was originally coined to describe an organism that benefitted from being on or in a host but did not have any direct detriment or benefit for the host [ ] . as the field of microbiome research blossomed, it became clear that many of the microbes that had originally been considered commensal truly did have direct effects on host health (reviewed in [ ] ). these organisms may be more accurately defined as atypically pathogenic or mutualistic (see sections above and below, respectively) if these effects are common or stable, but in general, scientists have followed the practice of innocence until proven guilty. however, it is likely that many commensals exist that either rarely or never have effects on the host. while at the local level some commensal viruses may have detrimental effects on individual cells, many eukaryotic and prokaryotic viruses are associated with healthy human tissues [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the prototypical examples of commensal viruses are phages that infect bacteria [ ] . no phages are known to infect human cells and therefore their presence may be primarily innocuous. the effect of phages on the composition of the microbiome, in that they may kill or inhibit growth of some bacteria, could be considered a possible detriment or benefit depending on the situation, albeit indirect. in fact, the targeted utilization of phages to eliminate unwanted bacterial pathogens (phage therapy) was introduced in the early s and has many potential benefits including, but not limited to, low toxicity, treatment of antibiotic resistant infections, and rapid discovery/generation [ ] . however, concern over unknown health implications and the rise of antibiotics stalled the field, though a renaissance has been building since the s [ ] . many different levels of this application exist, such as treatment of animals and plants with phages to ensure better yield as well as to kill human pathogens that may grow on or in these food sources [ ] . phages are also known to be able to stimulate and/or modulate immune action in humans at multiple levels including antibody generation, development of adaptive immune cells, and innate pattern recognition (reviewed in [ ] ), and the possible applications of phages in these contexts are intriguing. in the future, sections of this group may be able to re-classified as atypically pathogenic or mutualistic if their presence can be linked to roles in specific disease phenotypes, but for now they are most appropriately classified as commensal. viruses can also infect fungal members of the human microbiome (mycoviruses) and protozoa of the human macrobiome [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . while much less is known about these particular types of viruses, similar conjectures have been made as for phages in regard to their potential as both atypical pathogens and mutualists. direct effects on human health are likely rare, though they have been observed for protozoan parasite viruses (see atypically pathogenic viruses, above). the presence of these viruses may ultimately have an indirect effect on human health of unknown consequence. investigation of the breadth and impact of such viruses is an interesting and important area of future research, particularly for the benefit of the immunocompromised and those in developing countries who are most affected by diseases caused by fungi and protozoa. there is also an abundance of prototypically planttropic viruses found in the human gut [ ] . it is likely these viruses have been acquired via agricultural crops in the diet and the effect of their presence is unknown, but assembly of plant viral particles in escherichia coli has been exhibited, so it is possible that these viruses may interact with gut bacteria [ ] . some commensal viruses can infect human cells and use the human as a vessel for transmission. these viruses establish asymptomatic infections that do not result in a change in host health or behavior. this represents a veritable antithesis to the strategy of parasitic human viruses (see the parasitic virome, above), wherein viruses have prioritized trade-offs other than pathogenicity to ensure their transmission to a new host. the most well documented examples of asymptomatic infection of humans by commensal viruses are the rhinoviruses and other infections of the nasopharynx and upper respiratory tract [ , [ ] [ ] [ ] [ ] . many of the human papilloma viruses (hpvs) interact with human cells as an asymptomatic infection of mucous membranes or skin [ ] . anelloviruses are also well described human commensals [ ] [ ] [ ] [ ] [ ] . while immune detection of non-human tropic commensal viruses is avoided by physical separation, the human-tropic commensal viruses assuredly engage with host innate and adaptive immune responses, and so must avoid or subvert these mechanisms to maintain their immunological silence. description of the viral techniques enabling this evasion is an important direction for future research on these viruses, as this may point to novel avenues of therapeutic intervention for suppression or activation of immune responses. while commensal viruses do not have any direct effect on human health during homeostatic equilibrium, perturbation of homeostasis may cause them to contribute to pathogenesis (see atypically pathogenic viruses, above). contrarily, the presence of viruses from the final major group of the human virome, mutualistic viruses, exhibit positive effects on host health via an assortment of mechanisms. perhaps the most underappreciated portion of the virome are those viruses that have a positive health benefit for humans. a mutualistic interaction is one that benefits both organisms involved [ ] . it is reasonable to assume that it may be beneficial for a virus to encourage and aid in its host's health to facilitate a longer lasting and amenable environment for survival. but this is a bit counterintuitive considering that above i discussed that viruses are intrinsically parasitic. this can be rationalized with the assertion that nearly all mutualistic interactions were probably at some point a form of parasitism, then passed through a commensal stage, and finally adjusted to mutualistic harmony through co-evolution. this stipulation is based on the reasoning that the amount of evolutionary change necessitated in the virus and/or the host to switch interaction types requires significant adaptation and thus is more likely to proceed stepwise [ ] . a relationship must become sufficiently non-pathogenic to allow interaction without rejection as well as develop benefit for both sides, and an intermediate state seems a logical platform for this. indeed, this postulation seems bolstered by the spectrum of mutualistic relationships that exist, spanning obligate to conditional mutualistic benefits [ ] . a classic non-viral example that illustrates this point is the integration of the mitochondrion into the cells of ancient prokaryotes or eukaryotes. this kind of relationship is considered symbiogenic, meaning that once separate organisms have now fused into a distinct new species [ ] . either by phagocytosis or infection, an endosymbiosis was established in which the mitochondrial bacterial progenitor supplied some benefit to its host. millions of years later, mitochondria are part of all eukaryotic organisms with functions spanning energy production, innate immunity, and membrane potential, to name a few [ , ] . it is now appreciated from data on the microbiome that it would be to the host's benefit if the power of the genetic information in and around them could be harnessed as an evolutionary (and medical) tool for self-betterment [ ] [ ] [ ] . this could occur in a number of ways, either through encouraging colonization by beneficial microbes or even by integrating the genetic material of these microbes into the human genome. a small body of literature characterizing virus-human mutualism has given a glimpse into this facet of the virome. in the next sections, i will review two levels of symbiotic relationships and discuss important gaps in knowledge important for the understanding and possible utilization of these interactions for human health. symbiotic viruses that are transmitted vertically from parent to child in the germ line are considered primary symbionts [ ] . these relationships are often ancient and obligate for both parties [ ] . the most obvious example is the eres that have integrated themselves into human genomes at many points throughout millions of years of evolution. while some have been inactivated by recombination, deletions, and random mutations, many still appear to be expressed [ ] . it is also obvious the control of these elements is an important challenge for the host, as many strategies have evolved to control replication and reinfection of inserted eres [ , ] . to date, the role of most eres in the human genome is unknown, but a few human and mammalian examples indicate that their presence confers a number of benefits. some of the most important effects of ere colonization come in the form of genomic diversity and plasticity. eres can not only insert new genetic material, including protein coding genes, but also play roles in genetic mobility and control. the recombinatorial ability of eres is conferred upon insertion and has facilitated recombination and duplication of large swathes of genomic material [ , ] . where an ere inserts itself can be an important factor in how it affects the genome, as insertion into a protein coding gene could be innocuous or catastrophic. alternatively, insertion in or near promotion and regulatory elements can result in changes in transcriptional control [ ] , as can the native regulatory sequences in the eres themselves [ , ] . of course, all of these benefits can have similarly damaging consequences, as destabilization of the genome or particular genes could have profound negative effects on the host (see atypically pathogenic viruses, above). however, natural selection dictates such effects will not successfully continue in the host lineage. eres also have important immunologic and developmental roles. building off of the transcriptional control effects mentioned in the last paragraph, eres appear to have a role in gene upregulation via pattern recognition during the initiation of adaptive immune responses [ , ] . adaptive responses to eres can also play a role in restricting viruses by neutralizing incoming exogenous virus via antibody development against ere surface glycoprotein antigens [ ] . examples from mice also indicate that the expression of the erv glycoprotein env can competitively bind surface receptors important for exogenous virus entry and that a short product of an erv gag gene can inhibit the nuclear translocation of incoming capsids. [ , ] . developmentally, the acquisition of erv syncytins has been a critical step in the evolution of placentation in humans [ , , ] . the presence of retroviral nucleic acids in human cells also suggests that ere reverse transcription may be able to activate host innate immunity [ ] . whether humans can harness the beneficial properties of eres is of much scientific import. for years it has been debated whether gene therapy using retroviral vectors for insertion of genes into an individual would be of more help than harm. overall the utility of such methods have been tempered by the observed oncogenic potential of such therapies [ ] . recently, studies with retroviral-modified chimeric antigen receptor t-cells (car tcells) have shown impressive application for the activation of adaptive immunity against cancers, though there have also been morbid failures in this field [ ] . the application of retroviral-modified car t-cells could presumably be expanded to infectious disease as well. these progressive areas of medical science will continue to be explored and debated well into the future, and better understanding the relationship of eres with their human hosts will be indispensable for these studies. similar to the primary symbionts, secondary symbionts form mutualistic relationships with their host, but are instead acquired via non-germline vertical transmission or horizontally during the post-natal life of the host. a popular example is that of phages. beginning with the microbial colonization of a child during birth, phages hitch a ride with their host bacteria. phages can regulate bacterial species in the gut, as it is known that phages can be coopted into mucosal surfaces for this purpose [ ] . free phages may also serve a similar role as sentinels maintaining a balanced microbiota. these benefits have been studied extensively as possible medical tools in phage therapy (reviewed in [ , ] ) and there has recently been a resurgence of interest as the problem of antimicrobial resistance has become more dire [ ] . these same principles can likely be extended to viruses that parasitize other microbes and macrobes as well. other examples of secondary symbiotic viruses are isolated, but numerous. in general, these viruses likely compete for receptors, cellular resources, or niches within hosts to confer protection. hepatitis g virus (hgv) is known to slow development of hiv infection to aids pathogenesis [ , ] and human cytomegalovirus (hcmv) infection can suppress hiv infection by limiting availability of the coreceptor ccr on macrophages [ ] . infection with hepatitis a virus (hav) can suppress infection with hcv and may actually promote clearance [ ] . evidence from mice supports the assertion that viruses can confer resistance to non-viral disease as well, as infection with murine gammaherpesvirus results in protection from challenge with listeria [ ] and type diabetes can be prevented in mice infected with lymphotropic viruses [ ] . oncolytic viruses, which specifically target, infect, and kill tumor cells are also notable in both their novelty and their clinical utility (reviewed in [ ] [ ] [ ] ). viruses may even be able to provide developmental and immunological benefits not unlike that of the bacterial microbiota, as evidenced by the establishment of gut homeostasis in mice colonized with norovirus [ ] . endogenous retroelements could also presumably be included here in the case of their population of a nongermline subset of cells within an individual and conferral of benefit to the host. one of the main functions of secondary symbionts may be to help maintain a baseline of immune activation in order to give the host a head start upon challenge with incoming pathogens. it has been estimated that healthy humans are infected with > viral infections at any given time [ ] . this constant barrage of immunogenic material presumably drives a smoldering activation of both innate and adaptive immunity. from this, it may be concluded that a state of continual challenge may be integral for the immune system to be fortified against incoming pathogens. while there is a plethora of research about secondary symbiotic bacteria, similar attention must to be paid to the virome. the examples outlined above point to incredible benefit derived from the interaction of humans with these mutualists, and significant effort must be made to discover and harness such potentially impactful relationships. moving forward, consideration of the effects of the human virome will be crucial in disease treatment and discovery of disease etiologies. from purely pathogenic viruses to those mutualists helping humans adapt to their world, the human virome has the potential to lend insight into the basic and applied biology of human health. in light of this, further exploration into the composition, tropism, and evolution of the human virome will undoubtedly make way for innovations in the treatment of human disease. throughout this manuscript, i have assembled the components of the human virome into broad classifications of parasitic, commensal, and mutualistic. while these definitions are logical, the intermixing of the groups and exceptions to rules drives home the point that the virome has inherent plasticity. disruption of the yin and yang of virus-host interactions can contribute to this transitory nature and may blur the lines that delineate the three main groups. this acknowledgement of convention and the departures from it gives both structure and flexibility to this conception of the human virome. the culmination of these ideas provides a template that identifies gaps in our current knowledge, postulates interactions that are currently unknown, and describes future areas of research, the results of which can then be classified in this same manner. i look on with anticipation to the continuation of the scientific soul search that is the exploration of the virome, where as we learn more about viruses, we come to better know ourselves. acknowledgments: i would like to thank william ruff for thoughtful discussion, critical evaluation, and editorial comments that contributed to this manuscript. i would also like to thank clinton bradfield and john ventura for editorial comments and suggestions. this material is based upon work supported by the national science foundation graduate research fellowship under grant no. dge . mycoparasitism and plant disease control ome sweet 'omics--a genealogical treasury of words global screening for human viral pathogens. emerg infect dis. centers for disease control and prevention the ancient virus world and evolution of cells international human genome sequencing consortium. initial sequencing and analysis of the human genome the evolutionary genomics of pathogen recombination role of viruses in human evolution evolution of complexity in the viral world: the dawn of a new vision ten reasons to exclude viruses from the tree of life viral metagenomics 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infection prevention of type i diabetes in nonobese diabetic mice by virus infection recent progress in the battle between oncolytic viruses and tumours gene therapy progress and prospects cancer: oncolytic viruses apoptin, a tumor-selective killer an enteric virus can replace the beneficial function of commensal bacteria any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the national science foundation. key: cord- - u tdrj authors: geoghegan, jemma l.; duchêne, sebastián; holmes, edward c. title: comparative analysis estimates the relative frequencies of co-divergence and cross-species transmission within viral families date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: u tdrj the cross-species transmission of viruses from one host species to another is responsible for the majority of emerging infections. however, it is unclear whether some virus families have a greater propensity to jump host species than others. if related viruses have an evolutionary history of co-divergence with their hosts there should be evidence of topological similarities between the virus and host phylogenetic trees, whereas host jumping generates incongruent tree topologies. by analyzing co-phylogenetic processes in virus families and their eukaryotic hosts we provide a quantitative and comparative estimate of the relative frequency of virus-host co-divergence versus cross-species transmission among virus families. notably, our analysis reveals that cross-species transmission is a near universal feature of the viruses analyzed here, with virus-host co-divergence occurring less frequently and always on a subset of viruses. despite the overall high topological incongruence among virus and host phylogenies, the hepadnaviridae, polyomaviridae, poxviridae, papillomaviridae and adenoviridae, all of which possess double-stranded dna genomes, exhibited more frequent co-divergence than the other virus families studied here. at the other extreme, the virus and host trees for all the rna viruses studied here, particularly the rhabdoviridae and the picornaviridae, displayed high levels of topological incongruence, indicative of frequent host switching. overall, we show that cross-species transmission plays a major role in virus evolution, with all the virus families studied here having the potential to jump host species, and that increased sampling will likely reveal more instances of host jumping. emerging infectious diseases are often characterized by host switching events, in which a pathogen jumps from its original host to infect a novel species. however, given the ecological and genetic barriers a virus must overcome to jump species and adapt to new hosts, it might be reasonable to assume that successful cross-species transmission is a relatively rare occurrence and that viruses are instead more likely to co-diverge with their hosts. using a comparative co-phylogenetic analysis performed at the scale of virus family a a a a a emerging pathogens that cross the species barrier to infect new hosts can profoundly affect human and animal health, as well as wildlife and the agricultural industries. although most emerging diseases seemingly result from such a process of cross-species transmission, it is also the case that some viruses seem to rarely jump the species barrier and instead co-diverge with their hosts over long stretches of evolutionary time. for example, long-term virus-host codivergence has been suggested to play a key role in the evolution of vertebrate herpesviruses over periods of~ million years [ ] and insect baculoviruses over a time-scale of~ million years [ ] . indeed, it has been proposed that a number of families of dna viruses have codiverged with their hosts over long evolutionary time-scales [ ] [ ] [ ] , and do so more frequently than rna viruses, which in contrast display a combination of co-divergence and host switching [ ] . in particular, while phylogenetic trees for some rna viruses, such as particular retroviruses, are generally congruent with those from their hosts suggesting long-term codivergence [ ] , for others, such as flaviviruses, host jumping appears to be relatively frequent [ ] . in the case of flaviviruses this likely in part reflects the fact that many are transmitted by arthropod vectors and characterized by short durations of infection. the situation appears to be even more complex in cases such as the hantaviruses where there is evidence of both codivergence and host jumping [ ] . given the evolutionary and ecological barriers a virus must overcome to cross the species barrier and successfully establish itself in a new host, it might seem reasonable to assume that successful cross-species transmission is a relatively rare occurrence [ ] . indeed, many emerging diseases are in reality 'spill-over' infections, in which onward transmission between members of a new host species is limited such that extinction of the novel virus occurs rapidly [ ] . nevertheless, it is possible that an increased sampling of hosts and their viruses will reveal more instances of host jumping, in turn implying that cross-species transmission is a fundamental aspect of virus evolution [ ] . as a case in point, although there is strong evidence that hepadnaviruses have co-diverged with their vertebrate hosts over hundreds of millions of years [ ] , the recent identification of hepadnaviruses in fish and amphibians has revealed more instances of cross-species transmission, potentially including that from aquatic to terrestrial vertebrates [ ] . clearly, identifying the relative frequencies of co-divergence versus cross-species transmission is of central importance to understanding the basic mechanisms of virus evolution and disease emergence. in particular, it is important to determine whether some virus families have a greater propensity to jump hosts than others and, if so, what factors govern this pattern. currently, however, there is no quantitative or comparative measure of the frequency of crossspecies transmission versus co-divergence, so that determining whether one virus family is more likely to jump species boundaries than another is difficult to assess. one simple and powerful way to estimate these key evolutionary parameters is via 'co-phylogenetic' analysis that assesses the degree of phylogenetic congruence (i.e. similarity) between hosts and their parasites [ ] . in particular, a clear congruence between the host and virus phylogenies provides strong evidence for a history of co-divergence, whereas phylogenetic incongruence (i.e. discordance) is compatible with cross-species transmission. to date, co-phylogenetic studies of viruses have largely focused on the evolution of a subset of viruses within a particular virus family, and have not been performed in a comparative manner. for example, although there has been much work dedicated toward describing co-divergence in herpesviruses, these studies generally only encompass one particular host type (e.g. primates [ ] ) and so may fail to capture the broader picture of potential host jumps among more distantly related species. hence, there has been no attempt to use analyses of this kind to provide a broad-scale comparative and quantitative measure of the frequency of co-divergence and cross-species transmission in virus evolution. herein, we provide such an analysis. specifically, using a normalized tree topology distance metric based on the penny and hendy distance metric that enables comparisons between pairs of virus and host trees with different numbers of tips [ ] , which we now term the 'nph ' distance (where n = normalized), we compare phylogenies of virus families and their hosts. while this method does not explicitly model host-switching events, it does provide a simple means to compare multiple topologies of virus-host pairs, and accounts for differences in sample size and the fact that several viruses from a specific family can infect a single host species. to provide a quantitative measure of host switching we compared virus families, incorporating viruses infecting a diverse sample of eukaryotic hosts including mammals, birds, reptiles, amphibians, fish, plants and insects. under the measure we utilize here, when nph = between the virus and host trees it implies that their topologies are identical such that there is very strong evidence for co-divergence ( fig a) . conversely, if nph = , there are no clades in common such that co-divergence is implausible ( fig b) . crucially, this metric does not depend on where the mismatched clades are located in the tree. for example, for a pair of virus and host trees that differ in one clade, the nph is the same whether species jumping events were recent (i.e. shallow nodes fig c) or ancient (i.e. deep nodes fig d) . importantly, the nph distance increases as the number of incongruent nodes (i.e. nodes that differ) between the virus and host trees increases ( fig e) . a phylogenetic measure of the relative frequency of virus-host codivergence our analysis considered a total of seven dna and rna virus data sets that provided sufficient data to perform a quantitative co-phylogenetic analysis. hence, the study relied heavily on specific selection criteria (see materials and methods) that necessarily limited data availability. despite these rigorous criteria, the majority of data sets encompassed a diverse collection of viruses and host species, and hence can be regarded as illustrative of the broad-scale frequency of co-divergence versus cross-species transmission. these data contained no evidence for recombination. to determine the prevalence of host switching between different viruses, we inferred family-level viral phylogenies and compared these to phylogenies of their hosts. importantly, our analytical approach-which utilizes the nph distance-provides a relative measure of phylogenetic congruence that is directly comparable between data sets that differ in size (i.e. different number of viruses and host species). our method assumes that viruses that have codiverged with their hosts will share the same tree topology. in contrast, an increasing number of host jumping events should lead to greater phylogenetic incongruence. the reasoning behind this assumption is that there exists a very large number of possible phylogenetic tree topologies even for data sets with a few samples, such that similarities between a pair of virushost trees (i.e. congruence) are highly unlikely to arise by chance. of course, phylogenetic events other than cross-species transmission might also lead to phylogenetic incongruence and we test the validity of this assumption later in the manuscript. across the data set as a whole we found that all virus families displayed relatively large tree topological distances with nph values of ! . , suggesting that cross-species transmission is widespread, at least at the family-level (fig ; s table) . while all families showed distances at the upper end of the scale, the hepadnaviridae (double-stranded dna) had the shortest distance (nph = . ), indicating that this family experiences more frequent co-divergence than any other studied here. at the other end of the spectrum both the rhabdoviridae and picornaviridae (single-stranded rna) displayed nph > . , indicative of frequent host switching and hence little evidence for virus-host co-divergence. we also investigated when the species jumping events occurred in the evolutionary history of the virus families. to do this, we determined whether phylogenetic incongruences tended to cross-species transmission among viral families occur in deeper sections of the phylogeny or to more shallow nodes in the tree. accordingly, we considered the number of nodes subtending clades in the host tree that are not present in the virus tree, a metric known as 'node depth'. nodes that are deep correspond to clades that are more diverse, and often older, than those clades subtended by shallower nodes. for each pair of virus-host trees we calculated the depth of every node that differed within each virushost pair and divide each depth by the maximum node depth (fig ) . this normalized metric, which we term 'relative node depth', ranges between near for phylogenetic incongruences at shallow nodes, and for incongruences at deeper nodes. most incongruences corresponded to shallow nodes, which is expected because there are naturally more shallow nodes than deep nodes in phylogenetic trees. however, that incongruences were found in both shallow and deep nodes suggests that co-divergence is relatively rare in these virus families, even over long evolutionary time-scales. tanglegrams depicting pairs of rooted phylogenetic trees display the evolutionary relationship between each virus family and their host species (fig ; phylogenies with the individual tip labels visible are shown in s fig) . despite the obvious widespread occurrence of host jumping, a number of co-phylogenies reveal the occurrence of at least some co-divergence, as expected from the nph distances. for example, the tanglegrams for the hepadnaviridae and poxviridae exhibit some clear matches with the evolutionary histories of their respective hosts. most notably, their co-phylogenies show a clear segregation between distinct clades that are associated with a specific host type (mammals, birds, etc.). conversely, the phylogenies of most rna viruses appear to largely mismatch those of their hosts. our fundamental assumption is that incongruences between virus and host topologies imply the occurrence of cross-species transmission. to test the validity of this assumption, we reconciled the viruses with the phylogenetic history of their hosts. by associating 'event costs' with host-jumping, as well as with lineage duplication and extinction events, we found the range of optimal co-phylogenetic solutions for each virus family ( fig a) . as with the analysis of topological distances, this revealed that cross-species transmission was the most common evolutionary event in all virus families studied here, with co-divergence consistently less frequent (with the possible exception of the hepadnaviridae-see below), and lineage duplication and extinction playing a much more minor role. we next reconstructed the history of these evolutionary events in detail in the hepadnaviridae (i.e. the most co-divergent virus family). this revealed that under the most likely co-phylogenetic scenario the proportion of crossspecies transmission represents . of all events (i.e. co-divergence = events; duplications = ; extinction = ; host-jumping = ; fig b) . since the nph distance for the hepadnavirus data set was . , we suggest that our method generates results consistent with the reconciliation analysis. in addition, one important disadvantage of performing full reconciliation analysis is that co-phylogenetic methods such as that implemented in jane [ ] and tarzan [ ] are not straightforward since they offer many combinations of possible events and are difficult to compare between families, especially in cases with more than~ viruses where there are many possible co-phylogenetic scenarios. despite these limitations, our reconciliation analysis did reveal the possible causes of the topological incongruence between the virus and host phylogenies. we next determined whether there was any association between the relative frequency of codivergence and larger scale biological properties, such as the number of viruses per family and whether the viruses in question possess rna or dna genomes. to better display this analysis branches on the co-phylogenetic trees were colored according to host type, which comprised mammals, fish, birds, reptiles, amphibians, invertebrates, and plants (fig ) , such that each cophylogeny incorporated between one (i.e. potyviridae) and five (i.e. togoviridae) host types. notably, we found a significant association between the number of viruses per virus family and the nph (p< . ) (fig a) . importantly, because we expect no association between [ ] . boxplots illustrate the range of the proportion of possible events. the 'event costs' associated with incongruences between trees were conservative towards co-divergence and defined here as: for co-divergence, for duplication, for host-jumping and for extinction. virus families are ranked in order of highest mean co-divergence to lowest mean co-divergence. abbreviations on the x-axis are as follows: 'co-div' = co-divergence, 'dup' = duplication, 'hj' = host-jumping, 'ext' = extinction. (b) reconciliation of the hepadnaviridae phylogeny with that of their vertebrate hosts, again utilizing the co-phylogenetic method implemented in jane [ ] . the figure illustrates all possible codivergence, extinction and host-jumping events (no lineage duplication events were reconstructed in this case). the number of viruses and hosts per family and the nph under our tree distance metric, this result implies that sampling more viruses increases the likelihood of detecting host jumping events. in addition, we found that dna viral families had, on average, a shorter nph distance than families of rna viruses (p< . ) (fig b) . note that there is no significant difference (p = . ) between the number of viruses in families of dna viruses compared to those in rna virus families. in this context it is striking that the five families with the shortest topological distances all possessed dna genomes. this analysis also revealed that segmented viruses had a significantly larger nph distance than non-segmented viruses (p< . ), and that negative-sense rna viruses had a larger nph distance than positive-sense rna viruses (p< . ); however, the sample sizes within all these categories were small so that these results should be treated with caution. finally, we note that although the duration of infection (for example, the division between acute versus chronic infections) is clearly a parameter that would likely affect the frequency of host jumping [ , ] , we were unfortunately unable to perform any analyses of this variable on the data available here as it tends to be host-specific rather than a general characteristic of individual virus families. understanding how viruses and their hosts co-evolve is central to revealing the nature of virus evolution and the determinants of disease emergence. in particular, we lack a quantitative understanding of whether some types of virus, such as those classified into different families or that possess genomes of different nucleic acid types, are better able to jump species boundaries compared to others. to investigate the comparative prevalence of cross-species transmission among viruses we measured the congruence between virus and host phylogenetic trees using a normalized tree topological distance-based approach (nph , [ ] ). if taxonomically related viruses have an evolutionary history of co-divergence with their hosts the virus and host phylogenetic trees should be similar in topology, whereas phylogenetic incongruence is the signature of species jumping. overall, our analysis revealed absolute departure from co-divergence among all the virus families studied here (nph ! . and supported by the reconciliation analysis) suggesting that cross-species transmission occurs frequently, at least at the level of virus family. particularly striking was that even the most slowly evolving dna viruses, which have previously been suggested to represent exemplars of virus-host co-divergence [ ] , exhibit relatively common cross-species transmission. hence, at their most basic, these results indicate that viruses are often exposed to a variety of susceptible host species that provide opportunities for cross-species transmission. despite the overall large nph distances observed among all virus families, our data also revealed that the hepadnaviridae, polyomaviridae, poxviridae, papillomaviridae and adenoviridae had the shortest nph distances and were thus relatively more host-specific than the other virus families analyzed here. this is supportive of earlier suggestions that some dna viruses have a long history of co-divergence with their hosts [ ] , which in some cases may be a reflection of relatively long durations of infection. indeed, long-term virus-host associations have been observed in the herpesviridae [ ] , the poxviridae [ ] and the polyomaviridae [ ] . however, it is also important to note that we found these viruses contain more instances of host jumping than previously thought. for example, although the tanglegram shown in fig suggests co-divergence in the case of some primate hepadnaviruses, cross-species transmission seemingly occurs more frequently among those hepadnaviruses that infect birds. in addition, it was recently observed that a fish (bluegill) hepadnavirus clusters more closely with mammalian hepadnaviruses than to other fish viruses [ ] (see figs and b). similarly, early studies of rna viruses suggested that virus-host co-divergence was important in the evolution of two members of the flaviviridae that infect primates-the pegiviruses and hepaciviruses, [ ] [ ] [ ] . however, more recent phylogenetic analyses of expanded data sets have revealed multiple cross-species transmissions events, including the recent emergence of hepaciviruses in domestic dogs, horses and donkeys [ ] , and a newly described pegiviruses in rodents, bats and horses [ ] . despite the obvious caveat of sample size, it seems that rna viruses generally experience more frequent cross-species transmission than their dna counterparts. indeed, the rna viral families analyzed here had an overall mean nph distance of . , compared to dna viruses with a mean of . . this may, in part, be due to the fact that rna viruses are generally characterized by very high rates of mutation and replication [ ] . intuitively, high rates of evolutionary change should confer more rapid adaptation to new environments, which, coupled with the frequency of exposure to new hosts, will facilitate host-switching. in addition, many rna viruses are characterized by short durations of infection that will limit the opportunities for virus-host co-divergence [ ] . an informative exception among rna viruses are the simian foamy viruses (sfv), in which hosts may develop long-term latent infections and the virus has been associated with long-term co-divergence [ ] . indeed, it is notable that among the retroviridae analyzed here those assigned to sfv seem to display relatively similar evolutionary histories to those of their primate hosts (see s fig) . it is also possible that successful cross-species transmission occurs more frequently among phylogenetically related hosts, likely because it is easier to infect and replicate in genetically similar hosts that share less divergent cell receptors [ ] . in addition, related hosts may sometimes inhabit the same geographic region, increasing the probability of cross-species transmission through more frequent exposure [ ] . indeed, a useful generality in studies of disease emergence is that the closer the phylogenetic relationship between hosts, then, given appropriate exposure, the more likely that a pathogen will be able to jump between them, in turn leading to preferential host switching [ ] . if true, so that cross-species transmission results in a viral phylogeny that mirrors that of their hosts, then any phylogeny-based approach such as that utilized here will underestimate the true frequency of host jumping. as a case in point, although there is a general concordance between the phylogenies of simian immunodeficiency virus (siv) and their primate hosts, in which four species of african green monkey harbor distinct forms of siv that is clearly suggestive of co-divergence [ ] , it has been argued that the evolutionary history of siv may also have been shaped by preferential host switching [ ] , although these mechanisms are not mutually exclusive. in contrast, incomplete lineage sorting among closely related viruses may produce a false signal for cross-species transmission when co-divergence has in fact occurred [ ] . in addition, because there is growing evidence that viruses can have complex evolutionary histories with genes derived from multiple sources [ ] , it is important to note that our virus phylogenies are necessarily gene trees rather than species trees. it is therefore possible that other virus gene trees will exhibit a stronger topological match with host phylogenies than those presented here, and hence provide more evidence for co-divergence. finally, while our analysis was only based on robust phylogenetic patterns, because nodes that were topologically uncertain were excluded from the analysis, it is possible that our virus trees contain topological errors reflecting the use of sometimes small numbers of highly divergent sequences. another important aspect of assessing virus-host co-divergence is that the evolutionary time-scales of viruses and their hosts are consistent [ ] . although such a comparison is valuable, it is problematic for the present study because high rates of evolution lead to substitutional saturation in virus genomes at a much faster rate than in cellular organisms. indeed, it is likely that many of the cross-species transmission events implied here have occurred on timescales of many millions of years. as a result, temporal signal is rapidly lost, precluding accurate estimates of their long-term evolutionary time-scales, even though the topology is often accurately recovered [ ] . we therefore suggest that simpler topological comparisons such as those performed here may be a more informative way to proceed in family-level studies of cross-species transmission versus co-divergence. overall, we have observed frequent cross-species transmission across the virus families studied here, with relatively little evidence for virus-host co-divergence. hence, our study suggests that, at the virus family scale in the data analyzed here, host switching plays a major role in the evolution and diversification of viruses and, importantly, that it can occur in viruses of all types. interestingly, we found that increased sampling of viruses from different host species reveals more frequent species jumping events among viral families. as such, the discovery of new viruses is likely to reveal more instances of cross-species transmission. undoubtedly, the analysis presented here should be extended to a wider range of data sets as they become available, particularly because increased taxon sampling results in a larger tree space and increases the statistical power of these analyses. gene sequence data of viruses were obtained from genbank (table ; see s table for all gen-bank accession numbers). following a broad and comprehensive survey of all virus genomic data available on genbank, a total of family-level virus data sets passed our selection criteria and were included in the analysis. these selection criteria, which are independent of whether the viruses have evolved by co-divergence or cross-species transmission, were: (i) the availability of virus sequence data that included a wide range of distinct and diverse virus species that is representative of the virus genera currently available; (ii) the availability of data with informative genomic regions that can be used to reveal evolutionary relationships (e.g. the rnadependent rna polymerase-see table ) and that were not so divergent as to prevent reliable sequence alignment; and (iii) the virus sequence data met a minimum length requirement of amino acids following alignment and the removal of any ambiguously aligned regions. the virus families that passed these selection criteria were the adenoviridae, bunyaviridae, caliciviridae, coronaviridae, flaviviridae, hepadnaviridae, herpesviridae, orthomyxoviridae, papillomaviridae, paramyxoviridae, parvoviridae, picornaviridae, polyomaviridae, potyviridae, poxviridae, reoviridae, retroviridae, rhabdoviridae and togaviridae. each data set contained between - viruses from a diverse range of eukaryotic hosts, including mammals, birds, reptiles, amphibians, fish, invertebrates, and plants. for the purposes of this study we regarded a virus isolated from a particular host species as a distinct virus sample worthy of analysis: for example, rabies virus isolated from a human host was deemed distinct from rabies virus isolated from a canine host. the resulting virus and host data sets included in this study comprised a diverse sample of the available data (see s table for a summary of the virus and host diversity). most data sets contained more viruses than those from their corresponding hosts because they included multiple viruses from a family that can infect the same host. for each virus family nucleotide sequences were first translated to amino acid data using seqotron v. . . [ ] , aligned with muscle v. . [ ] , and poorly aligned regions then eliminated using trimal [ ] , ensuring that all remaining sequences were at least amino acids in length (table ) . amino acid sequences were aligned because there is widespread substitutional saturation at the nucleotide level. although our data sets utilize single genes, we ensured that they were free of inter-specific virus recombination using rat [ ] . to estimate phylogenetic trees for the virus data sets we selected the optimal amino acid substitution model identified using the bayesian information criterion as implemented in modelgenerator v . [ ] and analyzed the data using phyml v . [ ] , employing the spr branch-swapping tree search algorithm (see table for the substitution models used). we assessed the support for individual nodes using the approximate likelihood ratio test (alrt) implemented in phyml v . [ ] , with alrt values ranging between (no support) and (strong support). studies involving simulations and empirical data have demonstrated that this statistic has similar false-positive rates to other metrics, such as the non-parametric bootstrap [ ] . cladograms were constructed for all host species from which the viruses of interest were isolated. in each case the host tree topologies used were the most up-to-date available in the literature [ ] [ ] [ ] [ ] [ ] . for the vector-borne viruses studied here, in which viruses pass between arthropods and vertebrates, the appropriate vertebrate species were assigned as the hosts. in contrast, for insect-specific viruses, where there is no evidence for vertebrate involvement, the relevant invertebrate species were assigned as the hosts. since there were often multiple viruses that infected the same host species, multiple lineages within a single host (i.e. polytomies) were added to the host phylogenetic tree to ensure the number of hosts equaled that of the virus tree. the addition of these polytomies does not influence the nph distance metric (described in detail below) because the distance between a polytomous clade and one that is fully resolved is zero [ ] . all virus and host phylogenetic trees and virus sequence alignments are available at github. com/jemmageoghegan. we measured the extent of virus-host co-divergence (and by exclusion host-jumping) by comparing, in a quantitative manner, the tree topologies for viruses and their corresponding hosts. to this end we calculated a normalized ph tree topological distance [ ] , referred to here as the 'nph ' distance (this function has been included in nelsi v . [ ] ). specifically, the nph distance, which utilizes two phylogenetic trees as its input, describes the number of bipartitions (clades) that are not shared between two tree topologies. importantly, it does not depend on the nodes where the topological differences occur in the tree (fig ) . in addition, this metric considers the tree topology of unrooted trees, but not the branch lengths of the tree. first, the ph metric is calculated as the topological distance between a pair of unrooted trees. it can be understood in terms of the following: where t and t are the clades contained within the host and virus trees, respectively. let the expression t \ t denote the clades that are shared between both trees so that (t \ t ) corresponds to the clades that are not shared between the pair (i.e. those that are unique to each tree). the actual ph distance is twice the number of unique clades. to normalize this metric we divide ph by the maximum distance by considering the two tree topologies, randomizing the tips for one of the trees times, and calculating ph for each replicate (where randomizations was shown to be robust even for very large trees; see s fig) . the largest value of the randomizations is approximately the maximum ph distance in tree topologies. therefore, nph ranges between , for identical trees, and , for trees that have no clades in common (fig ) . the advantages of this method over other tree distance metrics is that it is comparable for pairs of trees with different numbers of tips, it maintains the backbone of the tree (i.e. the tree structure remains constant, unlike in [ ] ), and it is comparable for trees with polytomous nodes. to address phylogenetic uncertainty, we collapsed all nodes with alrt of less than . , which corresponds to a false-positive rate of < . [ ] . in such cases, we randomly resolved the polytomies times and calculated the nph . accordingly, we report the overall normalized topology distance, as well as the mean and % percentile range of values (s table) . to determine whether host jumping occurred more often toward the root or tips of the trees, we calculated the relative node depth for incongruent nodes between virus-host pairs of trees (see fig c and d ). this metric counts the number of nodes contained within each clade in the host tree that are not present in the virus tree. because this number can depend on the size of the tree, we divide each of the node depths by the largest value in the tree. accordingly, this metric is decreased if incongruent clades correspond to shallow nodes ( fig c) compared to deep nodes (fig d) . for example, the maximum node depth is if a pair of trees differs in the deepest node and approaches if they differ only in very shallow nodes. an important assumption of the current study is that incongruence between virus and host topologies is a result of cross-species transmission. in some instances, however, it might be possible to explain the lack of virus-host co-evolutionary history through multiple instances of lineage duplication and extinction, without such host-switching events. to address this issue, we reconciled the co-phylogenetic relationship between viruses and their hosts. in particular, we determined the optimal solutions for co-phylogenetic reconstruction for all families, including the possibility of lineage duplication and extinction, using the jane co-phylogenetic software package [ ] . this uses a polynomial time dynamic programming algorithm in conjunction with a genetic algorithm to find optimal solutions to reconcile cophylogenies. although this is a simple heuristic method, it is able to generate results on relatively large data sets (although it is most effective for trees with less that~ - tips). importantly, we used 'event costs' associated with incongruences between trees that were conservative towards co-divergence and defined here as: for co-divergence, for duplication, for host-jumping and for extinction. utilizing this reconciliation, we also examined the evolution of the hepadnaviridae in more detail as this family contains the best evidence for co-divergence (see results). finally, to assist in visualization of these data, tanglegrams for each virus family were constructed using treemap v . [ ] . lines between the trees connect the host (left) with its virus (right). we utilized the 'untangle' function, which rotates the branches of one tree, to minimize the number of crosses lines. if viruses and hosts have congruent topologies then the number of crossed lines, and hence cross-species transmission events, will obviously be reduced. required to obtain the maximum topological distance (black lines) for the hepadnaviridae and the parvoviridae phylogenies, which represent the minimum and maximum number of viruses in our data sets, respectively. the red, dashed line illustrates the ph distance of the non-randomized data, while the black, solid line is the ph distance after randomizing the data after n randomizations. (tif) s table. genbank accession numbers for the virus and host genetic sequence data utilized here. (docx) virus genera were excluded either due to lack of available data or because we were unable to obtain a reliable alignment of sufficient length for phylogenetic analysis (i.e. at least amino acids after trimal pruning). (docx) s table. overall nph distances, means and % percentiles between two unrooted phylogenetic trees for each virus family determined using the normalized penny and hendy [ ] topological distance method, implemented in in nelsi v . [ ] . the overall nph distances are illustrated in fig in integrating reptilian herpesviruses into the family herpesviridae paleozoic origin of insect large dsdna viruses acute and persistent viral life strategies and their relationship to emerging diseases evolution and emergence of rna viruses virological factors that increase the transmissibility of emerging human viruses the evolution and emergence of hantaviruses. current opinion in virology a cophylogenetic perspective of rna-virus evolution family level phylogenies reveal modes of macroevolution in rna viruses pathogen population bottlenecks and adaptive landscapes: overcoming the barriers to disease emergence early mesozoic coexistence of amniotes and hepadnaviridae distinct viral lineages from fish and amphibians reveal the complex evolutionary history of hepadnaviruses tangled trees: phylogeny, cospeciation and coevolution absence of frequent herpesvirus transmission in a nonhuman primate predator-prey system in the wild the use of tree comparison metrics jane: a new tool for the cophylogeny reconstruction problem reconstruction of the cophylogenetic history of related phylogenetic trees with divergence timing information time scale evolution of avipoxviruses comparing phylogenetic codivergence between polyomaviruses and their hosts evaluating the evidence for virus/host co-evolution. current opinion in virology convergent evolution of escape from hepaciviral antagonism in primates phylogenetic analysis of gb viruses a and c: evidence for cospeciation between virus isolates and their primate hosts. the journal of general virology differential infection patterns and recent evolutionary origins of equine hepaciviruses in donkeys bats are a major natural reservoir for hepaciviruses and pegiviruses viral mutation rates island biogeography reveals the deep history of siv cross-species virus transmission and the emergence of new epidemic diseases viral evolution and the emergence of sars coronavirus preferential host switching by primate lentiviruses can account for phylogenetic similarity with the primate phylogeny redefining the invertebrate rna virosphere substitution model adequacy and assessing the reliability of estimates of virus evolutionary rates and time scales model selection in phylogenetics seqotron: a user-friendly sequence editor for mac os x muscle: multiple sequence alignment with high accuracy and high throughput trimal: a tool for automated alignment trimming in large-scale phylogenetic analyses recombination analysis tool (rat): a program for the highthroughput detection of recombination assessment of methods for amino acid matrix selection and their use on empirical data shows that ad hoc assumptions for choice of matrix are not justified new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml . . systematic biology approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative survey of branch support methods demonstrates accuracy, power, and robustness of fast likelihood-based approximation schemes. systematic biology a comprehensive phylogeny of birds (aves) using targeted next-generation dna sequencing the tree of life and a new classification of bony fishes angiosperm phylogeny poster-flowering plant systematics resolving conflict in eutherian mammal phylogeny using phylogenomics and the multispecies coalescent model reevaluating the arthropod tree of life simulating and detecting autocorrelation of molecular evolutionary rates among lineages distributions of tree comparison metrics-some new results. systematic biology writing -review & editing: jlg sd ech. key: cord- -xfb lq authors: bull, james j; ebert, dieter title: invasion thresholds and the evolution of nonequilibrium virulence date: - - journal: evol appl doi: . /j. - . . .x sha: doc_id: cord_uid: xfb lq the enterprise of virulence management attempts to predict how social practices and other factors affect the evolution of parasite virulence. these predictions are often based on parasite optima or evolutionary equilibria derived from models of host-parasite dynamics. yet even when such models accurately capture the parasite optima, newly invading parasites will typically not be at their optima. here we show that parasite invasion of a host population can occur despite highly nonoptimal virulence. fitness improvements soon after invasion may proceed through many steps with wide changes in virulence, because fitness depends on transmission as well as virulence, and transmission improvements can overwhelm nonoptimal virulence. this process is highly sensitive to mutation supply and the strength of selection. importantly, the same invasion principle applies to the evolution of established parasites, whenever mutants arise that overcome host immunity/resistance. a host population may consequently experience repeated invasions of new parasite variants and possible large shifts in virulence as it evolves in an arms race with the parasite. an experimental study of phage lysis time and examples of mammalian viruses matching some of these characteristics are reviewed. virulence management is a modern field whose goal is to predict the consequences of social practices on the evolution of parasite virulence . by understanding how virulence is expected to evolve, it may be possible to encourage social practices that indirectly select lower virulence. of special concern in virulence management are environments that might favor evolutionary increases in virulence, such as leaky vaccines and high host densities (ewald ; gandon et al. gandon et al. , . typically, the models of virulence management are evolutionary. they consider how natural selection of parasites is expected to shape the evolution of virulence and other traits toward states that maximize fitness and the parasite optima. the argument put forth here is that this 'optimality' approach is not always suitable, because it assumes equilibrium conditions that some types of parasites may be slow to attain. even for parasites that are able to evolve an optimum virulence, once a host population has a high density of immune or recovered individuals, then that host population remains vulnerable to invasion by parasite genotypes that escape immunity. during invasion, a much wider range of virulence can spread, at least temporarily, than is evident from evolutionary optima. the frequency of new invasions and the rapidity with which parasites evolve to their optima will dictate how often the host experiences nonoptimal parasite virulence. we develop this argument in the context and framework of the standard models for the evolution of virulence, models that address virulence optima. two standard models are reviewed. they differ in the state of host-parasite dynamics when the virulence optimum is calculated, but they both illustrate the same general approach. those models are then extended to nonequilibrium conditions. our use of those simplistic models is for ease of illustration in a familiar context. our neglect of more complicated processes, such as within-host dynamics (ball et al. ), within-host combined with betweenhost dynamics (gilchrist and coombs ) , or of virulence models that lack optima (dieckmann ) is not to deny the importance of those alternatives. indeed, the concept of a delayed approach to equilibrium conditions can be applied to many of those and other types of models. furthermore, the process we describe is fully and properly encompassed in the new formalism of gandon ( , ) and day and proulx ( ) , albeit that their formalism encompasses a far wider range of possibilities than the ones we emphasize. optimal virulence theories r perspective the now conventional method for considering the evolution of virulence is about three decades old may , ) . it assumes that the parasite population has gone to its dynamic equilibrium (the endemic phase), such that the parasite population is no longer spreading in the host population. this model identifies the combination of transmission and virulence that leads to highest parasite fitness. the result is that selection maximizes the parasite reproductive number, r (or more properly, maximizes r; day and gandon ) . r is a dimensionless number that equals the number of new infections started during the lifetime of the first infected individual in a susceptible population (r is the number of new infections from an infected individual once the parasite has established itself in the host population.) the formula for r is simply the parasite fecundity rate times the average lifetime of the infection. although r is defined for the first infected host in a naive population, what maximizes r also maximizes r because the only difference between them lies in the value of s (ebert and herre ; day and gandon ; gilchrist and coombs ) . the result that selection at the dynamic equilibrium favors maximization of r parallels the usual demographic result that selection favors maximal lifetime reproductive output in a stable population (wilson and bossert ; charlesworth ) . we will expand on the implications of r maximization below, but it is most informative to do so in the broader context of the second virulence evolution model, considered next. lenski and may ( ) pointed out that selection on parasite parameters was different during the epidemic phase than at dynamic equilibrium: maximizing r no longer maximized fitness if the parasite population was expanding. again from demography, selection favors early births over late ones in a growing population, so during the epidemic phase, there is a benefit to early transmission and its consequent shorter generation time even when it lowers life-time reproductive success of the infection. in the model, earlier reproduction is achieved by a higher transmission rate, which in most models also results in higher virulence because of an assumed trade-off between transmission and virulence. the lenski and may model lays the foundation for the argument we develop here, so we offer a simple version in the form of an 'si' model. for a constant density of susceptible hosts, s, and an absence of genetic variation in parasite and host parameters, the epidemic is characterized by one equation for the change in abundance of infected individuals, i: where b is the transmission rate, and d is the combined death rate of infected individuals [d is the sum of an intrinsic host mortality rate and a virulence, or parasiteinduced mortality, and aside from this difference, equation ( a) is otherwise the same as equation ( ) in lenski and may ( ) and gilchrist and coombs ( ) ]. the total host population is simply n = s + i. a constant density of susceptible hosts is reasonable whenever the infection is so rare that most hosts have not yet been infected. the per capita rate of pathogen spread is then: this quantity is a measure of parasite fitness in the epidemic (a type of invasion fitness, metz et al. ) , because it defines how rapidly the parasite spreads; indeed, the quantity on the right of equality ( b) is the intrinsic rate of increase of i. the rate of spread is a function of parasite transmission (b) and the death rate of infected individuals (d), two parameters that are typically assumed to be subject to a trade-off boundary (fig. top; ebert and bull ) . a trade-off means that the parasite cannot increase b without incurring increasing death rates (d), although a more stringent criterion on the trade-off, such as the concavity used here, is required to select intermediate levels of virulence (sasaki and iwasa ) . following lenski and may ( ) for now, we let d be the sum of a constant host mortality plus virulence; we will refer to d as simply virulence without loss of generality. given that the parasite is confined to a trade-off but is allowed to evolve along the trade-off, the combination of b and d maximizing parasite fitness during invasion is given by which is graphically represented as the dot along the trade-off function whose slope is s (fig. , bottom; shown in comparison to the r maximum). in prior work, the fitness 'optimum' found in this way is usually regarded as the phenotype that the parasite will evolve toward (and will ultimately attain, if the environment does not change). as demonstrated by lenski and may ( ) , this optimum changes as the density of susceptible hosts is reduced by the parasite, ultimately coinciding with the r maximization (fig. ) . thus, the optimum stops changing when the epidemic is over and the population has reached dynamic equilibrium between host and parasite (and parasite virulence is optimal). both prior perspectives essentially restrict evolution to the trade-off function, with an emphasis on optima. in the lenski-may model, the optima are temporary, changing as the density of susceptible hosts changes. both prior perspectives differ from a new approach that incorporates dynamics, selection and genetics simultaneously (day and proulx ; gandon , ) . this latter approach allows the virulence and transmission parameters to vary anywhere within the zone of attainable phenotypes, and it maps changes in the mean phenotypes as a function of selection and of genetic covariances between the two traits. the optima in this new approach are not altered, but the model is extended to include the evolutionary and population dynamics. this newer method thus subsumes the former ones. our goal is to recognize a possible feature of virulence evolution in parasites that newly invade a host species. more importantly, the same feature may apply when an established-parasite evolves to overcome host resistance or immunity. the method we use in developing this argument is compatible with that of day and gandon ( ) , although our arguments are presented as if evolution proceeds via a succession of successful mutants rather than by obeying covariances of variation that is already present. either type of model can be used to make the same point, however. invasion criteria are permissive an optimum is best regarded as a long-term endpoint of evolution. prior to attaining an optimum, however, the evolutionary dynamics apply (as codified by day and gandon , for example) . even before dynamics are relevant, the issue is merely what range of parasite phenotypes/genotypes can invade the host population. the invasion threshold provides the boundary to the range of parameters that allows invasion. it is the set of all (mutant) parameter combinations for which the parasite neither invades nor goes extinct. thus, parameter values on one side of the invasion threshold will allow invasion, whereas those on the other side will lead to loss/extinction. . death rate is divided into intrinsic host death rate and virulence -parasite-induced host death rate. the intrinsic host death rate is assumed to be constant, so the virulence-transmission trade-off (dashed curve) emanates from the axis at this value and increases as transmission increases. the trade-off gives the minimum total parasite death rate (d) that could be achieved for a given transmission rate (b), so only points on and above the trade-off boundary are attainable by the parasite (shaded). for simplicity, we henceforth refer to d as virulence, but acknowledge that it is the sum of a constant mortality plus virulence. (bottom) optimum for a parasite invading a host population, when the susceptible host density remains constant at s. during the epidemic phase, the parasite's optimum lies on the dashed tradeoff function and satisfies the condition that the slope of the tangent along the trade-off function equals the current density of hosts, s (upper arrow). as the parasite epidemic reduces the density of susceptible hosts, the optimum shifts progressively further down the tradeoff curve, until dynamic equilibrium is reached, whereby parasite and host densities remain constant. in this state, the parasite optimum maximizes r = b/d, given by the point at which a line through the origin is tangent to the trade-off function (lower arrow). in this paper, we are concerned chiefly with conditions at and shortly after the time of invasion, before the population has reached dynamic equilibrium. the invasion threshold is easily found in our model. extending result ( b) above, the pathogen invades if di/ dt > , or the invasion threshold is thus found by replacing the inequality in equation ( ) with equality. the set of parameters for invasion of a host population whose density is s* can be represented in a two-dimensional coordinate system as the points falling below a line of slope s* passing through the origin (fig. , top) and above the trade-off boundary. this graph illustrates why the transmission and virulence state (b, d) that maximizes fitness gives an inadequate picture of the levels of virulence that can invade the population. any point inside the region between the invasion boundary line and the trade-off boundary represents a pair of parasite values (b, d) that can invade. thus, the population is susceptible to invasion by far more virulent parasites and by far less virulent parasites than those at the optimum, a point made by andre and hochberg ( ) . although all values below the line can invade, not all have the same fitness, so further evolution is expected whenever the invading genotype is not at the optimum. evolution following invasion may be slow to attain the optimum following day and proulx ( ) , if the invading parasite persists and becomes established, it is expected that virulence and transmission will begin to evolve toward values that improve parasite fitness. indeed, if evolution to the optimum is fast enough, the duration of nonoptimal parasite behavior could be ignored. yet, the approach to optimum virulence may be slow for a few reasons: (i) the optimum may be changing (lenski and may ) , (ii) virulence evolution will be influenced by the genetic covariance or mutational properties between virulence and transmission (day and gandon ) , and conditions for the invasion of a host population of density s* by a parasite with parameters (d, b) are s* > d/b, where d is virulence and b is the transmission rate. points satisfying s* = d/b lie a line radiating from the origin (of slope s*), so radii with progressively higher slopes represent parasite parameters that can invade only at progressively higher host densities. all parasites whose values fall in the darker shaded region below the line of slope s* and above the dashed trade-off boundary are attainable and can invade, whereas the lightshaded region above the invasion boundary represents values that the parasite can attain but do not allow invasion (for host density s*). the parasite population would eventually evolve toward the dot on the trade-off boundary (arrows), but it could create a potentially highly virulent epidemic from the start of the invasion until the evolutionarily equilibrium virulence was attained. (bottom) evolution following invasion may be slow to approach optimal virulence. the rightmost oblique line is a fitness isocline (of slope s*), and all points on it have equal fitness, superior to the fitnesses of all points to its left. evolution would tend to move parasites progressively toward right-most isoclines, but as a mutant's relative position on the fitness isocline does not affect its fitness, evolution at each step could move virulence further or closer to the optimum (solid point) until the optimum was approached closely. the input of mutations (or genetic covariance between transmission and virulence) thus has a major impact on the evolution of virulence in these early stages (day and gandon ) . improved fitness need not greatly restrict the range of virulence values allowed, and indeed, virulence could evolve to deviate further from its optimum during intermediate stages of the adaptation. these arguments apply to constant host density, and if susceptible host density is declining, the fitness isoclines will have progressively shallower slopes. (iii) there may simply be little genetic variation (see also day and proulx ) . as lenski and may ( ) pointed out, (i) the optimum will shift toward progressively lower virulence values as the density of the susceptible hosts is reduced by the epidemic. thus the optimum will not be static, and the parasite can do no better than adapt toward the current optimum. even in the absence of this effect, parasite evolution may be slow to reach the optimum because of (ii), pleiotropy or genetic covariances. selection favors mutations with a higher fitness than the currently dominant strains. these mutations may improve transmission but may result in virulence further away from its optimum. fig. (bottom) shows that fitness isoclines run parallel to the invasion boundary, so all points on a fitness isocline have the same fitness, assuming that s remains constant. as can be seen for the trade-off boundary drawn, there is a wide range of virulence associated with each step of evolution toward the optimum, until the optimum is neared (depending on the shape of the trade-off). thus, particularly during the initial phases of invasion, evolution of higher parasite fitness need not be accompanied by a change in virulence. of course, other factors may also slow the approach to equilibrium, but these two follow directly from our model. the invasion threshold model has been presented as though the only outcome of host infection is death. in such a system, the initial parasite invasion would be followed by a reduction in host density to the dynamic equilibrium, and the resulting low absolute host density would greatly limit opportunities for the re-invasion of any parasite mutants that had an even more devastating impact on the host. yet, host death is not the only outcome of infection. recovery and consequent immunity is typical of many parasites (not necessarily precluding the death of some infected hosts). how does recovery affect our model? here, we restrict ourselves to the extreme case that recovery is complete, so that a recovered individual cannot be reinfected by the same strain (see van baalen for models of virulence evolution with partial recovery). most basically, if a large fraction of the host population is immune, a large advantage can be gained by parasite mutants that can overcome the host immunity but also retain the ability to infect susceptible hosts (s). those mutants have their own invasion threshold, and depending on their parameters, those constraints may be much more lenient than the constraints applying to parasites unable to escape host immunity (fig. ) . thus, parasites that engender host immunity have the potential to cause repeated invasions, with potential high and devastating virulence each time. the dynamics of this process are sensitive to the nature of host recovery and virulence, however. a highly virulent parasite (most infections lethal) that reaches high abundance in the host population will crash the host population and not leave many recovered hosts, at least in the short term. until host numbers rebound, there would be little opportunity for new parasite invasions because of the low absolute density of hosts. if recovery rate from infection is instead high, virulence (host mortality) is necessarily low, and the parasite impact on host density will be slight. in this latter case, the host population is soon prone to invasion of mutants that escape immunity. thus epidemics that kill large numbers of hosts will experience longer lags before possible reinvasion than epidemics that do not kill many hosts. the addition of recovery does change the interpretation of the preceding model somewhat. the derivations above remain the same, but the d term (now denoted d¢) is no longer just host mortality. instead, the d terms are replaced by d¢, where and q is the recovery rate of infected hosts (to a state in which they can no longer be infected). thus, if recovery figure a population with a high density of recovered hosts is susceptible to invasion by highly virulent mutants. once a parasite has evolved close to its optimum along the trade-off boundary, there will be only a small set of mutants that can invade, because they are all subject to the same constraints, provided they can infect only the susceptible hosts; the invasion threshold for those mutants is a line of slope s (the rightmost line). a mutant capable of infecting both susceptible and recovered hosts will have a much larger set of possible mutants that can invade (the invasion threshold is a line of slope s+r, where r is the density of recovered hosts, hence lies to the left of the line of slope s) and thus have greater potential to evolve nonoptimal virulence. the graph is the same as in previous figures, except that the vertical axis (d¢) now includes the recovery rate of infected hosts as well as mortality rate (the d in previous figures was just mortality rate). from infection is allowed, the derivations and figures above merely substitute d¢ for d, and the trade-off functions now combine recovery with virulence, as shown in fig. . on intuitive grounds, parasite dynamics are sensitive to the loss of infected hosts, but it does not matter whether those infections are lost because the host died or because it recovered. the main implication of this change for evolution of virulence theories is that evolution of higher d¢ no longer implies evolution of higher virulence. without specifying the relationship between virulence and recovery, higher d¢ could mean a higher recovery rate, with no change in virulence or even a decline in virulence (fig. ) . this indeterminacy applies to all evolution of virulence theories, and it is not otherwise possible to predict an optimum virulence per se when recovery is involved in the trade-off with virulence. the indeterminacy poses little problem for the invasion threshold perspective, however: the invasion threshold perspective merely specifies a range of possible virulence levels that can potentially invade. this range may be reduced by the inclusion of recovery, but it will always be wider than the range spanned by the optima. before proceeding to empirical examples, it is useful to consider the distinction between the standard r maximization model, the lenski and may's ( ) dynamic optimum model, and invasion threshold as alternative perspectives on the evolution of virulence. there is obvious overlap among these models (and both are nicely subsumed in the covariance method of day and gandon ) , but the emphases from them have differed. the r maximization model offers a single optimum, albeit one that may be sensitive to environmental conditions. it assumes dynamic equilibrium exists between host and parasite, hence does not apply when new parasite mutants invade. the other two models, lenski-may dynamic optimum and the invasion threshold, are similar in that both apply to invasions. the only difference is that one emphasizes the change of virulence optima during the invasion, the other addresses the range of virulence values that can invade and the nonequilibrium states that may persist after invasion. without knowing the optima, the lenski-may and invasion threshold perspectives are difficult to distinguish, as either or both could obtain across repeated invasions. however, observing the evolution of an increase in virulence (instead of a decrease) as the epidemic matures would support the invasion threshold model for the initial virulence, because the lenski-may optima shift toward decreasing virulence as the epidemic matures. likewise, if hosts merely die from the infection and do not recover (and are not resistant), then the invasion threshold model cannot possibly apply after dynamical equilibrium has been reached, because there is no reservoir of immune or resistant hosts to be exploited by a mutant parasite. for many parasites, evolvability to escape existing immune and resistance profiles is highly adaptive. longterm parasite success may depend not so much on the ability to achieve evolutionary equilibrium, as in the classic virulence evolution models, but rather to keep changing, staying ahead of host defenses and continually jumping outside the parameter space confining the parasite's recent ancestors. such a process would ensure that the invasion threshold model is relevant at many episodes in the parasite's history. the applicability of the invasion threshold model at some times does not exclude attainment of optima at other times. thus, over the course of history, there can be a cycling between invasion, evolution toward an optimum (perhaps indirectly), and maintenance of an optimal state. the relative importance of figure indeterminacy of virulence (host mortality rate) when hosts recover and are immune to subsequent infection. when recovery is allowed as a component of d in equation ( ), such that d¢ = d + q is substituted for d, the evolution of higher d¢ no longer implies the evolution of higher virulence. the trade-off boundary (between d¢ and transmission) is given by the black line at the upper boundary of the gray area, and it can be seen in the shift from left to right graphs that d¢ increases somewhat but host mortality rate decreases. the sizes of the gray and clear areas are drawn for illustration and are not based on data. each phase will depend on details of the parasite and host. we offer the caveat that the invasion threshold model is not the only model for nonoptimal virulence (dieckmann ) . if host and parasite populations cycle, or if environmental factors cause host populations to fluctuate profoundly, there will be no single virulence optimum, and the best the parasite might do is to track a moving optimum gandon , ] . likewise, if hosts are infected by multiple strains of parasites, parasite-parasite competition within hosts will often lead to nonequilibrium evolutionary dynamics (frank ; bull et al. ) . it should also be noted that different mathematical definitions of virulence have different evolutionary consequences (day ) , but again, our point about nonequilibrium dynamics should hold under many of those alternatives. the general point of the nonequilibrium model is that optimality of a phenotype involved in a trade-off may be evolved only slowly; our specific context is virulence. there is no absolute threshold for rejection of this model, only a subjective one of whether evolution to the optimum is fast enough that the period of meaningful deviation can be neglected. a quantification of nonoptimality could be provided by the experimental adaptation of a parasite with a known optimum, with the approach to that optimum measured over time. a test satisfying several of these criteria was carried out for bacteriophage lysis time, analogous to phage virulence (heineman and bull ) . the optimum was predicted when assuming a linear trade-off between lysis time and number of progeny at lysis and that the linearity extended well beyond the normal lysis time. adaptation of phage t was performed under constant environmental conditions of both high and low host density; this constant host density provided for a fixed optimum and matches the models in this paper. at high host density, t attained a lysis time close to the optimum within generations of adaptation, but the phage failed to approach the optimum at low host density in two separate adaptations of $ generations each. one failure stemmed from a lack of evolution, likely because of small population size. in the other case, molecular evolution was observed, and the failure to attain the optimum was interpreted as a failure of the model used to calculate the optimum (that the assumed trade-off did not match the t trade-off). the failure to evolve is a special case of our nonequilibrium model, and presumably, the virus would have evolved after sufficient time. the failure to attain the optimum despite evolution in the other replicate is presumably a failure to calculate the true optimum, and thus not an illustration of nonequilibrium dynamics, unless it could be shown that the predicted equilibrium would eventually be obtained given sufficient time. overall, this experimental study illustrates how the measurement of deviation from optimality reflects on the nonequilibrium model, but only when the basis for nonoptimality is understood. it is no doubt rare that a parasite optimum is known and that evolution toward the optimum can be observed; repetition of this phage experiment seems especially difficult for parasites of multicellular hosts. the bacteriophage study benefited from the fact that the optimality model required only linearity of the trade-off, and that the slope of the trade-off did not affect the optimum (and even the assumption of this generality appears to have been wrong). such generality may be rare, and quantitative knowledge of the trade-off may be necessary to predict the optimum in most systems. even if an optimum cannot be calculated a priori, it may be determined empirically in an experiment, as a long-term equilibrium that is attained and maintained. the evolutionary approach to this optimum from different starting points then provides insight to the dynamical behavior relevant to the perspective given here. insight may also be gained from observations of parasites under natural conditions. some basic characteristics predispose a parasite toward delayed-equilibrium virulence, and the following examples of vertebrate parasites (all viruses) illustrate how one may begin looking for candidates. these examples illustrate that not all parasites exhibit nonequilibrium dynamics, and even among those that do, the frequency of the phenomenon varies considerably. the primary criterion that can be used in considering the invasion threshold perspective is that a host experiences periodic invasion by new parasite mutants. as noted above, this criterion does not rule out the lenski and may ( ) model. thus, a second important question, but one that can rarely be answered, is whether the parasites are far off the optimum. we start with two pathogens that elicit host immunity but clearly do not exhibit repeated invasions by mutants that escape immunity. humans are the only known natural hosts for measles and polio viruses. successful vaccines have greatly reduced the incidence of both infections, and the vaccines derived from decades-old isolates have remained effective without apparent evolution of novel antigens that escape vaccine-induced immunity (schrag et al. ; macadam et al. ) . both viruses, therefore, are good candidates for the r maximization model, but their virulence optima are difficult to establish, and alternatives to the r model have been proposed at least for polio (levin and bull ; frank ) . in addition, the virulence of measles is notoriously sensitive to host nutritional status, so it is not clear what level of virulence to apply to the model. even without knowing whether these viruses have achieved an optimum level of virulence, both viruses appear to be good candidates for viruses that violate the invasion threshold model (at least in contemporary populations) by virtue of their failure to generate escape mutants. as an aside, the lack of 'escape' mutants capable of infecting immune hosts is puzzling, as both viruses have rna genomes, which are thought to be prone to rapid evolution. furthermore, three antigenically distinct polioviruses are known (and each is included in the vaccine), yet no others have evolved in response to vaccine pressure (macadam et al. ). these two viruses highlight the point that, contrary to common perception, some parasites may not be able to adapt to altered environmental conditions. the flu virus is notorious for its recurrent epidemics in humans, and these epidemics arise from various forms of viral evolution to escape host immunity. flu thus exhibits one of the main features of the invasion threshold model. there is also documented variation in virulence, with the highest virulence associated with some of the new mutants (see below). flu dynamics are governed largely by the interaction of host immunity and viral surface antigens as well as evolution of the viral antigens (webster et al. ) . influenza is typed according to two viral antigens, the hemagglutinin ('h' type) and neuraminidase ('n' type); we will limit ourselves here to the different h types. there is a wide variety of h types in nature, due partly to the fact that different influenza a strains span a wide variety of warmblooded hosts. currently, h and h are circulating in humans, but h was abundant in the past. there are two classes of h mutants that invade humans. one class consists of simple point mutants of existing h types (referred to as subtypes, because of a process known as 'antigenic drift'). most annual epidemics consist of these subtype mutants, and subtype evolution is substantial enough to largely overcome immunity to the same ancestral type from several years in the past (ferguson et al. ) . the other class consists of h types introduced from other species (a process of 'antigenic shift'). flu virus with novel h types have a profound advantage because there is no prevailing immunity in humans against those types. three world-wide flu pandemics of the s were notorious for causing large numbers of infections and thus large numbers of deaths; all were due to viruses that had acquired h types that humans had not previously experienced. not all viruses with novel h types readily spread in humans, however, and many of these introductions have simply died out. we thus have a reasonable understanding of what types of molecular variation enable a flu virus to escape prevailing immunity, although this advantage by itself is not sufficient to enable spread in the human population. the virulence of influenza variants varies somewhat, although it is difficult to apportion mortality between the virus (its intrinsic virulence) and the host (whether it has any prevailing immunity to the strain). the case mortality rate of the h n flu that killed - million people worldwide was reported to be about %, somewhat more than a -fold excess of the typical mortality rate per infection of the viruses of today (taubenberger and morens ) . the virulence of this strain appeared to wane over a few years. h n disappeared in the s, and its accidental reintroduction in to naive hosts did not lead to the high mortality rate of , so it appears that the virulence per se evolved downward (kilbourne ) . although mortality rates of the usual antigenic shift strains remain relatively constant, some high virulence exceptions are known (e.g., an h n strain from ; o'donnell et al. ) , and there may well be many low-virulence variants that go unnoticed. however, the h n strain (bird flu) that is circulating widely in birds has a case mortality rate exceeding % in humans, far in excess of the mortality rate observed in any strain that established itself in humans (webster et al. ). h n has not established itself in the human population, but the obvious fear is that it can do so and maintain its high mortality rate, at least during its first round of global spread. the invasion threshold perspective is thus especially relevant here. ewald has used the public media to argue that the virulence of h n will quickly evolve to low levels, should it invade (orent ) ; this view has not been widely accepted (normile ) , and the perspective of this paper is that, even if the h n optimum in humans is low virulence, the epidemic could have devastating effects before it neared the viral optimum. feline calicivirus (fcv) is an rna virus of domestic cats. a large variety of fcv strains is known to circulate concurrently, with sequence divergence in the variable region of the capsid protein gene ranging as much as % between strains ). virulence varies among isolates, from asymptomatic to highly virulent, the latter being as high as % mortality (termed vsd strains, for 'virulent systemic disease'). multiple strains often circulate within a local cat population. vaccines are available, but vaccination neither prevents viral infection nor prevents viral replication within a cat, so it appears that cross immunity among strains is only partial . consistent with the invasion threshold model, nearly a dozen separate outbreaks of vsd have been reported. when subjected to a phylogenetic analysis, the different vsd strains appear to have independent origins from less virulent ancestors. additionally, the vsd outbreaks appear to have been terminated by viral extinctions, possibly from reducing the local cat density ). the die-outs could be interpreted as the failure to evolve optimal virulence (consistent with the invasion threshold model), or as evidence that near-optimal strains are replacing the vsd strains as host density wanes (consistent with the lenski-may model). most agricultural animals used as food sources, such as pigs, chickens, and cattle are housed at high densities and in conditions of stress that facilitate the spread of infectious diseases. even if a farm's livestock is wiped out by an infection, new animals may be brought in and maintained at high densities again. this artificial maintenance of high host density is in contrast to the usual pattern in epidemiological models that parasites regulate the densities of their hosts. these enforced high host densities have been suggested to be especially prone to the evolution of highly virulent parasites, because virulent parasites do not die out even when their hosts do. although cleanups following an outbreak are usually attempted, the same buildings and grounds are often used for restocking, allowing any remaining environmental source of parasite to reinvade. the difficulty of applying equilibrium theory to these situations is illustrated by the pig virus transmissible gastroenteritis coronavirus (tgev). historically, a highly lethal gut infection of piglets in pig farms, a mutant form evolved (porcine respiratory coronavirus, or prcv) that not only had altered tissue tropism but also apparently lower virulence (kim et al. ) . prcv differs from tgev by a small deletion and a couple point mutations, yet it infects the pig respiratory system and is often much less virulent than tgev. there is some antibody cross reactivity between tgev and prcv, thus the population of one form of the virus interferes with the other, and it is suspected that a low virulence prcv was responsible for the disappearance of tgev in some pig farm areas. prcv now exists in forms of high and low virulence. this example thus represents what may be a single mutant invasion in the recent history of tgev. to what extent prcv spread because it could overcome prevailing immunity (as assumed by the invasion threshold model) or because it had other advantages, is not clear. another viral scourge of agriculture is marek's disease virus (mdv) of chickens (hirai ; davison and nair ) . mdv has shown a progressive evolution toward increasing virulence over four decades, but again, many strains circulate and some have low virulence. live vaccines have been used for three decades, the virus used in them having been changed twice. as with fcv, the vaccines do not prevent infection and replication by other strains, and it is widely suspected that the vaccines may have fostered the evolution of progressively higher virulence (references within davison and nair ; hirai ) . (we lack the control of viral evolution at high chicken density in the absence of the vaccine to have any confidence that the vaccine was the cause in the higher virulence evolution.) with mdv, there is considerable ambiguity in the causes of virulence evolution as well as in the spectrum of virulence evolution itself. it may well be that vaccines have selected escape mutants time and again. whether escape mutants retained near-optimal virulence or not is difficult to establish, because there is a strong ascertainment bias -avirulent viruses are not noticed by chicken farmers. thus, without understanding the range of virulence levels that have evolved, it is not practical to discriminate among the models. such a system seems ripe for testing the models, however. there is sufficient evidence to suggest that the invasion threshold model is relevant to the evolution of many infectious agents, but little evidence to suggest how often it is important. recommendations for use of this perspective are thus accordingly muted. certainly, it seems wise in virulence management efforts to be aware that a broader range of virulence might invade than is optimal and that evolution to an optimum may not be rapid. it would be obvious folly to ignore a case in which the predicted optimum was low virulence but in which a highly virulent parasite could invade and persist. if the perspective offered here proves to be broadly relevant, then its impact is to support and even bolster any concerns about the evolution of high virulence but also to raise the specter of temporary high virulence in parasites expected to equilibrate at low virulence. the most useful work at this stage would be to contrast predicted virulence optima with actual virulence levels over time. calculating virulence optima has not met with much success, however, likely because the optimum invariably depends on an unknown trade-off and potentially on many environmental factors. thus, most tests of virulence evolution models have been relative, i.e., correlating virulence level with some environmental characteristic and determining if the correlation is in the right direction. direct, quantitative tests of virulence optima are rare, nonexistent for human pathogens. if optima cannot be calculated, then it is of course difficult to test any nonequilibrium model against an optimality model. but an alternative to testing whether virulence is optimal is to observe the dynamics of virulence evolution. if virulence does not change over long periods after invasion, despite a dropping susceptible host density, then it would seem that virulence has been nonoptimal during much of the time. coevolution of hosts and parasites population biology of infectious diseases: part i virulence evolution in emerging infectious diseases coevolution of recovery ability and virulence modeling within-host evolution of hiv: mutation, competition and strain replacement evolutionary feedback mediated through population density, illustrated with viruses in chemostats evolution in age-structured populations a longitudinal molecular epidemiological analysis of feline calicivirus infection in an animal shelter: a model for investigating calicivirus transmission within high density, high turnover populations on the evolution of virulence and the relationship between various measures of mortality applying population-genetic models in theoretical evolutionary epidemiology insights from price's equation into evolutionary epidemiology a general theory for the evolutionary dynamics of virulence adaptive dynamics of pathogen-host interactions adaptive dynamics of infectious diseases: in pursuit of virulence management challenging the trade-off model for the evolution of virulence: is virulence management feasible? the evolution of parasitic diseases evolution of infectious disease ecological and immunological determinants of influenza evolution models of parasite virulence imperfect vaccines and the evolution of pathogen virulence imperfect vaccination: some epidemiological and evolutionary consequences evolution of virulence: interdependence, constraints, and selection using nested models testing optimality with experimental evolution: lysis time in a bacteriophage. evolution marek's disease: current topics in microbiology and immunology influenza pandemics of the th century molecular characterization and pathogenesis of transmissible gastroenteritis coronavirus (tgev) and porcine respiratory coronavirus (prcv) field isolates co-circulating in a swine herd the evolution of virulence in parasites and pathogens: reconciliation between two competing hypotheses short-sighted evolution and the virulence of pathogenic microorganisms rational design of genetically stable, live-attenuated poliovirus vaccines of all three serotypes: relevance to poliomyelitis eradication how should we define 'fitness' for general ecological scenarios avian influenza. pandemic skeptics warn against crying wolf epidemiology and molecular characterization of co-circulating influenza a/h n virus variants in children chicken little feline calicivirus optimal growth schedule of pathogens within a host: switching between lytic and latent cycles spontaneous mutation rate of measles virus: direct estimation based on mutations conferring monoclonal antibody resistance influenza: the mother of all pandemics evolution and ecology of influenza a viruses h n outbreaks and enzootic influenza a primer of population biology we thank troy day, carl bergstrom, and robin bush for comments. two reviewers essentially did the literature work for this paper. our choice for some of the examples reviewed above was inspired by a seminar of a. read. this work was supported by nih gm to jb. jb also receives support as the miescher regents professor at the university of texas. de was supported by the swiss national fonds. key: cord- -re c e b authors: khan, junaid s.; provencher, jennifer f.; forbes, mark r.; mallory, mark l.; lebarbenchon, camille; mccoy, karen d. title: parasites of seabirds: a survey of effects and ecological implications date: - - journal: adv mar biol doi: . /bs.amb. . . sha: doc_id: cord_uid: re c e b parasites are ubiquitous in the environment, and can cause negative effects in their host species. importantly, seabirds can be long-lived and cross multiple continents within a single annual cycle, thus their exposure to parasites may be greater than other taxa. with changing climatic conditions expected to influence parasite distribution and abundance, understanding current level of infection, transmission pathways and population-level impacts are integral aspects for predicting ecosystem changes, and how climate change will affect seabird species. in particular, a range of micro- and macro-parasites can affect seabird species, including ticks, mites, helminths, viruses and bacteria in gulls, terns, skimmers, skuas, auks and selected phalaropes (charadriiformes), tropicbirds (phaethontiformes), penguins (sphenisciformes), tubenoses (procellariiformes), cormorants, frigatebirds, boobies, gannets (suliformes), and pelicans (pelecaniformes) and marine seaducks and loons (anseriformes and gaviiformes). we found that the seabird orders of charadriiformes and procellariiformes were most represented in the parasite-seabird literature. while negative effects were reported in seabirds associated with all the parasite groups, most effects have been studied in adults with less information known about how parasites may affect chicks and fledglings. we found studies most often reported on negative effects in seabird hosts during the breeding season, although this is also the time when most seabird research occurs. many studies report that external factors such as condition of the host, pollution, and environmental conditions can influence the effects of parasites, thus cumulative effects likely play a large role in how parasites influence seabirds at both the individual and population level. with an increased understanding of parasite-host dynamics it is clear that major environmental changes, often those associated with human activities, can directly or indirectly affect the distribution, abundance, or virulence of parasites and pathogens. parasites, defined as organisms that live at the expense of their hosts (combes, ; poulin and forbes, ) , form diverse and ubiquitous components of all ecosystems, and include both macroparasites (e.g. helminths, arthropods) and microparasites (e.g. bacteria, viruses, fungi, protists) . while most ecological studies focus on megafauna, these organisms can exert a significant influence on ecosystem composition and function via their diversity, abundance and impact on other members of the community (thomas et al., ) . while much is known regarding parasites in some taxa (i.e. captive mammals), considerably less is known in others. given that changing climatic conditions are expected to influence parasite distribution and abundance, understanding current levels of infection, transmission pathways and population-level impacts are integral aspects for predicting ecosystem changes (e.g. l eger et al., ) . seabirds are a particularly interesting group to study in this respect as they often traverse large distances along their migratory routes, and thus may be exposed to, and disperse, many more parasites when compared to other groups . additionally, seabirds are long-lived and typically breed in large, dense colonies, traits that can favour parasite maintenance within populations . finally, some species of seabirds frequently come into contact with humans (e.g. gulls or harvested species), and thus may be a source of zoonotic disease. although the impact of parasites on captive (e.g. poultry) and terrestrial (e.g. passeriformes) bird species has been relatively well-studied, less has been done on seabird parasites. recent research has provided an overview of some of these relationships (e.g. granroth-wilding et al., ; provencher et al., ) , whereas others, such as grimaldi et al. ( ) , focused on the impact of parasites in specific bioregions and orders/genera. except under extreme conditions where the presence of a parasite has a devastating impact causing widespread mortality (e.g. avian cholera; butler et al., ; descamps et al., ; friend and franson, ) , little is known about how interactions with these organisms alter seabird health, reproductive success, and ultimately, seabird population viability and evolution. the impact of a parasite will depend on the nature of the parasite in question, and may be influenced by interactions with other local environmental factors, such as the quantity and quality of food resources, levels of local pollution, habitat quality, and presence of predators, competitors and/or other parasites (e.g. butler et al., ; provencher et al., ) . understanding seabird-parasite interactions is particularly important in regions that are expected to experience great degrees of change in the near future. for example, polar regions are expected to warm at a more rapid rate than temperate or tropical regions (francis and vavrus, ) . generally, parasitism is thought to be relatively depauperate in polar regions because of the lower diversities of suitable intermediate hosts, coupled with conditions that may be unsuitable for certain parasite groups (brooks et al., ; davidson et al., ; kutz et al., ) . however, with changing climatic conditions, and corresponding distributional changes in different taxonomic groups, parasite diversity in the polar zones may increase substantially. there is already evidence to suggest increased parasitism in northern wildlife due to changing climatic conditions (brooks and hoberg, ; kutz et al., kutz et al., , . additionally, novel parasites may extend their ranges in polar regions, or potentially expand to new hosts introducing new interactions within ecosystems (davidson et al., ; galaktionov, ) . the purpose of the present work is to give an overview of known parasite taxa in eight seabird orders, to survey their current distributions, and examine the known effects on seabird populations worldwide. by doing so, we hope to identify the gaps in our current knowledge and shed light on the relationships between parasites and their seabird hosts, and how changing conditions and other stressors may alter the seabird host-parasite relationships. we used a literature scanning approach using web of science core collection and google scholar as our main databases. queries entered into both databases were a combination of general and taxon-specific search terms. all search results were scanned for relevance related to the diversity, abundance and relationships of parasites with seabird hosts. publications that did not achieve these goals, or had more specific research focuses were not included. all relevant journal articles were coded and entered in a spreadsheet for further examination. for each article, the following information was recorded: the parasitic organism studied, a short summary of the article, any effects of the parasite on the birds, the seabird order and species infected by the parasite, the location where the study was conducted, the methods used in the study, the year the study was conducted, and citation information. all search terms used for this review can be found in table . for the purpose of this study, we consider seabirds as defined by gaston ( ) , with updated classifications proposed by the international ornithological committee world bird list (gill and donsker, ) . this includes gulls, terns, skimmers, skuas, auks and selected phalaropes (charadriiformes), tropicbirds (phaethontiformes), penguins (sphenisciformes), tubenoses (procellariiformes), cormorants, frigatebirds, boobies, gannets (suliformes), and pelicans (pelecaniformes) as the main species. we also include, to a limited extent, seaducks and loons (anseriformes and gaviiformes) in this review, as they fulfill a very similar ecological niche. a total of journal articles on the taxonomy, distribution, prevalence and abundance of parasites in seabirds were reviewed. of these, articles housed information on the impacts of parasites on seabirds specifically. research results gathered on each of the parasite groups are summarised below. ticks are blood-feeding arachnids belonging to the order parasitiformes, sub-order ixodida and include approximately described species (guglielmone et al., ) . all species require at least one bloodmeal to complete the life cycle, feeding on a wide range of mammals, birds, reptiles and amphibians. among arthropods, ticks transmit the greatest variety of infectious agents, including viruses, bacteria, protozoa and even helminths, but most research to date on seabirds has focused on ixodes spp., which act as vectors of lyme disease bacteria (borrelia burgdorferi s.l.; dantas-torres et al., ; jongejan and uilenberg, ; table ). ticks can be divided into two distinct categories; hard ticks (ixodidae) and soft ticks (argasidae). hard ticks have a single long bloodmeal in each of their three life stage (larva, nymph, adult), whereas soft ticks take several short bloodmeals in nymphal and adult stages and have a variable number of nymphal instars (dietrich et al., ) . in both groups, the duration of attachment on the host determines the likelihood of disease transmission (dantas-torres et al., ) . of the identified species of hard ticks found on seabirds, all but one (amblyomma loculosum) belong to the genus ixodes. of these, ixodes uriae has the largest distribution, and is the most studied species (fig. ; coulson et al., a coulson et al., , b mccoy et al., ; muñoz-leal and gonzález-acuña, ; ols en et al., ) . hard ticks have been recorded feeding on seabirds belonging to the orders charadriiformes, phaethontiformes, sphenisciformes, procellariiformes, and suliformes (dietrich et al., ; gauthier-clerc et al., ; ramos et al., ) . however, no observations of hard ticks on wild individuals from pelicaniformes, gaviiformes, or anseriformes have been reported to date. the taxonomic status of most seabird hard ticks is well-established, though work has shown that these ticks may form isolated host specific populations, even when different seabird species breed in sympatry (e.g. mccoy et al., ) ; the potential for host-associated speciation may be high in this group and overall diversity as measured using traditional morphological criteria may underestimate true diversity (moon et al., ) . the known species of soft ticks exploiting seabirds all belong to the genera argas and ornithodoros (carios), and tend to be restricted to tropical and sub-tropical zones (fig. ) . soft ticks have been found parasitizing charadriiformes, phaethontiformes, sphenisciformes, procellariiformes, suliformes, and pelicaniformes (dietrich et al., ; duffy, ; feare, ; fig. ); we found no study that reported the presence of soft ticks on wild individuals of gaviiformes or anseriformes. however, due to the short time that these ticks require for blood-feeding (i.e. minutes) and their nocturnal activity, they are difficult to observe under natural conditions and their diversity, prevalence and local abundance in seabird colonies is surely underestimated. in addition, general questions related to their systematics remain open guglielmone et al., ) , and much basic observational work is still required to clarify their diversity, specificity and impact on seabird population dynamics. many studies on tick-seabird systems have focused on the prevalence of human pathogens such as soldado virus and b. burgdorferi within ticks, with few discussing the circulation of these agents among ticks and seabirds (e.g. gylfe et al., ; main et al., ; but see . as most research has targeted known pathogenic agents, the presence and diversity of other microparasites transmitted via ticks (or other potential vectors) remain to be investigated. the effect of known tick-borne pathogens on seabird populations is also poorly understood (dietrich et al., ) . we discuss these pathogens in more detail in later sections. some work has evaluated the direct impact of ticks on seabird health, reproductive success, and population dynamics in relation to hyperinfestations (e.g. duffy, ; monticelli et al., ; ramos et al., ramos et al., , . results vary greatly among species and orders, and are often related to other factors, such as food access and supply. for example, ramos et al. ( ) and monticelli et al. ( ) reported that in years of poor food supply, populations of roseate terns (sterna dougallii) experienced massive chick mortality and breeding failure in areas of high tick (amblyomma loculosum) densities. however, similar studies conducted on white-tailed tropicbirds (phaethon lepturus) in the same region found no significant effect of tick parasitism/interference competition on productivity (ramos et al., ) . there is evidence to suggest that species size may influence the effect that ticks have on individuals and populations. in a study conducted in the huaura islands, peru nest desertion due to ticks was more common for guanay cormorant (phalacrocorax bougainvilli) and peruvian booby (sula variegata) populations than for the larger peruvian brown pelican (pelecanus occidentalis ambius; duffy, ) . however, post-mortem investigations of three dead king penguins (aptenodytes patagonicus) found on the crozet archipelago in the sub-antarctic region led researchers to conclude that hyperinfestation by ticks during incubation (when birds do not feed) was the most likely cause of death (gauthier-clerc et al., ) . although no difference in body mass or behaviour of breeding king penguins was found with respect to more commonly observed levels of tick infestation , some impact on reproductive success has been suggested . no such observations have been made for smaller penguin species. a relatively well-studied aspect of tick-seabird systems is the cyclical dynamics of seabird populations in relation to tick abundance. as the number of ticks in a given colony increases, the likelihood of nest desertion and chick mortality increases duffy, ; monticelli et al., ; ramos et al., ; ramos and drummond, ) . conversely, breeding failures at the colony level also may also have a detrimental impact on local tick populations (danchin, ; dietrich et al., ; monticelli et al., ; ols en et al., ; ramos et al., ) . this type of dynamic interaction between seabirds and ticks will also have consequences for the local transmission and large-scale circulation of infectious agents . although these are also small arachnids unlike ticks, only a small fraction of mite species parasitize animals. species exploiting seabirds as hosts belong to the orders mesostigmata, trombidiformes, and sarcoptiformes. among the mesostigmata, it is species of the superfamily dermanyssoidea (e.g. dermanyssus gallinae, ornithonyssus bursa) that are among the most studied. species in this group have been reported to agitate adult birds, causing interruption in egg incubation, reducing nestling growth rates, and reducing overall reproductive success (clayton and tompkins, ; moller, ) . it is the cohort astigmatina (order sacroptiformes) however that houses the greatest diversity of bird-associated mites, including feather mites (psoroptidia). while there is no doubt on the parasitic status of blood-feeding mites, the status of feather mites as parasites is still debated. these mites are thought to mainly consume feather pith or skin, and in some cases, feather oils (stefan et al., ) . in domestic birds, mites that feed on feather pith can weaken feathers and cause premature breakage and skin lesions (proctor, ) . hyperinfestation can also result in severe itching, causing birds to pull feathers (proctor, ) . however, studies by blanco and frias ( ) and blanco et al. ( suggest that feather mites may help boost body condition by removing excess body oils and fungi. mite abundance on a bird host can be affected by season, temperature, light, humidity, and host body condition (proctor, ) . mites are typically transmitted between hosts by close body contact, which can be frequent in colonial nesting seabirds (proctor, ) . feather mites have been found on species belonging to the order procellariformes, gaviiformes, pelecaniformes, anseriformes, and charadriiformes (proctor, ; stefan et al., ) . a study by stefan et al. ( ) determined that a single bird can play host to multiple species of mites (microspalax brevipes and zachvatkinia ovate) due to resource partitioning and spatial segregation among the species. indeed, a more recent study found up to eight different mite species exploiting cape verde shearwaters (calonectris edwardsii) and bulwer's petrels (bulweria bulwerii; stefan et al., ) . however, the effects of mites on wild seabirds are largely unknown. studies on miteseabird interactions have focused on the phylogeny and co-evolution of mites and their hosts, rather than the impact of mite infestation itself (choe and kim, ; coulson et al., a coulson et al., , b stefan et al., ) . fleas are small (adults mm), flightless, insects of the order siphonaptera that parasitize mammal and bird populations worldwide. of the identified species, only those belonging to the genera ceratophyllus, parapsyllus, mioctenopsylla, and xenopsylla have been reported on seabirds (coulson et al., a (coulson et al., , b kane et al., ) . flea parasitism probably occurs in most seabird species and has been reported in wild seabirds belonging to the orders sphenisciformes, charadriiformes, anseriformes, procellariformes, and suliformes (alonso and garrido, ; choe and kim, ; kristjánsson et al., ) . the egg, larval, and pupal stages of most fleas live off the host's body, but adults are obligate blood-feeders. because a large part of the life cycle occurs in the habitat used by the host, the presence and abundance of fleas is closely related to the conditions of their breeding environment (gómez-díaz and gonzález-solís, ). fleas can act as disease vectors for several bacteria, viruses and helminths (bitam et al., ; kane et al., ) . although poorly studied in relation to seabirds, they are known mechanical vectors for avipoxviruses (discussed in a later section) (kane et al., ) . many studies on fleas in seabird colonies focus on examining the diversity and trophic relationships within arthropod communities, rather than the effects of flea parasitism itself (alonso and garrido, ; choe and kim, ; coulson et al., a coulson et al., , b . gómez-díaz and gonzález-solís ( ) analysed host-parasite relationships by reporting on nitrogen and carbon enrichment in fleas from host species; however, the effects of parasitism on birds themselves were not discussed. a direct effect of fleas on seabirds was reported in a recent study by kristjánsson et al. ( ) , examining the presence of the flea ceratophyllus garei in common eider (somateria mollissima) colonies. the authors noted a positive relationship between the age of nest bowls, and the number of fleas found within them. hyper-parasitism by fleas led to irritation in adults, which resulted in agitated egg incubation. blood cover on eggs was also positively correlated to flea infestation (kristjánsson et al., ) suggesting a potential cost in terms of blood loss. these results fall in line with observations in passerine birds (e.g. fitze et al., ) . lice are obligate parasites belonging to the insect order phthiraptera. lice are further subdivided into two main groups: sucking lice (suborder anoplura) and chewing lice (suborders ischnocera, amblycera and rhynchophthirina). anoplura are obligate blood-sucking parasites found almost exclusively on mammalian hosts. in contrast, species of chewing lice are commonly found on bird species, feeding on hair, feathers and epidermal skin/scales (clay and moreby, ) . unlike ticks and fleas, lice spend their entire life cycle on a single host. females lay their eggs, commonly known as nits, by gluing them to the hairs or feathers of their hosts through a secretion created in their accessory glands. lice are typically host-specific, rather than geographically-specific, which makes estimating their global distribution challenging (clay and moreby, ) . factors influencing the presence and abundance of lice on seabirds are not extensively studied. rivera-parra et al. ( ) sampled five seabird species across seven islands in the galapagos, and found a connection between host density and louse infestation levels. however, the study also pointed out the need for further research on the impact of local weather conditions and host density on lice loads; factors which have been found to affect louse loads in other bird groups (clayton et al., ) . louse-seabird interactions are generally studied by collecting lice present on individual birds or in bird nests (alonso and garrido, ; coulson et al., a coulson et al., , b gómez-díaz and gonzález-solís, ; rivera-parra et al., ) . the most common methods of louse collection include dust ruffling with an insecticide and agitating the feathers until parasites fall off rivera-parra et al., ) , or collecting invertebrates from active nests (alonso and garrido, ; coulson et al., a coulson et al., , b . although lice are prevalent in six of the eight main seabird orders that we consider here (procellariformes, charadriiformes, pelicaniformes, anseriformes, suliformes, sphenisciformes), most research on louse-seabird interactions has been focused on the co-evolution or phylogeny of lice species in relation to their hosts (page et al., ; paterson et al., paterson et al., , . although rarely studied in seabirds, evidence suggests that lice can reduce host fitness in numerous ways (thermoregulatory stress due to feather condition, grooming costs related to higher metabolic rates, predator vigilance, etc.; clayton et al., ) . we found one study that measured the impacts of louse loads on wild seabirds; daunt et al. ( ) measured loads of eidemanniella pellucida on breeding european shags (phalacrocorax aristotelis) and their chicks, and found no measurable effect of louse load on offspring growth. parasites of seabirds: a literature survey hippoboscid flies (order diptera), commonly known as louse flies, are the most common family of parasitic flies found on birds and mammals. the hippoboscidae are divided into three main subfamilies, only two of which (ornithomyinae and hippoboscinae) include obligate parasites of birds (duvallet and baldacchino, ) . as hippoboscid flies are highly mobile (some species are winged) and can spend part of their lifecycle off host, host to host transmission can be very high (bequaert, ) . factors affecting the parasitism by hippoboscidae in seabirds are not well understood, and many species may have co-evolved with their specific hosts (baker, ; levin and parker, ) . however, humidity and oily plumage can be related to the prevalence of louse flies on seabirds (bequaert, ) . the annual colonial nesting behaviour of seabirds may also be a key aspect supporting the maintenance and spread of hippoboscid flies levin and parker, ) . hippoboscid flies have been recorded on seabirds belonging to the orders suliformes, procellariformes, pelecaniformes, and charadriiformes bequaert, ; whiteman et al., ) . olfersia aenescens most commonly parasitizes charadriiformes, procellariformes, and pelecaniformes. however, information on hippoboscid flies in other avian orders is less clear. for example, no reports were found of hippoboscids on any members of anseriformes in this review; this is thought to be due to their oily plumage and damp nesting habitat preferences (bequaert, ) . within avian orders, the diversity of hippoboscids can vary greatly from family to family. among pelicaniformes, all members of the family fregatidae share the species olfersia spinifera, whereas o. aenescens, o. fossulata and o. sordida parasitize phalacrocoricidae, sulicidae and pelicanidae (bequaert, ) . the impact of hippoboscidae on seabirds is also sparsely understood. bequaert ( ) suggested that since many louse fly species are host-specific, they are more dependent on host survival than other, more generalist, ectoparasites. however, their relatively high mobility may limit constraints on virulence. recent studies on the impact of hippoboscidae on seabirds have focused on the transmission of microparasites such as haemosporidia, rather than the direct impact of parasitism itself levin and parker, ) . the impact of these microparasites on seabirds is discussed in a later section. there are approximately named species of mosquitoes (diptera: culicidae), of which seven known genera (aedes, anopheles, culex, mansonia, haemagogus, psorophora, and sabethes) transmit disease to animals, including humans (fang, ; gubler, ) . culicidae are the most prolific disease vectors within the order diptera, and can transmit a range of parasites (e.g. plasmodium relictum), viruses (alphaviruses, flaviviruses, etc.) and helminths within bird populations worldwide (more details outlined below). of the four stages of their life cycles, mosquitoes spend the egg, larval and pupal stages in aquatic habitats, and only parasitize hosts as adults. only the females bite and can transmit infectious agents (gubler, ) . mosquitoes are known to parasitize species belonging to charadriiformes, suliformes, procellariiformes and pelecaniformes. in studies relating to wild seabirds, only aedes taeniorhynchus has been identified as a parasite (anderson, ; anderson and fortner, ) . most studies conducted on mosquito-bird systems have focused on birds acting as reservoirs for, or direct transmitters of, viruses and diseases that affect humans (parkinson and butler, ; reed et al., ; tsiodras et al., ) . like other macroparasites, the direct impact of mosquitoes on their hosts will depend on their relative abundance. although factors affecting mosquito abundance are largely unknown, heat, humidity, and the availability of stagnant water for breeding are key determining factors (wegbreit and reisen, ) . heat has been identified as a particularly important factor affecting mosquito parasitism of seabirds (gaston and elliott, ; gaston et al., ) . the colonial and often synchronised nature of seabird breeding supports the spread of mosquitoes among hosts within populations; birds born later in the breeding season can be prone to higher degrees of mosquito exposure due to a reduction in nestling hosts (caillouët et al., ) . in seabird populations, mosquito parasitism has been positively correlated with egg neglect in waved albatrosses (diomedia irrorata) in the galapagos islands (anderson and fortner, ) . in hudson bay, adult mortality and egg loss in a population of thick-billed murres (uria lomvia) were also positively correlated with mosquito parasitism (gaston et al., ; gaston and elliott, ) . helminths are a group of worms that are often parasitic in birds and include flatworms-which further separate into cestodes (tapeworms) and trematodes (flukes)-nematodes (roundworms), and acanthocephala (thornyheaded worms; galaktionov, ; galaktionov and bustnes, ) . the life cycle and transmission pathways of helminths are often complex, with both direct transmission (i.e. from parent to chick during feeding), and indirect transmission via intermediate hosts or vectors. helminths are typically studied in seabirds through carcass collection (e.g. mallory et al., ) , although some studies have used regurgitation, endoscopic techniques, and anti-helminth treatments in seabirds, to assess parasitism in the gastrointestinal tract and their effects (fonteneau and cook, ; granroth-wilding et al., ; provencher et al., ) . information on the impact of helminths on some species are more available because they are hunted or often beach cast, but overall the need for carcasses poses a challenge to explore helminth-seabird relationships in wild populations, as lethal sampling is most often used. in situations where carcasses are available, a range of helminths have been found in host species (e.g. mallory et al., ; tourangeau et al., ) . cestodes and trematodes (phylum platyhelminthes) are characterised by their lack of a body cavity, hermaphroditism, and the use of suckers (borthridia) to attach themselves within their host (galaktionov, ) . flatworms can be most easily distinguished by the segmentation in their bodies; trematodes have unsegmented, smooth bodies, whereas cestodes have segmented bodies (galaktionov, ) . the lifecycle of trematodes and cestodes often involves two, sometimes three, intermediate hosts (e.g. copepod infected by feeding on cestode eggs, which in turn are ingested by cephalopods or fish) before reaching maturity within a seabird (galaktionov, ) . however, only trematodes require the obligatory participation of molluscs to complete their lifecycle (galaktionov, ) . the prevalence and impacts of cestodes and trematodes vary by species, region, and sex. in double-crested cormorants (phalacrocorax auritus) in southern canada, robinson et al. ( ) found that males had significantly higher levels of the trematode drepanocaphalus spathans when compared with females. mallory et al. ( ) found a higher frequency of occurrence of cestodes in northern fulmars (fulmarus glacialis) during the post-breeding season as compared with the pre-breeding season. the diversity of cestodes and trematodes has been shown to vary across a host species range. for example, birds inhabiting the southern extent of the common eider (somateria mollissima) range in the north sea have as many as trematode species, whereas at the northern extent of the range in franz josef land, only a single trematode species has been reported (galaktionov, (galaktionov, , . in terms of the impact of these worms on seabird health and condition, as for most macroparasites, it is expected that negative impacts may only occur at very high parasite loads (see galaktionov, ) , but detailed studies in seabirds are limited and difficult to attribute to cestodes and trematodes in particular. nematodes or roundworms (phylum nematoda) are characterised by their round body shape and the presence of a body cavity. nematodes also have distinct male and female organisms (galaktionov, ) , similar to cestodes and trematodes, however, the prevalence of nematodes in seabirds is highly variable. even individuals nesting at nearby colonies can exhibit strong differences in parasitism levels. fonteneau and cook ( ) found that kerguelen shags (phalacrocorax verrucosus) nesting approximately km from each other had significantly different prevalence and infection loads of the nematode contracaecum rudolphii. the authors attributed this result to differences in diet between the two colonies, highlighting how local diet can be a significant factor for parasitism in seabirds. sex bias was also identified by robinson et al. ( ) in double-crested cormorants in canada, where males had significantly higher levels of the nematode contracaecum spp. when compared with females. experimental removals of gastrointestinal nematode parasites was found to alter resource allocation in european shags in relation to local breeding conditions (granroth-wilding et al., , reed et al., ) , highlighting the importance of the interaction between parasites and local environmental factors in determining parasite impacts (granroth-wilding et al., ) . much like cestodes and trematodes, acanthocephalans are parasitic worms that lack a mouth, and instead attach to their hosts using a proboscis that is covered with spiny hooks (galaktionov, ; hoberg, ) . acanthocephalans also have distinct male and female sexes, and their life cycle requires intermediate hosts, most commonly thought to be benthic crustaceans (galaktionov, ) . although acanthocephalans have been identified in many antarctic and arctic seabird fauna (charadriiformes, sphenisciformes, anseriformes; brandão et al., ; hoberg, ) , they are best studied as one of the most prolific parasites of common eiders in the northern hemisphere (galaktionov, ; provencher et al., provencher et al., , fig. ) . a sex bias in the prevalence of acanthocephalans in seabird species has been identified, and may be attributable to differing life history strategies between males and females, especially during the breeding season, caring of young and feeding in distinct areas during the moulting season (skirnisson, ) . similar to the other parasitic worms, the distribution and number of species in acanthocephalans vary by region, with the number of species of acanthocephalans being higher in the sub-arctic regions than in arctic environments (galaktionov, ) . again, there is very little work done on how this particular group may negatively affect seabirds given the co-occurrence with other species, and the difficulty of examining this group in isolation in relation to potential impacts on species. while a single host can be affected by the number of helminths it carries, there is no clear relationship demonstrated in the literature on how helminths may interact with respect to each other within a host. for example, robinson et al. ( ) found no correlation between the most abundant helminths (nematodes and cestodes) in double-crested cormorants. in contrast, provencher et al. ( ) found that the abundance of cestodes and acanthocephalans were negatively correlated with each other in common eiders, and suggested a possible negative interaction between helminth species within a host, at least in some species. northern fulmars from the high canadian arctic harboured several helminth species including cestodes, trematodes, nematodes, and acanthocephalans in varying levels of infection (mallory et al., ) . tourangeau et al. ( ) similarly found that common eiders in northern canada to have several cestodes and acanthocephalans with varying levels of infection in relation to age and sex. due to the difficulty in obtaining reliable estimates of parasite diversity and loads, the impact of these species on seabird health and population dynamics have received relatively little attention. in some cases, only the presence or absence of helminths is reported without any additional information. for example, in provencher et al. ( ) , helminths are reported in arctic terns (sterna paradisaea), but specific parasite identification and enumeration were not possible during the dissection of the birds. globally, the lack of trained parasite taxonomists may also be hampering the identification and reporting of seabird parasites (hopkins and freckleton, ) . furthermore, some of the most comprehensive parasite publications are only available in russian where there is still a strong tradition of parasitologists interested in systematics and natural history (e.g. kuklin, ) . although there are cases where viruses are transmitted by direct contact with an infected individual, most viruses in wild seabird populations are vectorborne (see table ). arboviruses, or arthropod-borne viruses, comprise close to different viruses that have been identified in wild seabirds (beckham and tyler, ; mackenzie and williams, ; nuttall, ) . this section focuses on some of the most common types of arboviruses found in seabirds. avipoxviruses, a subgroup of poxviruses, are most notable for causing avian pox in birds around the globe (bolte et al., ) . ectoparasites such as mosquitoes are the most common mechanical vectors for this group of viruses in wild seabirds, although a flea species (parapsyllus longicornis) has been suggested as a possible vector as well (kane et al., ) . avian pox is a slow-developing disease, the effects of which vary by species. the most common form of avian pox affects the skin, where the development of warts around featherless regions of birds (face, legs, and feet) is a common occurrence (tripathy, ) . these lesions often disappear with time but can et al. ( ) leave behind scarred and damaged skin. however, in cases where these warts become enlarged and clustered, loss of sight, breathing impairment, and difficulty feeding are common side effects. it is in the case of secondary infections due to a dampened immune system, and the development of scar tissue, where avian pox can contribute to bird mortality (friend and franson, ; kane et al., ) . infections are also highest during wet seasons and virus transmission is mostly host-density dependent (van riper et al., ) . this is a key factor to note in the spread of avipoxviruses among seabird populations, many of which form dense breeding colonies. avian pox has been reported in sphenisciformes, anseriformes, charadriiformes, and procellariiformes. a study of magellanic penguins (spheniscus magellanicus) from two colonies in argentina revealed chicks with avian pox lesions; however, the prevalence of lesions was . infected chicks to every handled chick. of the afflicted chicks, were found dead, but whether the cause of death was due to avian pox or secondary infections is unknown (kane et al., ) . avian pox is also the most common viral infection in albatrosses and large petrels (uhart et al., ) . it has been reported from wandering albatross (eudyptes chrysocome) in the prince edward islands in the indian ocean, to laysan albatross (phoebastria immutabilis) on oahu, hawaii (young and vanderwerf, ) , suggesting that albatross populations may be exposed to these viruses worldwide. flaviviruses (family: flaviviridae) have been mostly studied for their impacts on human health, in particular, dengue fever, yellow fever, japanese encephalitis, zika virus, and west nile virus (gaunt et al., ; jaeger et al., ) . ticks and mosquitoes are the most common vectors for flaviviruses (gaunt et al., ; george et al., ) . although west nile virus has been well studied in some bird populations, it is important to note that the most common flaviviruses isolated from seabirds do not include west nile virus. most research on west nile virus in birds has focused on relatively common terrestrial species such as american crows (corvus brachyrhynchos), common grackles (quiscalus quiscula), blue jays (cyanocitta cristata), and house sparrows (passer domesticus; rappole and hubalek, ; ladeau et al., ) . in wild seabird populations, west nile virus has been identified in post-mortem analyses of american white pelicans (pelicanus erythrorhynchos), and is suggested to have caused mortality in adults and nestlings (rocke et al., ) . antibodies known to neutralize west nile virus were also detected in blood sera from adult great frigatebirds (fregata minor) in the western indian ocean, with no apparent sign of disease . in wild seabirds, flaviviruses such as meaban virus, usutu virus, saumarez reef virus, tyuleniy virus, and gadget's gully virus have been identified in species belonging to charadriiformes and sphenisciformes (arnal et al., ; gaunt et al., ; george et al., ; jaeger et al., ; mackenzie and williams, ) . only the pathogenicity of saumarez reef virus has been tested in seabirds through small-scale experimental infections of little blue penguins (eudyptula minor). wild-captured individuals inoculated with the virus succumbed to disease and mortality within - days of the infection (morgan et al., ) . although research on factors affecting the spread of flaviviruses among wild seabirds is lacking, one can presume that warm weather, damp/wet habitats, and host density favour the transmission of these vector-borne viruses (van riper et al., ) . there are at least other types of arboviruses identified from seabird ticks alone, with their pathogenicity and spread in wild seabird populations largely unknown (nuttall, ) . lab tests of the avalon nairovirus isolated from black-legged kittiwakes (rissa tridactyla) on suckling mice exhibited little to no effect (quillien et al., ; spence et al., ) . sakhalin virus, named after the location it was first discovered (sakhalin island, sea of okhotsk), was pathogenic in lab-tested suckling mice after intracerebral inoculation, but the effects of the virus on the host common murres (uria aalge) are unidentified (lvov et al., ) . other arboviruses isolated directly from wild seabirds include nugget virus, tagart virus, and an unnamed species identified in black-legged kittiwakes in brittany (chastel et al., ; doherty et al., ) . the effects of any of these viruses on their host populations are also unknown. the first isolation of influenza a virus (orthomyxoviridae) in wild birds was reported in , following an epizootic among common terns (sterna hirundo) in south africa (becker, ; rowan, ) . since that date, influenza a viruses have been detected in more than seabird species worldwide, mostly in gulls and terns (see arnal et al., ; lang et al., for review in gulls and other seabirds, respectively). birds of the anseriformes and charadriiformes orders are the main hosts for influenza a viruses . significant variation in virus prevalence have been found between closely related species, but also season and bird colony, suggesting that the transmission dynamics of avian influenza are strongly associated to the ecology of their hosts. annual epidemics have been found in black-headed gulls (chroicocephalus ridibundus), with peaks of transmission during the breeding season and, in particular, fledgling birds (verhagen et al., ) . this supports the idea that bird age is an important factor involved in influenza virus infection. investigations of virus transmission on tropical oceanic islands have also shown that terns could be involved in the introduction and maintenance of avian influenza viruses in these remote locations (lebarbenchon et al., ) . among the described avian influenza virus hemagglutinin subtypes, h and h are associated to seabirds . although low pathogenic virus subtypes do not induce clinical signs of disease (brown et al., ; verhagen et al., ) , infection with highly pathogenic avian influenza viruses (h and h subtypes) can induce severe clinical signs. these subtypes can cause cloudy eyes, ruffled feathers, weakness, a lack of coordination, torticollis, and mortality (brown et al., ) . to date, there is only limited evidence that these poultry-adapted viruses can be maintained in wild birds for long periods of time (krauss et al., ; lebarbenchon et al., ) . the increasing number of spillover events nevertheless highlights the need to further investigate the role of seabirds in the spread of both low and highly pathogenic avian influenza viruses during epizootics. although it is likely that other directly transmitted viruses affect seabird populations, particularly in dense breeding colonies where contact rate is very high, current knowledge on these pathogens remains very limited. in addition to avian influenza, other viruses have been documented in seabirds in the past, such as the one causing puffinosis in manx shearwaters (puffinus puffinus) in europe (harris, ) , or coronaviruses in gulls in the bering strait area (muradrasoli et al., ) . the epidemiology, ecology and evolution of these viruses would require further investigation in order to precisely assess the physiological and behavioural effects in their natural hosts. . . avian cholera avian cholera is a highly contagious disease resulting from infections of the pasteurella multicoda bacterium. dna fingerprinting has resulted in the identification of at least different serotypes of p. multicoda (friend and franson, ) . avian cholera has become the most prolific infectious disease of waterbirds, even though it first appeared in north america as recently as . since then, the disease has expanded globally, and outbreaks have increased in frequency since the s (friend and franson, ) . one of the key reasons avian cholera has been so successful in its spread is due to the multitude of ways p. multicoda can make its way into a host's system. bird-to-bird contact, ingestion of carcasses or contaminated food/water, insect bites, and even the aerosolisation of the bacteria in heavily contaminated regions can all lead to avian cholera in wild birds (friend and franson, ) . infections of p. multicoda can be acute or chronic, but in most species studied to date, infections result in death within - h. mortality in - h has also been reported in wild birds, with peak mortality exceeding more than birds a day in some cases (friend and franson, ) . avian cholera is identified in wild seabirds almost exclusively through necropsies, and has been reported in species belonging to anseriformes, procellariiformes, phaethontiformes, sphenisciformes, suliformes, pelicaniformes and charadriiformes (crawford et al., ; friend and franson, ; leotta et al., ) . environmental contamination from diseased birds is a primary source of infection. due to the dense nesting colonies formed by wild seabirds, and the highly contagious nature of the disease, avian cholera can lead to rapid colony death and even extinction. the only known way to control its spread in these populations is by removing afflicted and dead birds from the region (descamps et al., ; friend and franson, ) . avian cholera has been responsible for annual colony collapses in common eiders in nunavut (fig. ) , cape cormorants (phalacrocorax capensis) in south africa, common murres in the baltic sea, yellow-nosed and amsterdam albatrosses (diomedea chlororhynchos and d. amsterdamensis) on amsterdam island, and macaroni penguins (eudyptes chrysolophus) in the antarctic crawford et al., ; descamps et al., ; € osterblom et al., ; waller and underhill, ; weimerskirch, ) . long term the effects of these annual outbreaks is still being studied, but at least in some cases colonies persist but at reduced numbers. lyme disease, caused by the bacteria of the complex borrelia burgdorferi sensu lato is the most widespread tick-borne disease in north america and europe. the transmission of the infectious agent is best understood in terrestrial systems involving humans, wild birds and small mammals, and ixodes ricinus and i. scapularis ticks (anderson et al., ; kurtenbach et al., ) . lyme disease symptoms in humans and dogs range from complex mix of neurological disorders, cardiac-associated complications, joint pain, and skin rash in humans, to lameness, vomiting, diarrhea, lack of appetite, weight loss, and inflammation in dogs (klempner et al., ; littman et al., ) . despite having now been isolated from dozens of wild terrestrial birds, the effects of b. burgdorferi in these populations is not well understood. most research on bird-tick systems studying the disease focus on the role of wild birds as reservoirs, with the potential to spread it across large spatial scales (clow et al., ; smith et al., ) . in seabirds b. burgdorferi has been isolated from i. uriae ticks in the northern and southern hemispheres exploiting species belonging to charadriiformes, procellariformes, and sphenisciformes (duneau et al., ; gylfe et al., ; olsen et al., ) . more generally, evidence of the widespread presence of this infectious agent in seabirds is accumulating (gasparini et al., ; gómez-díaz et al., ; staszewski et al., ) . however, for now, no evidence suggests a pathogenic effect of infection for the birds; in particular, a long-term capture-mark-recapture analysis of breeding black-legged kittiwakes found no association between exposure to b. burgdorferi s.l. and survival (chambert et al., ) . bacteria belonging to the genus clostridium are responsible for an immense amount of wild bird deaths. clostridium botulinum is the most prevalent species of bacteria from this genus found in wild birds, causing mortality through infecting hosts with botulinum toxins. although there are seven types of botulinum toxins created by this bacterium-labelled a to g-it is the type c botulinum toxin that has caused the highest mortality in wild birds (friend and franson, ) . before avian cholera outbreaks increased in frequency in the past quarter century, avian botulism was considered the most destructive disease in wild birds (friend and franson, ) . botulism outbreaks can regularly result in the death of thousands of waterbirds and shorebirds in a region and up to a million birds have been lost in a single outbreak of the disease (friend and franson, ) . blood samples from live birds are needed to identify the toxin in populations, making the study of avian botulism in wild birds a resource dependant endeavour (friend and franson, ) . factors affecting the spread of botulism in wild birds include colony density, water-level changes, water quality, and the prevalence of c. botulinum toxins in food or water (friend and franson, ) . temperature also plays a critical role in the spread of botulism, with most outbreaks taking place during summer and fall (friend and franson, ) . in wild seabirds, botulism has been isolated from species belonging to charadriiformes, pelecaniformes, and anseriformes (friend and franson, ; neimanis et al., ; rocke et al., ) . the symptoms of the disease in wild seabirds (e.g. herring gulls; larus argentatus) include decreased physical coordination, paralysis of legs and wings, dehydration, and a decrease in food consumption. avian botulism has caused mass mortality in herring gulls in sweden, and american white pelicans in north america (neimanis et al., ; rocke et al., ) . chlamydiosis is an infection associated with bacteria belonging to the genus chlamydia, which typically live within animal cells. chlamydia psittaci is the most common species associated with the disease in wild birds. chlamydiosis can range in pathogenicity in wild birds from inapparent infections to an parasites of seabirds: a literature survey acute disease with high mortality, depending on the strain of c. psittaci affecting the individual/population (friend and franson, ) . symptoms in wild birds range from decreased food consumption, pus discharges from the eyes and nares, and bloody diarrhea, to becoming rigid and motionless (friend and franson, ) . chlamydia species can remain active in the tissues, faeces, discharges, and even plumage of affected birds, and can be transmitted between individuals by direct contact. the only known way to control the spread of the disease in wild birds is by the removal of dead or affected birds from colonies (friend and franson, ) . although waterfowl, pigeons, herons and parrots are the most commonly infected wild birds, chlamydiosis has been reported in over species belonging to orders. the most common wild seabirds infected by the disease include gulls and fulmars (charadriiformes and procellariformes; franson and pearson, ; herrmann et al., ) . studies have also isolated c. psittaci from red-footed (sula sula) and nazca boobies (sula granti; padilla et al., ) . however, since chlamydiosis in wild birds is often inapparent, the effect of the disease on these populations has not been discussed in any of the studies we found. in outbreaks recorded in wild gulls (species unidentified), chlamydiosis has been fatal for adults, though fledglings reportedly died at a much higher rate (friend and franson, ) . haemosporidia, or blood parasites, are vector-borne parasites that have been well-studied (e.g. padilla et al., ; valkiunas, ; vanstreels et al., ) . they are transmitted to wild birds primarily by midges (culicoides spp.), mosquitoes (culex and aedes spp.) and flies (simulium and hippoboscidae spp.; friend and franson, ; vanstreels et al., ) . there are currently four primary types of haemosporidia identified in seabirds, each carried by a different type of vector. plasmodium species are carried by mosquitoes, and are associated with diseases such as malaria in humans (friend and franson, ; vanstreels et al., ) . haemoproteus species are carried by midges, and leucocytozoon species are carried by flies (friend and franson, ) . it is unconfirmed what vectors carry babesia spp., but it is assumed to be ticks (earl e et al., ; yabsley et al., ) . although many groups of birds are known hosts of various haemosporidian parasites, often with high prevalence in populations (e.g. passerines, waterfowl), in detailed surveys of north america birds, greiner et al. ( ) considered seabirds to be "nearly hematozoan-free". haemosporidia have been found in wild seabirds belonging to the orders charadriiformes, suliformes, sphenisciformes, and procellariformes, and in some cases certain parasites (e.g. plasmodium relictum) have been associated with high mortality (e.g. wild penguins; friend and franson, ) . however, in the majority of studies, there is little evidence of mortality of wild seabirds caused by haemosporidia (padilla et al., ; parsons et al., ; peirce and parsons, ; yabsley et al., ) . temperature, migration, and local climatic conditions supporting ectoparasites are key considerations in determining the spread of haemosporidia among wild seabirds (friend and franson, ; van riper et al., ) , and help explain regional prevalence levels (e.g. bennett et al., ; greiner et al., ) . for example, vanstreels et al. ( ) found no evidence of haemosporidia in magellanic penguins in the sub-antarctic region of argentina, and mallory et al. ( ) also found no haemosporidia in norhern fulmars from the high arctic. it is also thought that many haemosporidia are host species specific. for example, babesia shortii is the only babesia spp. known to cause pathogenicity in birds (peirce, ) . however, it is best studied in falconidae, and experimental transmissions of the parasite into other terrestrial species have failed in producing pathogenic responses (peirce, ) . while our review focused on eight orders of seabirds (procellariformes, phaethontiformes, suliformes, charadriiformes, sphenisciformes, pelicaniformes, and select gaviiformes, and anseriformes), we found that species belonging to the order charadriiformes were the most represented in the parasite and pathogen literature (table ). although anseriformes have been studied more extensively in terms of parasites, our focus on parasites in seaducks resulted in far fewer study results. the research done in the eight orders is generally representative of the number of species within each order; charadriiformes and procellariformes have the most numerous species and resulted in more parasite studies found in the literature, while phaethontiformes and gaviiformes have the fewest species, and we found the fewest parasite studies ( table ) . the focus on parasites and pathogens in colonial nesting seabirds, such as those belonging to charadriiformes and procellariformes, may be because they are easier to study due to their high population densities during the breeding season. this colonial nature lends these species to parasite related studies as larger sample sizes can be accessed relatively easily. on the other hand, species belonging to gaviiformes are found in lower densities on the landscape, making population-wide analyses more resource and time intensive. important to note is that the colonial nature of some species of birds may also make them more vulnerable to higher levels of parasites and pathogens as compared with birds that breed in isolation because of the contact between individuals at bird colonies (e.g. boulinier et al., boulinier et al., , mccoy et al., ) . for example, in some cliff-nesting seabird species where there is little to no vegetation cover individuals may literally be covered in the faeces of those nesting above them (fig. ) ; these faeces are a potential vector for a number of parasites. the impact of ectoparasites on seabirds are studied through two main metrics; the impact on seabird populations through direct parasitism, and the potential for seabird ectoparasites to act as disease vectors. the impacts of direct parasitism on wild seabirds can dependent on the age, sex, and body condition of the host (e.g. granroth-wilding et al., ; provencher et al., ) . although adult mortality can be caused by ectoparasites (e.g. gaston and elliott, ) , this occurs most often in cases of hyper-infestations. the most commonly reported impact of ectoparasites on adult seabirds is that of reduced parental care due to parasitism (e.g. dietrich et al., ; duffy, ) . nest abandonment and inconsistent/slower offspring development are the most well-known outcomes. mortality in fledglings due to parasitism is much more likely, and has been reported in species belonging to six of the eight orders studied here ( table ) . the effect of endoparasites on seabirds is much more diverse (table ) . flaviviruses, chlamydophillia spp., haemosporidia and borrelia burgdorferi seem to be asymptomatic in seabirds, which makes understanding their relationships with their hosts difficult. in contrast, highly pathogenic avian influenza and avian cholera can cause mass mortality and colony collapses (e.g. allison et al., ; descamps et al., ) . except for these exceptional cases when such massive die-offs are reported, the physiological and behavioural effects of viral and bacterial infections in seabirds remain largely unknown, at both the individual and population levels. this limitation is mostly associated with the inherent properties of viruses and other microparasites. in particular, field investigation of infectious status and prevalence in populations usually requires strict conservation procedures of biological material. laboratory-based techniques (e.g. molecular biology, virus isolation) in sometimes high security laboratories such as for highly pathogenic avian influenza and west nile viruses is needed, and certain biological safety precautions are required by law in some regions (e.g. allison et al., ; lebarbenchon et al., lebarbenchon et al., , . effects of certain parasites and pathogens may also be subtle and difficult to measure with respect to the impact of other environmental factors, and choosing the appropriate traits to measure requires clear information on the biological system (sánchez et al., ) . for example, the effects of a parasite may only be detectable during times of physiological stress (e.g. breeding or migration), but not during other parts of the annual cycle (provencher et al., ) . identifying the links between such basic epidemiological information and host physiological and behavioural traits, in order to assess the precise effects of infection on host fitness, therefore remains very challenging. experimental approaches in the field can also be somewhat limited; ethics and biosafety considerations naturally preclude the manipulation of the infectious status of natural migratory hosts with parasites (although this may be easier for helminths than viruses and bacteria). understanding the interactions between different parasite types affecting seabirds is also lacking, but has been shown to be essential in other animal groups (e.g. graham, ) . additional observational and experimental studies from a larger range of seabirds aimed at specific parasitic groups would greatly enhance our understanding of these interactions. new methods for investigating the diversity of endoparasites using metabarcoding analyses of faeces may facilitate our understanding of the diversity and distribution of these organisms at different spatial scales (e.g. mcinnes et al., ) , and may enable us to incorporate parasite dynamics into long-term population survey data. although there are limitations to metabarcoding approaches to studying effects of parasites as metrics beyond presence and absence will be difficult to quantify. control measures for the spread of parasites are also rudimentary and resource/time intensive. one way to control their spread in seabirds is by the selective removal of afflicted or dead birds from colonies (e.g. friend and franson, ) . vaccination has also been recently proposed as an effective measure to protect endemic albatrosses from avian cholera , and may be an important conservation consideration for seabirds of particular conservation concern, but is not likely a viable solution for more metropolitan species. due to the biological connection between many endoand ectoparasites, it is important to note that the factors influencing their spread are intrinsically connected. research shows that abiotic conditions such as heat and humidity, and biotic conditions such as host body condition and colony density, are key factors determining in the spread of parasites within seabird colonies (caillouët et al., ; monticelli et al., ; proctor, ; ramos et al., ) . discussing the spread of these parasites on a global scale is more complex, however. factors such as migration, climate change, and range extension/change for seabirds and parasites alike need to be taken into consideration. seabirds are often long-lived and highly migratory, so it should come as no surprise that they can act as effective parasite reservoirs and transporters. there is already evidence showcasing range and species expansions for parasites, although the exact factors involved are as yet unknown (de souza petersen et al., ; kane et al., ) . changing seasonal temperatures and water levels due to climate change can also extend the time period that seabirds can be parasitised by ectoparasites, potentially resulting in increased local abundances and subsequently higher chances of pathogenicity. increasing anthropogenic pollution and higher water levels due to climate change can further contribute to the spread of devastating diseases such as botulism and cholera (friend and franson, ; garamszegi, ; parkinson and butler, ) . the interaction between different environmental stressors and parasitism is only recently starting to be considered for understanding the overall impact on seabird populations. there is clearly a bias towards research interest in seabird parasites that have the capacity to parasitize or infect human beings (i.e. zoonoses). evidence of this comes from the comparatively extensive research on west nile virus and lyme disease, which can be transmitted to humans through vectors such as ticks and mosquitoes. although direct transmission of pathogens from birds to humans is considered uncommon, little work has been done to assess seabird pathogens in humans and thus this relationship is likely underestimated. vectors carried by migratory birds can play a role in the transmission of at least different human pathogens (tsiodras et al., ) , but a much better understanding of this phenomenon is still required. recent work by fossádal et al. ( ) has shown a positive association between handling c. psittaci infected juvenile northern fulmars-a species hunted for human consumption-and human psittacosis disease in the fulmar islands. it is possible that other commonly harvested species, such as eiders and murres may present similar correlations in future works. the aforementioned control measures for diseases such as cholera and influenza also expose human beings to these parasites. with the increased risk of outbreaks in seabirds due to climate change and range expansions, pathogen spillover events will likely become more frequent. beyond their zoonotic implications, the impact of parasites on seabirds and human beings have economic and cultural implications. commonly harvested birds such as common eiders and murres have experienced pathogenicity, mortality, and even annual colony collapse due to increased parasitism (gaston et al., ; gaston and elliott, ; kristjánsson et al., ; lovvorn et al., ) . anthropogenic activities such as overfishing and oceanic pollution are also evolving factors contributing to reduced health in seabirds (braune et al., ; sydeman et al., ) . the long-term impacts of these changes can contribute to increased, additive mortality caused by parasites in seabird colonies. changing climatic conditions and food availability have also resulted in seabirds being preyed upon by novel predators (iverson et al., ; smith et al., ) . more research on cumulative effects from parasitism, reduced food availability due to anthropogenic activity, and changing predatory interactions is needed to understand the collective impact these factors can have on parasite-host relationships in seabird colonies (dey et al., ; iverson et al., ; ramos et al., ) . understanding these interrelations becomes even more crucial when we consider endemic or naïve species of seabirds that may be experiencing increases in multiple stressors due to changing conditions in their habitats. for example, increasing numbers of non-native species in hawaii have introduced parasites that have led to mass mortality in native songbirds parasites of seabirds: a literature survey (van riper et al., ) . expanding ranges for parasites and disease vectors have raised similar concerns in antarctic seabird populations (grimaldi et al., ) . it is clear that the colonial nature of seabirds makes them effective reservoirs and hosts for parasites. not all seabirds come in direct contact with each other in these colonies, however, because of strong territoriality. more research is needed to understand the impact of localised separation in colonial nesting seabirds on the spread of parasites. colonial nesting might indeed yield some benefits to hosts against certain parasites. for example, douglas et al. ( ) showed evidence of aldehydes created by crested auklets (aethia cristatella) can effectively repel ectoparasites. there are also studies to suggest that certain species of wild birds can slow the spread of pathogens like west nile virus (levine et al., ) . the role of seabird diversity and abundance in suppressing zoonotic disease is a worthwhile avenue for further study. concurrently, most ecosystems are warming under climate change (walther et al., ) , and evidence suggests that increased parasitism is linked with warmer weather (bellard et al., ; kutz et al., ). therefore, there is a need to understand current level of parasites in seabirds to understand changing patterns and trends in the future. in particular, we stress that studies examining parasitism in bird populations should publish results, regardless of the outcome (e.g. no infection found in seabirds). these baseline studies are essential for documenting the effect of shifting environmental conditions on bird-parasite relationships and teasing apart the questions 'is this new?' or 'new to us?'; a critical question to understanding how environmental changes will impact biota. cyclic avian mass mortality in the northeastern united states is associated with a novel orthomyxovirus arthropods in shag (phalacrocorax aristotelis) nests from the national park of the atlantic islands (nw spain): occurrence and abundance at different breeding phases differential responses of boobies and other seabirds in the galapagos to the - el nino-southern oscillation event waved albatross egg neglect and associated mosquito ectoparasitism involvement of birds in the epidemiology of the lyme disease agent borrelia burgdorferi circulation of a meaban-like virus in yellow-legged gulls and seabird ticks in the western mediterranean basin a review of the role played by the hippoboscidae (diptera) as vectors of endoparasites haemoproteus iwa in great frigatebirds (fregata minor) in the islands of the western indian ocean the isolation and classification of tern virus: influenza virus a/tern/ south africa/ arbovirus infections impacts of climate change on the future of biodiversity scarcity of haematozoa in birds breeding on the arctic tundra of north america the hippoboscidae or louse-flies (diptera) of mammals and birds. part i. structure, physiology and natural history fleas and flea-borne diseases symbiotic feather mites synchronize dispersal and population growth with host sociality and migratory disposition feather mites on group-living red-billed choughs: a non-parasitic interaction? 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central europe are microbes the source of a seabird's tangerine-like scent? mounting evidence for the presence of influenza a virus in the avifauna of the antarctic region investigating the effects of age-related spatial structuring on the transmission of a tick-borne virus in a colonially breeding host flanders strain, an arbovirus newly isolated from mosquitoes and birds of new york state massive infection of seabird ticks with bacterial species related to coxiella burnetii diseases of antarctic seabirds surveillance and molecular characterization of newcastle disease virus in seafowl from coastal areas of china in we thank the many researchers and teams that have and continue to explore and study parasties and seabirds globally. thanks grant gilchrist and thierry boulinier for allowing us to use their photographs. thanks also to anonymous reviewers for their constructive feedback. key: cord- -m ahicqb authors: romano, alessandra; casazza, marco; gonella, francesco title: energy dynamics for systemic configurations of virus-host co-evolution date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: m ahicqb virus cause multiple outbreaks, for which comprehensive tailored therapeutic strategies are still missing. virus and host cell dynamics are strictly connected, and convey in virion assembly to ensure virus spread in the body. study of the systemic behavior of virus-host interaction at the single-cell level is a scientific challenge, considering the difficulties of using experimental approaches and the limited knowledge of the behavior of emerging novel virus as a collectivity. this work focuses on positive-sense, single-stranded rna viruses, like human coronaviruses, in their virus-individual host interaction, studying the changes induced in the host cell bioenergetics. a systems-thinking representation, based on stock-flow diagramming of virus-host interaction at the cellular level, is used here for the first time to simulate the system energy dynamics. we found that reducing the energy flow which fuels virion assembly is the most affordable strategy to limit the virus spread, but its efficacy is mitigated by the contemporary inhibition of other flows relevant for the system. summary positive-single-strand ribonucleic acid ((+)ssrna) viruses can cause multiple outbreaks, for which comprehensive tailored therapeutic strategies are still missing. virus and host cell dynamics are strictly connected, generating a complex dynamics that conveys in virion assembly to ensure virus spread in the body. this work focuses on (+)ssrna viruses in their virus-individual host interaction, studying the changes induced in the host cell bioenergetics. a systems-thinking representation, based on stock-flow diagramming of virus-host interaction at the cellular level, is used here for the first time to simulate the energy dynamics of the system. by means of a computational simulator based on the systemic diagramming, we identifid host protein recycling and folded-protein synthesis as possible new leverage points. these also address different strategies depending on time setting of the therapeutic procedures. reducing the energy flow which fuels virion assembly is addressed as the most affordable strategy to limit the virus spread, but its efficacy is mitigated by the contemporary inhibition of other flows relevant for the system. counterintuitively, targeting rna replication or virion budding does not give rise to relevant systemic effects, and can possibly contribute to further virus spread. the tested combinations of multiple systemic targets are less efficient in minimizing the stock of virions than targeting only the virion assembly process, due to the systemic configuration and its evolution overtime. viral load and early addressing (in the first two days from infection) of leverage points are the most effective strategies on stock dynamics to minimize virion assembly and preserve host-cell bioenergetics. as a whole, our work points out the need for a systemic approach to design effective therapeutic strategies that should take in account the dynamic evolution of the system. virus cause multiple outbreaks, for which comprehensive tailored therapeutic strategies are still missing. virus and host cell dynamics are strictly connected, and convey in virion assembly to ensure virus spread in the body. study of the systemic behavior of virus-host interaction at the single-cell level is a scientific challenge, considering the difficulties of using experimental approaches and the limited knowledge of the behavior of emerging novel virus as a collectivity. this work focuses on positive-sense, single-stranded rna viruses, like human coronaviruses, in their virus-individual host interaction, studying the changes induced in the host cell bioenergetics. a systems-thinking representation, based on stock-flow diagramming of virus-host interaction at the cellular level, is used here for the first time to simulate the system energy dynamics. we found that reducing the energy flow which fuels virion assembly is the most affordable strategy to limit the virus spread, but its efficacy is mitigated by the contemporary inhibition of other flows relevant for the system. positive-single-strand ribonucleic acid ((+)ssrna) viruses can cause multiple outbreaks, for which comprehensive tailored therapeutic strategies are still missing. virus and host cell dynamics are strictly connected, generating a complex dynamics that conveys in virion assembly to ensure virus spread in the body. this work focuses on (+)ssrna viruses in their virus-individual host interaction, studying the changes induced in the host cell bioenergetics. a systems-thinking representation, based on stockflow diagramming of virus-host interaction at the cellular level, is used here for the first time to simulate the energy dynamics of the system. by means of a computational simulator based on the systemic diagramming, we identifid host protein recycling and folded-protein synthesis as possible new leverage points. these also address different strategies depending on time setting of the therapeutic procedures. reducing the energy flow which fuels virion assembly is addressed as the most affordable strategy to limit the virus spread, but its efficacy is mitigated by the contemporary inhibition of other flows relevant for the system. counterintuitively, targeting rna replication or virion budding does not give rise to relevant systemic effects, and can possibly contribute to further virus spread. the tested combinations of multiple systemic targets are less efficient in minimizing the stock of virions than targeting only the virion assembly process, due to the systemic configuration and its evolution overtime. viral load and early addressing (in the first two days from infection) of leverage points are the most effective strategies on stock dynamics to minimize virion assembly and preserve host-cell bioenergetics. as a whole, our work points out the need for a systemic approach to design effective therapeutic strategies that should take in account the dynamic evolution of the system. interaction between a (+)ssrna virus and the host cell and therefore addressing effective intervention strategies. starting from the knowledge of relevant processes in (+ss)rna virus replication, transcription, translation, virions budding and shedding and their energy costs (reported in supplementary methods table ) , we built up a systems-thinking (st) based energy diagram of the virus-host interaction. figure shows the stock-flow diagram for the system at issue, where each stock was quantified in terms of embedded energy of the corresponding variable. symbols are borrowed from the energy language , : shields indicate the stocks, big solid arrows the processes and line arrows the flows, whereas dashed lines indicate the controls exerted by the stocks on the processes. all stocks, flows and processes are expressed in terms of the energy embedded, transmitted and used during the infection. we used atp-equivalents (atp-eq) as energy unit referred to cellular costs, by using the number of atp (or gtp and other atp-equivalents) hydrolysis events as a proxy for energetic cost , . the dynamic determination of flows was based on the knowledge of characteristic time-scales of well-established biological processes (for more details see supplementary methods). the output flows j and j were set to be effective only if the value of the respective stocks q and q is higher than a threshold, as represented by the two switch symbols in the diagram. the stock q represents the embedded energy of resources addressed to protein synthesis in the host cell. the dynamics of allocation for protein synthesis depends on the cell bioenergetics, e.g., the number of mitochondria, ox-phos activity levels, and the cell cycle phase [ ] [ ] [ ] (for more details, see supplementary methods). in the absence of virus, energy flows from q (flows j , j a and j b) to produce short-half-life proteins (stock q a) and long-half-life proteins (stock q b), whose synthesis, degradation and secretion follow dynamic stationary conditions. in particular, typical of the specialized cells such as those of the pulmonary epithelium, there is a flow of proteins destined for degradation and recycling through the recruitment of autophagic receptors or organelles or proteasomes (identified in the diagram by the flows j a and j b, supplementary methods table ). the outflow of folded, fully functional proteins addressed to secretion or surface exposure is first, we investigated the system dynamics under different initial conditions, exploring the possible role of different initial viral loads (figure ). in the configuration of initial null viral load (q stock value= ) the value of stocks q , q a and q b were constant and the system behavior was stationary (figure a) . assuming a different viral loads (time zero) in the - , virions range, we found that there is a threshold in the initial viral load for triggering the progressive reduction of q , whose amount could in turn trigger the cell death in different ways. apoptosis is a cellular process requiring energy, and a deflection in q , as shown in figure when a (+)ssrna virus enters the single host cell, the q stock is fed, and its proteins can interact with the host proteome to sustain rna replication. based on previous works in the field - , we identified a time delay of to hours required to record changes in the q stock. moreover, the value of q varies over the time due to changes occurred at a different timepoints in the stocks q b, q and q . to be effective, a therapeutic strategy should limit the outflows of virions, j and/or j . however, minimization of j seems to be counter-effective in our simulation, due to the increase of q as consequence of the feedback action in the virus replication (vr) process. the minimization of j prevents the outflow (viral shedding) from q without stopping its growth. at the same time, this leads to increased resources diverting from q and q b that could promote the cell death, with consequent spread of the virions in the environment. arising from targeting j was mitigated by the combination with reduction of j or j (figure ) . the partial reduction of j , alone or in combination, did not change significantly the dynamic growth of q , induced increase of q at day , though it could prevent a reduction of q , to preserve the cell bioenergetics and preventing the cell death by atp lack. we also simulated the effect of applying the same external inputs at different times: after (extended data figure ), (extended data figure ) or days (extended data figure ) from the initial infection, alone or in combination (figure ) . application of external forcing factors at day , day or day could affect in a non-linear way the system response, with the largest efficacy targeting j (figures - ) . the combination of external driving forces acting on j and j did not result to be significantly synergic, while targeting j and j increased the q stock value instead of the expected reduction. it is worth stressing that this kind of behavior is a typical systemic feature, where an intervention on a specific local process may lead to counterintuitive rearrangements in the system dynamics as a whole. late suppression of j and j at day could reduce only the stock values of q . only early full reduction of j (by day ) could significantly limit the growth of q , while the combination of contemporary suppression of j and j could not prevent q growth (figure ) . thus, application of external driving forces at different timepoints is expected to model additional resilience configurations. in this work, we approached the host-virus interaction dynamics as a systemic problem and, for the first time in the field, we used combined systems thinking tools as a conceptual framework to build up a systemic description of the viral action and host response, critically depending on the existing metabolic environment. the complex dynamic behavior was described in terms of underlying accessible patterns, hierarchical feedback loops, self-organization, and sensitive dependence on external parameters, that were analytically computed by a simulator. the following novelties were addressed: i) the use of energy language as a common quantitative unit for different biological cells and used mass spectrometry to measure protein-protein interactions . with this experimental approach, they identified interactions between viral and host proteins, and noted existing drugs, known to target host proteins or associated pathways, which interact with sars-cov- , addressing the importance to target the host-virus interaction at the level of rna translation. there are known advantages of in silico modelling the action of therapeutic agents on known diseases through agent-based modelling . however, the literature evidenced some intrinsic limitations on the choice of parameters, like the size of investigated populations , while major problems are related to model validation , , also requiring to supplement the models with adequate formal ontologies . thanks to its abstract nature, stock-flow description can be used in a wide range of different fields, realizing the conceptual bridge that connects the language of biological systems to that of ecology. our approach unveils the potential of systems thinking for the study of other diseases or classes of disease, since it appears more and more clear how some incurable pathologies can be described only by adopting a more comprehensive systemic approach, in which the network of relationships between biological elements are treated in quantitative way similar to that applied in this paper. the method used to study the dynamics of the virus-host interaction system is structured in basic steps, namely, the development of a flow-stock diagram, that describes the virus-host interactions, the development of a virus-host systemic simulator, and, finally, its calibration and validation (extended data, figure ) . a typical systems thinking diagram is formed of stocks, flows and processes. stocks are countable extensive variables qi, i= , ,...,n, relevant to the study at issue, that constitute an n-ple of numbers that at any time represents a state of the system. a stock may change its value only upon its inflows and/or its outflows, represented by arrows entering or exiting the stock. processes are any occurrence capable to alter -either quantitatively or qualitatively -a flow, by the action of one or more of the system elements. in a stationary state of the system, stocks values are either constant or regularly oscillating. in the dynamics of a system, stocks act as shock absorbers, buffers, and time-delayers. processes are all what happens inside a system that allows the stationarity of its state, or that may perturb the state itself. to occur, a process must be activated by another driver, acting on the flow where the process is located. these interaction flows may be regarded as flows of information, that control the occurring processes and so the value and nature of the matter flows. the pattern of the feedbacks acting in the system configurations is the feature that utlimately defines the systems dynamics. we adopted an energyy approach, where stocks, flows and processes are expressed in terms of the energy embedded, transmitted and used, respectively, during the system operation. the equations, that characterize the flows relevant to the diagram, are typical of dynamic st analysis , , and their setting up is linked in many respects to the energy network language . in this approach, each flow depends on the state variables qi by relationships of the kind dqi/dt=kf(qj), i,j= ,...,n, where n is the number of stocks in the system. given a set of proper initial conditions for the stocks (i.e., the initial system state) and a properly chosen set of phenomenological coefficients k, the set of interconnected equations will be treated by standard finite-different method, taking care of choosing a time-step short enough to evidence the possible dynamics of any of the studied processes. the coefficients ki are calculated using data on the dynamics of any single stock, in particular, by estimating the flows and the stocks during the time interval set for the simulation steps (as described in supplementary methods). when different flows co-participate in a process, a single coefficient will gather all the actions that concur to the intensity of the outcoming flow(s). these phenomenological coefficients are not, therefore, related to specific biophysical phenomena or processes, but are set to describe how and how fast any part of the system react to a change in any of the other ones. in general, our model may be then regarded as based on a population-level model (plm) as defined by , which can be run at different 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(books on demand gmbh explanations of ecological relationships with energy systems concepts combining molecular observations and microbial ecosystem modeling: a practical guide formal aspects of model validity and validation in system dynamics the philosophy and epistemology of simulation: a review competing interests. authors declare no competing financial interests. the author to whom correspondence and material requests should be addressed is:dr. marco casazza key: cord- -io jmp o authors: roche, benjamin; guégan, jean-françois title: ecosystem dynamics, biological diversity and emerging infectious diseases date: - - journal: c r biol doi: . /j.crvi. . . sha: doc_id: cord_uid: io jmp o in this article, we summarize the major scientific developments of the last decade on the transmission of infectious agents in multi-host systems. almost sixty percent of the pathogens that have emerged in humans during the last – years are of animal origin and about sixty percent of them show an important variety of host species besides humans ( or more possible host species). in this review, we focus on zoonotic infections with vector-borne transmission and dissect the contrasting effects that a multiplicity of host reservoirs and vectors can have on their disease dynamics. we discuss the effects exerted by host and vector species richness and composition on pathogen prevalence (i.e., reduction, including the dilution effect, or amplification). we emphasize that, in multiple host systems and for vector-borne zoonotic pathogens, host reservoir species and vector species can exert contrasting effect locally. the outcome on disease dynamics (reduced pathogen prevalence in vectors when the host reservoir species is rich and increased pathogen prevalence when the vector species richness increases) may be highly heterogeneous in both space and time. we then ask briefly how a shift towards a more systemic perspective in the study of emerging infectious diseases, which are driven by a multiplicity of hosts, may stimulate further research developments. finally, we propose some research avenues that take better into account the multi-host species reality in the transmission of the most important emerging infectious diseases, and, particularly, suggest, as a possible orientation, the careful assessment of the life-history characteristics of hosts and vectors in a community ecology-based perspective. the pathogens responsible for the infectious diseases that affect human populations are seldom strictly confined to our species. among the about currently known human infectious diseases, c.a. to % have an established animal origin [ , ] . ruminants, carnivores, rodents, birds and primates constitute, in the order, the five major animal categories that have transmitted pathogens to human populations [ ] . this percentage increases to - % if only the ''emerging'' or ''reemerging'' diseases, which have appeared in the last to years, are considered [ , ] . the work by woolhouse et al. [ ] has also shown that these pathogens have an important range of host reservoir species as about % of them have or more host species (in addition to humans), thus highlighting the notion that pathogen dynamics driven by a multiplicity of hosts is a more realistic picture for emerging infectious diseases [ ] . without doubt, the origin of the most important pathogens and parasites that affect human populations is zoonotic (in this work, we will use the term ''parasite'' to in this article, we summarize the major scientific developments of the last decade on the transmission of infectious agents in multi-host systems. almost sixty percent of the pathogens that have emerged in humans during the last - years are of animal origin and about sixty percent of them show an important variety of host species besides humans ( or more possible host species). in this review, we focus on zoonotic infections with vector-borne transmission and dissect the contrasting effects that a multiplicity of host reservoirs and vectors can have on their disease dynamics. we discuss the effects exerted by host and vector species richness and composition on pathogen prevalence (i.e., reduction, including the dilution effect, or amplification). we emphasize that, in multiple host systems and for vector-borne zoonotic pathogens, host reservoir species and vector species can exert contrasting effect locally. the outcome on disease dynamics (reduced pathogen prevalence in vectors when the host reservoir species is rich and increased pathogen prevalence when the vector species richness increases) may be highly heterogeneous in both space and time. we then ask briefly how a shift towards a more systemic perspective in the study of emerging infectious diseases, which are driven by a multiplicity of hosts, may stimulate further research developments. finally, we propose some research avenues that take better into account the multi-host species reality in the transmission of the most important emerging infectious diseases, and, particularly, suggest, as a possible orientation, the careful assessment of the life-history characteristics of hosts and vectors in a community ecology-based perspective. ß acadé mie des sciences. published by elsevier masson sas. all rights reserved. refer to agents of infectious diseases). therefore, it is easy to recognize the importance of a better understanding of animal species as potential hosts and sources of novel contaminations in humans [ ] . the recent appearance and propagation of the highly pathogenic h n avian influenza (bird flu), west nile fever, lyme disease, rabies as well as of severe acute respiratory syndrome (sars), all of animal origin, have truly highlighted the role of animal species as major reservoirs for (new) human infections. understanding how they are transmitted in animal species communities and identifying the underlying ecological and evolutionary mechanisms is essential to lessen the transmission risk to humans [ ] . anthropogenic changes in landscapes, particularly habitat destruction and fragmentation [ ] and their subsequent effects on biological diversity have led to disease emergence in a number of situations [ ] . here, we discuss new insights gained from studies that explicitly acknowledge the contribution of multi-host species systems to the dynamics of emerging pathogens. throughout this review, we also argue on the necessity to better cross-fertilize the two separate research fields of epidemiology and ecology of infectious diseases if we want to anticipate future epidemics. finally, we recommend an increased attention to the role of the many epizootic host species and the weakly competent vectors in disease transmission not only among themselves, but also in driving the infection towards human communities. many definitions can be used to describe what an ''ecological community'' is. this profusion of definitions does not simplify communication within this research field. in this article, we have chosen to use the relatively neutral, but also vague, definition by putman [ ] . an ecological community is thus defined as ''a group of species that interact in a given geographical area and whose functions and dynamics are somehow interdependent''. classically, a community structure is described using a species abundance/richness curve that associates a number of species for each abundance ''rank'' (e.g., species involving between and individuals each). these relations can take different forms according to the animal communities under study. for instance, fisher et al. [ ] showed statistically that this relation follows a geometrical law ( fig. ) for butterfly communities. a few years later, preston showed that this relation takes more frequently the shape of a log-normal distribution in mammal communities [ , ] (fig. ) . these two explanations have been unified only recently following hubbell's demonstration that both relations correspond to two specific situations of another more general distribution called zero-sum multinomial [ ] . these species abundance/richness relationships are the results of all the interactions that occur within local communities (e.g., preys/predators, competition. . .) and as such they may constitute a good integrator of all these relations. since it is not sufficient to account for the observed community structure, trophic networks should not be dismissed as they might be another important contributing mechanism to the dynamics of ecological communities [ ] . however, within these networks, the importance of parasitic organisms (e.g., viruses, bacteria, protozoans, helminth worms.) has been frequently ignored due to sampling issues. parasites are surprisingly important in food webs and their role in food web stability is increasingly recognized [ ] [ ] [ ] . first, by inducing a differential mortality among the host species, parasites can positively or negatively influence the abundance and the spatial distribution of the host species they use as resources. second, and this aspect has been less studied, parasites can affect food webs and interacting host species especially by altering trophic cascades. the first ''visible'' effect exerted by parasites is the more or less important regulation that virulent infections can have on the size of host populations in wildlife [ ] . the literature in this domain is teeming with scientific evidences that describe the parasitic pressures on natural host populations. the case of the accidental introduction of bovine rinderpest in the wild ungulate communities of the serengeti national park in is here emblematic [ ] . the disease decimated different animal species. this, in turn, caused the decrease in the predation pressure on the vegetation, leading, at the same time, to a modification of the vegetation cover of the savannah that made it less favourable to fire development. although this example describes well the primordial role exercised by parasites in ecosystems, it is however necessary to distinguish the effects of generalist (nonspecific) and host-specialized parasites on host species communities. in the case of a generalist parasite that can thus infect a significant range of host species, the consequences are generally destabilizing for the host communities. indeed, if a parasite species can simultaneously attack many host species, the ones that are more tolerant to the parasite (i.e., the host species that can support the reproduction of the parasite while sustaining little damage) will engender an ''apparent'' competition with the less tolerant host species that will pay for the cost of the parasite virulence. in scotland, the parasitic nematode heterakis gallinarum infects both pheasants and partridges [ ] . this pathogen multiplies very well in pheasants without decreasing the size of their population that, as a result, shows a great tolerance towards this parasite. for partridges, the situation is the opposite because a large proportion of partridges infected by this nematode will rapidly die. the degree of abundance of pheasants in the community has thus the consequence of increasing locally the nematode incidence and this will directly affect the abundance of partridges, which are more susceptible to the parasite (fig. ) . although we cannot propose other examples as representative as this one, in nature such situations must be frequently encountered. on the other hand, the effects of host-specialized parasites on host species communities are the opposite of the ones previously described, because generally an important diversity in host species is maintained locally. indeed, by limiting the abundance of the species they infect, these host-specialized parasites also decrease the local competition pressure among host species and allow the rarest species to freely coexist in the environment [ ] . to sum up, parasites can influence the structure of species communities and the consequences of this on the ecosystems can be contrasting due to the leverage action they exert on the coexistence of host species within local communities. moreover, within natural ecosystems, parasites are undeniably key elements that contribute to the shaping of the observed variety of local species communities. in return, this diversity in species richness and composition can also influence the transmission mode of pathogens. asking about the role played by a community of hosts on the dissemination of a parasite boils down to consider the influence that a local change in the host species composition can have on its transmission. one or more host species can either disappear or appear locally, and this in turn can have important repercussions on the dynamics of parasite transmission. the consequences of greater host species richness may strongly depend on the transmission mode of the pathogen. for pathogens with ''density-dependent transmission'', in general any local addition of host species is likely to enhance disease transmission due to the increase in contact rates among individual hosts within the community [ ] . this situation is often observed in the case of directly-transmitted infectious pathogens, like rodent-borne hantaviruses or avian influenza viruses [ , ] . a second situation is that of pathogens with ''frequency-dependent transmission'' in which contacts among host individuals occur at a constant frequency and do not depend on the host population density. this type of transmission is assumed for sexually transmitted pathogens, like hiv, in which the number of sexual contacts does not increase with the size of the population. another example of frequency-dependent transmission concerns vector-borne diseases, e.g. malaria, in which the transmission is from host to host via a mosquito vector. this type of transmission is also encountered with pathogens transmitted by ticks, such as lyme disease for instance. here, fig. . example of indirect competition induced by a parasite within a local community of hosts. the introduction of individuals of a host species (here symbolized by a pheasant), which is tolerant to the parasite (here a heterakis nematode worm), leads to an increase of the parasite prevalence within the community. if this tolerant host species interacts with another host species, which is less or nontolerant (here partridges), the increase in the disease prevalence will then cause extra mortality in the non-or less tolerant host species, independently from the type of interactions between tolerant and nontolerant host species. the frequency of blood meals by the arthropod vectors generally does not increase with the abundance of host individuals [ ] . for the sake of simplicity, we consider here only the case of vector-borne transmission. during a blood meal from an infected reservoir host, the vector can be contaminated and transmit the parasite to healthy host individuals. in this context, the individuals challenged by the pathogen can be from a single host species (for a hostspecialized parasite) or can belong to different host species (for a generalist, nonspecialized parasite). in the case of a nonspecialized, multi-species system, the local conditions of species richness and composition in reservoir hosts will have important consequences on the disease transmission, as it has been shown by richard ostfeld et al. [ , ] . these authors have analysed the circulation of the bacterium borrelia burgdorferi, responsible of lyme disease in north america. this biological model, which involves numerous reservoir host species and only one vector species (the tick species ixodes scapularis), represents an interesting first step for the study of pathogen transmission in multispecies systems. each of these potential reservoir species has naturally a different competence to multiply the pathogen and to transmit it again during a blood meal by noninfected ticks. here, we will define as susceptibility the probability that a healthy host individual might become infected following a contact with an infected vector [ ] . hence, in animal species communities, each reservoir and vector species has a different susceptibility that is characteristic of that species. in reality, within a host (reservoir or vector) species, important differences in susceptibility can be observed, but it is difficult to integrate this intraspecies variability due to the simplicity of the epidemiological models that are currently used. by focusing on the interspecific variability of susceptibilities, ostfeld et al. have carried out a series of experimental and field studies that have demonstrated the importance of the local composition in reservoir host species on the transmission of the bacterium responsible of lyme disease [ ] . these authors observed a decrease of pathogen prevalence in ixodes ticks and a lower human prevalence with greater reservoir host species richness [ ] . further studies on the west nile virus, which is transmitted by different mosquito species and many bird species in the usa, have also highlighted a tendency to lower pathogen circulation in the presence of greater host bird richness [ ] . the results of the few empirical studies, which have been carried out, converge and indicate that local communities with low richness in reservoir host species tend to present higher levels of disease prevalence (fig. ) , whereas local communities that involve a larger number of reservoir hosts species see this level decreasing (fig. ) . ostfeld et al. [ ] [ ] [ ] ] have called this phenomenon the ''dilution effect'' in reference to the process of deviation to which is subjected the pathogen in communities which are rich in reservoir species and a significant proportion of which are less or not susceptible to infection. to the best of our knowledge, only two theoretical studies tried to formally explain the dilution effect. first, using an epidemiological model of the susceptible-infected-resistant (sir) type that relies on allometric laws to quantify the different parameters, dobson [ ] analysed the epidemiological consequences of host reservoir species inclusion (up to six, and progressively less susceptible) in a virtual host community. among other outputs, dobson [ ] studied the basic reproduction number (r ) that quantifies the mean number of new infections caused by a single infected host individual. he underlined that, in the case of frequency-dependent transmission, the value of r decreases when the richness in host species increases, which represents a direct application of the previously described ''dilution effect'' phenomenon. this first theoretical formalism had the merit of introducing a theoretical [ ( ) t d $ f i g ] fig. . schematic representation of the dilution effect proposed by richard ostfeld and his collaborators. for the sake of simplicity, it is considered that one vector individual can bite only twice during its lifespan. in the left panel, an infected vector transmits the pathogen to two individuals from a susceptible reservoir species. these two highly susceptible reservoir individuals will infect in turn two new vector individuals. on the right, the same initial conditions, but with the introduction of an individual from a nonsusceptible reservoir species. if the vector is generalist, there is a nonnegligible probability that the infected vector will bite this nonsusceptible reservoir individual. hence, only one reservoir individual will be infected and transmit the disease to one vector individual. overall this results in a decline of the pathogen prevalence in the vector populations. framework to explain the dilution effect observed empirically. nevertheless the low reservoir species richness involved ( species) and the fact that dobson's work considered only directly transmitted infections did not allow the generalization of these results to vector-borne diseases, especially when multiple vector species are involved. in a more recent theoretical work, begon [ ] has discussed the effects of the addition of a second, less competent host species on transmission pathways, pathogen persistence and abundance. in the case of louping ill virus, which is transmitted in red grouse by the vector tick species ixodes ricinus, begon [ ] concluded that in the presence of a second, less competent host species, a transmission event linked infectious individuals with incompetent susceptible host individuals that generated many fewer new infections than a competent host would have. the second host species therefore served only to dilute the transmission process, but in general, it is difficult to separate a dilution effect due to greater host species richness or composition. to continue the works by dobson [ ] and begon [ ] , a recent study by roche et al. [ ] has proposed a more general theoretical formalism by borrowing empirical observations derived from community ecology to mimic the relationships observed in nature between local species richness and relative abundance in species (see section , i.e., species richness-abundance curves). this study concentrated on the intensity of transmission of a pathogen in a local community of host species by studying the observed maximum prevalence of locally infected cases that represents a close estimate of the r [ ] . by taking into account that, in the case of vector-borne zoonotic diseases, two categories of animal communities interact, on one side, the reservoir species and, on the other side, the insect species, this study allowed highlighting the following findings [ ] . for two local communities with the same reservoir species richness and abundance but with different species compositions, an increase in the variation of the reservoir species susceptibility corresponded to a greater number of reservoir species with lower level of susceptibility. an immediate consequence is that an important number of vectors, all other proportions kept identical, will feed on reservoir individuals which are less able to transmit the pathogen. thus, the interspecific variation in susceptibility of the host reservoirs is an important criterion that on its own can explain the decrease in prevalence of a pathogen in vector populations. in practice, these pathogen dynamics are usually the result of a balance mainly between efficient transmission by competent hosts and abortive transmission by poorly competent hosts that can likely lead to a dilution effect (fig. ) . indeed, it is not, strictly speaking, the local richness in reservoir species that acts on the level of prevalence of the pathogen in the vectors, but the proportion of reservoir species with low susceptibility, which tends to augment in species-rich communities, that decreases the intensity of vector-borne transmission. so far, we have focused on the community of host reservoir species and its role in the transmission of pathogens in multi-host systems. nevertheless, the vector species community also plays an important part in disease transmission. the dilution effect has been most thoroughly studied in vector-borne diseases, especially lyme disease, which, in the usa, is transmitted by a single tick species, i. scapularis [ , , ] . this, therefore, might not be representative of the many indirectly transmitted zoonotic pathogens for which several vector species may be more or less competent in disease transmission. for zoonotic vector-borne infections, there are two types of contacts: (i) between infectious vectors and more or less susceptible reservoir hosts and (ii) between infectious host reservoirs and more or less susceptible vectors. indeed, the dilution effect requires that vector contacts must be wasted on poorly competent reservoirs, but also that vector species richness, by increasing their abundance, cannot compensate for these wasted contacts to overcome the dilution effect. in the case of vector-borne pathogens, the analysis of the species richness-abundance curves of the local vector communities in the different models allows the assessment of the effect of the number of vector species in a given community on the pathogen persistence and prevalence. this raises two important questions concerning: (i) the effects on local disease transmission of the accidental introduction or biological invasion by exotic vectors, even when they show a low competence to transmit the infection; and (ii) the role of low to very low in species-rich reservoir communities, generally a decrease in the prevalence of disease pathogens in the vectors is observed. this is illustrated by the case of borrelia burgdorferi that is transmitted by the ixodes scapularis ticks in many vertebrate hosts in the usa. the diversity of reservoir hosts dilutes disease transmission (dilution effect) and the pathogen dynamics drops. on the other hand, in species-rich communities of vectors, a larger proportion of vectors, even if poorly competent, can acquire the pathogen from infectious hosts and then amplify the transmission of the disease to susceptible hosts (amplification effect). in natural conditions, these two antagonistic effects can lead to very contrasting epidemiological situations depending on the characteristics of the local ecosystems and their biological diversity, as observed for the emerging disease caused by west nile virus (see text for further explanations). competent resident vector species on the general circulation of pathogens. moreover, it highlights what is perhaps a key issue in the understanding of vector-borne emerging pathogens. although medium to very high competent vector species are usually credited with the maintenance and transmission of a disease, the role of the many poorly to very poorly competent vectors should not be underestimated as recent evidences have shown for bovine catarrhal fever (also known as blue tongue disease) in northern europe [ ] , chikungunya in italy [ ] and reunion island [ ] , or malaria in corsica [ ] . it is interesting to note from the multi-host dynamic model by roche et al. [ ] that, while an increase in the number of reservoir host species generally dilutes the transmission of a pathogen, a local increase in vector species richness amplifies transmission (fig. ) . to understand why, it is necessary to consider the number of new additional bites that occur when local vector species richness increases, even though some vector species might be poorly to very poorly competent for the pathogen locally. these opposite effects of dilution and amplification of the pathogen circulation must be taken into consideration when analysing vector-borne disease transmission. indeed, depending on the local species richness and composition in host reservoirs and vectors, we may observe different disease trends ( fig. ; and see also begon [ ] ). this model clearly illustrates the mechanics and the potential role played by the biological diversity of host reservoirs and vectors in vector-borne infections. a possible example of this is represented by the transmission of west nile virus. west nile virus is a flavivirus commonly found in africa, west asia, middle east, eastern europe, northern and central america and west indies that causes encephalitis (inflammation of brain and spinal chord) in humans and horses. west nile disease is transmitted by different mosquito vectors, which bite and infect birds. many mosquito species have been tested positive for this pathogen [ ] , but in experimental studies the most susceptible one seems to be the culex species complex [ ] . infections have been identified mostly in wild birds (and even domestic ones), which are the primary reservoirs for the virus. although most wild bird species are not affected and rather act as more or less amplifying reservoirs, members of the corvid family, including crows, blue jays, magpies and ravens, are very susceptible to the effects of west nile virus. the virus can also infect many different dead-end hosts in terms of transmission, including humans and other mammals. in the case of west nile virus, the ''take home'' message from the multi-host/multi-vector models is that the local richness and composition in host reservoirs and vectors may lead to different combinatorial effects on disease transmission. in southern europe, like in the camargue area, where the bird reservoir species are poorly to moderately susceptible to infection and only two vector species, one being moderately and the other one poorly susceptible, are present, the prevalence of west nile virus is highly sporadic [ ] . conversely, in the usa, where there are a multitude of bird reservoir species (of which a large part appears to be very susceptible) and an important diversity in vector species (some vectors being highly competent), huge epidemics that have propagated to the entire country, from east to west, in only a few years have been reported [ ] . the absence of previous immunity in bird populations to this virus (west nile fever appeared in north america in ) cannot explain this pattern due to the fast demographic turnover within avian communities. in the simplified context of fig. , the combination(s) of local reservoir and vector species richness and composition that can either amplify or dilute the infection may help to explain many other (new) emerging disease spillovers [ ] . bearing in mind that, generally, pathogen dynamics are driven by the dynamics of the overall biological diversity of the community and not of one single reservoir or vector host species, then future research should concentrate on revisiting the idea of disease transmission using a broader community-scale perspective than the one generally applied. our understanding of the influence of reservoir host and vector diversity on pathogen epidemiology and disease dynamics is still rudimentary. the possibility of strong effects due to a local multiplicity of hosts and vectors is however high for a large number of emerging pathogens [ ] . for some pathogens, like for instance west nile virus, different species of reservoirs (birds) and vectors can be involved locally, and changes in both reservoir and vector diversity from place to place can result in functional changes in transmission that have strong effects on the disease dynamics [ ] . indeed, animal viruses with multiple hosts are more likely to be designated as emerging threats than the ones with single hosts, a finding that emphasizes the importance of better examining the role played by host and vector diversity [ , ] . acknowledging a multiplicity of hosts in many zoonotic diseases [ ] may also complicate the way these diseases should be studied and analyzed when compared to simple one host-one vector systems. our examination of emerging infectious diseases with a zoonotic origin indicates that an explicit assessment of the influence of multiple host reservoirs and vectors must be taken into account. undoubtedly, it is realistic to acknowledge that the local and regional heterogeneity in vector and reservoir diversity can more or less affect disease behaviour and spread in nonspecialized parasites. overall, the evidence that the local host diversity influences the outcomes of disease dynamics seems clear for both lyme disease and west nile virus. as a consequence, not considering a priori the potential role played by poorly to very poorly competent hosts -the immersed part of the iceberg -, and particularly of vectors, under the pretext of a low or very low supposed or proven participation in the transmission, can have important ecological and epidemiological consequences. in many situations, the onset of emerging infectious diseases might be due to the multiplicity of hosts and vectors and its local variability and the role of the many epizootic reservoirs and poorly competent vectors should not be underestimated, not only in driving the disease dynamics in the system, but also in increasing the risk of infection to humans. this highlights possible research key issues for understanding pathogens in multi-species systems. given the recent emergence of important infectious diseases in several complex communities [ ] , more attention should be given to generalist pathogens and to recording the number and identity of all potential vector and reservoir species locally, instead of focusing only on the more competent, well-accepted hosts and vectors. it is clear from this review that to better understand multi-species dynamics of diseases, we should rather collect qualitative information on the largest possible number of actors in disease dynamics than to obtain quantitative and substantial data but only for one or two reservoir hosts or vectors that are a priori suspected to be involved in the transmission. moreover, since we can observe both spatial and temporal variations in local species diversity, such analysis should be repeated in several different communities and for different periods of time in order to infer on disease regional dynamics and on its effects on host assemblages, and vice versa. in addition, the potential for an accidental introduction in an originally untouched geographical area of some vector specimens, even if they are not very competent in transmitting the pathogen, should not be underestimated especially because they might originate or exacerbate the onset of an epidemic. the same could be said also about resident vectors and reservoir hosts that are accepted as being putatively low to very low competent hosts, but might play a key starting role in the epidemiology of a disease. besides, it comes back also to better understand the links that exist between the relative competence of reservoir and vector species and their life-history strategies, particularly the fact that competent hosts and vectors are often ubiquitous and generalist organisms. other lifehistory characteristics of reservoir and vector species should be further explored, such as reproductive strategies, feeding habits or body features, for instance. from an evolutionary point of view, pathogens should select for the best host vessels that facilitate their own survival and dissemination. a multiplicity of hosts might thus complicate the evolutionary dynamics of pathogens, if it introduces conflicting selective pressure between being embarked into the main reservoir host(s)/vector(s) or into a more occasional host/vector [ ] . on the other hand, there is no clear evidence that the pathogen prevalence for vector-borne infections is lower in richer reservoir communities. indeed, by studying the transmission of a generalist plant virus in experimentally manipulated plant communities, power and mitchell [ ] showed that the pathogen prevalence was entirely driven by the single, most competent plant species. in this case, therefore, the link between pathogen prevalence and species richness was a reflection of the community composition rather than of the community richness. to what extent, this observation might be applied to other multi-species systems is still a matter of debate. nevertheless, this suggests again that it would be more fruitful to explore the links between biological diversity and emerging infectious diseases by focusing on community composition and species life-history strategies. the community epidemiology perspective we advocate here may present also some practical aspects. indeed, it could be envisaged to exploit the natural complexity of ecological systems by introducing locally animal baits in the form of reservoir species with low susceptibility that might thus divert vector species from their human targets. by choosing preferentially these baits, vectors will consequently reduce disease transmission to humans [ ] . this type of control is called zooprophylaxy. the development of livestock rearing in western countries, by increasing the herd of animals with low susceptibility, has been suggested to be responsible, according to bruce-chwatt [ ] , to the disappearance of malaria in europe. this method, which has been theoretically studied for malaria [ ] , is still considered for application in some regions of asia [ ] . this calls into question the importance, in practice, of the community function in the epidemiology of infections. of course, more complete theoretical and experimental studies must be carried out because the introduction of these animal baits could have other indirect and even more dramatic consequences. community epidemiology is still in its early days. limited so far by a too simplistic perspective (the triad ''one agent-one vector-one reservoir'') on the complexity of natural communities, its development in the next years should stem from a partnership between specialists of complex systems, especially those working on multi-agent systems, ecologists, evolutionary biologists and epidemiologists. we conclude this review with an idea that has been pervasive throughout this essay: different epidemiological and ecological outcomes for a same disease can occur in different places, and one important and probably missing ingredient in the understanding of emerging infectious disease is the role of biological diversity and its heterogeneity in space and time. the authors declare that they have no conflicts of interest concerning this article. population biology of multihost pathogens changing geographic distributions of human pathogens emerging pathogens: the epidemiology and evolution of species jumps host range and emerging and reemerging pathogens infectious disease ecology. effects of ecosystems on disease and of disease on ecosystems infectious history infectious disease ecology. effects of ecosystems on disease and of disease on ecosystems global trends in emerging infectious diseases community ecology the relation between the number of species and the number of individuals in a random sample of an animal population the commonness and rarity of species the canonical distribution of commonness and rarity: part i the unified neutral theory of 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epidemiology may drive disease patterns for vector-borne pathogens re-emergence of blue tongue in northern europe in infection with chikungunya virus in italy: an outbreak in a temperate region chikungunya disease outbreak, reunion island a case of autochthonous plasmodium vivax malaria west nile virus: a growing concern? vector competence of california mosquitoes for west nile virus west nile outbreak in horses in southern france, : the return after years pathogen spillover in disease epidemics the epidemiology and control of malaria zooprophylaxis or zoopotentiation: the outcome of introducing animals on vector transmission is highly dependent on the mosquito mortality while searching survey of adult mosquitoes (diptera: culicidae) during irrigation development in the mahaweli project, sri lanka key: cord- - hobwqpe authors: bier, ethan; guichard, annabel title: deconstructing host-pathogen interactions in drosophila date: - - journal: dis model mech doi: . /dmm. sha: doc_id: cord_uid: hobwqpe many of the cellular mechanisms underlying host responses to pathogens have been well conserved during evolution. as a result, drosophila can be used to deconstruct many of the key events in host-pathogen interactions by using a wealth of well-developed molecular and genetic tools. in this review, we aim to emphasize the great leverage provided by the suite of genomic and classical genetic approaches available in flies for decoding details of host-pathogen interactions; these findings can then be applied to studies in higher organisms. we first briefly summarize the general strategies by which drosophila resists and responds to pathogens. we then focus on how recently developed genome-wide rna interference (rnai) screens conducted in cells and flies, combined with classical genetic methods, have provided molecular insight into host-pathogen interactions, covering examples of bacteria, fungi and viruses. finally, we discuss novel strategies for how flies can be used as a tool to examine how specific isolated virulence factors act on an intact host. drosophila has emerged as an important model for examining the function of genes that are relevant to diverse human diseases affecting a broad range of cell types (for reviews, see bier, ; bier and mcginnis, ) . additionally, this model organism can serve as a host for a surprising variety of bacterial and viral pathogens. seminal discoveries in the field of host-pathogen interactions have been made in drosophila. for example, the toll signaling pathway, which plays a central role in innate immunity, was first identified in drosophila, and studies using this model organism have helped to identify and delineate fundamental conserved host genetic pathways involved in barrier formation and maintenance (mace et al., ; martin and parkhurst, ; pearson et al., ; ting et al., ) , innate immune signaling (agaisse and perrimon, ; dionne and schneider, ; ferrandon et al., ; igaki et al., ; ryu et al., ) , the rna interference (rnai) response (sabin et al., ) , pathogen engulfment (meister, ) , and the evolution of intracellular pathogens (haselkorn, ; serbus et al., ) . as discussed in detail below, drosophila has also been used to identify and analyze the function of pathogen-derived virulence factors (avet-rochex et al., ; avet-rochex et al., ; botham et al., ; guichard et al., ; guichard et al., ; shelly et al., ; guichard et al., ) . many genetic tools are available to address the mechanisms of pathogen action in drosophila. these include comprehensive genetic screens, or genome-wide rnai screens, in cell lines and intact flies that can identify host pathways required to defend against pathogens. reciprocally, it is also possible to search for pathogen-encoded factors that are required for virulence in flies. in vivo studies in flies are greatly facilitated by the ability to direct expression of transgenes encoding host or pathogen proteins in specific cell types using the gal -uas transactivation system (brand and perrimon, ) or the new independently acting lexa system, which allows for combinatorial expression of genes in distinct or overlapping patterns (yagi et al., ; pfeiffer et al., ) . moreover, it is possible to perform epistasis experiments using combinations of dominant and recessive mutations (or mutants with opposing phenotypes) in a given pathway to determine the sequence in which genes act in that pathway. these versatile tools, combined with the rapid drosophila life cycle, allow detailed genetic analysis of virulence factors that act on tissues or organs; such experiments would be much more difficult to conduct in vivo in mammalian systems. in this review, we first outline the basic host defense mechanisms used by drosophila to resist and respond to invading pathogens to provide context for our discussion of how flies can be used to deconstruct key mechanisms of host-pathogen interactions. we then focus on three related topics: ( ) genome-wide rnai screens in drosophila cell lines infected with pathogens to identify host pathways for defense or that are exploited by pathogens (e.g. bacteria, fungi, viruses); ( ) classical genetic and rnai screens conducted in intact flies to delineate host defense pathways that are active in specific tissues (e.g. the gut) or to identify important virulence factors produced by the pathogen; and ( ) analysis of the function of specific pathogen virulence factors in an intact organism. the studies reviewed here highlight the speed and power of drosophila genetics for uncovering new pathways and factors in host-pathogen interactions, as well as for characterizing unknown activities of specific virulence factors. identification of such elements in the host-pathogen relationship should help to guide studies in vertebrate systems and contribute to defining new targets for potential therapeutic intervention. host-pathogen interactions in drosophila perspective and an internal component comprising the gut (or endoderm). the formation of both the outer epithelial barrier and the inner intestinal barrier depends on the formation and maintenance of intercellular junctions, and many basic discoveries in this field have been made in drosophila (banerjee et al., ; furuse and tsukita, ; wirtz-peitz and zallen, ). such studies have delineated key mechanisms involved in establishing apical-basal polarity, including the assembly of distinct protein complexes at adherens junctions and septate junctions (claudin-dependent junctions that share important similarities with vertebrate tight junctions). one highly conserved feature of this process is the role of the exocyst protein complex in trafficking proteins such as cadherins and cell signaling components to adherens junctions (andrews et al., ; beronja et al., ; blankenship et al., ; jafar-nejad et al., ; langevin et al., ; mehta et al., ; murthy et al., ; murthy et al., ; murthy and schwarz, ; murthy et al., ) . at first glance, the mammalian epidermis seems very different from that of flies ( fig. a ), but there are striking parallels with respect to the formation and maintenance of epithelial barriers in the two species, illustrating a probable common ancestral origin. for example, claudin-family proteins forming the tight junctions between epithelial cells seem to have similar functions in both flies (behr et al., ; nelson et al., ; wu et al., ) and mice (furuse et al., ) [see the article by furuse for a review on the role of claudins and other tight junction proteins in mammalian epithelia (furuse, ) ]. similarly, the transcription factor grainyhead (grh) plays an important role in regulating the expression of genes that are required to form the cross-linked outer epidermal surface both in flies (bray and kafatos, ) and mice (matsuki et al., ; ting et al., ) (although the set of grh target genes seems to be different in each species). grh also regulates genes that are involved in wound repair both in flies (mace et al., ) and mice (ting et al., ) . it is noteworthy that drosophila and vertebrate intestinal epithelia are also similar in several respects. these parallels include: the fact that stem cells play an important role in replacing cells that have undergone pathogen-dependent apoptosis; the sequential deployment of wnt and hedgehog (hh) signaling during the differentiation of intestinal epithelial cells (pitsouli and perrimon, ; takashima et al., ) ; and the formation of the morphologically specialized brush border microvilli and the underlying cytoskeletal terminal web (li et al., ; morgan et al., ; phillips and thomas, ) . a challenge faced by intestinal cells is that they must tolerate commensal bacteria, with which they have a mutualistic relationship (backhed et al., ; dale and moran, ; sansonetti and medzhitov, ) , while also mounting a vigorous response to pathogens (for a review, see ryu et al., ) . one important pathway involved in this distinction controls the production of reactive oxygen species (ros) by the dual-oxidase (duox) transmembrane protein (ha et al., ; ha et al., a; ha et al., b) , which also plays a key role in the human gut (for a review, see ryu et al., ) . genetic analysis in drosophila has revealed bi-stable control of duox activity in the gut. in the presence of commensal bacteria and absence of pathogenic species, low-level activation of the immune deficiency (imd) pathway of the innate immune system (see later) induces negative feedback of the duox pathway (at both the level of expression and activity), resulting in low basal levels of ros production. by contrast, when invading pathogens are detected by host immune signaling, expression and activity of duox components is greatly increased, leading to destruction of the pathogenic bacteria (ha et al., ) . the inducible ros-producing duox system works in parallel with other immune pathways, such as the jun n-terminal kinases (jnk) pathway. jnk signaling is activated in intestinal epithelial cells of adult flies following ingestion of pathogenic pseudomonas aeruginosa , which leads to proliferation of intestinal stem cells to compensate for apoptotic loss of mature infected cells (for a review, see pitsouli et al., ). an interesting aspect of this pathogen in drosophila is that, in combination with an activated oncogenic form of ras, it can lead to overproliferation of stem cells to form tumors . whether the elevated incidence of human cancers of the intestinal tract as a result of associated bacterial infection (bornschein et al., ; selgrad et al., ) is similarly influenced by ras activation remains to be determined. another interesting emerging theme is the elucidation of host pathways involved in detecting cell damage in the intestine, which then regulate stem cell mediated repair of the damaged epithelium. these studies have revealed important contributions of the insulin (amcheslavsky et al., ) and tsc-torc (amcheslavsky et al., ) pathways, as well as of hippo (hpo)-mediated activation of the jak-stat and epidermal growth factor receptor (egfr) pathways (ren et al., ) . finally, experiments involving oral infection of flies with erwinia carotovora, a natural drosophila pathogen, suggest that gut homeostasis is maintained by active tissue repair of cell damage caused by bacteria (buchon et al., ) . the observation that ros can trigger apoptosis followed by repair in the larval gut (gupta et al., ) suggests that the duox pathway provides compensatory feedback to pathways controlling apoptosis and stem cells to ensure that host cells damaged by ros exposure are duly replaced. overall, these studies provide an excellent foundation for further analysis of how the gut responds to pathogens by repairing damage and differentially responding to commensal versus pathogenic bacteria. broadly speaking, the innate immune response consists of three parts: ( ) pathogen immobilization (fig. b) , ( ) core immune signaling pathways (toll, imd and jak-stat) (fig. c) and ( ) the rnai pathway (fig. d ). because there have been several excellent reviews describing these pathways, we only summarize here their key elements, as depicted in fig. , and refer the reader to other sources for more in depth descriptions (agaisse and perrimon, ; akira et al., ; bhavsar et al., ; brodsky and medzhitov, ; diacovich and gorvel, ; dionne and schneider, ; ferrandon et al., ; folsch et al., ; sansonetti, ) . the most basic innate response to bacterial or fungal infection is a cellular response (jiravanichpaisal et al., ) that immobilizes the invading microbe by phagocytosis, engulfment or a host-pathogen interactions in drosophila perspective melanization reaction that traps it (fig. b) . pathogens can also be immobilized in flies and other insects by a clotting reaction (dushay, ) . once immobilized, the pathogen can then be either destroyed extracellularly by antimicrobial peptides (amps) or eliminated intracellularly. three basic types of drosophila blood cell (known as hemocytes) perform these functions: plasmatocytes, which are professional phagocytic cells akin to mammalian macrophages; lamellocytes, which wrap themselves around invading microorganisms to form an enveloping capsule; and crystal cells, which contain the enzymes that catalyze melanization (meister, ) (fig. b) . as discussed later in more detail, many host genes that are required for phagocytosis have been identified using drosophila in a series of genome-scale cell-based screens. similar studies in the future might shed light on genes that are essential for lamellocyte and crystal cell function. autophagy is another general mechanism important for clearing bacteria (yano et al., ) and viruses (cherry, ; shelly et al., ) . it should be pointed out, however, that autophagy can also be hijacked for the benefit of the pathogen, as in the case of poliovirus, which derives its envelope membranes from autophagic vesicles (suhy et al., ) . the second part of the drosophila innate immune response comprises a set of core signaling pathways (fig. c) : the toll pathway, the imd pathway and the jak-stat pathway. the activities of these pathways are modulated by other pathways, such as that mediated by target of rapamycin (tor) or eiger-wengen [drosophila homologs of human tumor necrosis factor (tnf) and tnf receptor]. when induced following pathogen infection, innate immune pathways result in the production of amps such as drosomycin and diptericin (dionne and schneider, ; ferrandon et al., ; agaisse and perrimon, ; dionne and schneider, ; ferrandon et al., ; folsch et al., ) . the third part of the drosophila innate immune response is the double-stranded rnai pathway that is involved in defending against many types of viral infections, and which also protects against viral infection in plants and animals (for a review, see sabin et al., ) (fig. d ). the rnai pathway is activated by viral nucleic acids and can be broken down into two main steps: ( ) biogenesis of -base-pair double-stranded viral small interfering rnas (sirnas), which is accomplished by the dicer protein complex, and ( ) the silencing of viral rnas by the host-induced viral sirnas, which is accomplished by the rna-induced silencing complex (risc). this innate protective system has been highly amenable to analysis using genome-wide screening in drosophila cells (see below). one of the great recent technical advances in the field of drosophila cell biology has been the development of efficient whole genome rnai screens to identify genes required for specific cellular processes (mohr et al., ; perrimon and mathey-prevot, ; perrimon et al., ) . in such assays, drosophila cell lines such as hemocyte-derived s cells or kc cells (which can be induced by hormone treatment to differentiate into neurons) are grown in well plates and treated with a library of double-stranded rnas that have been designed for highly selective rnai-mediated knockdown of each of the predicted drosophila coding messenger rnas (mrnas). these cells are then assayed for performance of a cellular process such as cell viability, cell shape changes or bacterial uptake by phagocytosis. by screening such libraries in replicate and then re-screening rnai candidates that test positive for a specific effect, it is possible to approximate genome-wide coverage of all genes required in these cells for a given process [for an excellent, comprehensive review of such rnai screens, see cherry (cherry, ) ]. such screens have been used to identify many host response factors that are crucial during infection by bacteria, fungi and viruses. several straightforward rnai screens have been conducted to identify genes that are required for phagocytosis of various species of bacteria by s cells. for these experiments, ingestion of bacteria expressing green fluorescent protein (gfp) is monitored and host genes involved in phagocytosis are revealed on the basis of the identity of specific rnai molecules that inhibit uptake of fluorescence. these screens have revealed that distinct sets of host genes are essential during infection by various pathogens. for example, different pathogens are recognized by distinct cell surface receptors, such as peptidoglycan recognition proteins (pgrps) (ramet et al., ) , sr-c (ramet et al., ) , eater (kocks et al., ) , nimrod (kurucz et al., ) or dscam (watson et al., ) . however, these screens also defined a core set of intracellular uptake components that are regulated in all types of bacterial infection tested: these included genes required for actin remodeling (e.g. genes encoding proteins of the arp / complex) and endocytosis (e.g. copi and copii), as well as genes encoding factors that are required to recycle endosomes to the cell surface, such as proteins in the exocyst complex (agaisse et al., ; cheng et al., ; philips et al., ; ramet et al., ; stroschein-stevenson et al., ; stuart et al., ) . other genes involved in the response to bacterial infection that have been identified in rnai screens are required for host cells to clear ingested bacteria. again, these screens defined a set of generally required genes that limit bacterial survival or replication, such as genes encoding endosomal sorting complex required for transport (escrt) proteins (philips et al., ) , as well as genes preventing the growth of specific pathogens, such as lysosomal hexosaminidase, which restricts growth of mycobacterium marinum but not listeria monocytogenes or salmonella typhimurium (koo et al., ) . in other standard genetic studies, intracellular microorganisms such as wolbachia were found to also engage in mutualistic symbiotic relationships with the host, such as protecting the host against viral infection (hedges et al., ; teixeira et al., ) and nutritional supplementation (brownlie et al., ) , which presumably arose during co-evolution of the endosymbiont and host. rnai technology can also be used in a combinatorial fashion to knock down the activity of two or more genes at a time, which permits detection of genes acting in parallel in a given process or pathogenic infection. in one study, dorer and colleagues performed host-pathogen interactions in drosophila perspective a series of single and double gene knockdown experiments of genes in kc cells to test the hypothesis that legionella pneumophila, the agent of legionnaires' disease, recruits membrane material from endoplasmic reticulum (er)-to-golgi trafficking (dorer et al., ) . although few single knockdowns had much of an effect, the authors found evidence supporting their hypothesis in several double knockdown experiments. for example, double knockdown of the intermediate compartment and golgi-tethering factor transport protein particle (trapp) together with the er snare protein sec resulted in reduced pathogen replication efficiency. they also showed a requirement in bacterial replication for the cdc -p complex that is involved in er-associated degradation, and demonstrated that this complex is also important for legionella pneumophila replication in mouse bone-marrow-derived macrophages. these studies underscore the role of endocytosis in phagocytic host cells and, owing to the combinatorial power of the system used, revealed a role for endocytic steps carried out by parallel mechanisms. fungi generally activate the toll signaling pathway of the drosophila innate immune system via a specific set of pgrp detection peptides (fig. c ). rnai screens similar to those performed to identify host genes required for phagocytosis of bacteria have also been carried out to identify host factors involved in response to fungi such as candida albicans (stroschein-stevenson et al., ; stroschein-stevenson et al., ) . beyond identifying genes with broad expected functions, such as regulators of the actin cytoskeleton and vesicular trafficking, these studies also identified genes required for the uptake of specific fungal pathogens. one of these proteins, macroglobulin complement related (mcr), is a secreted protein that binds directly to c. albicans and promotes its internalization. interestingly, mcr is related to four other drosophila thioester proteins (teps), two of which are selectively required for phagocytosis of specific bacterial species (tepii for escherichia coli and tepiii for staphylococcus aureus), but not for phagocytosis of c. albicans (stroschein-stevenson et al., ) . in addition to being susceptible to infection by bacterial and fungal pathogens, drosophila is also a natural host for viruses such as drosophila c virus (dcv), drosophila x virus (dcx) and flock house virus, and, perhaps surprisingly, by a broad variety of viruses causing disease in humans such as sindbis virus, vesicular stomatitis virus (vsv; a virus of the rhabdoviridae family, which includes the well-known rabies virus), rift valley fever virus, dengue virus and west nile virus (cherry et al., ; cherry et al., ; cherry and perrimon, ; galiana-arnoux et al., ; van rij et al., ; wang et al., ) . genome-wide rnai screens have identified several important host factors that are exploited by viruses, such as factors required selectively for replication of influenza virus (hao et al., ) or propagation of dengue virus (sessions et al., ) . similarly, viruses such as dcv that have transcripts with internal ribosome-binding sites depend on several host translation factors that are not required for other types of viruses lacking these sites (cherry et al., ) . dcv also requires the host factor copi to generate a vesicular compartment, which is necessary for viral replication, and copi is also required for the replication of the related poliovirus in human cells (cherry et al., ) . as another example, infection by vaccinia virus (the prototypical poxvirus) was found to depend on the amp-activated kinase (ampk) complex, the master energy sensor of the cell, for endocytic entry and actin remodeling (moser et al., ) . the authors found a similar requirement for ampk in facilitating vaccinia infection of mouse embryonic fibroblasts and showed that this kinase was also involved in viral entry via the process of macropinocytosis. as mentioned above, the rnai pathway plays a key role in defending against viral infection. genome-wide and targeted rnai screens have contributed to the elucidation of this pathway (galiana-arnoux et al., ; nayak et al., ; otsuka et al., ; sabin et al., ; van rij et al., ; wang et al., ) (for a review, see sabin et al., ) and the importance of the systemic spread of an rnai activating signal (probably some large viral doublestranded rna) for stimulating rnai-dependent immunity throughout the organism (saleh et al., ) . interestingly, sirnas do not spread from cell to cell in drosophila (roignant et al., ) , in contrast to the mechanism by which rnai molecules are directly distributed in plants (palauqui et al., ; winston et al., ) and nematodes (fire et al., ; voinnet et al., ) to mediate systemic immunity. as a complement to cell-based screening methods, it is also possible to screen for host genes that are required to combat pathogen infection using intact flies. although these screens are more laborious than screens in drosophila cell lines, or wholegenome rnai screens in worms (i.e. caenorhabditis elegans screens can be done on plates), screens using intact flies can be accomplished either by classic mutagenesis or by screening high quality collections of stable uas-rnai stocks. a great advantage of the latter approach is that one can use the gal -uas expression system (brand and perrimon, ) to drive expression of uas-rnai constructs throughout the organism or in specific subsets of cells or stages of development (fig. b ). for such experiments, a strain of flies carrying a transgene under the control of the yeast upstream activating sequence (uas) is crossed to a strain of flies expressing the gal transcription factor (which binds to the uas sequence and activates transcription in a particular pattern, e.g. in the gut). the progeny then express the uas transgene of interest in the pattern determined by the gal 'driver' stock, permitting expression of genes in specific cell types at specific stages of development. this level of control permits investigators to identify the cells or organs in which gene functions are required [e.g. epidermis, fat body (the main source of systemic amps, and an approximate model of the mammalian liver), hemocytes or gut]. in one screen using adult flies, host defense factors that are required to protect against intestinal infection with the opportunistic broad-host-spectrum pathogen serratia marcescens were first identified by using a large collection of fly lines in which , individual rnai molecules were used to knock down target gene expression throughout the organism (cronin et al., ) . rnai molecules that caused increased lethality following infection were then tested further for their role in defending host-pathogen interactions in drosophila perspective against s. marcescens infection of the gut by expressing the relevant uas-rnai constructs with gut-specific and hemocytespecific drivers. these studies first confirmed the dependence on the imd (but not toll) innate immune pathway for responding to infection by s. marcescens (as would be expected for a gramnegative bacterium), and also revealed an important role for the jak-stat pathway in responding to infection in the gut (fig. c ). further analysis of jak-stat signaling showed that this pathway regulates stem cell proliferation and thereby intestinal epithelial homeostasis during infection. these results obtained in intact flies provide an important complement to screens performed in c. elegans, which also identified several signaling systems important for innate immunity (irazoqui et al., ) . studies of damage and repair by gut pathogens can be conducted in drosophila because flies, but not worms, have intestinal stem cells that replenish epithelial cells after they undergo programmed cell death during infection (see above). systematic screens such as that mentioned above (cronin et al., ) can also be used to identify host factors co-opted by a pathogen that, when mutated, render the host resistant to the pathogen. an example using a traditional genetic approach is the case of vibrio cholerae, in which investigators showed that feeding v. cholerae bacteria to flies caused rapid death (i.e. in - days) that required the function of the primary virulence factor cholera toxin (ctx) (blow et al., ) . ctx is an adp ribosyl transferase that specifically ribosylates the gs subunit of a host trimeric gs protein, resulting in constitutive activation of adenylate cyclase (middlebrook and dorland, ) . the dependence on ctx was host-pathogen interactions in drosophila perspective unexpected because flies lack the enzymes required to synthesize the gm ganglioside that serves as the ctx receptor and that is present in most vertebrates and a few invertebrates. accordingly, feeding flies purified ctx holotoxin had no effect (blow et al., ) . paradoxically, however, full virulence of ctx-mutant bacteria could be restored by feeding infected flies purified ctx, suggesting that, in the presence of the bacteria, a novel alternative route of ctx delivery to host cells in the gut might be employed. further analysis showed that several host target factors known from mammalian studies were required by v. cholera to infect flies, such as proteins mediating the dehydrating effects of ctxdependent camp production -including a gs subunit, adenylate cyclase and an sk-type potassium ion channel (blow et al., ) . having established flies as a model for v. cholerae infection, the authors then screened a large collection of stocks with mapped transposon insertions into the fly genome and identified mutations that either enhanced or reduced severity of infection. this strategy identified several host genes important for the response to v. cholerae infection, including those conferring resistance when mutated and that presumably are exploited by bacteria (e.g. components of the tnf and imd pathways) as well as those used in host defense (e.g. the apoptotic pathway) (berkey et al., ). adult flies and cells have also been used to screen for pathogenencoded factors that contribute to virulence. one particularly elegant screen for bacterial virulence factors was carried out for p. aeruginosa, an opportunistic human pathogen that can cause serious disease. over transposon insertion mutants of the bacterium were screened by injecting them into the adult fly hemolymph and measuring percent lethality. this resulted in the identification of different bacterial loci that contributed significantly to virulence (kim et al., ) . the authors examined the basis of virulence for one of these genes, hudr. hudr encodes a transcription factor that represses expression of the neighboring gene huda, which is involved in ubiquinone biosynthesis. on the basis of their genetic analysis, the authors hypothesized that the decreased virulence of hudr mutants resulted from overexpression of huda. they confirmed this hypothesis by showing that overexpression of huda in a hudr-mutant background resulted in attenuated virulence of p. aeruginosa in flies and that huda hudr double mutants had normal virulence. flies have also been used to differentiate virulence of p. aeruginosa strains, such as those isolated from the sputum of cystic fibrosis patients (who are particularly sensitive to infection by this pathogen) (lutter et al., ; salunkhe et al., ; sibley et al., ) . for these assays, flies are either fed different strains of bacteria obtained from burn wounds or from cystic fibrosis patients, or bacteria are inoculated by wounding flies. similar infection experiments can be performed to identify interactions between p. aeruginosa and other microbes present in sputum that could contribute to the virulence of this pathogen (sibley et al., ) . virulence factors of human fungal pathogens can also be identified in drosophila (ben-ami et al., ; chamilos et al., ; chamilos et al., ; lamaris et al., ) . for example, gliotoxin produced by the filamentous fungus aspergillus fumigatus is required for virulence of this pathogen in both flies and mice (spikes et al., ) , and cas has been shown to be a transcription factor regulating a set of genes required for integrity of the cell wall of c. albicans . adult flies have also been used as an intact organism to screen for drugs that block fungal infection (chamilos et al., a; chamilos et al., b; lamaris et al., ; lamaris et al., ; lionakis et al., ) . in the previous section, we discussed strategies by which flies can be used to screen for pathogen virulence factors. in this final section, we consider the advantages of drosophila as a model for analyzing virulence factor function, and for identifying the host proteins and pathways that they target (e.g. fig. c ). although cellbased expression systems and biochemical experiments performed with purified virulence factors can be invaluable for establishing mechanism of action, they do not necessarily predict how such factors will act in an infected organism -either systemically or in selected tissues, in which cell-autonomous and non-cellautonomous processes might be important. model systems such as flies and worms are ideal for this level of analysis owing to the great variety of genetic tools available to tease apart the effects that such factors might have on specific host pathways and biological processes. although flies and worms are only distantly related to humans, many virulence factors target host proteins and pathways that are among the most conserved in eukaryotes -thus, studying the effect of pathogens in these organisms is often highly relevant to human disease. in addition, as discussed below, studies in model organisms also enable examination of the combinatorial effect of two or more virulence factors, which is more challenging in intact mammals. finally, we highlight in box how studying the effect of toxins can shed light on basic cellular processes. drosophila is an excellent in vivo genetic system for analyzing toxin activities in a multicellular and organ context given the highly conserved nature of many host targets of these virulence factors. for example, flies have been used to study the activity of the virulence factor exos from p. aeruginosa, which encodes a factor one of the first uses of toxins in flies was to genetically ablate specific cells with cell-lethal toxins such as diphtheria toxin (kunes and steller, ) or ricin (moffat et al., ) . it is also possible to block the neuronal activity of cells without killing them, as with tetanus toxin (ttx), which was used to block synaptic transmission in the nervous system (allen et al., ; baines et al., ; reddy et al., ; sweeney et al., ) and activity-dependent regulation of synaptic size and function (nakayama et al., ) . ttx has been used in a myriad of drosophila studies to inhibit neurotransmission in various processes, including learning and memory, locomotion and courtship (for a review, see martin et al., ) , circadian rhythms (johard et al., ; kaneko et al., ) , and the serotonin-dependent response to light (rodriguez moncalvo and campos, ) . similarly, application of cholera toxin (ctx), an adp-ribosylation factor, was used to study the function of the g protein concertina, which is involved in initiating embryonic gastrulation (morize et al., ) . similarly, transgenic expression of a uas-ctx-a construct helped to distinguish which heteromeric g proteins contribute to wing maturation (katanayeva et al., ) . indeed, these and other toxins, which neutralize or alter the activities of multiple host proteins, can be used to perform a variety of in vivo pharmacological studies to complement classical genetic loss-offunction studies. containing a domain with rho-gap activity (which can inactivate host small gtpases of the rho/rac subfamily). during p. aeruginosa infection, exos is injected into host cells by a type-ii secretion system (ttss), and infection of flies with p. aeruginosa leads to rapid death that depends on ttss function (fauvarque et al., ) . when the gap domain of exos (exosgap) is expressed in fly hemocytes, phagocytosis is inhibited (avet-rochex et al., ) . in addition, expression of exosgap in flies increases their sensitivity to infection by p. aeruginosa (avet-rochex et al., ) , and this effect can be rescued by co-expressing host rac with exosgap (avet-rochex et al., ) . these studies provide evidence that host rac is inhibited by bacterial exosgap during infection. a second example illustrating the utility of drosophila for investigating toxin activities in vivo is provided by studies of helicobacter pylori (fig. c) , which is associated with the development of gastric ulcers and cancer in humans. under normal circumstances, ligand-initiated receptor tyrosine kinase (rtk) signaling in both fly and mammalian cells is mediated by a receptorassociated protein complex including grb (drk), gab (dos) and shp- [corkscrew (csw)] (drosophila protein names are shown in parentheses) that then activates signaling via the downstream components of the ras-mapk pathway. drosophila played a prominent role in discovering key components of this pathway and in establishing the order of molecular events that take place during signaling (simon, ) . in mammalian cells, the h. pylori virulence factor caga activates rtk signaling at the level of shp- , a tyrosine phosphatase that is homologous to drosophila csw (hatakeyama, ; hatakeyama, ) , which acts downstream of gab (dos in flies) (herbst et al., ; raabe et al., ) . studies in drosophila confirmed the hypothesis that caga can bypass the need for signaldependent activation of dos in an intact organism, because caga expression in drosophila embryos or in the adult eye was capable of rescuing dos-mutant phenotypes (botham et al., ) . furthermore, the ability to activate effectors of the sevenless rtk pathway in the eye was shown to be dependent on the downstream effector csw, validating the hypothesized role of caga in the rtk signaling pathway acting between gab (dos) and shp- (csw). beyond providing a multicellular model for assigning known biochemical activities to virulence factors, drosophila can also be used as a tool to discover completely new activities of virulence factors. for example, bacillus anthracis, the etiological agent of anthrax, produces two toxic factors required for systemic virulence (lacy and stevens, ; mourez, ; tournier et al., ; guichard et al., ) : lethal factor (lf), a zinc metalloprotease that cleaves mapkks (duesbery et al., ; vitale et al., ) , and edema factor (ef), a highly active calmodulin-dependent adenylate cyclase (leppla, ) . both lf and ef are essential for the lethal effects of anthrax (pezard et al., ) , which culminates in vascular failure and septic-shock-like death. an important unanswered question is, how do lf and ef, with such seemingly disparate enzymatic activities, collaborate during infection (particularly within vascular endothelial cells, which become leaky at advanced stages of disease, leading to death)? in initial studies, we showed that anthrax toxins act on drosophila homologs of their known targets in mammalian cells (guichard et al., ) . in addition to these known effects of lf and ef, we observed that both toxins also caused adult wing and bristle phenotypes similar to those caused by inhibition of the notch signaling pathway, and blocked expression of notch target genes in developing wing imaginal discs (guichard et al., ) . moreover, these toxins interacted in a synergistic fashion to block notch signaling (fig. a) . further analysis of this notch-like phenotype revealed that it resulted from failure to recycle the notch ligand delta to the cell surface (guichard et al., ) . ef was found to reduce the levels and activity of the small gtpase rab , whereas lf reduced cell surface levels of the rab binding partner, sec (fig. b ). sec is part of an octameric protein complex known as the exocyst, which targets proteins, including delta and the cell adhesion molecule decadherin, to adherens junctions (accordingly, de-cadherin trafficking to adherens junctions was also reduced by ef and lf). these results from flies were validated in human vascular and lung endothelial cells by our collaborators in victor nizet's laboratory (fig. c) , who also showed that ef reduced epithelial barrier integrity in a cell culture assay and in vivo in mice (guichard et al., ) . maintenance of vascular integrity depends on cell-cell adhesion (dejana et al., ) , and cell-cell communication mediated by notch signaling plays a role in promoting the formation of primary (or patent) vessels over more permeable microvessels (hellstrom et al., ; leslie et al., ; lobov et al., ; roca and adams, ; siekmann and lawson, ; suchting et al., ) . by inhibiting these two interrelated processes, and possibly interactions between endothelial cells and other vascular cell types such as mural cells, anthrax toxins might contribute to the latestage effects of anthrax infection when disruption of endothelial barrier function leads to lethal vascular collapse. once sufficient levels of anthrax toxins are produced, they can be fatal even if the bacterial infection is eliminated with antibiotic treatment. thus, these studies of anthrax toxins initiated in flies and validated in mammalian models might ultimately have therapeutic implication for treating humans infected with anthrax or for other conditions compromising vascular integrity. given the compact sizes of viral genomes, only few viral proteins fall into the category of bona fide virulence factors, similar to the potent bacterial toxins discussed above. by contrast, most viral proteins are dedicated to basic processes essential to the virus life cycle, such as entry or exit, replication, or manipulation of host processes such as transcription or translation. model organisms are useful for examining specific interactions between viral and host proteins to gain insights into their mechanisms of action. an excellent example of using the full complement of drosophila tools to study a viral pathogen was carried out by cherry and colleagues, who showed that fly cells can be infected with vsv. vsv can replicate in these cells to generate mature viral particles that can infect mammalian cells. they showed that infection of adult flies with vsv induces autophagy (shelly et al., ) and that autophagy was mediated by vsv-g, a pathogen surface protein that is recognized by drosophila cells. the authors found that induction of autophagy plays an important role in protecting against vsv infection and then asked what host pathways might mediate the autophagy response to vsv. a variety of elegant genetic epistasis experiments demonstrated that the pi k-akt pathway was attenuated by vsv infection, thereby relieving its constitutive (guichard et al., ) . wt, wild type. (b)analyze mechanisms of toxin action. the notch-like phenotypes caused by expression of lf or ef in the wing both result from inhibition of endocytic recycling of membrane cargo to the aj by the exocyst complex. ef acts by reducing the levels and activity of the rab gtpase, which indirectly results in a loss of large vesicles containing its binding partner sec -gfp, a component of the exocyst complex. lf does not seem to alter rab levels or function, but inhibits the formation of large sec vesicles (guichard et al., ) . (c)validate toxin mechanism in vertebrates. human brain microvascular endothelial cells were treated with purified ef toxin or lf toxin. as in fly cells, both toxins greatly reduce the number of sec -gfp vesicles in these cells and reduce cadherin expression (guichard et al., ) . (d)examine interactions between toxins. cooperative interactions between toxins or other virulence factors can be assessed by co-expressing them in specific cells and comparing the effects of both toxins to that of the action of either toxin alone. in the example shown, anthrax toxins were expressed alone or in combination using a weak gal driver to express low levels of the toxins. each panel consists of an adult wing (top) and a larval wing imaginal disc showing expression of the notch target gene wingless (wg) along the future edge of wing in third instar larvae (bottom). expression of lf or ef alone (+lf or +ef, respectively) has little or no effect on formation of the wing margin (compared with wt). when lf and ef are co-expressed, the wing margin virtually disappears, as does expression of wg along the primordium of the wing margin. (e)in vivo structure-function analysis of toxins. the systemic activities of mutant forms of toxins or other virulence factors can be assessed in drosophila. such activities include cell-non-autonomous effects mediated by intercellular signaling systems, which are difficult to screen for in cell culture. in the simple case shown in this panel, high levels of lf expression lead to reduced wing size (middle panel) and a single point mutation in the lf catalytic domain renders it inactive (right panel). panels a-d adapted from guichard et al. (guichard et al., ) with permission. panel e adapted from guichard et al. (guichard et al., ) , with permission. host-pathogen interactions in drosophila perspective inhibition of autophagy ( fig. a) . akt activation is also attenuated by expression of the sars-coronovirus membrane protein in flies, which in this case results in increased apoptosis (chan et al., ) . in another study, host factors required for the hiv accessory protein nef to downregulate expression of the human cd protein were identified by rnai screening in drosophila s cells expressing human cd . these factors included components of the clathrinassociated ap complex, which was then validated as an essential cellular component mediating a similar nef-cd interaction in human cells (chaudhuri et al., ) . classic genetic approaches in drosophila can also be applied to probing structure-function relationships of toxins or other virulence factors. one straightforward approach to define domains of a toxin that are important for producing the phenotype of interest is to mutagenize flies carrying a uas-toxin construct and to screen for loss of the phenotype that results from expression of the wild-type toxin (fig. e ). one can then pcr amplify the mutated uastransgenes and sequence the putative mutant allele to determine the molecular nature of the loss-of-function mutation. it is also possible to screen for mutations in the transgene that results in altered phenotypes caused by dominant gain-of-function mutations (guichard et al., ) . an important goal of this review has been to convince readers from other fields that flies provide a broad range of advantages for studying host-pathogen interactions at the level of the cell, tissue, organ and intact organism. as discussed, genome-wide rnai screens in drosophila cell culture have generated a wealth of new information regarding the genes involved in mediating basic host cellular responses to pathogens, such as those involved in innate immunity, phagocytosis and restriction of intracellular pathogen survival. these cellular studies can be complemented by studies that aim to identify host resistance factors and pathogen virulence factors using intact flies as infection models. studies in flies also provide the potential to explore mutualistic interactions with intracellular endosymbionts, and to conduct mechanistic analysis of specific virulence factors, and combinations of these factors, using the state-of-the-art genetic tools available in drosophila. a particular advantage of model systems such as yeast, c. elegans and drosophila is the potential to examine arrays of genetic combinations to identify factors produced by the host or pathogen that act redundantly (host) or that genetically interact (host or pathogen). these types of studies are inherently number intensive (known as 'the n problem'), because many combinations must be analyzed in a comprehensive fashion. however, there are excellent examples in which combinatorial genetic analysis has been used to investigate cellular processes involved in other types of human disease. for example, the interacting components of the dna mismatch repair machinery were first identified in yeast, and the same components were found to interact in humans in a dominant manner and to contribute to cancer (kolodner, ) . drosophila cells and intact fly mis-expression systems (e.g. combined rnai expression) are also well suited for such analyses, which would be prohibitively expensive and labor intensive in vertebrate models. it is of course important to validate results obtained in single cells or invertebrate model systems in vertebrates, a process previously referred to as 'closing-the-loop' (bier, ) . it is possible to envision a tiered system of analysis in which initial discoveries that are made using powerful model genetic systems, including yeast, worms and flies (in cells and in whole organisms), are then validated in vertebrate models, including zebrafish, mice and human cells, and finally are linked via human genetics to specific disease processes. for example, in a recent study of genes on human chromosome causing congenital heart defects when overexpressed in individuals with down syndrome, a combined genetic analysis in flies, mice and humans pointed to two interacting genes, dscam and col a , as contributing to formation of atrial septal defects (grossman et al., ) . in addition to assessing combinatorial contributions of host factors, drosophila is well suited for examining cooperative interactions between pathogen virulence factors, as presented in the examples above. many pathogens produce a complex cocktail of virulence factors, subsets of which are often co-expressed from neighboring genes in so-called pathogenicity islands. these coregulated virulence factors are typically delivered by a dedicated injection system and often act by unknown means in various combinations in different host cell types. such virulence factors from a given pathogenicity island can be expressed in various combinations in specific cell types to identify specific cellular contexts in which they interact. given the great success of the fly for analyzing the activities of single host or pathogenic factors in disease processes, it will interesting to see whether it also serves as a robust system to study more complex networks of interactions between host pathways or pathogen virulence factors. with the advent of whole genome rnai tools and comprehensive mutant collections, flies should also provide an important intact model system for identifying unknown activities of virulence factors that act in a multicellular context to inhibit specific signaling systems or to alter contact between neighboring cells in structured tissues and organs. combined use of drosophila cells and intact flies in moderate-to high-throughput drug screens is also emerging as an effective strategy to identify compounds, or combinations of existing compounds, that alter the activity of host pathways to counter the effect of pathogens. clearly, flies have a bright future as tools for further deconstructing human host-pathogen interactions. the roles of jak/stat signaling in drosophila immune responses genome-wide rnai screen for host factors required for intracellular bacterial infection pathogen recognition and innate immunity targeted expression of truncated glued disrupts giant fiber synapse formation in drosophila tissue damage-induced intestinal stem cell division in drosophila tuberous sclerosis complex and myc coordinate the growth and division of drosophila intestinal stem cells drosophila sec is required for hormone secretion but not general exocytosis or neurotransmission synergy between bacterial infection and genetic predisposition in intestinal dysplasia suppression of drosophila cellular immunity by directed expression of the exos toxin gap domain of pseudomonas aeruginosa rac is a major actor of drosophila resistance to pseudomonas aeruginosa acting in phagocytic cells host-bacterial mutualism in the human intestine postsynaptic expression of tetanus toxin light chain blocks synaptogenesis in drosophila organization and function of septate junctions: an evolutionary perspective the claudin-like megatrachea is essential in septate junctions for the epithelial barrier function in drosophila interstrain variability in the virulence of aspergillus fumigatus and aspergillus terreus in a tolldeficient drosophila fly model of invasive aspergillosis genetic analysis of drosophila melanogaster susceptibility to intestinal vibrio cholerae infection essential function of drosophila sec in apical exocytosis of epithelial photoreceptor cells manipulation of host-cell pathways by bacterial pathogens drosophila, the golden bug, emerges as a tool for human genetics chapter , model organisms in the study of development and disease the drosophila homolog of the exo exocyst subunit promotes apical epithelial identity vibrio cholerae infection of drosophila melanogaster mimics the human disease cholera helicobacter pylori and clinical aspects of gastric cancer a transgenic drosophila model demonstrates that the helicobacter pylori caga protein functions as a eukaryotic gab adaptor targeted gene expression as a means of altering cell fates and generating dominant phenotypes developmental function of elf- : an essential transcription factor during embryogenesis in drosophila targeting of immune signalling networks by bacterial pathogens evidence for metabolic provisioning by a common invertebrate endosymbiont, wolbachia pipientis, during periods of nutritional stress drosophila intestinal response to bacterial infection: activation of host defense and stem cell proliferation lovastatin has significant activity against zygomycetes and interacts synergistically with voriconazole drosophila melanogaster as a facile model for large-scale studies of virulence mechanisms and antifungal drug efficacy in candida species drosophila melanogaster as a model host to dissect the immunopathogenesis of zygomycosis candida albicans cas , a regulator of cell wall integrity, is required for virulence in murine and toll mutant fly models exploring the concordance of aspergillus fumigatus pathogenicity in mice and toll-deficient flies the sars-coronavirus membrane protein induces apoptosis through modulating the akt survival pathway downregulation of cd by human immunodeficiency virus type nef is dependent on clathrin and involves direct interaction of nef with the ap clathrin adaptor use of rna interference in drosophila s cells to identify host pathways controlling compartmentalization of an intracellular pathogen genomic rnai screening in drosophila s cells: what have we learned about host-pathogen interactions? vsv infection is sensed by drosophila, attenuates nutrient signaling, and thereby activates antiviral autophagy entry is a rate-limiting step for viral infection in a drosophila melanogaster model of pathogenesis genome-wide rnai screen reveals a specific sensitivity of irescontaining rna viruses to host translation inhibition copi activity coupled with fatty acid biosynthesis is required for viral replication genome-wide rnai screen identifies genes involved in intestinal pathogenic bacterial infection molecular interactions between bacterial symbionts and their hosts the control of vascular integrity by endothelial cell junctions: molecular basis and pathological implications bacterial manipulation of innate immunity to promote infection models of infectious diseases in the fruit fly drosophila melanogaster rna interference analysis of legionella in drosophila cells: exploitation of early secretory apparatus dynamics proteolytic inactivation of map-kinase-kinase by anthrax lethal factor insect hemolymph clotting role and activation of type iii secretion system genes in pseudomonas aeruginosa-induced drosophila killing the drosophila systemic immune response: sensing and signalling during bacterial and fungal infections potent and specific genetic interference by double-stranded rna in caenorhabditis elegans the ap- a and ap- b clathrin adaptor complexes define biochemically and functionally distinct membrane domains knockout animals and natural mutations as experimental and diagnostic tool for studying tight junction functions in vivo claudins in occluding junctions of humans and flies host-pathogen interactions in drosophila perspective claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin- -deficient mice essential function in vivo for dicer- in host defense against rna viruses in drosophila over-expression of dscam and col a cooperatively generates congenital heart defects a screen for dominant mutations applied to components in the drosophila egf-r pathway anthrax lethal factor and edema factor act on conserved targets in drosophila anthrax toxins cooperatively inhibit endocytic recycling by the rab /sec exocyst new insights into the biological effects of anthrax toxins: linking cellular to organismal responses. microbes infect chlorpyrifos induces apoptosis and dna damage in drosophila through generation of reactive oxygen species a direct role for dual oxidase in drosophila gut immunity an antioxidant system required for host protection against gut infection in drosophila coordination of multiple dual oxidase-regulatory pathways in responses to commensal and infectious microbes in drosophila gut drosophila rnai screen identifies host genes important for influenza virus replication the spiroplasma heritable bacterial endosymbiont of drosophila linking epithelial polarity and carcinogenesis by multitasking helicobacter pylori virulence factor caga helicobacter pylori and gastric carcinogenesis wolbachia and virus protection in insects dll signalling through notch regulates formation of tip cells during angiogenesis daughter of sevenless is a substrate of the phosphotyrosine phosphatase corkscrew and functions during sevenless signaling eiger and wengen: the drosophila orthologs of tnf/tnfr evolution of host innate defence: insights from caenorhabditis elegans and primitive invertebrates sec , a component of the exocyst, promotes notch signaling during the asymmetric division of drosophila sensory organ precursors cell-mediated immunity in arthropods: hematopoiesis, coagulation, melanization and opsonization peptidergic clock neurons in drosophila: ion transport peptide and short neuropeptide f in subsets of dorsal and ventral lateral neurons disruption of synaptic transmission or clock-gene-product oscillations in circadian pacemaker cells of drosophila cause abnormal behavioral rhythms competing activities of heterotrimeric g proteins in drosophila wing maturation drosophila melanogaster-based screening for multihost virulence factors of pseudomonas aeruginosa pa and identification of a virulence-attenuating factor eater, a transmembrane protein mediating phagocytosis of bacterial pathogens in drosophila mismatch repair: mechanisms and relationship to cancer susceptibility role for lysosomal enzyme beta-hexosaminidase in the control of mycobacteria infection ablation of drosophila photoreceptor cells by conditional expression of a toxin gene nimrod, a putative phagocytosis receptor with egf repeats in drosophila plasmatocytes sequence homology and structural analysis of the clostridial neurotoxins virulence studies of scedosporium and fusarium species in drosophila melanogaster does preexposure of aspergillus fumigatus to voriconazole or posaconazole in vitro affect its virulence and the in vivo activity of subsequent posaconazole or voriconazole, respectively? a study in a fly model of aspergillosis increased virulence of zygomycetes organisms following exposure to voriconazole: a study involving fly and murine models of zygomycosis drosophila exocyst components sec , sec , and sec regulate de-cadherin trafficking from recycling endosomes to the plasma membrane anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic amp concentrations of eukaryotic cells endothelial signalling by the notch ligand delta-like restricts angiogenesis myosin v, rab , and drip direct apical secretion and cellular morphogenesis in developing drosophila photoreceptors toll-deficient drosophila flies as a fast, highthroughput model for the study of antifungal drug efficacy against invasive aspergillosis and aspergillus virulence delta-like ligand (dll ) is induced by vegf as a negative regulator of angiogenic sprouting pseudomonas aeruginosa cystic fibrosis isolates from individual patients demonstrate a range of levels of lethality in two drosophila melanogaster infection models an epidermal barrier wound repair pathway in drosophila is mediated by grainy head targeted expression of tetanus toxin: a new tool to study the neurobiology of behavior parallels between tissue repair and embryo morphogenesis defective stratum corneum and early neonatal death in mice lacking the gene for transglutaminase (keratinocyte transglutaminase) mutations in drosophila sec reveal a function in neuronal targeting for a subset of exocyst components blood cells of drosophila: cell lineages and role in host defence host-pathogen interactions in drosophila perspective bacterial toxins: cellular mechanisms of action inducible cell ablation in drosophila by cold-sensitive ricin a chain genomic screening with rnai: results and challenges characterization of myosin-ia and myosin-ib, two unconventional myosins associated with the drosophila brush border cytoskeleton hyperactivation of the folded gastrulation pathway induces specific cell shape changes a kinome rnai screen identified ampk as promoting poxvirus entry through the control of actin dynamics anthrax toxins the exocyst component sec is required for membrane traffic and polarity in the drosophila ovary mutations in the exocyst component sec disrupt neuronal membrane traffic, but neurotransmitter release persists sec mutations and the drosophila exocyst complex sec , a member of the exocyst complex, mediates drosophila embryo cellularization activitydependent regulation of synaptic size in drosophila neuromuscular junctions cricket paralysis virus antagonizes argonaute to modulate antiviral defense in drosophila the drosophila claudin kune-kune is required for septate junction organization and tracheal tube size control hypersusceptibility to vesicular stomatitis virus infection in dicer -deficient mice is due to impaired mir and mir expression systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions multiple transcription factor codes activate epidermal wound-response genes in drosophila applications of high-throughput rna interference screens to problems in cell and developmental biology in vivo rnai: today and tomorrow contribution of individual toxin components to virulence of bacillus anthracis drosophila rnai screen reveals cd family member required for mycobacterial infection escrt factors restrict mycobacterial growth brush border spectrin is required for early endosome recycling in drosophila developmental biology: our fly cousins' gut homeostasis in infected epithelia: stem cells take the lead refinement of tools for targeted gene expression in drosophila dos, a novel pleckstrin homology domain-containing protein required for signal transduction between sevenless and ras in drosophila drosophila scavenger receptor ci is a pattern recognition receptor for bacteria functional genomic analysis of phagocytosis and identification of a drosophila receptor for e. coli mutant molecular motors disrupt neural circuits in drosophila hippo signaling regulates drosophila intestine stem cell proliferation through multiple pathways regulation of vascular morphogenesis by notch signaling role of serotonergic neurons in the drosophila larval response to light absence of transitive and systemic pathways allows cell-specific and isoform-specific rnai in drosophila innate immunity and gut-microbe mutualism in drosophila ars regulates both mirna-and sirnadependent silencing and suppresses rna virus infection in drosophila innate antiviral immunity in drosophila antiviral immunity in drosophila requires systemic rna interference spread a cystic fibrosis epidemic strain of pseudomonas aeruginosa displays enhanced virulence and antimicrobial resistance host-bacteria homeostasis in the healthy and inflamed gut learning tolerance while fighting ignorance jc virus infection in colorectal neoplasia that develops after liver transplantation the genetics and cell biology of wolbachia-host interactions discovery of insect and human dengue virus host factors autophagy is an essential component of drosophila immunity against vesicular stomatitis virus discerning the complexity of community interactions using a drosophila model of polymicrobial infections notch signalling limits angiogenic cell behaviour in developing zebrafish arteries receptor tyrosine kinases: specific outcomes from general signals gliotoxin production in aspergillus fumigatus contributes to host-specific differences in virulence identification of drosophila gene products required for phagocytosis of candida albicans phagocytosis of candida albicans by rnai-treated drosophila s cells a systems biology analysis of the drosophila phagosome the notch ligand delta-like negatively regulates endothelial tip cell formation and vessel branching remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles targeted expression of tetanus toxin light chain in drosophila specifically eliminates synaptic transmission and causes behavioral defects the behaviour of drosophila adult hindgut stem cells is controlled by wnt and hh signalling the bacterial symbiont wolbachia induces resistance to rna viral infections in drosophila melanogaster a homolog of drosophila grainy head is essential for epidermal integrity in mice contribution of toxins to the pathogenesis of inhalational anthrax the rna silencing endonuclease argonaute mediates specific antiviral immunity in drosophila melanogaster anthrax lethal factor cleaves the n-terminus of mapkks and induces tyrosine/threonine phosphorylation of mapks in cultured macrophages systemic spread of sequence-specific transgene rna degradation in plants is initiated by localized introduction of ectopic promoterless dna rna interference directs innate immunity against viruses in adult drosophila extensive diversity of ig-superfamily proteins in the immune system of insects systemic rnai in c. elegans requires the putative transmembrane protein sid- junctional trafficking and epithelial morphogenesis sinuous is a drosophila claudin required for septate junction organization and epithelial tube size control refined lexa transactivators and their use in combination with the drosophila gal system autophagic control of listeria through intracellular innate immune recognition in drosophila we thank bill mcginnis, victor nizet, steve wasserman, emily troemel, raffi aroian, margery smelkinson and beatriz cruz-moreno for helpful discussions and comments on the manuscript. the authors declare that they do not have and competing or financial interests. key: cord- -i okohf authors: hartigan, ashlie; kosakyan, anush; pecková, hana; eszterbauer, edit; holzer, astrid s. title: transcriptome of sphaerospora molnari (cnidaria, myxosporea) blood stages provides proteolytic arsenal as potential therapeutic targets against sphaerosporosis in common carp date: - - journal: bmc genomics doi: . /s - - -y sha: doc_id: cord_uid: i okohf background: parasites employ proteases to evade host immune systems, feed and replicate and are often the target of anti-parasite strategies to disrupt these interactions. myxozoans are obligate cnidarian parasites, alternating between invertebrate and fish hosts. their genes are highly divergent from other metazoans, and available genomic and transcriptomic datasets are limited. some myxozoans are important aquaculture pathogens such as sphaerospora molnari replicating in the blood of farmed carp before reaching the gills for sporogenesis and transmission. proliferative stages cause a massive systemic lymphocyte response and the disruption of the gill epithelia by spore-forming stages leads to respiratory problems and mortalities. in the absence of a s. molnari genome, we utilized a de novo approach to assemble the first transcriptome of proliferative myxozoan stages to identify s. molnari proteases that are upregulated during the first stages of infection when the parasite multiplies massively, rather than in late spore-forming plasmodia. furthermore, a subset of orthologs was used to characterize d structures and putative druggable targets. results: an assembled and host filtered transcriptome containing proteins, mapping to , contigs was mined for protease virulence factors and revealed that cysteine proteases were most common ( %), at a higher percentage than other myxozoans or cnidarians ( – %). two cathepsin ls that were found upregulated in spore-forming stages with a presenilin like aspartic protease and a dipeptidyl peptidase. we also identified downregulated proteases in the spore-forming development when compared with proliferative stages including an astacin metallopeptidase and lipases (qpcr). in total, transcripts were identified as putative proteases using a merops database. in silico analysis of highly transcribed cathepsins revealed potential drug targets within this data set that should be prioritised for development. conclusions: in silico surveys for proteins are essential in drug discovery and understanding host-parasite interactions in non-model systems. the present study of s. molnari’s protease arsenal reveals previously unknown proteases potentially used for host exploitation and immune evasion. the pioneering dataset serves as a model for myxozoan virulence research, which is of particular importance as myxozoan diseases have recently been shown to emerge and expand geographically, due to climate change. the relationship between parasites and their hosts is under constant pressure; parasites must invade, replicate and feed whilst avoiding the host immune system. proteases are the weapon of choice for parasites to overcome these challenges within the host, and can be specifically adapted for cleaving host proteins or modifying their own proteins for immune system avoidance [ ] [ ] [ ] . proteases are often high priority proteins for investigation as they have essential roles in development, invasion or feeding [ ] . however, proteases are involved in other cellular functions e.g. transport and activation of other peptidases, and it can often be unclear which peptidases are essential to parasite survival or success [ , ] . drug or interference targets can be difficult to identify in a wash of uncharacterized proteins, however proteases linked to an essential cellular pathway or localised to a particular organelle e.g. lysosome can be considered useful targets for life cycle or development disruption [ , ] . anti-parasite drugs currently available have been identified by screening sets of compounds in vitro culture systems and by borrowing compounds that have worked in other pathogens and applying those to a new parasite model [ ] . firstly, this limits progress to organisms and life stages that can be isolated and cultured; secondly it relies on applicable compounds having been found in related organisms; and thirdly it limits discovery as it looks at one target at a time for feasibility. in silico drug target discovery in contrast has the attractive attributes of speed, low cost and no requirement for living parasites. in the case of non-model organisms this is likely the first step before prioritising any protein for further experimentation with the aim of anti-parasite treatment development. myxozoans are parasitic cnidarians that are important pathogens to both wild and cultured fish populations and yet there are no drug targets specified for this group and limited proteolytic studies to examine activity or function of selected proteins [ , ] . myxozoans are suggested to have reduced genomes compared to their free living cnidarian relatives [ , ] which could have an impact on the range and diversity of the peptidases expressed. many aspects of myxozoan biology are still unknown or inferred by comparison with other parasites to infer biology such as their metabolism (thelohanellus kitauei - [ ] ), their replication [ ] or proteins interacting with the host immune system (reviewed in [ ] ). myxozoans are entirely parasitic in their life cycle, they alternate between a vertebrate and an invertebrate host with two entirely different types of transmissible spores in each developmental phase [ ] [ ] [ ] [ ] . myxospores are often hardy stages that are capable of being exposed to the environment for long periods of time waiting for uptake by their invertebrate hosts. the actinospores are generally more fragile and only viable for a limited period of time as they are released into the water column to encounter a suitable vertebrate host [ ] . there are two main sources of material for genomic and transcriptomic analysis, plasmodia or cysts of developing myxospores from the vertebrate [ , ] or actinospores released from their invertebrate host [ ] . spore development represents the final step prior to transmission with the genetic arsenal related to their production of durable spores often expressed in cysts, separated from the host immune response by connective tissue, while actinospores are collected from the environment, prior to infecting their vertebrate hosts. therefore, they do not provide many insights into what proteins are helping the parasite feed or replicate or evade immune detection. sphaerosporids are a major clade of the myxosporea, with a large proportion found in bony and cartilaginous fish, and amphibians [ ] [ ] [ ] [ ] [ ] . a specific trait that has only been identified in this clade is the presence of large, extracellular stages circulating in the blood stream of their fish hosts [ ] [ ] [ ] . the parasites not only use the blood for transport to their target organ but proliferate within it and are present almost all year round (fig. , [ , , ] ). sphaerospora molnari is a parasite of the common carp in central europe with motile blood stages that provoke a strong immune response [ ] and are a likely co-factor for developing swim bladder inflammation [ ] . s. molnari blood stages (smbs) are prime targets for parasite intervention therapy, as they are ) responsible for massive proliferation in the earliest stages of infection of fish, ) freely circulating in the blood and any drug targeting the smbs would not need to be applied to host tissue or taken up by host cells; ) they are circulating in the blood for an extended period and therefore there is a longer window for application of anti-parasite therapies. in addition, preliminary protein studies on smbs show a high level of sequence divergence even in highly conserved proteins such as actin [ ] and therefore smbs could potentially have proteases that are highly divergent from their hosts as well as other cnidarians which would aid protein target assay development. this study examines protease families and groups present in the transcriptome of smbs to investigate their diversity and divergence. we compare key protease groups with examples known from other parasites that have been successfully flagged as drug or anti-parasite targets. in addition, we provide gene expression data for selected candidates with the goal of identifying stage-specific proteases of interest for future functional studies. this transcriptome is the first next generation sequencing dataset from any sphaerosporid species, and also the first dataset from a highly proliferative, extrasporogonic developmental myxozoan stage. pooled host (cyprinus carpio) blood cells and sphaerospora molnari blood stages (smbs) from infected fish were used for this transcriptome. illumina hiseq sequencing yielded a total of , , clean paired reads, mapping these to the gene models of c. carpio removed , , reads (bioproject prjna ). a trinity assembly of these remaining reads gave , transcripts, (mean length nt, . % gc content). , of these transcripts ( . %) were found in the carp genome with an e-value of e − or more and a percentage identity of > %. the remaining , transcripts were therefore assumed to be s. molnari, and were translated into , proteins (table ) . to examine the presence of potential chimeric sequences, we amplified a substantial number (n = ) of our flagged proteases and ribosomal dna to verify their sequence according to our assembly. sanger sequencing of the complete ribosomal dna yielded , bp (genbank acc. nr mk ), and large fragments of all flagged proteases were also verified from cdna (supplementary data). screening with busco, identified that s. molnari retained and is expressing at least half of the benchmark metazoan genes [ ] ( % of the single copy metazoan genes). we completed the same analysis for myxozoans myxobolus cerebralis and kudoa iwatai, and non-myxozoans polypodium hydriforme, edwardsiella lineata, nematostella vectensis (table ). s. molnari had the highest number of complete buscos of all the myxozoan datasets, whereas n. vectensis had the highest overall ( / , %), similar results were found for the single copy buscos. s. molnari had the lowest number of missing genes ( / , . %) within the myxozoans, in comparison n. vectensis was only missing genes ( / , %) ( table ) . developmental cycle of sphaerospora molnari within its host cyprinus carpio. sphaerospora molnari blood stages and infected gill images by a.s. holzer, (blood stages modified from [ , ] we queried the transcriptomic dataset of smbs for proteases using representative protease sequences downloaded from the merops database. less than % of all transcripts had a strong sequence match. there were homologs identified in smbs representing peptidase families, the majority of the proteases were cysteine ( %), followed by metalloproteases ( %), serine ( %), threonine ( %) and aspartic groups ( %) (fig. ). families that were highly represented in smbs (table ) were c papain-like proteases, c and c ubiquitinyl hydrolases, m aminopeptidases and dipeptidyl peptidases, m ubiquitin releasing proteases often associated with proteasomal degradation and t proteasome proteases. there were many families that were absent in all examined cnidarians, and even more, missing from only the myxozoans compared to the free living species e.g. s lysosomal carboxypeptidase. to more closely examine the proteases present in smbs, we looked at enzymes that were in highly represented families, and were transcripts with a high number of reads mapping to them (tpm) or had high similarity to proteases known from other parasite species. in particular, we examined cysteine proteases in the merops family c cathepsin l, aspartic proteases in the family a -signal peptide and presenilin-like proteases, metallopeptidases in the m the metzincins, and s the prolyl oligopeptidase family. cathepsins: s. molnari's transcriptome revealed eight cathepsin-l like sequences by sequence homology, however, five were excluded from our further analysis due to ) incomplete transcript and ) missing or uncharacterised active sites at either substrate s or s sites, or ) a sequence homology that appeared to be closest to cathepsin l but in fact had chymotrypsin-like folds (ser-his-asn) or gly-his-asn catalytic triads. three cathepsin ls analysed (cathl - ) were all propeptides with signal peptides which may indicate later activation within the cell (e.g. lysosome) or extracellularly [ ] . all had conserved a glutamine for the oxyanion hole known for cysteine peptidases, however, sm_cl did not retain a stabilising asparagine close to the his active site, this was replaced by a negatively charged aspartic acid. we aligned the predicted tertiary structures of s. molnari cathepsin ls to a procathepsin l from a metazoan parasite with x-ray crystallography evidence (pdb: o x, [ ] ) ( fig. a-c) . two of s. molnari's cathepsin ls, sm_cl and were able to be aligned to the crystal structure with high confidence, however, there was a marked difference in the distribution of hydrophobic resides ( fig. b -c). in particular, there were higher numbers of hydrophilic residues at the active site compared to fasciola hepatica ( fig. d-f ). the number of charged residues were similar overall, however the distribution of positive and negatively charged amino acids was different between all three proteins ( fig. g -i). aspartic: we identified two aspartic proteases in the transcriptome of smbs, one presenilin-like and a signal peptide peptidase-like sequence (sm_sppl). the presenilin-like protease had a lower tpm value than sm_ sppl ( . compared to . ). we therefore focused our analysis on sm_sppl, which clustered with other invertebrates (fig. a) and conformed to the structure of a signal peptide peptidase including active residues in transmembrane domains , and in the predicted model (fig. b, c, d) . sm_sppl has the appropriate active site residues and was able to align the predicted tertiary structure of a sppl. sm_sppl was distinguished from a presenilin by the presence of qpally motif in its last transmembrane domain, like others in this group [ ] . metallopeptidases: seventy two metallopeptidases in of families were flagged in the s. molnari proliferative stage peptidases. all of the s. molnari metallopeptidase families were shared by one or both of the other myxozoan species (m. cerebralis or k. iwatai) examined as well as two or three of the free living species (table ). the highest proportion of smbs metallopeptidases were in the m family (adamalysins and reprolysins). we examine five of these metallopeptidases in more detail (sm_mp tpm = . ; sm_ mp tpm = . ; sm_mp tpm = . ; sm_mp tpm = . ; sm_mp tpm = . ). all except one of the five have the methionine turn and zinc binding motif hexxhxxgxxh with either a serine or an aspargine binding residue. two of the target metallopeptidases had signal peptides, and almost all had reprolysin and disintegrin domains. sm_mp has a potential c-terminal transmembrane tail that may anchor it similarly to other well-known adam proteins. serine: multiple serine proteases were identified within smbs transcriptome, the largest was merops family s . the transcriptome of smbs yielded a serine protease with sequence and structural homology to dipeptidyl peptidase (fig. b, c) . smbs_dpp contained the catalytic triad (ser- , asp- , his- ), a predicted transmembrane domain at the n-terminus. phyre was able to model the sequence with % confidence according to the models of other dipeptidyl proteases (dppiv and viiii). the sequence clustered to thelohanellus kitauei dppiv and hydra vulgaris pop isoforms (fig. a) however, both of the h. vulgaris isoforms contained a transmembrane domain and a high homology to dppivs rather than pops. we then examined the expression of eight key proteases in blood stages compared to spore forming gill stages by qpcr and also in silico expression (tpm). three cathepsins sm_cl , and ; a presenilin like aspartic protease (sm_sp ); a dipeptidyl peptidase (sm_ddpiv); a metallopeptidase (sm_mp ) and two lipases (sm_lip and ) were used as target proteases. half of our candidates were upregulated in blood stages rather than gill stages (fig. ) . expression of sm_cl was over x higher in sporogonic gill stages than presporogonic smbs (fig. a) . sm_ cl was predicted to contain a transmembrane domain and had a relatively low tpm value according to our assembly ( . ) compared to the other two cathepsins sm_cl and (tpm = . and . , respectively), both of which had signal peptides and were transcribed as procathepsins. both sm_cl and showed similar expression in the blood and the gills. an astacin metallopeptidase and two lipases were also upregulated in blood compared to gill samples (fig. b) . in contrast, both the aspartic and serine proteases, sm_sp and sm_dppiv respectively were significantly upregulated in spore forming stages in the gills. sm_dppiv did not have a high relative expression within our transcriptome (tpm = . ), however when we compared its expression in blood stages vs. gill stages, we found it was more highly expressed in sporogonic gill stages by almost fold. sm_sp had a higher tpm value than sm_dppiv ( . ) and was times more highly expressed in the gills than blood. this transcriptome analysis of s. molnari is the first from this entire clade of sphaerosporid myxozoans, this is the only group for which an invertebrate host is unknown and is the only dataset from an extrasporogonic stage of development. therefore, it offers a unique insight into the mechanisms of myxozoan development and host interactions. our focus on proteases, was primarily in the pursuit of identifying proteins that would be worthy of further investigation to understand their role in the host-parasite relationship. the transcriptome of s. molnari blood stages (smbs) appears to follow a similar trend as other parasitic and free living cnidarians in its heavy at richness for many of its genes [ ] and its overall reduction in genes [ , ] . gene divergence and at richness of the smbs dataset aids distinction of host and parasite genes in many cases and could also aid targeted gene therapy as seen in other anti-parasite drug design e.g. plasmodium falciparum [ ] . the number of proteins identified could have been limited due to the extensive host filtering we conducted on the dataset, divergent genes and/or the nature of the particular life stage of smbs. separation of the smbs from the host immune cells would significantly improve further transcriptomic analysis to combine physical and bioinformatic filtering. more than half of the known busco metazoan genes were identified in this dataset, similarly to m. cerebralis triactinomyxon stages and sporogonic k. iwatai transcriptomes. although using a different comparative dataset (cegma, previous benchmarking metazoan dataset) chang et al. [ ] also noted a reduced number of core eukaryotic genes in the transcriptomes and genomes of k. iwatai ( % of cegs, compared to our % of buscos) and m. cerebralis ( % of cegs compared to % of bus-cos) whilst p. hydriforme consistently had more than its myxozoan relatives and was closer in numbers to the free living species ( % of cegs and % of buscos). busco genes are also used to estimate the completeness of a genomic or transcriptomic dataset, whilst it is unlikely that we sequenced the extent of the expressed transcripts of s. molnari due to overwhelming host contamination, it is interesting to note an overall reduction in this gene set for myxozoans in general. for comparison, other early branching metazoans and cnidarians have retained a high percentage of these conserved proteins, e.g. placozoa species hoilungia hongkongensis had - . % [ ] ; sponge halisarca caerulea had . % [ ] . free living cnidarians have variable accounts of fig. real-time pcr of selected proteases in s. molnari blood stages (non-sporogonic) and gill stages (sporogonic). relative abundance to two housekeeping genes (elongation factor and glyceraldehyde- -phosphate dehydrogenase) in cdna samples of circulating blood stages (n = ) and spore forming gill stages in common carp (n = ) including % confidence intervals and average for each marker. a cathepsins (sm_cl , and ) in blood stages (red) and sporogonic gill stages (blue); b other proteases (a -signal peptide and presenilin like peptidases (sm_sp ); m metallopeptidase (sm_mp ); s -prolyl oligopeptidase (sm_dppiv ) and lipases (sm_lipase and ) in blood stages (red) and sporogonic gill stages (blue) their retained complete busco genes, e.g. acropora digitifera % ( / ) [ ] ; exaiptasia pallida . % ( / ) [ ] ; scleractinian coral porites lutea . % [ ] . other metazoan parasites have also retained a higher level of genes e.g. the trematode microphallus sp. maintained close to % of genes from the same metazoan dataset [ ] . the transcriptomes of parasitic nematode teladorsagia circumcincta were found to have only - % of the conserved gene set yet appeared to have good matches to the representative genome [ ] . furthermore, cestode parasites have a range of conserved eukaryotic genes from taenia multiceps ( . %) to echinococcus multilocularis ( . %) [ ] . additionally, despite removing protein sequences that were more than % similar to each other in the s. molnari dataset, there were a high number of duplicated single copy busco genes ( table ). this may be caused by the pooling of wild-type individuals sampled from different locations and populations. de novo assemblers such as trinity, designed to generate alternative transcripts, are more sensitive to sequencing errors and/or highly heterozygous datasets, and thus multiple loci may have been assembled into 'isoforms' of the same parent transcript increasing the amount of duplication [ , ] . we examined some specific examples of proteases of interest within the s. molnari transcriptome that were homologous to other known parasitic proteases involved in important host-parasite functions. there are examples of parasitic proteases that have been exploited as drug targets in all different groups and protease families. two of the aspartic peptidases we identified in the smbs transcriptome were also in groups that had been identified as potential drug targets. a presenilin-like and a signal peptide peptidase-like protease (sm_sppl) were found in smbs both are in the merops protease family a . the proteases are closely related, sppls are transmembrane proteases with their catalytic sites buried in bordering transmembrane domains, and have varied functions in eukaryotes depending on their localization within the cell [ , ] . in humans, they are involved in the processing of peptides for the mhc i epitopes and their central role in processing signal peptides indicates an important role in signaling and protein modification as part of the endoplasmic reticulum [ ] . in contrast to sppls, presenilins require a co-factor and multiple subunits to form a catalytic γ -secretase complex [ ] . both aspartic proteases have been identified as potential drug targets in plasmodium falciparum [ ] , and can potentially play a role in the cleavage of peptides for antigen presentation on a cell surface [ ] . sppl blockers have been shown experimentally in parasite-derived homologs e.g. ly , (reduced plasmodium berghei in mice and humans [ ] ), and bioinformatically as suggested in protozoan species toxoplasma gondii, leishmania infantum, and trypanosoma cruzi [ ] . both s. molnari proteases are divergent from their hosts and in the case of sm_sppl have been shown to be highly expressed during its sporogenesis development in the host's gills. this localization may be useful for targeting future therapeutic assays at such an aspartic protease in this system. another group of proteases that are commonly targeted for anti-parasite therapy are the metallopeptidases. these have been associated with many functions of parasitic development, they are linked to digestion e.g. falcilysin in plasmodium falciparum [ ] , host immune system disruption e.g. matrix and secreted metalloproteases [ , ] ; or host immune system evasion e.g. leishmania gp [ ] . smbs had a large variety of metallopeptidases, it is also important to note families that are missing in the myxozoan species, for example we did not identify any m homologues which have been originally characterised in leishmania to aid immune system avoidance in promastigotes [ ] . nor did we identify any matrix metallopeptidases (m family) which have also been previously identified as protozoan parasite drug targets [ , ] . one of the largest groups we identified in smbs was m , the metzincins. these have been found in other parasitic organisms feeding in their host's blood and are considered important for digestion or nutrient uptake which may be their role in smbs as they are circulating in the host's blood. the diversity of smbs metzincins also indicates varied roles for these metallopeptidases in these developmental stages. although the function of each specific protease are not known, and they were not considered to be highly expressed (tpm values range from . - . ), they share some valuable attributes that make them strong candidates for future research. primarily, they share a key domain and binding site and none of the potential target sequences (sm_mp - ) had "cysteine rich" domains identified in their sequence which has been discussed as a possible hindrance to recombinant protein production [ ] . recombinant protein application or other interference with parasite metzincins has been shown to be a successful anti-tick treatment [ ] and may potentially be employed with any of these smbs metallopeptidases. a recombinant protein with epitopes of multiple smbs metzincins, considering they are so divergent from each other as well as any recognisable fish host homolog, could hold potential for a therapeutic experiment. in the serine proteases, the largest family found in smbs was s . this family has multiple groups of which only some have been identified as putative drug targets. one such group is the dipeptidyl peptidases (dpps) and prolyl oligopeptidases (pops), which specialize in the cleavage of proline residues (dpps cleave at n-terminus, pops cleave at c-terminal). the s protease we looked at as a potential drug target in smbs had similarities to both groups however we determined it to be a dpp according to its sequence, phylogenetic position and its structural homology to dppiv (fig. ). dppiv has also been identified in bee and snake venoms and possibly influences vasodilation and constriction, something that may be useful to smbs in host blood vessels. dppiv exists as a homodimer (potentially tetramer) targets proline residues and has suggested roles in antigen presentation on the cell surface, cell adhesion and collagen binding activity [ ] . in protozoans, dppivs have been shown to play a role in the encystment of giardia, a process that can be blocked by the application of inhibitors [ ] . ddpivs have been associated and inhibited in other blood-feeding parasites including haemonchus contortus where it is suspected to play a role in fibrogen breakdown and coagulation in the blood [ ] . due to the choice and variety of dppiv inhibitors that are available for both human and veterinary medical applications, we propose that smbs dppiv could be a good target for assessing the impact of its inhibition on the proliferation and metabolism of s. molnari in its host. this is based partially on its unique sequence as evidenced from its relation to other homologs, its predicted structure which could aid the application of known inhibitors, and its expression in a developmental stage that could be feeding on blood. one of the most well researched protease groups in parasites are cysteine proteases, commonly investigated for their potential as therapeutic targets in parasites as they are known to be involved in a variety of pathways as well as being associated with parasitic development and proliferation [ ] . in myxozoans, cysteine proteases have been suggested to be involved in the proteolytic destruction of host tissue based on activity and substrate assays [ , , ] . they are also associated with host hemoglobin degradation in blood feeding parasites, e.g. many parasitic helminths [ ] and maybe the case for s. molnari which proliferates in host blood and interacts with erythrocytes [ ] . the replacement of a stabilising asparagine close to the his active site of sm_cl was also seen in a cathepsin l of the myxozoan kudoa thyrsites. k. thrysites is suggested to use its cathepsins for the degradation of host tissue, i.e. "milky flesh disease" [ ] . s. molnari cathepsins appear to have all of their disulfide bridges in the three isoforms we examined in contrast to the cathepsin ls reported from k. thyrsites [ ] . sm_cl , and had close homology to fasciola hepatica cathepsins, these proteases are expressed throughout f. hepatica's development with distinct roles in feeding and invasion based on their binding sites [ , ] . all sm_cls had higher numbers of hydrophilic residues at the active site compared to f. hepatica (fig. a-c) . the number of charged residues were similar to each other overall, however the distribution of positive and negatively charged amino acid was different between all three proteins (fig. ) . these changes could potentially impact the activity and substrate affinity for the proteases. cysteine proteases are particularly studied in parasites due to their roles in moulting, encysting and digestion across parasite taxa, and in particular they are often targeted for anti-parasite therapies or inhibition [ , , [ ] [ ] [ ] . here, we present the first expression analysis of cathepsins in smbs that are potentially important in the feeding, immune evasion or tissue penetration of this parasite in its host. characterisation of these proteases (substrate, inhibitors, abundance in the proteome) will advance our knowledge of the roles these cathepsins play in the development of this parasite and further inform our prioritization of protease targets for intervention and control assays. cathepsin ls were the most highly expressed (using tpm as indication) proteases identified within our transcriptome, that combined with the large number of in vitro and in vivo experiments on other parasite cathepsins and their effect on parasite survival make them prime candidates for further development. some examples use peptides mimicking compounds such as aziridine- , dicarboxylate-based inhibitors to inhibit cathepsin l activity e.g. trypansoma brucei rhodesiense [ ] or non-peptidic inhibitors e.g. chalcones in f. hepatica [ ] . s. molnari is unusual in the number of cathepsin l's expressed in its blood stages, not just isoforms but distinct proteases. until in vitro assays are able to better pinpoint which cathepsins are linked to smbs survival, we rely on sequence similarity and predictive modeling to infer homology to other cathepsins including those that have been inhibited successfully. sm_cl and as described above, had signal peptides for potential secretion and they differed in amino acid composition ( . % identity). sm_cl had more hydrophobic residues at the s active site and sm_cl was more highly expressed in the blood stage than sm_ cl . both had predicted structures that aligned to fhcl f. hepatica's cathepsin's structure which has been inhibited as well as another of its cathepsins fhcl with flavonoid compounds both experimentally and with computational docking [ ] . both f. hepatica cathepsins exhibited hydrophobic interactions with compound c , and were successfully inhibited although at different rates depending on the concentration of the compound. experimental evidence would be needed, however we propose sm_cl and as good candidates for inhibition with flavonoids such as c in future in vitro assays. the divergence in sequence identity from their host, their expression in both blood and gill stages and their similarity to proteases that have been successfully blocked in such assays are all good evidence that these could be the first drug targets for s. molnari. comparison of the expression of some of the proteases in non-spore forming blood stages and gill sporogonic stages gave further insights into the function of some groups. the cathepsin ls that we examined each had a different expression profile during smbs development, with sm_cl being the main cathepsin expressed in the gills. localisation and expression would influence which cathepsin to target for future work, and may indicate roles in sporogenesis for sm_cl compared to cl and cl in the blood. cl and may act as other cathepsins in blood feeding parasites and break down hemoglobin for sustenance. lipases and metallopeptidases are also associated with feeding in parasites and were comparatively higher in smbs than in the gills. astacin metallopeptidases have also been shown to break down complex proteins such as hemoglobin or migrate through tissues [ , , ] , and its upregulation in the proliferative developmental stage rather than spore forming could indicate a role in nutrient acquisition or penetration of target tissues (fig. ) in smbs. lipases have been suggested to play role in parasite cell surface protein presentation as well as digestion of host protein [ , ] , the two candidates we show are upregulated in smbs should be further investigated for their substrate and localisation within the smbs to further characterise if they are integral to parasite survival. disruption of such enzymes could be of interest to uncover the proteomic arsenal smbs use to digest and navigate its host for survival. conversely, the aspartic (sm_sp ) and serine protease (sm_dppiv) would be more informative to investigate where they appear to be highly expressedthe gills. these two proteases have been associated with antigen presentation and host immune system avoidance, it could be significant that they are relatively abundant when the parasite is bound within host target tissue, rather than its extracellular blood stage, where it would be directly in conflict with the host immune system. localisation and further characterisation will be vital to learning more about the role these proteases play in the developmental stages we identified them in and determine if they are interesting targets with anti-parasitic potential. we produced and explored the first transcriptomic dataset of early proliferative myxozoan stages to date and identified family expansions in cysteine, metallo and threonine clans. we did not identify any myxozoanspecific radiations in particular groups, however, all of the myxozoan proteases we examined were highly divergent from each other as well as from other cnidarians. vaccine development against a number of metazoan parasites is based on proteases as antigens of interest [ ] [ ] [ ] . however, with regard to myxozoans, the function and involvement of these enzymes in host-parasite interaction first need to be elucidated as a major lack of knowledge exists with regard to metabolomics and the molecular means of host interaction. the general strategy for therapeutically targeting proteases is to identify a specific inhibitorgenerally a small moleculethat blocks the active site. discovery efforts for new inhibitors have typically been based on the structure of known protease substrates, presenting a substantial challenge for the development of peptidomimetic compounds that have the pharmacokinetic characteristics needed to be suitable as a drug. this study advances our knowledge of myxozoan protease sequence, predicted structure and in some cases hydrophobicity and amino acid changes. this information furthers investigation into the potential role these proteases play in the development, sustenance and host immune evasion of these important parasite stages. vaccination plays an important role in commercial largescale fish farming and is a key reason for the success of salmon aquaculture, however, available vaccines are aimed at bacterial and viral pathogens, while parasite vaccines for fish are still inexistent [ ] , likely due to an insufficient knowledge of potential parasite target molecules of fish parasites, when compared with parasites of human or veterinary importance. it is now more than timely to explore new genomic data for such targets as epidemical models predict major emerging disease outbreaks and an increased geographic range of myxozoan species such as t. bryosalmonae, in relation to temperature increase [ , ] , with new records from northern european territories [ ] [ ] [ ] and recent reports of massive fish killings from the yellowstone river [ , ] , hutchins et al. ). exploring the enzymes expressed during early establishment and proliferation of myxozoan infections is essential to finding putatively relevant vaccine targets that can inhibit rapid multiplication of cryptic parasite stages in fish, long before the onset of disease. the proteases we discuss here are putative targets for further research, confirmation of their expression in different stages of s. molnari's life cycle (smbs vs. gill sporogonic stages vs. extracellular secretion) would be an invaluable method of testing their activity and function and therefore their use in anti-parasitic development. common carp (cyprinus carpio) were obtained from two localities, Štrmilov in czech republic ( . °n, . °e) and hortobágy in hungary ( . °n, . °e) during - . all fish were obtained commercially, were less than years old and were transported live to the laboratory. fish were anaesthetized with clove oil and blood was taken, all animal procedures were performed in accordance with czech legislation (section of protection of animals against cruelty act no. / ) and approved by the czech ministry of agriculture. fish were found to be infected with sphaerospora molnari blood stages (smbs), and whole blood was centrifuged for min at rpm in heparinized hematocrit tubes to isolate host white blood cells mixed with smbs. presporogonic s. molnari blood stages and sporogonic gill stages were isolated from experimentally infected fish (n = ) in the czech republic or recirculation system in hungary (n = ). specific parasite free fish tissues were selected from laboratory cultures within the czech republic (n = ). fish were euthanized according to the ethics license and methods above. with bowtie , using -very-sensitive parameter. all remaining reads were then assembled with trinity v . . and the transcripts were compared with the carp genome again for further host transcript identification (blast parameters: tblastx, e − ), those with a percentage identity of > % were removed to create a "non-host" dataset which was translated to protein using orfpredictor [ ] . redundancy of the translated non-host dataset was removed with cd-hit using . cutoff [ ] and blastp ( e − ) annotated with ncbi nr database. the non-redundant dataset was screened for assembly quality and completeness by identifying busco genes (http://busco.ezlab.org/) using the metazoan dataset [ ] . proteases and inhibitors were identified using the "meropsscan" dataset (https://www.ebi.ac.uk/merops/ index.shtml) using blastp, e − [ ] . signal peptides and transmembrane domains were predicted using signal p and tmhmm (http://www.cbs.dtu.dk/services/). transcriptomic sequences for other cnidarian species were downloaded from ncbi: myxozoans -myxobolus cerebralis triactinomyxon stages from tubifex host (prjna ), kudoa iwatai myxospores from cysts of sparus aurata tissue (prjna ). non-myxozoan cnidarians with endoparasitic life stages -polypodium hydriforme non-parasitic stolons (prjna ), and edwardsiella lineata (mix of parasitic and free living life stages) (downloaded from edwardbase site when was active in [ ] . finally, to compare with a completely free living relative, genome derived refseq mrna sequences of the anthozoan nematostella vectensis prjna were downloaded from ncbi. transcripts were translated into peptide sequences by orffinder [ ] and searched for proteases as above, the redundancy was removed ( . cutoff) and the non redundant dataset was screened for busco genes as above. target protease groups: four groups of proteases were more closely examined (a -signal peptide peptidases and presenilins, c -cathepsin ls, m metalloendopeptidases, s -prolyl oligopeptidases and dipeptidyl peptidases). representative s. molnari sequences with other sequences from genbank and uniprot (including fish sequences) were analysed phylogenetically with fish and other cnidarian and parasitic-derived sequences using raxml (l + g + i). tertiary structures were predicted for key proteases using the phyre server [ ] and models were compared and manipulated in pymol ver. sanger sequencing of key predicted proteases and rdna rna was extracted from biological replicates of s. molnari proliferative blood stages (pooled samples from several individuals) and spore-forming stages (individual fish). total host+parasite rna was isolated using the nucleospin rna kit (machery-nagel) including a dnase treatment step. rna concentration and purity was checked using a nanodrop (nd- ) spectrophotometer (nanodrop technologies) and cdna was synthesised using the transcriptor high fidelity cdna synthesis kit (roche). primers were designed to amplify full length sequences of selected proteases and ribosomal dna as single amplicons or with long overlaps between individual sections, to confirm the assembled transcriptome sequences. gene-specific primers were designed to amplify short, - bp regions suitable for qpcr (supplementary data). all primers were tested for functionality and specificity using conventional pcr prior to performing qpcr. qpcr was performed using the faststart universal sybr green master mix (rox) on lightcycler® real-time pcr system (roche). reactions contained . ul of faststart universal sybr green pcr master mix, roche, germany ( x conc.), μl of each forward and reverse primer ( μm conc.), . μl of pcr grade water, and approx. ng of cdna, resulting in a final volume of ul. cycling conditions were as follows: denaturation at °c for min, followed by cycles of °c for s, and °c for s and °c for s. melting curve analysis were performed after each qrt-pcr to ensure primer specificity. the relative expression ratio of each sample was calculated according to pfaffl [ ] , based on the take-off deviation of sample versus controls at each time point and normalized relative to elongation factor and glyceraldehyde- -phosphate dehydrogenase (housekeeping genes, [ ] ). confidence intervals, and box plots made in r. supplementary information accompanies this paper at https://doi.org/ . /s - - -y. additional file . supplementary data : 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tetracapsuloides bryosalmonae in fish tissue and environmental dna water samples orfpredictor: predicting protein-coding regions in est-derived sequences godzik cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences bateman merops: the database of proteolytic enzymes, their substrates and inhibitors production of a reference transcriptome and transcriptomic database (edwardsiellabase) for the lined sea anemone, edwardsiella lineata, a parasitic cnidarian the phyre web portal for protein modeling, prediction and analysis a new mathematical model for relative quantification in real-time rt-pcr publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors acknowledge bgi genomics for their sequencing services and are grateful for the support of our fish farmers in czech republic and hungary. authors' contributions ash conceived and planned the project; ah, ee and ash collected rna and dna samples; ah performed transcriptome analyses and structural modelling, ak and hp performed control sequencing of proteases and rdna, ak designed and performed qpcr assays; ah and ash wrote the manuscript. all authors agreed to the final version of it. the study was funded by the european commission horizon research and innovation action (project no. , parafishcontrol) for travel, reagents, sequencing and salary support, the czech science foundation (project no. - x; aquapara-omics) for travel and lab consumables and the hungarian national research, development and innovation office (project no. nn ) funded travel and lab consumables. no funding bodies played a role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. raw unfiltered sequence reads are deposited to genbank under bioproject prjna . primers used for qpcr are provided as supplementary material. host and parasite transcripts are available through dryad depository link sanger sequenced ribosomal dna was submitted to genbank under mk .ethics approval and consent to participate fish were obtained commercially, with the owners of the carp ponds providing us with the fish directly on site. fish were transported live to the laboratory and thereafter, fish were anaesthetized with clove oil and blood was taken. all animal procedures were performed in accordance with czech legislation (section of protection of animals against cruelty act no. / ) and approved by the czech ministry of agriculture. we declare that animal handling complied with the relevant european and international guidelines on animal welfare, namely directive / /eu on the protection of animals used for scientific purposes and the guidelines and recommendations of the federation of laboratory animal science associations. not applicable. the authors declare that they have no competing interests.received: october accepted: march key: cord- -nu ckeud authors: nieto-rabiela, f.; suzán, g.; wiratsudakul, a.; rico-chávez, o. title: viral metacommunities associated to bats and rodents at different spatial scales date: - - journal: community ecol doi: . / . . . . sha: doc_id: cord_uid: nu ckeud one of the main goals of community ecology is to measure the relative importance of environmental filters to understand patterns of species distribution at different temporal and spatial scales. likewise, the identification of factors that shape symbiont metacommunity structures is important in disease ecology because resulting structures drive disease transmission. we tested the hypothesis that distributions of virus species and viral families from rodents and bats are defined by shared responses to host phylogeny and host functional characteristics, shaping the viral metacommunity structures at four spatial scales (continental, biogeographical, zoogeographical, and regional). the contribution of host phylogeny and host traits to the metacommunity of viruses at each spatial scale was calculated using a redundant analysis of canonical ordering (rda). for rodents, at american continental scale the coherence of viral species metacommunity increased while the spatial scale decreased and quasi-clementsian structures were observed. this pattern suggests a restricted distribution of viruses through their hosts, while in the big mass (europe, africa, and asia), the coherence decreased as spatial scale decreased. viral species metacommunities associated with bats was dominated by random structures along all spatial scales. we suggest that this random pattern is a result of the presence of viruses with high occupancy range such as rabies ( %) and coronavirus ( %), that disrupt such structures. at viral family scale, viral metacommunities associated with bats showed coherent structures, with the emergence of quasi- clementsian and checkerboard structures. rda analysis indicates that the assemblage of viral diversity associated with rodents and bats responds to phylogenetic and functional characteristics, which alternate between spatial scales. several of these variations could be subject to the spatial scale, in spite of this, we could identify patterns at macro ecological scale. the application of metacommunity theory at symbiont scales is particularly useful for large-scale ecological analysis. understanding the rules of host-virus association can be useful to take better decisions in epidemiological surveillance, control and even predictions of viral distribution and dissemination. electronic supplementary material: supplementary material is available for this article at . / . . . . and is accessible for authorized users. the distribution of species can either be explained by random processes, as neutral theory explains (chave , hubbell , gonzalez ), or by environmental filters, as niche theory proposes (leibold et al. , lorencio , morand and krasnov . however, species distributions are influenced by the spatial and temporal contexts in which they occur, thus, neutral theory and niche theory should be tested across a different scale of analysis. there is evidence for the influence of different abiotic factors like temperature and elevation that can determine the organized distribution of species diversity, depending on the spatial scale studied (rahbek and graves ) . the differences in species distributions between scales depend on three factors: physical barriers, the ability to co-occur, and their ability to disperse (chase and myers ) . the diversity of symbionts has not been excluded from this type of analysis (guernier et al. ) . a large body of evidence suggest that the distribution of symbionts, defined as all organisms that must infect or inhabit hosts for at least part of their life cycle (mihaljevic ) , also responds to environmental filters determined by their hosts (morand and krasnov , mihaljevic , dallas and presley . host phylogeny can act as an environmental filter to symbionts communites due to their interaction in the evolutionary history, co-adaptation and ecosystem process in the community (streicker et al. , krasnov et al. , córdova-tapia and zambrano . similarly, the host functional characteristics can play a role as an environmental filter due to shared life histories or by spillover events pedersen , morand and krasnov ) . we used viral communities associated with rodents and bats to analyse the effect of environmental filters. rodents and bats are the most diverse orders of mammals thus they constitute suitable model taxa to explore the viral diversity providing relevant information to understand the dynamics of virus-host distribution (lorencio ) . these taxa have been recognized as the main reservoirs of a high number of zoonotic viruses, some of them with an enormous impact in public and animal health (luis et al. (luis et al. , . besides the individual approach, the study of the association between viral metacommunities associated to bats and rodents at different spatial scales f. nieto-rabiela , g. suzán , a. wiratsudakul and o. rico-chávez , host and virus requires a clear understanding of the ecological context of infection and transmission be required (woolhouse , suzán et al. , johnson et al. . because the host-virus system is intimately embedded within the communities, it is possible to recognize the existence of an organization in the distribution of viral diversity and later recognize the filters that allow or not to associate with a host (suzán et al. ) . the dispersion of a virus within a host community is accomplished through transmission events and may depend on the viral richness present in the community, so it is common to have multi-pathogen systems that can be considered as metacommunity (suzán et al. ) . metacommunity theory implemented in viral communities at different spatial scales in combination with a redundancy analysis allows identifying the factors that facilitate virus distribution among hosts (mihaljevic , dallas and presley , suzán et al. . based on the metacommunity structures proposed by leibold and mikkelson ( ) , and mechanisms for infection and prevalence proposed by suzan et al. ( ) , we can expect random, checkerboard and clemenstian viral structures. a widespread distribution of abundant reservoir species are related with random structures while a limited viral distribution or a high viral specificity are related with checkerboard or clementsian structures (suzán et al. ) . the factors that shape viral communities and their distribution through their host at different spatial scales have not been studied. to measure the influence of the host phylogeny and functional characteristics of the host on viral community structure we hypothesized that both the expression of clementsian structures based on the niche theory would prevail at different macroecological scales, and the host phylogeny will explain the viral metacommunity distribution as response of the shared host evolutionary histories and ecological relationships. we analyzed the contribution of phylogenetic and functional factors to the structure of viral metacommunities associated with rodents and bats. in our model, the viral community is defined as all viruses detected in each host species inside the geographic scale to analyze. in this analysis, the metacommunities are composed by viral communities linked by processes of dispersion and transmission between hosts. to consider the spatial scale, the first analysis of viral metacommunities was performed on a continental scale considering the ocean as the main geographic barrier for viral distribution. the subsequent macroecological scales were selected by their similarity on diversity composition as biogeographical scales, recognized by their similarity in plant diversity (cox ) . zoogeographical scales by their similarity in animal diversity (holt et al. ), and regional scales that contain local shared evolutionary histories (holt et al. ) . we constructed a database based on reports of viruses isolated or detected by molecular techniques in bats and rodents. we recorded the host species, the virus species, and viral family according to the international committee of the taxonomy of viruses (ictv) (https://talk.ictvonline.org/). the bat viruses information was collected from dbatvir database (http://www.mgc.ac.cn/dbatvir/), and data from rodents were collected by a literature search in web of science (https://webofknowledge.com), elsevier (https://www.elsevier.com/advanced-search) and world wide science (https:// worldwidescience.org/) with the keywords: "rodent", "virus", "pcr" and "wild". the registered information was collected from years to and only studies with molecular techniques were considered. the geographic location was registered and classified into four spatial scales ( afrotropical, indomalayan and australian (cox ) ) zoogeographical scale: nearctic, panamian, neotropical, palearctic, afrotropical, oriental, saharo-arabian, sino-japaness, madagascan and australian (holt et al. ) . ) regional scales: north american, mexican, panamian, south american, amazonian, palearctic, african, guine-congolian, madagascan, indomalayan, oriental, saharo-arabian, sino-japaness, novozelandic, and australian (holt et al. ). a presence-absence data matrix was constructed for each spatial scale and for each viral taxonomic level (species and family), where the virus was the column and the host the row. we obtained rodent and bat matrices, but due to the measures and the capacity of the algorithm performed in the metacommunity structure analysis, we could only obtain results of and matrices respectively. to evaluate the metacommunity structure of virus-host species form each matrix, we measure three properties. the coherence as the degree to which pattern can be collapsed into a single dimension, turnover counting the number of species replacements along the matrix and boundary cumpling measuring how the edges of species boundaries are distributed along this dimension (leibold and mikkelson ) . the analysis of metacommunity structure was performed with the metacom package (dallas ) implemented in r (r core team ), and the detection of metacommunity structure was using the presley's decisions tree (presley et al. ). we measured the influence of the host phylogeny and functional characteristics of the host on viral community structure. the phylogenetic component was estimated taking the two first components of the pcoa analysis on the phylogenetic distance matrix, which was obtained by extracting the host species from the mammalian super-tree hosts (bininda-emonds et al. ) with the "picante" package. we include the body mass, litter size, number of litters per year and trophic guild, as host functional characteristics that may explain variation among hosts in viral community composition and their influence on the viral transmission. the variables were obtained from pantheria database (jones et al. ), animal diversity web (http://animaldiversity.org) and encyclopedia of life (http://eol.org/). a redundant analysis of canonical ordination (rda) has been performed to detect the relationship between the host phylogenetic component and host functional characteristics in the metacommunities from each spatial scale to obtain the explaining percentage on each one of detected metacommunity structures. the rda was calculated with the algorithm varpart (peres-neto et al. ) of "vegan" package (oksanen et al. ) implemented in r. this function performs a partition of the variation in community data for the explanatory variables. the rodents' database has records, and includes rodent host species, virus species distributed in viral families, of which species and families are zoonotic. the bats' database has , records and includes host bats species, virus species and viral families of which virus species and nine viral families are zoonotic. rodents. we detected a quasi-clementsian structure in the metacommunities of viruses in the american continent, and the distribution of these virus species was partly explained by host phylogeny ( %). the big mass scale showed a clementsian structure explained by a low percentage of host functional characteristics ( . %) ( table ). oceania continent was not included in the analysis because only four records were obtained. at biogeographical level, a clemenstian structure was detected in the nearctic region, a quasi-clementsian structure in the neotropic and palearctic, and a random structure was found in the afrotropical region. at zoogeographical scale, only four regions were analyzed including afrotropical with quasi-clemenstian structure, nearctic with clementsian and palearctic and oriental with random structures. at regional scale, three regions were analyzed including north amercian, with clementsian structure, african and palearctic with random structures. no relevant results were observed at metacommunities of viral families, where the random structures dominated in all scales except for nearctic biogeographic and zoogeographic scale where a quasi-clementsian structure was detected. chiropterans. except for the big mass scale where a quasi-clementsian structure was detected, the rest of the viral species metacommunities analyzed showed random structures. oceania continent with records was not possible to analyze. at biogeographic and zogeographical scale, afrotropical and palearctic regions presented a quasi-clemenstian structure, while we detected a checkerboard structure in the neotropical zoogeographical region. only the quasi-clementsian structure of the palearctic zone is maintained up to the regional scale. at all scales, the host phylogeny and host functional characteristic explain the distribution of the viral families (table ). we observed different patterns at different scales of analysis. for example, in the american continent the coherence increases as the geographic scale decreases, as shown in regional scales. contrarily, in the big mass an opposite pattern was found; the coherence decrease with a decreasing geographic scale (fig. ) . in the american continent these pat- terns may be explaining by the high influence of the latitudinal gradient on the host distribution and by the island biogeography and the edge effect because the american continent has a higher border surface (lovejoy et al. ). instead, the big mass surface allows a homogeneous host distribution (buckley et al. ) . at the big mass scale there are not strict physical barriers between bioregions and viruses are widely shared at the edges, mainly at the palearctic-afrotropical edge. these boundaries merged many years ago, forming a large area that allowed this exchange of viral diversity, so they are now arbitrary limits (morand and krasnov ) . therefore, it was possible to detect a clementsian structure in the big mass metacommunity, but when the spatial scale decreased we observed a dominance of random structures. at smaller communities, the characteristics of the host species are more widely shared without delimiting niches, and therefore the viral distribution depends on its capacity of dispersion rather than local filters. in the nearctic biogeographic region, the clementsian structure was explained by host phylogeny in % (table ) , suggesting a phylogenetic signal and therefore, a higher specificity for host clades. the quasi-clementsian structure detected in the neotropical biogeographical region showed a weak response to environmental host filters ( . %). when we compare these two regions, they show a response to a latitudinal diversity gradient (kaufman , gaston , guernier et al. , suggesting a greater diversity of host and viruses in the tropics probably influenced by a constant temperature (morand and krasnov ) . this property facilitates the survival and viral mutations, facilitated by vectors proliferation, incrementing the chance of spillover, giving rise to generalist symbionts (harvell et al. ) . the coherence in the structure of the afrotropical zoogeographical region increases by the loss of the middle east region, which was included at biogeographical scale and prevented the potential viral dispersion. functional and phylogenetic characteristics explained the random structure detected in the african region by % (table ) . this percentage can be explained by the absence of thryonomys swinderianus and xerus erythropus, species in the african region who contained extreme values in their phylogenetic characteristics. it also shows a structure with a clementsian tendency that is disturbed by mastomys natalensis, a rodent associated with seven of the viruses in this scale, in comparison with the remaining rodents that host - virus species. also, it is the only species in this level with reports of banzi virus, gairo virus, mopeia and morongo virus. probably because of its anthropism and the most considerable sampling effort. in rodents we can assume that coherence decrease in viral families metacommunities may emerge due to the loss of information of viral species characteristics when viral families data was analyzed. the metacommunity structure of viral species associated with bats was dominated by a random structure, however, the distribution of the viruses is not aleatory because most of the metacommunities were explained by the host phylogeny and functional characteristics. besides some viruses associated with chiropterans are cosmopolitan, like rabies virus, which have a wide range distribution within the metacommunitiy preventing the detection of a coherent structure. in chiropterans, contrary to rodents, higher coherence at viral family scale suggests a clustering pattern of viral families. besides, their classification of viral species is more specific, they even take the name of the host in which they were isolated. the coherence of viral families metacommunities was only detectable at biogeographical and zoogeographical scales, while in the continental and regional scales the coherence cannot be observed. this result can be explained by removal of the host and viral families with a wide range of distribution, due that our model assumes a homogeneous distribution of host and viruses. at america continent scale, we detected several random and checkerboard structures in viral families metacommunities due to the presence of cosmopolitan viruses with a high sampling effort due to their public health relevance, such as the rhabdoviridae and coronaviridae families, found in % and % of the host species distributed in america respectively. in the palearctic and afrotropical biogeographic regions a quasi-clementsian structure was detected. when analyzing the palearctic region at zoogeographical scale, we can observe a coherence increase due to a separation of random structures which belong to the zoogeographical regions saharo-arabica and sino-japanese (fig. ) , with a low sampling effort. despite this, the sino-japanese region is explained by phylogenetic ( . %) and functional ( . %) characteristics but separately, because the region contains extreme climates (urteaga ) that could generate divergence of host and viruses (gorman et al. ) . the random structure observed in oriental regional scale was explained by a high value ( . %) of environmental host filter (phylogeny + functional characteristics). this result suggests that the absence of a coherent structure is resulted by the poor sampling effort in the area. the virus families' distribution in the quasi-clementsian structure detected in the afrotropical biogeographic region is explained by environmental host filters ( . %), however, when the geographic scale decreases the coherence increases with environmental host filter ( . %). this increase of coherence can be explained by the absence of madagascan region due to the geographic barrier that prohibits host migration. meanwhile, the host migration between african and guineo-congolian regions could be possible, explaining the decreases of coherence and the random structures detected in these two regions. in general, a higher number of clementsian and quasi-clementsian structures was observed in response to environmental host filters. the viral distribution responds primarily to dispersion filter by the geographic scale and secondly to the host characteristics, being affected by two types of simultaneous filters, at different spatial scales. random structures are explained by taking into account the information biases and the dynamism in which the ecosystems are involved, undergoing constant changes, of which we only manage to capture moments of their history. in spite of random spatial structures, host-virus relationships can still be highly specific, suggesting coevolution between hosts and viruses (drexler et al. ) including coronaviruses (cov, however our framework addressed to community scale cannot measure these events. even so, the rules of community assembly are not a law, and they are only one of several mechanisms that alternate, so it proposes the predominance of clements superorganism at macroecological level (jaisson ) . thus, the study of such viral community assembly rules must be deepened to understand these processes. clarifying the influence of these environmental host filters, enables us to address the effect of differential study efforts and also to plan surveillance systems and responds to situations like emergent diseases. to understand and predict viral transmission dynamics it is important to identify those variables that explain viral distribution through their hosts. if the viral distribution is explained by the phylogenetic component we will be able to predict new hosts based on the phylogenetic similarity. instead, if viral distribution is explained by the functional component we could predict new hosts based on functional traits similarity. we must not forget that multiple factors interact and affect the direction of changes in viral metacommunities so that their dynamism must be monitored and understood by multidisciplinary approaches. in general, it is more feasible to analyze the viral metacommunities associated with rodents at viral species scale by overlapping families, showing a weak phylogenetic signal between the host and the virus species. bats, on the other hand, showed more order at the viral family level due to viral taxonomic classification, but also present more cosmopolitan viruses. these inferences were based on the currently available data. unfortunately, the present data set is insufficient to analyze the virus assemblages of small regions like the australian and madagascan due to the scarcity of data. our data set is also likely to be biased because synanthropic species and viruses of public health relevance were sampled heavily, and this likely interferes with the influence of natural structures. however, at the macroecological level, viral metacommunities associated with rodentia and chiropterans showed clementsian structures or at least tended to them (quasi-clementsians). the viral metacommunities mainly respond to spatial abiotic 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the viral families metacommunities of rodents and results of rda analysis. abs, embedded absences; sd, standard deviation; df, degree freedom. results of the analysis of coherence, range turnover, and boundary clumping for the viral species metacommunities of bats and results of rda analysis. abs, embedded absences; sd, standard deviation; df, degree freedom.the file may be dowloaded from www.akademiai.com.open access. this article is distributed under the terms of the creative commons attribution . international license (https://creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, you give a link to the creative commons license, and indicate if changes were made. key: cord- -qhlatg authors: verma, anukriti; sharda, shivani; rathi, bhawna; somvanshi, pallavi; pandey, bimlesh dhar title: elucidating potential molecular signatures through host-microbe interactions for reactive arthritis and inflammatory bowel disease using combinatorial approach date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: qhlatg reactive arthritis (rea), a rare seronegative inflammatory arthritis, lacks exquisite classification under rheumatic autoimmunity. rea is solely established using differential clinical diagnosis of the patient cohorts, where pathogenic triggers linked to enteric and urogenital microorganisms e.g. salmonella, shigella, yersinia, campylobacter, chlamydia have been reported. inflammatory bowel disease (ibd), an idiopathic enteric disorder co-evolved and attuned to present gut microbiome dysbiosis, can be correlated to the genesis of enteropathic arthropathies like rea. gut microbes symbolically modulate immune system homeostasis and are elementary for varied disease patterns in autoimmune disorders. the gut-microbiota axis structured on the core host-microbe interactions execute an imperative role in discerning the etiopathogenesis of rea and ibd. this study predicts the molecular signatures for rea with co-evolved ibd through the enveloped host-microbe interactions and microbe-microbe ‘interspecies communication’, using synonymous gene expression data for selective microbes. we have utilized a combinatorial approach that have concomitant in-silico work-pipeline and experimental validation to corroborate the findings. in-silico analysis involving text mining, metabolic network reconstruction, simulation, filtering, host-microbe interaction, docking and molecular mimicry studies results in robust drug target/s and biomarker/s for co-evolved ibd and rea. cross validation of the target/s or biomarker/s was done by targeted gene expression analysis following a non-probabilistic convenience sampling. studies were performed to substantiate the host-microbe disease network consisting of protein-marker-symptom/disease-pathway-drug associations resulting in possible identification of vital drug targets, biomarkers, pathways and inhibitors for ibd and rea. our study identified na((+))/h((+)) anti-porter (nhaa) and kynureninase (kynu) to be robust early and essential host-microbe interacting targets for ibd co-evolved rea. other vital host-microbe interacting genes, proteins, pathways and drugs include adenosine deaminase (ada), superoxide dismutase (sod ), catalase (cat), angiotensin i converting enzyme (ace), carbon metabolism (folate biosynthesis) and methotrexate. these can serve as potential prognostic/theranostic biomarkers and signatures that can be extrapolated to stratify rea and related autoimmunity patient cohorts for further pilot studies. www.nature.com/scientificreports/ approach has advantages over the traditional approach for network analysis that can help to simultaneously characterize several protein interaction modules and has the potential to study complex diseases. the vital information obtained in our study from in-silico analysis is cross-validated through targeted gene expression experimental analysis on patient cohorts. this study will help us to obtain clinico-molecular informatics-based outcomes and expand our knowledge regarding the understanding of biological functions for ibd co-existent rea. text mining: data screening and selection. systematic data search and organization was carried out incorporating data identification, data screening and data selection to find target microorganisms involved in inflammatory bowel disease (ibd) and reactive arthritis (rea). data identification was carried out to obtain records through data sources utilising keywords (e.g. "microorganism and inflammatory bowel disease and reactive arthritis") incorporating boolean operators (and/or/not). data screening and selection were carried as part of the manual curation through primary and secondary screening scrutinizing collected data records to obtain organized records relevant for the autoimmune and enteric disorders triggered by microorganisms, especially ibd and rea and the microbial triggers implicated in ibd and rea that were utilised for further metabolic network reconstruction. bottom-up approach consisting of draft reconstruction and manual reconstruction refinement was followed to create metabolic networks of obtained target microorganisms. genome-scale metabolic models simulation, reconstruction and visualization (gemsirv) software that includes reciprocal basic local alignment search tool (blast) of target microorganisms against a template metabolic network of its phylogenetic neighbour and incorporates information from national center for biotechnology information (ncbi), kyoto encyclopedia of genes and genomes (kegg) and transport db was used for creating draft reconstructs. the manual curation of missing links or gaps in the draft reconstruct was done by mapping the incomplete information to other databases such as expert protein analysis system (expasy) and integrated relational enzyme database (intenz) . this fully connected and annotated network was used for further simulation studies . the metabolic networks thus obtained were visualized using celldesigner, a tool for modelling and editing biochemical and gene-regulatory networks. simulation analysis was carried by converting the metabolic networks obtained into a mathematical model and performing the gene deletion analysis to retrieve essential genes. model conversion was through generation of stoichiometric based matrixes consisting of reactions (columns) and metabolites (rows) corresponding to respective genes. upper boundary and lower boundary fluxes i.e. movement of matter across a system were generated for the gene associated reactions and metabolites that was extracted in systems biology markup language (sbml) format. the next step was gene deletion analysis done using the constraint based reconstruction and analysis toolbox (cobra) that runs in matrix laboratory (matlab) for finding the essential genes based upon the gene-reaction matrix and boolean relationship between genes and reactions . the purpose of data filtering is to remove repeats and homologs from essential genes of target microorganisms associated with ibd co-existent rea. the non-homologous protein sequences corresponding to the essential genes of target microorganisms were extracted from pathosystems resource integration (patric) database . refinement of protein sequences was further done using cluster database at high identity with tolerance (cd-hit) suite so as to have % identity non-repeat sequence tolerance stringency. blast-p was further used to remove the homologs from such non-repeats against human database at e-value of - to obtain nonhomologous protein sequences used for further in-silico analysis. essential host-microbe and microbe-microbe interactions. the host-microbe interactions of the non-homologous proteins for the selected target microorganisms were obtained using host-pathogen interaction database (hpidb) , . the host-microbe interactions were visualised using cytoscape. simulation analysis (gene essentiality) was done to obtain the essential host proteins interacting with common microbe proteins of microorganisms triggering ibd and rea utilising the human metabolic model hmr , a cobra compliant metabolic model of human consisting of around , genes, , reactions and , metabolites . this led to profiling of the common host-microbe and microbe-microbe interactions comprehending the complex 'interspecies communication' as complex interaction maps, executed using search tool for the retrieval of interacting genes/proteins (string) , . host-microbe disease network and molecular mimicry studies. the host-microbe disease network is a multilayered archetype that connects the protein-marker-symptom/disease-drug-pathway associations. the contributions of the microorganisms in the co-evolved ibd and rea as part of the disease network was created through the interactive maps of the essential host interaction proteins (verified using literature survey) and the information processed through gene expression data analysis . the information patronised here is mostly scored through the available non-specific protein diagnostic markers of both ibd and rea e.g. c-reactive protein (crp), interleukin (il ) and toll like receptor (tlr ), major histocompatibility complex, class i, b (hla-b) and major histocompatibility complex, class ii, dr beta (hla-drb ) with the essential host proteins determined using string . database genecards was used to assess the role of these interacting partners aka proteins further with symptoms/diseases associated with ibd and rea. the pathways of the above host interacting proteins were found out using kegg database that provides ontologies for proteins related to biological processes www.nature.com/scientificreports/ subsequently, the role of drugs or inhibitors used to suppress the effect of ibd and rea such as indomethacin, prednisone, ciprofloxacin, sulfasalazine, azathioprine, methotrexate and hydroxychloroquine was scored in the disease network through their docking studies against the potential targets (both host as well microbial targets) as per published methodologies , . the host-microbe disease network which is an amalgamation of all the above patterned associations was visualized using cytoscape software . molecular mimicry analysis between the vital targets triggering ibd co-evolved rea, essential human proteins including hla-b , hla-b and hla-drb was done using data repository expasy. this led to retrieval of microbe relayed protein sequences that have been implicated in disease development after sequence alignment performed using emboss . experimental evidences to identify the signature molecules in patient samples. the cross-validation of vital in-silico targets was done in rea patient cohort cases via targeted gene expression analysis. scientific and ethical clearance was taken from amity university ethics committee and institutional ethics committee, fortis noida for handling the patient samples. all experiments were performed in accordance with indian council of medical research (icmr) guidelines constituting the ethics committees. the study was carried out for months on the rare disorder rea patients, with the inclusion criteria as patients having rea according to european spondyloarthropathy study group (essg) and exclusion criteria as patients undergoing treatment from last - months and healthy controls (hc). the participants were inducted in the study design with an informed consent form along with a questionnaire containing information regarding symptomatic and diagnostic history of patient and linked disorders. blood ( ml) was drawn from participants in ethylenediaminetetraacetic acid (edta) vacutainers. these were transported to the laboratory for further analysis. the processing of the samples was done within - h of procurement . peripheral blood mononuclear cells (pbmc's) were isolated from blood using density gradient centrifugation . rna was isolated from pbmc's using trizol method . the quantification of rna was done using nano-drop . the high capacity cdna reverse transcription kit (applied biosystems™) was used for conversion of rna to single-stranded cdna as per the standard protocol . quantitative pcr analysis of target gene was executed using biorad cfx real time-pcr taking human housekeeping gene, gapdh as a reference. previously reported primers for qpcr analysis of target and reference gene were selected for this study , following the standard protocol . relative gene expression analysis from qpcr data was performed using the relative expression software tool (rest® ) that utilises the expression of reference genes to normalize expression of target genes in different samples. the schematic representation of methodology involved in our combinatorial analysis is provided in fig. . text mining: data screening and selection. a systematic literature mining and curation for our thematic connecting autoimmune disorders, inflammatory bowel disease (ibd) and reactive arthritis (rea) was carried out. data identification extracted , records (articles in journals, book chapters, conference papers etc.) corresponding to autoimmune and enteric disorders. data screening extracted records of autoimmune and enteric disorders triggered by microorganisms that belong to class of bacteria, fungi, protozoan, mites, virus, yeast and nematode. data selection yielded ibd, rea and ibd co-evolved rea records. data selection was directed towards the microbial contenders implicated here resulting in target microorganisms namely campylobacter jejuni, escherichia coli o :h , klebsiella oxytoca, salmonella typhimurium, shigella dysenteriae and yersinia enterocolitica, whose genome information was available. the etiopathogenesis in the co-evolved disorders have been documented through gut microbiome associated host-pathogen interactions studies, perpetuating where pathogen microorganisms involve in dysbiosis leading to autoimmunity. the results of text mining are provided in fig. . the list of microorganisms is provided in supplementary table s online. ing of genes along with their corresponding proteins, reactions and metabolites for the selected microorganisms serve as primary set of partial metabolic network information. the missing data persistent in the draft reconstruct obtained through genome-scale metabolic models simulation, reconstruction and visualization (gem-sirv) was manually refined. entirely associated metabolic networks of target microorganisms were obtained (genes, proteins and reactions). the essential genes of microorganisms (vital for survival. sustenance and growth) were obtained after performing simulation on mathematical models consisting of gene associated reactions and metabolites (metabolites, inner cell reactions, exchange reactions and essential genes). due to lack of availability of exchange reactions for campylobacter jejuni, simulation analysis on the partial metabolic network could not be carried out and essential genes could not be retrieved. an alternative approach for finding essential genes of campylobacter jejuni was carried out. the essential genes of campylobacter jejuni were taken from our previous published report and were found out to be . table portrays the results of metabolic network reconstruction and simulation of target microorganisms. the metabolic network and simulation analysis data of target microorganisms is provided in supplementary table s online. the proteins corresponding to essential genes, non-repeats and non-homologs were obtained as stated below according to the parenthesis {proteins corresponding to essential genes, non-repeats, non-homologs}. the essential genes, their corresponding proteins, reactions and metabolites from the curated dataset were refined to create a list of most relevant molecular indicators to assess their coveted role in disease establishment. the non-redundant filtered proteins were utilised further in the computational work-pipeline canvassing the drug targets and signatures in the interspecies communication. essential host-microbe and microbe-microbe interactions. the central mechanism of hostmicrobe/microbe interface conferred through gut microbiome was correlated for the selected microbial species and processed to obtain the common signatures so as to follow the core system of metabolic changes affecting the host harbouring them as either commensal or pathogenic loads. the interactors between human and target microorganisms were obtained. the interactors of escherichia coli o :h were ; klebsiella oxytoca were ; salmonella typhimurium were ; shigella dysenteriae were and yersinia enterocolitica were . there were no interactors for campylobacter jejuni (supplementary table s -s online). table shows the results of filtering and host-microbe interactions of protein sequences corresponding to essential genes of target microorganisms. www.nature.com/scientificreports/ the host-microbe interactors were analysed for all the target microbial species and processed to obtain the common signatures. proteins were found between all target microorganisms having interaction among themselves and with human proteins. the essential host correlative targets to the microbial gene targets were followed by obtaining host essential genes and corresponding proteins from human metabolic model hmr . there were , essential proteins (supplementary table s online) the essential human protein was found out to be kynu having interaction with essential microbial protein nhaa (fig. ) . nhaa was also having interactions with non-essential hcls associated protein x- (hax ), prolyl endopeptidase-like (ppcel), biogenesis of lysosomal organelles complex subunit (hps ) and eukaryotic translation initiation factor alpha kinase (e ak ) proteins of human host. kynu was further mapped with host proteins (direct and indirect) resulting in interactions. out of these the single connected essential protein interactions were and protein interactors were ( fig. and see supplementary table s online). the research design here followed to assess the interaction map of essential proteins in human host to indicate the clinical insights in pathophysiological trends in the autoimmune development. host-microbe disease network and molecular mimicry. the human essential proteome complement with its interacting proteins were analysed further as part of the disease network. human essential protein interactors were found to be associated with ibd and similarly essential protein interactors namely adenosine supplementary table s online) . these proteins can be postulated as probable contenders transcending their role in the simulated network as important regulators in the co-existent disorders. the composite associations of the above proteins with non-specific protein diagnostic markers of ibd and rea were obtained (see supplementary table s online) . this gave rise to a single connected protein network consisting of proteins and , interactions. the association of above with symptoms and diseases linked with ibd and rea were obtained (see supplementary table s online) . apart from non-specific diagnostic markers, the major protein linked with majority of symptoms/diseases is angiotensin i converting enzyme (ace). pathways of the proteins were obtained (see supplementary table s online) in total out of which the pathway associated with majority of proteins was carbon metabolism. another layer of disease network substantiates the role of therapeutic regime followed in the studied autoimmune diseases, so the docking analysis of drugs used to suppress the effect of ibd and rea against nhaa of target microorganisms and kynu of human host was done. the docking analysis resulted in docking scores that represent binding of drugs with host kynu and microbial nhaa of all microorganisms selected in our study. higher the negative docking score more is the binding . escherichia coli o :h nhaa shows highest and lowest docking score with methotrexate (− . ) and azathioprine (− . ); klebsiella oxytoca nhaa with methotrexate (− . ) and azathioprine (− . ); salmonella typhimurium nhaa with ciprofloxacin (− . ) and hydroxychloroquine (− . ); shigella dysenteriae nhaa with methotrexate (− . ) and azathioprine (− . ); yersinia enterocolitica nhaa with hydroxychloroquine (− . ) and azathioprine (− . ) and human kynu with hydroxychloroquine (− . ) and indomethacin ( . ). our results portray methotrexate to have highest docking scores with maximum proteins and therefore can be considered as a vital drug for ibd associated rea. the resultant docking scores are provided in fig. . the extensive interaction pattern of nhaa with kynu along with proteins, markers, symptoms/ diseases, pathways and drugs give rise to a host-microbe disease network of ibd co-existent rea (fig. and see supplementary table s online) . the final league of information processed in this study design was to accommodate the concept of molecular mimicry between the essential host proteins and selected microorganisms. nhaa protein of target microorganisms shows homology with human hla-b , hla-b and hla-drb (fig. ) . peptides homologous to hla-b : peptides homologous to hla-drb : experimental evidences to identify the signature molecules in patients. the in-silico analysis followed for the molecular signature identification till far through gene expression datasets and curated metabolic reconstructs strongly indicate the host protein, kynu being the singular common predictive markers for all pathogenic microbes. kynu has also been indicated in the expression data of inflammatory linked disorder, www.nature.com/scientificreports/ ibd. there is lack of data available regarding kynu differential expression in rea, therefore the experimental evaluation of kynu through targeted expression analysis in rea patients was carried out. a non-probabilistic convenience sampling was followed for our single blind study. this study encompassed individuals: % male with mean age of . and % female with mean age of ( males and females). out of these cases were: with rea and controls were: currently undergoing treatment, with poncet's disease (pd) and healthy control (hc). the clinical characteristics of the patients recruited in the study included inflammatory back pain in %, fatigue in %, fever in %, swollen joint in %, ankylosing spondylitis (as) that affects spine in %, dactylitis that is inflammation in finger or toe in % and poncet's disease (pd) in % of participants. the clinical characteristics of the recruits are provided in table . the expression of kynu in peripheral blood mononuclear cells (pbmc's) of rea cases vs controls was evaluated using relative expression software tool (rest) software that estimated a sample's relative expression ratio in relation to the control housekeeping gene (here gapdh) by calculating an intermediate absolute concentration value: where cp = point at which fluorescence escalates considerably above the background fluorescence. here the cp values for reference and target genes are collectively redistributed to control and sample groups and the expression ratios are calculated based on the mean value. a pair wise fixed reallocation randomisation test is followed for normalisation of the target genes with a reference gene and for calculating the statistical difference of variation between groups . it utilises a bootstrapping technique providing a % confidence interval for expression ratios. it uses a p(h ) test for testing the significance between the samples and controls. according to our analysis, kynu sample group is different to control group where p(h ) = . . kynu was found to be downregulated in sample group (in comparison to control group) by a mean factor of . (standard error range is . - . ) as depicted in the whisker-box plot (fig. ) . kynu expression showed a ~ ninefold decline in rea cases as compared to controls. gut microbiome is pitched to be the central theme housing enormous diversity of microbial species, characterizing the fine balance between healthy and diseased states. the physiological drifts from healthy to diseased and vice-versa is tuned to sophisticated interactive networks of human host and the microbial flora residing the gut. the autoimmune conditions reactive arthritis (rea) and inflammatory bowel disease (ibd) have been linked to prevalent dysbiosis of the gut, where disease development occurs as a perceptive reaction due invading population of microbes. to find out the basal networks of interactions at the host-microbe interface, common microbes affecting the co-evolved diseases with shared characteristics were studied. these involved comprehensive analysis of the bimolecular functional networks including the gene, protein, metabolite molecular signatures engraved at the host-microbe and microbe-microbe interface. this 'interspecies communication' have been linked now with immuno-pathogenesis of most human autoimmune disorders , . www.nature.com/scientificreports/ the etiopathology of these interactions have remained elusive leading to non-specific diagnostic criteria and therapeutic regimes. it is suggested that microbial dysbiosis, pathogenic infection and host-microbe interactions cause incidence of rea. in this study, utilising the combinatorial approach we have compiled a repertoire of microorganisms, biomolecules and pathways that are possibly involved in triggering co-evolved autoimmune disorders ibd and rea. in our study, text mining results convey the presence of microorganisms namely campylobacter jejuni, escherichia coli o :h , klebsiella oxytoca, salmonella typhimurium, shigella dysenteriae and yersinia enterocolitica implicated in both the disorders. the thematic concepts for microbe contribution in host immunity have been explored in our previous analysis of metabolic reconstruction and simulation of campylobacter jejuni and salmonella enterica , . in our current study, we used a designated work-pipeline for metabolic network reconstruction and simulation of target microorganisms. the analysis conducted extracted the information via constraint-based bottom-up approach that was filtered and utilised for further computational analysis. the essential genes, proteins and metabolites of microorganisms represent the promising drug targets as these are speculated to contribute towards infection triggered host physiological drifts leading to development of the co-evolved pattern of autoimmunity in ibd and rea. a thorough curation pattern followed led to provide robust molecular cues in terms of essential proteins and biological networks that are correlated to the 'interspecies communication' using the host-microbe and microbemicrobe interaction profiling. the most closely associated common protein observed in all the selected common microbial species involved in both ibd and rea is na (+) /h (+) antiporter (nhaa), microbial integral membrane protein, catalyzing the exchange of h (+) per na (+) and involved in processes crucial for cell viability. similarly, the common host interacting protein with nhaa is kynureninase (kynu), involved in tryptophan metabolism and whose differential expression (upregulation and downregulation based on the control samples) have been followed in ibd patient cohorts [ ] [ ] [ ] . as per the scientific discourse presented in the studied disorders, the pathological mechanism hypothesizes that after bacterial infection, antigen-presenting cells transport bacterial antigens/peptides into the synovial membrane, where the bacterial components persist causing inflammation. it is suggested that in host-microbe interactions, bacterial proteins entering host cells interact with host proteins and inject their effector components, but has not been proven in rea and ibd. so, this formed a basis of one of the parameters in our study design where we found the physical interactions between nhaa and kynu and predicted that these might be the early host-microbe interactors for establishing pathogenesis in ibd associated rea. this could assist to comprehend the very few reports indicated in the rare autoimmune rea, where gene expression datasets of the co-evolved disorder ibd can serve to incorporate the larger theme of gut-microbiome associations. the theme of gut-microbiome paradigm shifts thus contemplates the vital cues in triggering autoimmunity with indirect linkages to diet and environmental triggers. this is indicative of the identified target molecular signature, kynu, found to be differentially regulated in the patient cohorts with history of infection triggered or ibd co-evolved rea. kynu and nhaa could serve as the robust early and essential host-microbe interacting targets and molecular indicators involved in interspecies communication in ibd associated rea. the investigations further were targeted for parallel analysis of other host-essential protein partners enmeshed to have interaction with host protein kynu indicating the intricate details of host-microbe interaction information. the disease network constructed through our approach consists of single connected essential protein interactors of kynu, where human essential protein interactors are found to be associated with ibd, while of them (adenosine deaminase (ada), catalase (cat) and superoxide dismutase (sod )) are associated with both ibd and rea. ada protein has been reported in juvenile idiopathic arthritis and rea patient cohorts in serum samples . similarly, cat and manganese superoxide dismutase (sod) genes polymorphisms were observed in rea patient cohorts , . these become part of the host-microbe disease network where such molecular elements and co-regulatory pathways represent the intricate biological cross-talk followed during disease development. pathological conditions can also trigger immune cells such as il's and tlr's and various cytokines leading to immune cell infiltration in host and higher levels of inflammation. genetic factors such as hla alleles encode susceptibility, contribute to bacterial persistence and increase risk in rea cases. based on this we also found the interactions of important targets in our study with immunogenic and genetic factors. the host harboured assorted essential proteins were further probed for their association with non-specific protein diagnostic markers as well as with symptoms/diseases linked with ibd and rea, accruing towards a single connected network consisting of interdependent proteins. the reciprocation of these integrated protein indicators to the disease development is conveyed through metabolite monitoring as in the study, angiotensin i converting enzyme (ace) was found to be linked with maximum symptoms/diseases. ace is involved in catalyzing the conversion of angiotensin i into angiotensin ii that is a potent vasopressor and aldosterone-stimulating peptide that controls blood pressure and fluid-electrolyte balance . this could be the indicator of involvement of microbe triggered host physiological drifts. subsequently, the pathways associated with the proteins ramified into pathways of human host speculated to give details of metabolic regulatory checkpoints where carbon metabolism is found to be associated with majority of deduced proteins. carbon metabolism pathway implicated here as the vitally generic pathway for ibd co-related rea confers how diet, balance of gut microbiome, antibiotic exposures can have layered impact on autoimmune disease progression and remissions. kynu is found to be downregulated in rea patients as compared to controls through our targeted gene expression analysis. collectively, the disease network followed here confers interaction of microbial nhaa with host kynu, that is further correlated to proteins, markers, symptoms/diseases, pathways and drugs. docking analysis of drugs used to suppress the effect of ibd and rea predicts methotrexate as an important drug that could be useful for early treatment of ibd co-evolved rea. www.nature.com/scientificreports/ genetic factors found common in both rea and ibd are hla-b , hla-b and hla-drb . the most important mechanism of susceptibility of hla in rea is molecular mimicry that is microbial peptides mimicking hla autopeptides of human host leading to autoimmunity. this mechanism has been observed in rea where reports have predicted microorganism peptides such as chlamydial proteins (clpc, nqra and dnap) and yersinia pseudotuberculosis peptides (yoph) showing homology with human hla-b via bioinformatic analysis . similarly, molecular mimicry has also been observed in ibd cases having extraintestinal manifestations. we performed targeted molecular mimicry analysis in our study using our robust microbial protein (nhaa) with hla-b , hla-b and hla-drb , enhancing the importance of nhaa acting as a trigger for generating ibd associated rea. we generate a putative hypothesis amalgamating key findings with literature. we state that the initial hostmicrobe triggers for ibd associated rea is when pathogenic microbial protein nhaa interacts with host protein kynu that further interacts with human proteins ada, sod , cat and ace and carbon metabolism involving the above host proteins is hampered. methotrexate regulates carbon metabolism and the associated host-microbe proteins reducing effect of ibd associated rea. since carbon metabolism is the most basic aspect of life and therefore an extensive network consisting of sub-pathways, we narrowed down our findings towards a consequentially central and a significant pathway that embrace the carbon metabolism pathway involving the molecular signatures kynu, ada, sod , cat and ace, further is also effectuated by potential drug methotrexate and is associated with ibd/ rea/ ibd and rea cohorts. it is reported that methotrexate is incorporated intracellularly interfering with adenosine concentrations and affecting proinflammatory cytokines in ibd reducing inflammation . in inflammatory arthritis, the mechanisms reported by which methotrexate reduces inflammation include enhanced adenosine release, de novo synthesis of purines and pyrimidines, inhibition of transmethylation reactions, diminished accumulation of polyamines and nitric oxide synthase uncoupling. most of the mechanisms are associated with folate biosynthesis, a type of carbon metabolism . kynu, ada, sod , cat and ace are also found to be involved in folate biosynthesis and metabolism from genecards. apart from the above targets, parallel interactors, pathways and drugs for ibd co-evolved rea obtained in our host-microbe disease network can be utilised further as disease determinants. the experimental validation of these targets in patient cohorts need to be performed on a pilot scale in future to increase the robustness of this network. the intertwined information processed through the knowledge-base created for the linked disorders have given the most elaborate layout of patterns observed in disease diagnosis and analysis. the major information after processing the gene expression profiles, protein markers, molecular networks and metabolic networks involved here have led to chalk out as well as connect the strings for robust gut microbiome paradigm shifts. the current work on host-microbe interactions provides a starting point for researchers and clinicians to investigate inflammatory bowel disease (ibd) associated reactive arthritis (rea). in this study a combinatorial approach is utilised to reveal the interactions of gut microbes with human host extensively sketched through the work-pipeline providing the vital insights for the drug targets, biomarkers, pathways and inhibitors for etiology, prognosis, diagnosis and treatment attributes of pathogenic rheumatic autoimmunity. 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advanced studies, new delhi for providing the facility and technical support during the preparation of the manuscript. we also thank fortis hospital, noida for providing the patient samples. s.s. and b.r. conceived the study concept; s.s., b.r. and p.s. jointly designed and supervised the work; b.d.p. supervised the clinical setting and recruitment of participants; b.d.p. and a.v. recruited the participants and contributed to the sample collection and preparation; a.v. performed the experiments; s.s., b.r., p.s. and a.v. contributed to the analysis and interpretation of data; a.v. generated all figures and tables; a.v. wrote the first draft of the manuscript; s.s., b.r., p.s. and b.d.p. critically reviewed and edited the manuscript; all authors reviewed and approved the final version of the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be 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view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -gt uqfrl authors: casadevall, arturo; pirofski, liise-anne title: the damage-response framework of microbial pathogenesis date: journal: nat rev microbiol doi: . /nrmicro sha: doc_id: cord_uid: gt uqfrl the late twentieth century witnessed the emergence of numerous infectious diseases that are caused by microorganisms that rarely cause disease in normal, healthy immunocompetent hosts. the emergence of these diseases shows that the existing concepts of pathogenicity and virulence do not take into account the fact that both the microorganism and the host contribute to microbial pathogenesis. to address this impediment to studies of host–microorganism interactions, we propose a new theoretical approach to understanding microbial pathogenesis, known as the 'damage-response' framework. the 'damage-response' framework of microbial pathogenesis is based on three tenets , (box ). first, that microbial pathogenesis is the outcome of an interaction between a host and a microorganism, and is attributable to neither the microorganism nor the host alone. second, that the pathological outcome of the hostmicroorganism interaction is determined by the amount of damage to the host. third, that damage to the host can result from microbial factors and/or the host response. these tenets form a scaffold -or framework -on which a formal theory can be built and tested. (glossary terms within this article are defined as applied in the damage-response framework - .) it is likely that relationships have existed between hosts and microorganisms for as long as there have been interactions between host and microbial cells. indeed, ancient host-microorganism interactions, in which bacteria were incorporated into a primordial host as organelles, are likely to have resulted in the evolution of eukaryotes , . the outcome of many hostmicroorganism interactions can be either beneficial or detrimental to the microorganism, to the host, or to both the microorganism and the host (table ) . mutualism and commensalism are examples of interactions that are beneficial to both the host and the microorganism. in the case of microorganisms that replicate within their hosts, detrimental outcomes result in the inability to replicate further and/or death. although microbial replication can cause host damage, and possibly disease, host damage and/or disease are not essential for microbial survival. furthermore, microbial viability is not a requirement for microbial pathogenesis. for example, cysticercosis -a devastating neurological disease that is caused by the host inflammatory response to the cestode taenia solium -can be precipitated by the death of the parasite, such that anti-helminthic therapy is considered to be detrimental in certain clinical situations . host-microorganism interactions that result in the clearance and/or control of a microorganism without the development of clinically relevant host damage represent a basis for the development of vaccines and immune-response-based therapies for infectious diseases. however, host-induced cell and/or tissue damage can also produce detrimental outcomes, which can result in disease or death -although certain manifestations of host damage represent the outcome of a successful immune response to microbial infection. some interactions are highly complex and illustrate the difficulty that can be associated with discriminating between a host and a microorganism. for example, the ingestion of certain microorganisms by amoebae (the host) can be beneficial to the amoebae,as the microorganisms are a source of food, and detrimental the late twentieth century witnessed the emergence of numerous infectious diseases that are caused by microorganisms that rarely cause disease in normal, healthy immunocompetent hosts. the emergence of these diseases shows that the existing concepts of pathogenicity and virulence do not take into account the fact that both the microorganism and the host contribute to microbial pathogenesis. to address this impediment to studies of host-microorganism interactions, we propose a new theoretical approach to understanding microbial pathogenesis, known as the 'damage-response' framework. host an entity in which microorganisms reside and/or replicate; an entity in which microbial pathogenesis occurs. damage disruptions in the normal homeostatic mechanisms of a host that alter the functioning of cells, tissues or organs; for microorganisms, disruptions in the normal mechanisms that enable host entry, replication and/or the ability to establish residence in a host. infectious diseases that are caused by microorganisms that have been referred to as 'weak' , 'non'-pathogens or those that are 'less virulent'. similarly, the concept of microbial 'opportunism' was devised to explain the emergence of previously rare infectious diseases in individuals with impaired immunity , . however, the classification of such microorganisms as 'opportunistic' was often inadequate and misleading, as some so-called 'opportunistic' microorganisms can also cause disease in normal hosts . host failure, which is a 'host-centred' view of microbial pathogenesis, has also been used to explain the occurrence of diseases that are caused by microorganisms that do not fit koch's postulates because they lack virulence determinants. these microorganisms do not cause disease in all hosts and so cannot be explained by 'microorganism-centred' views. examples of such diseases are catheter-associated candidiasis, staphylococcus epidermidis endocarditis and pneumocystis carinii pneumonia. the study of microbial pathogenesis has its intellectual and scientific origins in microbiology and immunology -two fields that are largely 'microorganism-centred' and 'host-centred' , respectively. indeed, an inherent bias towards either the host or the microorganism is often central to the thinking and investigative approaches of immunologists and microbiologists, respectively.'microorganism-centred' investigators tend to test the influence of microbial manipulations (mutants) on virulence by holding the host variables constant -as exemplified by the frequent use of immunologically naive and genetically inbred hosts. the capacity of a defined characteristic, or mutation, to confer virulence in a host is required to fulfil molecular koch's postulates . by contrast, the approach of 'hostcentred' investigators, such as immunologists, generally involves manipulation of the host as the microbial variables are held constant. this is exemplified by studies that attempt to establish that a host component is essential for host defence by comparing the outcome of infection with the relevant microorganism in both normal (wild-type) and deficient (knockout) mice. notably, the field of vaccinology has elements of both views, as evidenced by 'microorganism-centred' approaches with 'host-centred' thinking -a distinction that can place it at odds with its parental discipline of immunology . although 'microorganism-centred' and 'hostcentred' views of microbial pathogenesis have each provided valuable insights into microbial pathogenesis, neither simulates the reality in which each hostmicroorganism interaction is unique and occurs against a background of constant changes in both immune function and microbial fitness. it might not always be possible to account for both 'microorganism-centred' and 'host-centred' views in experimental design. however, consideration of the possible contributions of both the host and the microorganism to host damage could focus studies of microbial pathogenesis around a common principle, and has the potential to unify the field of microbial pathogenesis and the allied disciplines of immunology and vaccinology. to the ingested microorganism. however, the ingestion of certain microorganisms, such as legionella pneumophila or cryptococcus neoformans, by the amoeba acanthamoebae castellanii can result in death of the host -a process with striking similarities to the interaction of these microorganisms with macrophages in mammalian hosts , . the same amoebae, however, can be microorganisms in human hosts and cause amoebic keratitis. therefore, the distinction between host and microorganism is not invariant, yet it is relevant to understanding the outcome of certain host-microorganism interactions. despite being the outcome of an interaction, microbial pathogenesis is often viewed as a 'microorganismcentred' process. there are also proponents of the view that pathogenesis is 'host-centred'. although 'microorganism-centred' advocates recognize the importance of the host, they generally attribute the capacity for pathogenicity and virulence to the activity and functions of microbial gene products and/or microbial replication. inherent in this view, is the belief that microorganisms possess certain attributes that make them pathogenic, and that microorganisms that do not possess these attributes are not pathogenic (non-pathogens). 'microorganism-centred' views have their intellectual origin in the discovery of virulins at the turn of the twentieth century , are strongly supported by the discovery of virulence genes and pathogenicity islands, and are common in the field of bacterial pathogenesis , . the existence of microbial factors that confer virulence, such as capsular polysaccharide or toxins, gives credence to the concept that individual gene products can determine pathogenicity. such microbial determinants validate molecular koch's postulates . however, 'microorganism-centred' views fail to account for the pathogenicity of common microorganisms, such as candida albicans and staphylococcus aureus, as the strains of these microorganisms that are isolated from symptomatic or asymptomatic individuals are often indistinguishable. 'host-centred' views of microbial pathogenesis subscribe to the concept that a failure of host defence mechanisms results in susceptibility to certain microorganisms and their ability to establish themselves in protected niches.'host-centred' views are based on the fact that not all individuals in a population become ill after infection with a pathogenic microorganism. these views find support in the observation that immunocompromised hosts have a markedly increased incidence of box | basic tenets of the damage-response framework • microbial pathogenesis is an outcome of an interaction between a host and a microorganism. • the host-relevant outcome of the host-microorganism interaction is determined by the amount of damage to the host. • host damage can result from microbial factors and/or the host response. as illustrated by the discovery that p. carinii is a fungus after many years of being considered a protozoan parasite . classifications based on the perceived capacity of a microorganism to cause disease are also inadequate, because changes in host immune function, ecology and/or behaviour can render them obsolete. for example, candida spp. were considered essentially non-pathogenic until the mid- s, when the introduction of antimicrobial chemotherapy and corticosteroids resulted in the recognition that they could cause disease in certain hosts . similarly, immunization can render a pathogenic microorganism a 'nonpathogen', as host immunity abrogates the ability of the relevant microorganism to cause disease. for example, variola virus is not pathogenic in individuals immunized with vaccinia virus. the central tenets of the 'damage-response' framework -that the outcome of microbial pathogenesis is the result of a host-microorganism interaction, and that the relevant outcome of this interaction is host damage -provide the basis for a new pathogen-classification scheme. this scheme is based on damage-response curves, which depict the host-microorganism interaction (fig. ). it consists of six different parabolic curves that represent the amount of host damage as a function of the intensity and degree of the host response. each type of curve represents a type or a 'class' of pathogen (fig. ) . in the absence of a host response, damage is the result of the ability of the microorganism to induce damage. however, it is likely that most, if not all, hosts generate some degree of response to microbial infection and that many mechanisms that produce this response have the capacity to induce host damage. the use of host damage as the common principle to classify pathogens allows them to be grouped according to common pathogenic outcomes. microorganisms that cause similar types of diseases can be grouped together -despite their differences at the level of phylogeny, growth characteristics or other criteria that are used for pathogen classification. the pathogens that are grouped in a single class might not cause similar diseases, but what they have in common is the extent to which the damage or disease they cause is a function of the host immune response. therefore, the pathogen classes convey the relative ability of a microorganism to cause disease based on the immune status of in the damage-response framework, a pathogen is defined as a microorganism that is capable of causing damage to a host , . this definition allows the terms that have been used to define microorganisms that do, and do not cause disease, such as commensal, saprophyte, non-pathogen, opportunist and primary pathogen, to be dispensed with. this is a less ambiguous definition of a pathogen than that used previously, as the outcome or possible outcomes of damage are used to define the pathogen, and it dispenses with the need for modification or qualification to encompass microorganisms that cause disease rarely, or only in certain hosts. in the past, the meaning of the word 'virulence' has also caused confusion . the damageresponse framework defines virulence as the relative capacity of a microorganism to cause damage to a host . the word 'relative' is included because virulence is frequently measured in comparison with another microorganism or a variant of the same microorganism. however, when a more complete understanding of quantitative measures of damage is available, it should be possible to dispense with the word 'relative'. the damage-response framework definitions of pathogen and virulence underscore the concept that only in a susceptible host is a microorganism a pathogen, and virulence can be expressed . consequently, neither the characteristics of a pathogen, nor virulence, can be considered independent microbial variables. at present, microbial classifications are largely based on phylogenetic groupings (for example, bacteria, viruses, parasites and fungi), the perceived capacity to cause disease (for example, primary pathogens, saprophytes, opportunistic pathogens or commensals) or, for bacteria, growth or other identifying characteristics (for example, whether it is an anaerobe, aerobe or facultative microorganism, or its appearance under gramstain). these classifications are confusing, because, in addition to being problematic and often inadequate, they are also overlapping. phylogenetic groupings are not very useful for pathogen classification because most of the members of any group are not pathogenic in a host. for example, of the more than , fungal species, only about are pathogenic for humans, and these are often genetically distant from each other. phylogenetic classifications are also subject to change, • the phenotype or property that is under investigation should be associated with pathogenic members of a genus or pathogenic strains of a species. • specific inactivation of the gene or genes that are associated with the suspected virulence trait should lead to a measurable loss in pathogenicity or virulence. • reversion or allelic replacement of the mutated gene should lead to restoration of pathogenicity. downwards and to the left, deteriorations in host immunity can shift them upwards, and exuberant immune responses, such as those elicited by some vaccines, can shift them upwards and to the right. finally, the damage-response curves can help in the classification of new pathogens and diseases. for example, based on the available information, the new coronavirus associated with severe acute respiratory syndrome (sars) could be classified as a class -, -, -or -like pathogen, depending on the information that is obtained from subsequent studies. pathogens in these classes all cause host damage in the setting of strong immune responses, and sars has been associated with lung damage in the setting of apparently robust immune responses. indeed, the proposed system is flexible and allows pathogenic microorganisms to be regrouped as new information becomes available. for example, saccharomyces cerevisiae (bakers' yeast) was considered to be non-pathogenic until it was associated with disease in certain patients with severe immune impairment , which would have led it to be categorized as a class pathogen. however, recent evidence indicates that certain mutations in s. cerevisiae increase its virulence in normal mice by altering its surface properties to elicit a potent pro-inflammatory response that damages tissues and causes disease . consequently, it might be more appropriate to characterize s. cerevisiae as a class or pathogen in mice. microorganism-mediated damage in a host can result from a variety of mechanisms, ranging from replication of the pathogen, the production of toxic substances and subversion of the normal host homeostatic and/or immune mechanisms ( (table ) . notably, each also has a polysaccharide capsule that is the key determinant of their capacity to induce host damage, and each causes a similar clinical syndrome. p. carinii is a class pathogen because it does not cause disease in hosts with normal immunity, and aspergillus fumigatus is a class pathogen because it causes disease in individuals with either weak or strong immunity. in the case of individuals with strong immunity, disease results from an exuberant response to aspergillus antigens that damages the host through hypersensitivity reactions. the use of host damage to classify the outcome of a host-microorganism interaction acknowledges and accounts for the contribution of the host immune response to microbial pathogenicity and virulence. therefore, the 'damage-response' framework can be used to characterize host-microorganism interactions that are either detrimental or beneficial to the host. a benefit to the host is indicated by the damage-response curve extending below the x-axis. the framework is a flexible system in that it can accommodate changes in both host and microorganism. for example, therapeutic interventions can shift the damage-response curves the arrow indicates that the position of the curve is variable, and depends on the particular host-microorganism interaction. the y-axis denotes host damage as a function of the host response. in this scheme, host damage can occur throughout the host response, but is magnified at both extremes. the host response is represented by a continuum from 'weak' to 'strong'. 'weak' and 'strong' are terms that can encompass both quantitative and qualitative characteristics of the host response and are used as the best available terms to denote the spectrum of host response as more precise terms are limiting. weak responses are those that are insufficient, poor or inappropriate -that is, they are not strong enough to benefit the host. strong responses are those that are excessive, overly robust or inappropriate -that is, they are too strong and can damage the host. when a threshold amount of damage is reached, the host can become symptomatic and if damage is severe, death can ensue. green, yellow and purple represent health, disease and severe disease, respectively. from the immune response of normal individuals to these microorganisms. for example, the chemokine and cytokine mediator response to s. pneumoniae is responsible for the clinical manifestations of meningitis. a long-lasting inflammatory response to microbial components can result in cell death and the replacement of viable cells and parenchyma with fibrosis. similarly, chronic inflammation can lead to amyloid production, as is observed in patients who have had tuberculosis (tb), and antigen-antibody complexes that form in the course of the immune responsesuch as those that develop in staphylococcal endocarditis or rickettsial diseases -can form deposits in vascular tissues and other organs, which trigger inflammation, organ damage and dysfunction. antigenic mimicry can evoke immune responses that destroy cells and tissue -as exemplified during the pathogenesis of acute rheumatic fever and the development of haemolytic anaemia in the course of mycoplasma pneumonia. host-microorganism contact is followed by two main outcomes, namely, the elimination of the microorganism from the host or infection, which can be defined as the acquisition of a microorganism by a host . following infection by the microorganism, four main outcomes, or states, are possible: commensalism, colonization, persistence (or latency) and disease (fig. ) . these states are a consequence of the outcome of the amount of host damage that results from hostmicroorganism interactions over time, and are generally continuous, such that when damage exceeds a threshold amount, another state becomes relevant . for example, the state of colonization becomes the state of disease when a critical amount of host damage has occurred; the state of disease can become the state of latency; and the state of latency can become the state of disease, again depending on the amount of damage resulting from the host-microorganism interaction. these definitions make no assumptions about the length of time that a state predominates, but instead indicate that as time progresses, different outcomes of infection are determined by the nature and degree of damage that results from the host-microorganism interaction. the role of the host response in the outcome of microbial infection is twofold. first, it defines the threshold that distinguishes host damage from clinical disease, and second, it represents a factor that mediates transitions between the different states. at present, the inability to precisely define both damage and the threshold between damage and disease, makes the distinction between certain states difficult. for example, distinguishing commensalism from colonization is not possible when the latter is accompanied by little or no damage , . in contrast to microorganism-based classification schemes, the damage-response framework is based on the concept that the fundamental difference between pathogenic and non-pathogenic microorganisms is a function of the host response to the microorganism. an effective immune response can abrogate the factor is entirely responsible for host damage -for example, a toxin that causes damage irrespective of the host response because toxin action is so rapid and/or the amount of toxin is insufficient to trigger an immune response. previously, we have proposed that toxin-producing microorganisms are a variant of class where the curve is flat at both ends , but here we suggest that this type of interaction might be unique and warrants a separate panel. as shown here, the damage-response classification scheme is flexible and makes it possible to postulate the existence of pathogens for which there are no known examples at present. such pathogens could be recognized in the future as 'emerging' pathogens as shown in c and d. c | the class curve is extended below the x-axis. such a theoretical microorganism would be a commensal in the setting of intermediate host responses, but pathogenic in hosts with either weak or strong responses. d | the inverted parabola represents a putative host-microorganism interaction that induces damage over a narrow and limited range of responses, but not in the presence of either strong or weak host responses. one example of such a phenomenon would be an antibody response to a hypothetical microorganism, whereby host damage is caused by antigen-antibody complexes. although we cannot think of a specific microorganism that fits this description at this time, examples of this type of host damage are the host-microorganism interactions characterized by the herxheimer reaction following treatment of syphilis, the similar reaction that can occur after the initiation of therapy for pneumocystis carinii pneumonia, and serum sickness following the injection of foreign protein. this we make no apologies, as the field of microbial pathogenesis seeks to understand how microorganisms cause disease, an outcome that can only occur in a susceptible host. the only components of the damage-response framework are microorganisms that can cause disease and hosts. similarly, the outcomes of a host-microorganism interaction after initial contact are simplified to infection, commensalism, colonization, persistence (latency) and disease. the use of damage as the common classification for microbial pathogenicity and virulence simplifies the lexicon of microbial pathogenesis and makes it possible to discard ambiguous terms, such as commensal, saprophyte, opportunist, exposure and carriage , , , . the damage-response framework was proposed based on clinical and experimental observations of the outcome of host-microorganism interactions. despite being supported by a large body of observational evidence, it should also be subjected to experimental validation and/or refutation. to accomplish this, it will be necessary to develop better quantitative and qualitative measures of the immune response and host damage, as, at present, the information needed to validate or refute the damage-response framework is unavailable. the damage-response framework plots damage as a function of the immune response and uses the qualifiers 'strong' and 'weak' to denote quantitative and qualitative characteristics of the immune response. we appreciate that these terms are vague and, as such, that they might seem to oversimplify the complex, diverse and multidimensional nature of the host response -which includes both humoral and cellular components of the innate and adaptive immune systems. pathogenicity of a microorganism. even highly virulent toxigenic microorganisms are not virulent in hosts that have been immunized with toxoid vaccines, such as tetanus toxoid. therefore, although the existence of microbial virulence factors is incontrovertible, for many microorganisms it is the ability of these factors to override the mechanisms of host defence that makes them virulence factors. the damage-response framework is neither microorganism-centred nor host-centred. it does, however, state that the outcome of host-microorganism interactions in a host is the defining aspect of microbial pathogenesis. in this regard, the damage-response framework might be considered to be biased towards the host. however, rather than holding the host responsible for pathogenesis, the damage-response framework places the context of microbial pathogenesis within the host. for colonization a state of host-microorganism interaction that leads to a variable amount of host damage, from minimal to great, thereby reflecting host immune responses that have the capacity to eliminate the microorganism or to promote the development of another state. latency a state of host-microorganism interaction in which a microorganism persists in a host and can be associated with damage that can be evident at the cellular or tissue level, but is not associated with disease. a microbial component that can damage a host. the shortcomings of the terms 'strong' and 'weak' are evident if the case of p. carinii pneumonia is considered. this disease occurs almost exclusively in severely immunocompromised individuals and, consequently, p. carinii was classified as a class pathogen. however, clinical experience has shown improved outcomes in patients that are treated with corticosteroids, which indicates that lung damage in aids patients with p. carinii pneumonia is mediated largely by the residual immune system. therefore, p. carinii pneumonia occurs in individuals with impaired ('weak') immune responses, but damage is likely to be immune-mediated. if treated as singular parameters, measurements of the immune response that are available at present, such as the amount and type of antibody response, delayed-type hypersensitivity, lymphocyte proliferation, cytokine levels or immunoglobulin e levels, are too limited and one-dimensional to be useful quantitatively on the x-axis of the damage-response curves. even in combination, these parameters are not, at present, useful as we lack the knowledge to devise formulas that could take their relative contributions into account. similarly, measures of damage, such as fever, organ dysfunction and cytokine levels in serum, are too insensitive to provide an accurate measure of the amount and quality of damage that results from the host-microorganism interaction. damage, like the immune response, is the result of multiple events and their contributions from cellular, tissue and organ toxicity. for example, it has been shown that the interaction of c. albicans with different host effector cell receptors can result in different inflammatory profiles, which, in turn, translate into differences in virulence . however, at present, we cannot predict the point at which subclinical damage becomes clinical disease or, indeed, the level of disease that results in the death of the organism. therefore, the correct parameters for plotting on the x-and y-axes should be functions of multiple variables that include the contribution of multiple components of the immune system and damage, respectively. the damage-response framework proposes classifications, statements and predictions that are, in principle, amenable to experimental testing. we anticipate that, in the future, accurate measurements of host damage and the immune response will be available, which will allow the generation of experimental curves to which mathematical functions can then be fitted to formally describe the host-microorganism interaction. any effort to investigate the validity of the damageresponse framework is likely to stimulate research into the relationship between host damage and the immune response. such endeavours will provide new scientific insights, irrespective of whether the framework is ultimately validated or falsified. we anticipate several important uses of the damageresponse framework. first, as has already been alluded to, efforts to validate or refute the framework will undoubtedly foster research into improved measures of damage and the immune response. second, the framework can lead to predictions that might enhance the development and predictability the host-microorganism interaction can be depicted by plotting host damage as a function of time. panels a-d show how plotting damage versus time can be used to denote the states of the host-microorganism interaction for four different pathogens. infection represents the acquisition of the microorganism by the host and is followed by the states of commensalism, colonization, latency and disease, depending on the amount of damage to the host , . these plots highlight the fact that for certain pathogens there is continuity between the various states. the colours green, yellow and purple denote health, disease and severe disease, respectively, and the relevant states for each host-microorganism interaction are highlighted in bold. www.nature.com/reviews/micro r e v i e w s blood donors . recently, several countries have been affected by coronavirus-associated sars . if disease in sars is found to be associated with a large viral burden, it might be anticipated that the process involves viralmediated damage and that antiviral therapy could be helpful therapeutically. however, if disease is found to be associated with inflammation and a paucity or absence of virus in tissues, then the pathogenic process might reflect immune-mediated damage for which antiinflammatory therapy could be helpful. initial reports that corticosteroid therapy might be helpful are consistent with the possibility that host-mediated damage is responsible for the disease. fourth, the damage-response framework promises to be a useful educational tool as it avoids the 'bug parade' that students of microbial pathogenesis find so unsatisfactory, and instead provides a more straightforward classification of pathogenic microorganisms that integrates the contributions of both host and pathogen. finally, the damage-response framework should provide a mechanism that brings together, under one umbrella, the different areas of microbial pathogenesis that, at present, are isolated from one another, such as viral and bacterial pathogenesis. such an approach would foster collaboration at all the interfaces between microbiology and immunology, and ultimately advance our understanding of infectious diseases. of rational vaccine design. consider tb, a disease for which there has been a major effort to develop a vaccine and/or immune-based therapy. the causative agent of tb, mycobacterium tuberculosis, is a class pathogen that induces damage at the extremes of the immune response. in immunocompetent individuals, damage reflects a robust, often helper-t-cell- -like response, whereas in immunocompromised individuals it reflects an insufficient inflammatory response. as the basis of host damage differs, depending on the immune status of the individual, the damage-response framework would predict that the use of different types of vaccines, vaccine antigens or vaccination strategies might be necessary to prevent tb in immunocompetent and immunocompromised individuals. similarly, immune therapies could be directed towards reducing the inflammatory damage in immunocompetent hosts and enhancing the immune function in immunocompromised hosts. third, the damage-response framework has the potential to characterize new and emerging infectious diseases, thereby facilitating a more rapid and focused response by the public authorities and research community. two recent emerging infectious diseases exemplify this point. the recognition that the disease-to-infection ratio for west nile virus in endemic areas is very low, led to the successful experimental use of passive antibody therapy with immunoglobulins from asymptomatic introduces the 'damage-response' framework and proposes a new classification for pathogens based on their ability to damage the host as a function of the host response expands the 'damage-response' framework to incorporate the variable of time and proposes that the outcome of the host-microorganism interaction can be reduced to four states that differ in the amount of damage incurred by the host as a function of time what is a pathogen? discusses limitations of the existing concepts of virulence and problems with definitions of virulence factors highlights how the damage-response framework can be used to classify the host-microorganism interaction in clinical practice on the definition of virulence the origin of plant and animal cells symbiosis and evolution principles and practice of infectious diseases legionella pneumophila pathogenesis: a fateful journey from amoebae to macrophages cryptococcus neoformans interactions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages common themes in microbial pathogenicity revisited. microbiol pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution landmark paper that proposed a rigorous approach to establishing whether a gene was involved in microbial virulence the role of opportunistic bacteria in human disease history of opportunistic infection in the immunocompromised host an informative story of how the concept of microbial opportunism evolved in clinical practice pathogenicity and virulence: another view an excellent discussion of the limitations of koch's postulates exploiting the redundancy of the immune system: vaccines can mediate protection by eliciting 'unnatural' immunity ribosomal rna shows pneumocystis carinii to be a member of the fungi antimicrobial therapy severe acute respiratory syndrome (sars): breath-taking progress saccharomyces cerevisiae bacteremia a saccharomyces cerevisiae mutant with increased virulence parasitealtered behaviour: is the effect of toxoplasma gondii on rattus norvegicus specific? dendritic cells and receptors: a host perspective of fungal virulence prophylactic and therapeutic efficacy of human intravenous immunoglobulin in treating west nile virus infection in mice colonization with caga-positive helicobacter pylori strains inversely associated with reflux esophagitis and barrett's esophagus the following terms in this article are linked online to: infectious disease information: http://www.cdc.gov/ncidod/diseases/index.htm candidiasis | cryptococcus neoformans | cysticercosis | legionella pneumophila | meningitis | mycoplasma pneumonia | pneumocystis carinii pneumonia | syphilis | tuberculosis access to this interactive links box is free online. key: cord- -pk pnmlo authors: hanley, brian title: an object simulation model for modeling hypothetical disease epidemics – epiflex date: - - journal: theor biol med model doi: . / - - - sha: doc_id: cord_uid: pk pnmlo background: epiflex is a flexible, easy to use computer model for a single computer, intended to be operated by one user who need not be an expert. its purpose is to study in-silico the epidemic behavior of a wide variety of diseases, both known and theoretical, by simulating their spread at the level of individuals contracting and infecting others. to understand the system fully, this paper must be read together in conjunction with study of the software and its results. epiflex is evaluated using results from modeling influenza a epidemics and comparing them with a variety of field data sources and other types of modeling. epiflex is an object-oriented monte carlo system, allocating entities to correspond to individuals, disease vectors, diseases, and the locations that hosts may inhabit. epiflex defines eight different contact types available for a disease. contacts occur inside locations within the model. populations are composed of demographic groups, each of which has a cycle of movement between locations. within locations, superspreading is defined by skewing of contact distributions. results: epiflex indicates three phenomena of interest for public health: ( ) r( )is variable, and the smaller the population, the larger the infected fraction within that population will be; ( ) significant compression/synchronization between cities by a factor of roughly occurs between the early incubation phase of a multi-city epidemic and the major manifestation phase; ( ) if better true morbidity data were available, more asymptomatic hosts would be seen to spread disease than we currently believe is the case for influenza. these results suggest that field research to study such phenomena, while expensive, should be worthwhile. conclusion: since epiflex shows all stages of disease progression, detailed insight into the progress of epidemics is possible. epiflex shows the characteristic multimodality and apparently random variation characteristic of real world data, but does so as an emergent property of a carefully constructed model of disease dynamics and is not simply a stochastic system. epiflex can provide a better understanding of infectious diseases and strategies for response. the most commonly used measure in public health, r , is estimated from historical data and derived from sis/sir type models (and descendents) for forward projection [ , ] r is the basic reproductive ratio for how many individuals each infected person is going to infect [ ] r is often used on its own in public health as an indicator of epidemic probability; if r < then an epidemic is not generally considered possible, for r > , the larger the value, the more likely an epidemic is to occur. r is a composite value describing the behavior of an infectious agent. hence, r can be decomposed classically, for example, as: p d c, where p is probability of infection occurring for a contact, d is duration of infectiousness, and c is number of contacts [ ] . however, r in the classical decomposition above, while it is one of the best tools we have, does not account for age segregation of response, existing immunity in population, network topology of infectious contacts and other factors. these observations were significant in the motivation for developing epiflex. the epiflex model was designed to create a system that could incorporate as much realism as possible in an epidemic model so as to enable emerging disease events to be simulated. there are limitations, described below in a separate section, but the model is quite effective as it stands. in most cases, the limitations of epiflex are shared by other modeling systems. there are a variety of methods used for mathematical modeling of diseases. the most common of these are the sir (susceptible, infected, recovered) of kermack and mckendrick [ ] , sis (susceptible, infected, susceptible), seir (susceptible, exposed, infected, recovered), and sirp (susceptible, infected, recovered, partially immune) as developed by hyman et al. [ ] and further developed by hyman and laforce [ ] . the sirp model was used as the starting point for development of the object model of epi-flex. in sirp, the sir model is extended to include partial immunity (denoted by p) and the progressive decline of partial immunity to allow influenza to be modeled more accurately. (see appendix.) there is a need for experimentation in more realistic discrete modeling, since the lattice type of discrete modeling is understood to skew in favor of propagation, as discussed by rhodes and anderson [ ] and haraguchi and sasaki [ ] . others such as eames and keeling [ ] and edmunds et al. [ ] have explored the use of networks to model interactions between infectable entities, and ferguson et al. [ ] and others have called for more balance in realism for epidemiology models. since epiflex was completed, lloyd-smith et al. [ ] have shown the importance of superspreading in disease transmission for the sars epidemic. epiflex is designed to take these issues into account. there are known weaknesses in sis-descended models, some of which are discussed by hyman and laforce [ ] . they suggested that a model dealing with demographics and their subgroups would be useful and described a start theoretical biology and medical modelling , : http://www.tbiomed.com/content/ / / toward conceiving such a model, creating a matrix of sirp flows for each demographic group within a "city" and modeling contacts between these groups. thus, the possibility of building an entirely discrete model using the object-oriented approach, essentially setting the granularity of the hyman-laforce concept at the level of the individual, together with monte carlo method, was attractive. the object method of design seemed to be a good fit, since object-oriented programming was invented for discrete simulations [ ] . an object-oriented (oo) design defines as its primitive elements "black box" subunits that have defined ways of interacting with each other [ ] . the oo language concept originally was conceived for the simula languages [ ] for the purpose of verifiable simulation. enforcement of explicit connections between objects is fundamental to oo design, whereas procedural languages such as fortran and cobol do not because data areas can be freely accessed by the whole program. oo languages wrap data in methods for accessing the data. if each "black box" (i.e. object) has a set of specified behaviors, without the possibility of invisible, unnoticed interactions between them, then the simulation can potentially be validated by logical proof in addition to testing. (it would take an entire course to introduce oo languages and concepts, and there is not space to do so here. interested readers are suggested to start with an implementation of smalltalk. there are excellent free versions downloadable. smalltalk also has an enthusiastic and quite friendly user community. see: http:// www.smalltalk.org/main/.) the design of epiflex is described more completely in the appendix. design proceeded by establishing the definition of a disease organism as the cornerstone, then defining practical structures and objects for simulating the movement of a disease through populations. the disease object was assigned a set of definitions drawn from literature that would allow a wide spectrum of disease-producing organisms to be specified. the aim was to minimize the number of configuration parameters that require understanding of mathematical models. the hosts that are infected became the second primary object. a host lives and works in some area, where hosts are members of some demographic group, which together determine what of n types of contacts they might have to spread an infectious disease. the hosts move about the area in which they live between locations at which they interact. in epiflex, an area contains some configured number of locations, and locations are containers for temporary groups of hosts. since people travel between metro areas, the model supports linkages between areas to move people randomly drawn from a configurable set of demographic groups. the remainder of this section presents the disease model adopted, an overview of each component, an overview of program flow, and a description of the core methods. this is followed by discussion of results from the epiflex software system. this model has up to four stages during the infection cycle: the incubation, prodromal, manifestation, and chronic stages; to this is added a fatality phase. i have named this 'extended-sirp'. fig. shows a diagram of this model. the model of fig. allows us to track the different phases of the disease process separately, and to define variable infectiousness, symptoms, fatality, recovery and transition to chronic disease at each stage as appropriate. this allows us to model the progress of a disease in an individual more realistically. for diseases that have no identifiable occurrence of a particular stage, this stage can be set to length zero to bypass it entirely. the contact types designed into epiflex are drawn from literature in an attempt to model spread of infection more accurately. these contact types are: blood contact by needle stick, blood to mucosal contact, sexual intercourse, skin contact, close airborne, casual airborne, surface to hand to mucosa, and food contact. the probability of infection for a contact type is input by the user as estimated from literature or based on hypothetical organism characteristics. durations of disease stages are chosen uniformly at random from a user-specified interval [r low , r high ]. random numbers, denoted by ξ, on [ , ] are used to seed the determination of the infected disease stage periods (denoted i incubation , i prodromal , i manifestation , i chronic ). r low and r high are taken from medical literature and describe a range of days for each stage of an illness. these calculations are simply: (ξ × (r high -r low )) + r low = d, where d is days for a particular stage. (this may be extended in the future to include ability to define a graph to determine the flatness of distribution and the normative peak. this will make a significant difference in modeling of diseases such as rabies, which can, under unusual circumstances, have very long incubations.) one of the following three equations describing immunity decay is chosen; l is the current level of partial immunity, p is the level of partial immunity specified as existing random values on [ , ] are then used to decide whether an infection occurs during the partial immunity phase p shown in the chart above. this decision uses the output of the immunity level algorithm, l, which is a number on [ , ], as is the random value ξ: epiflex uses a dynamic network to model the interactions between hosts at a particular location based on the skew provided and the demographic segments movement cycles. the networks of contacts generated in this version extended-sirp disease model of epiflex figure extended-sirp disease model of epiflex. s: susceptible i: infected r: recovered p: partially immune f: fatality extended sirp breaks the infected stage i into : i incubation , i prodroma l, i manifestation , i chronic , and adds a fatality terminating stage. of epiflex are not made visible externally; they can only be observed in their effects. (see: limitations of epiflex modeling.) their algorithms were carefully designed and tested at small scales, observing each element. a location describes a place, the activities that occur there, and the demographic groups that may be drawn there automatically. a location can have a certain number of cells, which are used to specify n identically behaving locations concurrently. this acts as a location repetition count within an area when the location is defined. the user sets an average number of hosts inhabiting each cell, and a maximum. there is also a cell exchange fraction specifiable to model hosts moving from cell to cell. the algorithm for allocating hosts in cells is semi-random. it randomly puts hosts into cells in the location. if a cell hits the average, then it does another random draw of a cell. if all locations are at maximum, then it overloads cells. interactions are within the cell. so a host must be exchanged to another cell in order to be infective. see the appendix for 'location component', and also with an open model look at how hospitals were defined. households are modeled at this time using a cell configuration. epiflex is implemented with a monte carlo algorithm such that each host in a location is assigned a certain number of interactions according to the cauchy distribution parameter setting for that location. this distribution describes a curve with the y axis specifying the fraction of the maximum interactions for the location and x axis specifying the fractional ordinal within the list of hosts in the location. the distribution can be made nearly flat, or severely skewed with only a few actors providing nearly all contacts, as desired by the user of epiflex. note that the structure of the network formed also depends on what locations are defined, what demographic groups are defined for the population, and how demographic groups are moved between locations. each location has a maximum number of interactions specified per person, which is used as the base input. initially, a gaussian equation was used, but it was discarded in favor of a cauchy function since this better fits the needs of the skew function and computes faster. the algorithm iterates for each infectious host, and selects other hosts to expose to the infected party in the location, by a monte carlo function. this results in a dynamically allocated network of interactions within each location. the exposure cycle also makes use of monte carlo inputs. each location has a list of contact types that can take place at a particular location, and a maximum frequency of interactions. this interaction frequency determines how many times contacts that can spread a disease will be made, and the contact specification defines the fractional efficacy of infection by any specific route. modeling the effect of different types of contacts has been discussed in the literature, e.g. song et al. [ ] . epiflex attempts to make a more generalized version. for each host infection source, target hosts are drawn at random from the location queue. a contact connection is established with the target as long as the contact allocation of that target has not been used up already. contact connections made to each target are kept track of within the location to prevent over-allocation of contacts to any target. thus, for each randomly established connection, a value is set on both ends for the maximum number of connections that can be supported. once the maximum for either end of the link is reached, the algorithm will search for a different connection. the location algorithm is described below in more detail. the user specifies the maximum number of connections for a location; the σ output from a cauchy distribution function determines how many connections an individual will have. this allows variations in the degree of skewness for superspreading in a population to be modeled, which has been shown to be of critical importance by lloyd-smith et al. [ ] . if p = position in queue, q = number of hosts in queue for location: x = p/q, where x denotes the proportional fraction of queue for position. if k is a constant chosen for the location to express skew distribution, the cauchy distribution function is: if κ is the number of contacts for a particular host and κ max is maximum number of contacts for any given host in the location: when hosts move from one location to another within the model, they tend to maintain a rough order of ordinal position. consequently, when there is a high σ for a location, the high connection host in one location tends to be a high connection host in another. this reflects real-world situations, (though not perfectly) and corresponds better than persistently maintaining high connection individuals from location to location, since host behavior changes from place to place. the cauchy distribution function is fairly fast in execution. the function can be used to approximate the often radical variations seen in epidemiology studies; as an extreme example, one active super-spreader individual might infect large numbers, when one or even zero is typical [ ] . this type of scale-free network interaction has been explored by chowell and chavez [ ] . the cauchy function allows networks to be generated dynamically within each type of location in a very flexible manner, such as corresponding to super-spreader dynamics [ ] . in addition to the specification of skew within a location, the network of contacts is also defined by (a) what locations are present and (b) the movement cycles defined for each demographic group within the model. processing time increases with population. this slowing is an expected characteristic of an object modeling system and is the price paid for the discrete detail of the epiflex model. the primary source of this increase in processing time is the sum of series of possible infectious events that are modeled for each iteration. it therefore scales as a series sum not as a log, based on the contagiousness of the disease and the number of potential hosts in a location with an infected host. this is minimized by only processing infectious host contacts. the increase stems from the characteristics of networks in which each node has n connections to other nodes. when iteration is done for a location containing infectable hosts, it is the number of infected hosts that creates an element of the series. the infected hosts are put into a list, and each one interacts randomly with other hosts (including other infected ones) in the location. thus, considered as a network with m nodes, each of the m nodes is a host. a temporary connection to another host is made to n other nodes where n <m, and n<k. the value of k is determined by a randomized input that returns the number of contacts of this infected host in this location. consequently, the series consists of all the temporary connections made for contact modeling for each cycle. in the interest of completeness, the limitations of the epi-flex model are described here. the plan is to address these elements for implementation in future versions. only one infectious disease can be occurring at a time. thus, competitive inhibition [ ] and synergistic effects will not be seen. hosts do not reproduce hosts do not reproduce within a model. removal and addition rates are defined for the population as a whole, and the basis is us census data. to meet the specifications for removal and addition within the model, hosts are removed from randomly chosen locations, and similarly added to randomly chosen locations. demographic group is also randomly assigned. for long-term modeling, and modeling of alternative short-lived hosts, a reproduction cycle is desirable. however, epiflex is a practical way of modeling periods of a few years. there is no explicit definition of an age distribution for the host population, which can be quite significant [ ] . to a degree, age is taken into account through the demographic segmentation of populations. a demographic can be defined with a fraction or multiple of baseline susceptibility. however, hosts do not age, nor do they move from one demographic to another as they age. no provision is made to define a previous exposure profile for hosts [ , ] . in real populations previous exposures can have significant effects on the spread of a disease and dramatic effects on mortality where infection does occur [ ] . proper implementation of previous exposure profiles is intertwined with age definition. there is no implementation of mutation rate for diseases. mutation rates vary considerably by type, particularly for viruses [ ] . decay of immunity is modeled, and immunity decay can act as a fair surrogate for antigenic change. pass-through events must be defined as part of surface contacts for efficiency, epiflex eliminates pass-through infection events from being modeled: for example, an infected a shakes the hand of a non-infected b, who then shakes the hand of another non-infected c, but b washes hands and does not become infected while c does. therefore, the model definition must account for this through "surface to hand to mucosa" contacts, where a person can also be a surface. the model does not at this time record the contact network that is dynamically created except in the log file at this time. those that are logged are only potential infectious contacts. to get at that data requires looking at the log file and writing an extract program. making the network visible is an item for the future. currently, epiflex has no way of accounting for seasonal damping. similarity of results is due to the settings of the rate at which immunity declines. addition of a seasonal damping function would be expected to cause epiflex results to synchronize with a yearly cycle. seasonal damping would result in loss of interesting epidemic behavior with an overriding function that would virtually be guaranteed to drown out other behaviors. public health response to epidemics is not optimally modeled a public health response definition component is present in epiflex. testing of this component, and more thorough review of literature, indicate that the method used is not optimal. current public health responses are centered on contact tracing, ring vaccination and quarantine [ ] , with mass vaccination as a backup when it is available. closures of schools, daycare and travel restrictions are also used. these methods are not modeled in epiflex' response component. their importance has recently been underscored by lloyd-smith et al. [ ] . as a consequence, results from the current system that defines across-theboard cuts in the probability of infection should be considered in this light. it is not clear whether any other object system can model all current techniques properly. diseases have ranges of times for each stage that can be drawn from literature. probability of a specific disease stage time period for an infected host being chosen within the range is equal. this is reasonably adequate for most diseases where times are measured in a few days, however, some, such as rabies have a quite unequal distribution, and their very long tail makes a difference in modeling. the discussion is presented in three parts: ( ) a brief set of examples of native epiflex displays to develop a better feel for the system; ( ) comparisons of epiflex results with real world data; ( ) a set of examples of observations made using epiflex. the purpose of these examples is to serve as a guide to others who may want to experiment and analyze results. different views of the epidemic data for simulated influenza in two different populations are shown in figure . figures a and b show graphs of the second and third epidemics in the population. these graphs show the kinds of commonly-seen deviations from a smooth curve that occur in real world data [ ] . in the epiflex model, this is attributed to less synchronization of immunity combined with the formation of small world networks among demographic groups as they move from location to location. figures c and d are alternate views of a simple influenza epidemic occurring within a naïve population (figure ). comparison of epiflex with who/nrevss surveillance comparing epiflex with surveillance data, we see that who/nrevss surveillance data [ ] have a qualitatively similar graph form to epiflex for influenza, as shown in figures and . the width of the primary curves per season for epiflex is . to . months while that of the nrevss data is approximately to months, which can be explained by the nrevss data being collected nationally from surveillance centers, whereas the epiflex data shown are for a single area. epiflex runs executed with multiple cities connected by transport, such as the . million population city model, have a combined graph for all cities showing self-similarity to the graphs for individual areas, becoming wider, matching the nrevss data graph formation. the nrevss data consist of diagnostics of samples sent in by physicians. comparisons of absolute numbers in terms of quantity are therefore not applicable. a percentage of population comparison is done below. in table , epiflex indicates that roughly % of the population has been infected before herd immunity stops the epidemic, though this depends on population size. total morbidity is obtainable from epiflex by adding maximum immune level to deaths, although deaths contribute such a small amount to influenza morbidity that for practical purposes the immune level is used as a proxy for morbidity. moreover, true morbidity itself is relatively prone to inaccuracy, whereas better measures of immune fractions for influenza are available. the california state average for - is . % infected in a range from . to . depending on county [ ] . thus, epiflex is above the high end of the state of california estimated morbidity range. dowdle [ ] gives serological influenza data categorized by age. for influenza a/swine/ / h , seroprevalence ranges from roughly % to over %. for influenza a/ hong kong/ h , the range is from % to %. epiflex figures fall within this latter pair of ranges, and epiflex immune fraction is more properly comparable. what is most notable in figure is the relationship between the rough sine wave form of the classically derived sirp [ ] once the initial startup period is over for influenza, a repeating wave develops that is similar in overall shape and variability to real world data such as those for milwaukee, at a roughly similar scale. these two graphs refer to populations that differ in size by about order of magnitude (i.e. milwaukee is times the size of the model run shown). we can also see a similar number of peaks. owing to the need to compare these two graphs natively, these two figures are not optimum. however, they show essential features. total morbidity rate linked to population size the smaller a population over the range , to ~ . million, the higher the total morbidity rate, given identical organisms ( figure ). it is intuitively expected that population size will affect morbidity since, for any given network of contacts connecting individuals in populations, the chance of the epidemic spreading during the window prior to the development of immunity in parts of the population increases as the population size decreases. this this is the simulation that was imported for comparison figure this is the simulation that was imported for comparison. vertical scale per demarcation. horizontal scale one year per demarcation. upper white line is total population with standard removal rate. effect is most striking when very small populations in the order of , are examined. literature data regarding this in real world populations are sparse. however, there are indications from historical accounts of small populations in the new world that a link between population size and morbidity is observable in real world populations [ ] [ ] [ ] [ ] [ ] [ ] . the most recent such account is from heyerdahl in the pacific in the mid th century [ ] . in the graphs of figure , the immune fraction at completion is used as a proxy for total morbidity on a log scale of population. the longer an epidemic takes to progress within an enclosed population, the greater the number of potentially infectious contacts that hit a dead end because the host is already immune. since very small populations will mostly function within the window when there is no host immunity, the infection will spread to a larger fraction. this effect has public health implications because, clearly, the structure of the network is highly significant in determining the likelihood that an infected host will contact naïve hosts. essentially what this epiflex result indi-cates is that during the period prior to the development of an immune subpopulation, a disease has a functionally higher r . (i.e. r is variable through the course of an epidemic.) in the figure graphs, epiflex is also suggesting that there are more asymptomatic infected spreaders of influenza in our populations than surveillance data estimate. this is also suggested by the discussion above regarding comparison with seroprevalence. a minor experimental result is that for a repeating illness such as influenza, when a continuously active initiating disease vector tries to infect people per day, it will develop higher peaks after the initial event than a vector that tries to infect people a day (where both are randomly distributed through the population.) this makes intuitive sense, because there is lower probability that a subpopulation of susceptible hosts will become large when there are more attempts to infect them. similarly, in a system of cities interconnected by transport linkages, later peaks tend to be smaller and more variable than earlier ones. this is due to two things. first, a degree of lowgrade infection linking back through the system provides a higher total level of infection events in the whole system than the formally defined initiating disease vector. second, as time passes, the mix of immune versus susceptible becomes unsynchronized for the population as a whole, since hosts that escaped infection during one epidemic may be infected during the next, and some whose immunity has declined may also become infected. thus, it is expected that we would see the development of a complex non-repeating waveform with some similarity. this type of waveform is what epiflex shows with longer simulations in large populations, as illustrated in figure . a variety of results is obtained when one or just a few index cases are provided to seed a single city's susceptible population when those index cases are not repeated. this is expected, owing to random interactions that break the chain of contacts in some percentage of cases. this effect would be expected to increase with a higher skew on super-spreaders. the significance of this for modeled epidemics, particularly in the light of recent work [ ] , is that in some cases (the proportion would be expected to vary in rough accordance with r ) the infection dies out owing to random chance. thus, a monte-carlo model such as epiflex, when used in multiple trials, clearly reveals the potential range of variation in epidemics given apparently identical conditions. epiflex shows wave propagation of epidemics through its transport network that are similar to real world epidemic studies such as those of viboud et al. [ ] . viboud et al. state a mean duration of . weeks to spread across the united states, with a range from . to . weeks. the epi-flex results shown using a simplified city configuration of major airline hub cities, with a total . million population among all cities, shows a propagation wave of . weeks. while this is shorter than real world data, several factors account for the difference. first, in the current epiflex "vanilla" configuration, the transport network is flat in terms of the numbers of persons moved from city to city. second, each city contains the same population of only , due to practical limitations. for the propagation histogram of figure a , manifesting cases or more was used as the data point. please note, however, this flatness of transport and population is purely a matter of the configuration of the specific model used. the epi-flex system allows separate specification of all parameters for each city, and any kind of transport level between any two cities that is desired. figure a shows a histogram of cities in which , manifesting cases are first occurring. figure b shows a histogram of cities in which the first occurrences of at least incubating cases of influenza appear. note that in figure upper graph shows graphed points for population versus total morbidity as estimated from immunity figure upper graph shows graphed points for population versus total morbidity as estimated from immunity. lower two graphs show sample graphs for (lower left) and (lower right). the green line in the lower two graphs shows immunity. one vertical demarcation on the x axis is one month in the lower two graphs. epiflex is useful for doing in-silico experiments with epidemic behavior and easy to configure. it can run on an ordinary computer without special configuration. data can be imported from it into other tools and worked with there. it is effective for showing factors that are invisible or difficult to access under normal conditions, such as visibility of incubation and prodromal stages, true morbidity and estimates of immunity. epiflex has capabilities that were not discussed owing to time and space constraints. this system is effective in duplicating the kind of multimodality and apparent stochastic variation [ ] that are seen in real populations. epiflex results can provoke thought about the nature of epidemics and infectious disease spread in interesting ways by providing an experimental test environment that is not as abstracted from its subject as most mathematical models are. items in bold below are entities that are objects in the epi-flex system. they are the "nouns", each of which became a class when the program was written in c++. items in italics are actions, the "verbs" that represent interactions between objects. hosts go through the infectious stages, are members of demographic groups and move between locations locations contain hosts (for example a hospital, home or workplace.) areas contain locations (a city, typically) and contain special locations that can move hosts to locations in other areas. disease vectors introduce disease into host populations at some location in some area, in either a limited way or on a regular cycle. epidemic responses modify the spread of an infection. they represent actions taken to respond to the epidemic that will damp the spread. sirp is a simple flow chart, which is then elaborated into a multi-city system. three graphics are reproduced in figure concluding with output of the model, because the sirp paper is in a specialty publication and the work was important to the conception of epiflex. as seen in the "sirp diagram" of figure , a host begins as susceptible (s) in the upper left. they can become infected (i), and then they either recover (r) or are removed (removal not shown). after recovery (r), they have an immunity level that starts at or near complete as in sir. this immunity declines, in the case of influenza, over a period of roughly years, with a steep drop-off beginning after the first year, and hence is termed partial immunity(p). (it is not actually the case that the immune system's response to influenza antigens degrades so rapidly; rather, the virus mutates rapidly, and host immunity declines. the net result is similar.) this decline in immunity models the real world antigenic mutation of influ-enza viruses. in a more perfect model, the influenza virus would change and multiple viruses would be modeled. at some point the immunity declines so that it is effectively zero in the model (which corresponds to large antigenic change) and the person is returned to the fully susceptible(s) population. a person can become reinfected (diagonal arrow), though their probability of reinfection is lower, during this period of declining immunity. in the "multi-city sirp" flows of figure center, m [x,y] denote movement between the cities in a four city example. moving further to the right of figure and looking at the "actual versus model graph", one can see how a sinusoid curve, with some general correlation with period, results from the conventional model. there are four files used or generated by epiflex. all three generated files have the name of the configuration from a . year, city simulation linked by transport corridors. , people per city comparison of distribution of days when , manifesting cases or more first appear in a city (upper graph - a) versus distri-bution of days when or more incubating cases first appear in a city (lower graph - b) figure comparison of distribution of days when , manifesting cases or more first appear in a city (upper graph - a) versus distribution of days when or more incubating cases first appear in a city (lower graph - b). model files end in .epdm. these files are an xml format. this is the file edited by epiflex using the various panels available from the menu. an epdm file can also be edited directly in wordpad or a good xml editor by more sophisticated users. reading through them should be selfexplanatory to most users who familiarize themselves with the software. run record files end in .rpx. these files are in text format, comma, and semicolon delimited. this format is intended to be as easy to import as possible. rpx files can be imported into excel, read by sas, spss, etcetera. at the top of this file is a descriptor of the fields. the most common problem is to import using spaces as a delimiter and have spaces in area names define new columns. log files end in .log. everything that happens in a model is logged. these files can get fairly large. potentially, they could be parsed by a secondary piece of software to match them up with rpx files. however, that is an exercise for the user at this time. there is a viewer for log files under the view menu. snapshot files end in .snap. whenever a run completes, a snap file is written that is read only. this file is a duplicate of the model that was used to run -as that model existed in memory at completion of the run. this is done as a "lab notebook" aid because it was recognized early on that remembering exactly what was contained in a model was well nigh impossible and a lot of work to document. note that if your model does not complete its run, the rpx file will still be there, but the snap file will not be. in that case it is up to the experimenter to make a record of the state of the model. the snap file can be copied outside of epiflex, manually changed from read-only and the extension renamed to .epdm. the resulting model can then be used and run just like any other. software engineering details such as internal class definitions and structure of components will not be presented since the ui does a better job of educating, and is much more compact. disease component looking at screen shots of the current ui in figure , one can see what the functional parametric elements of a disease specification are. this particular example is selected for influenza. the parameters set reproduce the known characteristics of the disease. the disease stages of figure correspond to the model diagram shown in figure . one can see that this definition is superior in several ways to what is available with sirp modeling as shown in the previous section. (see figure ). in addition to multiple stages, one can define the level of infectiousness for each type of contact that an infected host might have. on the lower right, the partial immunity stage is specified. here, one is able to define a wide variety of immune responses. asymptotic decline seems to fit available data and theory on immune system (from left) sirp diagram, multi-city sirp, actual versus model figure (from left) sirp diagram, multi-city sirp, actual versus model. function the best for many diseases. use of the slider allows one to "eyeball" the asymptotic curve to approximate what seems reasonable. in this case, immunity is set to begin dropping after approximately half the day period has passed. it makes sense to do this because data in this area are fairly sparse, and one may want to perform multiple runs with slightly different immune drop characteristics. keep in mind that the width of the graph is for the number of days entered on this panel. a host is implicitly one of the number in the population of an area. in the epiflex system, there is one object defining each disease. a host infected by a disease receives a pointer to this archetypal disease object, and stores their disease stages internally as the disease progresses. most of the functionality of hosts consists of an implementation of disease stage transition. figure is a screen shot of the ui for a set of demographic definitions selected from us census data for the nation as a whole. in the resulting model, one can over-ride the default fractions later when using them in populations for specific areas. no fractions are represented that could not be derived from recent census data. each demographic group can be set up to specify variations in overall susceptibility to disease, or modify it by specific type of contact if that is desired. in the example, no fractional modifiers have been created for this demographic group. there is no necessity to stick to the demographic groups defined here. one can add new ones as desired. a location describes a place, the activities that occur there, and the demographic groups that may be drawn there automatically. a location can have a certain number of cells, which are used to specify n identically behaving locations concurrently. this acts as a location repetition count within an area when the location is defined. a technical point with homes as cells is that the model cannot yet be set to create a number of homes based on population size with a distribution of household sizes. figure disease definition panel. consequently, one must, at present, define the number of home cells appropriate to hold the population for each area. if a distribution function were available to specify household sizes and households were definable by population size, it would then be practical to provide canned profiles of household sizes based on us census data. the types of contacts that are available among hosts in the location are defined, together with the average number of such contacts per host during one cycle of the model; see figure . to the right of the contact definitions is a skew function that enables one to specify the degree to which contacts vary. within any host subpopulation, there are high contact individuals and lower contact individuals. the example shown sets the difference rather high to describe the way that service workers interact more strongly than customers. when running the simulation, during determination of which hosts become infected in this cycle, the position of the host in the queue is used together with the contact frequency to decide how many contacts an individual will get in a particular cycle. think of this adjustable skew graph as the histogram of contact counts. for each demographic group, a cycle of movement between locations in an area is defined as illustrated in figure . in the model used as an example, days are divided into three cycles. so a sequence of movements defines a day week. once a cycle completes, it restarts at the top. this is how hosts in the model are moved from one location to the next. an area is made up of a list of locations that it contains, as shown in figure . an area specifies a particular population level, and the population fractions for each demographic group making up the area are allocated at runtime. demographic population, removals and additions can be overridden at the area level. an area also has linkages to other areas, through special locations that are eligible as links. in the example of fig- group demographic population definition panel figure group demographic population definition panel. ure , the anchorage airport is going to send % of its population to chicago, illinois, each cycle. in each cycle, % of the population of the anchorage airport location is going to be shipped off somewhere. a 'vector' is defined in epiflex as n infection events to attempt at a specific location. a vector can repeat each cycle, or have a limited input. a vector can represent an arthropod or snail, or it can represent people arriving on aircraft from hong kong. the vector shown in figure is a startup vector for influenza used for these modeling runs. this initiating disease vector will operate for the first cycles, then stop, attempting to produce cases at a location in chicago. an initiation disease vector can have delay parameters, run continuously, or run once. it can force the infection of a specific number of people or infect them at a particular rate. when a disease occurs, the medical system will respond to it in some way. some illnesses, such as influenza a, will result in very little intervention. others, such as sars or smallpox, would result in a great deal of intervention very rapidly. consequently, a reasonable modeling system will allow response to an epidemic occurrence to be modeled. in the example shown in figure , we see the response specification for an emergent illness. a response has an alert trigger, which in this case is the appearance of fatalities. this results in a period of heightened awareness of the disease and its symptoms, which is set in this example to days. the probability of noticing the triggering event is set, to unity in this example, which may be overly optimistic. this specification says that after three fatalities, an alert would be triggered. since this alert trigger is for fatalities, not symptoms or instruments, the detection fraction for each disease stage is not applicable. once the alert has been triggered, the detection method for the illness switches primarily to symptoms. we can then specify what fraction of each occurrence will be diagnosed and at what stage. if we detect an event, then we can mitigate it. in this implementation, one has to specify the degree of infectiousness remaining after intervention is in process. some methods, for some diseases, can stop disease spread completely. this is not true for all diseases, so it must be specifiable. figure location definition panel. note that this component is the one with which the author is least comfortable. none of the examples for this paper have used responses as a consequence. the primary reason is that the way response is modeled is over-simplified and does not conform well to the response that happens in the real world; see: limitations of epiflex modeling in the introduction. epiflex is now composed of major classes, of which are core internal model functionality, the rest being infrastructure and ui. a functional walkthrough of how the system operates is presented below. a model starts running by verifying the model data. it looks for any references to things that were deleted and definitions that are impossible. it writes an error log file with this information that can be audited after the run. if errors are found, you will be informed of their severity and given the option to cancel. many of these errors are non-fatal, and may be modified with a warning, but they may change the results of a run. the epiflex system iterates through all areas in a model and allocates hosts, putting them in their initial locations, per the movement definitions for the demographic group. each group steps through its location movement list to determine whether the area to which it is attached has the locations the group needs. if it does not, epiflex will increment the pointer when it comes to this location, but it will leave the hosts in their previous real location until a good one is found. this was done for ease of practical use, allowing the same demographic to be used when a location may not exist in an area. step -disease stage pass. for each infected host, it will update the stage of the illness for that host. this is also where the system checks to see if disease response conditions have been met yet. step -vector pass. epiflex will iterate through each vector and apply the disease to the locations in each area in the vector according to the rules defined. step -infection pass. iterate through each location in each area, and figure out the contacts between infected and uninfected hosts. for each contact between an group movement cycle definition panel figure group movement cycle definition panel. infected host and a host not infected with the disease, a probabilistic determination will decide whether or not this illness is communicated. step -iterate areas looking for locations that have group draw factors. randomly pull hosts from the groups specified, and move them to the group draw location. this feature is used to simulate various things; generally an airport will have this type of specification. step -iterate all areas looking at area links. area links push population from one location to another area's location as specified. step -areas will be iterated, and each location in each area will be iterated using the random exchange factor to move hosts assigned to cells of a location between cells in that location. step -normal addition and removal of population groups is applied to model. this allows the user to model normal birth and death rate plus immigration and emigration across the outer boundary of the model. step -iterate all areas, and iterate through each location in each area. for each location, it will iterate each host attached to the location. each host has a location pointer for its group that indicates where it is in its movement cycle. it increments the pointer, and if the location name is different from the one it is in, it will find a location of that name in the area to put itself into. if the location has n cells, it will put itself into the cells in a semi-randomized fashion according to the parameters defined. note that currently the hosts do not remember which cell they are put into from one iteration to another. so they will not return to the same cell next time they come back around in their movement cycle. this is a limitation at present, which would require greatly increased space allocations to change. step -an audit is done to verify that all hosts occur only once in a location. step -areas are iterated, locations are iterated, to total results. results are then either written to the log file and/ or displayed on the main window in a graph form depending on how parameters were specified. figure area definition panel. area linkages panel figure area linkages panel. initiating disease vector definition panel figure initiating disease vector definition panel. monte carlo simulation program for ecosystems indisim, an individual-based discrete simulation model to study bacterial cultures modelling disease outbreaks in realistic urban social networks awarded $ million grant for disease-fighting computer modeling research notice on the role of social clusters in the transmission of infectious diseases transmission and dynamics of tuberculosis on generalized households epidemic diseases and host clustering: an optimum cluster size ensures maximum survival the implications of network structure for epidemic dynamics who mixes with whom? a method to determine the contact patterns of adults that may lead to the spread of airborne infections why are epidemics so unpredictable a mathematical model for global spread of influenza a contribution to the mathematical theory of epidemics review. perspectives on the basic reproductive ratio superspreading and the effect of individual variation on disease emergence the differential infectivity and staged progression models for the transmission of hiv modeling the spread of influenza among cities persistence and dynamics in lattice models of epidemic spread the evolution of parasite virulence and transmission rate in a spatially structured population modeling dynamic and network heterogeneities in the spread of sexually transmitted diseases. proceedings of the national academy of sciences planning for smallpox outbreaks the birth of object orientation: the simula languages object-oriented computer simulation of discrete-event systems tuberculosis models with fast and slow dynamics: the role of close and casual contacts worst case scenarios and epidemics baseline criteria and evolution of hosts and parasites: d , r and competition for resources between strains the age distribution of excess mortality during a hong kong influenza outbreaks frequency of naturally occurring antibody to influenza virus antigenic variants selected in vitro with monoclonal antibody comparative analyses of the specificities of anti-influenza hemagglutinin antibodies in human sera bulletin of the world health organization genetic diversity of rna viruses upshur r: principles for justification of public health intervention atlas of disease distributions -analytic approaches to epidemiological data cdc: who/nrevss collaborating laboratories national summary influenza and pneumonia deaths california of plimoth plantation - their number become thinned: native american population dynamics in eastern north america europeans, indians, and the age of discovery in american history textbooks ecological imperialism: the biological expansion of europe, - puritans indians and manifest destiny american indian holocaust and survival: a population history since synchrony, waves, and spatial heirarchies in the spread of influenza thanks to tara laforce, birthe borup and kathryn dereimer for their comments and review. this work was supported by bw education and forensics. publish with bio med central and every scientist can read your work free of charge key: cord- -f jwdzj authors: geoghegan, jemma l.; holmes, edward c. title: the phylogenomics of evolving virus virulence date: - - journal: nat rev genet doi: . /s - - - sha: doc_id: cord_uid: f jwdzj how virulence evolves after a virus jumps to a new host species is central to disease emergence. our current understanding of virulence evolution is based on insights drawn from two perspectives that have developed largely independently: long-standing evolutionary theory based on limited real data examples that often lack a genomic basis, and experimental studies of virulence-determining mutations using cell culture or animal models. a more comprehensive understanding of virulence mutations and their evolution can be achieved by bridging the gap between these two research pathways through the phylogenomic analysis of virus genome sequence data as a guide to experimental study. despite many advances in our understanding of virus evolution, how virulence evolves in a virus, particularly following a jump to a new host species, continues to be contentious. will a virus become more or less virulent in a new host? what level of virulence is optimized by natural selection and why? is there any consistent association between host-jumping and virulence such that predictions can be made about how virulence might evolve following emergence? not only will the answers to these questions reveal fundamental aspects of virus biology, but they also may assist in infectious disease management and mitigation, particularly as humans, other animals and plants face a continual threat from emerging viruses. the term 'virulence' has different meanings depending on context, can be assessed in a variety of ways and is often only an operational measure . to be as general as possible, we assume a simple working definition of virulence: the harm caused by pathogen infection, particularly in terms of host morbidity and mortality. virulence is also a complex trait determined by a combination of pathogen, host and environmental factors. although it is obviously necessary to understand all three, we focus on the pathogen (virus) component as this is the most tractable, with the small genomes and rapid replication and evolution of viruses facilitating comparative and experimental studies, and because there is strong evidence for heritable virus genetic variation for virulence . although we purposely focus on the analysis of virus genomes, genome-wide association studies (gwas) promise to open up new ways to explore the evolutionary impact of viral infections on host genomes and hence the intimate interaction between host and virus . another complexity in studies of virulence evolution is that metagenomics is increasingly showing that mixed (that is, polymicrobial) infections are commonplace , and even seemingly healthy hosts can carry multiple microorganisms of the kind often thought to be pathogenic , . determining which microorganism is the cause of a particular disease syndrome can be troublesome, and it is possible that overt illness might result from synergistic interactions between multiple microorganisms that overwhelm the host. hence, the model of one pathogen-one disease that has dominated studies of human infections, and implicitly models of virulence evolution, may be overly simplistic. it is also possible that measurements of relative virulence vary between humans and wildlife populations. for example, whereas dengue is considered an important infectious disease of humans, the virulence of dengue virus is low in terms of overall mortality in humans, and infections of equivalent severity may go unnoticed in wildlife, particularly as there is a strong sampling bias towards the most virulent presentations. virulence evolution in viruses has traditionally been studied from one of two separate research paths -the theoretical and the empirical -that have largely been pursued independently. although theory and empiricism have each generated important and parallel insights, they have each been able to paint only a partial picture of virulence evolution. few attempts have been made to bridge this divide , . there is now a large body of long-standing evolutionary theory that considers what level of virulence maximizes pathogen fitness under variable conditions, such as differing modes of transmission, levels of co-infection, selection pressures, and both within and between hosts . although of great value, a drawback is that this work is unavoidably based on a small number of case studies, the most famous of which is the co-evolution of myxoma virus (myxv) and european rabbits following the release of myxv as a biological control . however, the insights virulence although virulence can be defined in a range of casespecific ways, the simplest definition is the severity or harmfulness of a pathogen. the study of genetic material recovered from microbial (including viral) communities in which genome sequence data are simultaneously generated for all the microorganisms present. the reduction in numbers or elimination of pest organisms through the introduction of a pathogen. from examples such as myxv may be insufficient to adequately inform theoretical models confronted with novel, real-world emergence events. by contrast, empirical studies involve laboratorybased methods to identify the mutations that affect virulence (that is, virulence determinants), usually on the basis of a combination of reverse genetics and cell culture and/or animal models , , [ ] [ ] [ ] [ ] [ ] . these studies are often very successful in pinpointing causal mutations (see are not considered in an evolutionary context, their relevance for general theories of virulence evolution is usually ignored. in addition, in vitro methods may not reflect real-world selection pressures, there may be little consideration about how virulence mutations affect inter-host transmission, and animal models commonly differ from the species infected in the field. for example, virulence determinants in myxv identified on the basis of in vitro studies and mouse models have often not been upheld in reverse genetic experiments using the natural rabbit host . similarly, despite the regularity of their use, there has been a long-standing debate over the validity of ferrets as accurate models for human influenza . bridging the gap between the theoretical and empirical approaches would bring a new impetus to studies of virulence evolution. in this review, we outline how this can be achieved within a phylogenomics framework. we show that virus phylogenies are being increasingly used to help identify virulence determinants and that the data obtained can be used to test general theories of virulence evolution. such a phylogenomic approach to studying virulence evolution is timely because of the rapidity with which virus genome sequence data are now being generated, including during ongoing disease outbreaks of emerging viruses [ ] [ ] [ ] , and because of the development of new phylogeny-based methods for studying and visualizing genomic data [ ] [ ] [ ] . however, the success of this approach also requires that phylogenomic data are combined with relevant clinical, epidemiological and experimental metadata so that a direct link can be made between virulence, virus genotype and phenotype, and population fitness. arguably the most interesting context of virulence evolution is following a host jump as this sits at the heart of virus emergence, and the question of how virulence will evolve is commonly asked following the appearance of a new virus or the emergence of an existing virus with altered host range (box ). to make meaningful inferences, it is important to compare virulence in both the reservoir (that is, donor) and novel (that is, recipient) host species. although this may sound straightforward and is tractable in some cases , in reality it faces a number of difficulties. in many cases, including common infections such as hepatitis c virus , as well as emerging infectious diseases such as zika virus (zikv), the reservoir species is unknown or is at best uncertain. even if a reservoir species is known, we generally know little, if anything, about virulence in that species, and there is likely to be an ascertainment bias towards the most virulent cases. for example, although species of fruit bats appear to be reservoirs for ebola virus (ebov) , little is known about its virulence in these animals , as is true of many wildlife infections. the identification of host species may also change with better sampling, which is invariably poor in wildlife. for example, it was long thought that the canine parvovirus (cpv) that emerged in dogs in the late s had jumped from cats infected by a closely related virus . however, more recent sampling of wild carnivore species has shown this to be incorrect, such that the true reservoir species for cpv is unclear . therefore, it is crucial to understand disease processes, including virulence, in reservoir hosts under natural conditions, which will require more detailed studies of animal ecology. although we currently know little about virulence in reservoir species, comparative data tell us that, on average, low-virulence infections have a greater chance of successfully establishing transmission cycles in humans than viruses with higher mortality . this greater chance is presumably because high virulence requires a greater supply of susceptible hosts during the early stages of emergence. evolutionary biologists have had a long fascination with virulence [ ] [ ] [ ] [ ] [ ] [ ] . because there is a very large literature base on this subject, we necessarily provide only a brief overview here. a straightforward interpretation of virulence evolution is that natural selection will optimize the level of virulence that maximizes pathogen fitness, expressed as the basic reproductive number (r ) , although in reality fitness is shaped by a complex set of host-pathogen interactions , . current evolutionary theory tells us that when a virus jumps to a new species, its initial virulence can vary from asymptomatic to highly pathogenic, and precisely where it lies on this virulence spectrum is difficult to predict. however, it is possible that the direction of virulence evolution can be anticipated, at least in part, if the key relationship between virulence and transmissibility, and hence fitness, is understood. importantly, there is also evidence from insect viruses that host phylogeny is able to predict some aspects of virulence evolution following species jumps, with related host species tending to have similar levels of virulence . a commonly stated idea is that there is often an evolutionary trade-off between virulence and transmissibility because intra-host virus replication is necessary to facilitate inter-host transmission but may also lead to disease, and it is impossible for natural selection to optimize all traits simultaneously. in the case of myxv, this trade-off is thought to lead to 'intermediate' virulence grades being selectively advantageous: higher virulence may mean that the rabbit host dies before interhost transmission, whereas lower virulence is selected against because it does not increase virus transmission rates. a similar trade-off model has been proposed to explain the evolution of hiv virulence . however, many doubts have been raised about the general applicability of the trade-off model , [ ] [ ] [ ] , virus fitness will be affected by traits other than virulence and transmissibility , , , contrary results have been observed in experimental studies and relatively little is known about evolutionary trade-offs in nature. for example, in the case of the second virus released as a biocontrol against european rabbits in australia -rabbit haemorrhagic disease virus (rhdv) -there is evidence that virulence has increased through time, probably because virus transmission often occurs through blow flies that feed on animal carcasses, making host death selectively favourable . similarly, experimental studies of plant rna viruses have shown that high virulence does not necessarily impede host adaptation and, in the case of malaria, higher virulence www.nature.com/nrg reverse genetics experimental method used to identify gene function by modifying the sequence of a target gene and analysing its phenotypic consequences. the inference of evolutionary patterns and/or processes through the comparison of whole-genome sequences. depiction of the evolutionary history of a genetically related group of organisms. contains branches and nodes. infectious diseases that have recently appeared in populations or known diseases that are rapidly increasing in incidence or geographic range. (r ). represents the number of secondary infections caused by an infectious host in an entirely susceptible population. occurs when a change in one trait increases fitness but simultaneously reduces fitness because of its impact on another trait, thereby preventing the organism from optimizing both traits. was shown to provide the plasmodium parasites with a competitive advantage within hosts . other factors in addition to evolutionary trade-offs can shape the level of virulence in an emerging virus. for example, 'short-sighted' virulence evolution within a single host may be detrimental for inter-host transmission , and newly emerged 'spillover' infections that have experienced only a limited number of transmission events are likely to have virulence levels that have not yet been optimized for transmissibility by natural selection . accordingly, for spillover infections, ongoing transmission may be largely at the mercy of random drift effects, including the severe population bottlenecks that routinely accompany such events . finally, it is possible that virulence may sometimes simply be a coincidental by-product of selection for another trait or selection for transmission in another species. theory therefore tells us that natural selection can increase or decrease pathogen virulence, depending on the particular combination between host, virus and environment , , , , , , . although providing a useful framework, theory can provide only useful generalities because the relevant factors vary substantially and need to be assessed on a case-by-case basis. virulence evolution could, however, be better understood if its genomic basis were known. phylogenetic studies of viruses, including those that consider whole-genome sequences, are commonplace and are often used to understand a variety of aspects of virus evolution. in particular, virus phylogenies are being increasingly used to understand the evolution of key phenotypic traits such as virulence (and see the examples described below). phylogenomics provides an informative way to help understand virulence evolution and establishes a set of hypotheses that can be tested using appropriate experimental assays , . we also believe that phylogenomics provides valuable information on how natural selection acts on virulence and can be used to test general models of virulence evolution, thereby providing a key link between theoretical and empirical approaches to studying virulence evolution. the crux of this approach involves mapping mutations there is no clear understanding on whether emerging viruses become more or less virulent following a jump to a new host. before a host jump, pathogens are likely to have been selected to optimize their virulence for onward transmission in the reservoir host. however, immediately following a host jump, the pathogen is likely to be initially poorly adapted to the new host, which may inhibit successful emergence , and the host is likely to be poorly adapted to the new pathogen . indeed, high host mortality is associated with a reduced likelihood of successful onward transmission in the case of human viruses . there are three possible scenarios for virulence evolution immediately following a host jump (the filled nodes in the figure): • some of the most devastating epidemics have been associated with an increase in virulence following a cross-species transmission event (see the figure, part a). for example, whereas hiv almost invariably progresses to aids if untreated in humans, in african non-human primates, the closely related simian immunodeficiency virus (siv) persists without causing disease , or is associated with less overt disease than in humans . similarly, the (deliberate) transfer of myxoma virus (myxv) from the south american tapeti, where it was largely benign, initially resulted in mortality of ~ % in european rabbits . • no apparent change in virulence following a host jump can be seen in h n influenza a virus that jumped from horses to dogs , (see the figure, part b). disease is mild in both cases, and there are minimal biological differences between these viruses . similarly, rabies virus (rabv) causes an equally severe, ultimately fatal encephalitis in foxes and dogs and following spillover to humans . • cases of decreased virulence may be largely overlooked because avirulent viruses will often persist in a host population without detection. an example of decreased virulence following a host jump may be that of the infectious haematopoietic necrosis virus (ihnv) that spread from sockeye salmon to rainbow trout in north america and japan (see the figure, part c). upon the initial cross-species transmission event in the s to s, the virus seemed at first to be avirulent in the new host in contrast to the mortality seen in sockeye salmon. since then, however, virulence in some genotypes of ihnv in rainbow trout has increased, itself associated with an increase in transmissibility , . for many diseases, however, the extent of changes in virulence upon emergence is not known, in part because little is usually known about disease epidemiology in reservoir hosts, and there is likely to be a sampling bias towards the cases of highest virulence. nature reviews | genetics r e v i e w s volume | december | the initial and sometimes transient appearance of a pathogen in a new species following a host jump. onto phylogenetic trees of viruses sampled within and/or between disease outbreaks and from reservoir and novel hosts. the phylogenetic location of these changeswhether they fall on shallow or deep nodes (branches) and/or singularly or in parallel -makes it possible to infer, at least in broad terms, the selection pressures acting on virulence mutations and from this infer important aspects of virulence evolution (box ; fig. fig. | phylogenomics of virulence evolution. a | a model phylogeny with virulence determinants mapped to a fairly deep node suggesting that higher virulence has increased virus fitness. b | a model phylogeny with virulence traits mapped to shallow nodes suggesting that higher virulence reduced pathogen fitness so that viruses with these mutations are purged from the population or require compensatory mutations. c | a model phylogeny with a high-virulence mutation arising multiple times independently owing to parallel or convergent evolution. the occurrence of parallel/convergent mutations that occur more frequently than by chance is likely to reflect adaptive evolution (fig. ) . d | the relationship between virulence, fitness and host jumps. a virus is assumed to be at a fitness peak (high r ), in this case high virulence, in the reservoir host, so that the mutations determining both virulence and host range are expected to be subject to strong purifying selection (for example, a low value of d n /d s ). as the virus emerges in the new recipient host, it will initially be maladapted (that is, reside in a fitness valley) and subject to genetic drift as the population is small. as it adapts to the new host, virulence will be selectively optimized (in this case declining), increasing r and resulting in positive selection (for example, d n /d s > , although other measures of selection pressure are available). once the virus becomes adapted to the new host, the virulence determinants are again subject to purifying selection. the greater the fitness of a virulence determinant, the more rapidly it will spread through the virus population and the deeper it will fall on a virus phylogeny (that is, closer to the root of the tree), including on the branch linking reservoir and novel hosts. of particular importance are repeated occurrences of the same mutation falling on deep branches across multiple outbreaks, or multiple cross-species transmission events, as both parallel evolution and convergent evolution can be signatures of adaptive evolution , - (fig. ) . for example, in the case of west nile virus (wnv), a single mutation in the virus helicase protein repeatedly evolved in high-mortality outbreaks in birds, which is indicative of a selective advantage . similarly, the reversion to virulence in oral polio vaccine (opv) strains of poliovirus has been associated with extensive parallel evolution , and parallel evolution was also associated with hostspecific adaptation in experimental studies of crossspecies transmission involving drosophila virus c . because adaptive evolution has been at play, phylo genies of the sequence in question may have a characteristic shape , and the sequences associated with selected branches may also contain genomic signatures indicative of positive selection, such as the rapid fixation of amino acid changes or an increased rate of nonsynonymous to synonymous substitutions per site (ratio d n /d s ) , . in the case of frequent parallel or convergent evolution for specific virulence mutations, it is also possible that the amino acid sites involved will have signatures of positive selection, such as an elevated d n /d s (as was the case in wnv; see below). following the same logic, mutations that fall on shallow branches in virus phylogenies (that is, closer to the tips) are present in a smaller proportion of the population and are therefore more likely to be of lower fitness such that they may be removed by purifying selection. hence, virulence-determining mutations that repeatedly fall on tip branches alone are likely to inhibit some other aspect of pathogen fitness, thereby reducing r at the population scale. although this approach has a solid theoretical and empirical basis , , a complicating factor is that a virulence-determining mutation that has very recently emerged will necessarily fall towards the tips rather than on an internal branch even if it is selectively advantageous. similarly, although popular, d n /d s measures are less robust over short timescales, such as during outbreaks, because mutations may not have reached fixation by positive selection or had time to be purged by purifying selection, and it can be difficult to detect selected mutations that occur only once , . approaches to detect positive selection that do not rely on d n /d s , such as those based on tree shape , or tracking mutations that are increasing in frequency compared with those thought to be evolving neutrally , , may therefore add analytical power. the phylogenetic mapping of virulence mutations can proceed in two ways depending on the extent of a priori knowledge. in a 'top-down' approach, in which virulence determinants are unknown, a virus phylogeny is inferred, mutations are mapped onto this phylogeny and the mutations on key branches are then identified. such 'key branches' include those directly associated with cross-species transmission events, invasions of new geographic areas, increases in rates of transmission, spikes in morbidity and/or mortality or clear instances of positive selection. the mutations identified in this way are candidates for virulence determinants that can be tested in an appropriate experimental framework . an example of this approach is shown in fig. . the second, 'bottom-up' , approach utilizes existing knowledge of virulence determinants, such as that determined by an experimental study. the putative virulence determinant is then mapped onto the phylogeny, and its phylogenetic location (that is, deep or shallow branch, singular or parallel/convergent evolution) is used to infer how it affects virulence evolution, whether it is associated with reciprocal mutations that reflect evolutionary trade-offs and the selection pressures it faces. although this phylogenomic approach is being increasingly used to identify virulence determinants, and we discuss a number of real data examples below, it can be used to make general statements about the nature of virulence evolution. specifically, a virulence mutation that falls deep in the phylogeny such that it is inherited in all subsequent branches, and one evolving in parallel or with evidence of positive selection, necessarily implies that virulence is selectively advantageous. conversely, a virulence determinant that occurs sporadically on shallow branches and is subject to strong purifying (negative) selection suggests that virulence is not directly beneficial, probably because it inhibits some other component of overall fitness. in such cases, each instance of high virulence may represent an independent and transient evolutionary event. if only a single mutation is associated with a change in virus virulence, as in the case of wnv, then this change in virulence is likely to be selectively advantageous without an evolutionary trade-off with transmissibility, as a reduction in transmissibility would probably need to be compensated for by additional reciprocal mutations located elsewhere in the genome. hence, if multiple mutations fall on a branch associated with a change in virulence, it is possible that some are virulence determinants and the others are associated with evolutionary trade-offs on other traits. although we have described it in terms of emerging viruses, this phylogenomic approach can, in theory, be applied to any system in which a phylogeny can be inferred and in which it is possible to experimentally assess the impact of individual mutations on virulence. similarly, it can be used to study other virological traits associated with disease emergence, particularly host range. for example, the repeated evolution of the same amino acid changes following the cross-species transmission of avian influenza virus to humans strongly suggests that they directly affect host range , and a similar approach has been used to elucidate the nature of the evolutionary arms race between viruses and their hosts , . critically, however, the approach described here should also be considered an idealized one that works best when a limited number of genomic mutations act independently to shape virulence. virulence determinants nature reviews | genetics r e v i e w s volume | december | the transmission of a pathogen from one host species to another. also called hostjumping or host-switching. an evolutionary process by which two or more separate lineages develop identical characteristics independently. the descendants of unrelated ancestors that have evolved similar traits independently. natural selection that leads to advantageous mutations spreading through a population. mutations in coding regions can be either synonymous (which do not change the amino acid; measured as d s ) or non-synonymous (which change the amino acid; measured as d n ). the d n /d s ratio > is sometimes used to infer the occurrence of positive selection, although the accuracy of this measure depends on various factors, including the timescale of sampling. natural selection that acts to remove low-fitness (including deleterious) mutations from populations. it is the most common form of natural selection and gives a d n /d s < . also called negative selection. may be harder to identify when there are more complex interactions between mutations , which appears to be true of myxv (box ). although epistasis is likely to be commonplace in rna viruses , little is currently known about whether virulence mutations interact epistatically . similarly, this approach may work best for rna viruses because their constrained genome sizes mean that there are probably a limited number of virulence determinants, increasing the likelihood that they are subject to parallel and/or convergent evolution, and rates of recombination (which complicate phylogenetic relationships) are often fairly low within species . to illustrate how a phylogenomic approach can shed light on the evolution of virus virulence, we now briefly outline a number of cases in which it can be or has been applied. we begin by considering cases in which virulence determinants have been successfully mapped (wnv and avian influenza a virus (aiv)), move on to those in which revealing the mutations that underpin changes in virulence has been more complex (myxv, marek's disease virus (mdv) and hiv) and end by examining virulence evolution in two recent disease outbreaks (ebov and zikv). the evolution of virulence in strains of oral polio vaccine (opv) . opv is an attenuated form of poliovirus that can occasionally revert to a virulent form and cause outbreaks of poliomyelitis. a | phylogenetic analysis of opv strains in nature reveals that some mutations associated with high virulence have experienced more frequent parallel evolution than expected by chance (and occupy well supported nodes) and hence are likely to be seletively favoured . b | computational evolutionary analysis then reveals that this parallel evolution for high virulence is associated with a hypothetical threonine-to-proline (t-to-p) amino acid change that is subject to significant adaptive evolution (which can be detected in a variety of ways) , . c | the virulence impact of these mutations is then confirmed in both in vitro (cell culture; part ca) and in vivo (mouse; part cb) experimental studies. in all cases, the red shading signifies increased virulence. the interaction among genes at different loci. the function and evolution of one gene may be dependent on the presence of one or more other genes. analysis in each case has told us about the evolution of virulence in general. in , a new lineage of wnv became the leading cause of arthropod-borne viral encephalitis in humans and horses in north america, spreading from east to west across the continent and causing severe mortality in many bird species, particularly the american crow . phylogenomic analysis revealed that a single thr pro (t p) amino acid substitution in the virus ns helicase protein was associated with high-virulence wnv outbreaks in corvids on multiple continents. experimental analysis in captive crows then showed that this mutation was sufficient to explain the high fatality rates in american crows, perhaps because it increased the rate of virus replication . wnv therefore provides an important example of where a single genetic switch controls virulence, which is obviously the easiest scenario to detect using a phylogenomic approach. of more general importance was that t p evolved in parallel and experienced an elevated rate of nonsynonymous change, suggesting that high virulence was selectively favoured in the absence of an evolutionary trade-off as no reciprocal mutations were observed elsewhere in the viral genome the co-evolution of european rabbits (oryctolagus cuniculus) and the myxoma virus (myxv) released as a biological control against them is arguably the most famous case study in virulence evolution. fenner and colleagues were able to identify 'grades' of myxv virulence using laboratory assays in european rabbits. the most lethal grade i strains (shaded red in the figure) were characterized by almost % mortality, whereas the most 'attenuated' grade v strains had mortality < % and longer survival times. grade i strains were released in both australia (starting strain, sls) and europe (starting strain, lausanne) in the early s, and soon after lower virulence viruses began to appear in parallel in both continents, with intermediate virulence grades (for example, grade iii) becoming the most common in the field. although of large importance, the studies of fenner were necessarily limited in that the genetic basis of virulence evolution was unknown. that the trajectory of virulence evolution was the same in both australia and europe meant that the same virulence determinants might be involved on both continents. therefore, it was a surprise when the first large-scale genome comparisons of myxv revealed that virulence grades changed frequently across the virus phylogeny, as depicted in the phylogeny, and that different mutations appeared in the australian and european epidemics (depicted as different filled shapes at branch tips), with no parallel evolution of possible virulence determinants within australia and europe , . for example, there were no mutations that were unique to the most attenuated grade v viruses, nor to the highest virulence grade i viruses. hence, there are multiple genetic routes to attenuation or virulence in myxv, such that there has been convergent evolution for phenotype but not genotype, and both attenuating and virulence-restoring mutations have been fixed in australian myxv. therefore, there is no simple way to predict virulence evolution from genome-scale comparisons in the case of myxv and no parallel or convergent evolution to guide experimentation. indeed, experimentally verified virulence determinants in rabbits remain elusive . the ongoing evolution of myxv virulence reflects both mutations acquired in the virus and resistance evolution in the rabbit host . this co-evolutionary process has also resulted in a marked increase in myxv virulence . unlike viruses sampled in the s before the evolution of any host resistance, most virus isolates sampled in the s that have evolved in the face of host resistance induce a lethal immune collapse syndrome with similarities to septic shock . may have been responsible for a substantial proportion for the cross-continent spread of the virus , in which case t p may have been selected to increase replication (transmissibility) in that species, a coincidental by-product of which was heightened virulence in crows. ever since the emergence of the highly pathogenic h n subtype of influenza virus, there has been concern over whether this influenza virus could establish sustained transmission in humans, in which it causes only sporadic spillover infections at present , . more recently, highly pathogenic h n has sporadically infected humans and continues to spread through poultry populations in china , evolving from a low-virulence ancestor . although the true number of human cases, and hence accurate mortality, is difficult to ascertain, it is clear that both h n and h n cause fairly high mortality in humans and could have serious consequences were they to trigger a large-scale human epidemic. this concern has led to attempts to use genomic data to help in pandemic risk assessment . at the virus subtype level, the presence of a run of polybasic amino acids in the hinge region between the ha and ha subunits that make up the haemagglutinin (ha) protein of influenza virus helps it establish a systemic, and subsequently more serious, infection and thereby acts as a useful marker of high-virulence strains of the h and h aiv subtypes , , . this marker makes it relatively easy to distinguish between potentially lowvirulence and high-virulence aivs, although what triggers the evolution of the high-pathogenicity variants in these subtypes is unclear . other individual amino acid changes, affecting a variety of gene functions, have also been proposed as specific virulence determinants for h n (refs - ) as well as in those viruses that circulate in human populations such as seasonal h n (ref. ), the h n virus responsible for the global pandemic of - (ref. ) (in which host inflammatory and cell death responses to infection appear to play a key role) , and influenza b virus (table ) . the key unresolved question is how natural selection will shape both virulence and transmissibility if an aivlike h n or h n virus is eventually able to develop sustained transmission in humans. an added complexity is that phylogenomic analyses reveal a consistent set of mutations that distinguish human and avian influenza viruses, although whether these affect host range alone, or both host range and virulence, is unclear , . the canonical study of virulence evolution following a species jump is myxv in european rabbits, with a body of classic work undertaken by fenner and colleagues - (box ). in both australia and europe, highly virulent strains of myxv were used as a biological control against the european rabbit population, with releases beginning in the early s. in both continents, the same trajectory of virulence evolution was observed: virulence declined from the highly virulent (that is, grade i) release strains to encompass a far wider range of virulence grades, including the most attenuated grade v strains, with strains of 'intermediate' virulence the most commonly sampled in the field. this pattern, reflecting a combination of the virus evolving more attenuated strains and the host developing resistance, fuelled the idea of a trade-off between virulence and transmissibility. sixty years after the initial release of myxv, the first large-scale genomic studies of its spread were performed (box ). phylogenomic analysis revealed that the virulence phenotype has changed on a regular basis . however, a major surprise was that each change in virulence was associated with a different set of mutations across multiple genes , . although which mutations had the greatest impact on virulence is still unclear and requires further experimental analysis, such a phylogenomic pattern indicates that there are multiple routes to achieving the same levels of virulence, including attenuation, such that there has been convergent evolution for phenotype but not genotype. it is likely that this evolutionary flexibility in part reflects the fairly large genome size of myxv (a double-stranded dna virus of ~ , bp), which may mean that there is a large number of potential virulence determinants that can interact through epistasis , in turn complicating any phylogenomic analysis. marek's disease virus. whether 'imperfect' (that is, 'leaky') vaccination against infectious disease, in which disease symptoms are reduced but there is less impact on virus replication and transmission, will change the selection pressures acting on the pathogen and affect virulence evolution has been the source of debate , . although still contentious, particularly in the case of human disease, there is good evidence that imperfect vaccination has increased virulence in the case of mdv, a dna herpesvirus that poses a major problem to the poultry industry . in the s, the appearance of virulent mdv strains forced the development of the first generation of marek's disease vaccines. however, because these vaccines were imperfect, 'very virulent' mdv began to appear within years, necessitating a second-generation vaccine. this very virulent mdv was followed, more rapidly, by the appearance of 'very virulent plus' mdv, requiring a third-generation vaccine (fig. ) . imperfect mdv vaccines enhance virulence by elongating the infectious periods and hence transmission potential of virulent strains that would have been removed by natural selection before transmission in the absence of vaccination . although the genomic basis to mdv virulence evolution is currently uncertain, with some causative amino acid changes proposed , initial phylogenomic studies suggest that, as in the case of myxv, there are multiple genetic pathways to high virulence (and which again may reflect the fairly large size of the viral genome). not only does virulence evolution in mdv have important implications for vaccination strategies against other diseases in which vaccine efficacy is fairly low , but it also shows that in some circumstances increased virulence can be selectively advantageous. given the importance of hiv to human health and that it ignited much of the research on disease emergence, it is no surprise that there has been considerable discussion on the evolution of hiv virulence [ ] [ ] [ ] [ ] [ ] . indeed, it is striking that hiv in humans is markedly www.nature.com/nrg r e v i e w s | december | volume influenza a viruses are categorized into subtypes on the basis of the diversity in two proteins present in the viral envelope: haemagglutinin (h or ha) and neuraminidase (n or na). more virulent than the closely related viruses that naturally infect non-human primates in africa (box ). although there have been suggestions that hiv has begun to evolve reduced virulence , discussions of the trajectory of virulence evolution are necessarily complicated by the fact that antiviral therapy has greatly extended life expectancy. hiv virulence is often approximated as the degree of variation in the set point viral load (spvl) that is established soon after initial infection . the higher the spvl, reflecting greater levels of virus replication, then the more rapidly the patient will progress to aids in the absence of antiviral therapy, although other studies have suggested that the replicative capacity of the virus itself is a more informative marker of virulence and is also a direct measure of virus fitness . indeed, some 'controller' individuals are able to control levels of hiv in the absence of antiviral therapy, and it has been shown that this is in part due to infection with viruses of reduced replicative capacity . importantly, viral genetic variation may play a more important role in shaping hiv virulence than host factors, with approximately one-third of the observed variability in spvl assigned to virus factors and only ~ % seemingly due to the host . this observation also implies that spvl, and hence virulence, can be selectively optimized . in support is evidence that spvl, and hence virulence, has declined in some african hiv subtypes, even accounting for the use of antiviral therapy, and that this reflects a trade-off between virulence and transmissibility . importantly, however, despite many studies into the determinants of hiv virulence, the virus genomic mutations responsible for determining spvl are still uncertain and multiple genes may be involved . the difficulty in assigning the genetic determinants of spvl may be in part due to genetic variation across viral populations . for example, heritability in spvl was highest (~ %) between individuals in the swiss hiv cohort, which also represents the most homogenous viral population marek's disease virus (mdv) of chickens present on poultry farms. this imperfect vaccine reduced disease symptoms but did not prevent virus replication, thereby extending the infectious periods, and hence potential for transmission, of virulent strains that would have been removed by natural selection before transmission to a new host in the pre-vaccine era . because of this, 'very virulent' mdv began to appear within years, necessitating the development of a secondgeneration vaccine that was also imperfect. this was followed, in an even shorter period, by the appearance of 'very virulent plus' mdv, requiring a third-generation vaccine. although the genomic basis of mdv virulence is currently unknown, the phylogenies at the bottom of the figure hypothetically assign virulence to multiple causative mutations (as in the case of myxoma virus). the dashed arrows indicate the evolution of viruses to the next virulence grade. the amount of phenotypic variation in a population that is attributable to individual genetic differences. the - outbreak of ebov (makona variant) in west africa was the largest and longest described in humans since the first description of the disease in , with approximately , cases and some , deaths. in addition to hindering attempts at disease control, this elongated period of transmission in humans may have resulted in different selection pressures from those faced in the animal reservoir. this outbreak also raised key questions about virulence evolution, particularly whether natural selection would have favoured ebov variants causing higher or lower human case fatality rates had the virus not been stamped out by public health intervention . phylogenetic analysis of ebov during the - outbreak revealed an ala val (a v) substitution in the virus glycoprotein to be of particular importance , . a v is notable as it falls on a deep internal branch of the ebov phylogeny, compatible with adaptive evolution, whereas other amino acid changes are associated with individual or only small clusters of sequences. moreover, a v improves binding to the human npc receptor utilized by ebov , which would increase infectivity in humans, at the same time reducing infectivity in cells from the bat reservoir species , . intriguingly, the appearance of a v on the ebov phylogeny is associated with two key epidemiological features: an increase in case numbers and an increase in mortality (fig. ) . if there was indeed an increase in both ebov transmissibility and virulence, then higher virulence is likely to have directly increased viral fitness, and in the absence of an evolutionary trade-off as only a single substitution was identified. however, these apparent changes in phenotype also coincided with the movement of the virus from guinea to sierra leone, such that any change in case numbers and mortality could in fact be due to a change in epidemiological factors (such as access to health care or differing human demographics and/or transmission networks), and recent studies using animal models suggest that a v has no direct impact on virulence . zikv is the most recent emerging virus to lead to a major public health scare and is puzzling because a seemingly benign virus suddenly increased in virulence, causing severe neurological disease in humans. before , there were fewer than human cases of zikv reported and all were mild infections restricted to africa and asia , . consequently, neither the disease caused by zikv nor the molecular determinants of zikv virulence were well characterized, and it is likely that there was systematic under-reporting of infections, including those associated with severe disease. in , the pacific islands reported the first major outbreaks of zikv before the virus spread to the americas in . although the majority of human infections range from asymptomatic to mild, the virus was associated with the neurological guillain-barré syndrome in french polynesia in (ref. ), and those cases from the americas, particularly brazil, were linked to more severe diseases, including congenital abnormalities such as microcephaly . phylogenetic analysis revealed that the most recent zika epidemics are due to the asian lineage of zikv, rather than the african lineage , and that the virus spread cryptically in brazil for at least a year before its detection . although there are multiple amino acid differences between the african and asian lineages , , it has been claimed that those in the asian lineage that spread through the americas may be directly linked to both increased infectivity in aedes aegypti mosquitoes and microcephaly, most notably a ser asn (s n) amino acid change in the prm protein . however, there is still considerable uncertainty in this area, with others arguing that viruses from both lineages can cause neurovirulence but that cases often go unreported , . hence, the case of zikv highlights the difficulty in assessing virulence evolution within a background of sparse and biased sampling even with phylogenomic data and shows the importance of collecting reliable, real-time epidemiological data even in low-incidence situations. , . maps above the phylogeny show the spread of ebov over the timeline of the outbreak in the three affected countries in west africa, where blue-shaded regions correspond to the wild-type virus variant (a ) and redshaded areas correspond to mutated virus variant (v ). it is possible that a v was also associated with an increase in both ebov case numbers and mortality (that is, virulence) as the outbreak progressed, such that increased virulence is directly selectively advantageous, although this is confounded by epidemiological factors. virulence evolution has been one of the longest-standing issues in evolutionary biology. although a strong body of theory has been developed, there are few cases in which we understand the forces that have shaped particular instances of virulence evolution and even fewer in which we have successfully linked evolutionary theory with individual genomic changes. we believe that a synthesis of experimental studies of virulence determinants and long-standing theory of virulence evolution set within a phylogenomic framework will generate a more comprehensive understanding of virulence evolution. in particular, not only does a phylogenomic approach enable potential virulence determinants to be identified, which is being increasingly used in the case of emerging viruses, but this analysis also sheds light on the models of virulence evolution that have occupied theoreticians for decades. recent advances in real-time genomics during disease outbreaks , and the increased demand for precision in public health interventions may help in the development of a new understanding of the evolution of pathogen virulence. we contend that this can be achieved within a phylogenomic framework as long as relevant data are available and strong links are made between genomics, phylogenetics, epidemiology, and experimental studies of virus virulence and fitness. therefore, it is critically important to collect clinical (that is, disease symptoms and severity) and epidemiological (that is, time and place of sampling) metadata concurrently with the sequencing of virus genomes and to sample across a range of clinical syndromes, not just those associated with severe disease. we also stress the value of gathering concurrent and historical data from likely reservoir species as these will provide a more complete insight into virulence evolution and determining the full range of microorganisms that infect a particular species, as well as their interactions, as assigning disease syndromes to individual pathogens may often be difficult. thankfully, advances in metagenomics now make the latter task feasible , , . similarly, there is a marked lack of good virulence grading schemes among viral infections. although such schemes can sometimes be simplistic, assuming discrete virulence categories that may not exist in nature and incorporating degrees of subjectivity, the case of myxv shows that they are key to considering the relationship between genotype and phenotype that is essential to understanding virulence evolution. finally, it is possible that an increased understanding of virulence evolution drawn from a phylogenomic approach may contribute to new strategies for pathogen control and eradication, and there is a clear potential for this framework to inform and improve the fields of disease management and the biological control of invasive pests. although predicting where and when a new disease might emerge is clearly unfeasible because of the immense complexities involved , , predicting the overall trajectory of the virulence evolution of a virus in a novel host may be more achievable. once again, biocontrol presents a compelling example. although controversial [ ] [ ] [ ] , the proposed release of cyprinid herpesvirus (cyhv- ) as a biological control against invasive common carp (cyprinus carpio l.) in australia may present a unique opportunity to follow, in real time, the co-evolution between host and virus at both the genotypic and phenotypic scales. both theory and virus natural history predict that cyhv- virulence will decline with time , and it will be interesting and informative to see how any such virulence evolution is manifest in phylogenomic data. this review provides insight into how virulence theory might be united with empirical data and what is needed to better understand the relationship between virulence and transmissibility elevated virulence of an emerging viral genotype as a driver of honeybee loss microbial genome-wide association studies: lessons from human gwas genome-to-genome analysis highlights the effect of the human innate and adaptive immune systems on the hepatitis c virus unbiased parallel detection of viral pathogens in clinical samples by use of a metagenomic approach redefining the invertebrate virosphere the evolutionary 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antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus m r and m r are two virulence factors for myxoma virus pathogenesis in the european rabbit genetic and structural analysis of a virulence determinant in polyomavirus vp sheeppox virus kelch-like gene sppv- affects virus virulence related links national geographic: leaky vaccines enhance spread of deadlier chicken viruses both authors contributed to all aspects of the manuscript. the authors declare no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. nature reviews genetics thanks s. alizon and the other, anonymous reviewer(s) for their contribution to the peer review of this work. key: cord- -b r cyp authors: maritz, julia m.; land, kirkwood m.; carlton, jane m.; hirt, robert p. title: what is the importance of zoonotic trichomonads for human health? date: - - journal: trends parasitol doi: . /j.pt. . . sha: doc_id: cord_uid: b r cyp trichomonads are common parasites of many vertebrate and invertebrate species, with four species classically recognized as human parasites: dientamoeba fragilis, pentatrichomonas hominis, trichomonas vaginalis, and trichomonas tenax. the latter two species are considered human-specific; by contrast, d. fragilis and p. hominis have been isolated from domestic and farm mammals, demonstrating a wide host range and potential zoonotic origin. several new studies have highlighted the zoonotic dimension of trichomonads. first, species typically known to infect birds and domestic mammals have been identified in human clinical samples. second, several phylogenetic analyses have identified animal-derived trichomonads as close sister taxa of the two human-specific species. it is our opinion, therefore, that these observations prompt further investigation into the importance of zoonotic trichomonads for human health. the trichomonad lineage in phylum parabasalia trichomonads are anaerobic, flagellated protists belonging to the large and diverse groups trichomonadea and tritrichomonadea of phylum parabasalia [ ] . they are characterized by the presence of three to five anterior flagella, hydrogenosomes -hydrogen-producing organelles corresponding to anaerobic versions of mitochondria [ ] , a parabasal body (a large golgi), and a complex cytoskeleton. a few species have been isolated from environmental samples and may represent free-living species; however, the majority of species form symbiotic interactions (see glossary) with various animal hosts. among the parasitic trichomonads, several species inhabit the oral, digestive, and urogenital tracts of invertebrate and vertebrate hosts, including livestock, pets, and humans. historically, phylum parabasalia was divided into two groups based on morphological characteristics; however, the recent inclusion of molecular data recovered six groups: trichomonadea, tritrichomonadea, hypotrichomonadea, cristamonadea, spirotrichonymphea, and trichonymphea [ ] . the trichomonadea, tritrichomonadea, and hypotrichomonadea are of primary concern to parasitologists; however, the evolutionary relationships within and between these groups are unclear [ ] . several molecular phylogenies have attempted to resolve these evolutionary relationships using phylogenetic markers such as ribosomal rna (rrna) and protein coding genes (figure ), which give inconsistent phylogenies [ , ] . four species of trichomonad are considered human parasites: trichomonas vaginalis (found in the urogenital tract) [ ] , trichomonas tenax (localized to the oral cavity) [ ] , and pentatrichomonas hominis and dientamoeba fragilis (located in the digestive tract) [ , ] . only one species has wellestablished pathogenic potential: t. vaginalis, the cause of the most prevalent non-viral sexually transmitted infection in humans, trichomoniasis [ ] . only t. vaginalis and t. tenax are considered human-specific, with the former characterized by the richest, although still limited, epidemiology data [ ] , but very little is known about the latter. p. hominis and d. fragilis can cause gastrointestinal symptoms in some patients, such as abdominal pain and diarrhea [ , ] , d. fragilis has also been proffered as a potential causative agent of irritable bowel syndrome (ibs) [ , ] , but debate surrounds its pathogenicity, infection route, and epidemiology [ ] . in addition, several trichomonad species are of veterinary importance, such as the avian pathogens trichomonas gallinae, tetratrichomonas gallinarum, and histomonas meleagridis [ ] [ ] [ ] [ ] , and tritrichomonas foetus, the causative agent of a venereal disease in cattle [ ] . this extensive host range, along with the isolation of d. fragilis [ ] and p. hominis [ ] from various animal hosts, suggests that certain species of trichomonads may exhibit the characteristics of zoonoses. although the question of zoonotic trichomonads has been considered for some years (e.g., [ , ] ), recent results from several different sources have highlighted this potential. here we summarize the clinical and phylogenetic studies that suggest a zoonotic potential for trichomonads, discuss their implications for human health, and the next steps required for investigation into their epidemiology, pathobiology and evolution. new evidence supports the zoonotic potential of trichomonads human trichomonad infections are not body site-specific the four trichomonad species recognized as human parasites were initially thought to be site-specific [ ] (table ) . however, various clinical studies have shown that they can also be found in atypical locations. for example, t. tenax, a commensal of the human mouth found in patients with poor oral hygiene [ ] , has been identified by microscopic and molecular methods in the upper and lower respiratory tracts [ , ] . one possibility that could account from this 'aberrant' location is inhalation of the parasite from the oral cavity into the respiratory tract. however, in some cases where t. tenax was identified in the respiratory tract, no parasites were found in the mouth [ ] . other human trichomonad species have also been identified in the respiratory tract including the sexually transmitted species t. vaginalis [ , ] and the gut parasite p. hominis [ ] , which suggests that these species too can proliferate outside their usual body sites. at least five species of trichomonad, including p. hominis, t. tenax, t. vaginalis, t. foetus, and t. gallinarum, have been identified in the human respiratory tract and as causative agents of pulmonary trichomoniasis (table ) . they have been found in up to % of patients with pneumocystis pneumonia (pcp) and in up to % of patients with acute respiratory distress syndrome (ards) [ ] . because trichomonads are microaerophilic it is unlikely that they initiate and cause these diseases themselves, but may represent secondary and opportunistic infections that could exacerbate symptoms and prolong illness [ ] . these trichomonad respiratory infections seem to depend upon: (i) the presence of bacteria on which to feed and (ii) local anaerobic conditions caused by pcp or ardsassociated infections [ ] but not necessarily upon immunosuppression, because drugs against pcp consistently cure patients of pulmonary trichomonosis and, in one study, treated ards patients were not found to be immunocompromised [ ] . thus, the presence of an increasing number of distinct trichomonads in a broader range of clinical samples from patients with diverse diseases, such as aids, rheumatoid arthritis, prostate cancer, pulmonary infections (empyema and pneumonia in addition to pcp and ards), and digestive conditions such as diarrhea and ibs [ ] [ ] [ ] , is becoming increasingly apparent. indeed, the frequency of pulmonary trichomonosis infections may be higher than reported because transformation of parasites from the motile, pear-shaped stage to the amoeboid stage renders microscopic identification in clinical samples difficult [ ] , highlighting the importance of molecular data to identify such infections [ ] . non-human species of trichomonad have been isolated from clinical samples trichomonads were thought to have strict host specificity [ ] ; however, trichomonad parasites not previously reported as infecting humans have recently been found in human clinical samples (table ) . for example, parasites belonging to the genus tritrichomonas can be isolated from the reproductive tract of cattle (tritrichomonas foetus), the nasal mucosa and intestine of pigs (tritrichomonas suis), and the intestine of non-human primates (tritrichomonas mobilensis) [ ] . another example is t. foetus, historically considered specific to cattle [ , ] . nonetheless, experimental cross-infections of the parasites between pigs and cattle in addition to analysis of molecular data suggest that these three species should be considered glossary commensal: : a form of symbiosis between two organisms where one derives benefit, whereas the other is unaffected. some gut trichomonads are thought to represent commensals. disease incidence: : the number of new disease cases that occur in a population for a given time period (typically per year). dysbiosis: : an imbalance of the microbiota (the microbial populations at a particular body site of an animal host) that leads, or predisposes, the host to disease conditions [ ] . emerging infectious disease: : outbreaks of previously unknown diseases or known diseases that show an increase in incidence, expansion in geographical range, or spread to a new population. emerging infections can be caused by previously unknown or undetected infectious agents, newly evolved strains, environmental changes, and changes in human demography [ ] . a recent review found that over % of human emerging infectious diseases are zoonotic in origin [ ] . examples include influenza, hiv/aids, and severe acute respiratory syndrome (sars) coronavirus. intermediate host: : a host in or on which a pathogen spends a part of its life, usually a transition period, but does not reach sexual maturity. mutualism (mutualist): : a form of symbiosis between two organisms in which both benefit from the relationship. some gut parabasalids from termites are thought to represent mutualists [ ] . opportunistic: : a potential pathogen that typically does not cause disease in a healthy host, but can in particular situations cause disease, for example, owing to the compromised immune system (e.g., attributable to aids, chemotherapy, or malnutrition) of the host. lung trichomonads represent probable opportunistic infections -see main text. parasitic (parasite): : a non-mutual symbiosis between two species where one, the parasite, benefits at the expense of the other, the host. parasites typically do not kill their hosts but exploit them for resources necessary for their survival. obligate parasites cannot complete their life cycles and reproduce without a suitable host. pathogenic (pathogen): : a broad term that refers to the ability of an organism to cause disease. it is typically used to describe an infectious agent or microorganism, such as a bacterium, protist, virus, etc., that causes disease in its host. some pathogens, for example, protists acanthamoeba spp. and naegleria fowleri and fungi aspergillus spp. are free-living species thriving in the environment and occasionally infect humans, often in an opportunistic manner in compromised hosts [ ] . pathogenicity: : the ability of a pathogen to overcome host defenses and cause disease. re-emerging infectious disease: : the reappearance of a historically known infectious disease after a significant decline in incidence. acquired resistance of pathogens to antimicrobial medications is an important factor in the reemergence of many diseases. examples include west nile virus, cholera, mrsa (methicillin-resistant staphylococcus aureus). reservoir: : the habitat or host that harbors an infectious agent, where it can live, grow, and multiply. reservoirs can include humans, animals, and serve as a source of potential disease outbreaks. symbiosis: : a close and often long-term relationship between two or more different biological species. these relationships can be obligate or facultative, mutualistic, commensalistic, or parasitic. transmission: : the passing of an infectious agent from one host to another host. direct transmission routes include: physical contact, contact with a contaminated environment or surface, airborne transmission, and fecal-oral transmission. indirect transmission routes involve another organism such as an insect vector or intermediate host. vector: : an organism that carries and transmits a pathogen from an infected individual to another individual. virulence: : a property of a pathogen, such as specific structural elements or biochemical compounds commonly called virulence factors that cause a reduction in host fitness or damage to the host. it is now recognized that virulence is multifactorial and involves characteristics of both the pathogen and its host, which influence the outcome of their interaction and hence the observed virulence (e.g., an opportunistic pathogen in immunocompromised patients) [ ] . zoonosis: : an infectious organism, such as a bacterium, virus, parasite, or fungus, transmissible between wildlife or domesticated animals and humans. examples include: (i) the lyme disease bacterium borrelia transmitted to humans by ticks from a natural reservoir in rodents; (ii) the malaria parasite plasmodium knowlesi transmitted by anopheles vectors that causes malaria in monkeys and humans; and (iii) cryptosporidium parvum, a parasite found in cats, dogs, and farmed animals and transmitted as a cyst in contaminated water, food, or through the fecal-oral route. zoonoses are the leading cause of emerging infectious diseases worldwide, responsible for devastating disease outbreaks, mortality, and serious socioeconomic consequences [ , , ] . zoonotic potential: : the potential for infectious diseases of wildlife or domestic animals to be transmitted to humans. trends in parasitology july , vol. , no. strains of the same species [ , ] . in addition, several different genotypes of t. foetus have been identified as causing diarrhea in cats in countries [ , ] and have also been isolated from dogs with diarrhea [ ] . moreover, in several new clinical cases, t. foetus or t. foetus-like organisms have unexpectedly been identified in the lungs of human patients [ ] . such findings suggest that t. foetus is a zoonotic parasite capable of colonizing an extensive range of hosts and body sites. other examples of species of non-human trichomonads recently found to infect humans are members of the genus tetratrichomonas, currently the largest genus in phylum parabasalia. tetratrichomonas species are found in the small intestine of a wide spectrum of invertebrate and vertebrate hosts, such as leeches, birds, and rodents [ ] . indeed, some species of tetratrichomonad are known to infect a wide range of unrelated hosts, such as tetratrichomonas prowazeki, which has been found in species of amphibians and reptiles [ ] . another example, tetratrichomonas gallinarum, is primarily thought of as an avian parasite of the digestive tract in domestic and wild birds [ ] , although its pathogenicity is not well established [ ] . however, several recent studies have identified tetratrichomonas strains isolated from human lungs or the human the primary host may not represent the true natural history of the species, which may have a broader host range. c pulmonary infections include pcp, ards-associated infections, pneumonia, and can lead to empyema. trends in parasitology july , vol. , no. oral cavity as t. gallinarum or t. gallinarum-like organisms [ , , ] . studies have also shown that genus tetratrichomonas is much more diverse than previously thought and that t. gallinarum comprises at least three cryptic species with variable host specificity, some that represent human isolates [ , ] . notably, experiments failed to transmit two tetratrichomonas of human origin to birds, although the authors suggest this result could be explained by either adaptation of the t. gallinarum-like trichomonads to the human host or extensive in vitro culturing, so that infection of birds was no longer biologically achievable [ ] . molecular phylogenies reveal close relationships between human and avian trichomonads recent molecular phylogenetic analysis of trichomonads using rrna and protein coding genes (e.g., rpb ) has begun to answer important questions regarding trichomonad phylogeny. rpb is a ubiquitous eukaryotic gene coding for the largest subunit of rna polymerase ii and is present as a single copy in many eukaryotes. a recent analysis of rpb generated a fully resolved phylogeny of trichomonadea, tritrichomonadea, and hypotrichomonadea, and species and isolates within these groups (figure ) [ ] . interestingly, the phylogeny recovered some avian isolates of trichomonas spp. as sister taxa to t. vaginalis, and t. tenax as closely related to t. gallinae; these findings are consistent with previous phylogenies based upon rrna and other protein coding genes [ , [ ] [ ] [ ] . the common ancestor to this complex is also related to the avian t. gallinarum (e.g., [ ] ). these new phylogenies complicate the inferred relationship between t. vaginalis and t. tenax, which on the basis of host specificity might be expected to be sister taxa [ ] . in this scenario, an ancestor of both species infected humans and subsequently differentiated into two distinct species with different body site preferences. however, the lg+g+i model, bootstrap replicates, based on unambiguously aligned sites (the alignment is available upon request)], based on rpb illustrating the relationship between human-and animal-specific trichomonad species and one isolated from the environment (pseudotrichomonas keilini). note the high similarity of sequences derived from the human trichomonas vaginalis isolates and trichomonas sp. (hmo ) isolated from a dove. this sequence and other trichomonas gallinae sequences are clearly distinct from the recently defined trichomonas stableri (kf ) species isolated from band-tailed pigeons [ ] . taxa in red were isolated from humans and those in blue isolated from birds; accession numbers of each sequence are shown. adapted from [ ] . trends in parasitology july , vol. , no. phylogenetic data suggest the zoonotic transfer of trichomonad parasites from humans to birds, and/or vice versa, on at least two occasions, or a combination of these events. other studies investigating outbreaks of avian trichomonosis in wild birds, primarily attributed to t. gallinae infections, have also confirmed the close relationship between avian and human trichomonads [ ] . epidemic infections by t. gallinae of passerine species in europe have recently been described and associated with high mortality and population decline [ ] . this dramatic case of disease emergence demonstrates the potential for a trichomonad to jump host species (columbiform to passerine) and spread rapidly through populations. the disease is thought to have been initiated and spread through transfer of the parasite via contaminated water and bird feeders, as well as through direct contact between passerines [ ] . furthermore, the recent isolation and preliminary characterization of trichomonas stableri associated with epidemic mortality in california band-tail pigeons suggest that avian trichomonosis may be caused by pathogens other than t. gallinae and h. meleagridis [ , ] . genetic analysis at multiple loci indicated t. stableri to be more closely related to t. vaginalis than to the bird-associated t. gallinae [ ] (figure ), further supporting the hypothesis that trichomonads are crossing host boundaries. generating genetic markers and whole genome sequences of t. stableri and other t. vaginalis-like isolates derived from cases of epidemic avian trichomonosis will provide important insight into the evolution and origins of these pathogens. for example, does the trichomonas sp. isolate from a whitewinged pigeon extremely closely related to isolates of t. vaginalis represent a case of human to bird transfer (accession hm in figure )? the implications of zoonotic trichomonads for human health according to current disease dynamic models, the zoonotic emergence of parasitic diseases in humans is typically associated with several characteristics, including broad host range, genetic variability, presence of genotypes better suited to the parasitism of humans, and modified pathogenic potential [ ] . emergent zoonoses are thought to appear through a number of different stages, for example, some develop as animal 'parasitoses' that are newly transmissible to humans, although the source of the disease remains the animal reservoir [ , ] . in other cases, parasites are able to cross the species barrier, modify their specificity, and become sustainably transmissible from human to human [ , , ] . the evolution of these emergent parasitoses is not linear, and an explanation for such a complex process requires consideration of the multi-host ecology and complex dynamics of zoonotic infections [ ] . based on these models and the clinical and molecular evidence discussed previously, it may be that several trichomonads are at different stages of zoonotic emergence ( figure ). these observations raise important questions regarding the implications of the potential widening pathological spectrum of trichomonads in humans and suggest that owing to links with other diseases these parasites may be of greater medical importance than previously thought. historically, trichomonads have not been considered as emerging infections because of their site-and host-specific occurrence. nonetheless, the presence of trichomonads in a diverse array of clinical disorders suggests that they may exhibit a form of opportunism and multiply when local conditions are favorable. for example, the diseases in which trichomonads are found as co-infecting agents in respiratory infections are probably not limited to pcp and ards-associated infections, but may include other pulmonary diseases such as cystic fibrosis [ , ] . overall, the high prevalence of pulmonary diseases globally [ ] combined with the higher burden of both lung conditions and zoonotic diseases among people in resource-limited settings [ , ] suggest that only the 'tip of the iceberg' of pulmonary trichomoniasis may currently be known. digestive tract infections by trichomonads are also increasingly recognized as being common. although the exact clinical profile of d. fragilis is still poorly understood some consider this species to have pathogenic capabilities [ ] , and recent studies have associated the rise of ibs with a high prevalence ( %) of d. fragilis in europe [ ] . however, the pathogenicity of d. fragilis has been questioned owing to the asymptomatic nature of many infections, and it is considered by some as a commensal of the intestinal flora [ ] . indeed, treatment of d. fragilis-infected children with metronidazole was not associated with better clinical outcomes [ ] . because association is not evidence for causality, additional data are required to establish the pathogenicity of trichomonads in the digestive tract and 'aberrant' body sites such as the lungs. in this context, it is important to consider characteristics of both host and parasite with regard to the outcome of their interactions. for example, one extreme is represented by severely immune-compromised patients that are more susceptible to a wider range of microbial infections compared with immunocompetent hosts [ ] . when studying the outcome of human-microbe interactions, a complex interplay among viruses, bacteria and archaea, microbial eukaryotes, and animal parasites influence the health status of the human host, with mucosal microbiota playing a key role influencing health and disease status [ , ] . based on these considerations and examples, trichomonads may be more prevalent and have a wider pathological spectrum in humans than currently recognized, influencing human health through direct pathologies but also indirectly through dysbiosis of the mucosal microbiota and local inflammation, facilitating transmission of pathogens -a prime example being t. vaginalis infection and bacterial vaginosis contributing to hiv transmission [ , ] . the potential influence of gut trichomonads to human health will also have to consider their potential impact on the gut microbiota, which might explain observations of the association between d. fragilis and ibs through inducing gut dysbiosis [ ] . indeed, the ability of trichomonads to live on a variety of mucosal tissues may be the key to their wide host range and ability to develop infections at different body sites, as well as contribute directly or indirectly to pathologies. once the capacity to thrive on vertebrate mucosal surfaces has developed, there may be less of a barrier to cross both species and mucosal sites. for example, in the case of review trends in parasitology july , vol. , no. t. foetus, this sexually transmitted species may represent a recent transfer from the digestive to the urogenital tract, with a capacity of the parasite to thrive in the gut in different species (e.g., pigs, cats, and dogs). trichomonads provide a unique system for the study of the origins and pathobiology of zoonotic and emerging infectious diseases. in addition, they have attracted interest as model systems for evolutionary biology and comparative genomics [ , ] , and for biochemical, molecular, and cell biology investigations [ , , ] . the t. vaginalis genome sequence published in was the first species of trichomonad to be sequenced [ ] , and others are currently underway including isolates of t. foetus, p. hominis, t. gallinae, and t. tenax. these sequences will enable comparative analysis of common and unique parasitic modes of life cycle, and possible adaptive mechanisms. for example, t. vaginalis and t. foetus appear to have evolved independently to colonize the urogenital tracts of different mammalian hosts [ ] . in addition, t. foetus has been isolated from the digestive tract of cats and dogs, indicating that it is capable of colonizing a broad range of hosts and environments [ ] . these two species of sexually transmitted trichomonad probably represent cases of convergent evolution and provide an opportunity to compare the derived similarities and the origins of these traits that coincide with a shared niche. species of trichomonad exhibit a range of genome sizes, from mb for the p. hominis genome to mb for the t. foetus genome [ ] . the t. vaginalis genome sequence revealed the -mb genome to be a result of expanded transposable elements and protein coding gene families, . speculative models of zoonoses caused by trichomonads.trichomonads are listed on the right and colored according to primary hosts assigned historically in the literature. unbroken lines represent known infections or transmission routes, and broken lines represent speculative infections or transmission routes for which data are lacking. the relationships are represented as follows: (blue box) trichomonads identified in wild bird species (e.g., green finch [ ] and toucan [ ] ) in partially domesticated species (rock dove) and in fully domesticated species (chicken) circulate within these populations with variable host specificity [ ] (blue unbroken circle with arrow). two of the four avian trichomonads listed (tetratrichomonas sp. and tetratrichomonas gallinarum) have been identified in human lungs [ ] , and trichomonas gallinae and trichomonas stableri are also included owing to their close relationship to trichomonas tenax and trichomonas vaginalis [ , ] . (red box) t. vaginalis and t. tenax are the two species considered human-specific, with known human-to-human infections (unbroken red circle). the close genetic relationship of the human and avian trichomonads ( figure ) suggests either independent zoonotic acquisitions from avian sources (broken blue arrow) or transfer of the parasites from humans to birds through environmental contamination (broken red arrow). (green box) tritrichomonas foetus has been isolated from a variety of pets and farm animals, with the same strain known to infect cattle and pigs (unbroken green arrow) [ ] , but different genotypes infecting cattle and cats [ , ] ; the origins of dog infections remain unclear [ ] . thus, there are at least two t. foetus genotypes capable of colonizing an extensive range of hosts, including humans [ ] (broken green circle and arrow). the lack of precise epidemiological data is indicated by '?'. (purple box) pentatrichomonas hominis has been isolated from a variety of pets and farm animals [ ] , but little is known about its infection route and epidemiology; the same strain could be circulating between all identified hosts (broken purple circle). (orange box) dientamoeba fragilis has been isolated from farm animals (pigs) and non-human primates (gorillas), with the same strain known to infect pigs and humans [ ] (unbroken orange arrow). recent evidence suggests that household pets do not play a role in transmission [ ] ; however, the origins remain unclear and multiple strains could be circulating in animal hosts (broken orange circle and arrow). additionally, given recent prevalence and transmission data it seems unlikely that transmission from non-human hosts represents a significant proportion of infections. contaminated surfaces and water [ ] , uncooked meat, or direct contact with pets and farm animals could lead to animal-to-human transmissions of trichomonads. initial infections were presumably through the digestive tract (via oral ingestion) with further progression to the lungs for some (various) species or the urogenital tract (t. vaginalis). trends in parasitology july , vol. , no. including those responsible for interaction of the parasite with its immediate environment [ ] [ ] [ ] . it has been hypothesized that the large genome size may be a recent event that occurred when the most recent common ancestor of t. vaginalis underwent a population bottleneck during its transition from the digestive tract to the urogenital tract. because genome size is positively associated with cell volume, an increase in genome size and concomitant increase in cell size might have increased its phagocytic potential as well as surface area for the interaction of the parasite with host cell tissue [ , ] . in this context, it will be particularly interesting to compare the genomes of t. vaginalis with its closely related isolates and species derived from birds ( figure ) to gain detailed insight into correlations between genome evolution and how this might relate to parasite pathobiology in humans and birds. lateral gene transfers from bacterial donors sometime in the evolutionary past have also importantly influenced the evolution of t. vaginalis protein coding genes [ , ] . for example, the parasite has gained an almost complete pathway for the degradation of complex glycans present in host mucosal secretions, factors which may contribute to its adaptive potential and pathogenicity [ , ] . comparison of a broad range of trichomonad genomes will help test this hypothesis and may pinpoint gene families whose acquisition and/or expansions correlate with pathogenicity and facilitated or mediated transitions from: (i) an ancestral animal-only stage to human-inclusive infections or (ii) from the digestive to urogenital tracts. trichomonads also provide a unique system to study the features of a zoonotic lifestyle via comparative examination of molecular and cellular characteristics. for example, successful t. vaginalis infections are probably favored by virulence mechanisms such as cytoadherence and phagocytosis [ , ] . the predicted protein coding genes of t. vaginalis [ ] includes a plethora of candidate genes for surface molecules mediating interaction with host tissues and membrane trafficking and signaling, important processes involved in parasite pathobiology [ , ] . cysteine proteases in particular have been identified as virulence factors central to the host-pathogen interface in t. vaginalis [ , [ ] [ ] [ ] [ ] . transcriptomic studies have shown upregulation of some of these t. vaginalis virulence factors in response to contact with host cells in vitro [ ] and have also documented their expression under in vitro growth conditions in t. foetus [ ] . similar to t. vaginalis, recent studies have shown the presence of cysteine proteases in the cell-free filtrate of t. gallinae and demonstrated their involvement in its in vitro cytopathogenic effects [ ] . mining other trichomonad genome data to identify important virulence proteins will improve our understanding of the molecular and cellular basis of infections and can be used to test hypotheses, such as whether zoonotic organisms show greater diversity in key virulence proteins underlying their capacity to parasitize a variety of host species and mucosal sites [ ] . concluding remarks and future perspectives although we have discussed several recent studies that provide strong evidence for the zoonotic origin and potential of trichomonads, regular and sustained zoonotic transmission of these microbes has yet to be definitively established. to improve our knowledge of the zoonotic origins of trichomonads, detailed investigations including systematic surveys of trichomonads in humans and animals will be required. molecular methods have been instrumental in our understanding of the biology and complexity of trichomonads so far; however, more data and novel approaches are needed to resolve evolutionary relationships and to improve diagnostic tools. wide sampling and whole genome sequencing of trichomonads, with subsequent comparative genomic investigations, will facilitate identifying the closest relatives of human trichomonad pathogens, providing a solid evolutionary framework for how these diseases have emerged and forming a basis for epidemiological studies across both animal (wild and domestic) and human hosts. environmental studies such as the 'microbes, sewage, health and disease' metagenomics project in new york city (http://www.nyu.edu/ about/news-publications/nyu-stories/video-mapping-nycs-metagenome.html) will provide important information on potential transmission routes and the patterns, nature, and occurrence of trichomonad infections in humans, animals, and birds; and establish the importance of trichomonads zoonotic transmissions -as has been established for species of trypanosoma, cryptosporidium, and toxoplasma [ ] . furthermore, studies investigating the potential pathogenicity of these parasites in various mucosae (respiratory, digestive, and urogenital) are needed to determine the clinical significance and public health implications of trichomonads. 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early infection of vaginal epithelial cells and amoeboid transition cysteine peptidases, secreted by trichomonas gallinae, are involved in the cytopathogenic effects on a permanent chicken liver cell culture data-mining approaches reveal hidden families of proteases in the genome of malaria parasite oxford textbook of zoonoses symbioses of flagellates and prokaryotes in the gut of lower termites molecular confirmation of trichomonas gallinae and other parabasalids from brazil using the . s and its- rrna regions evaluation of the easyscreen enteric parasite detection kit for the detection of blastocystis spp., cryptosporidium spp., dientamoeba fragilis, entamoeba complex, and giardia intestinalis from clinical stool samples persistence of two trichomonas gallinae isolates in chlorinated and distilled water with or without organic material we thank four anonymous referees for their constructive comments that allowed us to improve the manuscript. j.m.m. is supported by the maccracken program in the graduate school of arts and science, and by a new york university grand challenge project. r.p.h. acknowledges past wellcome trust funding for his work on trichomonas vaginalis. key: cord- - fhg o authors: mull, nathaniel; jackson, reilly; sironen, tarja; forbes, kristian m. title: ecology of neglected rodent-borne american orthohantaviruses date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: fhg o the number of documented american orthohantaviruses has increased significantly over recent decades, but most fundamental research has remained focused on just two of them: andes virus (andv) and sin nombre virus (snv). the majority of american orthohantaviruses are known to cause disease in humans, and most of these pathogenic strains were not described prior to human cases, indicating the importance of understanding all members of the virus clade. in this review, we summarize information on the ecology of under-studied rodent-borne american orthohantaviruses to form general conclusions and highlight important gaps in knowledge. information regarding the presence and genetic diversity of many orthohantaviruses throughout the distributional range of their hosts is minimal and would significantly benefit from virus isolations to indicate a reservoir role. additionally, few studies have investigated the mechanisms underlying transmission routes and factors affecting the environmental persistence of orthohantaviruses, limiting our understanding of factors driving prevalence fluctuations. as landscapes continue to change, host ranges and human exposure to orthohantaviruses likely will as well. research on the ecology of neglected orthohantaviruses is necessary for understanding both current and future threats to human health. due to their direct noticeable impacts on humans, certain viruses tend to receive relatively large amounts of research attention. members of the coronaviridae (sars-cov, mers-cov, and now sars-cov- ), filoviridae (ebola and marburg virus), flaviviridae (west nile and zika virus), lyssaviridae (rabies), and paramyxoviridae (hendra and nipah virus) families contain several dangerous human pathogens that have emerged in recent decades and have resulted in extensive research attention. while studying such viruses is important, there are an untold number of other pathogens that persist among humans and wildlife that receive little to no attention [ ] . even in high-profile viral groups, a disproportionate amount of attention is given to the viruses that are known to cause disease in humans, highlighted by the current global response to sars-cov- . due to unforeseeable circumstances, such as host-switching events (e.g., influenza virus, human immunodeficiency virus [ ] ), exposure to new viruses via landscape encroachment (e.g., hendra virus [ ] , nipah virus [ ]), and changes in host or virus geographic range due to climate change, species introduction, or migration events (e.g., zika virus [ ] , west nile virus [ ] ), less-significant viruses can quickly become significant human health concerns. therefore, viruses that are disproportionately under-studied require research focus, and they may ultimately aid understanding of related viruses and increase awareness of current and future threats. a key example of research bias within a virus group is the hantavirus family (bunyavirales: hantaviridae). recent taxonomic restructuring of hantaviruses was necessitated by the discovery of non-rodent-and non-mammal-borne viruses [ , ] . however, mammals, particularly rodents, are still the most common natural hosts of hantaviruses, encompassing viruses in the largest subfamily (mammantavirinae) and genus (orthohantavirus) [ ] , and only rodent-borne orthohantaviruses have been linked to human disease [ ] . human infections caused by spillover of old world and new world orthohantaviruses can result in hemorrhagic fever with renal syndrome (hfrs) or hantavirus cardiopulmonary syndrome (hcps or hps), respectively [ ] . the international committee on taxonomy of viruses (ictv) lists unique orthohantaviruses distributed throughout the world, with distinct viruses within virus species endemic to north and south america [ ] [ ] [ ] . although the first known american orthohantavirus, prospect hill virus (phv), was described in [ ] , most viruses were found shortly after the outbreak of sin nombre virus (snv) [ ] in north america and the cases of andes virus (andv) [ ] in south america (table ) . new orthohantaviruses and genotypes continue to be identified via broad surveillance. some discovered genotypes are suggested to be distinct viruses, but a lack of sequence data and virus isolation prevents formal taxonomic placement. for example, phylogenetic analyses show up to distinct branches within the andes orthohantavirus clade [ , ] , but only four strains meet all ictv criteria as distinct viruses (tables and a ) [ ] [ ] [ ] . despite an increasing number of described hantaviruses, andv and snv are disproportionately studied when compared to other orthohantaviruses in the americas (table ) . such bias may be the reason for inadequate information to discriminate between potentially different viruses, and the lack of distinction may discourage the collection of additional data, creating a negative feedback loop. muleshoe virus (mulv), for instance, is a genotype of black creek canal virus (bccv), and evidence supports mulv being a separate virus based on genetic differences [ ] . however, the necessary ictv criterion of mulv isolation has not been accomplished, which keeps mulv from being distinguished as a distinct virus strain and may limit the amount of research conducted on this genotype. until more virus-specific information is known, we must infer characteristics of under-studied orthohantaviruses using other available information. in this review, we summarize current knowledge on neglected orthohantaviruses and highlight areas where future research is necessary. to determine the potential range of these viruses, we report evidence regarding the rodent hosts of each american orthohantavirus and the potential for various host-virus relationships and communities based on existing evidence. information regarding transmission for well-studied orthohantavirus systems is used to postulate the transmission characteristics of neglected american orthohantaviruses, including direct transmission routes, environmental persistence, and spillover risk to humans. as the number of described orthohantaviruses increases, so does the number of suggested reservoir hosts (table a ) . reservoir hosts typically have asymptomatic and persistent infections [ , ] , although there is evidence of negative effects associated with orthohantavirus infection on the survival of young animals [ ] and possibly decreased weight gain in newly-infected individuals [ ] . most studies that identify orthohantavirus infections in rodents have not evaluated the pathological or demographic consequences of infections. the ability of rodents to be infected with an orthohantavirus without noticeable effects does not alone implicate them as a reservoir. virus isolation is generally deemed the gold-standard evidence to support a reservoir role, followed by positive polymerase chain reaction (pcr) results. while orthohantavirus isolation from rodent hosts is rare-even for well-established virus-host relationships (e.g., snv and peromyscus maniculatus [ ] )-recent advances in establishing rodent cell cultures, such as those of the bccv host sigmodon hispidus, may aid future isolations [ ] . in contrast, positive rt-pcr results for a particular virus in multiple rodent species are common (table a ) . orthohantavirus infections are generally considered single-host-single-virus systems [ ] [ ] [ ] , and viruses tend to co-diverge with their hosts [ ] . the term "primary host" is sometimes used for the most common reservoir host [ , ] , but this wording retracts from the idea that orthohantaviruses could persist in multiple hosts with the same propensity. evidence increasingly suggests that some american orthohantaviruses do not follow the single-host-single-virus paradigm as strictly as their old world counterparts (table a ). for example, the reservoir for lechiguanas virus (lechv) is considered to be oligoryzomys flavescens, but results from a recent study found lechv-positive reverse transcriptase pcr (rt-pcr) samples from oligoryzomys nigripes in argentina, while all o. flavescens samples were seronegative [ ] . similarly, a study in texas found snv-positive rt-pcr samples from five seropositive peromyscus attwateri, four p. leucopus, one p. laceiarus, and one reithrodontomys fulvescens, but all of the sampled p. maniculatus, the reservoir of snv, were seronegative except a single rt-pcr negative individual [ ] . it is unknown whether such instances are caused by frequent spillover events or the persistence of the virus within or among multiple species. multiple-host systems are also more common than generally acknowledged when considering virus genotypes that are not classified as separate viruses by the ictv. in such cases, the reservoirs for a virus strain would be the combination of reservoirs for all genotypes. for example, limestone canyon virus (lscv) is a genotype of snv that is associated with peromyscus boylii and other peromyscus species [ , ] instead of p. maniculatus, the reservoir of snv; isla vista virus (islav) is a genotype of phv that is associated with microtus californicus [ ] instead of m. pennsylvanicus, the reservoir of phv; and rio mearim virus (rimev) and anajatuba virus (anajv) are genotypes of rio mamoré virus (riomv) that are associated with holochilus sciureus and oligoryzomys fornesi, respectively [ ] , instead of o. microtis, the reservoir of riomv (table a ). in some circumstances, distinct orthohantavirus genotypes are also host subspecies-dependent. for instance, oryzomys couesi is suggested to be the reservoir for catacamas virus (catv) and playa de oro virus (orov), a genotype associated with a clade composed of catv, bccv, and bayou virus (bayv), but orov and catv are associated with different subspecies of o. couesi [ , ] . choclo virus (chov) and maporal virus (mapv) are both associated with oligoryzomys fulvescens, although a distinction in the mitochondrial cytochrome-b gene suggests that these viruses are host subspecies-specific, infecting o. f. costaricensis and o. f. delicatus, respectively [ ] . another technical issue to consider is the concept of host-switching events among orthohantaviruses. evidence of historical host switching has resulted in hantavirus lineages among disparate mammal taxa [ ] [ ] [ ] . more recent host-switching is supported by several mismatches in the cophylogeny of orthohantaviruses and rodent hosts. for example, monongahela virus (mglv) is an orthohantavirus genotype primarily carried by p. maniculatus nubiterrae, a subspecies of the mouse associated with snv, despite mglv often showing a closer phylogenetic relationship to new york virus (nyv), which is associated with p. leucopus [ ] [ ] [ ] (although the true relationship is still pathogens , , of unknown (figure ) , and mglv has also been reported in p. leucopus [ ] ). additionally, orov and catv are often found in the same species, o. couesi, despite orov being more closely-related to bccv, which is associated with s. hispidus. minimal range overlap between o. couesi and s. hispidus and minimal genome sequencing prevent conclusion of a host-switch event among these viruses. additional host-switching events have been proposed as the reason for the multitude of reported hosts in american orthohantaviruses (table a ) , such as from oligoryzomys flavescens to o. nigripes for lechv [ ] . however, while reports of orthohantaviruses infecting multiple species supports multiple hosts for many orthohantaviruses and, therefore, a plethora of host switching events, there is a shortage of research examining the competence of many putative hosts and therefore the classification of true reservoirs. information regarding the relative transmissibility of virus from each host to humans and other wildlife is also lacking, with the exception of several case studies involving focused trapping around areas of assumed exposure (e.g., [ , ] ). additionally, no orthohantavirus has been isolated from more than one rodent species (table a ) , although few studies have reported such attempts. further research is, therefore, necessary to determine if frequent documentation of american orthohantaviruses in multiple species represents host switches or spillover. phylogenetic tree demonstrating relatedness among american rodent-borne orthohantaviruses. the evolutionary history was inferred using the maximum likelihood method implemented in mega . the percentage of trees in which the associated taxa clustered together is shown next to the branches; values over % are shown. the tree is drawn to scale, with branch lengths measured in the number of substitutions per site. triangular branches represent multiple closely-related sequences. the analysis involved orthohantavirus s segment nucleotide sequences retrieved from genbank. additional host-switching events have been proposed as the reason for the multitude of reported hosts in american orthohantaviruses (table a ) , such as from oligoryzomys flavescens to o. nigripes for lechv [ ] . however, while reports of orthohantaviruses infecting multiple species supports multiple hosts for many orthohantaviruses and, therefore, a plethora of host switching events, there is a shortage of research examining the competence of many putative hosts and therefore the classification of true reservoirs. information regarding the relative transmissibility of virus from each host to humans and other wildlife is also lacking, with the exception of several case studies involving focused trapping around areas of assumed exposure (e.g., [ , ] ). additionally, no orthohantavirus has been isolated from more than one rodent species (table a ) , although few studies have reported such attempts. further research is, therefore, necessary to determine if frequent documentation of american orthohantaviruses in multiple species represents host switches or spillover. in addition to the potential for multiple hosts, the number of sympatric viruses must also be considered. propensity for coexistence of different orthohantaviruses within a rodent community appears to vary spatially and temporally. in one texas study, viruses, and even virus genomes, appear to segregate at the county level [ ] . similar results were found in mexico, with most states containing only one orthohantavirus [ ] , although another study examining a smaller portion of the same mexican region found viruses to commonly coexist [ ] . sympatric riomv genotypes, anajv and rimev, were also found in the same area but in distinct host species [ ] . in california and nevada, elmcv, phv, and snv were also found in the same area, indicating that viruses hosted by diverse rodents can exist in sympatry [ ] . thus, multiple orthohantaviruses may exist together in rodent communities, but separation based on habitat type and species distributions likely play a role in structuring their presence. in the absence of data on orthohantavirus presence in a particular area, host distributions may be useful as proxies, as rodent ranges and habitat types are often well-documented [ , , ] . several orthohantaviruses have been found throughout large extents of their host range, including bccv [ , ] , bayv [ , ] , and others, indicating that orthohantaviruses have the potential to be present throughout the entire range of host species. however, the use of virus genotypes causes confusion when determining the range of orthohantaviruses. for example, bccv is used in florida, united states [ ] while mulv is used in texas [ ] . similarly, chov is used in panama [ ] while its genotype jabora virus (jabv) is used in brazil [ ] . until such genotypes are considered distinct viruses by taxonomists (i.e., ictv), acknowledgement of these relationships may be helpful in minimizing confusion and aiding understanding of orthohantavirus distributions. without analyzing positive samples throughout species ranges for new viruses, incorrect assumptions may also be made regarding orthohantavirus distributions. for example, riomv infects oligoryzomys microtis throughout most of its range in south america [ ] , so hcps cases in french guiana were thought to be riomv [ ] . however, virus sequencing from an hcps case in french guiana found that maripa virus (marv), a then-new virus closely-related to riomv found in o. fulvescens and zygodontomys brevicauda, was the responsible agent [ ] [ ] [ ] . difficulty in estimating virus range via host range also increases when one species can host several viruses. both necocli virus (necv) [ ] and marv [ ] have been found in z. brevicauda via positive rt-pcr, but the range of each particular virus is unknown. a similar situation was found for o. longicaudatus, the most common host of andv and also the host of oran virus (ornv), although the increased attention given to andv revealed which populations of o. longicaudatus host which virus [ ] . therefore, host distribution can be useful in estimating virus distribution, but caution should be applied. hantaviruses are likely to spread to new areas and vanish from existing areas due to changes in rodent host distribution and abundance. changes in grassland habitats caused by land-use changes and climate change [ ] [ ] [ ] have been strongly associated with rodent distributional changes. for example, range expansion of a north american grassland rodent species, baiomys taylori, was recently found in new mexico, united states, likely due to an increase in grassland areas, particularly along roadsides, due to climate change and habitat disturbance [ ] . thus, the grassland rodents that host orthohantaviruses may show similar patterns in the future. much of what we know about the transmission of american orthohantaviruses among conspecific rodent hosts is derived from studies of andv and snv [ ] . both viruses are primarily shed in saliva, occasionally in urine, and apparently not in feces, suggesting that behaviors such as grooming and biting are the primary routes of transmission [ , , ] . such transmission contrasts with old world orthohantaviruses such as puumala virus (puuv), which are commonly shed in feces as well [ , ] . older males are more commonly infected with orthohantaviruses than other much of what we know about the transmission of american orthohantaviruses among conspecific rodent hosts is derived from studies of andv and snv [ ] . both viruses are primarily shed in saliva, occasionally in urine, and apparently not in feces, suggesting that behaviors such as grooming and biting are the primary routes of transmission [ , , ] . such transmission contrasts with old world orthohantaviruses such as puumala virus (puuv), which are commonly shed in feces as well [ , ] . older males are more commonly infected with orthohantaviruses than other demographic groups [ ] , including snv [ , ] , laguna negra virus (lanv) [ ] , lechv [ ] , and bccv [ ] . compounding more exposure opportunities for older individuals, higher prevalence in older males is assumed to result from increased aggression and competition, primarily for access to mates [ , ] . associations of bayv-infected male o. palustris with receptive females and non-infected males with non-receptive females [ ] further supports the concept of reproductive behaviors as a primary driver of orthohantavirus transmission among wild rodents. thus, some females likely become infected via allogrooming during copulative behaviors common in rodents (e.g., [ , ] ). the occasional shedding of the virus in urine may be important for transmission among conspecifics, and perhaps heterospecifics as well. urine is used by rodents for various reproductive and territorial behaviors [ ] , creating ample opportunities for exposure of virus in aerosolized urine via oropharyngeal routes. however, information pertaining to virus persistence outside the host in american orthohantaviruses is limited to circumstantial evidence regarding spillover infections, and such transmission may be mitigated by uncommon virus shedding in urine. relatively frequent rodent spillover events (i.e., transmission from one species to another) [ ] suggests that other variables, including overlap in habitat use such as shared runways, burrows, and nests, is necessary for transmission among species. during the breeding season, many rodents compete for mates, food, space, and protection of offspring, so there is little overlap in space use by conspecifics, and often congeners [ , ] . however, in the non-breeding season, these territories break down and space overlap increases [ , ] . during this time, many rodents also share burrows within [ , ] and occasionally among [ ] species. during warmer months, some species may also use the burrows of other species who have since vacated [ , ] . burrow-sharing behavior in rodents has been associated with the spread of several other diseases, including plague (yersinia pestis) [ ] , tick-borne relapsing fever (borellia spp.) [ ] , and possibly valley fever (coccidioides spp.) [ ] , and the stable cool, humid microclimates of burrows [ , ] may allow orthohantaviruses to persist in the environment. this phenomenon would also help explain why multiple species can be infected by the same orthohantavirus, potential opportunities of spillover to non-muroid rodents [ ] , and original host-switching events to other rodents, shrews, and moles ( figure ). further research is necessary to determine the role of habitat overlap on conspecific orthohantavirus infection via competition, excrement exposure, and other potential sources of virus shedding and routes of transmission. regardless of the routes of transmission, population density appears to play a role in orthohantavirus maintenance. experimental modeling of snv prevalence in peromyscus maniculatus populations and hcps cases indicates that climate-mediated fluctuations in host abundance are linked to orthohantavirus outbreaks [ , ] . high seroprevalence in p. maniculatus is found after a time lag following high rainfall events, particularly those associated with the el niño-southern oscillation (enso) [ , ] . although this phenomenon has been relatively well-studied in snv, data demonstrating similar patterns among other american orthohantaviruses is lacking. however, such lag times in other systems may explain why less-abundant species in a rodent community may occasionally be the primary carriers of orthohantavirus [ , ] , as population sizes could have been larger in a recent season. theoretical models indicate that orthohantavirus transmission among rodents also has aspects of frequency-dependent transmission. infection prevalence is greatly influenced by contact rates [ ] , which increase as population density increases. however, increases in prevalence are greater in males than in females [ ] , likely due to increased competitive encounters among males, but not females, at higher densities. high seroprevalence among overwintering animals [ , ] are assumed to be caused by persistently infected animals infecting susceptible individuals. population sizes generally crash during this time period [ ] (although p. maniculatus populations remained stable prior to the hcps outbreak of - [ ] , likely due to a strong enso event), suggesting that winter infections may be caused by frequent interactions despite low host density. it is unclear how the stable winters of tropical regions impact orthohantavirus transmission systems in northern south america and central america. further attempts to imitate such systems in a controlled environment are necessary to better understand how orthohantaviruses persist and proliferate through rodent populations. most american orthohantaviruses have been associated with at least one human case of hcps ( / ) , and approximately half ( / ) were discovered following an hcps case (table ) . practically all hcps cases are thought to be caused by spillover events from rodents to humans [ , ] . the exception comes from andv in argentina and chile where some evidence supports transmission from infected patients to family members and medical workers [ ] [ ] [ ] . however, these instances are limited to outbreaks in small, rural communities, and regional medical staff that cared for hcps patients had similar seroprevalence to the general population [ , ] . several orthohantaviruses were originally discovered through broad surveillance of rodent tissues but were later implicated with human disease. for example, riomv was originally discovered while studying the host associations of andes orthohantavirus strains in [ ] , and was connected to hcps eight years later [ ] . other hcps cases were attributed to the incorrect orthohantavirus until the actual virus was described, such as marv cases originally diagnosed as riomv, as mentioned previously [ ] . similarly, due to regional variation in virus prevalence, elmcv was suggested to be the etiological agent of several hcps cases ascribed to snv, but the virus in these cases was never tested [ ] . without verification via sequencing of hcps cases, elmcv is considered to not cause disease in humans. thus, certain orthohantaviruses may be infectious to humans but incorrectly dismissed due to a lack of sequencing. conversely, additional orthohantaviruses or viral genotypes that are pathogenic to humans may exist that have not yet been linked to any hosts, such as tunari virus (tunv), which was discovered following an hcps case but the reservoir is still unknown [ ] . while understanding host ecology may help explain the maintenance of orthohantaviruses in wild rodent populations, it can also inform spillover threats to humans. snv and andv are both found most commonly in generalist rodent species that can be locally abundant. these host characteristics allow viruses to be present in most habitats throughout a large geographical range, increasing the likelihood of encounters between infected rodents and susceptible humans. however, due to the large number of described orthohantaviruses and their hosts, most regions and habitats have the capacity to contain multiple viruses of human health concern. on the other hand, some species and their viruses are common in a variety of habitat types. for example, in west virginia, united states, where peromyscus are the dominant muroid rodents, hcps cases were attributed to exposure of airborne particulates of p. maniculatus secretions within cabins [ , ] . such cases indicate an infection risk in seasonally-used buildings in rural areas in the northeast, similar to initial assessments in the southwestern united states [ ] . therefore, these generalist species appear to be capable of transmitting virus to humans regardless of habitat. urban areas may pose a risk for human exposure to orthohantaviruses and their hosts as well. for example, in addition to their abundance in forested habitats, peromyscus mice are common in green urban spaces, such as the park system in new york city [ , ] , and nyv was discovered on shelter island near new york city [ ] . notably, homeless residents may be at increased risk, as sleeping near rodent activity was associated with european orthohantavirus infections [ ] , although empirical evidence is lacking for american viruses. due to limited migration of wild rodents throughout urban areas [ , , ] , green spaces may also be protected from orthohantavirus invasion. orthohantaviruses carried by invasive rodents, such as seoul virus (seov) in rattus norvegicus, may pose a risk as well. seov has been documented in the united states and canada due to the pet trade [ ] , while wild rats can also carry this virus. one study found a seroprevalence for orthohantavirus in r. norvegicus of . % overall and - . % in green spaces in baltimore, maryland [ ] . although broader documentation of orthohantaviruses in urban areas is lacking, these findings as well as observations of a range of other disease-causing pathogens in urban rodents (e.g., [ , ] ) suggest that this may be an area of major human health concern. while current threats would likely be documented already, misdiagnoses, failure to seek medical attention, and the potential for future outbreaks warrant attention. it appears that hcps risk is greatest in areas where humans infiltrate rodent habitats, rather than vice versa, such as areas of landscape fragmentation and encroachment caused by urbanization and development. in uruguay, oligoryzomys flavescens infected with lechv were more common in disturbed habitats than in undisturbed habitats [ ] . relationships between habitat encroachment and infection risk occur for other zoonotic diseases, such as nipah virus [ ] and ebola virus [ , ] , suggesting a possible pattern in orthohantaviruses other than lechv as well. many forms of habitat encroachment can increase risk of exposure to orthohantaviruses. ranching and farming activity in the midwest united states prairies, such as construction of new barns and field plowing, could expose individuals to sigmodon hispidus (bccv); construction of rice fields and other encroachments into marsh habitat in the southern united states could expose individuals to oryzomyz palustris (bayv); the creation of edge habitat via development in the amazon basin provides additional habitat for oligoryzomys microtis (riomv) and increases contact with humans. all of these rodent species carry orthohantaviruses that cause disease in humans (table ) [ , , ] . estimating the risk of exposure to most orthohantaviruses with various human activities in south america is more difficult due to minimal information about the ecology of the rodent hosts; although some evidence indicates that habitat disturbance, particularly construction of domiciles in rural areas, appears to increase the risk of human exposure to hantaviruses there as well [ , ] . interestingly, ornv-positive oligoryzomys longicaudatus were found in orán, argentina, outside of the reported distribution range of o. longicaudatus (figure ) [ ] , showing the ability for agricultural development to expand orthohantavirus presence. despite the discovery of at least different new world orthohantaviruses carried by rodents, most orthohantavirus studies in the americas focus on andv and snv. while the majority of hcps cases are attributed to these viruses [ , ] , recent evidence suggests that such statistics may be skewed due to misdiagnosis of either the causative orthohantavirus or of the disease itself [ , ] . we show that despite having many similar characteristics, american orthohantaviruses differ from their old world counterparts and from each other in several ways. in the absence of empirical data, we shed light on the diversity, transmission, and risk of spillover for neglected american orthohantaviruses and viral genotypes using the ecology of their hosts and information on andv and snv. additionally, comparisons were occasionally made to old world orthohantaviruses. the ecological approach from this review may also be useful in implicating transmission and spillover risk of old world orthohantaviruses not yet examined. a key constraint to inferring information about each orthohantavirus system is the complexity between the taxonomy of orthohantaviruses and their hosts. related viruses appear to interact with hosts similarly, as shown by the comparable phylogenies of orthohantaviruses and their natural rodent hosts [ ] , their affinity to cause disease in humans (table ) , and frequent spillover or multiple related hosts (table a ) . however, confusion in orthohantavirus taxonomy and the number of distinct virus strains limits further conclusions. in particular, surveillance of related rodent species may produce additional genetic samples that allow clearer orthohantavirus phylogenies to be constructed. additional information regarding mole-borne orthohantaviruses, such as oxbow virus (oxbv) and rockport virus (rkpv) [ , ] , and shrew-borne genotypes, such as ash river virus (arrv), camp ripley virus (rplv), and jemez springs virus (jmsv) [ , ] , which have similar taxonomical issues due to minimal research and have some overlap in rodent phylogeny (figure ), may aid in understanding rodent-borne orthohantaviruses. ultimately, broader surveillance will aid in understanding which genotypes constitute distinct viruses and which represent genetic diversity of single orthohantaviruses. in addition to the controversy over viral taxonomy, the ability for multiple orthohantaviruses and their hosts to persist in the same environment and region [ , ] (figure ) further limits conclusions on orthohantavirus samples that are not sequenced, whether rodent or human. since multiple rodent species are commonly found rt-pcr positive for particular american orthohantavirus strains (table a ) , virus-host relationships are unclear. although orthohantaviruses are difficult to isolate, attempts to isolate these viruses from rodent samples is necessary to determine which rodents are reservoirs and which species experience frequent spillover events. these results will aid in determining whether american orthohantaviruses follow a single-host system like their old world counterparts. empirical data on the ecology of neglected american orthohantaviruses are crucial to understanding transmission and persistence of such viruses and threats to human health. few studies have examined the impacts of new world orthohantaviruses on rodent populations, with the exceptions of variation in prevalence between sexes and age classes [ ] [ ] [ ] , survivorship of age classes [ ] , and reproduction-dependent spatial variation [ ] . additional information regarding transmission routes and environmental persistence is also necessary, as the minimal data currently available using snv and andv show mixed results [ , , ] . although hcps cases are often associated with snv and andv, changes in the landscape, climate, and host switching may cause particular orthohantaviruses to increase in severity. each orthohantavirus may have the capability to become more significant to human health in the future, and insight into each virus is necessary for adequate preparation. various viral families have existed amongst humans with little to no impact until recent decades. therefore, research regarding neglected american orthohantaviruses is crucial for a holistic understanding of orthohantavirus epidemiology and to enable preparation for future risks. the authors declare no conflict of interest. table a . evidence supporting natural infections of orthohantaviruses in american rodents. bolded viruses represent strains accepted as distinct by the international committee for taxonomy of viruses (ictv), and non-bolded viruses indicate genotypes not accepted as distinct viruses by ictv. genotype placements are based on published phylogenetic analyses. all studies first found rodents to be seropositive for orthohantavirus antibodies and then performed reverse transcriptase polymerase chain reaction (rt-pcr) prior to sequencing or virus isolation (except for phv, where isolation was attempted without rt-pcr). orthohantaviruses have tri-segmented genomes-s, m, and l segments. studies including only seropositive rodents without additional diagnostic evidence of infection were not included in this a strategy to estimate unknown viral diversity in mammals cross-species virus transmission and the emergence of new epidemic diseases. microbiol the impact of 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panama genetic diversity and epidemiology of hantaviruses in argentina a global perspective on hantavirus ecology, epidemiology, and disease acute sin nombre hantavirus infection without pulmonary syndrome, united states host switch during evolution of a genetically distinct hantavirus in the american shrew mole (neurotrichus gibbsii) shared ancestry between a newfound mole-borne hantavirus and hantaviruses harbored by cricetid rodents hantavirus in northern short-tailed shrew, united states this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -hxxizipk authors: roberts, katherine e.; hadfield, jarrod d.; sharma, manmohan d.; longdon, ben title: changes in temperature alter the potential outcomes of virus host shifts date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: hxxizipk host shifts–where a pathogen jumps between different host species–are an important source of emerging infectious disease. with on-going climate change there is an increasing need to understand the effect changes in temperature may have on emerging infectious disease. we investigated whether species’ susceptibilities change with temperature and ask if susceptibility is greatest at different temperatures in different species. we infected species of drosophilidae with an rna virus and measured how viral load changes with temperature. we found the host phylogeny explained a large proportion of the variation in viral load at each temperature, with strong phylogenetic correlations between viral loads across temperature. the variance in viral load increased with temperature, while the mean viral load did not. this suggests that as temperature increases the most susceptible species become more susceptible, and the least susceptible less so. we found no significant relationship between a species’ susceptibility across temperatures, and proxies for thermal optima (critical thermal maximum and minimum or basal metabolic rate). these results suggest that whilst the rank order of species susceptibilities may remain the same with changes in temperature, some species may become more susceptible to a novel pathogen, and others less so. emerging infectious diseases are often the result of a host shift, where a pathogen jumps from one host species into another. understanding the factors underlying host shifts is a major goal for infectious disease research. this effort has been further complicated by the fact that host-parasite interactions are now taking place in a period of unprecedented global climatic warming. here, we ask how host shifts are affected by temperature by carrying out experimental infections using an rna virus across a wide range of related species, at three different temperatures. we find that as temperature increases the most susceptible species become more susceptible, and the least susceptible less so. this has important consequences for our understanding of host shift events in a changing climate as it suggests that temperature changes may affect the likelihood of a host shift into certain species. temperature is arguably the most important abiotic factor that affects all organisms, having both indirect and direct effects on physiology and life history traits [ ] [ ] [ ] . there is much to be learned about the impact of climate change on infectious diseases [ , , ] . changes in temperature can impact both host and parasite biology, leading to complex and difficult to predict outcomes [ , ] . host shifts, where a parasite from one host species invades and establishes in a novel host species, are an important source of emerging infectious disease [ ] . a successful host shift relies on a number of stages occurring [ ] . firstly, exposure of the host to the new pathogen species must occur in such a way that transmission is successful. secondly, the pathogen must be able to replicate sufficiently to infect the novel host. finally, there must be sufficient onwards transmission for the pathogen to become established in the new host species [ , , ] . some of the most deadly outbreaks of infectious diseases in humans including ebola virus, hiv and sars coronavirus have been linked to a host switch event [ ] [ ] [ ] [ ] and many others have direct animal vectors or reservoirs (e.g. dengue and chikungunya viruses) [ , ] . the potential for novel host shifts may increase with changing temperatures due to, fluctuations in host and/or parasite fitness, or changes in species distributions and abundances [ , ] . distribution changes may lead to new species assemblages, causing novel contacts between parasites and potential hosts [ ] [ ] [ ] . susceptibility to infection is known to vary with temperature, due to within individual physiological changes in factors such as the host immune response, metabolic rate or behavioural adaptations [ ] [ ] [ ] [ ] . thermally stressed hosts may face a trade-off between the resource investment needed to launch an immune response versus that needed for thermoregulation, or behavioural adaptations to withstand sub-optimal temperatures [ ] [ ] [ ] [ ] . temperature shifts could also cause asymmetrical or divergent effects on host and parasite traits [ ] . for example, changes in temperature may allow differential production and survival of parasite transmission stages, and changes in replication rates, generation times, infectivity and virulence [ ] [ ] [ ] . temperature is also known to impact vector-borne disease transmission through multiple effects on both vector life cycles and transmission behaviours [ , [ ] [ ] [ ] [ ] . host shifts have been shown to be more likely to occur between closely related species [ ] [ ] [ ] , but independently of this distance effect, clades of closely related hosts show similar levels of susceptibility [ , ] . thermal tolerances − like virus susceptibility − are known to vary across species, with groups of closely related species having similar thermal limits, with a large proportion of the variation in these traits being explained by the phylogeny [ ] [ ] [ ] [ ] . previous studies on host shifts have assayed the susceptibility of species at a single temperature [ , , , ] . however, if the host phylogeny also explains much of the variation in thermal tolerance, then phylogenetic patterns in virus susceptibility could be due to differences between species' natural thermal optima and the chosen assay temperatures. therefore, for experiments carried out at a single temperature, phylogenetic signal in thermal tolerance may translate into phylogenetic signal in thermal stress. any apparent phylogenetic signal in susceptibility could potentially be due to the effects of thermal stress, and may not hold true if each species was to be assayed at its optimal temperature. if this was indeed the case this would have implications for species distribution models that aim to use estimates of environmental conditions to predict host and pathogen ranges [ , , ] . here, we have asked how species' susceptibilities change at different temperatures and whether susceptibility is greatest at different temperatures in different species. we infected species of drosophilidae with drosophila c virus (dcv; dicistroviridae) at three different temperatures and measured how viral load changes with temperature. viral load is used here as a measure of dcv's ability to persist and replicate in a host, which has previously been shown to be tightly correlated to host mortality [ ] . we are therefore examining one of the steps ("ability to infect a novel host") needed for a host shift to successfully occur [ , , ] . we also examine how proxies for thermal optima and cellular function (thermal tolerances and basal metabolic rate) relate to virus susceptibility across temperatures, as increasing temperatures may have broad effects on both host and parasite [ ] [ ] [ ] . dcv is a positive sense rna virus in the family discistroviridae that was originally isolated from drosophila melanogaster and in the wild has been found in d. melanogaster and d. simulans [ ] [ ] [ ] . dcv infected flies show reduced metabolic rate and activity levels, develop an intestinal obstruction, reduced hemolymph ph and decreased survival [ ] [ ] [ ] [ ] . this work examines how temperature can influence the probability of host shifts, and looks at some of the potential underlying causes. we used drosophila c virus (dcv) clone b a, which is derived from an isolate collected from d. melanogaster in charolles, france [ ] . the virus was prepared as described previously [ ] ; briefly dcv was grown in schneider's drosophila line cells and the tissue culture infective dose (tcid ) per ml was calculated using the reed-muench end-point method [ ] . flies were obtained from laboratory stocks of different species. all stocks were maintained in multi generation populations, in drosophila stock bottles (dutscher scientific) on ml of their respective food medium at ˚c and % relative humidity with a hour lightdark cycle (table a in s text). each day, two vials of - day old male flies were randomly assigned to one of three potential temperature regimes; low, medium or high ( ˚c, ˚c and ˚c respectively) at % relative humidity. flies were tipped onto fresh vials of food after days, and after days of acclimatisation at the experimental temperature were infected with dcv. flies were anesthetized on co and inoculated using a . mm diameter stainless steel needle that was bent to a right angle~ . mm from the end (fine science tools, ca, usa) [ , , ] . the bent tip of the needle was dipped into the dcv solution (tcid = . × ) and pricked into the pleural suture on the thorax of the flies. we selected this route of infection as oral inoculation has been shown to lead to stochastic infection outcomes in d. melanogaster [ ] . however, once the virus passes through the gut barrier, both oral and pinpricked infections follow a similar course, with both resulting in the same tissues becoming infected with dcv [ ] . one vial of inoculated flies was immediately snap frozen in liquid nitrogen to provide a time point zero sample as a reference to control for relative viral dose. the second vial of flies were placed onto a new vial of fresh cornmeal food and returned to their experimental temperature. after days (+/- hour) flies were snap frozen in liquid nitrogen. this time point was chosen based on pilot data as infected flies showed little mortality at days post infection, and viral load plateaus from day at ˚c. temperatures were rotated across incubators in each block to control for incubator effects. all frozen flies were homogenised in a bead homogeniser for seconds (bead ruptor ; omni international, georgia, usa) in trizol reagent (invitrogen) and stored at - ˚c for later rna extractions. these collections and inoculations were carried out over three replicate blocks, with each block being completed over consecutive days. the order that the fly species were infected was randomized each day. we aimed for each block to contain a day and day replicate for each species, at each temperature treatment ( species × temperatures × experimental blocks). in total we quantified viral load in , flies over biological replicates (a biological replicate = change in viral load from day to day post-infection), with a mean of . flies per replicate (range across species = - ) . of the species, had biological replicates and three species had biological replicates. the change in rna viral load was measured using quantitative reverse transcription pcr (qrt-pcr). total rna was extracted from the trizol homogenised flies, reverse-transcribed with promega goscript reverse transcriptase (promega) and random hexamer primers. viral rna load was expressed relative to the endogenous control housekeeping gene rpl (rp ). rpl primers were designed to match the homologous sequence in each species and crossed an intron-exon boundary so will only amplify mrna [ ] . the primers in d. melanogaster were rpl qrt-pcr f ( '-tgctaagctgtcgcacaaatgg - ') and rpl qrt-pcr r ( '-tgcgcttgttcgatccgtaac - '). dcv primers were f ( '-gacactgccttt gattag- ') and r ( 'ccctctgggaactaaatg- ') as previously described [ ] . two qrt-pcr reactions (technical replicates) were carried out per sample with both the viral and endogenous control primers, with replicates distributed across plates in a randomised block design. qrt-pcr was performed on an applied biosystems steponeplus system using sensifast hi-rox sybr kit (bioline) with the following pcr cycle: ˚c for min followed by cycles of: ˚c for sec followed by ˚c for sec. each qrt-pcr plate contained four standard samples. a linear model was used to correct the cycle threshold (ct) values for differences between qrt-pcr plates. any samples where the two technical replicates had cycle threshold (ct) values more than cycles apart after the plate correction were repeated. to estimate the change in viral load, we first calculated Δct as the difference between the cycle thresholds of the dcv qrt-pcr and the rpl endogenous control. for each species the viral load of day flies relative to day flies was calculated as -ΔΔct ; where ΔΔct = Δct day -Δct day . the Δct day and Δct day are a pair of Δct values from a day biological replicate and a day biological replicate. calculating the change in viral load without the use of the endogenous control gene (rpl ) gave equivalent results (spearman's correlation between viral load calculated with and without endogenous control: ρ = . , p< . ) we carried out two assays to measure the thermal tolerances of species; a cold resistance measure to determine critical thermal minimum (ct min ) under gradual cooling, and a heat resistance measure through gradual heating to determine critical thermal maximum (ct max ). - day old males were collected and placed onto fresh un-yeasted cornmeal food vials. flies were kept for days at ˚c and % relative humidity and tipped onto fresh food every days. in both assays individual flies were placed in ml glass vials (st , ampulla, uk) and exposed to temperature change through submersion in a liquid filled glass tank (see fig a in s text). for ct max the tank was filled with water and for ct min a mixture of water and ethylene glycol ( : by volume) was used to prevent freezing and maintain a constant cooling gradient. five biological replicates were carried out for each species for both ct max and ct min . temperature was controlled using a heated/cooled circulator (txf , grant instruments, cambridgeshire, uk) submerged in the tank and set to change temperatures at a rate of . ˚c/min, always starting from ˚c (the rearing temperature for stock populations). flies were monitored continually throughout the assay and the temperature of knock down was ascertained by a disturbance method, whereby a fly was scored as completely paralysed if on gentle tapping of the vial wall the fly did not move any of its body parts. to examine how cellular function changes with temperature, we estimated the resting metabolic rate of each species at ˚c, ˚c and ˚c to examine if changes in general cellular processes were related to changes in viral load. following the same methods as the viral inoculation assay, groups of , - day old male flies from species were acclimatised at the three experimental temperatures for days (d. pseudoobscura was excluded as not enough individuals could be obtained from stocks for sufficient replication). every days flies were tipped onto fresh vials of cornmeal food. this was repeated in three blocks in order to get three repeat measures of metabolic rate for each of the species, at each of the three experimental temperatures. flies were collected in a randomly assigned order across the three blocks. closed system respirometry was used to measure the rate of co production (vco ) as a proxy for metabolic rate [ ] . flies were held in ml - airtight plastic chambers constructed from bev-a-line v tubing (cole-parmer instrument company, uk). all measures were carried out during the day inside a temperature controlled incubator, with constant light, that was set to each of the experimental temperatures that the flies had been acclimatised to. the set up followed that of okada et al. ( ) [ ] . compressed air of a known concentration of oxygen and nitrogen ( % o : % n ) was scrubbed of any co and water (with ascarite ii & magnesium perchlorate respectively) and pumped through a sable systems rm eight-channel multiplexer (las vegas, nv, usa) at ml/min - (± %) into the metabolic chambers housing the groups of flies. the first chamber was left empty as a reference cell, to acquire a baseline reading for all subsequent chambers at the start and end of each set of runs, therefore seven groups of flies were assayed in each run. air was flushed into each chamber for minutes, before reading the previous chamber. readings were taken every second for minutes by feeding the exiting air through a licor li- infrared gas analyser (lincoln, ne, usa). carbon dioxide production was measured using a sable systems ui analog-digital interface for acquisition, connected to a computer running sable systems expedata software (v . . ) [ ] . the metabolic rate was calculated from the entire -minute recording period by taking the co reading of the ex-current gas from the chamber containing the flies and subtracting the co measure of the incurrent gas entering the chamber. these values were also corrected for drift away from the baseline reading of the empty chamber. volume of co was calculated as vco = fr (fe co -fi co ) / ( -fi co ). where fr is the flow rate into the system ( ml/ min - ), fe co is the concentration of co exiting and fi co is the concentration co entering the respirometer. species were randomly assigned across the respiration chambers and the order in which flies were assayed (chamber order) was corrected for statistically (see below). to check for any potential effect of body size differences between species on viral load, wing length was measured as a proxy for body size [ ] . a mean of (range - ) males of each species were collected and immediately stored in ethanol during the collections for the viral load assay. subsequently, wings were removed and photographed under a dissecting microscope. using imagej software (version . ) the length of the iv longitudinal vein from the tip of the proximal segment to where the distal segment joins vein v was recorded, and the mean taken for each species. the host phylogeny was inferred as described in longdon et al ( ) [ ] , using the s, adh, amyrel, coi, coii, rpl and sod genes. briefly, any publicly available sequences were downloaded from genbank, and any not available we attempted to sanger sequence [ ] . in total we had rpl sequences for all species, s from species, adh from species, amyrel from species, coi from species, coii from species and sod from species (see www.doi.org/ . /m .figshare. full details). the sequences of each gene were aligned in geneious (version . . , [ ] ) using the global alignment setting, with free end gaps and a cost matrix of % similarity. the phylogeny was constructed using the beast program (version . . , [ ] ). genes were partitioned into three groups each with their own molecular clock models. the three partitions were: mitochondrial (coi, coii); ribosomal ( s); and nuclear (adh, sod, amyrel, rpl ). a random starting tree was used, with a relaxed uncorrelated lognormal molecular clock. each of the partitions used a hky substitution model with a gamma distribution of rate variation with categories and estimated base frequencies. additionally, the mitochondrial and nuclear data sets were partitioned into codon positions + and , with unlinked substitution rates and base frequencies across codon positions. the treeshape prior was set to a birth-death process. the beast analysis was run twice to ensure convergence for million mcmc generations sampled every steps. the mcmc process was examined using the program tracer (version . , [ ] ) to ensure convergence and adequate sampling, and the constructed tree was then visualised using figtree (version . . , [ ] ). all data were analysed using phylogenetic mixed models to look at the effects of host relatedness on viral load across temperature. we fitted all models using a bayesian approach in the r package mcmcglmm [ , ] . we ran trivariate models with viral load at each of the three temperatures as the response variable similar to that outlined in longdon et al. ( ) [ ] . the models took the form: where y is the change in viral load of the i th biological replicate of host species h, for temperature t (high, medium or low). β are the fixed effects, with β being the intercepts for each temperature, β being the effect of basal metabolic rate, β the effect of wing size, and β and β the effects of the critical thermal maximum (ct max ) and minimum (ct min ) respectively. u p are the random phylogenetic species effects and e the model residuals. we also ran models that included a non-phylogenetic random species effect (u np:ht ) to allow us to estimate the proportion of variation explained by the host phylogeny [ , , ] . we do not use this term in the main model as we struggled to separate the phylogenetic and non-phylogenetic terms. our main model therefore assumes a brownian motion model of evolution [ ] . the random effects and the residuals are assumed to be multivariate normal with a zero mean and a covariance structure v p � a for the phylogenetic affects and v e � i for the residuals (� here is the kronecker product). a is the phylogenetic relatedness matrix, i is an identity matrix and the v are × (co)variance matrices describing the (co)variances between viral titre at different temperatures. the phylogenetic covariance matrix, v p, describes the inter-specific variances in each trait and the inter-specific covariances between them. the residual covariance matrix, v e, describes the within-species variance that can be both due to real within-species effects and measurement or experimental errors. the off-diagonal elements of v e (the covariances) can not be estimated because no vial has been subject to multiple temperatures and so were set to zero. we excluded d. pseudoobscura from the full model as data for bmr was not collected, but included it in models that did not include any fixed effects, which gave equivalent results. diffuse independent normal priors were placed on the fixed effects (means of zero and variances of ). parameter expanded priors were placed on the covariance matrices resulting in scaled multivariate f distributions, which have the property that the marginal distributions for the variances are scaled (by ) f , . the exceptions were the residual variances for which an inverse-gamma prior was used with shape and scale equal to . . the mcmc chain was run for million iterations with a burn-in of million iterations and a thinning interval of , . we confirmed the results were not sensitive to the choice of prior by also fitting models with inverse-wishart and flat priors for the variance covariance matrices (described in [ ] ), which gave qualitatively similar results ( . /m .figshare. ). all confidence intervals (ci's) reported are % highest posterior density intervals. using similar model structures we also ran a univariate model with bmr and a bivariate model with ct min and ct max as the response variables to calculate how much of the variation in these traits was explained by the host phylogeny. both of these models were also run with wing length as a proxy for body size as this is known to influence thermal measures [ ] . we observed significant levels of measurement error in the metabolic rate data; this was partially caused by respiratory chamber order during the assay. we corrected for this in two different ways. first, we fitted a linear model to the data to control for the effect of respiratory chamber number and then used this corrected data in all further models. we also used a measurement error model that controls for both respiratory chamber number effects and random error. both of these models gave similar results although the measurement error model showed broad cis suggesting the bmr data should be interpreted with caution. all datasets and r scripts with the model parameterisation are provided as supporting information (s text). to investigate the effect of temperature on virus host shifts we quantified viral load in , flies over biological replicates, from species of drosophilidae at three temperatures ( fig ) . dcv replicated in all host species, but viral load differed between species and temperatures (fig ) . species with similar viral loads cluster together on the phylogeny (fig ) . measurements were highly repeatable (table ) , with a large proportion of the variance being explained by the inter-specific phylogenetic component (v p ), with little within species or measurement . we also calculated the proportion of between species variance that can be explained by the phylogeny as v p /(v p + v s ) [ ] , which is equivalent to pagel's lambda or phylogenetic heritability [ , ] . we found the host phylogeny explains a large proportion of the inter-specific variation in viral load across all three temperatures, although these estimates have broad confidence intervals due to the model struggling to separate the phylogenetic and non-phylogenetic components (low = . , % ci: . to examine if species responded in the same or different way to changes in temperature we examined the relationships between susceptibilities across the different temperatures. we found strong positive phylogenetic correlations between viral loads across the three temperatures (table ). our models showed that the variance in viral load increased with temperature, however the mean viral load showed no such upward trend (table ). this suggests that the changes in variance are not simply occurring due to an increase in the means, that is then driving an increase in variance. the high correlations suggest the rank order of susceptibility of the species is not changing with increasing temperature. however, the change in variance suggests that although the intercepts are the temperature-specific intercepts when the other covariates (e.g. wing size) are set to their temperature specific means. they can be interpreted as the expected viral loads at the root of the phylogeny at each temperature. v p is the variance in between-species effects, which are structured by the phylogeny, and v r is the variance in within species effects attributable to between individual differences and measurement error. reaction norms are not crossing they are diverging from each other as temperature increases i.e. the most susceptible species are becoming more susceptible with increasing temperature, and the least susceptible less so [ ] . for example, d. obscura and d. affinis are the most susceptible species at all three temperatures. the responses of individual species show that some species have increasing viral load as temperature increases (fig , e.g. z. taronus, d. lummei) , while others decease (e.g. d. littoralis, d. novamexicana). the changes we observe could be explained by the increase in temperature effectively increasing the rate at which successful infection is progressing (i.e. altering where in the course of infection we have sampled). however, this seems unlikely as at days post infection at the medium temperature ( ˚c), viral load peaks and then plateaus [ ] . therefore, in those species where viral load increases at higher temperatures the peak viral load itself must be increasing, rather than us effectively sampling the same growth curve but at a later time point. likewise, in those species where viral load decreased at higher temperatures, viral load would need to first increase and then decrease, which we do not observe in a time course at ˚c [ ] . to check whether this also holds at higher temperatures we carried out a time course of infection in a subset of six of the original experimental species at ˚c, where we would expect the fastest transition between the rapid viral growth and the plateau phase of infection to occur (fig b in s text) . this allowed us to confirm that the decreasing viral loads observed in some species at higher temperatures are not due to general trend for viral loads to decline over longer periods of (metabolic) time. we quantified the lower and upper thermal tolerances (ct min and ct max ) across all species with replicates per species. neither ct max nor ct min were found to be significant predictors of viral load (ct min - . , % ci: - . , . , pmcmc = . and ct max . , % ci: - . , . , pmcmc = . ). when treated as a response in models we found the host phylogeny explained a large proportion of the variation in thermal maximum (ct max : . , % ci: . , ) and thermal minima (ct min : . , % ci: . , . , see s text fig c) . we also measured the basal metabolic rate of flies from species, across the three experimental temperatures, to examine how cellular function changes with temperature. bmr was not found to be a significant predictor of viral load when included as a fixed effect in our model (slope = . , % ci = - . , . , pmcmc = . ). bmr increased with temperature across all species (mean bmr and se: low . ± . , medium . ± . , high . ± . co ml/min - , see s text fig d) . when bmr was analysed as the response in models, the phylogeny explained a small amount of the between species variation (low . , % ci: × − , . , medium . , % ci: × − , . , high . , % ci: × − - . , s text fig e) indicating high within species variation or large measurement error. consequently the mean bmrs for each species, at each temperature, were used in the analysis of viral load will be poorly estimated and so the effects of bmr will be underestimated with too narrow credible intervals. to rectify this we ran a series of measurement error models, the most conservative of which gave a slope of - . but with very wide credible intervals (- . , . ) . full details of these models are given in the supporting information (s text). we found that susceptibilities of different species responded in different ways to changes in temperature. the susceptibilities of different species showed differing responses as temperatures increased (fig ) . there was a strong phylogenetic correlation in viral load across the three experimental temperatures (table ) . however, the variance in viral load increased with temperature, whereas the mean viral load did not show the same trend. this suggests that the rank order of susceptibility of the species remains relatively constant across temperatures, but as temperature increases the most susceptible species become more susceptible, and the least susceptible less so. changes in global temperatures are widely predicted to alter host-parasite interactions and therefore the likelihood of host shifts occurring [ , , , , ] . the outcome of these interactions may be difficult to predict if temperature causes a different effect in the host and pathogen species [ , , [ ] [ ] [ ] . our results show that changes in temperature may change the likelihood of pathogens successfully infecting certain species, although they suggest that it may not alter which species are the most susceptible to a novel pathogen. the increase in phylogenetic variance with temperature is effectively a form of genotypeby-environment interaction [ , [ ] [ ] [ ] . however, it varies from the classically considered ecological crossing of reaction norms, as we do not see a change in the rank order of species susceptibly across the range of experimental temperatures. instead, we find the species means diverge with increasing temperatures and so the between species differences increase [ , ] . it is also important to note that temperature may not simply be causing a change in effect size when considering the biological processes occurring during host-parasite interactions [ , ] . for example, virus replication may plateau at higher temperatures due to resource limitation. the observed level of susceptibility may be the combined outcome of both host and parasite traits, which may interact nonlinearly with temperature. we also note that by using a limited range of temperatures for practical reasons we may have not captured all unimodal relationships between viral load and temperature. as temperature is an important abiotic factor in many cellular and physiological processes, we went on to examine the underlying basis of why viral load might change with temperature. previous studies that found phylogenetic signal in host susceptibility were carried out at a single experimental temperature [ , ] . therefore, the patterns observed could potentially be explained by some host clades being assayed at sub-optimal thermal conditions. we used ct max and ct min as proxies for thermal optima which, due to its multifaceted nature, is problematic to measure directly [ ] [ ] [ ] . we also measured basal metabolic rate across three temperatures to see if the changes in viral load could be explained by general increases in enzymatic processes. we found that these measures were not significant predictors of the change in viral load with temperature. this may be driven by the fact that all temperature related traits are likely to be more complex than what any single measure can explore. traits such as host susceptibility are a function of both the host and parasite thermal optima, as well as the shape of any temperature-trait relationship [ , ] . the host immune response and cellular components utilised by the virus are likely to function most efficiently at the thermal optima of a species, and several studies have demonstrated the outcomes of host-pathogen interactions can depend on temperature [ , , , ] . however, the mechanisms underlying the changes in susceptibility with temperature seen in this study are uncertain and a matter for speculation. our results show that in the most susceptible species, viral load increases with temperature; this may be due to the virus being able to successfully infect and then freely proliferate, utilizing the host cells whist avoiding host immune defences. in less susceptible species viral load does not increase with temperature, and in some cases it actually appears to decreases. here, temperature may be driving an increase in biological processes such as enhanced host immunity, or simply increasing the rate of degradation or clearance of virus particles that have failed to establish an infection of host cells. we have investigated how an environmental variable can alter infection success following a novel viral challenge. however, temperature is just one of the potential environmental factors that will influence the different stages of a host shift event [ ] . using a controlled method of viral inoculation allows us to standardize inoculation dose so we can ask, given equal exposure, how does temperature affect the ability of a pathogen to persist and replicate in a given host? however, in nature hosts will be faced with variable levels of pathogen exposure, infected through various modes of transmission and often by multiple strains or genotypes [ ] . such variables may have consequences for the establishment and subsequent infection success of any potential host shift event. it is known that oral infection by dcv is stochastic and immune barriers such as the gut are important [ , , ] , therefore establishing the relevance of infection in the wild in this system would require further study using different potential routes of infection. the geographical distribution of a host will also influence factors such as diet and resource availability [ , [ ] [ ] [ ] [ ] , and so further work on the role of nutrient and resource availability would therefore be needed to further explore the impact of these on potential host shifts. in conclusion, we have found changes in temperature can both increase or decrease the likelihood of a host shift. our results show the rank order of species' susceptibilities remain the same across temperatures, suggesting that studies of host shifts at a single temperature can be informative in predicting which species are the most vulnerable to a novel pathogen. changing global temperatures may influence pathogen host shifts; for example changes in distributions of both host and pathogen species may generate novel transmission opportunities. our findings suggest that increases in global temperature could increase the likelihood of host shifts into the most susceptible species, and reduce it in others. climate change may therefore lead to changing distributions of both host and pathogens, with pathogens potentially expanding or contracting their host range. understanding how environmental factors might affect broader taxonomic groups of hosts and pathogens requires further study if we are to better understand host shifts in relation to climate change in nature. climate warming and disease risks for terrestrial and marine biota global warming and temperature-mediated increases in cercarial emergence in trematode parasites climate change and evolutionary adaptation climate change and infectious diseases: from evidence to a predictive framework environmental-mechanistic modelling of the impact of global change on human zoonotic disease emergence: a case study of lassa fever. freckleton r, editor climate oscillations and the structure of natural communities emerging pathogens: the epidemiology and evolution of species jumps the evolution and genetics of virus host shifts host phylogeny determines viral persistence and replication in novel hosts population biology of emerging and re-emerging pathogens virus rna structure specialization facilitates host adaptation rapid spread of emerging zika virus in the pacific area ebola in west africa: the outbreak able to change many things impact of climate change on global malaria distribution chikungunya virus emergence is constrained in asia by lineage-specific adaptive landscapes the many projected futures of dengue rapid range shifts of species associated with high levels of climate warming host and parasite thermal ecology jointly determine the effect of climate warming on epidemic dynamics how will global climate change affect parasite-host assemblages? global temperature constraints on aedes aegypti and ae. albopictus persistence and competence for dengue virus transmission. parasit vectors evolution in action: climate change, biodiversity dynamics and emerging infectious disease thermal biology in insect-parasite interactions host thermal biology: the key to understanding host-pathogen interactions and microbial pest control? variation in the immune state of gammarus pulex (crustacea, amphipoda) according to temperature: are extreme temperatures a stress? environmental temperature variation influences fitness trade-offs and tolerance in a fish-tapeworm association the influence of ambient temperature on the course of myxomatosis in rabbits some like it hot: the effects of climate change on reproduction, immune function and disease resistance in the cricket gryllus texensis host-parasite and genotype-by-environment interactions: temperature modifies potential for selection by a sterilizing pathogen environmental stressors alter relationships between physiology and behaviour empirical evidence that metabolic theory describes the temperature dependency of within-host parasite dynamics parasites and global warming: net effects of temperature on an intertidal host-parasite system some (worms) like it hot: fish parasites grow faster in warmer water, and alter host thermal preferences global change, parasite transmission and disease control: lessons from ecology understanding uncertainty in temperature effects on vector-borne disease: a bayesian approach detecting the impact of temperature on transmission of zika, dengue and chikungunya using mechanistic models short title: temperature predicts zika, dengue, and chikungunya transmission impact of human mobility on the emergence of dengue epidemics in pakistan rethinking vector immunology: the role of environmental temperature in shaping resistance phylogenetic signal in plant pathogen-host range phylogenetic determinants of potential host shifts in fungal pathogens host phylogeny constrains 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viruses associated with drosophila melanogaster twenty-five new viruses associated with the drosophilidae (diptera) physiological and metabolic consequences of viral infection in drosophila melanogaster drosophila c virus systemic infection leads to intestinal obstruction the novel genome organization of the insect picorna-like virus drosophila c virus suggests this virus belongs to a previously undescribed virus family the toll-dorsal pathway is required for resistance to viral oral infection in drosophila existence in drosophila of groups of picornavirus with different biological and serological properties host shifts result in parallel genetic changes when viruses evolve in closely related species a simple method of estimating fifty per cent endpoints measuring metabolic rates: a manual for scientists longevity, calling effort, and metabolic rate in two populations of cricket genetic architecture of metabolic rate: environment specific epistasis between mitochondrial and nuclear genes in an insect sexual size dimorphism in a drosophila clade, the d. obscura group geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data bayesian phylogenetics with beauti and the beast . mcmc methods for multi-respoinse generalized linear mixed models: the mcmcglmm r package r: a language and environment for statistical computing. vienna, austria: r foundation for statistical computing the phylogenetic mixed model maximum-likelihood estimation of evolutionary trees from continuous characters phylogenetic analysis and comparative data: a test and review of evidence inferring the historical patterns of biological evolution the role of genotype-by-environment interactions in sexual selection how will global climate change affect parasite-host assemblages? identifying climate drivers of infectious disease dynamics: recent advances and challenges ahead complex effects of temperature on mosquito immune function infection risk decreases with increasing mismatch in host and pathogen environmental tolerances the thermal mismatch hypothesis explains host susceptibility to an emerging infectious disease genotype-environment interaction and the evolution of phenotypic plasticity quantitative genetics and the evolution of reaction norms genotype-by-environment interactions and adaptation to local temperature affect immunity and fecundity in drosophila melanogaster coexistence of similar genotypes of daphnia magna in intermittent populations: response to thermal stress temperature checks the red queen? resistance and virulence in a fluctuating environment phenotypic variance, plasticity and heritability estimates of critical thermal limits depend on methodological context making sense of heat tolerance estimates in ectotherms: lessons from drosophila validity of thermal ramping assays used to assess thermal tolerance in arthropods the evolution of transmission mode costs and benefits of sublethal drosophila c virus infection entry is a rate-limiting step for viral infection in a drosophila melanogaster model of pathogenesis impact of environmental variation on host performance differs with pathogen identity: implications for host-pathogen interactions in a changing climate host nutrition alters the variance in parasite transmission potential measuring parasite fitness under genetic and thermal variation immunity in a variable world many thanks to darren obbard and frank jiggins for useful discussion and vanessa kellerman and johannes overgaard for discussion about thermal assay methods. thanks to dave hosken for use of bmr chambers and to the drosophila species stock centre for supplying flies. thanks to two anonymous reviewers for constructive comments. key: cord- -vyiuv qu authors: bataille, arnaud; levin, iris i.; sari, eloisa h. r. title: colonization of parasites and vectors date: - - journal: disease ecology doi: . / - - - - _ sha: doc_id: cord_uid: vyiuv qu colonization comprises the physical arrival of a species in a new area, but also its successful establishment within the local community. oceanic islands, like the hawaiian and the galapagos archipelagos, represent excellent systems to study the mechanisms of colonization because of their historical isolation. in this chapter, we first review some of the major mechanisms by which parasites and vectors could arrive to an oceanic island, both naturally or due to human activities, and the factors that may influence their successful establishment in the insular host community. we then explore examples of natural and anthropogenic colonization of the galapagos islands by parasites and vectors, focusing on one or more case studies that best represent the diversity of colonization mechanisms that has shaped parasite distribution in the archipelago. finally, we discuss future directions for research on parasite and vector colonization in galapagos islands. hawaiian and the galapagos archipelagos, represent excellent systems to study the mechanisms of colonization because of their historical isolation. ecosystem isolation was one of the most important premises used by macarthur and wilson ( ) for postulating the theory of island biogeography. in this landmark theory, they proposed that the number of species present in an isolated natural community is determined by both immigration and extinction rates. immigration rates depend on the distance separating the island from potential sources of colonization (distance effect), while the size of the island limits resource and habitat availability and influences species extinction rates (species-area effect). the theory of island biogeography has also been applied to the colonization of hosts by parasites (poulin ; reperant ). in this case, host mobility or density are considered distance effects influencing parasite immigration rate, and host body size and life-span represent species-area effects on extinction rate. however, the observed patterns of parasite species richness do not always fit these simple general laws of biogeography due to the complexity of host-parasite interactions and co-evolution (poulin ; strona and fattorini ) . it is important to note that the term colonization usually refers to natural establishment. humans have deeply changed the distribution of species and their environment, and human-aided spread of species to new areas is typically called invasion or introduction. although this chapter is entitled colonization, it explores both natural and anthropogenic colonization of the galapagos archipelago by parasites and disease vectors. throughout this chapter, a broad definition of parasite will be used to include viruses, bacteria, protozoans, fungi, as well as more traditionally defined parasites such as arthropods or helminths. in this chapter, we first review some of the major mechanisms by which parasites and vectors could arrive to an oceanic island, both naturally or due to human activities (see table . ), and the factors that may influence their successful establishment in the insular host community. this introduction is not intended as an extensive review of species colonization, but rather as a way to place our understanding of parasite and vector colonization in galapagos in a global context. in the following sections of the chapter, we then explore examples of natural and anthropogenic colonization of the galapagos islands by parasites and vectors, focusing on one or more case studies that best represent the diversity of colonization mechanisms that has shaped parasite distribution in the archipelago. some parasites may cover large distances without the help of a host or vector. such direct immigration can be the result of active dispersal, and this mechanism is typically restricted to larger arthropod parasites or parasitoids with strong flying rats and trypanosoma in christmas island wyatt et al. ( ) infected domestic animals pigeon and trichomonas in mauritius bunbury et al. ( ) avian plasmodium and avian pox in hawaii warner ( ) , van riper et al. ( ) , van riper et al. game animal diamond and veitch ( ) introduction of vector a. by commercial air transport culicid species in pacific islands lounibos ( ) b. by commercial ship culicid species in pacific islands lounibos ( ) pathogen as a biocontrol agent myxoma virus in australia fenner et al. ( ) capacity such as parasitic wasps (iqbal and austin ; masner and johnson ) . other parasitic microorganisms have motile, free-living stages, but the distances they can travel are measured in centimeters rather than kilometers, and these short dispersals generally involve host-seeking behaviors. for instance, skin-penetrating nematodes such as hookworms can move quickly in the soil as they search for hosts using chemical cues (castelletto et al. ) , and uniflagellated zoospores of the highly lethal batrachochytrium dendrobatidis-a widespread amphibian pathogenic fungus-can only swim for h over few centimeters in still medium (piotrowski et al. ). thus, we do not consider host-seeking movements as active dispersal, at least not at a scale relevant to colonization of remote islands. parasites with limited or no active dispersal can still directly travel long distances under favorable environmental conditions. the aerial dispersal of plant pathogens, especially fungal spores, has largely contributed to the global spread of important crop diseases like the sugarcane rust or coffee leaf rust (brown and hovmoller ) . some animal pathogens can also survive several days or weeks outside of their host and can be spread by aerosols and reach naive hosts. notably, wind dispersal has played a role in various outbreaks of foot and mouth disease virus and avian influenza virus in europe (keeling et al. ; ypma et al. ) . other parasites may use water currents to reach new areas. pathogenic bacterial species such as vibrio cholera can form biofilms in marine and freshwater environments, facilitating their persistence and dispersal (hall-stoodley and stoodley ) . the protozoan toxoplasma gondii can survive in seawater for up to months and remain infective, and oocysts shed with felid feces in freshwater runoff are a likely source of infections in marine ecosystems (conrad et al. ; lindsay and dubey ). long-distance travel of infected hosts is probably the most frequent natural mode of parasite immigration to islands. ancestors of endemic species may have brought their parasites with them, leading to their coevolution in isolation. such patterns can be found in some malaria parasite lineages restricted to endemic avian hosts in the lesser antilles (fallon et al. ) , or in hepatozoon parasites of endemic reptiles in seychelles or new zealand (godfrey et al. ; harris et al. ) . newly arrived parasites can also switch to local hosts, sometimes even distantly related to their original host. this is the case for native helminth parasites of hawaiian stream fishes that most likely colonized the islands with native fish-eating birds and marine fishes (font ) . many seabird species form large breeding colonies on islands for several months each year. the breeding grounds and foraging areas of multiple species can overlap, favoring parasite exchange (mccoy et al. ) . although seabirds often show high breeding site fidelity, colonies can be visited by transient birds (e.g., immature birds, failed breeders) or by infested birds deserting their colonies, which facilitates the colonization of new locations by parasites (brown and brown ; dietrich et al. ) . many bird species also naturally travel long distances and may use islands as stop-over sites during their migration or foraging, carrying along parasites. wild migrating birds and seabirds play an important role in the global spread of avian influenza and lyme disease, respectively (olsen et al. ; olsen et al. ; kilpatrick et al. a ). some disease vectors such as mosquitoes or midges are also capable of traveling long distances, usually with the help of aerial currents, and may bring parasites with them to new areas. for example, wind dispersal of infected midges is thought to have played a role in the spread of bluetongue virus in the mediterranean basin and northern europe, and wind dispersal of infected mosquitoes is implicated in the spread of japanese encephalitis virus to australia (kay and farrow ; ritchie and rochester ; carpenter et al. ). infected vectors can also travel on their hosts, such as ticks (dietrich et al. ) and hippoboscid flies found on seabirds, but in such cases it is hard to determine the relative importance of the host or the vector in parasite colonization. human activities have a continuously increasing impact on the environment in direct relation with their unique capacity to innovate and harvest natural resources. human colonization has been followed by species extirpation since prehistoric times (milberg and tyrberg ) . the introduction of invasive species and diseases by humans is a major cause of disturbance of natural communities around the globe, and islands have not been spared (blackburn et al. ; bellard et al. ) . parasite invasions of islands can originate from accidental transport of invasive hosts and vectors (wyatt et al. ) , or the deliberate introduction of domestic animals (bunbury et al. ) and game (diamond and veitch ) . probably one of the most famous examples of the impact of introduced parasites on insular wildlife is the decline of the hawaiian endemic avifauna following the introduction of the avian malaria plasmodium relictum, avianpox virus, and their mosquito vector culex quinquefasciatus (warner ; van riper et al. van riper et al. , . in some rare cases, parasites are deliberately introduced in new areas as a mean to control invasive species. the most notorious example is the introduction of myxoma virus in australia in the s to control invasive rabbit populations (fenner et al. ) . the globalization of human activities in the last decades has multiplied the risks of parasite emergence to an unprecedented scale (daszak et al. ; jones et al. ) . the ever-expanding air transport network has dramatically increased the risk of global epidemics by facilitating the movement of infected hosts and disease vectors (mangili and gendreau ; tatem et al. a, b) . commercial ships have also contributed to the spread of many invasive species, including parasites and disease vectors across the globe (ruiz et al. ; lounibos ) . habitat destruction and intensification of agriculture have also led to human encroachment into wildlife habitat and loss of biodiversity, increasing the risk of pathogen spill-over between humans, domestic animals, and wildlife (daszak et al. ; keesing et al. ). due to their isolation and high level of endemism, species on oceanic islands are especially at risk in the face of these modern challenges (kier et al. ; bellard et al. ). again we can refer to the example of hawaii, where human activities have permitted secondary introduction of different strains of culex quinquefasciatus in the archipelago, which has been associated with an increase in the altitudinal range of the mosquito and in malaria virulence, further increasing the impact of the disease on the endemic fauna (fonseca et al. (fonseca et al. , . the ecological processes associated with island colonization by hosts and parasites, such as the island syndrome and host switching, will be the subjects of chaps. - . here, we will briefly mention some key factors influencing the success of parasite establishment in island communities. parasite colonization fundamentally depends on host colonization success (see chap. ), and on availability of suitable native hosts. during host colonization, the probability of parasite establishment and co-evolution with their host will also depend on parasite transmission efficiency. parasites will have a higher risk of extinction when founding host populations are small, when stochastic events result in host extinction, and sometimes, when the host population front moves faster than the disease transmission front (bar-david et al. ; macleod et al. ) . parasite colonization will also depend on introduction effort, also called propagule pressure (lockwood et al. ) . parasite establishment is more likely in situations where there are larger numbers of individual parasites arriving to the new area and more numerous arrival events. for parasites, introduction effort can be measured at the level of host individuals, populations, or species (poulin ) . notably, parasites in aggregated distributions among few hosts are more susceptible to "missing the boat" and going extinct (paterson et al. ; macleod et al. ). if its host fails to establish, parasites go extinct with them, unless other suitable hosts are present. large and dense breeding colonies of seabirds are good host candidates for parasites because they provide a large number of potential hosts and regular opportunities of transmission (mccoy et al. ) . in comparison to continental species, endemic insular species may have a lower diversity of evolved immune defenses, probably as a consequence of their reduced parasite assemblages, which can increase their susceptibility to arriving parasites (frankham ; matson ) . infecting naive hosts allows parasites to remain in the new ecosystem even if the original, colonizing host goes extinct or is only transient (smith and carpenter ) . therefore, the capacity of the parasite to infect a wide range of hosts contributes greatly to its persistence after colonization. it has been argued that all parasites, except true specialists, can switch hosts rapidly if they can exploit newly available resources without having to evolve novel capacities (hoberg and brooks ) . lower prevalence and smaller distributions of hostspecific avian blood parasites in island systems compared to widespread hostgeneralists provide support for this idea (beadell et al. ; ewen et al. ; clark and clegg ) . for vector-borne parasites, parasite establishment will also depend on the presence of a suitable vector. parasites with high vector specificity have more restricted ranges than parasites with a wide range of vectors (ishtiaq et al. ) . other important factors are vector habitat preferences, host specificity, and dispersal capacity (ishtiaq et al. ) . for this reason, the study of insular vector populations is critical to understand vector-borne parasite colonization. based on data compilations by deem et al. ( , and sari et al. ( ) , a total of species of parasites and disease vectors have been identified in the galapagos archipelago (see table . , fig. . ). of those, could have arrived naturally, based on taxonomic or phylogenetic data, and their distribution in the native fauna. most of these natural colonizations are directly transmitted parasites or arthropod disease vectors, although a relatively large number of native vector-borne blood parasites have been identified (see fig. . ). these numbers are unlikely to accurately reflect the absolute or relative abundance of native and introduced parasites, as some host-parasite systems have been more heavily studied than others (e.g., parasites of the galapagos avian fauna). in galapagos, there are several examples of parasites that have naturally colonized the islands with their hosts (see table . ). these are obligate parasites that are commonly found in close association with their bird hosts, usually with high prevalence and high densities on any given host. this happens because when hosts colonize a new area, less common parasites may have a higher chance of "missing the boat" (paterson et al. ) . feather mites (acari: astigmata), lice (insecta: phthiraptera), and blood parasites (apicomplexa: haemosporida) represent good examples of colonizers that arrived to galapagos with their vertebrate host. in order to make inferences about parasite arrival, we need to study the parasites from galapagos animals and from their closest related continental species, which requires an understanding of the colonization history of the hosts themselves (see chap. ). this list is based on compilations done by deem et al. ( , and sari et al. ( ) . the type « fly » includes insects of the order diptera, and the type « louse » regroups insects of the order phthiraptera. "und", undescribed species. numbers between brackets in front of species names indicates the number of species identified a species confirmed to be native by taxonomic or phylogenetic studies the galapagos flycatcher (myiarchus magnirostris) colonized the galapagos islands about , years ago, and their closest relatives are the brown-crested flycatchers (myiarchus tyrannulus) from central america (sari and parker ) . in order to investigate which parasites colonized the archipelago with their hosts, sari et al. ( ) sampled mites, lice, and blood parasites from galapagos flycatchers on seven galapagos islands and also from brown-crested flycatchers at four locations in costa rica. these authors described a suite of six mite and louse species infecting galapagos flycatchers, of which five species are shared with the brown-crested flycatchers, leading to the conclusion that these mites and lice arrived to galapagos together with their bird hosts when they naturally colonized the archipelago. taking a closer look at the morphology of this ectoparasite assemblage, sari et al. ( ) reported that only one of these species, a mite from the genus nycteridocaulus (family proctophyllodidae), has evolved evident morphological differentiation between the two host species, leading to the conclusion that the nycteridocaulus from the galapagos flycatcher is probably not conspecific with that from the browncrested flycatcher. the other mite and louse species have the same morphological characteristics and taxonomic classification, for both host species. however, genetic distance between ricinus marginatus lice collected from both host species was found to be almost ten times larger than the genetic distance between their hosts . both hosts and their lice have been evolving in allopatry for about , years (see chap. ), but evident speciation was only observed in the bird hosts ). these findings add to the body of knowledge indicating that morphological evolution tends to be more conservative in parasites than their hosts (klassen ) and suggest that the process of speciation for lice can take much longer than it takes for their hosts, as mentioned by mcdowall ( ) . a similar pattern was also observed for the galapagos hawk (buteo galapagoensis) (whiteman et al. , ), a more recent arrival to galapagos than the galapagos flycatcher. the galapagos hawk colonized the archipelago about , years ago and its closest related continental species is the swainson's hawk (buteo swainsoni) (bollmer et al. ; amaral et al. ) (see chap. ). five species of ectoparasites have been found on hawks on several of the galapagos islands: one hippoboscid fly (icosta nigra), three species of lice (degeeriella regalis, colpocephalum turbinatum, and craspedorrhynchus sp.), and one skin mite (myialges caulotoon) (whiteman et al. , . all these species are also found on swainson's hawks and are thought to have colonized galapagos together with their hawk host (price et al. ; parker et al. ) . whiteman et al. ( ) looked at variation in both morphology and dna sequences for one of these species, the head louse craspedorrhynchus sp., in populations of galapagos hawks and swainson's hawks in north and south america. they found approximately % genetic divergence between lice from the two host species, while almost no genetic differentiation was found between the galapagos and the swainson's hawks used in this study (only one base pair in nucleotides sequenced of coi). in contrast, hawks from galapagos and swainson's hawks are strikingly different in morphology but the craspedorrhynchus lice found on the two hosts showed a lot of overlap in morphological characteristics. subtle morphological differences, however, were observed in the head and genitalia of lice between the two host species, allowing the identification of their geographical origin and possibly lineage diversification. parasites that colonized the galapagos with their vertebrate hosts share patterns of distribution and evolutionary history with their hosts. rivera-parra et al. ( ) showed that the species identity of the host was more important than sampling location for determining the phylogenetic relationships within each species of louse parasitizing two related seabird species, despite the fact that the hosts breed together in dense mixed-species colonies. however, these lice have different prevalence on different islands, demonstrating that the relationships among parasites, hosts, and islands are idiosyncratic (rivera-parra et al. ) . nematode parasites that colonized galapagos together with the founding population of the galapagos tortoises (chelonoidis nigra species complex) also show a similar island-dependent distribution (fournié et al. ) . each galapagos tortoise species is found only on one island, and each island has a different combination of nematodes. fournié et al. ( ) suggested that the observed pattern could have resulted from a neutral process of founder events following the arrival of tortoises to galapagos. parasites might also colonize the galapagos archipelago via migratory or vagrant birds. for example, plasmodium parasites detected in galapagos endemic passerine bird species may have arrived with migratory bobolinks (dolichonyx oryzivorus; see fig. . ) (levin et al. ) . four lineages of plasmodium parasites have been identified in the galapagos (levin et al. (levin et al. , . one lineage was repeatedly detected in galapagos penguins and occasionally in yellow warblers and the other three were only detected in one or a handful of galapagos birds at one location, at one given time (levin et al. ) . a very large sampling effort (nearly birds molecularly screened for blood parasites) was needed in order to reveal these few rare lineages. interestingly, two of the rare lineages were perfect dna matches with plasmodium lineages amplified from north american breeding bobolink samples (levin et al. ) . although this is not confirmation that migratory bobolinks introduced these rare lineages to the galapagos, it is evidence that the islands may be exposed to more potential parasite introductions than previously thought. it is not currently known whether these rare lineages have established in the islands. because bobolinks spend time in both the southern and northern hemispheres during mosquito breeding season, they probably acquire blood parasites in both locations. by comparing the blood parasites found in north american breeding bobolinks to those found in the brown-headed cowbird, a short-distance migrant that breeds in sympatry, the authors were able to begin teasing apart the potential origins of the rare galapagos plasmodium lineages (levin et al. ). if the galapagos plasmodium lineage is shared between bobolinks and cowbirds and pre-dominantly matches dna sequences from other north american hosts, the colonizing lineage is likely of north american origin. if this lineage is detected in bobolinks but not cowbirds and matches predominantly south american sequences, then the origin is mostly likely south american. bobolinks stop over in galapagos only during their southward migration, but could still harbor chronic infections by parasites acquired many months before, while overwintering. indeed, one of the plasmodium lineages found in galapagos birds was likely of north american origin, while the other match was likely from south america (levin et al. ). more work is needed to further understand the probability of colonization by blood parasites (and other parasites) via migratory birds. recently, several bobolinks have been sampled in galapagos in october during migration (p. parker, personal communication). by far the most abundant migratory birds in galapagos are shorebirds, and to our knowledge, none have been tested for blood parasites. multiple blood-feeding arthropods with the capacity to disperse and transmit parasites may have naturally colonized the galapagos archipelago (see table . , fig. . ) . in all the cases, we have little knowledge of either their origin and native status, or their exact role as disease vectors in galapagos. the origin and phylogenentic relationships of the only mosquito native to the galapagos islands, aedes taeniorhynchus, are probably the best studied (bataille et al. a ), although its role as a disease vector is still not clear. conversely, the role of hippoboscid flies (four genera and seven species in galapagos; see table . and fig. . ) in the transmission of avian blood parasites has been well studied (valkiunas et al. ; levin et al. levin et al. , , but their origin is uncertain. these two contrasting cases will be explored further below. two ticks of the genus ornithodoros infecting reptiles are endemic to the galapagos islands (wikelski ) . two other amblyomma and three vatacarus ticks parasitizing reptiles and one avian ornithodoros species are also found in the archipelago and probably arrived naturally with their hosts. all these arthropods could be involved in the transmission of hepatozoon parasites, mainly through ingestion by the vertebrate host (smith ; bataille et al. ) . one horse-fly species of the genus tabanus has also been classified as endemic to galapagos (sinclair ) and can feed on galapagos reptiles (philip (philip , ), but its role in disease transmission is completely unknown. as argued in the introduction of this chapter, it is hard to determine whether a native vector, a native host, or both brought a colonizing parasite to galapagos (see sect. . ). combined phylogenetic studies of parasite, vector, and host may help resolve this issue. this type of study has been carried out with mites vectored by hippoboscid flies and infecting endemic galapagos birds , although this study focused on parasite population structure and host specificity rather than on their origin (see chap. ). aedes taeniorhynchus is widely distributed in temperate and tropical coastal areas of the new world (lang ) . it breeds primarily in temporary-water habitats, producing huge broods after flood or heavy rains. the species is widely distributed across the galapagos islands. the presence of a. taeniorhynchus in the archipelago was first recorded in the late s (howard ) . it was suggested early on that a. taeniorhynchus might have naturally reached the archipelago before the arrival of humans (hardy ). on the other hand, pirates and later whalers frequently visited the archipelago between the sixteenth and nineteenth centuries, giving multiple opportunities to introduce a. taeniorhynchus. to tackle this question, bataille et al. ( a) performed phylogenetic analyses using mitochondrial and nuclear dna markers on a. taeniorhynchus specimens collected across the galapagos archipelago and across the species' continental range. all analyses placed the galapagos population of a. taeniorhynchus within one single coherent cluster clearly separated from the continental mosquito populations. this result suggests that the galapagos population of a. taeniorhynchus origi-nated from a single colonization event and now represents a distinct evolutionary unit divergent from the continental populations. molecular clock analysis estimated that the galapagos and continental clades diverged , years ago ( % confidence interval: , - , years). although imprecise, this estimation demonstrated that the colonization by a. taeniorhynchus was not human-driven. some characteristics of the biology of a. taeniorhynchus support the hypothesis that this mosquito had the capacity to naturally colonize the galapagos islands and successfully establish itself across the archipelago. first, the strong flying capacity of a. taeniorhynchus is well known, and long-distance dispersal has been observed various times in this species (provost ; bello et al. ) . arrival of a. taeniorhynchus in galapagos could have been facilitated by the strong wind and oceanic current found in the intertropical convergence zone during cyclic climatic fluctuations such as el niño events (peck ) . second, a. taeniorhynchus lays desiccation-resistant eggs, which has been significantly associated with the success of invasive mosquito introductions in new areas (juliano and lounibos ) . third, larvae of a. taeniorhynchus successfully grow in water with a wide range of salinity (clark et al. ) , which gives them the possibility to breed in a wide range of habitats across the archipelago, notably in the mangroves found throughout the coasts of the archipelago. the role of aedes taeniorhynchus as a disease vector in galapagos is still poorly understood, but is likely to be important (see fig. . ) . this species has a wide distribution, high population density, and strong dispersal capacity (bataille et al. (bataille et al. , ) (see chap. ), so it represents an ideal vector for native and invasive mosquito-borne parasites. moreover, it feeds opportunistically on a wide range of vertebrate hosts including birds, mammals, and reptiles (bataille et al. ). this species could thus act as a bridge vector across most of galapagos endemic wildlife (kilpatrick et al. ) . swarms of mosquitoes can be an important nuisance for galapagos wildlife. for example, waved albatrosses (phoebastria irrorata) on española island were observed deserting their nests and neglecting their eggs due to mosquito harassment (anderson and fortner ). aedes taeniorhynchus is a competent vector for the transmission of various viruses, including west nile virus (hardy et al. ; turell et al. turell et al. , , but, fortunately, none of those are currently present in the archipelago. aedes taeniorhynchus is also considered an important vector of the dog heartworm (dirofilaria immitis) (labarthe et al. ; labarthe and guerrero ) . the disease was introduced with dogs in galapagos islands ), but the role of a. taeniorhynchus in its transmission, notably to galapagos sea lions and fur seals (dunn and wolke , sato et al. ) , has not been verified. pcr-based parasite screening identified the presence of haemoproteus parasites and of microfilarial nematodes in pools of mosquito thoraces collected in fernandina and isabela islands (bataille et al. ). however, these results do not provide direct evidence for the role of a. taeniorhynchus in the transmission of these parasites. mosquitoes are not the typical vectors of haemoproteus spp., although some studies have supported this possibility (ishtiaq et al. galapagos penguins (merkel et al. ). they may be transmitted by multiple vector species, but aedes taeniorhynchus is thought to be one important vector for this parasite, because microfilariae prevalence across its hosts' ranges correlates with ecological factors suitable for a. taeniorhynchus populations (siers et al. ) , and this mosquito was shown to feed on cormorants (bataille et al. ) . hepatozoon parasites were also detected by pcr in both thoraces and heads of mosquitoes. aedes taeniorhynchus could be involved in the transmission of this parasite by accidental ingestion of infected mosquitoes, but maybe also by mosquito bites (telford et al. ) . this mosquito could also be a mechanical vector (i.e., transfer of the parasite without passage within the vector necessary for the parasite's life cycle) of avipoxvirus between birds in the archipelago (thiel et al. ). hippoboscid flies are obligate, blood-feeding ectoparasites found on birds and mammals. although some are wingless (e.g., sheep ked), most hippoboscids have fully functional wings. despite the ability to fly, hippoboscids tend to remain closely associated with their hosts, with only one off-host life stage: female hippoboscids lay a single, late-instar larva, which pupates in the ground. hippoboscid flies are common on galapagos seabirds, the galapagos hawk, and the galapagos dove valkiunas et al. ; levin et al. ) (see fig. . ). the flies tend to be very host-specific, with one fly species per host species (or a few closely related host species). for example, over the course of sampling > flies from great and magnificent frigatebirds and blue-footed, red-footed, and nazca boobies, the species of hippoboscid that infects boobies, olfersia aenescens, was never found on a frigatebird and vice versa, despite the fact that these seabirds often breed in dense, multi-species colonies with ample opportunity for flies to hostswitch (i. levin, personal observation.) . hippoboscid flies are definitive hosts for fig. . hippoboscid fly on a nazca booby (sula granti; photo: iris i. levin) blood parasites in the subgenus haemoproteus haemoproteus (valkiunas ; levin et al. ) . because of the host-specificity of the flies, lineages of h. haemoproteus are often specific to a particular host and fly pair (valkiunas et al. ; levin et al. levin et al. , . for reasons already mentioned, it is difficult to determine the order and combination of arrival in galapagos: did colonizing birds arrive infected with flies and haemoproteus? or did the flies and haemoproteus arrive later? perhaps the most parsimonious explanation is that colonizing birds were infected with both parasite and vector, but we have little evidence to back up this claim. the various dna lineages of haemoproteus multipigmentatus found infecting galapagos doves were also detected in continental south american dove species ). these lineages do not appear to have diversified recently from one single lineage, which would be evidence of differentiation within the galapagos archipelago since arrival. instead, it seems plausible that this parasite was introduced recently and multiple times to galapagos, probably via a vagrant dove host like the eared dove (zenaida auriculata) or with introduced rock doves, both of which have been found to be hosts of h. multipigmentatus valkiunas et al. ) . rock doves were introduced to galapagos in the early s and are now completely eradicated from the islands, but h. multipigmentatus was also detected in rock doves once collected in galapagos (p. parker, personal communication). interestingly, rock doves in continental areas are infected with the hippoboscid fly pseudolynchia canariensis, while the flies found infecting galapagos doves belong to the species microlynchia galapagoensis, so the role of the supposed vector of h. multipigmentatus in galapagos in these multiple colonization events is currently unknown. in contrast, galapagos frigatebird species are infected with just one lineage of haemoproteus iwa, which is probably vectored by the hippoboscid fly, olfersia spinifera (levin et al. ) . vector confirmation for both m. galapagoensis and o. spinifera involved dna amplification of haemoproteus from fly thorax without amplification of avian host dna, indicating developing parasite (sporozoite) in the thorax (valkiunas et al. ; levin et al. ) . although galapagos frigatebirds are genetically isolated from frigatebirds in the rest of their tropical range (hailer et al. ; see chap. ), only one haemoproteus iwa lineage has ever been recovered both in galapagos birds and frigatebirds in other locations (levin et al. ) . magnificent frigatebirds colonized the galapagos archipelago approximately , years ago ( % confidence intervals: , - , ) (hailer et al. ). using the estimated dna sequence divergence rate for the hemosporidian cytochrome b gene ( . % per million years, ricklefs and outlaw ) , the lack of sequence divergence within galapagos is consistent with the parasite and vector arriving with the colonizing host (levin et al. ) . however, it is still possible that the parasite and fly vector are more recent arrivals. frigatebirds are philopatric to their breeding site, but travel great distances during the non-breeding season (dearborn et al. ; weimerskirch et al. ) . we know that hippoboscid flies do regularly move between host individuals at a local scale; interestingly, the flies that do move between birds are less likely to be infected with haemoproteus iwa . in order to fully understand the colonization history of hippoboscid fly vectors in galapagos, large-scale phylogenetic and phylogeographic studies of haemoproteus parasites, bird hosts, and hippoboscid flies are needed, with an effort to estimate arrival dates where possible. the introduction of new parasites and disease vectors due to human activities represents a major threat to galapagos islands biodiversity (wikelski et al. ; gottdenker et al. ; kilpatrick et al. b ). this threat has substantially increased in the last two decades due to the rapid growth of the resident and visitor population associated with the booming tourism industry (peck et al. ; unesco ; unesco ) . as for isolated islands elsewhere, the principal routes of introduction of disease vectors and parasites to the galapagos islands are transport by boat and airplanes (wikelski et al. ; causton et al. ; kilpatrick et al. b ). there are two airports in galapagos (baltra and san cristobal islands) connecting the archipelago to guayaquil and quito in mainland ecuador, and one cargo route from guayaquil stopping at san cristobal, santa cruz, and isabela islands (fig. . ) . private jets have flown to galapagos directly from places as varied as florida, brazil, and the middle east without any systematic quarantine measures (cruz martinez and causton ) . in , more than commercial flights, cargo ships, and privately-owned jets or boats made trips to the galapagos islands (galapagos quarantine and inspection system-sicgal annual report ). a quarantine and inspection system (sicgal) is in place to control the movement of goods to and between islands (causton et al. ), but presently sicgal does human activities have directly affected the health of the native galapagos fauna through deliberate or accidental killing and injuries by humans or by other species they have introduced since the discovery of the archipelago (dowler et al. ; gottdenker et al. ; poulakakis et al. ; denkinger et al. ) . beyond physical injuries, human presence can also have a direct stress-related effect on the health of galapagos wildlife. notably, it has been shown that even low levels of human disturbance like ecotourism can increase levels of the stress hormone corticosterone in marine iguanas and modify some of their immune response capacities such as bacterial killing ability or cutaneous wound healing (french et al. ) . in galapagos sea lions, the immune activity and body condition of individuals living in the urban colony of puerto baquerizo moreno on san cristobal island differ from those in the colonies located in the protected zones of the national park (brock et al. ) . human-related impact on this urban sea lion colony includes close contact with humans and domestic animals, contact with pollutants from sea vessels, and with bacteria of human origin (wheeler et al. ; brock et al. ; denkinger et al. ) . free-living, motile parasites that can survive a long time in the environment may have arrived directly to the galapagos archipelago by hitchhiking on human transports. the parasitic botfly philornis downsi represents the most likely case of such stowaway introduction in the archipelago. this fly lays eggs in bird nests and the larvae feed on blood of nestlings, with negative consequences on nestling survival (fessl et al. ; koop et al. ) . philornis downsi was introduced to the galapagos islands from mainland ecuador in the s (causton et al. ; bulgarella et al. ) , maybe with imported fruit or in the cargo holds of planes. alternatively, it could have been introduced with infected vertebrate hosts, for example chickens or pigeons, or with their nest material. the case of this fly and its impact on galapagos avifauna will be discussed in detail in chap. . most invasive parasites detected in the native galapagos terrestrial vertebrate fauna probably originated from introduced vertebrate hosts, especially domestic animals. parasites introduced with chickens have been particularly well studied. several surveys carried out on chickens from inhabited islands of the archipelago (gottdenker et al. ; soos et al. ; deem et al. ) indicated infection by mycoplasma gallisepticum, a globally distributed bacterium that causes chronic respiratory disease in poultry and conjunctivitis in wild birds (williams et al. ) , and by nine different types of viruses, including the contagious newcastle disease (avian paramyxovirus ) that infects many domestic and wild avian species (alexander et al. ) . antibodies to newcastle virus and to an adenovirus (adenovirus- ) were also detected in galapagos finches on floreana island, suggesting potential spillover from poultry to wild birds (deem et al. ) . multiple nematode, cestode, trematode, and protozoan parasites were also identified, including a dyspharynx nematode that has been associated with mortalities in galapagos dark-billed cuckoos (gottdenker et al. ). marek's disease, caused by a herpesvirus, is also present and caused mortality in galapagos domestic poultry in / , but risks for native galapagos avifauna are considered low because there are no susceptible native galliform species in the archipelago (miller et al. ; gottdenker et al. ) . galapagos poultry are also infected by avian pox, caused by an avipoxvirus of the fowlpox lineage (gottdenker et al. ) . however, this strain of avipoxvirus seems restricted to chickens, whereas other strains of the canarypox lineage have been affecting the galapagos passerine birds since at least (thiel et al. ; parker et al. parker et al. , . this canarypox virus lineage can have a major impact on the survival of some endemic passerine populations during stressful environmental conditions like el niño events (curry and grant ) . examination of museum specimens suggested that this avipoxvirus arrived to the archipelago in the late s, possibly with early settlers, or via a natural colonization with migrating passerines like bobolinks (see sect. . ) ). despite the difference in hosts between fowlpox and canarypox viruses, the sympatry of the two pox lineages in some areas may allow for recombination and virulence alteration of avian pox viruses in galapagos (thiel et al. ) . the transmission of avian pox in the archipelago is probably facilitated by endemic and introduced mechanical vectors (see sections on vectors above and below). importation of broiler chickens for industry in galapagos increases the risk of introducing avian parasites to the archipelago. strict biosecurity protocols are not implemented in galapagos broiler houses, permitting direct contact of wild birds with poultry (gottdenker et al. ) . however, backyard chickens may represent a greater threat of disease spillover to galapagos wildlife than broiler chicken, because they harbor more parasites and are more frequently in contact with wildlife . rock doves introduced to the galapagos islands facilitated the arrival of the flagellate protozoa trichomonas gallinae, and the subsequent infection of galapagos doves by this parasite (wikelski et al. ) . domestic cats and dogs also brought their parasites with them to the galapagos archipelago. high prevalence ( - %) of canine distemper virus and the dog heartworm dirofilaria immitis have been detected in galapagos dog populations, which has led to major concerns of spillover to marine mammals diaz et al. ) . of the parasites infecting cats in galapagos islands, the high prevalence of toxoplasma gondii represents the most significant threat to the endemic wildlife ). this protozoan parasite infects all warm-blooded animals, including humans, but felid species are their only definitive hosts, necessary for their sexual reproduction (tenter et al. ) . infected cats can notably transmit the parasite to other hosts via ingestion of contaminated feces. infection by t. gondii was observed in many species of birds across the globe (dubey ) , including the endemic bird 'alala from hawaii (work et al. ) . in galapagos, low prevalence ( - %) of antibodies to t. gondii was detected in galapagos penguins and flightless cormorants (deem et al. ) . apart from the potential spread of parasites through the introduced domestic species, a few introduced species that now live in the wild could also have brought parasites to the galápagos with them. the smooth-billed ani and the cattle egret are abundant birds in galapagos that were brought by humans and could represent a source of new parasites, but they have not yet been well studied in this respect. several disease vectors have been introduced by humans to the galapagos archipelago, but, for most of them, the exact mode and frequency of introduction has not been studied in detail. the yellow fever mosquito, aedes aegypti, has been present in urbanized areas of santa cruz and san cristobal islands since , and is associated with cases of dengue fever since then (causton et al. ) . the biting midge culicoides pusillus, a potential vector for blue tongue virus, is established in santa cruz island since at least (causton et al. ) . the blackfly, simulium bipunctatum, vector of the river blindness worm, was detected in and is distributed on three islands, including the uninhabited santiago island (causton et al. ) . lastly, the southern house mosquito, culex quinquefasciatus, vector of multiple parasites such as avian malaria parasites and west nile virus, was first recorded in in santa cruz, and is now distributed in all inhabited islands of the archipelago (peck et al. ; causton et al. ) . all these vectors have restricted distribution in the archipelago because they require fresh water for breeding, which is mostly accessible in the humid areas of the islands and in human-inhabited areas, where open fresh water tanks and manmade cavities (e.g., discarded tires and containers) containing fresh water are readily available (fig. . ). the only introduced disease vector that has been studied in further detail is c. quinquefasciatus. culex quinquefasciatus is a member of the globally distributed culex pipiens species complex, and is found in tropical and sub-tropical regions where it breeds in freshwater with high organic content, depositing egg rafts on the water surface. it is extremely successful in human-inhabited areas because of the abundance of stagnant freshwater bodies, but it also breeds in forest environments. it is an important vector for a wide variety of diseases, such as west nile virus (sardelis et al. ) , filariasis (farid et al. ) , avian pox and avian malaria (van riper et al. ; fonseca et al. ) . it feeds readily on mammals and birds, so it can play the role of a bridge vector, notably for the transmission of west nile virus (sardelis et al. ). the nineteenth century introduction of this vector and of avian pox in hawaii followed by a later introduction of avian malaria is thought to be the main reason for the dramatic decline of hawaiian endemic birds throughout the twentieth century (warner ; van riper et al. van riper et al. , . the presence of c. quinquefasciatus in the galapagos islands is considered to be a serious threat to its endemic fauna because of the role of this mosquito in wildlife disease transmission elsewhere causton et al. ) . multiple parasites vectored by this mosquito are already present in galapagos, most notably avian pox viruses, plasmodium parasites, and the nematode dirofilaria immitis (see sect. . . ). however, its exact role in the transmission of these parasites remains unclear. the capacity of the galapagos c. quinquefasciatus to transmit parasites has only been demonstrated for west nile virus (eastwood et al. ) . additionally, it is worth noting that a pool of c. quinquefasciatus heads tested positives to avian pox virus in a pcr assay (bataille a, cruz m, cedeno v, cunningham aa, goodman sj, unpublished data), supporting its potential role in the mechanical transmission of this virus (thiel et al. ) . a worldwide genetic survey of c. quinquefasciatus, including samples from the galapagos islands, was conducted using microsatellite markers (fonseca et al. ) . the galapagos samples were genetically very similar to specimens from mainland ecuador, supporting the idea of a recent colonization of the archipelago fig. . eggs of the introduced southern house mosquito (culex quinquefasciatus) deposited in an abandoned plastic container filled with water (photos: arnaud bataille) from this country. however, the study included only a small number of samples from one island (santa cruz), which was insufficient to fully understand the history of its presence on the archipelago or the risks of current and future introductions. during the - airplane monitoring program, eight live c. quinquefasciatus mosquitoes were collected in airplanes arriving in baltra and san cristobal airports, evidence of their on-going introduction to the archipelago (bataille et al. b ). the same authors sampled c. quinquefasciatus specimens across the galapagos islands and in mainland ecuador, and used microsatellite markers to further assess the genetic similarity between the mosquito populations in the archipelago and mainland ecuador, and to determine the pathways and frequency of introduction of this mosquito from the mainland to the archipelago. they showed that the c. quinquefasciatus populations in baltra and san cristobal (the two islands hosting airports connected to mainland ecuador) were genetically more similar to mainland mosquito populations than to populations from santa cruz, isabela, or floreana islands (bataille et al. b) . such a pattern is most likely the result of frequent introductions of mosquitoes via airplane, and of their successful integration into already-established populations. culex quinquefasciatus could also arrive in the archipelago with cargo boats arriving to santa cruz, san cristobal, and baltra islands. however, bataille et al. ( b) did not observe a strong genetic similarity between c. quinquefasciatus populations of santa cruz island and mainland ecuador, suggesting that c. quinquefasciatus introduction by boat is much less important than by airplane. although airplane disinfection has been implemented for commercial flights bound to galapagos since following world health organization guidelines, the increase in flights to the archipelago associated with tourism development still represents a major risk of parasite introduction with infected disease vectors. our understanding of parasite and vector colonization in galapagos is incomplete. however, there are several well-studied examples (e.g., mosquitoes, avian hemosporidians, avian pox), which emphasize that all routes of introduction including natural colonization, colonization with a vertebrate host (a colonizer or migrant/ vagrant), and anthropogenic colonization have been documented. we know the most about colonization of a few organisms of conservation concern, such as the mosquito species known as disease vectors elsewhere in their distribution. we lack specific information on colonization for the majority of parasites identified in galapagos (see table . ) and therefore can only speculate on arrival mode and evolutionary history. in order to properly study parasite and vector colonization, research must extend to continental sister taxa or, in cases of non-endemic species, research must include organisms sampled outside their galapagos range. the best approaches for studying colonization include detailed phylogeographic and population genetic approaches. when possible, researchers should sample the entire parasite community in and on galapagos animals and their closest continental relatives. by sampling parasite assemblages, we can reconstruct more accurate hypotheses about parasite arrival and divergence within galapagos. for example, by examining all species of lice and mites found on the galapagos flycatcher and their most closely related continental sister taxa, the brown-crested flycatcher, sari et al. ( ) concluded that the suite of ectoparasites probably arrived with the ancestor of the galapagos flycatcher and that only one mite species had diverged in morphology since arrival to galapagos. sampling only the divergent mite could have led to a different conclusion. furthermore, vertically transmitted and closely host-associated parasites can be used as additional evidence for revealing host evolutionary history . thus, studying parasite colonization adds more information about the evolutionary history of galapagos than simply an answer to how that particular parasite colonized the islands. when inferring whether a parasite arrived by natural colonization or by human introduction, it is important to predict which ones may represent a greater threat to the galapagos native fauna. endemic fauna have been isolated in the archipelago for a long time, and may have lost their ability to mount immune responses against recently introduced parasites. so knowledge about arrival and transmission of introduced parasites is essential and urgent for proposing conservation strategies and the prevention management in galapagos. continued-and perhaps expanded-monitoring programs are needed to prevent further human-aided parasite introduction. generalist parasites with robust freeliving stages or free-living vectors are of greatest concern, as they could be brought in on boats or in cargo holds of planes. host-specific parasites are of less concern because their most plausible route of introduction is with the host, and even if they arrived, they are less likely to establish on a novel host. there is still concern about the introduction of parasites via migratory birds, but there is little that can be done to prevent parasite spread from migratory sources, except regular avian screening (see fig. . ). for example, there is concern about the potential arrival of plasmodium relictum, the pathogenic hemosporidian species that has contributed to the decline and extinction of many hawaiian honeycreepers. plasmodium relictum could arrive to galapagos via an infected bird, but in order for the parasite to establish, a competent vector is needed. unfortunately, the vector of this lineage in hawaii, culex quinquefasciatus, is well established in galapagos. preventing the colonization of p. relictum would require eliminating the introduced c. quinquefasciatus, which is not a simple task. both c. quinquefasciatus and native aedes taeniorhynchus mosquitoes are competent vectors for west nile virus, which has not yet colonized the archipelago. collaboration should be a top priority as more research is conducted on parasites in galapagos. research teams working in galapagos do not always interact or coordinate during their research expeditions. there are many local and international groups collecting samples in the islands and many of these samples could be used for multiple purposes, beyond the original reason for the collection. for example, blood collected for a 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wildlife management, and related fields. virus emergence requires overlap between host populations, alterations in virus genetics to permit infection of new hosts, and adaptation to novel hosts such that between‐host transmission is sustainable, all of which are the purview of the fields of ecology and evolution. a firm understanding of the ecology of viruses and how they evolve is required for understanding how and why viruses emerge. in this paper, i address the evolutionary mechanisms of virus emergence and how they relate to virus ecology. i argue that, while virus acquisition of the ability to infect new hosts is not difficult, limited evolutionary trajectories to sustained virus between‐host transmission and the combined effects of mutational meltdown, bottlenecking, demographic stochasticity, density dependence, and genetic erosion in ecological sinks limit most emergence events to dead‐end spillover infections. despite the relative rarity of pandemic emerging viruses, the potential of viruses to search evolutionary space and find means to spread epidemically and the consequences of pandemic viruses that do emerge necessitate sustained attention to virus research, surveillance, prophylaxis, and treatment. despite their limited genetic repertoires and typically specific host tropisms, viruses have a remarkable ability to infect new, often highly unrelated host types. the incidence and spread of novel viruses in host populations is termed virus emergence. in recent years, the incidence of newly identified viruses in human populations appears to be increasing, but whether this is a consequence of improved detection or more emergence events is a topic of considerable debate. [ ] [ ] [ ] nevertheless, there are some general patterns for virus emergence. emergence events typically occur when novel host and reservoir populations experience significant changes in range, demographics, aggregation and dispersal behavior, contact rates, environmental and climatic conditions, or vector distributions. in humans, there appear to be several hotspots for emergence, typically regions of high human population activity and density. these emergence hotspots include the eastern united states, western europe, japan, and southeastern australia. sources of emerging viruses tend to be phylogenetically closely related organisms. for example, zoonotic viruses of wildlife are overrepresented among the emerging human viruses. in addition, there is a tendency for emerging viruses to have rna genomes. this pattern may be a consequence of the high evolvability of rna viruses. despite these generalities, efforts to predict potential emerging viruses have not progressed as fast as hoped. mechanistically, virus emergence is a three-step process. in the first step, a virus acquires the ability to infect new host cells. the second step consists of virus adaption to the novel host such that transmission between hosts is facilitated. finally, to achieve full emergence, the virus gains the ability to spread epidemically through the host population. while the first two steps entail genetic changes in the virus, the third step may require changes on the part of the vector or host populations, such as through increased contact rates, range changes, or other doi: . /nyas. ecological or environmental shifts. virus emergence is, therefore, by definition, an ecoevolutionary process. in this review, i address our current understanding of the evolutionary ecology of virus emergence, highlight new approaches to its study, and assess the future prospects for the management of risks associated with pandemic infectious viruses. examples selected to illustrate basic concepts tend to come from medically and agriculturally important viruses, primarily because these viruses are the best studied (table ). however, it should be noted that virus emergence is a biologically universal phenomenon and occurs across all phyla. a major future challenge is to broaden our perspective of emerging viruses to include bacterial, archaeal, fungal, and invertebrate viruses. the study of virus emergence necessarily touches on a broad range of interdependent ecological and evolutionary phenomena. conceptually, i begin by addressing changes concomitant with emergence in viruses themselves, then expand to consider broader population, ecosystem, and global changes associated with virus emergence. since gaining the ability to infect a novel host and the initial adaptation to that host is an inherently evolutionary process, i begin this discussion by focusing on virus genetics and evolution. i then link evolutionary changes to ecological processes in the context of host and virus population dynamics. virus quasispecies may facilitate host range expansion viruses are among the smallest nucleic acid-based replicating entities and possess characteristics associated with exceptionally fast evolutionary change: small genomes, short generation times, high mutation rates, large population sizes, high levels of genetic diversity, and strong selection pressures. , because viruses often lack nucleic acidproofreading mechanisms, virus mutation rates are commonly on the order of - - - per nucleotide each time the genome is copied. given the small sizes of viral genomes (typically ß - kb), viruses with high mutation rates can generate genetically different progeny each time the genome is replicated. this error-prone replication can produce viral quasispecies (i.e., vast populations of closely related genotypes). , although sequence spaces are unfathomably large (the sequence space of a -kb virus genome is , ), the immense virus populations can contain many alternative variants; thus, viruses can rapidly sample sequence space and locate high fitness combinations of mutations. the quasispecies nature of viruses may facilitate virus emergence. the initial gain-of-function mutations permitting infection of novel hosts tend to be associated with host entry, namely the binding of the virus to a specific molecule on the host cell's surface. in many cases, changes in cell tropism can be accomplished through a few (or even one) nucleotide substitutions. [ ] [ ] [ ] [ ] [ ] [ ] a typical example is provided by the alphaviruses, a group of nonsegmented, positive-sense, singlestranded rna (+ssrna) viruses mainly vectored by mosquitoes. , examples of alphaviruses include eastern equine encephalitis virus, chikungunya virus (chikv), ross river virus, and sindbis virus. despite the striking architectural similarities among the different strains, these viruses can infect an exceptionally broad range of hosts, including mammals, fish, amphibians, reptiles, birds, and insects. , emergence events can result in hundreds of thousands of human infections. , the molecular basis of this broad host range stems from the malleability of the alphavirus host attachment protein, the e envelope glycoprotein, and its ability to bind the highly conserved laminin receptor. single amino acid substitutions in the e envelope glycoprotein projecting from the alphavirus capsid surface have been linked with host range expansion. many of these substitutions were shown to occur in a specific domain of e , approximately spanning amino acid residues - (part of domain b; see fig. ). [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] mutations in this region are also associated with escape from monoclonal antibody-mediated neutralization. [ ] [ ] [ ] smith et al. used cryoelectron microscopy to show that fragment antigen-binding (fab) fragments bind the outermost tip of the e glycoprotein of sindbis and ross river viruses, roughly corresponding to amino acids - of the e protein. a class iii pdz domain binding motif was identified at residues - . pdz domains are protein-interaction modules that recognize short amino acid motifs at the c-termini of target proteins. it is this region that presumably binds the laminin receptor, and changes in the amino acid sequence constituting this region likely permit binding to different laminin receptor variants. furthermore, alphavirus host shifts may be facilitated by the indispensability and lack of structural flexibility of the highly conserved laminin receptor. , the e host receptor binding site has likely been shaped by natural selection for flexibil-ity to bind different host laminin receptors, providing alphaviruses with the ability to shift hosts easily. another well-studied example of changes in binding avidity concomitant to virus host shifts comes from influenza a viruses (iav). in birds, iav host attachment hemagglutinins prefer to bind oligosaccharides that terminate with a sialic acid linked to galactose by ␣ , -linkages. by contrast, human iavs prefer sialic acids with ␣ , -linkages. as few as one or two mutations in hemagglutinin can significantly alter binding avidity to favor either human or avian sialic acids. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] several studies have suggested that subtle changes in the electrostatic properties of the virus host receptor domain and increased opportunities for hydrogen bonding with complementary domains on host sialic acids permit host range expansion. [ ] [ ] [ ] [ ] [ ] crucially, mutations permitting binding to human receptors do not preclude binding to avian receptors, and vice versa, although binding affinities are typically reduced. , , [ ] [ ] [ ] the ability to bind both human and avian receptors allows the possibility that a broad host range virus could be amplified in one host while periodically infecting the other in what is described in ecological theory as a source-sink scenario, , source-sink evolution and its relevance to viral ecology will be discussed later in this paper. the small number of mutations required to shift hosts has significant implications for virus emergence. given small viral genomes and large population sizes, the number of potential host range mutants in a sample can be surprisingly large. a -kb genome has a maximum of × potential independent single mutants, which is well below the average population size of viruses. given that a considerable proportion of these mutants are not biologically feasible owing to lethal mutations, two or three rounds of replication of a single virus can generate every possible viable mutation one step away from the parental genotype. a simple thought experiment illustrates the ease of generating host range mutants in high mutation rate viruses. a single sneeze can expel , droplets, with each droplet containing virus particles. assuming that a single nucleotide substitution a→t in a -kb genome provides the ability to infect a novel host, there could be , virus particles in that sneeze that possess mutations allowing them to infect a novel host. moreover, while most virus mutations are highly deleterious, even lethal, host range-expanding mutations often incur minimal impacts on viral fitness on native hosts, although this is by no means universal. [ ] [ ] [ ] [ ] since the effectiveness of purifying selection is proportional to the strength of selection, one consequence is that slightly deleterious mutations may persist for long periods in quasispecies swarms in primary host populations. , due to genetic drift and other stochastic processes, their frequencies in host populations could even increase. in our example, , host range mutants per sneeze could be a significant underestimate of the number of particles able to infect a novel host. it is clear that the first step of virus emergence is not the rate-limiting step. virus host shifts can also occur via large-scale genomic rearrangements among related viruses, that is, through the recombination of homologous genetic sequences, the acquisition of nonhomologous sequences (i.e., illegitimate recombination), or the reassortment of virus genome segments (fig. ) . , recombination is relatively common among the dna viruses. [ ] [ ] [ ] while recombination frequencies vary considerably, some dna viruses have extremely high rates of recombination. in one study of cauliflower mosaic virus, . % of the genomes recovered after a single passage were recombinants. in the brome mosaic virus, practically every virus is a recombinant. nonreplicative recombination uses enzyme-mediated breakage-repair mechanisms, similar to dna-based genomes. thus, the function(s) of recombination in dna viruses may be similar to the hypothesized functions of recombination in other clonal organisms. that is, recombination in dna viruses may serve to repair dna, remove mutations, break down linkage disequilibrium, and maintain genome integrity. moreover, recombination may allow viruses to capture and use host-encoded genes to manipulate hosts and increase infectivity (fig. ) . for example, the epstein-barr herpesvirus likely acquired, via recombination, a homolog of human interleukin- , called brcfi. brcfi inhibits the synthesis of antiviral cytokines and stimulates the production of b cells in which the virus replicates. in contrast to dna virus recombination, rna virus recombination mainly occurs through a different mechanism, termed copy-choice recombination, although some rna virus recombination can occur through nonreplicative mechanisms. [ ] [ ] [ ] in copy-choice recombination, an rna-dependent rna polymerase molecule switches from one template rna molecule to another during the process of genome replication (fig. ). if the two templates contain divergent genetic information, these differences can be recombined into the same rna molecule. recombination frequency varies widely among the rna viruses. this variation in recombination frequency may reflect the diverse nature of rna genomes. rna virus genomes may consist of +ssrna, negative-sense, single-stranded rna (-ssrna), or double-stranded rna (dsrna). recombination is rare in -ssrna viruses, but frequent in retroviruses, particularly in hiv, where recombination rates can exceed mutation rates. recombination frequencies in the +ssrna viruses are mixed. some groups show high recombination rates (e.g., picornaviridae), while, in others, recombination is rare or nonexistent (e.g., some flaviviridae and leviviridae). some of these differences may derive from mechanistic constraints on recombination. for example, -ssrna viruses are often bound in ribonucleoprotein complexes, which limits opportunities for hybridization between complemen-tary sequences. encapsidation of dsrna viruses in nucleocapsids during genome replication prohibits recombination. however, in other cases, the rarity of recombination may have more to do virus and host life history. for example, recombination has not been observed among the tick-borne flaviviruses. ticks usually feed once during each life history stage (as larva, nymph, and adult). therefore, the likelihood of an infected tick feeding on a host infected by a different virus strain is low. since recombination requires coinfection of the same host by multiple virus strains, the rarity of tick coinfection may explain the failure to detect recombination among the tick-borne flaviviruses. virus recombination has been shown to facilitate virus emergence. , - a recombination event may provide a host receptor gene with a new genetic background, providing the recombinant virus with the ability to gain access into a host cell without triggering host active immunity. for example, the spike glycoprotein (host receptor) gene of avian coronavirus infectious bronchitis virus, an upperrespiratory virus of chickens, was replaced by a spike gene from an unknown source. the chimeric virus gained the ability to infect and cause disease in turkeys, but as an enteric, not respiratory, virus. moreover, this new virus, now called turkey coronavirus, no longer possessed the ability to infect chickens. a good case study of recombination-facilitated emergence is provided by the geminiviruses, a large group of vector-transmitted viruses infecting plants. geminiviruses are unique in the virus world by virtue of encapsidating their ß -kb single-stranded dna genome in two incomplete icosahedra to form a twinned particle. most geminiviruses, with the exception of the bipartititegenomed begomoviruses, are monopartite. these viruses have been implicated in a number of devastating plant diseases, including maize streak, cassava mosaic, cotton leaf curl, and tomato yellow leaf curl diseases. [ ] [ ] [ ] [ ] [ ] [ ] , interspecies recombination rates among the geminiviruses are among the highest of all viruses. , , some of these recombination events have given rise to destructive new variants. for example, in , an extremely virulent form of mosaic virus appeared in uganda, destroying crops of the staple food cassava and causing food shortages and famine. the new pathogen spread from its source at a rate of - km/year. the uganda variant (eacmv-ug ), as it was called, was shown to be a recombinant of african cassava mosaic virus (acmv) and east african cassava mosaic virus (eacmv), which despite their similar names are not closely related. , specifically, the eacmv-ug strain was generated when the eacmv strain acquired a fragment of the acmv coat protein. given the role of geminivirus coat proteins in intraplant movement and between-plant vector transmission, it should not come as a surprise that the protein is linked to an emergence episode. similar recombination-mediated emergence events may have occurred in other geminiviruses, including eacmv emergence in cameroon, tomato yellow leaf curl emergence in spain and the mediterranean basin, , , cotton leaf curl virus in pakistan, , and sugarcane yellow leaf virus in hawaii. the frequency of recombination among the geminiviruses may be due to its significant role in virus biology. geminivirus genomes are replicated using recombination-dependent mechanisms. , recombination may also be a mechanism for dna mutation repair. despite this, recombination may play a role in the generation of virus genetic diversity and the facilitation of host shifts. , , , , [ ] [ ] [ ] [ ] in fact, there is considerable evidence that recombination in the geminiviruses occurs at specific locations in the genome, termed hotspots. , , , [ ] [ ] [ ] many of these hotspots tend to preserve gene integrity because the break points are found in intergenic regions. by contrast, gene regions, such as that encoding the coat protein cp, are recombination coldspots. one common recombination hotspot in geminiviruses is located at the coat protein/small intergenic region (cp/sir) interface. , , , it is plausible that the genome architecture of geminiviruses has been selected to expedite the swapping of the coat protein module among different virus strains, thus facilitating host switching. reassortment only occurs in segmented or multipartite viruses. when two or more segmented viruses infect the same cell, their replicated segments can be packaged into procapsids regardless of the parent of origin, producing viruses with a mixture of segments acquired from both parents (fig. ) . , , alternatively, such as in the multipartite viruses, each genome segment is inde-pendently packaged into a separate virus particle, but all particles are required for successful host infection. reassortment is common in some viruses, including the reoviruses (e.g., rotavirus), orthomyxoviruses (e.g., influenza virus), [ ] [ ] [ ] bunyaviruses (e.g., hantavirus), [ ] [ ] [ ] and bromoviruses (e.g., cucumber mosaic virus). , it may be that reassortment fulfills a similar role to recombination by generating genetic diversity and facilitating host shifts. perhaps the most famous case of reassortment leading to the expansion of virus host range comes from the iav h n pandemic. this virus is believed to have originated from a triplereassortment event in , where iav segments from birds, swine, and humans were combined into one h n iav, which circulated in swine. [ ] [ ] [ ] this virus contained nucleoprotein (np), matrix protein (m), and nonstructural protein (ns) segments from a swine influenza strain; hemagglutinin (h ), neuramidase (n ), and polymerase subunit (pb ) segments from the seasonal human influenza strain; and polymerase subunits (pb , pa) from an avian influenza strain. subsequently, this virus reassorted with and acquired hemagglutinin (h ) and neuramidase (n ) segments from classical swine influenza. , , in , this h n swine strain reassorted with an avian-derived h n swine strain from europe, acquired the m and n segments, and subsequently shifted hosts and emerged in human populations (fig. ). by the time the pandemic subsided, the world health organization reported , laboratory-confirmed deaths, but studies suggest the total death toll was -fold higher. adaptive landscapes, pleiotropy, epistasis, and emergence a virus can be said to occupy a position on an adaptive landscape corresponding to its host. an adaptive landscape is defined as a network of genotypes connected by mutational paths. , this network of genotypes is visualized as a three-dimensional topology of peaks and valleys corresponding to the fitness values of the associated genotypes. the relevance of adaptive landscapes for virus emergence stems from the fact that most emergence events require a virus to significantly increase its fitness on the novel host such that between-host transmission can be sustained. when a virus emerges in a new host, it transitions to a new adaptive landscape. unlike the landscape corresponding to its previous host, it is exceedingly unlikely that the emerging virus is preadapted to the new host; thus, the virus would need to ascend a new fitness peak in order to fully emerge in the novel host. an example of the challenges a virus faces when emerging on a novel host comes from the bacteriophage . one or two mutations allow the bacteriophage to infect a novel host, pseudomonas pseudoalcaligenes. however, the phage's absolute fitness on the new host is approximately an order of magnitude lower than its fitness on the original host (fig. ) . , moreover, each mutation incurred up to a % reduction in absolute fitness in the original host, pseudomonas phaseolicola (fig. ) . in order to increase their fitness on p. pseudoalcaligenes to a level comparable to the original host, these mutants need to navigate a new adaptive landscape of unpredictable topology before going extinct. when a virus mutates to a genotype that allows infection of a novel host, the exact topology of the adaptive landscape on the new host will depend on the fitness values of genotypes adjacent to this new position. in order to ascend a fitness-peak host from its current position on the landscape, the mutant virus needs to navigate an adaptive trajectory that passes through these intervening genotypes. several adaptive landscape characteristics can affect a virus's ability to ascend a new fitness peak, including the genetic distance from the present location to the genotype representing the new fitness peak, any interactions between mutations along this adaptive trajectory, and the ruggedness of the adaptive landscape. genetic distance refers to the number of mutations separating any two genotypes on an adaptive landscape. there are at least two factors limiting the likelihood of traversing greater distances in adaptive trajectories. first, since the probabilities of acquiring specific mutations are additive, the greater the genetic distance between two genotypes, the lower the probability that the virus can navigate from one to the other. assuming no recombination and equal probabilities of mutations across all loci, the probability (p) of acquiring multiple mutations in the same lineage is (u) n , where u is the per generation mutation rate and n is the number of mutations that need to be acquired. therefore, if a virus with a mutation rate of - needs to acquire five mutations in order to fully emerge on a novel host, then the probability of acquiring all five mutations in the same lineage is - . by comparison, the probability of a coin coming up heads in consecutive coin flips is ß × - . this example clearly illustrates that these five mutations would need to be acquired sequentially rather than simultaneously, which brings about a second problem. if mutations are acquired sequentially, many mutational trajectories may not be accessible. while the five mutations may have high fitness together, other intermediate combinations would not necessarily have higher fitness than the starting genotype. in fact, they could have lower fitness than the starting genotype. in this situation, a virus would need to cross a fitness valley or find an alternative path to reach the fitness peak. what this means is that mutations may have to be acquired in a specific order, which further reduces the probability of traversing the intervening genetic distance. experiments exploring mutational trajectories have seldom been performed, but one example comes from the acquisition of ␤-lactamase resistance in escherichia coli. five point mutations provide to e. coli a high degree of ␤-lactamase resistance. in principle, there are five or possible mutational trajectories. weinreich et al. showed that of trajectories were inaccessible to natural selection, and many of the remaining were unlikely to occur. thus, it was demonstrated that the acquisition of ␤lactamase resistance in e. coli could only occur via a limited number of mutational paths. the specific order in which mutations need to be acquired is a function of epistasis and pleiotropy. pleiotropy refers to situations where mutations in a single gene affect multiple traits. a mutation that increases fitness in one context may simultaneously reduce fitness in a different context, a phenomenon termed antagonistic pleiotropy. an example of antagonistic pleiotropy was introduced earlier in this paper when i described how bacteriophage mutations permitting infection of a novel host decreased fitness in the original host (see also fig. ) . while the prevalence and magnitude of antagonistic pleiotropy in viruses have been largely unexplored, antagonistic pleiotropy may make some mutational trajectories nonviable because mutations along that trajectory may severely reduce fitness in some contexts. the compactness, multifunctionality, and lack of redundancy characteristic of virus genomes is expected to make antagonistic pleiotropy a frequent consequence of mutation. high frequencies of antagonistic pleiotropic effects among viral mutations will limit adaptive evolution in viruses infecting novel hosts. epistasis is the phenomenon where the fitness effects of a mutation depend on the genetic background in which it occurs. sign epistasis refers to a specific class of epistatic interactions where the fitness effect of a mutation is beneficial in some backgrounds but deleterious in others. reciprocal sign epistasis occurs when mutations are individually deleterious, but beneficial when they appear together. in the absence of sign epistasis, the adaptive landscape is smooth, fitness effects are additive, and mutations can be incorporated in any order. in the presence of sign epistasis, adaptive trajectories are constrained, and fewer paths to increased fitness are available. as reciprocal sign epistasis increases, the adaptive landscape becomes more rugged, and it becomes increasingly difficult to ascend the highest fitness peak without being trapped on local maxima. experimental studies have shown that epistasis is pervasive among viruses. [ ] [ ] [ ] [ ] the prevalence of epistasis, particularly reciprocal sign epistasis, restricts virus adaptive evolution by limiting accessible evolutionary trajectories and by making it more difficult for viruses to ascend global fitness maxima. an example of the limiting effects of epistasis comes from chikv, which exploits the vector aedes aegypti to facilitate its own transmission. a single nucleotide substitution (e -a v) allows chikv to infect aedes albopictus, but despite the high abundance of ae. albopictus in southeast asia, chikv has never become established in this vector in this region. the failure of the asian chikv lineage to exploit ae. albopictus as a vector can be attributed to a negative epistatic interaction between the e -a v mutation and a threonine residue at position e - . another chikv lineage, the east/central/south african genotype (ecsa), has alanine at position e - , which has no negative epistatic interaction with e - v. in fact, the e -t a substitution generated a nearly -fold increase in infectivity of ae. albopictus by e - v. thus, this ecsa lineage was able to acquire the a v mutation, adapt to ae. albopictus, and invade the southeast asia niche. since ae. albopictus has a broader range than ae. aegypti in temperate zones, this lineage switch could lead to the expansion of chikv into new regions. together, epistasis, pleiotropy, and genetic distance may help explain why, despite the ease of acquiring host range-expanding mutations, pandemic emerging viruses are relatively rare. these features of adaptive landscapes may explain why some viruses, such as severe acute respiratory syndrome virus (sars), fail to emerge in novel host populations. in , an outbreak of sars in guangdong, china eventually infected thousands of people in southeast asia and north america. however, relatively low fitness of sars (basic reproductive number, r = . ), a high degree of host population heterogeneity in transmission potential (k), and the rapid mobilization of containment procedures led to the eradication of the outbreak by late . the implication here is that sars was unable to effectively navigate the adaptive trajectory required to increase its basic reproductive number and cause a wider outbreak. rugged adaptive landscapes associated with host switches may make host adaptation too difficult to achieve in the light of the other ecological and environmental constraints on virus reproduction and transmission. the ability of organisms to navigate adaptive trajectories is referred to as evolvability. evolvability is defined as the capacity of a population to increase its fitness over time in response to changes in the environment. , components of evolvability include high mutation rates, high levels of standing genetic variation, large population sizes, facility for genetic rearrangements, modular genomes, and short generation times, all of which are characteristic of viruses. , since evolvability can be expressed as an organism's speed in responding to environmental change or, more formally, an organism's nonsynonymous substitution rate, viruses are exceptionally evolvable. virus nucleotide substitution rates estimated through phylogenetic analyses typically range between - and - substitutions per site per year, , although there is considerable variation in substitution rates. viruses and their genomes show many signs of having been selected for increased evolvability, and this may be a consequence of the ecological demands placed on viruses to change rapidly. high mutation rates and rapid adaptation were cited as the primary factors driving the emergence of parvovirus in canines. in hiv, the envelope (env) gene fixed adaptive mutations, on average, every . months or every viral generations, which was the fastest adaptive rate ever recorded. this exceptionally fast rate of evolution is testament not only to hiv's evolvability, which facilitates evolutionary change, but also to the strength of selection imposed by the human immune system, which necessitates evolutionary change. the capacity for rapid evolution makes viruses especially treacherous, as they can quickly escape host immunity, host resistance, and antiviral treatment and can rapidly adapt to increase their fitness on novel hosts. this feature of virus evolution is especially prominent in within-host hiv evolution, where virus phylogenies show a ladder-like structure over time. here, successive waves of virus genotypes expand and contract in response to host-imposed diversifying selection, particularly in the envelope (env) gene. these phylogenies dramatically contrast with between-host hiv evolution, which show more branched distributions as different lineages survive and differentiate over time. high viral mutation rates, however, may be a double-edged sword. while facilitating the acquisition of beneficial mutations, the high frequency of deleterious mutations may "sandbag" virus populations and slow adaptive evolution. , the effects of mutation load are more pronounced in small populations or when populations pass through bottlenecks. , by nature, viruses experience severe bottlenecks during between-host transmission. only a tiny fraction of a virus population's within-host growth is expected to successfully encounter a new host. these bottlenecks can result in a loss of genetic diversity, mutation accumulation, and fitness losses. for example, in an evolutionary study of the potyvirus tobacco etch virus, strong bottlenecks imposed on serially passaged virus populations produced fitness losses of ß % per passage. since the number of viruses transmitted between hosts is proportional to its within-host growth, viruses that grow poorly within a host have a lower probability of being transmitted. this point is especially pertinent for emerging viruses, because they are expected to be poorly adapted to novel hosts. because emerging viruses are often poorly adapted, within-host replication is reduced, and, correspondingly, the number of viruses released from the host is also reduced. this has two consequences. first, the number of viruses in transmission vehicles (e.g., sneeze droplets, fomites, etc.) is decreased, reducing the chance that they will be picked up by another host. second, if a new host is encountered, the number of particles entering the host is also decreased, reducing the probability that the virus can establish itself in that host without being eliminated by the immune system, inactivated, or washed out of the organism. in fact, these effects are likely compounded during each transmission event in a vicious cycle, and likely give rise to stuttering transmission chains that eventually go extinct. , in this sense, the population dynamics of preemerged viruses can be described by the source-sink paradigm of ecology, which will be discussed in greater detail later in this paper. an enduring paradox in evolutionary biology is the harm done to hosts by the parasites that depend on them for survival. the impairment and/or death of a host would appear to be disadvantageous to the parasite that depends on it for growth and transmission. early theory suggesting that viruses evolve to a benign coexistence with hosts has matured into theory emphasizing the benefits and costs of virulence (trade-offs) for virus transmission. in brief, every virus has an optimal virulence that maximizes between-host transmission. increases or decreases in virulence will evolve insofar as they also increase virus transmission. with respect to virus emergence, the relevance of virulence evolution largely stems from the supposed mismatch between virus virulence in a novel host and its optimal virulence in that host. since an emerging virus has, by definition, not adapted to its host, it is unlikely that its virulence in that host is optimal. we can speculate that many virus spillover events do not progress to full-blown emergence because of less-than-optimal virus virulence. for example, ebola virus (ebov) is remarkable for its extreme virulence in humans. mortality rates due to ebov are approximately %. given that ebov is spread through direct contact with infected individuals, ebov's excessive virulence curtails the duration of infectivity and person-to-person contact rates, thus reducing the transmission potential of the virus. this reduction in transmission may be the reason why ebov outbreaks are, so far, spatially limited to certain regions of africa. however, ebov outbreaks have been trending upward in both numbers of individuals infected and area affected. the most recent outbreak in west africa resulted in , cases of infection and , deaths across three nations, which exceeds the totals for all previous ebov outbreaks combined. reports examining ebov evolution during the latest outbreak caution that the virus might eventually evolve greater transmission and fully emerge in human populations. , excessive virulence is not the only reason why a virus may fail to emerge. an insufficiently virulent virus may suffer the same fate because within-host growth is not high enough to effect transmission to a new host. in addition, the weakly virulent virus may lose in direct competition for resources with a more virulent genotype. an example of the competitive replacement of a weakly virulent virus by a more virulent genotype comes from the dengue virus serotype (denv- ). before , denv- was endemic to the indian subcontinent, but caused mild or no disease in humans. after , the virulence of denv- changed and caused outbreaks of dengue hemorrhagic fever, a severe, often fatal form of the disease. messer et al. showed that the sudden onset of dengue hemorrhagic fever was caused by the competitive displacement of group a denv- strain by group b denv- strain in the indian subcontinent. subsequent work showed that the group b denv strain infects and disseminates in ae. aegypti more efficiently than the group a denv strain, suggesting that the invasive strain is transmitted more effectively. in a source-sink scenario, a primary host population (the source) contributes virus "migrants" to a secondary host population (the sink), where the virus is poorly adapted. , , by definition, populations existing in sinks have a negative population growth rate; thus, they are expected to go extinct in the absence of migration. the influx of migrants can provide genetic diversity and contribute to densitydependent growth in the sink population, allowing the acquisition and fixation of mutations increasing fitness in the secondary host. eventually, fitness gains are such that the virus is able to escape the sink by achieving positive population growth, even in the absence of migration. , the difficulties encountered in the evolutionary transition required to turn an ecological sink into a source are probably the strongest constraining factors in virus emergence. in a sink, a virus needs to increase in fitness fast enough to counteract the relentless downward pressure imposed by negative population growth. the synergistic effects of genetic drift and erosion, positive densitydependent growth, and mutational and migrational meltdown all act to limit the fixation of beneficial alleles. , [ ] [ ] [ ] moreover, sink populations are more susceptible to chance fluctuations and demographic stochasticity, , leading to broken transmission chains and virus extinction. ultimately, these factors may help explain the discrepancy between the relative ease with which host rangeexpanding mutations are acquired and the relative lack of pandemically spreading emerging infectious diseases infecting humans. one means of evading these difficulties is the possibility that viruses can migrate from sink hosts to source hosts for further amplification and spread. to use iavs as an example, imagine a mutant iav in waterfowl containing the ability to bind both avian and human sialic acids. despite being competitively inferior to other iav genotypes, this mutant does not necessarily go extinct in waterfowl. in fact, it is common for multiple competing isogenic variants of similar relative fitnesses to circulate in an adapting asexual population. , this mutant iav may epidemically spread through a waterfowl population with occasional spillover into human populations. the mutant virus may initially be a poor fit in humans but able to replicate in immunocompromised individuals, for example. while human-tohuman transmission is not observed at this point, the high affinity for avian receptors may lead to human-to-waterfowl transmission of iav genotypes that have increased infective/replicative ability in humans. in this manner, a broad host range iav is amplified by repeated transmission between humans and waterfowl until sustained transmission between humans is achieved. though it is recognized that host populations must overlap in order for virus emergence to take place, virus host alternation has seldom been explored outside of a few laboratory and theoretical studies. however, these studies show that, contrary to the prevailing perception, natural selection can simultaneously improve virus fitness on multiple different hosts. [ ] [ ] [ ] [ ] in the vernacular of evolutionary ecology, there seem to be few barriers to evolving to be a host generalist, and viruses are not necessarily constrained to specialize on certain hosts. the broader issue of parasite generalismspecialism has a long and complicated history. , commonly, it was believed that adaptation to a specific host necessitated a reduction in fitness in other hosts due to between-host trade-offs (i.e., antagonistic pleiotropy). such between-host trade-offs can and do occur, , but the frequency of observing no trade-offs has caused the question to be inverted to ask why broad host range viruses are not more common. , it may be that, while adaptation to multiple hosts is possible, ecological circumstances usually promote virus adaptation to a single host type. in other words, host specialization is caused by the absence of selection in favor of maintaining host generalism. if there are no alternative hosts for a virus in a particular habitat, then the virus will adapt to the currently available host (i.e., specialize). during the process of adaptation to a single host, mutations are fixed that are either beneficial or neutral to the present host but are deleterious to the alternate host. once host specialization has begun, linkage between loci affecting host preference and performance on that host may make it increasingly difficult to reverse direction and reevolve broad host ranges. by contrast, if multiple hosts are available, then a virus may adapt to both hosts simultaneously in the absence of fitness-reducing trade-offs. it is likely that, in most emergence situations, antagonistic pleiotropy is not a factor, and alternating exposure to multiple hosts can lead to increased virus fitness on both hosts. when host ecology changes, it may result in increased contact between primary and secondary host species, providing opportunities for multihost evolution. in humans, relevant ecological changes include altered local demographics, changing travel or immigration patterns, intensified agricultural practices, changing land-use patterns and encroachment into wildlife habitat, climate change in human occupied areas, and range expansion of virus vectors or reservoirs. the reciprocal transmission of viruses following increased interspecies contact rates may select for virus variants with increased fitness in one or both species. an interesting and open question is how the relative sizes and dynamics of the two host populations affect emergence probabilities. host utilization, host range, virus life history, transmission routes, and kindred subjects are all aspects of virus ecology; that is, interactions between viruses and the biotic and abiotic environment. environmental heterogeneity across space and time affects the incidence of disease in vectors and reservoir hosts and can lead to emergence events. an illustrative example comes from outbreaks of an arbovirus-bluetongue virus (btv)-among ruminants in europe. in its native range, btv is vectored by culicoides imicola midges. in recent years, a sub-saharan strain of the virus expanded northward from africa, first to southern and eastern europe and then into northern and western europe, leading to widespread mortality among livestock. changes in climate have been implicated as a major driver in the expansion of btv's range. the most compelling evidence supporting this hypothesis is the observation of btv and c. imicola in regions that have warmed the most since the s, but not in regions that stayed cool during this period. the northward shift in btv's range likely stemmed from the expansion of the african-asian vector c. imicola northward, where it came into contact with indigenous european culicoides species. , , btv then switched vectors from c. imicola midges to c. obsoletus and c. pulicaris midges adapted to the cooler, wetter climes of northern europe. , the overlapping culicoides populations may have allowed btv to increase its infection competence in the latter vectors. environmental variables of elevation, temperature, and rainfall had the biggest impact on btv spread because they affected vector abundance and host availability. , temperature, in particular, affected the magnitude of btv's basic reproductive number (r ), a measure of the transmission potential of a disease. r was maximal when temperatures ranged from to °c, but declined outside that range. the connection likely stems from the fact that culicoides midges show increased activity, reproduction, and feeding behavior at higher temperatures. , in addition, milder winter temperatures promote midge overwinter survival and are crucial for the maintenance of btv presence in any particular region. in the spring of , an unknown pathogen infecting humans appeared in the southwestern united states, resulting in death in more than % of cases. the causative agent was soon identified as a previously unknown hantavirus (bunyaviridae), subsequently named sin nombre virus (snv). snv's reservoir host was subsequently identified as the deer mouse peromyscus maniculatus, a common inhabitant of the deserts and dwellings of the southwest. peromyscus population densities were shown to have increased threefold to -fold between and . the peromyscus population surge was linked to the el niño-southern oscillation event of , which resulted in increased temperatures and precipitation in the region during the fall, winter, and spring of - . the outcome was a trophic cascade. the increased precipitation recharged the thirsty soil and increased primary production. more plants led to increased rodent forage, such as seeds, berries, nuts, and insects. improved forage augmented rodent survival and reproductive output. greater rodent densities led to increased contact rates among rodents and pushed them to their range margins, such as human dwellings. the higher rodent densities likely facilitated increased snv infection rates, triggering spillover infections in consanguineous human populations. rodent populations in the area subsequently crashed, reaching a nadir during the la niña event (a dry and cold period) in the spring of . human snv infections diminished as well. in - , another el niño episode appeared, and rodent populations resurged. however, fewer human snv infections were reported in than in . curiously, human cases doubled the following year in . while rodent densities in were lower than in , the density of snvinfected rodents in was an order of magnitude higher than at any other time during this study. this pattern was repeated in subsequent el niño events. the precise causes of this time-lagged infection prevalence are unclear, but it is hypothesized that prevalence of infection in peromyscus populations is proportional to the population densities reached during the previous reproductive season. the greatest snv risk to humans, therefore, occurs - years following the increased precipitation event. however, strictly speaking, snv has not yet fully emerged according to the three-step process established earlier in this paper. sustained human-to-human transmission has not yet been observed. in fact, person-to-person transmission of hantaviruses has been documented only for the andes virus. given that snv is mainly transmitted to humans by exposure to rodent urine and feces, human sanitary habits may reduce the opportunities for human-to-human transmission of snv. while the specific mechanism of andes virus human-to-human transmission has not been established, some evidence suggests that close exposure among sex partners and family members may be responsible. it stands to reason that hantaviruses may be optimized for indirect transmission but can gain the ability to be spread by bodily contact, as the andes virus may have done. if a hantavirus, such as snv, gains the ability to spread via direct human contact, the prospects for emergence are troublesome. the specific obstacles to hantavirus human-to-human transmission urgently need to be determined in order to assess the likelihood that hantaviruses can eventually fully emerge in human populations. the greater lesson from these studies is that climatic disruption alters species ranges and demographics and can have unforeseen consequences for virus dynamics. it is plausible that other viruses that episodically appear in human populations, (e.g., ebov) may be driven by as-yet unknown environmental and ecological changes, but many virus outbreaks are not well documented, especially in underdeveloped countries. particularly worrisome is the encroachment of tropical climates in typically temperate areas, along with the spread of vectors and reservoirs that are climate limited in their distributions. animal and human populations in temperate regions are fertile ground for virus emergence and spread. virus emergence poses tremendous challenges to human society, and ecological and environmental trends suggest that the problem will get worse. urbanization, globalization, encroachment of wildlife habitat, intensified agricultural practices, and climate-driven range shifts will tend to increase the incidence and spread of infectious viruses. exhortations in favor of more stringent epidemiological surveillance, improved predictive modeling, enhanced diagnostic tools, increased vaccination, and better public health infrastructure are common. while these approaches are valuable, their long-term economic and logistic feasibility as primary means to interdict emerging viruses is questionable. there are simply too many potential emerging viruses, and containment measures are too porous. more than human pathogenic viruses have been described. if similar numbers exist for each mammalian species, we can liberally estimate roughly , viruses among the ß known mammals. this figure suggests that enormous numbers of viruses are at least superficially similar enough to infect our phylogenetic relatives, and thus are likely suspects for potential emergence events. add the viruses of other animals, and potential pathogens may number in the millions. simply identifying these viruses would cost billions. actively tracking their frequencies and evolutionary trajectories is a daunting, perhaps impossible task. if surveillance of the viruses of economically important animals and plants are included, it is clear from these rough numbers that wholesale virus surveillance to prevent new viruses from entering new host populations will never be feasible. while a more limited triage approach may be possible, the ability of viruses to escape interdiction and spread globally (e.g., iav h n ) does not inspire much confidence that this approach will generally work. moreover, a surveillance approach does not account for laboratory biocontainment failures or even the possibility of deliberately engineered and released pathogenic viruses. the ease of dna synthesis, genome sequencing and editing, and cell culture have frightening implications in the hands of state-sponsored actors or terrorist organizations. can we rule out that a dedicated research effort with sufficient funds and expertise could produce a highly virulent and transmissible synthetic virus? one priority should be the development of broad-spectrum antiviral drugs. while the "one bug-one drug" approach has many successes, it is not well suited to countering new emerging viruses, as it suffers from a "closing the barn door after horse has bolted" problem because of the time required to develop and implement a new treatment regime. broad-spectrum antivirals will allow rapid treatment of newly infected individuals before the pathogenic agent is identified and epidemiologically characterized. the highly conserved nature of many virus structures and functions may aid the discovery of broad-spectrum antiviral agents. broad-spectrum drugs targeting shared virus mechanisms, such as membrane fusion, capsid assembly, receptor binding, metal nanoparticles, transcription, and translation have already been developed. from an evolutionary perspective, antiviral drug resistance will always be an issue, but they can be the knockout punch that interdicts an emerging virus before it goes pandemic. another promising approach is to find improved means of training immune systems to broadly recognize and defend against pathogenic viruses. leblanc et al. developed the vaxcelerate platform to generate self-assembling vaccines against specific pathogen targets in less than days. other efforts rely on computational and bioinformatic tools to identify highly conserved segments of antigenic genes or proteins and their binding epitopes (e.g., conservatrix, epimatrix). well-developed platforms can be modified for speedy use against novel viruses. since the majority of emerging viruses are arthropod-borne, special attention should be paid to developing gene-driven mechanisms to eliminate the most common vectors of emerging pathogens. the development of crispr-cas genome editing techniques has permitted the introduction of gene-drive mechanisms into insect vectors that disrupt reproduction. for example, hammond et al. identified, in the malaria vector anopheles gambiae, three genes that, when disrupted, confer recessive female sterility. crispr-cas gene-drive constructs designed to target and edit each gene were introduced into a. gambiae. the drive was transmitted to offspring at rates ranging from . % to . %. the drive successfully induced infertility in homozygous mutant females, while males and heterozygous females showed normal fertility, which is necessary for the drive system to spread within the population (fig. ) . while it is unlikely that gene drives can result in vector extinction because of the evolution of gene-drive resistance, such gene drives could suppress vector populations below levels required for sustained virus transmission, breaking epidemics and preventing further virus dissemination. finally, education should 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fate of wild populations a crispr-cas gene drive system targeting female reproduction in the malaria mosquito vector anopheles gambiae key: cord- - j euvc authors: williams, ernest h.; bunkley-williams, lucy title: life cycle and life history strategies of parasitic crustacea date: - - journal: parasitic crustacea doi: . / - - - - _ sha: doc_id: cord_uid: j euvc different parasitic life strategies are described including four new life cycles: complex rebrooding, micro-male, mesoparasite and prey-predator transfer. four new life cycle behaviours are named: nursery hiding, mid-moult stage, positive precursor (intraspecific antagonism) and negative precursor (ambush strategy). further strategies discussed are opossum attack, double parasitism (doubling of the normal reproductive set), duplex arrangement (separated male-female pairs), simple rebrooding, and describing how displaced parasites and superinfections may partly elucidate life cycles. proportional stunting masks life history effects of parasitism; cuckoo copepods are true parasites and not just associates; burrowing barnacles (acrothoracicans) are not parasites. further findings based on life cycle information: branchiurans and pentastomes are possibly not related; firefly seed shrimp are not parasites; copepod pre-adult life cycle stages are common in the western pacific but rare in caribbean; harpacticoids on vertebrates are not parasites; cuckoo copepods are true parasites; explained the importance of pennellid intermediate hosts. crustacean parasite life cycles are largely unknown ( % of species). most crustacean life cycles represent minor modifications from the ancestral free-living mode. crustacean parasites have less complex and less modified life cycles than other major parasite groups. this limits their exploitation of, and effectiveness, in parasitism. however, these life cycles will be an advantage in global change. most metazoan parasites will be eliminated while crustaceans (and nematodes) will inherit the new world of parasites. life cycles and life histories are arguably the most significant functional traits of all organisms (e.g. stearns ; roff ; mcgill et al. ) , and understanding those life cycles and life histories is essential to understanding the autecology and evolution of each organism, as well as its impact on community and ecosystem processes (e.g. heppell et al. ; mcgill et al. ) . parasitism is the most common consumer strategy, and parasites are estimated to account for approximately half of all biodiversity (reviewed by hatcher and dunn ) . carlson et al. ( and references) considered that conservation of parasites is essential to maintain the diversity in major ecosystems, particularly during global climate change. because parasites are typically small and cryptic and often infect multiple hosts and/or host species during their life cycle, unravelling the life cycles of even a small percentage of parasite species has proven to be challenging. indeed, while life cycles and life histories have been extensively investigated for a small number of model species (mostly species that impact the health of humans and/or plants and animals of economic importance to humans), we know virtually nothing about the vast majority of species. the phylum arthropoda is the most diverse animal phylum, with more than , , described species. while insects are the most diverse class within this phylum, the class crustacea has more than , described species (zhang ) . the crustacea also includes the greatest diversity of parasitic forms, with over one quarter of the described species. as with parasites generally, details of the life histories for most of these remain unknown. the goal of this chapter is to provide an overview of the current state of knowledge regarding life cycles of parasitic crustacea and offer insights and suggestions for future research; our suggestions and analyses are based on the authors' combined years of experience working with this group. we consider each parasite group separately in phylogenetic order, giving a brief summary of the life cycle(s) with recent discoveries and new details regarding life history strategies and concluding with some new information. we only present a brief overview of the impacts on hosts and on broader ecological aspects as these are reviewed in chaps. and , respectively. general larval descriptions (unless new) have been kept to a minimum because martin et al. ( ) presented an extensive description and collection of drawings and photographs of larval forms. the classification given in chaps. and has been followed. to improve readability and referencing, an annotated glossary has been included with less familiar terms in italics in the text. common names, with accompanying scientific names, are at the end (sect. . ). this is a small group of~ (worms ) species in four genera that largely ectoparasitise freshwater fishes (poly ) , with a few taxa on coastal marine fishes (< ; møller ) , and some attach to tadpoles, salamanders, and even alligators (møller ). most non-fish infections appear to be incidental or accidental, but poly ( ) described a species from a mexican salamander. because of their temporary association with hosts, they may more appropriately be referred to as 'micropredators' (e.g. lafferty and kuris ) ; however, we feel that serial parasites * is more appropriate. much of the interest in this group stems from their negative effects on aquaculture and aquarium fishes (lafferty et al. ) and vector fish viral diseases (møller ) . the japanese fish louse, argulus japonicus thiele, , is probably the most famous and has been spread around the world with cyprinids (bunkley- williams and williams ) . however, the common fish louse, argulus foliaceus (linnaeus, ), is also widespread, and occurs from fresh to marine waters (møller ) . in two cases, these parasites have infected humans. hargis ( ) found argulus laticauda smith, , in the eye orbit of a child in virginia; and an argulid has also been observed in the orbit of a tilapia aquaculturalist in venezuela (williams and bunkley-williams, personal observation) . the life cycles of only~ branchiuran species have been examined, mostly in argulus müller, , and a few species of dolops audouin, . most males transfer sperm directly to the females using a variety of modified structures on the third and fourth thoracic legs; however, in dolops, sperm are transferred in chitinous spermatophores. sperm morphology originally linked fish lice with tongue worms (see sect. . ) . molecular evidence also supports their similarities. only the life cycle of argulus is well known (poly ; neethling and avenant-oldewage ) , and no life cycles of marine species are known. a mature female argulus leaves its host and lays eggs in rows on a hard, submerged surface ( fig. . ). as many as eggs are laid at any one time and are cemented to the substrate. the eggs hatch - days later, varying by species and water temperature. eggs hatch into ( ) free-swimming metanauplius-like larvae ( species of argulus), ( ) freeswimming juvenile-like larvae ( argulus, dolops) or ( ) non-swimming larvae ( chonopeltis thiele, ). the three larval stages (above) moult into secondstage juveniles, which are parasitic and repeatedly change hosts (serial parasite). the - stages before the adult are also parasitic and host-hopping*. the maxillule undergoes a profound metamorphosis around the fifth stage, changing from a long limb bearing a powerful distal claw, into a short but powerful circular sucker (martin et al. ). this is a remarkable transformation. møller et al. ( ) described swimming and self-cleaning in the hatching, free-swimming stage and the subsequent juvenile stages of argulus foliaceus. wilson ( ) and reproduced with permission from benz and bullard ( ) . mature adults copulate on or off the host, and gravid females will then swim and find hard inanimate objects (such as stones, walls, equipment, etc.) on which to deposit eggs. hatching times vary according to temperatures and can take from weeks to months to occur. once hatched, infective larvae will search for a host, attach to the suitable host, and undergo multiple moults before reaching maturation. image modified from benz and bullard ( ) neither larvae, nor juveniles, nor adult chonopeltis can swim and are therefore referred to here as 'non-swimming fish lice*'. adults leave the host, mate, and deposit eggs, and how, or if, they return to a host is unknown. they have seven to eight free-living, developmental stages and are said to have intermediate hosts, but, as in other fish lice, these are really transfer hosts*. all host infection and reinfection are through host contact with the bottom. intermediate hosts are small, bottomdwelling fishes, and the definitive hosts are larger, bottom-dwelling fishes (grundlingh ) . how they infect or attach to hosts is still unknown, possibly only floating to a new host (fryer ; piasecki and avenant-oldewage ) . few parasite embryos have ever had mutualistic symbionts, but banerjee et al. ( ) have found one. a rotifer, philodina roseola ehrenberg, , feeds off the jelly coat of the egg strips of argulus bengalensis ramakrishna, . this makes the coating thin enough for larvae to break out. if the rotifer does not feed, the larvae cannot emerge. they suggested this knowledge of the life cycle could afford parasite control. van as and van as ( ) found adult and larvae chonopeltis lisikili van as & van as, , on the same host specimens and suggested host-change does not occur in c. lisikili, as was reported for other chonopeltis spp. these stages, and freeswimming adults, are attracted by bright objects, light, and motion, as mikheev et al. ( ) demonstrated in aquarium studies. in daylight, the parasite employed hoverand-wait tactics with low swimming speed and an inclined position of the body. in the dark, cruising tactics were employed, characterised by a much higher swimming speed and a horizontal position of the body. vision, olfaction and mechanoreception are used in daylight, whereas only the latter two are used at night. swimming speed was - times greater at night than in the daylight. host-induced cues increased mean swimming speed by a factor of . - . in adults starved for - days, the swimming speed was - times greater than those freshly detached from the host. a longer starvation caused a decrease in swimming activity (mikheev et al. ) . mikheev et al. ( ) found receptive females largely rested on hosts and attracted free-swimming males with pheromones. they also found females deposit eggs in fish spawning or nursery areas, where their offspring will have access to many fish. mikheev et al. ( ) suggested fish lice manipulate the behaviour of hosts for their benefit: ( ) when one attaches to a fish, the host reaction attracts other lice; ( ) injuring a host by attacking it may cause a predator-attack tightening of a school, favouring more parasite attachment. however, these effects seem too inadvertent, reactive, and temporal, to be called parasite-induced host behavioural changes. they do not help the individual parasite causing the reaction, possibly even harming it, similar to a positive precursor. they are certainly nothing like the host behaviour changes such as found in parasitic barnacles. mikheev et al. ( ) also suggested microbial pathogens were changing the behaviour of hosts and fish lice to spread their infection. they found 'sick' fish had more fish lice and stated that the parasites vector these diseases (see chap. ). banerjee's et al. ( ) mutualistic rotifer has obviously co-evolved with argulus bengalensis. we suggest that at one point, the rotifer appears to have been a hyperparasite (many in its genus are parasites) or predator harming the egg strip. the parasite gradually thickened the coating on its egg strip, protecting it from the rotifer. finally, the embryos were sufficiently protected, to turn the rotifer into a mutualist, and the organisms became inexorably linked. our suggestion is that this is first evidence of a hyperparasite evolving into a mutualist. while fish lice metamorphosis of suckers is an interesting change from the freeliving forms, they show no other major morphological developments towards parasitism. thus, while unusual, their larvae are strange, but not necessarily modified well for parasitism. there are~ extant species mostly parasitising the respiratory tracks of terrestrial vertebrates (christoffersen and de assis ; siveter et al. ) . they are of little commercial importance even though they may be found on crocodile and alligator farms, and the eggs of ten species can infect humans with nymphs (li et al. ). their phylogenetic relationship was once mysterious because they only have reduced parasitic morphologies. until recently, no fossil forms (~ . billion years ago) were known. molecular work has suggested a relation to fish lice (e.g. li et al. and references) , although this is still debated. williams ( ) suggested that these parasites were important and were once parasites of dinosaurs since the remaining species parasitise many extant close relatives of dinosaurs (e.g. crocodilians and birds). bunkley- williams and williams ( ) found sebekid nymphs in freshwater largemouth bass and peacock bass in puerto rico and speculated that spectacled caiman was the final host (williams and britton ) . we now identify these nymphs as sebekia oxycephalum (diesing, ) , and they are common in puerto rico (williams and bunkley-williams unpublished data) . reported nymphs in coral reef fishes in okinawa and suggested the final hosts were sea snakes. males fertilize females soon after they mature in the respiratory tract. males do not live long, and often only females are found in the definitive host. stored sperm fertilize ova released continuously from the ovaries of mature females. fertilized eggs mature as they descend the uterus of porocephalids. gravid females of armillifer sambon, , and linguatula frölich, , species may contain millions of eggs. the eggs of cephalobaenids are stored in a saccate uterus until they contain - % fully mature primary larva and are infectious; then egg deposition begins. the vagina is equipped with a sieve-like mechanism only allowing mature eggs to escape. they lay eggs in the respiratory track of vertebrates, which are either coughed or sneezed out by the host or leave the host body through the digestive system. usually, an insect or vertebrate ingests the eggs. the larva hatches into a nymph, penetrates the intestinal wall, and forms a cyst in the intermediate host's body. the nymph is rounded in form, with - short legs. the final host is infected when it eats the intermediate host, and the nymph crawls into the respiratory tract from the oesoph agus or stomach. it moults several times to become a post-larval juvenile and finally an adult. a few species, mostly in birds, have direct life cycles. subtriquetra subtriquetra (diesing, ) , in south american crocodiles, is the only tongue worm known to have a free-swimming larva. it searches for fishes as its intermediate hosts (winch and riley ) . they occur worldwide but mostly in the tropics and subtropics. very few reliable taxonomic characters exist, even in adults, and these few characters change in different adult stages (supra-adults). the long cherished hope of fossil forms revealing clues to relations with other groups has not been realised. siveter et al. ( and references) found these forms as nearly characterless and enigmatic as the extant species. the few fossil forms known are isolated larvae, which appear to have been freeliving. siveter et al. ( ) found adults ectoparasitic on a marine ostracod. these life cycle forms are completely different from the present-day endoparasites of terrestrial, semiterrestrial, and vertebrates. there has been either a monumental and complete change between the fossil and extant life cycles, or, which is more likely, the fossil forms are not in the same lineage as extant tongue worms. the fossils may be related to extant tongue worms, but do not represent their ancestors. furthermore, without any interconnecting forms over half a billion years, it is difficult to try to join these fossils in a lineage with the extant tongue worms. more likely, the fossil and extant forms represent parallel evolution. chapter does not recognise siveter et al. ( ) fossil as a tongue worm. if correct, this leaves tongue worm fossil forms without an adult and without a host. these apparently free-swimming and unattached forms do have a modern equivalent in the larvae of subtriquetra subtriquetra. sanders and lee ( ) suggested that these larval forms parasitised conodonts (early, eel-like organisms, famous for first teeth in the fossil record). this would agree with this only modern analogue, which parasitises fishes as intermediate hosts. however, they considered the small fossil forms adults, not larvae, with direct life cycles. large forms with indirect life cycles only developed after the air-breathing tetropods were available~ million years ago. while these are interesting life strategy hypotheses, they lack any supporting evidence. tongue worm life cycles are like those of any other crustacean parasite. in their life evolution, they have invaded the land and colonised all four classes of terrestrial vertebrates. they are completely endoparasitic, with the exception of a free-living stage in one species. no other crustacean parasite is even similar. their drastically different life cycles suggest they may not be crustaceans. many other analyses agree (e.g. de assis ( , ) , place them in their own phylum), but most place them with the fish lice (branchiura). sebekia oxycephalum is a generalist having little specificity in fish intermediate, and crocodilian and snake definitive, hosts (silva et al. ) . vague reports of nymphs in lizards and reports in snakes probably represent paratenic hosts. it has the greatest range (southeastern usa to southern south america) of any tongue worm and the greatest host diversity. seed shrimp are a large group of largely free-living, marine species. a few are commensal on invertebrates, and extremely few are apparently parasitic on a shark, a ray, pacific sea urchins, one polychaete, groundwater isopods, and gammaridean amphipods (smith ) . many host records are based on few observations and specimens, and some relationships are unclear. their shells in sediments and extensive fossils are very useful indicators of past conditions, climate changes, oil deposits, and crustacean sexual development. they are well known to scuba divers for their painful bites at night, for their bioluminescent glow and for their nocturnal attacks on injured fishes (stepien and brusca ) . mating typically occurs in swarms with large numbers of females swimming to join the males. however, some are partially or wholly partheno-genetic. all seed shrimp, except punciids (no shell), brood their eggs between the upper (dorsal) part of the body and the shell. most ostracods shed eggs directly into the water as plankton or attach them to vegetation or the substratum. in some groups, one or two larval moults occur before the larvae are shed. eggs hatch into nauplius larvae with a hard, bivalve shell, except punciids that have a single headshield. a nauplius stage is usually followed by - metanaupliar moults. kretzler ( ) described the seven instars in the life cycle of echinophilus xiphidion kretzler, , in pacific sea urchins. he also found intense wave action inhibited the infection of sea urchins. males and females occur together on hosts. most adults do not moult. often, only a few specimens of parasitic seed shrimp are reported, although they can be very abundant. kretzler ( ) found specimens in host specimens of four species of sea urchins. he reported no damage; therefore, even heavy infections do not obviously affect hosts. bennett et al. ( ) found of epaulette sharks examined had sheina orri harding, , ostracods attached in the gills. light and scanning electron microscopy showed ostracods were anchored to gill tissues with their mandibular and maxillular claws. they damaged host tissues and were often located in distinct pockets, formed by local distortion of shark respiratory lamellae, strongly suggesting that they had been attached to the gills for considerable time. these details were presented because of some controversy whether sheina orri was parasitic. it has also been found in the bluespotted ribbontail ray, skogsbergia squamosa (mueller, ), and may be a bony fish parasite (monod ) , but this was not clear. wilson ( ) found , , and photeros parasitica (wilson ) in the gills and nasal tubes of three smooth hammerheads, one on the gills of a rock hind, and three on a blue runner in jamaica. hypothesised that p. parasitica was specific to sharks and rays and only accidental on bony fishes in the caribbean. cohen and morin ( ) reported that p. parasitica is a luciferin bioluminescent carrion feeder, not a parasite. brian ( ) found cypridina sp. on the gills of dolphinfish (coryphaena linnaeus, ) and called them parasites. however, this form is another luciferin bioluminescent seed shrimp, like p. parasitica, and is unlikely to be a parasite . it is related to the famous sea firefly. thus, 'firefly seed shrimp' do not appear to be parasites. bioluminescent seed shrimp are sometimes reported as gill parasites because they feed on detritus, and the gills of an organism are the first part to deteriorate. most of the~ , described copepod species (worms ) are free-living, some are commensal of invertebrates, and many parasitise invertebrates and fishes (~ described species,~ species in fishes alone). some parasites are little changed from the free-living form and even capable of free swimming between hosts (serial parasites). at the other end of the copepod, parasite spectrums are highly modified forms, which are fully embedded inside their hosts and can only be recognised as copepods by their larval forms. the basic life cycle of copepods has two phases (naupliar and copepodid) (fig. . ). the egg usually hatches into a nauplius larva with a small, unsegmented body, and three pairs of functional appendages (antennules, antennae and mandibles). a maximum of six naupliar stages can occur, and all six are found in most freeliving copepods and in some parasites. nauplii may be planktotrophic (feed on plankton) or rely on its yolk (lecithotrophic). parasitic nauplii are usually lecithotrophic, have reduced setation on the three limb pairs, and no naupliar feeding process on the coxae of the antenna. in many parasites, the naupliar phase is abbreviated or occasionally lost. the final nauplius stage moults to become the first copepodid with a segmented body, a full adult set of cephalic appendages, and the first and second swimming legs. free-living copepods have a maximum of five copepodid stages with one body somite added at each moult. in almost all copepod parasites, copepodid i is a free-swimming stage. only parachordeumium aphiurae (hérouard, ) and internal sea cucumber copepods have copepodid ii hatching from their eggs (martin et al. ). copepodid i is in the typical crustacean form with two pairs of biramous swimming legs, each with -segmented rami. it begins free-living but is usually the infective stage. the copepodid stages provide a gradual transition from the copepodid body form to adult morphology, however transformed. in the more derived families, successive copepdid stages have increasing modifications in body form and limb structures. the fifth copepodid stage moults into an adult male or female. following this moult, the female becomes sexually receptive. adult males may conduct precopulatory mate guarding and holding pre-adult females until the final moult. males use an array of chemosensory aesthetics on their antennules to detect pheromones produced by females. mating takes place soon fig. . the generalised life cycle of an ergasilus von nordmann, , species showing the freeliving naupliar and copepodid stages as well as the parasitic adult female. image from smit and hadfield ( ) after the female becomes sexually receptive and consists of mate detection, mate recognition, and mate capture and culminates in copulation. sperm-containing spermatophore(s) are transferred to the female and usually discharged via copulatory pores, into seminal receptacle(s) within the genital region of the female. sperm are stored for fertilization, which occurs as egg batches are laid. females may produce several batches of eggs during her life. most parasitic copepods extrude their eggs into paired egg sacs or uniseriate egg strings, although some are stored internally. copepods have a great diversity of invertebrate and fish hosts but are remarkably limited among other vertebrate groups with a single species on whales and dolphins, none on reptiles or birds, and only a very few, almost accidentally, on amphibians. their simple life cycles may inhibit them from colonising more diverse vertebrates since their only mammal parasite has their most complicated life cycle. the life strategies of copepods suggest the simpler the host, the easier it is to parasitise. reported, based on decades of research, that copepodids, chalimus, and immature adult copepods were very rare on caribbean coral reef fishes but rather common on western pacific coral reef fishes (a -year study). one possible explanation for this difference is that caribbean, small cleaner gobies (elacatinus jordan, ) , are much more efficient in locating and removing these small, life cycle stages than are the larger indo-pacific cleaner wrasses (labroides bleeker, ). thus, life strategies of caribbean and indo-pacific parasitic crustaceans may operate under quite different selective pressures. brusca ( ) found adult cymothoid isopods of the genus nerocila leach, , with damaged pleotelsons and uropods and speculated these injuries might represent predation by cleaner fishes. williams and williams (unpublished data) have found numerous injuries and missing parts of fish lice, fish-parasitic copepods, fish isopods, and gill worms (monogenea) on, and in the gills, or mouths of fishes, and have observed copepods on fishes scurrying away from cleaner fishes, even though they were obviously too large to be removed. cleaners may bite and injure crustacean parasites that are too large for them to remove (cleaner nipping*). cleaner nipping is a widespread, important, but hitherto unrecognised, life history peril for ectoparasites. mahmoud et al. ( ) experimentally induced nipping and removal of fish parasitic isopods by portunid crabs. cleaner shrimp similarly snip off the legs of small crustacean parasites to remove and eat them (williams and bunkley-williams b, unpublished data many copepod parasites of invertebrates also have direct life cycles, but some have endoparasitic larvae and free-swimming adults, mesoparasitic larvae and ectoparasitic adults, and abbreviated or no larval stages. some endo-and mesoparasitic forms can be quite modified. most are small and free-living, but rarely planktonic, in marine and freshwaters. some are commensal with invertebrates and only rarely damage their hosts (williams and wolfe-walters ) . many parasitise molluscs, sea anemones, sea squirts, fishes, and a caridean shrimp (conradi et al. ) . the most well known is the anchor worm-an economically important fish pest, which was globally spread on goldfish and asian carp and is now common worldwide. species of the family ergasilidae cause the most important problems in aquaculture and are distributed globally (garcia and williams ; williams et al. a williams et al. , b, thatcher and williams ; . eggs are usually carried in paired or single sacs attached to first abdominal somite. however, some notodelphyids and pectinophilus nagasawa, bresciani, & lutzen, , store eggs internally. the full life cycle occurs in many copepods parasitising invertebrates and in ergasilid fish parasites. ergasilids are also unusual in having naupliar stages feeding on unicellular algae. thaumatopsyllids have a life cycle similar to that of the monstrilloids with parasitic nauplii inhabiting the gut of brittle stars and nonfeeding adults living in the plankton. the copepodid phase in thaumatopsyllus paradoxus sars, comprises the full five stages preceding the adult, and the entire phase from final nauplius to adult is completed without further food intake. parachordeumium amphiurae (hérouard, ) hatches directly as an infective copepodid ii, having passed through the first within the egg. in the tunicate parasite, gonophysema bresciani & lützen, , the infective copepodid larva settles on the host and moults into an onychopodid larva, which is reduced to a simple elongate sac-like body provided with grasping antennae used for attachment. the onychopodid penetrates the skin of the tunicate and transforms into an amorphous, lobate adult (rohde ) . the life cycle of anchor worms (lernaeids) has been described as direct with only one host, indirect with an intermediate host, or with a transfer host. the confusion lies in the apparent occurrence of all three cycles in the same species of anchor worm. the first copepodid usually attaches and develops through copepodid stages on the gills of a fish host. this may occur on the definitive host specimen, on a different specimen of the same species, or on a different species of fish. in the final copepodid stage, the female usually leaves the gills and attaches on the body of the same fish specimen (direct) or on a different one (indirect). thus, a real intermediate host can occur, but this is not obligatory. some of these species may be evolving towards an obligate intermediate host. this would represent a third method of developing, through an intermediate host, for which we propose the term parallel incorporation*. deep-sea copepods have been found resting and feeding on the mucus of gelatinous plankton (humes ) . gasca et al. ( ) found mating males and females and early to late copepodid stages of pseudolubbockia dilatata sars, , in the subumbrella cavity of deep-sea hydromedusae. we suggest this copepod is another protelean parasite in the short-antennae copepods. recent reports and descriptions of additional copepodids or of pre-adults in shortantenna copepods were probably growth stages, not moult stages (martin et al. ). the free-living stages in the life cycles of ergasilids and many of the copepod species parasitising invertebrates suggest that they have more recently evolved a parasitic lifestyle. the most modified female of any of the fish-parasitic copepods are species of sarcotaces olsson, . osman et al. ( ) described and pictured the naupli of possibly a new species of sarcotaces apparently host specific to a brownspotted grouper in the arabian gulf. surprisingly, they found no females in six gall cysts, even though nauplii and males were present. eggs were attached to the inner wall of the galls. nagasawa et al. ( ) found eight cysts with females, males, nauplii, and eggs in a blacktip grouper (fig. . a, b) in the ryukyu islands. some short-antenna copepods occur in the musculature and sinus canals of fishes. rosim et al. ( ) reported a new genus of ergasilid in the urinary bladder of fishes and considered the process of becoming an endoparasite. the muscle parasites are mesoparasites; however, their host positions do complicate their life cycles. forsskal, ) , collected in okinawa. (a) embedded (skin has been removed to expose large, sack-like cysts), (b) removed large, sack-like cyst. nagasawa et al. ( ) found eight cysts with females, males, naupli, and eggs in this host specimen. images © kazuya nagasawa . . harpacticoida: wormlike copepods* these are a very large group of mostly benthic copepods. very few associate with other organisms, and almost none are known to be parasitic. in the family tisbidae, species parasitise deep-sea octopuses. red bug, an aquarium pest, has often been called a parasite of corals and is caused by the copepod tegastes acroporanus humes, . neoscutellidium yeatmani (zwerner ) was also said to be a parasite of fish, and other wormlike copepods were noted to be parasites of whales, sea turtles, and manatees (e.g. aznar et al. ) . however, others have disagreed with these statements (e.g. suárez-morales et al. a; domènech et al. ). an apparent commensal species, the ochre copepod*, balaenophilus manatorum (ortíz, lalana & torrez, ) , has been observed in caribbean manatees and sea turtles (badillo et al. ; williams and bunkley-williams unpublished data) . haracticoids probably follow the usual copepod life cycle of six naupliar stages and five copepodids found in their family. dahms et al. ( ) discussed all that is known about the naupliar stages in tisbidae. no development stages of parasitic forms had ever been found until lópez- gonzález et al. ( ) described copepodids iii and v. they suggested similar copepodid stages occurred internally (actually mesoparasitic) for the other adult forms found in other octopuses. therefore, their discovery completed the life cycle of these parasites (see additional information below). they hypothesised copepodid i was the infective stage and all these occurred internally. they did not discuss the naupliar stages, but these are expected to be the usual, six, free-swimming forms. adult males have only been found in four of the known haracticoid species. they may not live very long, not stay on the host very long, or even move to new hosts to copulate with other females. the more complete life cycle suggested by lópez- gonzález et al. ( ) is interesting and possibly correct. the only problem is that it is in disjunct halves. until the external portion is matched with the internal portion in a single species, ockham's razor would suggest these are portions of two different life cycles of tisbids on octopuses. the complete life cycle is thus still unresolved. ogawa et al. ( ) suggested balaenophilus aurivillius, , species on sea turtles spend their entire life cycle on one host and cannot swim, like whale lice. domènech et al. ( ) experimentally found nauplii can only crawl, but copepodids and adults can swim, albeit only for short distances. zwerner's ( ) discovery of neoscutellidium yeatmani in the gills of antarctic eelpout has been uncritically repeated so many times that it appears to be widely believed. however, this form has never been reported again. he found a mere seven specimens in the gills of fish. these numbers are too low to sustain a viable life cycle for a parasite. this was very likely an accidental infection. this deep-water fish is known to eat molluscs, and species of parasites in this family infect the gills of deep-water octopuses. these copepods could have spilled from an octopus to the fish consumed (see prey predator transfer). more specimens with this association, with higher numbers per host, would need to be observed before this species could be confidently regarded as a fish parasite. many authors, even very recently (e.g. aznar et al. ) , have called the ochre copepod on sea turtles and manatees an ectoparasite, but it appears to be only a commensal. suárez-morales et al. ( a) found that it was a harmless epibiont. this copepod formed ochre-coloured patches on the skin with no positive precursor relationship with barnacles or algae. badillo et al. ( ) explored the possibility of parasitism of wormlike copepods on whales, sea turtles, and manatees in detail and believed that they ate keratin. they therefore claim this makes them commensals of whales, yet ectoparasites of turtles. this interpretation does not appear convincing, and we assert that they are all commensals. suárez-morales et al. ( a) also dismissed, another harpacticoid, harpacticus pulex humes, , associate. it had been reported on a manatee only once, in captivity, and appears to be a non-associated predacious species. thus, we surmise wormlike copepods do not parasitise vertebrates or corals (red bugs). some may be obligate commensals. some may also harm vertebrates in captivity but are not parasites. larval parasitic copepods are wholly parasitic and occur worldwide (tropical, temperate, polar) in marine waters and infect benthic gastropod and bivalve molluscs, polychaete worms, and sponges (martin et al. ) . approximately species are known in five genera in a single family. monstrilla dana, (latin for monster) is the best-known genus. they are not abundant anywhere but more often found in coastal and coral reef areas. their biology and ecology are poorly known. the only mortalities attributed to larval parasitic copepods was a partial die-off of cultured brown mussels (suárez-morales et al. b) caused by copepodids, the most damaging stage. suárez-morales ( ) reviewed the diversity, as well as the life cycles of larval parasitic copepods. they have a protelean life history unique among metazoan parasites (martin et al. ). the first naupliar stage is free-living, but the rest are endoparasitic. all the copepodid stages are parasitic. copepodid v, called subimago by suárez-morales et al. ( ) , is the emergent stage. it moults rather quickly into an adult after it leaves the host. the adults are nonfeeding, free-swimming, reproductive, and pelagic. most copepods produce egg sacs or spawn freely in the water column, but larval parasitic copepod females attach their eggs on their long, ovigerous spines with mucous secreted by the terminal part of the oviduct. egg masses are produced iteratively corresponding to when the ovigerous spines grow. eggs hatch into lecithotrophic nauplii that locate a mollusc or polychaete host and burrow into its tissues. they metamorphose into sac-like naupliar stage in the host's blood system. two antero-ventral root-like processes absorb nourishment from the host. this stage is like no other crustacean larvae. development continues endoparasitically until the last copepodid escapes from the host and undertakes a single moult into a reproductive adult. suárez-morales et al. ( ) described the first copepodids (iii, iv, v) in detail, finding copepodid v to be pre-emergent and emergent. adults spend very little time in the plankton; therefore, they are rarely found and usually in low numbers. suárez-morales ( ) described one mass aggregation in the caribbean sea off mexico. pelagic adults lack all cephalic appendages except antennules. the known hosts include pyramidellid and vermetid prosobranch gastropods, bivalves, and polychaete worms. pairing the same species of free-swimming males and females morphologically has been difficult. the few morphological characters have also made taxonomic work difficult and often inconclusive, and descriptive standards have only relatively recently been upgraded (grygier and ohtsuka ) . suárez-morales ( ) summarised the morphologies of the group to aid in identifying adults and life cycle stages. suárez-morales et al. ( b) were the first to find a monstrilloid in a commercial bivalve mollusc and to document the consequent harm and mortalities. a radical placement of the larval parasitic copepods within the sea lice, based on sem data, and antenna and caudal rami morphology, was proposed by huys et al. ( ) . the differences suárez-morales et al. ( ) found between these groups' copepodids suggested such a combination would be incorrect. their life histories are also completely different in almost every respect. a subimago refers to a pre-adult mayfly with wings, but no functional genitalia. it can fly, but cannot mate, and can be morphologically very similar to the adult. it moults into an adult. the copepodid v from suárez-morales et al. ( ) does not appear sufficiently different to warrant the use of this borrowed term. kuris et al. ( ) suggests larval parasitic copepods are parasitoids. they do have a life cycle similar to parasitoids; however, we believe they are parasites because they do not kill their hosts. only species (~ %) descriptions are based on both sexes, on females only, and on males. molecular studies might help pair females and males of the species (suárez-morales ). siphon-mouth copepods have siphon-like mandibles and a frontal filament that attaches to the hosts. these attributes have contributed to their great diversity (~ species in families) and success. sea lice (caligoids) are well known fish copepods and are very damaging to fishes in cage culture, salmon being particularly damaged by the salmon louse. cuckoo copepods*, or nicothoids (fig. . ) , are the most famous of this group of parasites of invertebrates that harm commercially important lobsters and spider crabs. this order holds % of the known copepod parasites of fishes ( species in families). most species are marine, but a few are freshwater (garcia and williams ; williams et al. a williams et al. , b, ). the full copepod life cycle occurs in many families of siphon-mouth copepods, especially those utilising invertebrates as hosts, such as asterocherids and cancerillids. in parasitic copepods, the infective larva is, with rare exceptions, the first copepodid, and life cycles are direct, involving only a single host. in fish parasites, the nauplius phase is reduced to two lecithotrophic stages and has uniseriate egg strings in which disc-shaped eggs are closely packed into one row extending the length of the string; and most nauplii have a single pair of modified caudal setae known as balancers (function unknown). related families with fig. . cuckoo copepods (nicothoids) parasitise the eggs of lobsters and spider crabs in asia and brazil. they mimic the eggs of their host and are not removed from the host's eggs, which they eat. the copepod (arrow) has egg strings. see life cycle in otake et al. ( ) . image © kaori wakabayashi multiseriate egg strings, such as lernaeopodids, sphyriids, and two genera of hatschekiids, have nauplii lacking balancers. in some lernaeopodids (allela leigh-sharpe, ; clavella oken, ; and nectobranchia hesse, ) , the nauplius phase is reduced to one stage. in other lernaeopodids and some pennellids (salmincola wilson, ; cardiodectes wilson, ; and peroderma heller, ) , it is lost completely, and eggs hatch directly into the infective first copepodid. life cycle abbreviation also occurs in some parasites of invertebrates. the herpyllobiidae and the genus trochicola dollfus, , have only two naupliar stages, and only one nauplius is known for gonophysema bresciani & lützen, , and for some genera of nicothoids. in other genera of nicothoids, some chordeumiids, and cucumaricolids, there is no nauplius stage. izawa ( ) experimentally showed that there could be five naupliar stages in gangliopus pyriformis gerstaecker, . in most fish parasites, the first copepodid secrets a chitinous frontal filament from an anteriorly located gland, soon after it settles on the host. this filament anchors the developing chalimus larva securely to its host (rohde ) . the life cycles of sea lice have been the topic of much research and debate of late. this attention is due to the damage that they cause cage cultured fishes, particularly of salmonids (lafferty et al. ) . knowing the correct life cycle is critical in determining when to treat for the damaging stages. caligid sea lice were thought to have four chalimus stages and one or two pre-adult stages. several recent papers have challenged this scenario (e.g. hamre et al. ; venmathi maran et al. ) . they found that the typical caligid life cycle comprised eight stages: two naupliar, one copepodid, and four chalimus stages preceding the adult in caligus müller, , but with the four chalimus stages represented by two chalimus and two pre-adult stages in lepeophtheirus heegaard, . this is a profound change with significant implications for the aquaculture industry. as the typical caligid life cycle may not exist, it may be necessary to determine the life cycle of every species of damaging sea louse. again, only . % ( ) of life cycles are known for caligid species (venmathi maran et al. ) . the new stingray laser gun has only been used to shoot adult sea lice (bevanger ) . considering the flexibility and accuracy described for the gun, it could probably be used to shoot multiple life cycle stages. pre-adults also secrete a frontal filament during moulting but soon detach and become motile. frontal filaments and chalimus larvae occur in most fish parasites for which the larvae are known, but none occur in the lernanthropids. nicothoids use a similar filament to attach their developing larva to the exoskeleton of a crustacean host. the basic copepodid stages, as primitively retained in cancerilla dalyel, , comprise five stages plus the adult. one pre-adult stage in caligus clemensi parker & margolis, , or two pre-adult stages in sea lice, as true moult stages, have been added to the basic life cycle. the general trend in parasites is to simplify or reduce ancestral free-living life cycles. these additions are quite unusual and have only been found in this order. pennellids (marine anchor worms*) differ from all other copepods by needing (obligate) intermediate hosts (fish, squid, pelagic gastropod) in order to develop. the copepodid larva becomes a chalimus larva stage on the intermediate host. some male and female chalimus individuals mate on this host. others leave the host and mate in the water column. males soon die; females have a short planktonic period, find a final host, and metamorphose into an adult (poulin a) . brooker et al. ( ) reviewed the literature and thoroughly described the life cycle and life history of the famous pennellid copepod lernaeocera branchialis (linnaeus, ) . not all marine anchor worms have intermediate hosts. okawachi et al. ( ) suggest peniculus minuticaudae shiino, , has a direct life cycle, unlike most pennellids, because copepodids, chalimi, adult males, premetamorphic adult females, and post-metamorphic adult females of the parasite were all found on a single fish. they also describe adult male, copepodid i, and late chalimus stages and redescribe post-metamorphic and premetamorphic adult females. ismail et al. ( ) described a complete, direct life cycle of a pennellid, peniculus minuticaudae shiino, . the hatching stage was an infective copepodid followed by four chalimi and adult instars. males associated with various pre-adult females, but copulation only occurred between adults. fertilised premetamorphic adult females carrying spermatophores may detach from the host and settle again before undergoing massive differential growth into the post-metamorphic adult female. many marine anchor worms (pennellids) have intermediate hosts. this is the first life cycle of the group in which the female remains in the same position on the same host specimen (ismail et al. ) . otake et al. ( ) had named a new cucukoo copepod* in and described its abbreviated nicothoid life cycle of free-living nauplius i (ni) observed hatching from female egg sac, copepodid i (ci) found on body of host, and copepodid ii+ (cii+) and adults found on host eggs. they surmised ni develops into infective ci in the water column, ci settles on the body of host, and ci moults to cii, migrates to host egg masses, and develops into cii+ and then adults. adults mate on host egg masses. brazenor and hutson ( ) examined the effects of temperature and salinity on the life cycle of lernanthropus latis yamaguti, , on the euryhaline barramundi in australia. nauplii hatched best at - c and ‰. none hatched in freshwater and only a few in brackish water. lernanthropus latis is euryhaline, but freshwater can be used to break its life cycle. Økland et al. ( ) described two new rhabdovirid viruses, which occurred in all life cycle stages of the salmon sea louse in norway. the viruses caused tissue necrosis in adult copepods but did not infect fish. they speculate the copepod injects the virus in the fish to confuse its immune system as part of its purposeful life cycle strategy. we find their suggestion interesting but rather astonishing. copepods do not purposely use viruses; theirs was not the first report of copepod viruses but the fifth; many other fish-parasitic viral vector mutualists* exist to the benefit of crustacean infective stages; and we designated the first viral crustacean mutualists*. sea lice are notorious for causing problems in marine aquaculture, particularly of salmonids (e.g. gonzález and carvajal ; lafferty et al. ) . the contamination of the environment by salmon sea lice from fish farms is a politically and economically important question that has received recent attention. serra-llinares et al. ( ) found that farms increase the infection of local, wild salmonids. they also noted thorstad et al. ( ) , and others strongly suggested the transmission of lice from farm salmon to wild salmonids in systems where the fish occur in close vicinity. these adult caligids can freely swim between hosts (host hop*) and build up in fish cages. few parasitic copepods have this life-history advantage (serial parasites). the filtering effect of cages tends to concentrate sea lice. adult male and female caligids are frequently found in plankton samples (venmathi maran and ohtsuka ) . they must spend considerable periods free-swimming off hosts. some species have even been described only from the plankton, and their hosts remain unknown. ohtsuka et al. ( ) previously described a dajid isopod and a nicothoid copepod parasitising the marsupial lumen of a mysid in japan. the adults eat mycid eggs and drastically reduce the population of mysids. infective stages of the copepod penetrate host body tissues, feed, and grow. infective isopods penetrate the space between the carapace and the dorsal tergites. remarkably, isopods and copepods rarely occur together, but alternately, albeit continuously, parasitised the same host at different times of the year. this life history association is unique in parasitology. we will call it alternate host sharing*. this allows both parasite species to use all available resources of the host. three species of copepods are known to fully encyst in intermediate host fish tissues. only one has been named. lewis ( ) found the first pandarids to encyst in the fins of bony (teleost) fishes in hawai'i. lewis only found male copepodids and immature males, which he tentatively identified as nesippus cf. costatus wilson, . amaterasia amanoiwatoi izawa, , was described from female copepodids i, iii, iv, and v and a female escaping from a copepodid v. izawa found , , , and copepodids in fin galls on a single striped triggerfish from the eastern pacific (izawa ) . a new species of amaterasia izawa, , was found in individual cysts, as lewis ( ) had found, not galls, on the body and dorsal fin of species of fishes in puerto rico (williams and bunkley-williams unpublished data). tang et al. ( ) found that lewis' ( ) younger early encysted stage was a copepodid iv, the older early encysted stage was a copepodid v, and the lateencysted and recently excysted stage was an immature adult male. izawa ( ) and tang et al. ( ) speculated about the life cycle of their species but made no descriptions. tang et al. ( ) suggested encystment could be protection from coral reef cleaners. we propose a new life cycle for these encysted copepods*: there are five planktonic nauplii (izawa ) and a first copepodid stage. the ci is infective (found in galls; izawa ) and settles from the plankton onto a host and forms a cyst or gall under the skin on the fins or body of a variety of different bony, coral reef fishes. lewis ( ) found surgeonfishes (acanthurids) were preferred in hawai'i. however, parrotfishes were preferred in puerto rico (williams and bunkley-williams unpublished data) . the cysts are open posteriorly for respiration (mesoparasite). after feeding, developing, and moulting through ci-civ, the fifth copepodid emerges from the cyst leaving a moulted exoskeleton behind. lewis ( ) found these forms were soft and pliant, which he interpreted to be of value in leaving their cysts. it was actually because they had recently moulted. the cv then swims out and searches for a shark definitive host. there could be some predator transfer involved since the copepodids are in hosts preyed upon by sharks. the encysted copepod life cycle* is the only mesoparasitic life cycle known in parasitology and only the second obligate intermediate host life cycle discovered in copepod parasites of fishes. many tapeworm (cestode) shark parasites have bony fish intermediate hosts, but this is the first crustacean one ever discovered. the evolutionary usefulness or necessity of pennellid intermediate hosts has never been explained (poulin a; martin et al. ) . we can discern at least seven, nonmutually exclusive explanations: . . faster attachment-when the female settles on the final host, water currents, host movements, and sometimes host cleaning or cleaner organisms make her stay perilous (loose on the host). nutrients from the intermediate host can be used to permanently attach without waiting to feed on the final host and digest. . widen host range-the only crustacean parasite to infect mammals cannot do so without an intermediate host. kik et al. ( ) found lepeophtheirus acutus heegaard, , was a potentially dangerous sea louse of elasmobranchs in captivity. not only did it damage sharks and rays but could complete its life cycle in an aquarium. muñoz et al. ( ) found early and late copepodids of two species of caligus, two of trifur wilson, , and two of unknown families, on juvenile fishes. they examined thousands of nearshore, planktonic fishes, found % infected, and % infected by multiple species. they thus opened a completely new dimension into parasitic life strategies, which we have termed planktonic juvenile fish infection*. juveniles of a single host species have sometimes been examined for parasites (e.g. nielson et al. ; herrera ), but masses of juveniles have seldom been studied (e.g. herrera ; felley et al. ; cribb et al. ) . muñoz et al. ( ) concluded these juvenile fishes were intermediate hosts for these copepods. however, caligids are not known to have intermediate hosts, and pennellid intermediate hosts are adult fish or squids. they had also concluded the copepods would mature too soon to develop with the host fishes. however, we believe the hosts and the parasites will grow up together in these cases. alternatively, these may be small predators* feeding on juvenile fishes as a part of their life cycles (table . ). whichever is the case, this extends and prolongs life cycles further than we had imagined. interestingly, only copepods are taking advantage of this resource, not only among crustacean parasites but also among all parasites. muñoz et al. ( ) also searched for internal parasites and found none. venmathi maran et al. ( ) cuckoo copepods are named after the nest-parasitic birds by that name (cuculidae). these copepods parasitise the eggs of lobsters and spider crabs in asia and brazil. they mimic the eggs of their host ( fig. . ) and are thus not removed by the host, as they resemble the real eggs, which they eat. otake et al. ( ) called these copepods 'associates'. kuris et al. ( ) calls them 'symbiotic egg predators'. however, we consider them to be true parasites, damaging and feeding off their hosts, with adults that never leave the host. flyingfishes are food for many large offshore predators, which host pennella spp. høeg et al. ( ) found that cypris larva morphologies of the barnacles reinforced the concept that this larva was a prerequisite to the tremendous success of that taxon. the evolution of parasitism, obligatory in three major taxa, was the result of convergent evolution. thecostraca was distinct from tantulocarida (sect. . ) because they differed in the life cycle stages that penetrated their hosts (høeg et al. ). these very small, naked barnacles bore into calcareous material such as animal shells and inanimate hardgrounds. burrowing barnacles produce a slit-like hole in the surface known by the trace fossil name rogerella saint-seine, . they feed on plankton ). they do not feed on their associates. burrowing barnacles are only found in their hosts' shells and never touch their flesh, except possibly in the case of corals. they are not obligates, perfectly happy living on inanimate hard ground, and do not harm their hosts. we do not understand how they can be called parasites? williams et al. ( ) called a burrowing barnacle a parasite and showed that it fed on its host hermit crab's eggs. however, this was predation, not parasitism. murphy and williams ( ) suggested burrowing barnacles in hermit crab shells were 'transient parasites' because they somehow consumed hermit crab eggs and preferred female hermit crab shells. in our opinion, burrowing barnacles are not parasites, as least when considering the current available information. the parasitic barnacle is the 'poster child' for gross modification of parasitic forms. adults are unrecognisable as crustaceans, let alone barnacles. only their larval forms resemble those of normal barnacles (see sect. . . ). they are also famous for controlling the behaviour and morphology of their hosts. they damage commercially important crustaceans. about species are known, about a quarter of all barnacle species. they infect crustaceans, mostly true crabs (brachyuran) and anomuran crabs (hermit crabs, squat lobsters, etc.). a few parasitise caridean shrimp, mantis shrimp, peracarids, and even other barnacles. we worked on the button-crab parasite* on the blue crab in the gulf of mexico but have not found such obvious parasitic barnacles in the caribbean. unlike most barnacles, parasitic barnacles have separate sexes. adults are sessile, with females consisting of a sac attached to the crab host (externa) (fig. . ) with rootlets of tissue flowing cancer-like through the host's body (interna) and dwarf males inside the female. some females sequentially only produce male larvae from large eggs, female larvae from small eggs or mixed sexes. they have the usual naupliar instars and infective cyprid; however, in some species, embryos develop directly into cypris larvae before adult females release them. the nauplii are smaller than those found in other barnacles, which may be necessary to produce them in much greater numbers. the larvae are lecithotrophic. the cypris are usually at least days old before they settle on a host. in kentrogonids, male cyprids are larger than female ones. some have a naupliar eye, and others have compound eyes. injection of the vermigon (the migratory internal stage) happens within - days after settlement. when many male larvae exist, only the fastest and strongest will succeed. when there are few males, the female remains receptive longer. when a virgin is found, the male cyrid must settle close to the orifice, enter the brood chamber, and inject a trichogon stage. the trichogon looks like a verogon, except with a spiny collar. it becomes a dwarf male and reaches one of two male receptacles. once established, the dwarf male undergoes spermatogenesis and is nourished by the female parasite for the duration of its life (cryptogonochorism). the male cypris of akentrogonids penetrate the host or the virgin female with their antennules and without a kentrogon or trichogon. a single male can fertilise all the broods of the female. a female externa produces several batches of larvae and drops off the host just before the host moults. a new, young externa is produced from the interna and emerges from the host body (waiho et al. ) . , ("greater than" symbol-shaped mass-red arrow), externa of this rizocephalan parasitic barnacle, which, in turn, was hyperparasitised and rendered sterile by four liriopsis pygmaea (rathke, ) (pearl shapes-black arrow) epicarid parasitic isopods (cryptoniscidae). a different black-and-white photograph of these associations was in lovrich et al. ( ) . associates in this present colour photograph have been misidentified in several popular sites online. image © gustavo a. lovrich the externa takes the place of the crab egg sac. the host's behaviour is chemically altered causing sterilisation and only moults when the aged externa drops off. the host treats the externa as if it were its egg sac. male crabs, which would never have carried eggs, care for the externa. they are even more affected since their tail shape changes to the female configuration to better protect the externa ( feminization). an externa may last for several years. these life cycles show parasitic barnacles are the most parasitic of the crustaceans. they totally penetrate all the tissues of their host, control the behaviour of the host, and are drastically modified for the parasitic existence. glenner and hebsgaard ( ) made a comprehensive phylogenetic analysis of the evolution of life history strategies in parasitic barnacles. they found they were monophyletic with a filter-feeding barnacle-like ancestor. the host-infective kentrogon larva, inserted in the life cycle of kentrogonida, was ancestral, and a homologue of the juvenile thoracican barnacle. the host inoculation in akentrogonida (last pelagic larval stage directly injects into haemolymph) is derived and has evolved only once within the parasitic barnacles. the ancestral host is anomuran (hermit crabs, squat lobsters, etc.). alverez et al. ( ) described the externae of the button-crab parasite in detail. unlike most parasitic barnacles, they found only a single male receptacle but two implanted males. they questioned what sorts of male-male competition occurs when they are not separated. glenner et al. ( ) used light and sem microscopy of cypris larvae to supplement molecular data showing that parasitic barnacles, thought to be the most primitive, were actually the most advanced, along with many evolutionary extrapolations. these parasitic barnacles are sometimes hyperparasitised by cryptic isopods (fig. . ). just as the barnacle sterilises its crab host, the hyperparasite sterilises its fellow parasite. there are some indications that swimming decapods, which must remove epibionts, are more resistant to the attachment of settling crustacean parasites; however, no experimental evidence exists (boyko and williams ). in the reverse, li et al. ( ) found a species of crab parasitised by a rhizocephalan had many more barnacles, and other epibionts, than those not parasitised. this is an example of our positive precursor*. several authors have recently suggested parasitic barnacles could be host-specific control agents for nonindigenous crabs, such as the problematic green crab. however, parasites seldom make effective controls, and well-intended introductions have often been disastrous. this group containing the normal acorn and gooseneck barnacles has four parasitic species in three families and three genera. the shark barnacle* is little modified for parasitic existence except for the loss of its shell and adding a tough tegument. polychaete barnacles* are moderately modified. the jellyfish barnacle* is the least modified with small, thin plates. the cirri (feeding legs) are still used for obtaining food. these free-living-to-parasitic transitionals are, of course, of great scientific interest (rees et al. ) . the shark barnacle is famous for promoting charles darwin's interest in barnacles. they are hermaphroditic with all individuals possessing a penis, and no dwarf males are known in shark barnacles and polychaete barnacles. adults are receptive as females for fertilisation after moulting. they use their penis to copulate with an adjacent individual as other hermaphroditic free-living barnacles. barnacles have the longest penises, relative to body size, in the animal kingdom. oddly, dwarf males have been found attached to larger hermaphrodites in some species (androdioecy; sawada et al. ) . the fertilised egg hatches into a standard, barnacle nauplius, a one-eyed, pearshaped larva with a head, a naupliar eye, a pair of horns, and a telson. nauplii are usually brooded by the parent and released after the first moult swimming freely with setae. towards the end of the sixth instar, they begin to develop compound eyes and a globular shape. they undergo months of growth, passing through five instars, before transforming into the cyprid stage, which has a carapace, is torpedo-shaped and is the stage before adulthood. it does not feed and only searches for a host. this may last for a period of days to weeks. it explores potential hosts with modified sensory antennules. once it finds a host, it undergoes metamorphosis into a juvenile barnacle. shark barnacles are usually found in pairs near the dorsal fin of their shark host; therefore, the cypris larvae must not only find a host but also a partner (see founder pair*). shark barnacles breed through the year and live on their host for at least a year. both genera feed on the host by roots formed from their peduncles. they mostly infect small specimens of sharks ( %), and incidence is reduced ( %) in large ones. gonads of infected sharks never develop (nutritional sterilisation*). reported a prey-predator transferred isopod in a lantern shark but found no barnacles. some epiphytic normal barnacles attach to hosts or parasites (e.g. williams ; williams and williams b; mignucci-giannoni et al. ) , and the sea turtle barnacle has been found on the carapace of a speckled crab at dauphin island, alabama (williams and bunkley-williams unpublished data). shark barnacles parasitise at least seven deep-sea lantern sharks and dogfish (ommundsen et al. ). furthermore, two species of polychaete barnacles and a jellyfish barnacle are known . the jellyfish barnacle is an obligate associate of jellyfishes, and pagès ( ) found it attached in an area with few nematocysts near the gonads of the jellyfish host. we believe the lack of stinging cells may have originally attracted the barnacle infective stage to this area, and the gonads became a convenient food source later. a simple development of parasitism is still in progress. the species in the larval genus hansenocaris Îto, of y-parasites remain a mystery in parasitology. y-larvae have been known for years, but their assumed parasite adult forms and hosts have never been found. these parasites are both everywhere and nowhere. they must be both parasitologically and ecologically very important, but how remains a mystery. life cycle y-nauplii are egg-shaped, with a faceted cephalic shield and carapace (reticulated cuticular ridges, forming plates), from which the group derives its name, and a relatively long, ornamented abdomen. they have a characteristic bobbing motion when swimming that makes them easy to distinguish in plankton samples. the function and homology of the naupliar horn pores and dorsocaudal organs have been much debated but remain uncertain. they are either planktotrophic or lecithotrophic. only lecithotrophic nauplii have been raised through all five instars. planktotrophic nauplii have food visible in their stomachs. unlike barnacle larvae, the y-cyprid is constantly swimming. this larva is distinctively the costracan. the y-cyprid has a univalved carapace that only partially covers the larval body and resembles an inverted boat but with elongated sharp posterior ends (fig. . ). paired compound eyes lie anteriorly in the body with antennules, labrum, paraocular processes, postocular filamentary tufts, and two pairs of rudiments of antennae and mandibles underneath (ventrally). the antennules have four segments. all larval stages are free-living and semitransparent. the cyprid does not feed. a number of species have been described based only on a y-cyprid (n ¼ ) or even a y-nauplii (n ¼ ). as with barnacles, the cyprid seeks a host to infect. it has compound eyes, is ambulatory with its antennae, and can produce an adhesive glue. recently, possible juvenile forms have been produced by treating y-cyprid with the hormone -hydroxyecdysone to stimulate ecdysis and the transition to the next life cycle phase. the resulting slug-like, unsegmented, and limbless form was called 'ypsigon' (fig. . ). it is formed in the cypris and escapes from its body (glenner et al. ). this may be a juvenile. see høeg et al. ( ) for more detailed descriptions, illustrations, and photographs. molecular studies support the present phylogeny of the y-parasites . they remain mysterious parasites with unknown adults and hosts. glenner et al. ( ) suggest the ypsigon is the 'vermigon of the y-parasites'. just like the barnacle vermigon, it is the injected form that will become the parasitic adult. this may be true, but just because formless structures are produced by similar methods and appear similar does not make them the same. their notion is courageous, albeit premature. glenner et al. ( ) found that + morphological types of y-larvae occurred very abundantly around sesoko island in okinawa. this unusual situation may indicate a centre of origin or, possibly, a diversity hot spot. yet, the adults remain unknown. the 'phantom' adults are neither too rare to discover, nor in some obscure unexamined hosts, nor in some restricted localities. the larvae are just too abundant, widespread, and diverse for such restrictions. this is an adult that parasitologists may be encountering all the time but just cannot recognise. the adult y-parasites may be morphologically similar to another parasite, with which they are confused, or so morphologically indistinct as to be unrecognisable as a parasite. glenner et al. ( ) came to a similar 'highly simplified structure' conclusion. showing the carapace, the thorax with six pairs of natatory legs and the segmented abdomen, (b) an ypsigon (red arrow) within minutes of leaving the empty cuticle of the spent y-cyprid (black arrow). the cuticles of the carapace, thorax, appendages and abdomen are clearly visible, but no tissues remain in the spent y-cyprid. the worm-shaped ypsigon exits by amoeboid bending and peristalsis movement of the body and is believed to be the initial parasitic stage that enters into the tissue or body cavities of a still unknown host. details in glenner et al. ( ) . images © jt hoeg, m grygier, y fujita, h glenner, j olesen . ascothoracida: copebarnacles* these are uncommon parasites that cause little damage and infect non-commercially important hosts. copebarnacles are a small group of~ species in genera, families, and orders, which are ecto-, meso-, and endoparasites. they occur from the intertidal to the deep sea around the world. coral copebarnacles are ecto-, meso-, and endoparasites of corals (scleractinia, zoantharia, antipatharia and alcyonacea) and ectoparasites of crinoids (waginella). the suggested common name is based on their bodies that are similar to copepods but are related to barnacles. echinoderm copebarnacles are meso-and endoparasites of echinoderms (asteroidea, echinoidea and ophiuroidea). sexes are separate except for the hermaphroditic petrarcids. sex determination is genetic, and some male and female larvae differ in the armature of chemosensory aesthetascs (as in most parasitic barnacles). a-nauplii have an oval, bowl-shaped head shield, which is broader anteriorly, and setiform frontal filaments. a-cypris have a bivalve carapace and antennules with hooked claws. echinoderm copebarnacles brood their larvae and only release a-cypris, while coral copebarnacles release nauplii. usually, six naupliar instars (sometimes two brooded) are followed by one to two cypris-like stages (a-cypris, ascothoracid larvae) (similar to the cirripede single cypris and the y-cypris of y-parasites). many are lecithotrophic, but some species are planktotrophic. coral copebarnacles a-cypris occur in the plankton. the a-cypris (ascothoracid-larva) attaches by grasping antennules rather than by glandular secretions as in cirripede cyprids. when two a-cyprid instars occur, the second is the settlement stage. many species have an abbreviated ontogeny, and the entire naupliar phase is sometimes brooded or embryonised. no complete life cycle is known for any copebarnacle. neither host infection, nor copulation has ever been observed. most females have seminal receptacles on their legs, but not in dendrogastrids. some male a-cyprids have testes with mature sperm and possibly fertilise the females through the pore in the host. some dwarf males sit close to the aperture, and others live in the mantle cavity of the female. the transition from a bivalved stage to a sac-like carapace is suspected to occur in one moult, but this remains unresolved. a 'post-larval' stage of females and males has been discovered, and second-stage a-cyprids, ready to moult to the male stage, have been observed. the cypris for the modified, enlarged female, and dwarf males of gorgonian copebarnacles (gorgonolaureus utinomi, ) are not known. the complete life cycle is equally not known (kolbasov et al. ) . threefive naupliar instars occur. gorgonolaureus muzikae grygier, has been noted to be frequently infected by hyperparasitic cryptoniscoid isopods (kolbasov et al. ) . some ectoparasitic males and females can swim from host to host. others are semipermanently glued in place. some are endoparasites in galls in corals and sea stars. others even start as an endoparasite but eventually create an opening to the outside and become mesoparasitic. parasites of cnidarians occupy individual polyps or nodules formed from several polyps. many of those in echinoderms sterilize their hosts. feeding methods are uncertain. many have piercing-sucking mouthparts, and the cuticle of at least one species seems capable of absorptive feeding (as in parasitic barnacles). hyperparasitism of ascothoracidans by cryptoniscid isopods is not uncommon, occurring in four of the six families. some sterilise their copebarnacle hosts. in a spectacular case, a copebarnacle, which had sterilised its host, was, in turn sterilised by an isopod. their body is enclosed by a bivalve carapace often modified and enlarged for brooding and possibly food absorption in females. minute-crustacean parasites are a highly specialised, small ( species in genera and families) group of minute (< . mm) ectoparasites on small benthic crustaceans (copepods, isopods, tanaids, amphipods and ostracods). they occur from the subtidal to the abyssal, widespread in the southern and northern hemispheres, and in both cold and warm waters. they claim the fame of the world's smallest arthropod ( μm). minute-crustacean parasites have asexual and sexual life cycles. the first is when a parthenogenetic female develops from a tantulus larva and remains permanently attached to its larva, and the host, as sort of a 'frankenstein' female. it feeds off the host and produces numerous eggs. this adult female has a large, sac-like trunk attached by the larval head. the larval trunk is sloughed leaving a scar, but no complete moult occurs. eggs develop within the trunk sac and hatch directly into the infective tantulus larval stage. the second life cycle occurs when a free-living semelparous female is produced by an attached tantulus larva, escapes from the larva, and swims away to copulate with a similarly produced free-swimming male. these minute-crustacean parasite life cycles are unique. instead of the standard moulting cycle of all other crustaceans, the mature adults develop in the attached parasitic tantulus larva. the extreme brevity of early ontogeny seems to be an adaptation to parasitism in situations where a high dispersal ability is not advantageous. minute-crustacean parasites have a dramatic reduction in body form compared to other crustaceans, with an unsegmented sac-like thorax and a much reduced abdo men. the attached larvae and parthenogenetic females are permanently attached to their host by the oral disc with an adhesive. in the centre of the disc, they make a minute puncture, through the host integument, with their cephalic stylet. this is their only access to the body fluids of the host. the free-swimming, nonfeeding adults lack cephalic appendages but possess two clusters of aesthetascs on its anterior margin. they are free swimming and have six pairs of large thoracopods without endites. the first two thoracic somites are incorporated into the cephalothorax. the male abdomen bears a posteriorly directed, median stylet and intermittent organ. it originates on the first abdominal somite. the parthenogenetic females live longer than their sexual doppelganger and are responsible for the majority of the reproduction of their species. knudsen et al. ( ) described tantulus larvae, developing males, parthenogenetic females, and only the third developing sexual female ever found. they noted that the taxonomy of the group is based mainly on the tantulus larvae, which is quite an unusual role for a larval life cycle stage. some suggest these minute crustacean parasites, with no larval stages, are the adults of the orphan mysterious y-parasites (facetotectans), which have no known adults, only larvae. there are myriad reasons why these life cycles are, unfortunately, unlikely to fit together. scuds are a minor group of known parasites, but they have many species commensal on ascidian, bryozoan, cnidarian, echinoderm, mollusc, sponge, and crab hosts. many of these may be parasitic or at least well on their way to parasitism. they are called commensals because we just do not know enough about most of them to make a proper categorisation. whale lice and jelly parasitoids are known to be parasitic, and we believe a bivalve scud is also an unrecognised parasite. there are species in genera and one family (cyamidae) of whale lice. they have no carapace, and their bodies are dorsoventrally flattened instead of laterally compressed as in other amphipods. they are one of the few aquatic crustaceans that cannot swim in any part of their life history. molecular studies of whale lice have determined the evolution of their host whales (kaliszewska et al. ) . infections can be heavy on whales and other marine mammals (mignucci-giannoni et al. ; colón-llavina et al. ), and superinfections can harm whales. whale lice will also attach to humans handling whales. there is an easily discernible life cycle. eggs are held in the marsupium of the female. females produce more eggs in each brood as they age. a quarter to half of the eggs die in the marsupium. the eggs hatch directly into a juvenile form with no larval stages, similar to fish isopods and all peracarida. they have clawed pereopods and immediately attach to the cetacean skin. sexual maturity is usually reached after six moults. some eat their exuvia (exoskeleton remains) after moulting. the complete life cycle may take or months. whale lice parasitise cetaceans (whales, dolphins, porpoises). since whale lice cannot swim, they can only be spread by direct contact among hosts such as during mating, nursing or care giving. they feed on dead cetacean skin and algae and are attracted to wounds on hosts but more for attachment than actual feeding. they may even clean up wounds and so speed up healing. whale lice also like creases, crevices, and barnacles for attachment. by eating algae, they control its growth on their host. in general, slow swimming whales have more whale lice, than fast swimming whales. some suggest that host jumping or breaching is done in order to knock off whale lice. some online videos show human divers easily brushing whale lice off whales with only their fingers. heavy infections reportedly harm humpback and gray whales. we found a heavy infection on a sperm whale that may have contributed to its death (mignucci-giannoni et al. ). melita anmyeonensis shin, coleman, & kim, , is found between the gills and mantle cavity of the broad angel wing bivalve, barnea dilatata (soulelet, ), in south korea. scuds in six families associate with bivalve mollusc hosts but never in high numbers of adults. shin et al. ( ) found no damage to the host. kretzler ( ) could not detect obvious physical damage to sea urchins even by very heavy infections of scuds. they could not determine if the association was obligate. they called it commensalism. we feel the high prevalence ( %) and intensity [ - (av. . )] in the broad angel wing, and the lack of free-living collections suggests that it is an obligate parasite. this bivalve is no stranger to crustacean parasitism having two species of parasitic copepods. shin et al. ( ) noted host abandonment* of m. anmyeonensis from damaged and captured broad angel wings. host abandonment is a frequent strategy of crustacean parasites for finding another host. they are often called parasites or parasitoids (lafferty and kuris ; kuris et al. ) but are not exactly parasites either. we term them serial parasitoids*. in various parts of their life histories, they may be free-living, kleptoparasites, minipredators*, parasitoids, serial parasitoids, or a mix. they have an obligatory association with jellies (salps and jellyfishes) as a nursery for their young (parasitoidism), often rest on jellies (phoresis), but spend most all of their lives as minipredators (see table . ). jelly parasitoids have~ species in families. they damage and kill some gelatinous zooplankton but have no known ecological or commercial importance. now that jellyfish are held and reared in major aquaria, jelly parasitoids have become a problem. effective chemical treatments have been developed (e.g. boonstra et al. ) . mating occurs on a host jelly, males depart, and ovigerous females remain on the host while brooding the eggs. brood sizes range from to several hundred eggs. the eggs are relatively small for amphipods. larval stages are in the female marsupium. the first stage is the pantochelis larva with four cheliform pereopods and unsegmented and limbless metasoma and urosome. the pantochelis stage metamorphoses into a 'protopleon' larva (often divided into three sub-stages), having a segmented metasome and imperfect pleopods. in many species, there is no pantochelis stage, and the egg hatches directly into a first protopleon stage. the last (or only) protopleon stage gives rise to the first juvenile stage (a miniature adult) and marks the demarsupiation or the deposition by the female of the larvae (in rare cases the juveniles) into a host. during demarsupiation, the gravid female swims out to find a host and deposits one or a few larvae upon the host specimen (depending on host size and capacity). she continues infecting one host after another. some females penetrate a host, split a gonad with its mouthparts, and inserts the larvae deeply into the organ. as they grow, the juveniles leave the gonad and start feeding on the prey trapped by the host. some females deposit pantochelis larvae on the surface of salps with their specialised seventh pereopods. when the larvae moult a few hours later, the ensuing protopleon larvae enter the branchial cavity and eat its wall or feeds on the collected suspended matter. once the host can no longer support the young as they mature, they leave it for another salp in the chain. species of phronimids excavate solitary salps or pyrosomes into 'barrels' open at both ends and in which they hide and use as a nursery. the larvae are demarsupiated into the barrel where they soon bunch together into a tight cluster that slowly moves around on the inner barrel wall. the female stays with the barrel and prevents the young from passing to the outer surface. at intervals, she makes short excursions into the water and returns with prey to feed her brood. the pereopods are used to maintain the position of the animal within the barrel, and beating of the pleopods propels the combined barreljelly parasitoids through the water (bio-jet ski). some adults feed on small plankton, at least part of the time; others steal small plankton out of jellies; and some eat jelly tissues as minipredators. they certainly use jellies as places to rest. jelly parasitoids seem immune to the stings of medusa. males are better swimmers than females. gasca and browne ( ) found ten hyperiid amphipods, a copepod, and a pycnogonid in jelatinous plankton in the gulf of california. they also named a new species, megalanceoloides aequanime gasca, , based on a redescription of m. remipes (barnard ) in gasca and haddock ( ) , distributions, sizes, and a few morphological differences. we name megalanceoloides gascae n. sp. based on the descriptions by vinogradov ( ) and vinogradov et al. ( ) of 'm. remipes'. we also distinguish m. gascae from m. remipes on the basis of geographical distributions in the northern indian ocean (vinogradov ) vs southwest atlantic (barnard ; usnm , ) , south-east pacific (usnm , ), and antarctic ocean (usnm ). the former three records represent two new locality records. the two species are further distinguished based on the sizes of females, mm female (vinogradov ) vs mm male (barnard ) , and morphological differences discussed by gasca and haddock ( ) . the holotype is the specimen collected by vinogradov ( : - , by monotypy iczn : art . . ) and also in illustrated ions by vinogradov ( : figs. , ) and vinogradov et al. ( : fig. ) (iczn : art . . , . . ) . the new species is the third in the megalanceoloides remipes species complex and in genus megalanceoloides zeidler, . . isopoda: isopods . . anuropidea: jelly isopods* jelly isopods are giant, blind isopods that occur in all oceans except the indian ocean (to date) and parasitise large scyphozoans in the deep sea of the eastern pacific and japan (ohtsuka et al. ). ten species in one genus, anuropus beddard, , are known, but only two have been associated with scyphozoans. they live in and feed on their host, but very little else is known (ohtsuka et al. ). our lack of knowledge may be an artefact of destructive net collections (gasca and browne ). fish-associated isopods are relatively large as adult parasites, in comparison with most other parasitic crustacea, and are often seen by divers on the outside of marine-reef fishes (figs. . and . ) and by fishermen in the mouths and gill chambers. fish gnats* (see below) are much smaller ( - mm) and highly mobile ectoparasites on marine and estuarine fishes (smit and davies ; tanaka ) . fish gnats are not true parasites but serial parasites. four other families have been variously stated to have parasitic species. no cirolanids are parasites, some corallanids may be parasites (gentil-vasconcelos and tavares-dias ) , and, very likely, some salve bugs (aegidae) and nasal isopods* (tridentellidae) are parasites (e.g. bruce and wong ) . the life cycles of fish-associated isopods and fish gnats are so completely different that their sharing a superfamily seems incongruous. fish-associated isopods are permanently parasitic as adults and fish gnats only as juveniles. fishassociated isopods hold their eggs in a marsupium and fish gnats in pouches in the female body. fish-associated isopods attach with their pereopods and fish gnats by their mouthparts. we have collected these isopods on fishes but have not considered them to be true parasites, because so little is known about their associations. some species do feed on fish blood. gentil-vasconcelos and tavares-dias ( ) considered excorallana berbicensis boone, , to be a parasite of south america freshwater fishes and e. tricornis (hansen, ) a facultative parasite of many marine fishes. they may be correct about e. berbicensis, but we are not ready to accept e. tricornis as a true parasite (bunkley-williams and williams a). we would call it a serial parasite. very little is known about any life cycles of excorallana stebbing, . the most famous cymothoid is the so-called tongue-replacement isopod (brusca and gilligan ) , which was also featured as the monsters in the horror movie 'the bay' ( ). fish isopods may cause some problems in aquaculture (williams ; woo ) . they drastically affected fisheries in a large lake in egypt (mahmoud et al. ) . juveniles may kill juvenile fishes, and adults may seriously stunt and slow the growth of hosts (bunkley- williams and williams a; mladineo ) . fish isopods have also been shown to increase swimming drag and metabolic demand of their hosts (Östlund-nilsson et al. ). more than species of fish isopods exist in genera (see chap. ). they occur in and on fishes around the world, but mostly in the tropics and subtropics, in coastal waters, with some in freshwaters largely in south america with a few species in africa and asia (bunkley- williams and williams a; smit et al. ) , and even the deep ocean (quattrini and demopoulos ; williams and bunkley-williams ) . some almost complete life cycles are known (e.g. williams and bunkley-williams ; aneesh et al. ) . klompmaker and boxshall ( ) listed many fossil fish parasitic isopods but dismissed all due to insufficient evidence. nagler et al. ( ) claimed the oldest fossil parasitic isopod based on sucking mouthparts and legs suited for attachment, but these could be just as indicative of a minipredator. we are also in the process of describing a parasitic fossil isopod (williams and bunkley-williams , unpublished data) , which may prove equally disputable. fish isopods may go through four parasitological life cycle stages: free-living, smaller*, serial parasite, and true parasite (see table . ). brood pouch development may include egg, oblong embryo predator, curled embryo with enlarged cephalic end and tapered posterior, uneyed embryo, eyed embryo, pre-manca, and manca juvenile (williams and williams a, b) . embryology is often neglected in species descriptions and even in life cycle studies. embryos in brood pouches number from to (adlard and lester ) but are usually in the low to mid-hundreds. larger and older supra-females have more offspring. although some brood mortality is expected, bakenhaster et al. ( ) found none in glossobius hemiramphi williams & williams, . pre-manca in the marsupium of some species are larvae. mancae in, and escaping from, the marsupium are juveniles. to avoid confusion, we here refer to this as a manca juvenile*. once the manca juvenile begins to form the seventh pair of legs in - moults, we consider these instars juveniles. the few juvenile stages that we know spend the daytime in the surface plankton. they descend at night, finding transfer or definitive hosts. the first to settle on a definitive host develops through the male stage and directly into a female. the second becomes a male. this was the traditional infective assumption based on little data. however, they may actually be infected by founder pairs. mladineo and valic ( ) and mladineo ( ) found only two ceratothoa oestroides (risso, ) manca infected each annular sea bream even when more mancae were available. they attached to the fish body and migrated under the operculum, rather than being swallowed as previously suspected (e.g. bunkley-williams and williams a). supposedly, a few complete life cycles of fish isopod are known, but none are complete. first, a natural release of mancae has only been closely observed once (williams and williams c) . a female on a brown chromis in a coral reef reared her posterior from the surface of the host and released a juvenile from the rear of her marsupium, slowly lowered back down, and reared up again to release another. this process was slowly and methodically repeated, although the complete release was not observed. adlard and lester ( ) found it took - h in the lab. the juveniles swam upwards towards the surface. some authors have mistaken our burst release (williams and williams c) with the normal release process. burst release occurs when a host is caught or struck. all juveniles are released at once even if they are not quite mature (pre-manca and manca; williams and williams c) . adlard and lester ( ) caused this response in the lab with pressure on the dorsal surface of a female and found it took - min. the second problem with most 'complete' life cycles is they do not include the free-swimming juvenile stages. we have found up to six stages ; and see below). the fish isopod manca juvenile has six pairs of legs. most manca juveniles are much more setose than later juvenile stages. the final juvenile has seven pairs of legs. intermediate juveniles may have . -, . -, and/or . -leg pairs. some suggest escaping mancae require a free-swimming period before they can attach to hosts; however, thatcher ( ) found they were ready to attach to fishes as soon as they left the brood pouch, and williams and williams ( c) reported that anilocra chromis williams & williams, , and cymothoa oestrum (linnaeus, ) could attach immediately. large samples of all post-manca juvenile stages (c. oestrum) have only been examined by williams and bunkley-williams ( ) . juveniles are positively phototaxic and can be captured in light traps at night. they swim to the surface light when released from the female. they can also be captured with surface plankton nets during the day. they thus appear to avoid the diurnal planktivores on the reef. adlard and lester ( ) found they rested at the surface of the water with their hooks (dactyls) through the day, but this was demonstrated in aquaria and not in the field. they descend back to the reef at night and can be caught near the bottom with diver-towed plankton nets. some have suggested juveniles must feed within - days to survive (lester ) . however, manca juveniles, unfed, for more than a week, are still capable of infecting hosts. adlard and lester ( ) reported that only half of the mancae were infective after days. juveniles of glossobius hemiramphi and livoneca ovalis (say, ) use resting hosts before locating their final hosts. cook and munguia ( ) found mancae of cymothoa excisa perty, had a window of days to infect hosts. juveniles of l. ovalis are micro-males since an adult male has never been found. resting hosts are commonly used by many species (thatcher ) and may be a part of the normal life cycle. this predisposes them to becoming micro-males. thatcher ( ) described an interesting life cycle strategy. a manca juvenile swimming right-side up (dorsal up), stops swimming, falls to the bottom landing upside-down (ventral up), and does not move. when a small fish comes near to investigate or eat it, the manca springs to life and attaches to the hapless fish. the isopod now has either a resting host to feed on or a final host on which to mature. this 'dead bug' or 'playing opossum' behaviour, we will call the opossum attack*. thatcher ( ) found mancae could feed on and kill up to four small fishes in h. this behaviour was in freshwater isopods. mancae attach all over the body of the host and move to the normal attachment site (adlard and lester ) as we have seen (williams and bunkley-williams unpublished data). legrand ( ) suggested mancae of anilocra physoides (linnaeus, ) were attracted to the motion of fish fins. some mancae fall off when they attempt to move to the normal attachment site (williams and bunkley-williams unpublished data). cook and munguia ( ) found that manca of cymothoa excisa located hosts by visual and chemical clues. the number of juvenile moults have not been determined for most species; however, williams and bunkley-williams ( ) reported finding six post-manca juveniles in cymothoa oestrum (fig. . ) . a juvenile attaches to a host and begins to develop through a series of instars: juvenile-male transitional, immature male, male, male-female transitional, immature female, virgin female (no oostegites), and ovigerous female. the second juvenile that arrives will cease developing and remain as a male, with development hormonally controlled by the female. if the female dies, the associated male resumes developing into a female. anilocra leach, , juveniles replace solitary dead females. williams and bunkley-williams (unpublished data) have often seen a juvenile attached in the attachment scar where a female was formerly located. micro-males may be attracted to dying females and begin to develop into females protected underneath the 'cougar' (old female). williams and bunkley-williams (unpublished data) have reared juvenile anilocra haemuli williams and williams, , to females and a. chromis to male-female transitionals, on their natural hosts, in the lab. adlard and lester ( ) found anilocra pomacentri bruce, , recruited from july to december, with a peak in september to october, at heron island, great barrier reef. they never saw males with females despite intense efforts in the field and laboratory. one of their hypothetical scenarios is essentially our micro-male life cycle. aneesh et al. ( ) gave the 'complete' life cycle for cymothoa frontalis milne edwards, . however, they have the same problems discussed above: ( ) omitting how the manca are naturally released (demarsupiation) and ( ) omitting the number of free-swimming juvenile stages. they did recognise six female stages. however, their stages fs- to fs- appear to be within the first female instar, which is confusing. furthermore, size alone does not determine supra-stages or instars (see size discussion below). their fs- would be our vegetative* supra-female (sf- ). their fs- to fs- would be the second marsupial stage (sf- ). overall, this does agree with our assertation that females have more than one brood with feeding vegetative stages in between. however, we think most fish isopods have more than two broods. when all brown chromis hosts with anilocra chromis on seven m segments of a linear coral reef were eliminated, and recovery followed for a year, the hosts recruited evenly from the plankton, but the isopod recruitment was significantly higher on the ends of the linear reef adjacent to areas still populated with isopods. the same result was obtained in a subsequent year (williams and bunkley-williams unpublished data). thus, the swimming juvenile stages only appear to disperse over very short distances. several studies on cymothoid-host associations have examined impacts of anilocra on components of host fitness (reviewed in chap. ). adlard and lester ( ) found that anilocra pomacentri reproductively compromised its female host. (meadows and meadows ; welicky and sikkel ) . brown chromis males infected with anilocra chromis appear unable to maintain a spawning site (williams and bunkley-williams unpublished data). contrastingly, meadows and meadows ( ) and robinson ( ) found little to no effect on host mating success or fecundity. a tenet in the strategy of fish isopod infection has always been that the first manca juvenile to arrive becomes a female and the second remains a male (epigametic sex determination). mladineo and valic ( ) and mladineo ( ) found a pair of ceratothoa oestroides manca juveniles became established simultaneously together in the mouth of a host and excluded all other mancae. aneesh et al. ( ) found something similar with cymothoa frontalis. possibly almost all fish isopod juveniles infect hosts in juvenile pairs, which we will call founder pairs*. this may change all cymothoid infection methods. life strategies sometimes consistently 'deposit' the male and the female of a pair on different sides of a host. have described these duplex arrangements for males and females in opposite gill chambers and on different sides of a fish caudal peduncle (williams and williams ) . this allows space for larger females and larger brood pouches, allows smaller fishes to be parasitised , prevents double parasitism, and separates areas of isopod feeding and damage. female hormonal fixing of their male appears to remain intact over these distances. thatcher ( ) found this arrangement with anphira xinguensis thatcher, , in the gill chambers of the beaked pacu. occasionally, site-specific isopods are found in a different location on their hosts, and they do not migrate to their normal site. when these parasites are kept from their site because of preoccupation, we call them displaced parasites*; when their normal sites are available, we say they are accidental attachments*. for example, we have seen normally under-eye anilocra attach above the eye or on top of the head when both of their normal sites were occupied. these are the normal attachment sites for other species of isopods and may help to explain how utilisation of different sites evolved. we found longsnout bullhead sculpins*, most with female elthusa sp. isopods in their gill chambers and males underneath the females. however, we also found three males, of the same size and species, attached in two of the same hosts on the body near the opercular openings (fig. . ) . the evolution of gilldwelling isopods into external attaching isopods has been previously hypothesised (brusca ) but never demonstrated. these normal female-male gill-chamberdwelling isopods with externally attaching cohorts represent the first ever observation of an example of this event (fig. . ). when two reproductive sets of the same species occupy the same host, when normally only a single set occurs, we call it double parasitism. this often occurs with the micro-male life cycle. it is common in anilocra chaetodontis williams & williams, , but less so in other anilocra spp. we found it was detrimental with a. brillae welicky, hadfield, sikkel & smit, on red hinds and coneys, and with livoneca redmanii leach, in cero (williams and bunkley-williams unpublished data). it is rare in gill-chamber isopods, and, of course cannot occur in oral cavity ones. the duplex arrangement also prevents this situation. as is typical of isopods generally, adult females continue to grow and moult on the host. older, larger females can be quite different morphologically from males and immature females (supra-females, bunkley-williams and williams ). these differences have caused many taxonomic problems. adult females moult on the host in two parts. this is probably necessary to have some hardening of the pereopods to maintain attachment on the host. we have seen this many times on anilocra spp. where the posterior part is shed usually posterior to the rd pereon, sometimes the th. there must be a considerable delay between moult halves, or we would have never seen so many. we call these mid-moult stages. the delay allows the new hooks (dactyls) to harden before the last anterior segments are shed. we have seen mid-moult stages with a large posterior body and a much smaller anterior body (williams and bunkley-williams unpublished data). we have also seen mid-moult stages in four wild cymothoa oestrum juveniles . discerning the different juvenile stages of cymothoids is most difficult. mid-moult specimens clearly mark the lower size range of each juvenile stage. when enough specimens exist to find mid-moults, the complete juvenile life cycle can be discerned. four mid-moults in c. oestrum allowed us to discern six postmanca juvenile stages ; williams and bunkley-williams unpublished data). as far as we are aware, this is first time all juvenile stages have been identified in the wild. post-juvenile mid-moults allow the elucidation of what changed in the moult and to positively identify instars. some supra-females regain their marsupium in one moult. spectacular, mid-moult stage half-female/half-male forms have been observed (williams and bunkley-williams unpublished data), as have supra-females with half formed marsupiums, indicating two moults are needed to form a marsupium (williams and bunkley-williams unpublished data). as noted above, isopod effects on hosts often cannot be measured with fish condition factors. we studied specimens ( infected) of doctorfish with - specimens of two species of adult isopods and noted there was no difference in condition factors (williams and bunkley-williams unpublished data). the isopods cause the host to grow more slowly, but they remain proportional (proportional stunting). Östlund-nilsson et al. ( ) also found no conditional factor difference and summarised the literature on this topic. a malefemale pair of livoneca redmanii in each gill chamber of mackerel will cause a decline in condition factor and often kills the host . lanzing and o'connor ( ) also found host condition was only reduced in multiple isopod infections. however, robinson ( ) found a reduced condition in bicolor damselfish infected with single anilocra partiti williams, females, and sala-bonzano et al. ( ) found no condition factor effect of ceratothoa italica schioedte & meinert, , on sand steenbras in a protected area, but severe effects in a similar overfished (unprotected) area. the prevalence of infection was also different ( vs %, respectively). obviously, the life cycle success rate was improved by stresses on the host, particularly when the lower abundance of host specimens should have had the opposite effect. the sizes of gill-and mouth-dwelling cymothoids are closely correlated with their host size due to space constraints; externally attaching ones are slightly less coordinated. however, isopod and host sizes are related suggesting they grow simultaneously. this also suggests most hosts are infected young, by young parasites, and the host and parasite grow up together. some have suggested that females only have one brood and others that they have one brood immediately after another. in many cases, the so-called virgin female (no oostegites) was the largest of the female specimens collected williams , a) . obviously, these females are in a feeding (vegetative) stage between broods. the number of adult moults and their morphological stages are not known for any cymothoid. however, aneesh et al. ( ) found six adult female stages corresponding to two of our supra-females, including a vegetative one (see above). adlard and lester ( ) found spent (demarsupiated) females could moult, feed, rejuvenate, and lay new eggs, all in one instar. no intervening vegetative instar was necessary. evidence exists to support two life cycle portions in these fish isopods: simple and complex rebrooding. simple rebrooding* with relatively small brood sizes, where the female internal organs are only flattened by the marsupium, not atrophied, and no vegetative moult is necessary to re-establish feeding. complex rebrooding* with large brood sizes, where female internal organs are atrophied, a moult to a vegetative supra-female is necessary to re-establish feeding, and a second moult is necessary to re-establish a supra-female with a marsupium. simple rebrooding is more economical and faster but limited numerically in offspring. complex rebrooding is slower and uses more resources but produces many more offspring. adlard and lester ( ) found simple rebrooding in an externalattaching isopod. found complex rebrooding in a gill chamber-dwelling isopod, williams and williams ( a) in an externally attaching isopod, and aneesh et al. ( ) in a buccal cavity-dwelling isopod. isopods producing larger broods may require the energy resources afforded by vegetative supra-female instars. of course, many other, undiscovered, reproduction scenarios probably exist. external isopods do not live as long as their hosts, as evidenced by empty attachment scars. additionally, some isopods become covered with encrusting organisms. these are likely old isopods which have ceased moulting and are about to die. bakenhaster et al. ( ) found that glossobius hemiramphi live for a year in south florida (usa). adlard and lester ( ) found anilocra pomacentri lived for a maximum of . months. bakenhaster et al. ( ) found monthly . - . % (ave. . %) prevalence throughout a year, in ballyhoo with the highest prevalences in the summer, with small, young-of-the-year hosts. with larger hosts, the prevalence exponentially declined. adlard and lester ( ) found anilocra pomacentri changed the behaviour of the great barrier reef chromis making them not migrate with uninfected cohorts. we (williams and bunkley-williams unpublished data) observed a similar phenomenon in brown chromis infected with anilocra chromis. the infected adults stay under coral heads with the juvenile brown chromis, while the uninfected adults go out above the reef slope to feed on plankton. meadows and meadows ( ) similarly found foureye butterflyfish infected with anilocra chaetodontis stayed in low-energy areas where their offspring were more available to young potential hosts. the diel migratory reef fish, french grunt, usually migrates from reef to seagrass habitat at dusk. those infected with anilocra haemuli were less likely to migrate than their uninfected schoolmates (welicky and sikkel ) . adlard and lester ( ) interpreted this as a depression of the host reproductive response to move to spawning areas on the reef slope. we interpret the observed behaviour of brown chromis to be because the infected adults cannot feed and survive in open waters (williams and bunkley-williams unpublished data). meadows and meadows ( ) suspected this was also a host behavioural change caused by the isopod. welicky and sikkel ( ) were not certain if infected fish had less energy to migrate or if uninfected cohorts harassed them. however, all four behaviour modifications, caused by the isopods, accomplished the same availability of manca juveniles to infect juvenile and young fishes. we will call this effect nursery hiding. these are newly recognised life cycle innovations caused by parasite modification of its host behaviour. among the nine species of anilocra described by williams and williams ( ) and two from japan (williams and williams a) , no males have been reported. after juveniles were found in apparent copula with females, we assumed that juveniles were functioning as males. to test this assumption, individual juveniles were raised on their host, periodically preserved, sectioned, and stained. the infective, -leg juvenile was a functional male as were all intermediates up to and including the -leg juvenile. however, as soon as a juvenile began to obtain an adult shape (juvenile-male transitional), it began to lose its male characters. only juveniles were reproductive males. this resolved the 'mystery of missing males'. we refer to this reproductive juvenile as a micro-male. juveniles with full male characters have been found in several species, but these have never been suspected to be sexually active. many species are known to use paratenic hosts (thatcher ; bakenhaster et al. ) , which may predispose them to becoming micro-males. six-legged manca juveniles are released from the marsupium (williams and williams c) , swim to the surface in the daytime, and descend near the bottom at night. they find small hosts, which do not go to cleaner fish or shrimp (blennies, gobies, cardinalfishes), attach, feed, and moult once, or several times, into -legged juveniles. in off-reef areas in venezuela, these juveniles were found on adult glasseye (bunkley- williams et al. ) . they may sense the pheromones of receptive females, leave this micro-male host, swim to the female, crawl under her, and mate belly-to-belly. we have observed them in copula and found a semen string when they were separated. they may hide under the female either before or after copulation, return to their micro-male host, or find another small host. they may eventually locate a juvenile of their definitive host and develop into a female as their host develops. no adult male stage exists. juveniles develop directly into juvenile-female transitionals, immature females, and females. we sometimes found juveniles under females or very close to females on the final host, but usually we found them on cardinalfishes, gobies, and other small fishes (williams and bunkley-williams unpublished data) . adlard and lester ( ) found anilocra pomacentri juveniles on the final host, blennies, and cardinalfishes. they apparently spend most of their time attached and feeding on resting hosts and only visit the female to mate. this life cycle is unique, not just among isopods, but in parasites in general. we are not aware of anything similar. it is a modification of juveniles on resting hosts probably driven by cleaner pressure. it seems to be widespread and highly successful. while we have not seen males associated with many other anilocra, some anilocra do have associated males. possibly, this life cycle and other host specificity differences will place the micro-male anilocra in a different genus. we are exploring this possibility. other cymothoids share this life cycle, e.g. livoneca ovalis (williams and bunkley-williams unpublished data), anilocra apogonae (see fogelman and grutter ) , and a. pomacentri (see adlard and lester ) . the advantages of this live cycle have allowed anilocra spp. to be the only large, external cymothoid isopod of larger caribbean coral reef fishes (fig. . ) . nerocila spp. occur around but are unable to penetrate any distance into the caribbean (bunkley- ; and renocila spp. only infect a few small species (fig. . ; williams and williams ) . the apparent advantages of this life cycle include the following: . it allows the female to grow larger on the host because no resources are used by a male partner (fig. . ). . two females may be supported by a host, instead of a male-female pair (fig. . ). . larger females produce more offspring and two females twice as many. . it protects vulnerable juveniles from cleaners. . it produces greater genetic diversity as many micro-males are available at different times to fertilise each female, instead of one permanent male partner. . the abundance and close proximity of numerous micro-males solve the problem of finding a mate. . micro-males form a quick reserve available for developing new females. . micro-males become sexually mature quicker than true males. . the energy necessary for a female to hormonally control a male partner is unnecessary. . larger-growing females quickly become too large for cleaner organisms to threaten. . micro-males can parasitise smaller hosts that do not seek out cleaner organisms. . micro-males can easily change host specimens and/or species and are thus more flexible and resilient. . micro-males could potentially use up the resources of one host and just move to another with little effort or danger. . the lack of host specificity in micro-males allows them to exploit a broader and more available food supply and to be more flexible and resilient. . the time a planktonic reproductive form has to locate a final host is almost infinitely extended by being able to exploit a broad range of more available smaller fishes. . copulation while on the outside of a fish host is easier and safer with a tiny micro-male than with a larger and more bulky 'true' male. . this life cycle also resolves the classic question of the expense of sexual reproduction, since all adults produce eggs. . it maintains, or even multiplies, the advantage of diversity in sexual reproduction while not sacrificing the productivity advantage of asexual reproduction. new life cycle: prey-predator transfer life cycle* many intriguing questions regarding the cymothoid life cycle still exist. these include the following: how can little, slow, juvenile fish isopods (cymothoids) possibly chase and infect fast-swimming pelagic fishes? why do juvenile isopods infect and develop into non-swimming forms in the mouths of fishes too small for them to develop into adults? juvenile isopods may not chase large, fast hosts, but rather rest and wait for small, slow ones. are little hosts dead-ends or 'bait'? apparent prey-to-predator transfer has been observed in king mackerel , shortfin smooth lanternshark , and red lionfish (aguilar-perera et al. ). connors et al. ( connors et al. ( , experimentally showed sea lice would abandon their host, when it was attacked by a predator (~ % of the time), and reattach to the predator. juvenile isopods infect the mouths of common, small, easily infected, 'bait' fishes. they feed and pass through moults from the -leg juvenile to a post-juvenile, non-swimming stage on this transfer host. at any point during this development, if the host is eaten by a predator, they flee their (prey) host and attach in the mouth of the predator host (final host). the first isopod to arrive becomes the female and the second a male, as was once thought to happen in all other cymothoids (mladineo and valic ; mladineo ; aneesh et al. ) . isopods in trap, net, and trawl fisheries are well documented to frequently abandon their host and sometimes enter a different fish host. three cases of natural prey-to-predator isopod transfers have been described aguilar-perera et al. ) , and adult and juvenile isopods have successfully, experimentally, been transferred between the same and different host species (e.g. . the attack of a predator or its feeding action may dislodge an isopod from a transfer host or the isopod may abandon the host. swallowing a single prey fish may be too fast for a transfer, but with a mouthful of prey fishes, adequate time may exist for a transfer. all transfers need not be successful, just enough. cymothoa spp. are quite common (~ %) in the two most abundant bait fishes in the caribbean and in five small species of cardinalfishes in okinawa. can all these juveniles be 'wasted' in dead-end hosts? they cannot develop into adults in these small fishes, and they can no longer swim. we have collected these juveniles from the plankton and found they attach to any available fish in aquarium experiments. is this a desperate survival mechanism or something more? we have described burst release (williams and williams c) , which is female isopods reacting to predator attack by dumping, and thus saving all her juveniles in her marsupium. these juveniles immediately attach to anything including humans. if juveniles react this way to attack, and adults are known to switch hosts, why would juveniles in a prey host not transfer to a predator? in field experiments, we found juvenile isopods first attached all over host fishes and then crawled to their adult positions. transferring juveniles could attach anywhere in the mouth or throat of predators and then crawl to their adult position. in contrast, adult transfers attach wherever they can and do not migrate aguilar-perera et al. ). infecting small, slow-swimming, schooling, abundant fishes seems rather easy as evidenced by~ % success rate. infecting fast-swimming, pelagic fishes seems almost impossible. we have observed many diverse cases where the isopods appeared to slow down their hosts and make them more susceptible to predation. in this case, it is a benefit for the completion of the parasitic life cycle and another form of a parasite modifying the behaviour of its host. the proposed life cycle occurs in cymothoa spp. and livoneca ovalis and may also occur in others. it may also occur as a supplemental life cycle to isopods that also have normal life cycles and could explain some of their unusual hosts. isopods of most predacious fishes infect juvenile hosts and mature with them. only a few older host specimens are infected. these could have been parasitised by preypredator transfer. this life cycle is unique, not just among isopods, but in parasites in general. we are not aware of anything similar, although pascual et al. ( ) reported an accidental prey-predator transfer in decapod isopods. the transfers may have begun as accidents and then gradually evolved into an important pathway. this allowed isopods to infect a variety of hosts that they could not have possibly otherwise reached. fish gnats are small ( - mm) ectoparasites on marine and estuarine fishes (smit and davies ; tanaka ) . as protelean parasites, they are only parasitic as juveniles and, due to their typically brief associations with hosts, may best be termed 'micropredators' (e.g. lafferty and kuris ) . fish gnats are perhaps best known as the main food of cleaner fishes on coral reefs (grutter ) . approximately species of fish gnats (most in gnathia leach, ) in genera and a single family are known around the world from the marine shallows to the deep sea. they occur at all latitudes but are more diverse and abundant in the tropics. the study of fish gnats has had a 'split personality' until recently, with benthic ecologists studying the adults and parasitologists the juveniles. only recently has the morphology of juveniles been included in the taxonomy (hadfield et al. ; farquharson et al. ) . however, the life cycles of six species are known (smit et al. ; tanaka ; hadfield et al. ; hispano et al. ) . even the most basic life cycle of fish gnats has only very recently been discovered (fig. . ; smit and davies ) . zuphea are the first juvenile stage of fish gnats that leave the female, find a host, gorge and swell up with blood, and become the second stage called 'praniza'. the praniza eventually drops off the host and finds a secluded place on the bottom to develop into the next zuphea (z ). z swims up, finds a host, and repeats the cycle (p ). zuphea feeding times vary from a few hours to a few days with z s taking the longest. they can be as short as an hour on coral reef fishes. zuphea may attack, feed off (snack*), and kill larval or juvenile fishes as smaller predators*. eventually, p moults into an adult (fig. . ) . in some species, in genera elaphognathia monod, , gnathia, and paragnathia omer-cooper & omer-cooper, , this occurs in one moult. in one species in genus caecognathia dollfus, , the first moult results in a pre-adult, which later moults into an adult. we call these feeding and metamorphing units serial parasites, which is somewhat similar to the life cycle found in ticks (arachnids). praniza may only stay on bony fishes for a few hours but on sharks and rays for weeks. complete life cycles vary from short to lengthy, directly in relation to seawater temperature: polar (~ - years), temperate (~ years males,~ year females), and tropical (~ months) (smit et al. ) . life cycle fluctuations appear to be seasonal in most species but could be dependent on host availability in some. adults are benthic, nonfeeding, and semelparous. global warming could make them more successful and more damaging (hispano et al. ) . some z have mouthparts too small to swallow blood cells and must feed on lymph. at z , they can feed on blood (hispano et al. ) . ota et al. ( ) appear to have solved the mystery of why p (praniza iii) were only found on elasmobranchs. they collected hundreds of praniza i and ii of gnathia trimaculata coetzee, smit, grutter, & davies, on bony fishes (teleosts) and hundreds of praniza iii only on sharks. this host switch seems to be the life strategy of all fish gnats so far found on sharks and rays. praniza iii takes the most time to feed. attaching to an elasmobranch protects this stage from cleaners. several recent studies on australian and caribbean recently settled, larval, coral reef fishes found gnathiid micropredation [¼ our smaller predator] damaged and/or killed them (artim et al. , and references therein) . this can have great importance in fish recruitment (artim et al. ) . sikkel et al. ( ) suggested gnathiids are micropredators [¼ our minipredators*] but act like parasites by not leaving the host during each larval life cycle instar (protelean). barnard, on its host fish, clinus superciliosus (linnaeus, ). image from smit et al. ( ) fish gnats are also implicated in the spread of a parasitic protozoa such as haemogregarina bigemina laveran & mesnil, , between hosts (smit and davies ) . juvenile fish gnats on fishes often cannot be identified morphologically as the taxonomy is based on adult males, although this is beginning to change in recent studies (jones et al. ) . fish gnats on coral reefs appear most active at night (sikkel et al. ) , although this may not be the case in other environments. at least in the eastern caribbean, diel activity appears to be related to both life history stage and sex (sikkel et al. ) . fish gnats appear to be generalists. they are known to parasitise families in orders of fishes; however, some fishes are infected more often and more heavily (tanaka ; coile et al. ). this could be attributable to some combination of preference, host susceptibility, and/or host behaviour. fish gnats may even appear on pelagic fishes . for example, amberjack (carangidae), which fed near the bottom, have been infected with fish gnats, and fish species which did not feed near the bottom were free of gnathiids (williams and bunkley-williams unpublished data). coile et al. ( ) found fish gnats, which fed on more susceptible hosts, produced larger, presumably more successful offspring. fish gnats can attach all over the body of fishes. smit et al. ( ) suggested where they first attach is where they stay. some studies suggested site preferences, but whether this is by selective settlement or migration is unknown. fish gnats are repelled from the skin of toxic gobies by their poison glands but do attach to their fins. it seems likely that they also avoid the toxic skin of trunkfishes (ostraciids, fishkill toxin) and also puffers (tetraodontids, fugu toxin). adult fish gnats do not feed. males attract females, usually young females with pheromones, and there are some reports of males defending or acquiring harems of females (smit and davies ) . in reality, of the few relationships known, in only two species have males been reported with many females. males do appear to fight with the big, impressive, giant mandibles, at least not under lab conditions. brood sizes vary from a few to almost and may vary by female size and environmental conditions (coile et al. and citations) . we suggested that another reason mudskippers leave the water during low tides was because fish gnats are concentrated in the remaining, small, tide pools (williams et al. ). the small atlantic cleaner gobies, with small mouths, swallow fish gnats whole. in contrast, the bigger, indo-pacific cleaner wrasses, with larger mouths, appear to bite them into pieces . some of these cleaners selectively feed on larger gnathiids (grutter ). with implications for our understanding of cleaning symbioses, cleaner fishes often do not eat the fish gnats that are not gorged with blood. is this because the blood-swollen ones are more easily found? or is this due to preference by cleaners for fish blood rather than gnats without blood? the category 'epicardeans' was formerly considered a suborder but is now an infraorder placed in suborder cymothoida. it contains described species of crab isopods and cryptic isopods. crustacean isopods are almost unique in using crustaceans as their intermediate and final hosts, with the exception of a few corallanids and a cirolanid (bruce pers. comm.) . williams and boyko ( ) call them partial castrators because reproduction is often not completely blocked. this is nutritional sterilisation, not hormonal sterilisation. boyko and williams ( ) reviewed the methods to find, collect, and preserve crustacean isopods. cryptic isopods are poorly studied, but interesting, with most of their species hyperparasitic on other isopods in their own order, or in parasitic barnacles (fig. . ) . they also parasitise a variety of free-living crustaceans. there are species in genera and nine families. they are of little commercial interest, except as potential hyperparasitic controls for other crustacean parasites. adult female cryptic isopods usually have neither pereopods nor oostegites. their epicaridian larvae must develop within the female since there is no marsupium. her body ruptures to free the larvae. they find and attach to copepods, and their life cycle is the same as in decapod isopods, except the males stay in the cryptoniscus larval form. cryptic isopoda are ecto-, meso-, or endoparasitic. liriopsids are hyperparasites of parasitic barnacles and parasites of other symbiotic crustaceans. lovrich et al. ( ) found liriopsis pygmaea (rathke, ) ( fig. . ) infected . % of the externa on the parasitic barnacle, briarosaccus callosus boschma, , parasitising false king crabs in argentina. these were mostly ( of ) cryptonicus larva. this suggests to us that most of the infective larvae, even finding a correct host, fail to infect the host. larvae were highly aggregated with . % inside empty externae, suggesting these sites attracted cryptonici. only a few early subadult females, late subadult, and one late subadult were found along with adult females. parasitic barnacles recovered from hyperparasitic sterilisation once the cryptic isopods died. cabiropsids, with species, are parasitic on free-living isopods and hyperparasitic on other crustacean isopods. the family may also include a few parasites of cumaceans. hemioniscids (barnacle isopods*), with eight species, are parasites of barnacles. dajids (backpack isopods*), with species, are external parasites of krill (euphausiids and mysids) and midwater shrimp; however, ohtsuka et al. ( ) found an endoparasitic species alternate host sharing with a copepod (see copepod section above). the parasites look like backpacks on their hosts and are often mistaken as fleshy growths or tumours. a few occur in the gills. cyproniscids, with species, are hyperparasitic on parasitic barnacles (some formerly liriopsids), occasionally directly parasitic on decapod hosts, and parasites (hypersymbiotic) on symbiotic crustaceans. podasconids (amphipod isopods*), with four species are parasites of amphipods. asconiscids only have a single species parasitic on a mysid. crinoniscids, with three species, also only have one species on a cirripede barnacle. they are parasitic on sessile and pedunculate thoracican barnacles. entophilids, with two species, are endoparasitic in callianassid shrimp and munidid squat lobsters. other cryptic isopods, besides the one we found (williams and williams ) , hyperparasitically infect the brood pouches of isopods. stone and heard ( ) found a new cryptic isopod in the serial fish isopod* excorallana delaneyi stone & heard, . many species of cryptic isopods remain undescribed. there are more than species of decapod isopods (bopyroidea). members of families bopyridae and ionidae cause a noticeable swelling of the gill chamber or carapace . the bulges they cause in the carapace of decapods make them among the most impressive and distinctive crustacean parasites. the deformities named kanthyloma crusta klompmaker, artal, van bakel, fraaije, & jagt, (ichnotaxa-trace fossil evidence), date these parasites in the fossil record back to the lower jurassic ( . - . million years ago; klompmaker et al. ) , and they have been found in~ species of fossil decapods (klompmaker and boxshall ) . they slow the growth and nutritionally sterilize some commercially important crabs and can cause the collapse of a population but have also been used as bioindicators (williams and boyko ) . eggs develop into free-swimming larvae within a ventral brood pouch (marsupium) formed of lamellar outgrowths of the female pereopodal coxae (oostegites). larvae of a single brood mature synchronously and are released simultaneously as microniscan larva (epicaridium, microniscus or microniscid). the microniscan attaches externally to a pelagc calanoid copepod, pierces its cuticle to feed on its blood, and undergoes six moults and becomes a cryptoniscan (cryptoniscus and cryptoniscid). when it drops off its copepod host, it seeks a crab or shrimp definitive host in the earliest post-larval stage. on the host, it develops into a juvenile (bopyridium) and then into a female. the first cryptoniscan arriving at a host becomes a female and the second, a male (epigametic sex determination). the female attracts a male with pheromones and hormonally controls it to remain a male similar to cymothoids. the female grows large, while the male remains a dwarf attached to the female. they are ectoparasites. females look nothing like isopods, except for some vague segmentation. males look more like isopods. they attach in the branchial chamber of their hosts. some adults do not moult to grow. partial or complete sterilisation of hosts is due to energy loss from parasitic feeding, not hormonal control as in parasitic barnacles. it is temporary and reversible once the parasite is gone. williams and boyko ( ) summarise the papers following the introduction of a nonindigenous decapod isopod, a drastic population decline of a mud shrimp, and possible collapse of a north-west pacific ecosystem. williams and boyko ( ) found double parasitism of two species in the gills and abdomen of a specimen of hermit crab in indonesia. only eight species are known and are ectoparasitic on the gills or under the abdomen of ghost shrimp. similar to bopyrids, they cause a noticeable swelling of the gill cavities. in most respects, they are like the decapod ectoparasitic isopods (above). there are species of crab mesoparasitic isopods. they are mesoparasites in the haemocoel of brachyuran and anomuran crabs. they make a small, chiselled hole through the host's exoskeleton to communicate with the environment. they release larvae, through an exit pore near the base of the fourth pereopods of the host, which follow the typical bopyrid life cycle. apparently, this also involves a copepod intermediate host, and they presumably settle as cryptoniscid larvae in the branchial chamber and then penetrate their hosts. they first become endoparasitic and later mesoparasitic. however, their life cycle is poorly and incompletely known (williams and boyko ) . females produce a posterior stalk that extends to the external environment of the host through the branchial region or eyestalks. as in bopyrids, females look nothing like isopods, except for some vague segmentation. males look more typical. some adults do not moult to grow. partial or complete sterilisation of hosts is due to energy loss from parasitic feeding, not hormonal control as in parasitic barnacles. they have been suggested as biological control agents for introduced crabs (williams and boyko ). kuris et al. ( ) suggested portunion maenadis (giard, ) could be used as a biological control of the green crab. squid are intermediate hosts for marine anchor worms and have been reported to attract many forms of crustacean parasites accidentally. pascual et al. ( ) surmised a prey-predator transfer of larval isopods when patagonian squid ate the intermediate host copepods. their cryptoniscus larvae successfully penetrated and colonised the squid. they were entirely embedded in the oral bulb at the beginning of the oesophagus of two female squid. they suspected this was only accidental parasitism; however, this could represent a strategy for host switching, new resting host, or even eventual speciation. a shift of phyla in hosts is always of interest. tanaids are a large, free-living group with only one species, exspina typica lang, , often found in the intestine and body cavity of deep-sea holothurians, assumed to be a parasite (e.g., kudinova-pasternak ) . alvaro et al. ( ) confirmed it was a parasite. many species associate with invertebrates and hexapleomera robusta moore, , even with sea turtles and the caribbean manatee from which we have collected it. most are similar to fish cymothoid isopods with a free-swimming manca leaving from the marsupium. two manca life cycle stages of one species were found in the gut of a deep-sea polychaete in the florida straits (suárez-morales et al. ). these stages are probably endoparasitic. the adults show few modifications to a parasitic life. however, the full reduction of maxillule setation only occurs in e. typica, and the bifurcated and sharply tipped dactyli in the pereopods is probably a parasitic adaptation for anchoring the crustacean in the soft tissue of the host (alvaro et al. ). the unmodified life cycles would suggest a parasitic mode of life is in the early developmental stages. many tanaidacean species make sand tunnels. this behaviour could have predisposed them to making tunnels in the body wall of holothurians. several families of shrimp are well known as associates of other invertebrates, notably sponges and corals, also bivalve molluscs and echinoderms. these species are generally categorised as commensal and not considered parasites as such. a review of these taxa is beyond the scope of the present work. shrimps* shrimp that inhabit sponges are usually referred to as commensals largely because very little is known about their habits. only a few parasitic species have been determined from two superfamilies; however, many more species are parasitic and probably not just in sponges. small, eusocial synalpheus spence bate, , carry one to a few dozen large eggs that hatch directly into benthic juveniles (duffy and macdonald ) . large, malepaired synalpheus sp. females release several hundred small, planktonically dispersing nauplii from a clutch (duffy and macdonald ) . eggs hatch into nauplii, which are feed on yolk reserves (lecithotrophic larvae), and metamorphose into zoeae. zoeae feed on algae (planktotrophic larvae) and metamorphose into myses, which look like tiny adults, and feed on algae and zooplankton. the final instar is post-larvae. typton carneus holthuis, , form heterosexual pairs and exclude conspecifics and other shrimp (negative precursor). duffy et al. ( ) found synalpheus regalis duffy, , excluded heterospecific shrimp. he also demonstrated this species had a colony hierarchy with only one reproductive female and hundreds of helpers. this was the first case of eusociality noted in a marine animal. six more, probably parasitic, species in the same genus have been found to practice eusociality. we suggest these are the first known eusocial parasites*. williams and boyko ( : fig. a ) illustrated a crab isopod, probably bopyrella harmopleon bowman, , on a sponge shrimp, synalpheus fritzmuelleri coutière, , from panama. this may be a hyperparasite, but we cannot be certain. we do not know if the shrimp was collected from a sponge and this shrimp species is not an obligate parasite. anker ( ) published a photograph of a decapod isopod on synalpheus brevicarpus (herrick, ), which is definitely a hyperparasite. several other species of sponge shrimp have hyperparasitic decapod isopods. duffy et al. ( ) showed that shrimp of the synalpheus gambarelloides group, and s. regalis, in particular, were sponge parasites. Ďuriš et al. ( ) studied typton carneus in caribbean fire sponges in belize and found it was parasitic. they also examined two other species of typton costa, , and three species in three other genera of palaemonids (pontoniinae) from the western and eastern atlantic and indo-pacific and one alpheid from the indo-pacific and found them to be parasites. their results suggested parasitism by sponge shrimp was widespread. zitzler and cai ( ) reported the first obligate sponge shrimp*, caridina spongicola zitzler & cai, , in freshwater. this spectacularly coloured, now popular aquarium, shrimp infects an undescribed spongillinid sponge in an ancient lake in indonesia. we believe it is also the first obligate parasitic sponge shrimp reported from freshwater. zitzler and cai ( ) only found diatoms in six shrimp stomachs and called them commensal. however, they have a life cycle like eusocial sponge shrimp [a few ( - ) black eggs directly develop and are released as immature adults with no planktonic phase]. have numbers (up to /host) like eusocialists. we doubt diatoms alone would support any shrimp, much less this many. all marine obligate sponge shrimp, studied thus far, are parasitic. true crabs do not make very good parasites. we would call them semi-parasites or kleptoparasites, at best. we believe this is because their basic adult structure is so very unsuited for parasitic adaption and modification. coral gall and pea crab parasites have a long fossil record but remain low in diversity. their life cycles also show no adaption for parasitism. they mostly steal their host's food, may slow host growth, but do little, if any, physical damage to the host. many reside at the uneasy border between parasitism and commensalism. we have seen indicators of the potential for the development of 'better' parasitism, for example, a superinfection of crab zoea living in the gills of a gray angelfish at mona island, puerto rico, and adult burrowing crabs living in the gill chambers and feeding on the gill filaments of two gray snappers in colombia . however, these examples were rare, in incapacitated hosts, and proved impossible to duplicate. jelly crabs benefit by their life cycles associating with gelatinous plankton through protection, transportation, food, and development faster in warmer waters and saving energy (towanda and thuesen ) . their relationships with their hosts are more complicated. ohtsuka et al. ( ) summarised the symbionts of gelatinous plankton. they found crab larvae were only associates, not parasites. however, at least four jelly crabs feed on their hosts, and they spend their entire life cycles on one host. this seems to us to be protelean parasitism. they also steal the food of their hosts, which seems to us kleptoparasitism. we know too little about most jelly crabs to determine their kind of symbiosis. towanda and thuesen ( ) closely studied one species, graceful rock crab, but did not diagnose their type of symbiosis. not much is known. adults are benthic and planktonic zoea search for and attach to jellyfish. the megalopae and juveniles develop on the host. eventually, the cypris drops off the host and develops into adults. sometimes hundreds of megalopae occur on the host. fewer juveniles are seem, which might suggest some cannabilism occurs (towanda and thuesen ) . jelly crabs steal food and eat tissues of their hosts; however, they eat jelly parasitoids, which greatly benefit their hosts. is parasitism rated as a proportion of good vs evil? we do not think so. this is mutualistic parasitism*. klompmaker et al. ( ) objected to the existing common name 'gall crab' because so few actually form real galls. whether these crab are parasites or commensals, if they damage corals, and even what they eat remains unknown (vehof et al. ). there are species in genera and a single family, which form galls in shallow reef corals and in some deep-water corals (castro ) . we have seen these crabs on coral reefs all over the world. they have separate sexes, different internal fertilization, and mate belly-to-belly. mating takes place just after the female has moulted and is still soft. females only mate once and store the sperm to fertilise all their batches of eggs. the eggs are released onto the female's abdomen, below the tail flap, secured with a sticky material, and protected there during embryonic development. females with eggs are called 'berried' (as are all egg-carrying decapods) because the eggs resemble round berries. when development is complete, the female releases the newly hatched larvae into the gall; they pass out into the water and become part of the plankton. zoea have a tall dorsal spine and may have additional spines for predator deterrence. the zoea of most species must find food (planktonic), but some crabs provide enough yolk in the eggs that the larval stages can live off the yolk (lecithotrophic). larval development is scarcely known for coral enveloped crabs but is thought to consist of at least five, and possibly seven, planktonic larval stages (van der meij ). different species may have various numbers of zoeal stages, separated by moults, before they change into a megalopa stage. this last larval stage resembles an adult crab, except for having the abdomen (tail) sticking out behind. megalopae settle in hole, cracks, or creases in living corals. after one more moult, the crab is a juvenile. the coral grows around the crab, and somehow the crab forms a gall to its particular specifications. females become sexually mature after the th instar in the gall but continue to enlarge until the th instar. they somehow manage to enlarge the gall. they produce multiple broods of eggs fertilised by the first mating. many feed on mucus secreted by the corals, inadvertently a little coral tissue, and detritus. some filter feed. males are smaller than females. females cannot leave their gall, but males can. sometimes pairs live together in one gall, in adjacent galls, or even in interconnected galls. they cause no real harm to corals. these crabs form their gall to their own size and design. related crab species form similar galls; therefore, the galls have phylogenic importance (wei et al. ) . some galls are too closed to be accessible to males. these females copulate before the gall closes, store sperm, and produce up to eight broods over the next months (vehof et al. ) . they found evidence of recent mating of females in more open galls. castro ( ) discussed all the publications concerning the food habits of gall crabs. they were largely based on supposition, and castro concluded, like vehof et al. ( ) , that we still do not know what they eat. he also found the question of their types of symbiosis unresolved. badaro et al. ( ) observed mucus feeding in the laboratory and suggested all enveloped crabs fed this way and that they are not parasites. we disagree with badaro et al. ( ) because corals are their obligate hosts and they feed off coral-produced products, if not tissues, which the corals need. that they cause little harm to corals may be true but is not relevant to their type of symbiosis. the most famous species in this group is the oyster pea crab. they are cosmopolitan, but more common in the tropics and subtropics, and speciose with species in genera and families. they are tiny soft-bodied crabs that live parasitically largely in the mantle of bivalve molluscs and in a few large gastropods, sometimes inside sand dollars and sea urchins, in the rectum of sea cucumbers, tubes of parchment worms, burrows of mud shrimp, and gills of sea squirts (castro ) . some have been reported commensally in, on, or in tubes or burrows of various invertebrates. many of these relationships are not well studied, and some may be parasitic. they retard the growth of some commercial molluscs by % causing serious problems and millions of dollars in losses in aquaculture (trottier and jeffs ) . many males venture out of their hosts to visit females in other hosts, mostly at night. this is because hosts are more active and sensitive during the day and can squash males. trottier and jeffs ( ) observed males being crushed. additionally, they are subject to predation less in the dark, than in the light of day. however, we documented the first record of predation on a male, and probably at night, since it was by a cardinalfish, on a coral reef in okinawa (williams and williams c) . pea crabs find females by their pheromones. male crabs sometimes must rub, or tickle, bivalve mollusc hosts for hours to make them open up (trottier and jeffs ) . once in the host, they copulate with the female, who never leaves the host. thus, females can become relatively larger (still only pea-size) to produce eggs, while the polygamous males are smaller and flatter to sneak in and out of hosts. in hosts with more roomy accommodations, males may reside with females. females carry egg masses attached to the pleopods, where they develop until they hatch into zoeae. usually, there are five zoeal stages, but some only have one. the zoeal and one megalopal stages are usually separated by only a few days. hernández et al. ( ) found extended parental care and the suppression of the free-living megalopa in a species associated with western atlantic ascidians. they undergo a complex metamorphosis during the post planktonic development. the male passes through two forms after the invasive stage, the pre-hard stage with a soft bare, carapace, and no swimming setae on the pereopods and the hard-stage with a hairy, hard carapace and natatory setae on some legs. the female goes through two very similar stages, only differing in the number of abdominal appendages, and five more feminine stages. population biology of few species has been studied, those in tropical and subtropical regions reproduced year round and in temperate regions seasonally during the summer. reproduction and the presence of juveniles were not related to water temperature or salinity. the greatest abundance of juveniles (pleopods poorly developed) occurred just after the peaks of abundance in gravid females. very few recruits (megalopal i) were found (peiró and mantelatto ) ; therefore, they must pass through this stage quickly. the female-juvenile correlation also suggests they do not disperse very far. de bruyn et al. ( ) examined dissodactylus primitivus bouvier, , ectoparasitic on two spatangoid echinoid (heart urchins) hosts, meoma ventricosa (lamark, ) and plagiobrissus grandis (gmelin, ), which have the entire life cycle on m. ventricosa and only adults on p. grandis, but with more fecundity. crabs detected hosts by olfactory cues. crabs from p. grandis were more attracted by this host (where the entire life cycle can be fulfilled, possible imprinting). crabs from m. ventricosa are equally attracted to either host. host switching may explain asymmetrical infection rates and specialisation on p. grandis may be in progress. jossart et al. ( ) characterised pea crabs ectoparasitic on sea urchins at discovery bay, jamaica, in which both sexes changed hosts, searched for sexual partners, and had a polygamous mating system. most mate by polygynandry between large females and wandering small males, although some by monogamy, or temporary monogamy between adults of similar sizes, and a few by swarming of males (castro ) . ambrosio and baeza ( ) found the pea crab, tunicotheres moseri (rathbun, ), did not attempt to infect previously, conspecific infected ascidian hosts, styela plicata (lesueur, ), to avoid conflict, even though this host was scarce and defence of the host was minimal. this is another variety of negative precursor. we wonder if defence of the host was once fiercer, incited this avoidance, and then faded with non-use. castro ( ) listed many damages attributed to pea crabs. the most common was slowed growth, and the most harmful, sterilization and sex reversal. he concluded that most associations were parasitic and only some more loosely associate forms in tube-or burrow-dwelling hosts might be commensal. the california bay pea crab has the distinction of being one of only two marine crustaceans on the iucn red list (wikipedia), and it is the only possible parasite on this list. the life strategies of most parasitic crustaceans are not very modified from their freeliving ancestors. with a few notable exceptions, their life cycles suggest they just do not make very good parasites. most would seem to have recently evolved into parasitism, yet fossil evidence shows otherwise. the most grossly modified females still metamorphose from simple life cycles. even the most successful group, the copepods, is still hindered in exploiting different classes of hosts by the simplicity of their life cycles. the rigidity of their life cycles seems a major limitation of crustacean parasites. part of this apparent situation may be a result of our lack of knowledge and understanding. here we described four new and innovative life cycles, complex rebrooding, mesoparasite, micro-male, and prey-predator transfer; four instances of a new life cycle host behaviour modification, nursery hiding*; a common, but undescribed, life cycle form, mid-moult stage*; two instances of parasite intraspecies facilitation, positive precursor*; parasite intraspecies antagonism, negative precursor*; an ambush life cycle strategy, opossum attack*; doubling of the normal reproductive set on a host, double parasitism*; and separated male-female pairs, duplex arrangement*. we also named and redescribed a known life cycle, simple rebrooding*. possibly, many more life cycles and modifications remain to be discovered. however, these still represent rather minor modifications. crustacean parasites lag far behind the other major parasite groups in both complexity and modification of their life cycles. mid-moult* individuals are an important new means to identify juvenile life cycle instars. they are also very useful in determining exactly what changes occur in a moult and can also be used to distinguish between supra-female instars. our term displaced parasites* refers to life cycle forms which matured in the wrong locations on their host due to their normal sites being occupied. this is also linked to superinfections*, which are mass infections of many parasites on or in a host (often resulting in the death of the host and the parasites). these occurrences strain the fabric of normal parasite relations, and life cycles, often revealing unimagined changes and trends madinabeitia and nagasawa ; ismail et al. ) . we used the term proportional stunting* to describe the slowing of growth in fishes caused by fish isopods (cymothoids). this cannot be evaluated by host condition factors, which have confused many into believing these parasites do little harm. actually, they are quite detrimental and cause major economic loses to aquaculture and commercial fisheries. the hypothesis of the first crustacean cymothoid being external attaching and then forms moving into the gills or mouth has not been supported by molecular phylogeny. the gill chamber appears to be a much more inviting and less hostile habitat for initial colonisation, as we have seen in two cases of unusual crustacean infections . our displacement cases seem to also suggest this scenario. schmid hempel ( ) did not find well-adapted parasites became harmless but rather more efficient at countering the defences of their host. many recent authors have suggested parasite evolution favours virulence. however, we report the first hyperparasite ever known to evolve into a mutualist and explain the pathway. poulin ( a) thought that parasites with few adaptions could revert to a free-living existence but cited few examples. he found no parasite reversals* to mutualism. predation has only recently been shown to occur within parasitic crustacean life cycles and cause damage and death of hosts. parasitism and predation are difficult enough to distinguish when isolated, much more so within a life cycle. as we learn more details of life cycles, predation may become important phases. we attempt to define the types involved (table . and annotated glossary (sect. . ) below). cleaners feeding on fish gnats (gnathiids) may be accessory vampires. they may more easily find and/or select larger, swollen fish gnats filled with blood. eckes et al. ( ) suggested cleaners benefited more from consuming fish mucus than fish gnats. we certainly believe they benefit more from eating blood-filled gnathiids, than ones without fish blood. we found copepod pre-adult life cycle stages were common on western pacific fishes but very rare on caribbean ones. this indicates that smaller cleaner gobies may be more efficient than larger cleaner wrasses and a factor in parasites completing life cycles. contrary to the literature, we find the wormlike copepod on sea turtles, manatees, and whales are not parasites. we described how pennella exocoeti may have speciated. flying fishes are food for many offshore large predators, which host pennella species. flying fishes were probably a downward incorporated intermediate host for pennella spp. at one point. eventually, a form became isolated and speciated into pennella exocoeti on flying fishes. fish isopods (cymothoids) seem on the verge of evolving a real intermediate host. fish gnats (gnathiids) may also be exploring intermediate hosts through micropredation. a new ergasilid copepod seems to be becoming an endoparasite. tongueworms are completely parasitic with no free-living stages, endoparasitic in an intermediate and a final host, and so modified to parasitism we cannot even equate their life cycle stages to free-living ones. all other parasitic crustaceans are incompletely parasitic in one way or another. therefore, these life cycles suggest tongue worms are not crustaceans. they further suggest tongue worms are not even related to crustaceans, and this needs further investigation. fish lice and tongue worms have long been suspected to be related on the basis of their sperm morphology. recent molecular work also finds them similar. their life cycles are the same in having no free-living larval stages but are otherwise worlds apart. fish lice are not even good ectoparasites, flitting around from host-to-host as juveniles and adults with females free-living off hosts. tongue worms are good endoparasites every step of their way. the life cycles of fish lice and tongue worms suggest they have no phylogenetic relationship. the few fossil 'tongue worms' only recently discovered are larval parasites of marine invertebrates. equating these with tongue worms of present terrestrial vertebrates with no larval stages is impossible. these fossil tongue worms may be related to extant tongue worms, but they are not their ancestors. a parallel, and completely separate, evolution is more likely. octopus copepods (harpacticoida) and tunnelling tanaidaceans (tanaidacea) have similar life histories tunnelling through the tissues of octopuses and sea cucumbers, respectively. they also represent the rare parasitic forms in their largely free-living orders. both also have commensal species on sea turtles and manatees. however, their life cycles are quite different (lópez-gonzález et al. ; alvaro et al. ) , and they reside in different classes of crustaceans. their modes of living and feeding must represent parallel evolution. anchor worms (lernaeids) and marine anchor worms (pennellidae) are an astonishing example of parallel evolution. so much so that they were originally classified together in lernaea linnaeus, . they are also the only copepods to make major host group switches: amphibia and reptilia by the anchor worm and mammalia by the marine anchor worm. even their life cycles are similar with intermediate hosts, except that it is not an obligate intermediate in the anchor worm. furthermore, the anchor worm is freshwater, and marine anchor worms are marine, and they are classified in different orders. lafferty and kuris ( ) recognised four life cycle strategies (a bit too simple) and ten trophic strategies. poulin ( b) arranged all parasites into six major life strategies (see table . ). all categories, except vector transmission, apply to crustaceans. poulin and randhawa ( ) further defined and defended the categories but made little more use of them. half were named for transmission methods and half for effects on the hosts, which seems confusing as they are not, necessarily, mutually exclusive. in order to standardise his terms, we rephrase parasitoid to 'adult injection transmitted', parasitic castrator to 'larval penetration/injection transmitted', and table . ). we also add a seventh strategy. most microbial parasites have no animate transmission agent. they contaminate potential hosts in incidentally shed host products or by long-lived free-living stages. some symbionts on sea turtles and manatees spend their entire life histories on their hosts, never leaving, never transmitted. poulin and randhawa ( ) call their categories 'dead ends', but we do not believe micropredator is necessarily a parasitological dead end. if we use the seven new transmission-standardised names to categorise crustacean parasites: . adult injection transmitted-larval parasitic copepods (sect. . . ) have a life cycle similar to parasitoids but do not kill the host, and jelly parasitoids (sect. . . ) are almost parasitoids. . larval penetration/injection transmitted-some parasitic barnacles (sect. . . ), some echinoderm copebarnacles (sect. . . ), and some crab barnacles sterilise their hosts. sterilisation is not one strategy but two. hormonal sterilisation is a permanent, parasite chemical control of a host. nutritional sterilisation is a temporary parasite use of the host resources to the extent that host reproduction cannot occur. . directly transmitted parasite-represents almost all of the crustacean parasites. poulin and randhawa ( ) found these forms were the simplest and least parasitically evolved of the parasites, which agrees with our analysis. . trophically transmitted parasite-is only found in tongue worms (sect. . , which are probably not crustaceans). marine anchor worms (pennellids) have this strategy, except the intermediate host is not eaten by the trophically higher predator. our prey-predator life cycle follows this strategy, except the parasite juvenile only moults to an adult in a paratenic (not intermediate) host, before it is eaten. . vector-transmitted parasite-does not occur in crustacean parasites. . micropredator transmitted parasite-occurs in fish lice (sect. . ), sea lice (sect. . . , caligids), jelly parasitoids (sect. . . ) and some juvenile fish isopods (sect. . . ) that are micropredators. . inanimate transmitted parasite-occurs in non-swimming fish lice (sect. . ) and whale lice (sect. . . ). williams and bunkley- made the first, large-scale comparison of caribbean and western pacific parasites of coral reef fishes using the same collection and examination techniques. in terms of crustacean parasites, they found less aegid associates and more tongue worms in the pacific. cymothoids, fish gnats, copepods, and barnacle associates were approximately equal. in contrast, non-crustacean parasites were less diverse and abundant in the pacific than the caribbean. fish lice, tongue worms, larval parasitic copepods, isopods, and whale lice lack larval dispersal stages. fish lice and isopods have free-swimming juveniles to make up for this limitation, somewhat. larval parasitic copepods, fish lice, and sea lice have free-swimming adults. many recent authors seem to equate, or even confuse, the complexity of parasitism with its severity. sometimes these do go hand-in-hand, but they are different. complexity makes parasites more resilient, adaptable, and in the case of marine anchor worms more able to switch major host groups. severity is how voraciously and efficiently parasites use host resources to successfully reproduce the most. some of the most severely parasitic crustaceans actually have rather simple life cycles (e.g. parasitic barnacles and sarcotaces). the elaborate modification of adults is also sometimes equated to severity. again, these may co-exist but are different. many crustacean parasites, in general, seem to infect young hosts and 'grow up' with them. this has been shown in many parasites where the younger hosts, even planktonic juveniles, are much more often parasitized than the larger, older ones. in addition, host tissues growing around their parasites indicate long-term association. younger hosts are easier to find and infect often occurring in inshore schools. our prey-predator transfer may be one of the only ways older host can be infected. to evolve towards greater parasitism, the life cycle stages of crustaceans must be less and more. they must be less like the free-living crustacean stages (e.g. fish lice and fish isopods) and/or must add more parasitic stages in real intermediate hosts (e.g. tongue worms). they can either convert their free-living stages to parasitism or metamorphose new parasitic stages. those few that have developed parasitic larval or juvenile parasitic stages are progressing. however, the free-living adults many retain must become parasitic. adults must also become more modified and adapted to a parasitic existence. crab barnacles have done a pretty good job of this, and a few copepod adults are well modified, but in general, crustaceans have done a terrible job of adapting to and exploiting parasitism. currently, we know only a minute fraction of the crustacean life cycles. additional studies may turn what we think we know on its head, upside down, or throw it out the window. we are fooling ourselves that the little we know is typical. we expect most known group life cycles are not only unknown but probably false. not only do we know few life cycles, but we also know few of the species in most groups, and these may have even different life cycles. also, major groups are still springing into existence. life cycle study is a field where magic is still awaiting discovery. crustacean and nematode parasites will soon dominate metazoan parasitology. we call the present dominating parasites (flatworms, tapeworms, thorny-head worms, etc.) as "climax parasites". they are ancient, stable, and perfect to take maximum advantage of the current conditions. unfortunately, they are also practically unchangeable in having no free-living forms to become parasitic, no adults in invertebrates, major parasitic modifications, and low species abundance. we call crustaceans and nematodes (ecdysozoans) as "transitional parasites". they would eventually evolve into climax parasites but now possess just the opposite of the climax characters stated above. the climax parasites have traded flexibility and the ability to innovate for stability and the maximum parasite experience. they are rigid and vulnerable with exposed multiple hosts and complex life cycles, unable to make major host group shifts, life cycle reductions or additions, and too involved in, and dependant on, stability. major global changes will leave them behind, at worse in extinction or, at best, in remnant triviality. crustaceans and nematodes will inherit the new world of parasites. an astonishing number and variety of recent authors misapply and misuse common life cycle terms. this inaccuracy creates misunderstanding and undermines scientific precision. we here precisely define these terms. abandon host-when parasites evacuate a host that has been captured, injured, incapacitated, or poisoned. accidental attachment-(a) a host-specific parasite rarely attaching to an accidental host or (b) a site-specific parasite rarely attaching in a different position. accidental (incidental) host-(a) a host on which the parasite cannot complete its life cycle; (b) also used for very rarely infected hosts on which the life cycle can be completed. aegathoa-a juvenile genus used for cymothoidae juveniles that could not be identified to genus and as such is a form genus. this should no longer be used, but inexplicably, recent uses exist. aesthetascs-chemosensory organs on the antennae and other structures of larval crustaceans used to locate appropriate hosts, virgin females, etc. aggregation-(a) usually refers to the distribution of parasites within hosts. often, these are not uniformly distributed among host but aggregated in a few hosts; (b) can also refer to a group of hosts or to a cloud of parasite infective stages. alternate host sharing-a life history strategy where two different species of parasites infect the same host, but not at the same time. ambushing-a manner of searching for hosts used by larvae and free-swimming adults, generally, resting still in the water column arched upwards at a body angle and then suddenly attacking an unaware host. used in the daytime by some forms. opossum attack is a form of ambushing. see cruising and hover and wait. androdioecy-having dwarf males and larger hermaphrodites. the small male is usually attached on or near a larger hermaphrodite. antennae-the second pair of antennae, usually longer than the first. antennulae-the first pair of antennae, which are usually shorter than the second. biological control-an agent killing or sterilising unwanted organisms. biphasic moult-a moult occurring in two posterior (first) and anterior (second) parts. this occurs in all peracarida. see moult, mid-moult stage. body-is in three sections: the head (cephalon), thorax (pereon), and abdomen (pleon). see metasome and protosome. bopyridium (juvenile)-the third life cycle stage of epicaridian crustacean isopods. brood mortality-loss of some individuals in developmental stages in the marsu-called a capsule. in some cases, both contain a parasite. some life cycle forms are encysted and/or encapsulated. see gall. definitive host (final host, primary host)-the usual, or normal, host of the parasite and the one on which it can complete its life cycle. demarsupiation-the releasing of the swimming or infective or reproducing stage from the marsupium (brood pouch). direct life cycle-involves a single host. direct develop larvae (crawl-away larvae)-larval stages that have very low dispersal potential and usually appear like the adult form of the animal. eusociality-a colony system where only the queen is reproductive and is served and protected by relatives. life cycles are direct lacking a planktonic stage. sponge shrimp (carids) are the first known eusocial parasites. exoskeleton-(a) the outer covering of crustaceans; (b) the old shell left after moulting or the exuviae. externa (plural: externae)-the outside, egg-, larvae-holding, sac of a female of parasitic barnacles (rhizocephalans). exuviae (formerly only plural, but exuvia is coming into use)-see exoskeleton. facultative intermediate host-infecting the host is not necessary to complete the life cycle. facultative parasite-may parasitise a host, but the host is not necessary to complete its life cycle. feeding stage-see vegetative stage. feminisation-parasitic barnacles (rhizocephalans) change the pleon configuration of male crabs they infcct into the female configuration to better protect their externa. cryptic isopods (cryptoniscoids) residing in the marsupium of other isopods may control their host to retain this brood pouch. final host-see definitive host. founder pair-two infective stages attaching to a host together, excluding other cohorts, and successfully developing into a male and female pair (e.g. cymothoids). fugu toxin-tetrodotoxin (ttx) in puffers, which repels gnathiids from the skin of puffers, and we believe some other crustacean parasites. gall-a plant term. it is sometimes misused for large, parasitic cysts. usually galls contain more than one parasite, often both sexes and life cycle stages. see cyst. good parasite-see true parasite and strict parasite. growth stage-a distinctly different morphological form not resulting from a moult. some pre-adults may qualify. many crustaceans have different growth stages as adults age or grow in size. see supra-female. hover and wait-a form of ambush host seeking employed by fish lice (branchiurans) during the day. see ambushing. hormonal sterilisation-a permanent, parasitic chemical control of a host. see nutritional castration and partial sterilisation. host hopping-is adults, and/or larval life cycle stages, leaving one host and finding the same, or another, within a life cycle stage. host switching-can be (a) a long-term evolutionary change usually to quite a different kind of host; (b) part of the prey-predator transfer life cycle sequence; (c) casual parasite survival (e.g. connors et al. connors et al. , ; or (d) even within a life cycle (ota et al. ) . hyperparasite (epiparasite)-a parasite parasitising another parasite. hypersymbiotic (episymbiotic)-a symbiont, usually a parasite, associating with/ infecting another symbiont, usually a commensal. immature-a young individual and another name for post-larva or juvenile. indirect life cycle-see complex life cycle. infective stage-the larval stage, which attaches to or injects infective material into a host. in many forms a cypris. infection-(a) parasitologically, a parasite or microbe invading a host (formerly referred only to endoparasites; see infestation); (b) medically, invasion of endoparasites; and (c) microbiologically, invasion of an organism that multiplies within the host (e.g. bacteria, fungi, protozoans, viruses). infestation-(a) parasitologically, ectoparasite on host can still be refer to parasites in the environment; (b) invasion of any parasite; (c) medically, sometimes used for ectoparasites; (d) medically, sometimes refers to the initial stage of parasite invasion followed by infection; and (d) common usage, aggregations of pests (e.g. mosquitoes, rats). inquiline-an animal living in another species of animal's nest, burrow, den, or resting place. instar-a larval stage between moults. for example, there are usually five naupliar instars. intermediate host-a host that a larval stage infects, feeds on, and undergoes at least one moult upon. see micro-male host, resting host, and transfer host. interna (plural: internae)-the internal and anastomosing part of a female parasitic barnacle (rhizocephalans). iteroparous-females have multiple broods, e.g. cymothoids. juvenile (immature, immature adult, post-larva)-a miniature adult. in cymothoids, the juvenile escaping the female marsupium has six pairs of legs (six-legged juvenile). see larva and manca. juvenile-like larvae-look like adults with four pairs of active thoracopods, but no suction disks. they are larvae of fish lice (branchiurans). kentrogon-an infective stage, metamorphosed from a crypis, of parasitic barnacles (rhizocephalans). the kentrogon injects a vermigon. kleptoparasites-steal the food or food stores of its host. larva (plural: larvae)-an immature greatly differing from the adult form, which must go through a metamorphosis to be an adult. some authors use the terms juvenile and larvae interchangeably; however, these are different and distinct stages, which should not be confused. lecithotrophic larva-do not feed but use their yolk for nourishment. leg-see pereopod. lice (singular: louse)-technically phthirapteran insects; fish lice are branchiurans; and sea lice are either micro stinging jellyfish (since ), bird schistosomes (since ), or caligid copepods (since~ ); and whale lice are cyamid amphipods. tongue-eating louse is an incorrect name for the famous tonguereplacing isopod. life cycle-development from conception until the organism produces its own offspring. its study often emphasises stages of development. see life history. life cycle abbreviation-the brooding or elimination of normally free-living larval stages. life cycle truncation-the elimination of parasitic larval life stages. life history-reproductive strategies and traits plus other key events in the life of an organism. see life cycle. lifestyle-the mode of life. habit (behaviour) and habitus (form). mobility, feeding, nutrition, habitat, activity period, etc. major host group switch-parasitising a new class of host. manca juvenile (plural: mancae) (aegathoa, manca, micro-male, pullus ii)-the post-larval juvenile of many pericardians (not amphipods), which leaves the brood pouch. see aegathoa. marsupium (brood pouch)-formed of lamellar outgrowths of the female pereopodal coxae (oostegites). this pouch is under the ventral body surface of the female. see complex rebrooding and simple rebrooding. marsupium stages-various egg, embryo, and larva stages. this embryology is seldom considered in life cycle studies. megalopa-a post-larval crab. extreme transformations occur in this stage. mesoparasite-(a) inside their host but retains a pore or hole connecting to the outside; (b) half-in-and-half-out as anchor worms (lernaea); and, quite confusingly, (c) also used for parasites that enter host orifices (ear, nares, etc.). the latter might be less confusingly called orifice parasites. metanauplus (pleural: metanauplii)-see nauplius. metanauplus-like larvae-are similar to crustacean nauplii, but not metanauplii because of post-mandibular appendages and differentiated first thoracopods. they are larvae of fish lice (branchiurans). metasome (tail)-the posterior part of the protosome. micro-male-a -, . -, . -, . -, -leg juvenile functional male of anilocra spp., livoneca ovalis, and possibly other cymothoids. micro-male host-a resting host for some juvenile fish isopods (cymothoids) on which moults from the -leg to -leg micro-male stage can occur. this is not an intermediate host because no larval stages are involved. the micro-male leaves this host to fertilise a female on the final host. it may return to this host specimen or another micro-male host. microniscan larva (epicaridium, microniscus or microniscid)-the first larval stage of epicaridian crustacean isopods. micropredator-like a mosquito; a small predator feeding on a big host and not spending much time with its macroprey. predator less than times size of prey. see minipredator, smaller predator, and table . . mid-moult stages-a form of half delayed moulting found in some parasitic forms that must stay firmly attached to a host. the first half moults as usual, but the second half moult is delayed until the first half hardens. minipredator-a small predator that may, or may not, kill its prey and spends little time with it (feeds and leaves) but is greater than / of its host's size, but less than / (e.g. gnathiids, cirolanids, some corallanids, some leeches). see micropredator, smaller predator, and table . . molt (apolysis, ecdysis)-the splitting of the outer covering (cuticle carapace, etc.) in order for a larger and/or different form to emerge in the life cycle. outside the usa, the preferred british spelling is moult. multiple parasitism-when one host is infected with two, or more, different species of parasites representing, three or more reproductive sets. see double parasitism. mutualistic parasitism-when the symbiont is both harmful and helpful. similar to sickle cell anaemia and malaria. mutualistic symbolism-when both host and associate benefit from the other. natural history-the interactions of an organism with its environment that influence behaviour, forms, function, and abundance. see life history, life cycle, and lifestyle. nauplius stage (plural: nauplii)-is characterised by the use of the appendages of the head (antennae) for swimming. this first series of larvae has been shared by almost all crustaceans for the last half billion years. negative precursor-when one species of parasite or commensal infects a host first and causes a second species of parasite to be less successful in infecting the same host. see positive precursor. non-swimming larvae-the odd larvae of chonopletis, a fish louse (branchiurans). nursery hiding-infected adult hosts staying with juveniles or young of the same species instead of migrating elsewhere with non-infected adults. nutritional sterilisation-is a temporary parasite use of the host resources to the extent that host reproduction cannot occur. see hormonal castration and partial castration. nymphs-are small - -legged, larval forms of tongue worms (pentastomids). obligate parasite-is a species that must infect a particular host to complete its life cycle. onychopodid larva-replaces the on-host copepodid larva in gonophysema gullmarensis bresciani & lützen, , a copepod parasite of a tunicate. oostegites (marsupium coverlets)-see marsupium. opossum attack-when a manca juvenile swimming dorsal side up, stops swimming, falls to the bottom, lands dorsal side down, and does not move. when a small fish comes near, the manca springs to life and attaches. see ambushing. orifice parasite-a term we prefer in place of mesoparasite for parasites living in natural orifices or openings in hosts. orthonauplius-a nauplius but has a shorter head, antennula, and two additional pairs of limbs. ova-eggs. overdispersed (aggregated, clumped)-in parasitology is a distribution with a higher variance than expected (e.g. when most hosts have a few parasites, but a few have many). pantochelis larva-the first larva of jelly parasitoids (hyperiideans) with four cheliform legs (pereopods) and an unsegmented and limbless metasome and urosome. it metamorphoses into a protopleon larva. prey-predator transfer host (prey host)-a resting host for some juvenile fish isopods (cymothoids) on which moults from the six-legged manca juvenile to a post-juvenile stages can occur. primary host-see definitive host. proportional stunting-parasites slowing the growth of their host in a way that cannot be discerned in length-weight condition factors. protelean organisms-have larvae that are parasites, usually endoparasites, and free-living adults. protopleon larva-the second larval series of jelly parasitoids (hyperiids) with a segmented metasome and imperfect pleopods. it metamorphosed from a pantochelis larva and gives rise to a juvenile. pseudoparasite-our term for something that almost everyone accepts as, and calls, a parasite, but is not (e.g. most leeches). pullus ii (plural pulli)-see manca larva. pupa-the unique, cigar-shaped, endoparasitic nauplii of larval parasitic copepods (monstrilloids). reproductive stage-see vegetative stage. recently encysted (copepodids - )-pandarid copepodid stage (lewis ) . see late encysted. resting host-a host on which infective larvae or juveniles rest and feed. larvae do not go through a metamorphosis on this host. see intermediate host, micro-male host, and transfer hosts. sea lice-caligoid copepods that infect marine aquaculture fishes, especially salmon. many recent authors have called them fish lice, which is incorrect. see lice. secondary host-a host less often infected than the preferred host or definitive host but on which the parasite may complete its life cycle. semelparous-females that have only one brood. serial parasitoid-similar to a parasitoid in brooding its young in a host, but may use and kill more than one host as a nursery, and also may feed on, and kill, multiple hosts, as an adult. serial parasite-an obligate parasite that largely depends on host, or hosts, for nutrition. it is not simply a minipredator, because it associates with a host longer than to feed and leave and has one or more life cycle stages that exist multiple times potentially on and off the same or different hosts. see temporary parasite. simple life cycle-see direct life cycle. simple rebrooding-new marsupial reproduction after a single moult. the internal organs are only flattened by a brood pouch with a moderate number of off spring. the female can feed as soon as she moults. see complex rebrooding. site specific-when a parasite almost always attaches in the same position on or in a host. see accidental attachment. smaller predator-not a great deal smaller than its prey, attaches to, feeds on, and kills the host. it is an, hitherto, unrecognised life cycle phase (minipredator phase) in parasitic crustacean life. see micropredator, minipredator, and table . . spillover-a term for the parasites, principally salmon sea lice, produced in fish farms, infecting native fishes. 'spillover' suggests the pen is so full of free-swimming, infective parasites (copepodids) that a few are forced out. see filtering. sterilisation-the act of making an animal unable to reproduce. see partial sterilisation, hormonal sterilisation, and nutritional sterilisation. strict parasite-see true parasite. superinfection-a mass infection with about as many parasites that can fit on or in a host. this usually results in the death of the host and the parasites. supra-females (post-adult stages, supra-adult)-stages after the first adult female stage with size and/or morphological differences with the first adult and each other. these can be growth stages or instars and often occur in crustaceans. supra-males-see supra-females. tantulus larva-the only larval stage of minute crustacean parasites (tantulocarids) with a dorsal head shield, six-segmented thorax, and biramous urosome. temporary parasite-(a) a facultative parasite; (b) a parasite that survives for a time after ingestion by a host species other than its customary host; (c) a minipredator, associated with a prey item just long enough for it to be called a host but largely free-living and rarely found associate with a host; or (d) a parasite with any freeliving stage. thoracopod-see pereopod. transfer host (prey host)-a resting host for some juvenile fish isopods (cymothoids) on which moults from the -leg manca juvenile to a post-juvenile stages can occur. this is not an intermediate host because no larval stages are involved. see paratenic host, micro-male host, and prey-predator life cycle. transitional parasite-a crustacean or roundworm (nematoda) in the clade ecdysozoa. they are relatively new parasites with many free-living relatives and little evolved towards a climax parasite state. see climax parasite. transport host-see paratenic host. trichogon-injected into a female by a male crypis, of parasitic barnacles (rhizocephalans), and becomes a dwarf male. trophic transmission-see parasite-induced trophic transmission. true parasite (good parasite, strict parasite)-a small organism, which feeds from and harms a larger organism. true parasites have a definitive host, which they never leave. true predator (carnivore)-an organism that hunts, kills and eats other organisms (prey) . see ambushing, opossum attack, and vector mutualists-when a disease or parasite transmitted to a host also benefits the vector. when crustacean parasites are vectors, the infective stage of the crustacean benefits by the invasive transferred organism confusing the immune system of the host and increasing infective crustacean survival. see viral crustacean mutalists. vegetative stage-is a term we borrowed from botany to describe a feeding and nonreproductive female stage of fish isopods (cymothoids) between stages with a marsupium. see complex rebrooding. vermigon-a migratory internal stage injected by the kentrogon stage of a parasitic barnacle (rhizocephalans). viral crustacean mutualists-a virus benefiting its crustacean host and doing little or no harm to the host. virgin female-a term used in the literature for an adult female lacking a marsupium. a vegetative stage is a better term since these stages can occur both before and after stages with a marsupium. whale lice-see lice. y-cypris (plural: y-cyprii)-see y-nauplius. y-nauplius (plural: y-nauplii)-the first larval stage of y-parasites (facetotectans) and y-cypris the second. ypsigon-a slug-like, unsegmented, and limbless form, which may be the infective stage for y-parasites (facetotectans). zoea stage (plural: zoeas or zoeae)-the first larval stage of a decapod characterised by the use of the thoracic appendages for swimming and a large dorsal spine. zuphea-the first juvenile of fish gnats (gnathiids) with obvious segmentation, which leaves the female, finds a host, feeds, and becomes the second stage, a praniza (p ). common names are listed alphabetically in bold with and family names in square brackets: and the larvae feed off the host and eventually kill it and escape as adults. larval parasitic copepods (monstrilloids) have a similar life cycle, but do not kill their host. paratenic host (transfer host, transport host)-like an intermediate host parthenogenesis-asexual reproduction by females without the need of males. partial sterilisation-a term used by williams and boyko ( ), as 'nutritional castration', where some reproduction still occurs. pereon-the thorax of the thorax (cephalothorax, pereon) of crustaceans. periodic parasite-acting like a facultative parasite, on a host for short periods of time, but free-living most of the time. does not obtain most of its nutrition from a host or hosts. not a serial parasite, temporary parasite, or true parasite. permanent parasite-is an obligate parasite, which spends more than one generation without leaving a host. see temporary parasite. planktotrophic larva-feeds on plankton ) the abdomen (pleon) of crustaceans. positive precursor-when the presence of one parasite facilitates infection by another. the interspecific form is fairly common; however, intraspecific forms are rare. this is when one species of a parasite or commensal infects a host first and causes a second species of parasite to be more successful in infecting the same host post-larva (plural: post-larvae) (immature, juvenile, pre-adult)-resembles the adult and characterised by the use of abdominal appendages (pleopods) for propulsion. praniza (formerly pranzia)-see zuphea predisposition-(a) some hosts of crustacean parasites appear to be predisposed to the presence of parasites (williams et al. ); (b) also may refer to a pre-existing condition or behaviour preferred host-see secondary host and definitive host pre-larvae (pre-zoae)-the first stage after an egg hatches. usually held in a marsupium in isopods pre-manca-the isopod brood-pouch stage after eyed embryo before manca and is less developed and has less setae. prevalence collection effect-when infected hosts are impaired by their parasites and are more readily to be collected (e.g. dip net, seine, trawl), or less likely to be collected (e.g. hook and line, baited traps), than uninfected ones. prey-predator transfer-fish isopods (cymothoids) from prey fishes can transfer to the predators that eat them brown mussel, perna perna (linnaeus, ) epinephelus chlorostigma (valenciennes, ) california bay pea crab, parapinnixa affinis holmes caribbean fire sponges, tedania spp trichechus manatus manatus linnaeus scomberomorus regalis (bloch, ) common fish louse, argulus foliaceus (linnaeus, ) epinephelus fulvus (linnaeus, ) cressey's sea louse, caligus rogercresseyi caranx hippos (linnaeus, ) acanthurus chirurgus (bloch, ) coryphaena hippurus (linnaeus, ) epaulette shark, hemiscyllium ocellatum (bonnaterre, ) false king crab, paralomis granulosa (hombron & jacquinot, ) chaetodon capistratus linnaeus haemulon flavolineatum (desmarest, ) heteropriacanthus cruentatus (lacepede, ) [priacanthidae]; goldfish, carassius auratus (linnaeus, ) graceful rock crab, metacarcinus gracilis (dana, ) (cancer sometimes still used) serranus tigrinus (bloch, ) japanese louse (also goldfish louse), argulus japonicus thiele jelly isopod, anuropus spp pomacanthus arcuatus (linnaeus, ) lutjanus griseus (linaeus, ) eschrichtius robustus (lilljeborg, ) green crab, carcinus maenas (linnaeus, ) king mackerel, scomberomorus cavalla (cuvier, ) micropterus salmoides (lecepede, ) marine anchor worm, pennella spp non-swimming fish lice, chonopeltis spp balaenophilus manatorum oyster pea crab, zaops ostreus (say, ) loligo gahi (orbigny, ) cichla ocellaris (bloch & schneider, ) [cichlidae]; pink salmon, salmo gorbuscha (walbaum, ) tegastes acroporanus humes epinephelus guttatus (linnaeus, ) pterois volitans (linnaeus, ) holacanthus tricolor (bloch, ) epinephelus adscensionis (osbeck, ) salmon sea louse, incorrectly called 'salmon louse', lepeophtheirus salmonis (krøyer, ) sea firefly, cypridina hilgendorfii (müller, ) 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(copepoda: philichthyidae), a parasite of a blacktip grouper, epinephelus fasciatus, from off the ryukyu islands, southern japan million years old "marine lice" and the evolution of parasitism within isopods branchiura -a compendium of the geographical distribution and a summary of their biology interactions of caligid ectoparasites and juvenile gadids on georges bank a new species of balaenophilus (copepoda: harpacticoida), an ectoparasite of a sea turtle in japan symbionts of marine medusae and ctenophores symbiosis of planktonic copepods and mysids with epibionts and parasites in the north pacific: diversity and interactions copepoda: pennellidae) from aquarium-held marine fishes in japan, with notes on its occurrence and life cycle in captivity genomic characterization and phylogenetic position of two new species in rhabdoviridae infecting the parasitic copepod, salmon louse (lepeophtheirus salmonis) caught in the act: phenotypic consequences of a recent shift in feeding strategy of the shark barnacle anelasma squalicola (lovén, ) studies on sarcotaces sp. (copepoda, philichthyidae) infestation (black bag disease) among some marine fish species of arabian gulf, saudi arabia parasitic isopod anilocra apogonae, a drag for the cardinal fish cheilodipterus quinquelineatus third-stage larva shifts host fish from teleost to elasmobranch in the temporary parasitic isopod copepoda: siphonostomatoida: nicothoidae) with the morphological descriptions of the nauplius, copepodid and adult male biological associations between barnacles and jellyfish with emphasis on the ectoparasitism of alepas pacifica (lepadomorpha) on diplulmaris malayensis (scyphozoa) first report of an endoparasitic epicaridean isopod infecting cephalopods population dynamics of the pea crab austinixa aidae (brachyura, pinnotheridae): a symbiotic of the ghost shrimp callichirus major (thalassinidea, callianassidae) from the southwestern atlantic diseases caused by crustacea argulus ambystoma, a new species parasitic on the salamander ambystoma dumerilii from mexico (crustacea: branchiura: argulidae)(pdf) global diversity of fishlice (crustacea: branchiura: argulidae) in freshwater the many roads to parasitism: a tale of convergence evolution of parasitism along convergent lines: from ecology to genomics ectoparasitism on deep-sea fishes in the western north atlantic: in situ observations from rov surveys on the origin of a novel parasiticfeeding mode within suspension-feeding barnacles ceccarelli ps ( ) a novel microhabitat for parasitic copepods: a new genus of ergasilidae (copepoda: cyclopoida) from the urinary bladder of a freshwater fish impact of a mouth parasite in a marine fish differs between geographical areas arthropod molecular divergence times and the cambrian origin of pentastomids dwarf males in the epizoic barnacle octolasmis unguisiformis and their implications for sexual system evolution evolutionary parasitology. the integrated study of infections, immunology, ecology, and genetics discovery of a new species of melita (amphipoda: melitidae) associated with barnea dilatata (bivalvia, mollusca) from south korea diel infestation patterns of gnathiid isopod larvae on caribbean reef fishes nocturnal migration reduces exposure to micropredation in a coral reef fish first record of sebekia oxycephala (pentastomida: sebekidae) infecting helicops infrataeniatus (reptilia: colubridae) a -million-year-old silurian pentastomid parasitic on ostracods the curious life-style of the parasitic stages of gnathiid isopods a systematic survey of parasites of freshwater fish in africa -crustacea life cycle of the temporary fish parasite, gnathia africana (crustacea: isopoda: gnathiidae) global diversity of fish parasitic isopod crustaceans of the family cymothoidae ostracod research at the lake biwi museum nocturnal attacks on nearshore fishes in southern california by crustacean zooplankton excorallana delaneyi, n. sp.(crustacea: isopoda: excorallanidae) from the northeastern gulf of mexico, with observations on adult characters and sexual dimorphism in related species of excorallana stebbing an aggregation of monstrilloid copepods in a western caribbean reef area: ecological and conceptual implications diversity of the monstrilloida (crustacea: copepoda) the copepod balaenophilus manatorum (ortiz, lalana, and torres, ) (harpacticoida), an epibiont on the caribbean manatee occurrence and histopathological effects of monstrilla sp. 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(copepoda: harpacticoida) from the antarctic fish rhigophila dearborni dewitt key: cord- - qck j w authors: martin, jennifer h.; clark, julian; head, richard title: buying time: drug repurposing to treat the host in covid‐ h date: - - journal: pharmacol res perspect doi: . /prp . sha: doc_id: cord_uid: qck j w in fedson stated …. “for almost two decades, leading scientists and health officials have warned that we must prepare for a potentially devastating global pandemic of an infectious disease. initial concern was focused on …h n …. more recently…a devastating outbreak of ebola virus..(and) several other emerging viruses are believed to seriously threaten global health and global security. to prepare, scientists have been urged to discover new vaccines and treatments for these emerging viruses. at the same time, political leaders have been urged by global health experts to invest millions in a “top down” restructuring of the global health system. this article takes a different view. it focuses on an alternative approach to the scientific discovery of treatments for individual patients, reviews the mechanisms of action and clinical experience with specific drugs that might be useful, and considers whether or not recent lessons regarding this “bottom up” approach to treatment have been learned”. now with a new virus and pandemic upon us, fedson's comments appear chilling, are cause for reflection on what we have learnt and importantly offer focus on an immediate opportunity in the area of treating the host (fedson ds, ann transl med, ; : ). has driven huge morbidity and mortality globally, destroying much of the social and economic fabric of societies. the immediate application of evidence-based research and its strategic timing (distinct from evidence-based medicine) is essential to enhance patient survival. for this to occur, a strategy must correctly be aligned with unmet need and this is where an international research strategy can build on success. currently there is discussion on two key issues in this covid pandemic; first, physical isolation, testing (current), and barrier isolation, which is fundamental; second, vaccine and/or antiviral development (possible long-term future). in physical isolation and impact, some countries appear to be doing very well. likewise, there is outstanding global research capability and scientists in epidemiology, modeling, immunology, antiviral development, and diagnostics. within the urgency of a pandemic we are still not achieving treatments with actual efficacy data for prevention, treatment or cure. realistically, a vaccine may never be developed as planned, or potentially take in fedson stated …. "for almost two decades, leading scientists and health officials have warned that we must prepare for a potentially devastating global pandemic of an infectious disease. initial concern was focused on …h n …. more recently…a devastating outbreak of ebola virus..(and) several other emerging viruses are believed to seriously threaten global health and global security. to prepare, scientists have been urged to discover new vaccines and treatments for these emerging viruses. at the same time, political leaders have been urged by global health experts to invest millions in a "top down" restructuring of the global health system. this article takes a different view. it focuses on an alternative approach to the scientific discovery of treatments for individual patients, reviews the mechanisms of action and clinical experience with specific drugs that might be useful, and considers whether or not recent lessons regarding this "bottom up" approach to treatment have been learned". covid , drug repurposing, global collaboration, host response, renin-angiotensin of | commentary a further - years to complete necessary studies, and finalise the regulatory pharmaceutics dossier, but even then, time is still needed to find funding to manufacture, upscale, and develop supply lines to roll it out globally. these future hopes are happening against a backdrop of the extremely urgent need to have effective treatments on the wards here and now. we also need an approach to provide a therapeutic insurance with the inevitable easing of social isolation in the absence of vaccines and antivirals. finally, in seeking effective antivirals one of the barriers to a rational drugs design program for humans is the disconnect between the time and resources required to conduct well-controlled preclinical and clinical studies and those of biomedical researchers dealing with a global healthcare crisis in real time. the arguments for buying time by utilizing the principles of treating the host are compelling. in the first instance, we would suggest the world needs to have a much more effective approach to using well-tested drugs and their dosing schedules that can block a dysregulated innate immune response at clinically tolerated doses. an approach based on treating the host built on sound physiology and pathophysiology, together with thorough administrative data input and accepted principles of drug repurposing based upon pharmacology and clinical pharmacology is needed. it follows that the immediate unmet need and opportunity sits between these two domains of social isolation and antiviral/vaccine development. it involves pharmacological-based approaches to treating the host (not necessarily the virus) in an acute setting. treating the host in a viral pandemic context has been highlighted at least years ago. , a "treating the host" approach has the potential to enable infected people to survive an acute pulmonary/vascular inflammatory dysregulation with a decreased call on high end health resource. this approach is increasingly recognized as being important to patient care. a critical aspect of covid- is the constellation of inflammatory processes that become grossly distorted, particularly in the lung, that can be life threatening. the unmet need and opportunity involve the identification of pharmaceuticals to dampen the inflammatory storm in infected individuals with a view to lessen the acute need for intubation and ventilation. it is this need that is displayed vividly in some infected societies across the globe. the key is therefore not to get overly distracted by the virus, rather the acknowledgment of collateral immediate immune exuberance that is the mortality and health care resourcing problem. and knowing that cytokine storms are quick and overwhelming, we must also focus on therapeutic repurposing for immune modulation. repurposing itself is a broad discipline; for covid, drug repurposing to treat the host is the focus of this commentary; in distinction, repurposing to identify a novel antiviral effect requires a much longer time frame which will reach fruition outside of this pandemic. drug repurposing to treat the host includes re-reviews of previously repurposed drugs in sars or other treatments for adult respiratory distress syndrome, for example. in this context, the details of repurposing and its limitations in the current era been comprehensively discussed recently. in order to convert to reality, the opportunity of repurposing drugs to treat the host, we suggest an urgent international drug repurposing clinical research program, governed from a central site, and which in the time of covid- is based on treating the host. the features of such a program would be: • an international approach to rapidly identify drugs that treat the host in a pandemic to permit time for vaccine and antiviral development. • an international approach to coordinating the repurposing of existing drugs in a pandemic based on the principles of treating the host and repurposing existing drugs for new indications. • focusing on treating the host, utilizing existing pharmacology and physiology knowledge and observational administrative data, as is in several countries currently but is unlinked to other data and other countries, and needs to be at scale. • a fast-moving program driven by a sense of urgency with the ability to disseminate information to minimize unwanted duplication. overall then we would thus urge attention be given immediately to coordinated clinical evaluation of existing pharmaceuticals in an international repurposing program targeted toward managing the host, via antagonizing the innate immune pathway or inhibition of the ras at appropriate dose and timing, in this pandemic. such a program can provide adequate time for the development of vaccines, serum-based approaches or antiviral drugs, and separately will provide a therapeutic insurance with the inevitable easing of social isolation. there are no conflicts of interest to declare. treating the host response to emerging virus diseases: lessons learned from sepsis, pneumonia, influenza and ebola clinician-initiated research on treating the host response to pandemic influenza drug repurposing in the era of covid -a market failure needing leadership and government investment a sars-cov- protein interaction map reveals targets for drug repurposing renin-angiotensin system inhibitors improve the clinical outcomes of covid- patients with hypertension treatment with ace-inhibitors is associated with less severe disease with sars-covid- infection in a multi-site uk acute hospital trust association of inpatient use of angiotensin converting enzyme inhibitors and angiotensin ii receptor blockers with mortality among patients with hypertension hospitalized with covid- a call for the appropriate application of clinical pharmacological principles in the search for safe and efficacious covid- (sars-cov- ) treatments rapid repurposing of drugs for covid- buying time: drug repurposing to treat the host in covid- h key: cord- -gq se authors: das, piyanki; choudhuri, tathagata title: decoding the global outbreak of covid- : the nature is behind the scene date: - - journal: virusdisease doi: . /s - - -y sha: doc_id: cord_uid: gq se the sudden emergence of sars-cov- causing the global pandemic is a major public health concern. though the virus is considered as a novel entity, it is not a completely new member. it is just a new version of previously emerged human sars corona virus. the rapid evolving nature by changing host body environment and extreme environmental stability, collectively makes sars-cov- into an extremely virulent genetic variant. the evolution of the virus has been occurred by the continuous process of molecular genetic manipulation, through mutation, deletion and genetic recombinationevents. different host body environment acts as the supportive system for the pathogen which creates extreme selective pressure. by the process of genetic evolution the pathogen developes new characters. then the new version of the virus has been naturally selected by susceptible human host and adapt itself inside the host body causing deadly effect. moreover, extreme environmental stability helps in the process of viral survival outside the host and its transmission. thus both the host body or internal environment and the external environment performs equally as a source, responsible for shaping the genetic evolution of the sars-cov- towards thecovid- disease fitness in nature in a pandemic form. after so many years of the discovery of dna/rna as the genetic base of organisms, we are not capable to fully understand the newly emerging diseases based on the genetic study. as the human population has been evolved, it is exposed to new environmental conditions in terms of migration, change in food habits and lifestyle pattern. parallely they have interacted with wide range of new micro biome. now in this host-pathogenic interaction, both of them are in a track for winning a race to evolve and establish itself by creating immense selective pressure over each other. here comes the role of the key player, the nature or environment. from the origin of life, the nature ultimately selects the prevalence of the genetic makeup and decides the winner beyond the game of neucleotides. the interplay between the environment and the genetic material actually creates new or evolved genetic variety and selects its existence both for pathogen and the host and decides who will dominate in the nature [ , ] . this ancient story of nature's selection rule has been observed for the development of novel diseases like, small pox, cholera, tuberculosis and in recent past like, aids, different flus and the most related newly emerging sars (severe acute respiratory syndrome) and mers (middle east respiratory syndrome) outbreak [ ] . in the current global covid- outbreak, which is the major health concern in the world, it is very important to understand how this novel entity suddenly emerged and capable enough to create a global pandemic with its fetal form. in this regard prevention in form of vaccination is the only way to combat the disease and genome study is the basic thing. but beyond this, the effect of nature over it needs to be understood to evade the origin and evolution of the new genetic character and its virulent establishment in nature by population distribution. it has been observed that % of the new emerging diseases is caused by virus, and rna virus are too much progressive in this case. thus how these rna based fetal pathogen with their upgraded genetic setup has been originated and selected by nature with its disease outcome must be analyzed well [ , ] . covid- is a viral disease caused by human rna virus sars-cov- . in this review we will focus on how the viral pathogen is evolved and potent enough to cause infection at pandemic level from nature's point of view. in case of infectious disease, the host-pathogen interaction must be understood where the host environment attributes a major role in the establishment of disease. in case of any pathogenic disease outcome both the host and the pathogen struggles with each other with their unique genetic setup but the gene function is not the only factor for disease establishment or the healthy body maintenance. the environmental conditions during this struggle play influential role in the action of genome, more specifically, collective action of environment and genetic mutation affects the regulation and function of genes [ ] . moreover when we consider a virus, it needs a living body to functionally active and thus we can say that, the environment for the virus to work is its host body environment. on the other hand the host body where the virus survives also needs the external environment as its survival resource during transmission. now in the following discussion we will try to understand how these two kinds of environment interplay in the development of the virus, causing current covid- outbreak. nature is the key player of origin, evolution and pathogenicity of the virus to understand the root of the novel viral pathogen and role of environment for its development, we need to explain it with the molecular basis of darwin's theory of evolution and natural selection by the continuous process of genetic permutation and combination. sars-cov- is associated with respiratory syndromes showing flue like symptoms. if we recall the emergence of different other deadly viral flue pandemics, specifically the sars and mers in past, all of them are of similar kind with different version and originated by species spill over and zoonotic transfer, which is occurred once in a while over a period. all of these evidences indicate towards the origin of the pathogens from a common ancestral form which needs to be investigated. the probable line of development for such pandemic outcomes happened by continuous evolutionary procedure within different species or host environment exposure, by mutation during replication or genetic recombination between two different viral species and ultimate adaptation to a susceptible host by natural selection of the new version of the viable pathogen resulting infection [ , ] . in case of these pandemics, beyond the genetic mechanisms, varied permissive host exposure (intra or inter species spill over/intermediate host) seems an important determining factor for pathogenic evolution among human population [ , ] . but the natural way of genetic adaptation of the current sars-cov- is not understood yet. in this part, based on its published sequence analysis we will try to figure out how the molecular genetic variation leads to viral adaptation to the human host body environment. out of the corona virus society, the sars-cov- can cause infection to humans like other human cov. but we have to think about the fact that, its infection severity is deadly enough compared to all the other human covid. this fact can be explained by the unique disease causing genetic character of sars-cov- . receptor recognition is the initial and important genetic determinant for a viral infection. evidence shows that viral spike protein structural specificity decides the host range in human cov like, sars and mers by exerting human cell receptor recognition [ , ] . the sars-cov and sars-cov- both shows same binding affinity with the ace (angiotension converting enzyme ) receptor in human cells. so it is important to understand the molecular genetic property of the spike protein receptor recognition site of sars-cov- which genetically isolates this variant from other corona virusand decides the human cell specificity and infection in more severe form. the human cell receptor binding domain of the spike protein of sars-cov- is mutated in - amino acid residues and shows high specific ace human cell receptor binding affinity. the spike protein has two subunits (s and s ) and at the junction, there is a polybasic cleavage site present which allows furin or protease mediated cleavage and insertion of prolin like molecule which adds -linked glycan molecules resulting the flanking of the two cleavages [ , ] . thus it is clear that, the uniqueness of spike protein is crucial for the genetic isolation of sars-cov- and its high affinity binding to human cells. but it has been observed from receptor homology study that, the sars-cov- also has affinity to the ace receptor in other animals like the humans. now the next question come in mind that, how sars-cov- particularly becomes pandemic for human beings with high mortality rate compared to the other one. for a specific host pathogen interaction and disease establishment, except the receptor recognition by the unique spike protein domain of the virus, the receptor variation of the host cell also is an important genetic character. the human ace cell receptor is genetically identical from other animals for the recognition by the corona virus as observed for mers-dpp receptor binding property [ ] . moreover it has been observed that, there are several ace receptor variants present in different human populations and several variant out of it may restrict or reduce the cov specific association with it. from the recent genetic analysis of the sars-cov- ace human receptor variation in different population shows that, the expression pattern of ace receptor is an important determining factor for the pathogen susceptibility. for example, among the asians specifically, the chinese population don't have the natural resistant mutated gene against cov infection and express high level of ace receptors in tissue [ ] . so it is revealed that, not only the genetic uniqueness of the spike protein of pathogen, but also the receptor of host cell is equally important for the successful establishment of covid- outbreak. now the next question is how this unique host-pathogen interactive characteristic has been developed under the influence of host environment. from different recent research evidence based on phylogenetic analysis of complete genome sequence of sars-c v- shows indication of the evolution of a rapid, diverse and sequential genome variability whichwe will analyze by molecular genetic perspective from four different angles. first we will analyses the genome variability in terms of the diversity of the sars-cov- genome in different host species. this is revealed that sars-cov- show overall % sequence similarity with the bat sars-cov like corona virus but the similarity differs in the spike protein receptor binding domain (rbd) [ , ] . we got this kind of sequence similarity in other human corona virus where bat have been found as the common ancestral reservoir of the pathogen [ ] . on the other hand, a similar kind of corona virus with sars-cov- has been identified in malayan pangolins, which shows strong and almost identical similarity with sars-cov in the spike protein rbd. thus these pangolins can be interpreted as the intermediate host for sars-cov- [ ] . these evidences indicate toward the sequential evolution of the spike protein rbd by species spill over nature of the virus. the variability of sars-cov- viral genome in different species confirms of its animal origin and subsequent evolution of by jumping species nature. but none of the sequence similarity of sars-cov with bat or pangolins has been found based on the unique polybasic cleavage site, which can be considered as next level of determining criteria for sars-cov- human cell receptor specificity and pathogenic severity as observed in case of mers an avian influenza virus [ , ] . the insertion of unique poly basic furin cleavage site in sars-cov- is probably acquired during human to human transmission. thus the second indication of molecular diversity and evolution will be analyzed in terms of different subtypes of the virus, which has been found in different human population throughout the world. most recent research work suggests three different genetically close sub types of the sars-cov- with small amino acid sequence variations which can also be linked with continental and ethnic specificity. this research shows that the a subtype is considered as the ancient form of sars-cov- which shows close similarity with bat corona virus and generates the other two different subtypes, the b and c. the sub type b, which is mainly profound in china and east asian continents is the genetic variant of the subtype a by two mutations, (synonymous and nonsynonymous) and not found outside asia. the b subtypes has been again mutated and generated a daughter subtype the c variant, which dominates in europe and represented among american populations. the c subtype differs from b by single nonsynonymous mutation [ ] . this evidence of sequential development of different sub types from a single variant among different human population in different continents indicates the continuous single lineage evolution of the virus during human to human species spill over. moreover, the different subtypes of sars-cov- can be linked with different genetic setup of the host population in the world. now, as previously described, different sars-cov- infected population in the world shows genome variation and expression of ace receptor coding sequence which can affect the binding and susceptibility of the sars-cov- , it can be predicted that, this ace receptor variation created intense selective pressure to the circulating sars-cov- genome and as a result of this the virus adapted to establish infection with its different genetic variants in a pandemic form throughout the world [ ] . every disease when it becomes a pandemic it is revealed sequentially through several stages, which also indicates toward the evolutionary nature of the pathogen with the host body. so the third molecular genetic evolution of the covid- will be analysis based on the different phages of the pandemic. if we categories the covid- outbreak into different stages, it has been found that at the initial stage it is reported in some individuals in hunan market of wuhan, in hubei province of china. in the second phase, it has started to found as clustered form in different regions of china. then in the third stage, the disease has been disseminated in different parts of europe, america and other asian countries and resulted as a pandemic. now this different phase of disease progression covid- outbreak till now, can be linked with the evolutionary pattern of sars-cov- and its different subtypes (as previously described the a,b,c) emerged in different population of the world. the first emergence and case reports of sars-cov- is found in china which represents the b subtype genome setup and specific to this region only. the b type is reported to be originated from the ancestral betacorona virus showing close sequence similarity. the association of the virus to the live stock animal market in china supports decoding the global outbreak of covid- : the nature is behind the scene this origin [ ] . now the pangolin theory as intermediate host for the development of the unique receptor binding site (rbd) in the sars-cov- can be linked with the report of sars corona virus in dead pangolins from guangdong wild life resource in china during the period of covid outbreak in china [ ] . this b subtype with the evolved genetic setup caused clustered infection in different parts of south asia soon after it infected a large population inside china. later, after a short lag period, the virus is mutated and emerged as a pandemic in different parts outside asia with another variant, the c subtype, profound in european and american countries. this step indicates another stage of variation with probable acquisition of the polybasic cleavage site under the influence of unique ace receptor specificity in these population. thus it is clear that the viral lineage of sars-cov- is constantly associated with evolutionary process that leads to the generation of different subtypes of the same species which is gradually revealed through different phases of the disease and spreaded among different population of the world causing pandemic. evidence form our fourth and last analysis, it has been observed that mutation of the viral genome and genetic recombination is another process which confirms the molecular genetic variability of the pathogen. several mutation and deletion of sars-cov- genome has been observed from different sars-cov- genome sequence references. it has been observed that mutation has been occurred in the different structural and non-structural glycoproteins like ' orflabpolyprotein, envelop protein, matrix protein and most important determining factor the viral spike surface glycoprotein in rbd domain. on this note, no such change has been observed in the envelope protein. such type of mutation has been also observed in betacorona viruses. not only genetic mutation or deletion, events of genetic recombination has been observed in case of origin of sars-cov- spike protein rbd. genetic recombination events among bat, pangolin and human corona virus confirms this [ , , ] . so it is clear from the above study that, generation of new versions of viral diseases in humans is acquired due to the continuous molecular genetic operations inside host body by continuous species transmission. this event can be supported by the evidence of acquisition of adapted character during repeated cell culture passagingof the sars-cov [ ] . but on this note it should be pointed that, new disease may appear at any time, in any places, among any population which is unpredictable, because evolution is a collective action of genetic operation and environmental effect. in case of virus the host body is the key factor which actually gives this opportunity or favorable environment for these genetic operations to happen because the virus needs a host body as a resource to be functionally active. it is the normal tendency of a microbial species to find a convincible way to transmit and adapt itself to new environment for evolution. in case of virus, more specifically for the rna virus, the tendency is high where there is continuous circulation of the viral genome among different host species [ ] . during this period, host environment creates intense selective pressure to the pathogen. the host body environment is acting as an influential factor for the virus to take a do or die decission.when the virusis successful,it is naturally selected and adapted to establish its character by gettingthe control over the host's genetic construction by exerting virulent effects. when this process continuously goes on by exposure to new environments, it can lead to an extent where it can cause a pandemic, otherwise it is the end of line for the pathogen to evolve. this is a gradual process, when the virus accumulate the required genetic makeup or prepare itself and takes several attempt for the big jump. during this preparatory phase the pathogen can generate mild infection or infection to a limited bunch of population or create silent carriers depending on the host body environment. these carriers don't show any symptoms but the infectious pathogen with its new genetic makeup is maintained here and can be transmitted to different suitable host environment for disease establishment. thus these carriers are very crucial, and might be another strategy of the virus for the evolution of new genetic setup. an evolved character is developed under environmental pressure [ ] . in this case, the protective immune system of the host body environment helps the virus to evolve under selective pressure of infection rejection. identification of several asymptomatic carriers for sars-cov- can be a result of the above fact. thus virus is the player, but its' optimum molecular genetic fitness, distribution and evolution are shaped by the host body environment [ , , ] . in case of sars-cov- its diverse genetic variability and evidence of its existence among different host species indicates towards its rapid ''jumping species'' nature which probably makes it extremely diverse and virulent. the host body environment gave the virus the opportunity to prepare for the battle where both the genetic manipulation and the natural selection process plays major role for its adaptability resulting the current uncontrolled pandemic. from another angle if we look over the two extremities of the origin and exposure of the sars-cov- , they are bats and humans. both of the host environments can be considered as large population. bats is the reservoir of a large number of viral pathogens and inhabit in a giggling manner in a close contact thus enhances the chance of cross transmission and giving the pathogen a field to evolve. similarly, the modern human population is highly dynamic and exposed to various extreme environmental conditions. the large co habitation and lifestyle pattern enhances the chance of cross transmission of virus and make a susceptible environment for emerging new viral disease. after considering the impact of molecular genetic diversity and host body environment in the origin and development of a virulent strain, we need to understand the role of the external environmental factors in the stability and survival of the virus outside the host body conditions. during the process of evolution, intra or inter species transmission among the susceptible host community, external environment plays a critical role. though in the external environment, outside the host body, the virus cannot multiply and cause infection but different factors are crucial in the stability of the virus outside its host environment for the process of its transmission. specifically when we are dealing with a respiratory tract associated virus, the role of environmental factors must be emphasized because the respiratory outputs are directly exposed to the environment through cough, sneeze, and sputum. the role of different environmental factors like temperature, salinity, p h etc. for viral stability in aerosol, inert surface, water etc. needs to be discussed, because external environment is the connecting media between various host species during the viral transmission. in case of the other two common human corona virus super spread, sars-cov- and mers air transmission is well reported in form of droplet and aerosol. the evolutionary pattern of sars-cov- is associated with geographical continent specific subtype diversity with a tendency of rapid species spill over which indicate towards its huge external environmental adaptation. moreover social distancing is emphasized for covid- spread due to its highly contagious nature. thus understanding the role of external environmental factors in maintaining viral stability and capability to survive in different patient samples as well as certain inert or abiotic environmental substances, the fomite, by which it can spread needs to be analyzed very well. if we consider the clinical samples by which the virus is exposed to the environment, research shows that sars-cov- may present in different types of patient samples. high viral load is found mainly in the respiratory samples. presence of virus in blood samples proves its systemic spread. detection of live viral particle in feces indicates its fecal-oral route of environmental exposure [ , ] . after the virus is exposed to environment by these clinical samples, sars-cov- shows various degree of fomite stability for different surfaces associated to those patient samples. in case of steel surface, the stability is maximum (approximately - days). it is also stable in polypropylene, card board and copper (show less degree of stability). in aerosol the virus shows three hours of stability [ ] . in connection with the fecal-oral route the stability and sustainability of sars-cov- has been found in waste and sanitary water in infected area,is recently reported [ ] . the stability of virus in different contaminated surfaces and in different patient sample outputs depends on the action of temperature and relative humidity percentage. it has been observed for corona virus that low temperature ( degree) and low humidity ( %) is supportive for the viral stability, where gradual increase of both the parameters inactivates the virus more rapidly. it has been observed that lipid virus like covid can be inactivated by the interference of two mechanisms, the air water interphase and desiccation process. when the relative humidity is nearly %, the virus is accumulated by the air water interphase. then the desiccation process excretes water from the lipid layer by oxidation and simultaneously breaks the cross linking thus which effectively damage the lipid layer of the pathogen much rapidly [ ] . on this note it must be pointed that mucus acts as the first protective barrier for invading respiratory virus. interaction of mucus layer glycoprotein with viral surface proteins have been well reported in influenza virus. recent research shows that the saliva or mucus substance may act as a protective barrier for the virus when it is exposed to the external environment. the aerosol also goes with the same flow, where it helps the virus from the hazardous effect of varying external environment by retaining its infectivity [ , ] . so temperature inactivation or the air water desiccation mechanism, are not effective on the virus when the organic barriers acts as insulator. thus in case of sars-c v- , it is received by the environment through different clinical sample outputs mainly the respiratory secretions from an infected person whether it is a susceptible host or carrier, in form of droplets or aerosol and can survive in this form in various fomites. after the virus is received in the external environment, the patient sample itself protects it from adverse environmental conditions and helps it to survive or sustain for long time acting as maintaining media for the virus outside the host body. this is the probable reason why sars-cov- shows high degree of external environment stability. from the above analysis it is clear that the outside host environment acts as the intermediate player which receives, maintains, protects and transfers the pathogen using its various factors as weapon for covid- pandemic. decoding the global outbreak of covid- : the nature is behind the scene from the above study it is clear that rapid evolving nature and high environmental stability of sars-cov- is responsible for the current outbreak of covid- . the evolutionary process of this new version of the virus indicates the 'synthetic theory of evolution' or the 'neo-darwinism theory' where, the virus has gone through sequential multistep thorough process of molecular genetic variation which is shaped and established by the natural selection process. in this review the role of both the host body environment and the external environment in disease establishment is discussed from different angle (fig. ) . the host body is the important factor for the covid- host-pathogenic interaction establishment where the molecular evolution happens. the rapid host body changing pattern helps the virus to be continuously exposed to new environmental conditions, which creates selective pressure to the virus. in order to struggle for existence and infection establishment inside host body, the virus undergo through several genetic manipulations and achieve its mutated characters. then genetically close different subtypes of sars-cov- develops unique spike protein receptor binding domain with high degree of receptor binding property to human cells and adapt itself to fit the character inside the host body. ultimately by the action of natural selection, specific host-pathogen interaction happens where the virus can establish the infection. the external environment acts as intermediate player in between one host to another, by providing suitable environment for stability and survival for sars-cov- outside its host body resulting high viability and higher transmissibility compared to other corona virus. it has been reported that the external environment also interacts with the host body environment and helps the stability of the virus inside the host body system and its infectivity. changing temperature and relative humidity contributes to the degree of host susceptibility to a certain pathogen, pathogen infiltration and survival inside the host body [ ] . thus the outcomes from the whole study will help to understand the sudden emergence of covid- pandemic and its character. the findings emphasize to consider the interactive role of both of the two environments, the host body environment and the external environment as the source of genetic evolution of emerging viral diseases and its control. history of highly pathogenic avian influenza. revue scientifique et technique (international 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structural studies emergence of sars-cov- through recombination and strong purifying selection the consequences of human actions on risks for infectious diseases: a review sars-cov- in wastewater: potential health risk, but also data source evolutionary potential of an rna virus trypsin treatment unlocks barrier for zoonotic bat coronavirus infection genetic diversity and evolution of sars-cov- changes in temperature alter the potential outcomes of virus host shifts pathways of cross-species transmission of synthetically reconstructed zoonotic severe acute respiratory syndrome coronavirus animal virus ecology and evolution are shaped by the virus host-body infiltration and colonization pattern. biorxiv the evolution of covert, silent infection as a parasite strategy epidemiology, genetic recombination, and pathogenesis of coronaviruses aerosol and surface stability of sars-cov- as compared with sars-cov- viruses switch hosts to evolve structure, function, and antigenicity of the sars-cov- spike glycoprotein detection of sars-cov- in different types of clinical specimens social and environmental risk factors in the emergence of infectious diseases a new coronavirus associated with human respiratory disease in china the interaction between respiratory pathogens and mucus pangolin homology associated with -ncov a pneumonia outbreak associated with a new coronavirus of probable bat origin acknowledgements we would like to thank mr. laltu hazra, regarding his general laboratory help and csir for their financial assistance by csir srf fellowship for pd (council of scientific and industrial research, govt. of india, senior research fellowship). tc is a fellow of indian virological society. the authors also thank iit delhi hpc facility for computational resources. conflict of interest the authors declare that they have no conflict of interest. key: cord- - mkadtp authors: duffy, siobain; burch, christina l.; turner, paul e. title: evolution of host specificity drives reproductive isolation among rna viruses date: - - journal: evolution doi: . /j. - . . .x sha: doc_id: cord_uid: mkadtp ecological speciation hypotheses claim that assortative mating evolves as a consequence of divergent natural selection for ecologically important traits. reproductive isolation is expected to be particularly likely to evolve by this mechanism in species such as phytophagous insects that mate in the habitats in which they eat. we tested this expectation by monitoring the evolution of reproductive isolation in laboratory populations of an rna virus that undergoes genetic exchange only when multiple virus genotypes coinfect the same host. we subjected four populations of the rna bacteriophage φ to generations of natural selection on a novel host. although there was no direct selection acting on host range in our experiment, three of the four populations lost the ability to infect one or more alternative hosts. in the most extreme case, one of the populations evolved a host range that does not contain any of the hosts infectible by the wild‐type φ . whole genome sequencing confirmed that the resulting reproductive isolation was due to a single nucleotide change, highlighting the ease with which an emerging rna virus can decouple its evolutionary fate from that of its ancestor. our results uniquely demonstrate the evolution of reproductive isolation in allopatric experimental populations. furthermore, our data confirm the biological credibility of simple “no‐gene” mechanisms of assortative mating, in which this trait arises as a pleiotropic effect of genes responsible for ecological adaptation. the origin of species has captured the imagination of evolutionary biologists from the time of darwin ( ) , through the modern synthesis (dobzhansky ; mayr ) , until today (schemske ) . yet scientists are still elucidating and debating the relative importance of different mechanisms for speciation (coyne and orr ) . in the traditional dobzhansky-muller model of speciation, genetic incompatibilities are thought to accumulate over time between allopatric, spatially separated populations, eventually resulting in postmating barriers to reproduction such as hybrid inviability and sterility (dobzhansky ; muller muller , muller , . although natural selection can play a role in the evolution of genetic incompatibilities, in this model of speciation the role of natural selection in the evolution of reproductive isolation is not direct. that is, reproductive isolation evolves as a consequence, not of the phenotypes, but of the genotypes produced by natural selection (templeton ; doebeli et al. ) . there is, however, a growing consensus that reproductive isolation often evolves as a direct consequence of the phenotypes produced by natural selection. in this scenario, natural selection could facilitate speciation in both sympatry and allopatry (schluter ; coyne and orr ) . of particular interest is the process of ecological speciation, in which assortative mating evolves as a pleiotropic consequence of divergent natural selection for ecologically important traits (schluter ) . for instance, assortative mating should evolve as a pleiotropic consequence of selection for divergent habitat preference as long as organisms mate in their preferred habitat. in this simplest mechanism of ecological speciation, the only genes required for the evolution of reproductive isolation are genes involved in ecological adaptation. because no genes for mate choice are required for the process of ecological speciation, it has been referred to as a "no-gene" mechanism of assortative mating (rice ; coyne and orr ) . host shifts may represent the most likely scenario for assortative mating to arise via a no-gene mechanism because mate choice is often determined by host preference. for example, assortative mating between host races of the apple maggot fly, rhagoletis pomonella, may have evolved via selection acting directly on host preference (feder et al. ; filchak et al. ) . in rhagoletis, a new host race emerged following the introduction of apples to north america. regardless of whether the host race formation occurred in allopatry before the introduction of apples (feder et al. ) or in sympatry after the introduction, the divergent host preference, itself, may have conferred an ecological adaptation by allowing the new host race to escape resource competition on hawthorns, the ancestral host plant (feder et al. ). thus, assortative mating (host preference) and ecological adaptation in rhagoletis might have a shared genetic basis. however, the genetic intractability of ecological models such as rhagoletis has made it difficult to confirm that the same gene underlies both traits. investigations of more tractable phytophagous insects have localized genes involved in host preference and host performance to shared regions of the genome (hawthorne and via ) , but no study has confirmed that an individual locus affects both traits. evolution experiments with phytophagous insects that attempted to demonstrate the plausibility of no-gene models of assortative mating have also met with limited success. although numerous laboratory evolution experiments have demonstrated the evolution of moderate levels of assortative mating in response to selection for habitat preference (e.g., rice and salt ; rice and hostert ) , none has achieved complete reproductive isolation. to date, the only laboratory evolution experiments to achieve high levels of reproductive isolation (leu and murray ) selected directly for assortative mating rather than ecological adaptation. viruses share the key characteristic that has made phytophagous insects popular models for studying ecological speciation-viruses engage in sexual reproduction only when two or more viruses coinfect the same host (delbrück and bailey ; malmberg ) . therefore, mate choice in viruses is determined by host specificity (relative ability for a virus to infect one or more host types). however, viruses offer three experimental advantages over insects for monitoring speciation processes in the laboratory. first, viruses evolve rapidly, facilitating the direct empirical observation of speciation, which is more difficult in most other systems (rice and hostert ) . in particular, the high pernucleotide mutation rates of rna viruses (drake ) provide extensive genetic variation that fuels evolution by natural selection, making the study of reproductive isolation and speciation especially feasible (holmes ) . second, in viruses it is possible to identify the individual mutations responsible for host performance and host specificity through whole genome sequencing and classical genetic crosses. last, it is easy to measure the fitness effects of the individual mutations responsible for reproductive isolation to determine whether these changes occurred via natural selection versus genetic drift. previous experiments have shown that viral adaptation to a single host is often accompanied by a reduced ability to infect alternative hosts (reviewed in fenner and cairns ; ebert ) . for example, adaptation of the bacteriophage x to salmonella enterica was accompanied by a reduced ability to infect escherichia coli (crill et al. ) . one might logically conclude, therefore, that adaptation to a novel host-a host that is not infectible by the wild-type virus-should often produce an evolved virus that no longer infects the ancestral host, so that the host ranges of the closely related wild-type and evolved viruses no longer overlap. however, this conclusion has yet to be experimentally validated. to our knowledge, no study has shown that viral adaptation to a single host led to nonoverlapping host ranges between closely related viruses. we examined the plausibility of the no-gene model of ecological speciation by monitoring the evolution of bacterial host specificity in lineages of the rna bacteriophage during selection for improved performance on a novel host. phage possesses all of the general advantages of virus model systems, and has proven to be a tractable experimental system for answering evolutionary questions that are difficult to address in natural populations (e.g., chao et al. ; turner and chao ; burch and chao ) . an expanded-host-range genotype ( broad ; mutant e g [duffy et al. ]) was obtained as a spontaneous mutant of the wildtype ( wt ; strain atcc- -b , american type culture collection, manassas, va). duffy et al. ( ) examined different pseudomonas hosts and determined that four bacteria comprise the known host range of wt : pseudomonas syringae pathovar phaseolicola hb y (atcc- ), and p. syringae pathovars persicae, savastanoi, and tagetis (generously provided by g. martin, cornell university, ithaca, new york). the mutant broad infects these same four bacteria, and additionally can infect two novel hosts: p. syringae pv. tomato (g. martin) and p. pseudoalcaligenes era (east river isolate a; generously provided by l. mindich, public health research institute, newark, new jersey) . detailed methods are previously described (duffy et al. ) . briefly, we used lc medium (luria bertani broth at ph . ) to culture bacteria. phage were grown by mixing viruses with a bacterial lawn in ml of lc top agar ( . % agar), overlaid onto an lc plate ( . % agar). incubation of all cultures and plates occurred at • c. phage populations and single plaques were archived in % glycerol/ % lc broth and stored at − • c. we used phage broad to found four replicate populations (e -e ) subjected to experimental evolution via plaque growth on p. pseudoalcaligenes era lawns (seeded using ∼ . × stationaryphase bacteria, measured in colony-forming units). each day, an evolving lineage was allowed to form ∼ plaques (always between and plaques), where each plaque initiates from a single virus particle. viral titers were thus measured in plaqueforming units. after -h incubation, plaques were harvested and filtered to obtain a bacteria-free lysate containing the virus progeny for the lineage. to maintain the population bottleneck size of ∼ individual viruses, the lysate was subsequently diluted and plated again in the presence of naïve (unevolved) bacteria. use of naïve bacteria that were freshly grown from frozen stock prevented evolution of bacterial resistance to phage, as well as any possibility for coevolution between bacteria and phage. the -h propagation scheme was repeated for consecutive days, to achieve passages per virus lineage. as overnight plaque growth corresponds to approximately five generations of viral evolution, the populations experienced ∼ generations of experimental evolution ( passages × generations per passage). over the course of the experiment we monitored the host range of individual genotypes in each population by plating phages on lawns containing mixtures of the unselected hosts p. syringae pathovar phaseolicola (the standard laboratory host) and p. syringae pathovar tomato. in contrast to the broad ancestor that formed clear plaques by killing both hosts on these plates, evolved phages that lost the ability to infect one of these hosts formed turbid plaques, and evolved phages that lost the ability to infect both hosts failed to form plaques at all. this method revealed whether an experimental lineage became dominated by narrowed host-range genotypes. to more closely examine host range of an evolved virus clone, we first obtained a high-titer lysate (∼ virus particles per ml) of the clone on the selected host, p. pseudoalcaligenes era. using the method described by duffy et al. ( ) , samples from the lysate containing ∼ virus particles were then spotted onto lawns of the six different bacterial hosts comprising the host range of the common ancestor, broad . these assay plates were incubated for up to h, and the method was repeated three times using independently grown host bacteria cultures. plaque formation indicated that the host bacterium was within the virus' host range. if the results were ambiguous, host range was confirmed by standard plating of ∼ viruses on a bacterial lawn to visualize individual plaques. using this method, from each end point (day ) evolved population we isolated at random and plaque-purified a single clone that possessed the majority phenotype observed in the population. this process yielded clones e narrow , e , e narrow , and e narrow from populations e , e , e , and e , respectively. in addition, we isolated and plaque-purified a random clone from end point populations e , e , and e that showed the minority broad host-range phenotype: e broad , e broad , and e broad . last, for some analyses it was necessary to isolate clone e narrow(day ) , a narrowed host range virus from population e at day of the study. fitness of an evolved test phage was determined by estimating its growth rate on p. pseudoalcaligenes era, relative to a reference strain of (paired growth assays [turner and chao ; duffy et al. ] ). this reference strain is denoted "era common competitor," or ecc. ecc is descended from wt but has a mutation that allows it to infect the relevant host range of the evolved viruses, as well as host p. syringae pv atrofaciens. the fitness assay began with mixing together a test phage and ecc at a : ratio in lc medium. a diluted sample of this initial mixture was then plated on a mixed-host lawn containing a : ratio of p. syringae pv phaseolicola to p. syringae pv atrofaciens. on the mixed-host lawn ecc produced clear plaques, whereas a test phage produced turbid plaques because it could not infect p. syringae pv atrofaciens. in this way, the plaques appearing on the mixed-host lawn after h were used to estimate the true ratio of test phage to ecc in the initial mixture (r ) at the beginning of the fitness assay. a diluted sample of the initial mixture containing ∼ plaque-forming virus particles total was also plated on p. pseudoalcaligenes era. after h, the resulting ∼ plaques were harvested and filtered to obtain a bacteria-free lysate containing the progeny of the two viruses. a diluted sample of the lysate was then plated on a mixed-host lawn to estimate the ratio of test virus to ecc after h of growth on era (r ). thus, fitness (relative growth rate) was determined on era, but the relative ratio of viruses was tracked by platings on the mixed-host lawns. fitness (w) is the ratio of the two observed ratios: w = r /r (duffy et al. ) . we used log w in all statistical analyses, because ( ) this approach is preferred in microbial evolution experiments (bennett et al. ; chao et al. ; burch and chao ) , and ( ) log w exhibits homogeneous variance. all test viruses were competed six times versus ecc. a two-tailed t-test (microsoft excel, redmond wa) was used to compare the fitnesses of broad and narrow host-range genotypes drawn from the same experimental population. when an evolved virus strain was observed to no longer infect the standard laboratory host p. syringae pv phaseolicola, we were forced to use a modified version of the paired-growth assay to estimate its fitness. the alternate fitness assay employed e broad as the common competitor. as in the standard fitness assay, the test phage and the common competitor were compared for relative growth on p. pseudoalcaligenes era. to track ratios of viruses (r , r ) in these modified fitness assays, we used mixed-host lawns containing p. syringae pv phaseolicola and p. pseudoalcaligenes era in a : ratio. on these discriminative plates, the e broad common competitor formed clear plaques, whereas the evolved test phage formed turbid plaques because it failed to infect p. syringae pv phaseolicola. as we employed multiple common competitors in our fitness assays, statistical analyses of fitness data involved only comparisons among strains that were assayed relative to the same reference strain. genomic rna was extracted from high-titer lysates of the endpoint clones as previously described (duffy et al. ). the genome consists of three double-stranded rna segments, designated small, medium, and large (mindich ) . the entire genomes (excepting the ends of each segment) were sequenced and deposited in genbank (accession numbers dq to dq ). we used classic genetic crosses to identify which molecular substitution led to narrowed host range in an evolved virus. to perform a genetic cross, we placed ∼ × virus particles of each of two virus genotypes (∼ × viruses total) into lc medium containing ∼ × exponentially growing p. pseudoalcaligenes era bacteria. thus, the mixture resulted in a multiplicity of infection of roughly five viruses per bacterium. here, the vast majority of cells experience coinfection by multiple viruses, which allows for reassortment: the formation of hybrid genotypes containing a combination of the rna segments present in the two coinfecting parent viruses (turner and chao ) . (we note that true recombination [template switching between rna segments] is rare or nonexistent in , allowing each of the three rna segments to be treated as a single locus [mindich et al. ].) after -min incubation to allow virus attachment to cells, a diluted sample of the mixture was plated to obtain a high-titer lysate containing the viral progeny. we then identified progeny with the narrow host-range phenotype (growth restricted to p. pseudoalcaligenes era), double or triple plaque-purified single genotypes with both the broad and narrow host ranges, and sequenced the regions containing mutations to identify the hybrid genotypes. in this manner, we isolated narrow or broad host-range genotypes containing the relevant combinations of evolved rna segments. we tested the plausibility of the no-gene mechanism of speciation by examining the consequences of adaptation to a novel host in laboratory populations of the rna phage , which infects a number of pseudomonas species. we founded four replicate populations (e -e ) with a broad host-range phage ( broad ) that differs from the wild-type ( wt ) only by the mutation e g in the host attachment gene p (duffy et al. ) . this mutation confers the ability to infect two host strains that do not permit infection of wt , including the novel host p. pseudoalcaligenes era ( table ) . each of the populations founded by broad was subjected to selection on the novel host p. pseudoalcaligenes by plating the phage population on a lawn of the novel host, incubating for h to allow individual phages to form plaques, harvesting progeny phages from ∼ of the resulting plaques, and plating these phages on a fresh lawn of the novel host. this cycle was repeated for days, corresponding to ∼ virus generations. the presence of only a single host in the habitat ensured that selection was not acting directly on host range (i.e., host-use specificity; the mechanism by which viruses achieve assortative mating). rather, if host specificity evolved, it could only occur as an indirect consequence of selection for improved performance on the novel host. we gauged fitness improvement on the new host via pairedgrowth assays (see methods). here, a representative virus clone from each endpoint (generation ) population was assayed for growth rate, relative to a common competitor virus. results showed that after generations of evolution on the novel host all four populations responded to selection, showing significantly higher fitness than the common ancestor, broad , on the novel host (table ) . by the end of the experimental evolution on the novel host, three populations became dominated by genotypes with a narrowed host range (fig. ) . these data strongly suggest that narrowed host range genotypes were generally favored by selection, such that viruses featuring this trait either fixed or were on their way to fixation by generation . individual narrow hostrange genotypes isolated from populations e and e ( e narrow , e narrow ) lost the ability to form plaques on one of the six hosts (p. syringae pv. tomato) within the host range of the ancestral phage broad ( table ). the narrow host-range genotype isolated from population e ( e narrow ) lost the ability to form plaques on five hosts, including all four hosts within the host range of wt ( table ). the disjoint host ranges of e narrow and wt cause complete reproductive isolation between these genotypes, among the group of bacterial hosts is known to infect (duffy et al. ) . to identify the individual mutations responsible for the narrow host-range phenotypes, we sequenced the genomes of one majority genotype with a narrow host range (e.g., e narrow ) and one minority genotype with a broad host range (e.g., e broad ) isolated from populations e , e , and e after days of evolution (genbank dq -dq , fig. ). from sequence data alone, it was possible to identify the individual mutation responsible for the narrow host range of e narrow . the only mutation that differed between e narrow and e broad was the substitution of a t in the host attachment gene p (fig. , e ) , unambiguously identifying this mutation as the sole cause of the narrow host-range phenotype. e narrow and e broad differed by this same mutation, a t in p , and by two additional mutations in p , one synonymous and one nonsynonymous (fig. , e ) . two observations suggest that the a t mutation in p is also responsible for the narrow host range of e narrow : ( ) it is the only convergent mutation shared by e narrow and e narrow , and ( ) e narrow and e narrow show identical narrowed host ranges. it was more difficult to identify the mutation responsible for the narrow host range of e narrow because this phage had acquired three nonsynonymous mutations since its divergence from the common ancestor of e narrow and e broad : a v in the nucleocapsid shell protein p , and t s and g a in the host attachment gene p (fig. , e ). an additional mutation was found in e narrow , in the untranslated region of the medium segment, but it was discounted as the cause of the change in host range because none of the noncoding regions in are known to impact function (mindich ) . to narrow our focus, we first isolated a narrow host-range genotype, e narrow(day ) , from the evolved population at day , when the narrow hostrange phenotype was starting to sweep through the population (fig. ) . sequencing only the regions corresponding to the nonsynonymous mutations acquired by the e narrow lineage, we determined that e narrow(day ) differed from e broad by two of the three mutations: a v in p and g a in p . because these mutations existed on different genome segments, we used classic genetic crosses between e narrow(day ) and e broad to generate genomes that contained these individual mutations, but were otherwise isogenic to e broad. in this manner, we confirmed that the p :g a mutation was the cause of the narrow host-range phenotype. addition of this mutation to the e broad genome recapitulated the narrow host-range phenotype of e narrow , whereas addition of the p :a v mutation did not (table ) . to confirm that host adaptation and host specificity had a shared genetic basis in our experiments, we determined whether the mutations responsible for the narrow host-range phenotypes also conferred a fitness advantage to the evolving populations. we measured the fitness effect of individual mutations that narrowed host range, using paired growth assays under culture conditions identical to the selection experiment on novel host p. pseudoalcaligenes. for each population, we measured the fitness of a genome with the mutation ( e broad+p :g a , e narrow , or e narrow ) relative to the appropriate broad host-range genome without the mutation ( e broad , e broad , or e broad ). both mutations in p (a t and g a) were tested against otherwise isogenic competitors, and both significantly improved fitness, that is, growth rate, on the novel host p. pseudoalcaligenes (fig. ) . thus, in all three populations showing reduced host range (increased host specificity), the phenomenon was caused indirectly by a single mutation that conferred a selective advantage on the novel host p. pseudoalcaligenes (increased host performance). our laboratory experiment with rna phage shows reproductive isolation can evolve via natural selection. by investigating the process of speciation in laboratory populations of a genetic model system we addressed the role of natural selection in speciation in ways that have not been possible in natural populations, nor in ecological model systems (e.g., stickleback fish [vines and schluter ], apple maggot flies [filchak et al. ], walking stick insects [nosil et al. ] ). we confirmed that in assortative mating (host range) and ecological adaptation (performance on the novel host) had a shared genetic basis. but this result was not due to reproductive incompatibilities that fixed in a population via drift or through genetic hitchhiking. rather, our results showed that assortative mating evolved via a biologically simple "no-gene" mechanism in which assortative mating arises as a pleiotropic effect of mutations that produce ecological adaptation. in addition, we demonstrated that the action of natural selection can produce reproductive isolation over a short time period, causing incipient viral speciation in . our study demonstrates the power for microbial evolution experiments to bridge the study of microevolution (change within a species) and macroevolution (change from one species into another). the same microevolutionary processes of mutation and natural selection, which led to the adaptation of populations to a novel host also resulted in a macroevolutionary event: the evolution of a new virus species that is reproductively isolated from the ancestral phage wt . although we are not the first to use microbes to investigate speciation (friesen et al. ; maclean ; rozen et al. ) , we recognize that our unique success in achieving reproductive isolation in the laboratory likely resulted from our unorthodox choice of an rna virus as a model for studying speciation. however, only two characteristics of the system were critical to our success-the short generation time and the high mutation rate. other characteristics of the system (e.g., viruses mate in the habitat where they eat) make our results highly relevant to speciation processes in a variety of organisms that share these characteristics (e.g., walking sticks, apple maggot flies, and sticklebacks). however, the pleiotropy between improved performance in a new habitat and increasing specificity has rarely been as clearly observed as it was in our study (e.g., trait associations can also be explained by linkage disequilibrium [via and hawthorne ] ). we attribute our success to the ability to completely dissect the genetic basis of adaptation and to measure fitness instead of fitness components. we observed an identical nucleotide substitution, a t, in the p attachment gene of e narrow and e narrow . evidence strongly suggested that this single molecular change was solely responsible for the similarly narrowed host range experienced by these two independently evolving lineages. thus, the result demonstrates the repeatability of adaptive evolution when phage undergoes selection while shifting from its original host onto the novel host p. pseudoalcaligenes, as well as the shared consequence of this improved performance for the unselected trait, host breadth. this parallel evolution, where multiple lineages "find" common mutational solutions, is predicted to occur in experimental and natural populations when there are small numbers of beneficial mutations available (orr ) . such observations are not uncommon in microbial evolution studies conducted under these populationgenetics conditions (e.g., bull et al. ) . interestingly, our study showed that beneficial mutations other than a t could lead to improved performance on the novel host, but that these changes either did not affect host range (in the case of population e ) or narrowed it even more dramatically (in lineage e ). we designed our laboratory experiments in a manner that mimicked the process of host shifts occurring in nature. it is generally believed that viruses can progressively evolve by first infecting only a reservoir host, then infecting both a reservoir and novel host, and finally infecting only the novel host (e.g., kuiken et al. ) . consistent with this idea, laboratory experiments suggest that viruses shift hosts by experiencing an intermediate broad-host-range genotype, rather than instantaneously shifting from one host range to another, nonoverlapping host range. thus, the vast majority of identified host-range mutations are shown to expand the host breadth of a virus, instead of causing an immediate host-shift (e.g., zarling et al. ; aytay and schulze ) . extensive evidence from animal, plant, and bacterial viruses shows that these broad host-range viruses readily lose host range when allowed to adapt to a single host (e.g., reviewed in fenner and cairns ; reddy and black ; marchette et al. ; wichman et al. ; crill et al. ; ebert ) . therefore, our laboratory results showing two reproductively isolated phages ( wt and e narrow ) bridged by a broad host-range intermediate ( broad ) encompass this complete progression. for this reason, our study may reflect how host shifts lead to reproductive isolation in natural virus populations. beyond uniquely demonstrating the evolution of reproductive isolation in the laboratory, our study extends the literature describing the evolutionary genetics of narrowed host range when viruses adapt to a single host. such experiments suggest a causal relationship between the observed reductions in host range and the general role of host attachment proteins in virus adaptation to new hosts, both in the laboratory (e.g., phage x [crill et al. ], vesicular stomatitis virus [zárate and novella ] , and sars coronavirus [poon et al. ] ) and in nature (e.g., influenza virus [parrish and kawaoka ] , and canine parvovirus [shackelton et al. ] ). because many viruses seem capable of changing host range via only one or two mutations (baranowski et al. ; rainey et al. ; parrish and kawaoka ; duffy et al. ) , it hints that other rna viruses have the potential to evolve reproductive isolation through no-gene mechanisms as rapidly as we observed in . the choice to conduct adaptation experiments in the absence of gene flow from the ancestral population (i.e., in allopatry) likely facilitated the evolution of reproductive isolation in our experiments. however, because reproductive isolation evolved via a no-gene mechanism in which recombination cannot separate ecological adaptation from assortative mating, we would not expect to obtain a different outcome in the face of gene flow (i.e., in sympatry). in other words, our finding of reproductive isolation via a no-gene mechanism suggests that viral populations could evolve reproductive isolation regardless of whether they were evolving in allopatry or sympatry. in reality, natural virus populations probably often evolve in parapatry. for example, a virus that enters a human host may persist and replicate in that host for tens, hundreds, or perhaps even thousands of generations before it is transmitted to the next human or nonhuman host (fu ; drummond et al. ; wilson ) . although parapatry may facilitate the evolution of viral host races, as in phytophagous insects (feder et al. ; hawthorne and via ) , parapatry would nonetheless provide substantial opportunity for gene flow to influence the divergence of rna virus populations. on the one hand, gene flow between the ancestral and emerging virus populations is expected to increase the adaptive genetic variation available to the emerging lineage (morgan et al. ) . for instance, gene flow may allow antigenic recombination between ancestral and emerging viruses, aiding the emerging virus' ability to elude the novel host's immune defenses (hay et al. ; garcia-arenal and mcdonald ) . in this scenario, the evolution of reproductive isolation would eliminate a source of beneficial genetic variation for the emerging population. we suspect this consequence of reproductive isolation would not have a major impact on rna virus disease emergence because the high mutation rate of rna viruses (drake ) ensures that genetic variation is rarely a limiting factor in adaptation. on the other hand, gene flow between the ancestral and emerging populations may slow adaptation of the emerging lineage to the novel host, if the process occurs often and continually reintroduces alleles that are deleterious for growth on the new host (lively ; cuevas et al. ; kawecki and ebert ) . in this case, reproductive isolation that separates the gene pools of ancestral and emerging populations may hasten adaptation of the emerging virus to the 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in the virion envelope antigen we gratefully acknowledge discussions with m. ferris and members of the burch and turner lab groups. we thank j. bull, m. doebeli, m. noor, m. servedio, and an anonymous reviewer for improving earlier versions of this manuscript. sd was a predoctoral fellow of the howard hughes medical institute. this research was supported by national science foundation grant deb- to pet and sd, national science foundation grant deb- and a yale center for genomics and proteomics pilot grant to pet, and national institutes of health grant gm to clb. key: cord- -kq rsuxj authors: etienne, lucie; emerman, michael title: the mongoose, the pheasant, the pox, and the retrovirus date: - - journal: plos biol doi: . /journal.pbio. sha: doc_id: cord_uid: kq rsuxj paleovirology is the study of ancient viruses. the existence of a paleovirus can sometimes be detected by virtue of its accidental insertion into the germline of different animal species, which allows one to date when the virus actually existed. however, the ancient and the modern often connect, as modern viruses have unexpected origins that can be traced to ancient infections. the genomes of two species of mongooses and an egg-laying mammal called an echidna show that a virus currently present in poultry, the reticuloendotheliosis virus (rev), is actually of ancient exotic mammalian origin. rev apparently spread to poultry through a circuitous route involving the isolation of malaria parasites from a pheasant from borneo housed at the bronx zoo that was contaminated with rev. repeated passage of this virus in poultry adapted the virus to its new host. at some point, the virus got inserted into another virus, called fowlpox virus, which has spread back into the wild. although rev may still exist somewhere in a mammalian host, its modern form links an million-year-old infection of the ancestor of a mongoose to a virus that now is circulating in wild birds through malaria studies in the mid- (th) century. these lessons of ancient and modern viruses have implications for modern human pandemics from viral reservoirs and for human interventions that may come with unintended consequences. the existence of a paleovirus can sometimes be detected by virtue of its accidental insertion into the germline of different animal species, which allows one to date when the virus actually existed. however, the ancient and the modern often connect, as modern viruses have unexpected origins that can be traced to ancient infections. the genomes of two species of mongooses and an egg-laying mammal called an echidna show that a virus currently present in poultry, the reticuloendotheliosis virus (rev), is actually of ancient exotic mammalian origin. rev apparently spread to poultry through a circuitous route involving the isolation of malaria parasites from a pheasant from borneo housed at the bronx zoo that was contaminated with rev. repeated passage of this virus in poultry adapted the virus to its new host. at some point, the virus got inserted into another virus, called fowlpox virus, which has spread back into the wild. although rev may still exist somewhere in a mammalian host, its modern form links an million-year-old infection of the ancestor of a mongoose to a virus that now is circulating in wild birds through malaria studies in the mid- th century. these lessons of ancient and modern viruses have implications for modern human pandemics from viral reservoirs and for human interventions that may come with unintended consequences. outbreaks of ''new'' viruses are real causes of concern for human and animal health. but where do they come from? the vast majority of emerging viruses in the human population originate from other animals [ ] . in fact, when a new virus appears, it is often a virus that already exists in one species, but then becomes established in another. for example, the transmission of a coronavirus (cov) from bats to palm civets and raccoon dogs and ultimately to humans led to the emergence of severe acute respiratory syndrome (sars-cov), which created an epidemic in , with , people infected and deaths before it was finally eliminated in humans [ ] . another example is the transmission of influenza virus from birds to mammals (pigs and humans) in that was responsible for over million deaths worldwide [ ] , and many of the genes from this virus continue to circulate in human influenza to this day. whether or not a virus will ''emerge'' in new species depends on a complex set of requirements including contact, mode of transmission, and adaptation to the new species [ , ] . moreover, human interventions can sometimes accidentally facilitate the transfer of infectious agents to an animal species where they did not previously exist. for example, the spread of ''mad cow'' disease from sheep to cattle (and then subsequently to humans) in the s occurred through changes in how cattle feed was prepared [ ] . in about the same time-period, the spread of a relative of hiv, the simian immunodeficiency virus (siv), from an african monkey species to a previously uninfected asian monkey, took place in primate centers in the united states [ ] . even more dramatically, the epidemic appearance of seemingly ''new'' viruses can be unwittingly aided by human activities that amplify previously rare viruses, as was seen with the reuse of hypodermic needles in campaigns against schistosomiasis (snail fever) that led to the exponential spread of hepatitis c virus in egypt in the mid- th century [ ] , as well as similar, but more speculative, theories for the amplification of hiv in central africa during campaigns to eliminate trypanosoma brucei (sleeping sickness) in the s [ ] . ultimately, though, where did these viruses come from? fossils of animals and plants have been used to understand the age of groups of species. although no physical fossils exist for viruses, clues about virus history can be gleaned from the remnants of viral sequences (called ''viral fossils'') that have been accidently inherited in the genome of the host they infected in the past. that is, although viruses typically spread between hosts by transmission from one individual to another (called horizontal transmission), on rare occasions viral nucleic acids can get integrated into the host germline and become endogenous viral elements (eves) [ ] . these eves can then be transmitted from parent to offspring (called vertical transmission) by inheritance of chromosomes that contain the viral dna. at some rare frequency, these inherited eves become present in all members of a species (called ''fixation'') and will evolve along with the host genome over extended periods of time ( figure ). the analyses of the host genomic data can thus reveal the presence of these eves, which represent ''viral fossil records'' and can provide information on ancient viruses; together these analyses are known as ''direct'' paleovirology (in contrast to ''indirect'' paleovirology, which instead looks at the consequences of ancient viruses on the evolution of host genes) [ ] . determination of the presence or absence of shared eves in different species can be used to estimate the date of the viral insertion into the host genome [ ] and to infer the presence of an ancient circulating virus among certain hosts over a very long period of time ( figure ). for example, the analyses of eves among different species showed that a relative of the hepatitis b virus that currently infects humans was present in the passerine birds at least million years ago [ ] . importantly, because integration in the host cell chromosome is an obligate part of the lifecycle of retroviruses, the catalog of endogenous retroviral elements provides an especially rich record of past retroviral infections (for example, [ ] ). among retroviruses, a group of viruses related to the reticuloendotheliosis virus (rev) has especially interesting ancestral origins. rev currently circulates in chickens, turkeys, pheasants, ducks, and geese, where it sometimes causes disease. however, its genome is not related to other avian retroviruses; rather, it is most closely related to some mammalian retroviruses [ ] . this is unusual because retroviral transmissions between different classes of animals (e.g., between birds and mammals), called inter-class transmissions, are much more difficult than those between closely related species and are often dead ends [ ] . indeed, the virus needs to adapt to large differences between the host species that will include, among many other things, differences in the host cell receptors to allow the virus to enter the cells, differences in the host cell environment (e.g., temperature) that the virus needs to replicate, and different sets of immune defense proteins. one hypothesis is that rev may have been aided in its ability to move between very different species because it is a an unusual recombinant virus between two different retrovirus genera; some of the rev genes are related to gammaretroviruses, while the viral gene that encodes the protein that allows it to enter cells is derived from beta-retroviruses [ ] . there are several important biological differences between gammaretroviruses and beta-retroviruses, but one that may be important in understanding the origins of rev is that the gammaretroviruses do not generally undergo inter-class transmissions, while the fossil records of beta-retroviruses show more propensity for large species jumps [ ] [ ] [ ] . finally, rev is unusual in having integrated itself into two other large dna viruses: a herpesvirus, which replicates in the nucleus, and a poxvirus, which replicates in the cytoplasm [ , ] . the concept is illustrated with the example of the viral fossil record and evolutionary history of the reticuloendotheliosis virus (rev). the tree is a timescaled representation of a host genome phylogeny. hosts for which genomic sequences are available are depicted in white, while hosts that do not have genome data available are represented in plain black. the hosts that harbor a rev-like endogenous element are depicted, as well as those from which information is not available (question marks) and others from which the eve is absent. regions in green represent the eve flanking region. the time frame in which an exogenous mammalian virus ( ), here rev, integrated in the host germline ( ) and reached fixation ( ) is represented. the presence of related eves at the same locus in both mongoose species shows that the insertion occurred before their divergence, approximately million years ago (mya), while the absence of eve at this site in the cat species indicates that the insertion occurred after mya. although not definitive, pcr for rev in fossa was negative [ ] . here, a more recent inter-class transmission from an unknown mammal was the origin of rev in birds. image credit: lucie etienne. doi: . /journal.pbio. .g in this issue of plos biology, niewiadomska and gifford elucidate the complex evolutionary history of rev and related viral strains by using paleovirology and a historical review of the literature to infer the viral origin, the successive inter-species transmissions, and the adaptive events that led to the circulation of revs in wild birds [ ] . along the way, the story takes us from a mongoose, to a pheasant at the bronx zoo, to a duck model of malaria that might have led to contamination of vaccines of large dna viruses of birds, which may have subsequently spread rev around the world. the link between this avian retrovirus and endogenous elements in mammals was first documented in a screen of available animal genomes in which the most closely related sequence to rev was from a fragment of the genome of an echidna, an egg-laying mammal from australasia [ ] . niewiadomska and gifford were characterizing the diversity of endogenous retroviral elements in malagasy carnivores using previously frozen tissue samples, and fortuitously happened upon rev-like sequences in the genome of the ring-tailed mongoose and the narrow-striped mongoose [ ] . additional dna sequencing identified viral sequences that were similar to rev across the entire viral genome, including the same recombination breakpoint found in modern rev. this crucial finding showed definitively that the endogenous retrovirus from mammals and the currently circulating avian exogenous revs share a common ancestor. to estimate the age of this ancient retroviral lineage, the authors showed that the flanking regions of the newly characterized eves were common to the two mongoose species [ ] , thus showing the rev-like retrovirus entered the germline and reached fixation before the split between these two species during the miocene period ( figure ) . therefore, an exogenous recombinant rev-like virus was circulating in mammals more than million years ago. how then did this mammalian retrovirus get into birds? from here the story gets spectacular and strange (yet still very plausible). in the s, the need for an experimental model system for malaria research led lowell coggeshall to isolate plasmodium parasites from a pheasant, originally from borneo, that was housed at the bronx zoo. for experimental vaccine and drug research purposes, the plasmodium stock was passaged over time in poultry until the s and was distributed to many us laboratories. however, the culture of plasmodium lophurae was contaminated with retroviruses from the rev lineages (contamination may have existed in the pheasant, or might have been acquired during passage). the authors depict a compelling scenario in which the zoological park, an environment in which exotic animal species from diverse lineages are brought together in unusually close proximity, could have favored the inter-class transmission of revs from mammals to the pheasant from which plasmodium was isolated. however, as inter-class transmissions often result in deadend infections, the authors surmise that serial passages of the retrovirus-containing plasmodium stock in poultry favored the adaptation of the mammalian virus to bird hosts. sometime along the way, the rev virus integrated into two large dna viruses: a poxvirus (fwpv) that contained a full-length insertion of rev and a herpesvirus (ghv- ) containing only a fragment of rev [ , ] . because live attenuated fwpv vaccines harboring replicative competent revs were used in vaccination campaigns of poultry, it may have further played a role in the spread of revs into wild birds around the world [ ] . thus, rev is now a bird virus that was once an ancient mammalian virus (box ). further efforts are needed to determine if rev still has a modern mammalian host; the authors of this study speculate it could be a bat species [ ] . indeed, the reservoir of viruses is vast, and recent field studies and metagenomic screens are finding relatives of human and domestic animal viral pathogens in unexpected places (for example, [ ] ). moreover, it will be important to determine if the ancient exogenous rev had a wider reservoir by investigating more animal genomes ( figure ) . importantly, the increasing number of full-length genome data obtained by next-generation sequencing from various animal species will reveal an even greater number and genetic diversity of eves, which will allow us to understand better the ancestors of the modern viruses and estimate with greater precision the date of their viral insertion into the host germline ( figure ) . thus, the answer to the question ''where did these viruses come from?'' will increasingly become ''they came from ancient forms of other viruses,'' although in some cases, such as those described for rev [ ] , they come through a thoroughly circuitous route. while the main text describes the origins of the reticuloendotheliosis virus (rev) and its close relatives as pathogens, the study of these retroviruses has also contributed to several areas of basic science and basic virology. the original description of rev was from a tumor taken from a turkey that contained two viruses. one, rev-a, was replication-competent, and the other, rev-t, was defective, but carried an oncogene called rel that allowed it to transform cells [ , ] . rel, it turns out, is a key component in the innate immune signaling pathway [ ] , as a component of the nf-kb transcription factor [ ] . the late howard temin adopted rev and related viruses as model systems to describe, among many other phenomena, the kinetics of retroviral infections [ ] , the derivation of cell lines for making gene therapy vectors [ ] , and retrovirus mutation and recombination rates [ , ] . global trends in emerging infectious diseases sars-cov and emergent coronaviruses: viral determinants of interspecies transmission the great influenza: the story of the deadliest pandemic in history. penguin books crossspecies virus transmission and the emergence of new epidemic diseases emerging virus diseases: can we ever expect the unexpected? how the cows turned mad: unlocking the mysteries of mad cow disease. schneider e, translator molecular epidemiology of simian immunodeficiency virus sivsm in u.s. primate centers unravels the origin of sivmac and sivstm the epidemiology and iatrogenic transmission of hepatitis c virus in egypt: a bayesian coalescent approach the origin of aids endogenous viral elements in animal genomes paleovirology -ghosts and gifts of viruses past genomic fossils calibrate the long-term evolution of hepadnaviruses a transitional endogenous lentivirus from the genome of a basal primate and implications for lentivirus evolution interclass transmission and phyletic host tracking in murine leukemia virus-related retroviruses origins of major human infectious diseases multiple groups of endogenous betaretroviruses in mice, rats, and other mammals identification of diverse full-length endogenous betaretroviruses in megabats and microbats a novel endogenous betaretrovirus group characterized from polar bears (ursus maritimus) and giant pandas (ailuropoda melanoleuca) retrovirus insertion into herpesviruses field and vaccine strains of fowlpox virus carry integrated sequences from the avian retrovirus, reticuloen dotheliosis virus the extraordinary evolutionary history of the reticuloendotheliosis viruses bats are a major natural reservoir for hepaciviruses and pegiviruses characterization of reticuloendotheliosis virus strain t dna and isolation of a novel variant of reticuloendotheliosis virus strain t by molecular cloning nucleotide sequence of v-rel: the oncogene of reticuloendotheliosis virus regulated nuclear import of rel proteins in the drosophila immune response nf-kappa b, kbf , dorsal, and related matters replication of reticuloendotheliosis viruses in cell culture: acute infection construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors lower in vivo mutation rate of human immunodeficiency virus type than that predicted from the fidelity of purified reverse transcriptase retroviral recombination and reverse transcription we thank kristopher azarm, jesse bloom, patrick mitchell, cristina wittkopp, and janet young for their comments. key: cord- -wwaa ls authors: potgieter, leon n.d. title: pathogenesis of viral infections date: - - journal: veterinary clinics of north america: small animal practice doi: . /s - ( ) - sha: doc_id: cord_uid: wwaa ls the article considers factors that influence pathogenesis, initiation of infection, dissemination of virus within a host, lytic viral infections, viral immunosuppression, viral immunopathology, and viral oncogenesis. genetic characteristics of the host · · or environmental influences. genetic host factors include animal species, breed, and organ or tissue susceptibility. · · · such restrictions function at the cellular level either as the presence or absence of appropriate cell surface receptors (in some instances, they have been shown to be inherited as dominant alleles in a mendelian manner) · · · · · ·u · or the intracellular hospitality of the cell (several genetic host restrictions on virus replication have been identified). · · · · · · · · restricted growth of several dna viruses in some cells results in transformation without production of progeny viruses. to a degree, the animal's ability to respond immunologically or by interferon production is an innate property of the host. , , . , , , , . nongenetic factors include passive immunity, acquired immunity, age, stress, trauma, hormonal levels, nutritional status, environmental temperature, pregnancy, and concurrent infections (which may either enhance or interfere with a virus infection). , , , , o, , , , , virus factors certain virus properties materially affect the manner in which disease is elicited. cytocidal viruses such as alphaherpesviruses effectively inhibit cellular metabolism, · · · · ·ii · · · and others result in pathophysiologic permeability of cell membranes. certain viruses contain cytotoxins, which may be structural proteins (such as the penton adenovirus protein) or induce production of cytotoxic substances. , ,u,us, , , , noncytocidal viruses may result in steady-state infections, · · · · • but these in turn can result in perturbations of the immune response. , , , , , s-so,oo, · · · , ·m· cell-mediated destruction of tissues, immune complex disease, autoimmunity, and disseminated intravascular coagulation are possible sequelae. the presence of the enzyme reverse transcriptase in virions of members of the family retroviridae imparts two important characteristics to these viruses-the capacity for latency as "proviruses" and for transformation of the cell. • · · strain variation in virulence and in tissue tropism is well recognized. · · ·uo.u · these phenomena are undoubtedly related to variations in the nucleotide sequences of the viruses. ·u · some viruses, as a result of their genomic structure or their particular replication strategy, induce interferon production in the host more efficiently, which in turn may result in greater resistance to the virus. · several instances have been documented in which viruses modify cell function without detectable cell injury. · examples include disease as a result of virus modulation of immunocompetent cells and cells with endocrine functions. · · - b,lll of particular concern in viral pathogenesis is the tissue tropism of the virus, which is determined by complex interactions of virus structure, cell receptors, cell metabolism, intracellular hospitality, and virus replication strategy. , , , , , , ,oo, , , o, , the host is more vulnerable if cell destruction occurs in vital tissues or organs such as the heart, central nervous system, immunocompetent cells, liver, endothelium, and certain endocrine tissues. , , , , , , , , , whether a virus initiates a successful infection in a particular host is often dependent on the amount of inoculum that the host is exposed to and the route by which virus gains entrance into the body. · · · for such viruses, a certain threshold amount of virus is required for successful infection, and, in some instances, the severity of the ensuing disease may be determined by dose of the virus. as alluded to earlier, the first barriers that viruses must penetrate are various epithelia of the skin and its mucosal extensions, alimentary canal, respiratory tract, and the urogenital tract. · · · these epithelia may have various products that aid in resistance to virus, such as film, mucus, keratin (skin), acidity (stomach), and so forth. these barriers must be overcome by viruses to initiate infections. some viruses (orthomyxoviruses, for example) penetrate mucus by enzymatic action (neuraminidase); others are more phresistant and may more readily infect the intestinal tract, and some viruses initiate infection after mechanical injury to epithelia. · · ·u · cell surface receptors characteristic for a particular species and for a particular tissue may determine species (or breed) and organ susceptibility. · · · ·u · these receptors are genetically specified. in most instances, the chemistry of cell receptors is not well characterized. it is best known for the ortho-and paramyxoviruses for which it is a neuraminic acid-containing mucoprotein. ·u others include lipoglycoproteins (picornaviruses), histocompatibility complex antigens (some alphaviruses), and fe and complement receptors on macrophages. · · a · · · the latter receptors on some flavi-, alpha-, bunya-, rhabdo-, and reoviruses result in enhanced infections in the presence of subneutralizing concentrations of antibody or heterologous strain antibody that promotes attachment to fe receptors. receptors that are ubiquitous on the surface of cells of various species (such as neuraminic acid-containing glycoproteins) are not important determinants of host range or tissue tropism. · receptors for picornaviruses often do determine these phenomena. · a receptor map has been made for four groups of nonenveloped viruses, and it is clear that even unrelated viruses may share the same receptor. viruses that share the same receptor can interfere with one another since they compete for the receptor (homologous interference). · host range and receptor specificity are particularly important in the retroviridae family. · · for instance, the susceptibility of certain genetic strains of chickens to various oncovirinae depends on the nature of cell surface receptors for the largest viral envelope glycoprotein. these receptors are specified genetically and are inherited as dominant alleles in a mendelian manner. the viral protein and cell receptor have clinical significance because occupation of a receptor by an avirulent oncovirus will block infection with a highly oncogenic virus. another factor in the initiation of infection should be considered. attachment of ortho-and paramyxoviruses to receptors is not important in determining host range and tissue tropism, but it appears that the step of penetration does determine these phenomena. · · · · certain peplomeres of paramyxoviruses have a fusion (f) protein that, after attachment, results in the fusion of the viral envelope with the plasma membrane, al-lowing the viral rna to enter the cell to initiate virus replication. the fusion protein exists as a precursor and is not activated until cleaved by specific cellular proteases probably on the surface of the plasma membrane. cell susceptibility is determined by the presence of the appropriate proteases. a similar event occurs with the orthomyxoviruses, but in this virus group, cleavage of the hemagglutinin facilitates penetration (but does not affect attachment to cells). • • · however, the cleavage of the hemagglutinin occurs during assembly of the virions late in the replication cycle. thus, virion infectivity is dependent on the virus undergoing a full replication cycle in susceptible cells that contain the appropriate protease. a novel concept with regard to receptors on cell surfaces is that cells infected with viruses may permit adherence of certain pathogenic bacteria, leading to bacterial colonization. the phenomenon appears to be mediated by virus-induced receptors on the surface membrane of cells and may be one mechanism of the often-encountered secondary bacterial infections associated with viral diseases. some viruses initiate infections as a result of mechanical injuries of epithelial barriers. • • this can occur as a result of insect bites, which is particularly important for the arthropod-borne viruses (many of the togaviridae, reoviridae, bunyaviridae, and so on). iatrogenic introduction of viruses with needles and so forth is not uncommon. rabies virus constitutes a classical example of a disease initiated by a wound and influenced by dose of virus. • · the latter virus is deposited in a wound usually as a result of a bite from an animal with salivary secretion of virus. if sufficient virus is present, nerve endings are immediately penetrated. however, often the virus first replicates in local muscle cells, which allows for amplification of the infectious dose and penetration of the nerve endings followed by centripetal spread along the axoplasm of nerves to the central nervous system. variations in pathogenesis occur when this general progression of virus is delayed or stopped at some point. replication of virus in muscle cells at the inoculation site may be responsible for variations in incubation period. survival may occur when infection is localized for some reason at the inoculation site or peripheral nervous system. for a variable period of time (before the virus enters the nerves), it is susceptible to antibody. the respiratory tract is a very common site at which virus infections are initiated, usually as the result of airborne infections. • • • • droplets often originate from the respiratory tract and mouth of infected animals, but aerosols of urine, fecal material, and so on can infect the respiratory tract. small droplets of less than j.lm in diameter dry to droplet nuclei of i to j.lm in diameter that remain suspended for long periods and easily gain entrance to bronchioli and alveoli. viruses that resist desiccation remain viable in droplet nuclei for extended periods of time. film and mucus afford some protection, but myxoviruses that are entrapped by receptor-like mucoproteins in mucus may be released by the peplomere enzyme neuraminidase, thereby allowing the virus to move on and finally become attached to a cell surface receptor. , ,ns, , a number of viruses remain localized in the respiratory tract during the course of infection-some in the upper respiratory tract (rhinoviruses, some herpesviruses) and others in the lower respiratory tract (parainfluenza virus). · · although restricted to the respiratory tract, some viruses (influenze, for example) still cause generalized clinical signs such as fever, malaise, and muscle pains as a result of the products and inflammation associated with cell injury and destruction. · · many viruses become rapidly disseminated throughout the body after the primary infection of the respiratory tract (canine distemper virus is a good example). • · · usually there is no evidence of respiratory tract infection in the initial phase of these diseases and the animal usually is not infectious during this phase. the virus spreads via macrophages or lymphatics to regional lymph nodes before spreading further. generally, viruses initiate infection in epithelial cells of the respiratory tract, but some viruses achieve this by successfully infecting alveolar macrophages. · · infection of alveolar macrophages has important sequelae in the pathogenesis of viral respiratory tract disease. · · viral respiratory tract disease is a consequence of mechanical and biochemical injury to epithelial cells and alveolar macrophages, which can, in the most severe instances, result in secondary bacterial infection, pneumonia, and death. · · · · · · denuded respiratory tract, impaired mucociliary escalator, and the growth medium provided by exudate have been thought to enhance bacterial growth and colonization in the respiratory tract. however, these mechanisms may not be as important as originally thought. injury to various biocidal mechanisms may be of greater consequence. · · · the alveolar macrophage, which is of critical importance in pulmonary resistance to bacterial colonization, may be affected in several ways either as direct consequence of virus replication in this cell or as a result of immunemediated cytotoxicity directed at the virus-infected macrophage. the latter is of particular consequence. it occurs late in the disease process when the immune response is initiated and when macrophages are ingesting virusladen cell debris. not only is the number of alveolar macrophages reduced by viral infection, the function of the remaining cells may be impaired. · · - · · · · evidence suggests that such macrophages have suppressed immunologic (fe) and nonimmunologic membrane receptor binding activity, fe and nonspecific receptor-mediated phagocytic ingestion, phagosome-lysosome fusion, intracellular killing, and bacterial degradation. some of these observations are the result of low levels of lysosomal enzymes and impaired biochemically mediated killing mechanisms. the alveolar macrophage may be impaired also because of hypoxia (it is an aerobic cell) and reduced surfactant levels. · the latter facilitates phagocytosis and is produced by type-ii alveolar pneumocytes. viral injury to the latter causes reduced production of surfactant in the lung. as alluded to earlier, certain viruses (such as influenza virus) promote bacterial colonization by altering the plasma membrane of infected cells, which facilitates bacterial adherence to the surface of such cells. the phenomenon appears to be the result of virus-induced receptors on the cell surface. some viruses appear to produce disease primarily by suppressing immunity, which is relatively sequestered from the systemic immune func-tions. • • • • • apart from the pulmonary macrophages, pulmonary cellmediated immunity is derived from bronchus-associated lymphoid tissue and augmented by the influx of blood monocytes and neutrophils. locally synthesized lgg, primarily synthesized in the lung, or transudated serum immunoglobulins function as opsonins, whereas secretory iga (primarily from the upper respiratory tract) prevents bacterial adherence and colonization and probably aggregates bacterial particles to facilitate mucociliary clearance. various viruses attach and replicate in epithelia of the mouth, pharynx, tonsils, and (or) the gastrointestinal tract. · • however, the stomach, abomasum, and intestine are not very hospitable to viruses because of an unfavorable ph and the presence of bile. enteric viruses usually are tolerant of a low ph. certain viruses (rotaviruses, for example) remain restricted to the gastrointestinal tract. · • • secretory immunity (and not humoral immunity) is protective against such viruses. • • many other viruses that invade intestinal epithelia subsequently become disseminated with varying frequency (enteroviruses). • • certain parvoviruses (canine parvovirus ii and feline panleukopenia virus) initiate infection in the pharynx-tonsil area, spread by the blood stream to various organs, and finally infect and destroy the crypt cells of the intestinal tract. the intestines thus become infected by a very circuitous route. villous atrophy results because of impaired replacement of enterocytes, which are derived from the crypt cells. central nervous system infection by the coronavirus, hemagglutinating encephalomyelitis virus of swine, occurs as a result of nerve tract migration from the gastrointestinal tract to peripheral ganglia. infection of neurons that regulate peristaltic functions of the intestinal tract results in gastrointestinal disease and subsequent starvation. certain viruses such as rinderpest and african swine fever initiate submucosal infection without infecting epithelial cells and appear to have the capacity to pass through the epithelial barrier. ·m some enteric viruses destroy the absorbtive columnar enterocytes on the tips of intestinal villi. • • the upper portion of the intestine is first affected, but infection often spreads throughout the entire length of the intestine. the affected cells are desquamated and are replaced by cuboidal or even squamous cells, resulting in a dramatic atrophy of the villi. these target cells are important for the digestion of disaccharides and the absorption of macrosaccharides, and contribute to osmoregulation. replacement of these cells occurs by m'igration of undifferentiated cells from the crypts, which are resistant to infection. however, the new villous tip cells are immature (contain thymidine kinase instead of sucrase, which is an enzyme profile similar to crypt cells) and result in a disturbed sodium transport system with a net extracellular fluid-to-lumen flux of sodium ions. pathophysiologic changes include loss of water, sodium, chloride, bicarbonate, and potassium. metabolism of glucose and lactate becomes severely disturbed, and the hypoglycemia, lactic acidosis, and an elevated efflux of potassium to hypovolumic plasma may lead to acute shock, heart failure, and death. the disease is more severe in neonates because of their milk diet and their dependence on readily available nutrients, and because replacement of sloughed epithelial cells is slower. the inert superficial protective layers of the intact skin usually are impervious to virus infection. • • • injury to the integument can allow several viruses to penetrate into susceptible tissues. trauma, insect bites, needles, and so on may be responsible for the introduction of virus (see earlier discussion). viruses that commonly initiate infection in this manner include most of the arboviruses, rabies, papular stomatitis, and some herpesviruses. some viruses penetrate directly through mucosal epithelia. • infection initiated in the urogenital tract few viral venereal diseases, affecting primarily the genital tissues, have been described. • the principal diseases in this category include those caused by herpesviruses and perhaps genital papillomaviruses of various species. • • however, the potential of infection occurring in the genital tract is high because many viruses may be present in semen. several viruses are capable of establishing infection in the placenta of pregnant animals and subsequently result in congenital infections. vertical infection occurs when a virus is transmitted to progeny with the germ cell. the latter occurs primarily with retroviruses in which viral genetic material becomes incorporated in host dna. it is difficult to consider the establishment of viral disease in a fetus without considering the infection of the dam, because the latter usually is infected for a variable time before the fetus. transplacental infection is perhaps the principal route of fetal infection by a virus. • it may occur by a variety of mechanisms depending on the nature of placentation of the dam and the nature of the virus. • in some instances, the virus appears to have selective pathogenicity for the fetus, because disease may not occur in the dam (for example, some strains of bluetongue virus). • • perhaps a primary infection of the fetus is one that is transmitted with the germ cells. this apparently occurs only with retroviruses, in which viral genetic material (provirus) is passed along as a cellular gene insert. . , fetal infection is a prominent feature of several viruses. the outcome of the disease in the fetus depends on both virus and host factors. some cytolytic viruses (for example, some herpesviruses and parvoviruses) uniformly result in fetal death, whereas other viruses (bluetongue virus, bovine virae diarrhea virus) may not. • • • the nature of the disease in the fetus with some of the latter viruses may depend on the age of the fetus. • this has been quite well documented with porcine parvovirus, bluetongue virus, and bovine viral diarrhea virus. the fetus is very vulnerable in the first third of pregnancy, and infection at this gestational age often results in death, but the fetus becomes progressively resistant to the effect of virus infection. infection in the last third of pregnancy may not have serious consequences. however, frequently the fetus is partially resistant only during the middle third of pregnancy, and infection during this stage may result in various lesions. the latter may include malformations of the central nervous system such as cerebellar hypoplasia (feline panleukopenia, bovine viral diarrhea virus), hypomyelinization (bovine viral diarrhea virus, border disease), hydranencephaly (bluetongue virus, bovine viral diarrhea virus), porencephaly (bluetongue virus, bovine viral diarrhea virus), and hydrocephalus (parainfluenza virus, japanese encephalitis virus). porencephaly is a later manifestation than hydranencephaly in lamb fetuses infected with bluetongue virus. stillbirths, weak neonates, and skeletal malformations may also occur. the progressive development of resistance in a fetus is correlated with the ontogeny of the immune response. some viruses (such as rubella virus) reduce the mitotic rate of infected fetal tissues and thereby affect organ development. • • offspring may be stillborn or runted. infection of the fetus at certain stages of gestation by a number of viruses (pestiviruses, arenaviruses) may result in "immunotolerant" offspring that are persistently viremic and without a detectable immune response. • • • in some instances, superinfection of such animals with the same virus or certain strains of the same virus results in serious, often fatal, systemic disease that may be immune-mediated. fetal disease can occur as a result of viral infections of the dam, even if the fetus itself is not infected. • • • changes in the placental circulation (vasoconstriction, congestion, and hemorrhage) as a result of viral placentitis can rapidly and severely affect the fetus. coxsackie b virus-induced pancreatic acinar atrophy in pregnant mice results in malnutrition owing to the mices' inability to digest and metabolize protein. fetal wastage and growth retardation are two sequelae of this condition. the hyperthermia associated with some viral infections in pregnant animals (for example, influenza) can cause abortions, stillbirths, and malformations (anencephaly, microencephaly, hydroencephaly) of fetuses. local extension of viral lesions occurs in susceptible hosts. · • • it may occur by virus release and dispersal from infected cells to neighboring susceptible cells. in some instances, lysis of infected cells is necessary before virus is liberated. herpesviruses, paramyxoviruses, and others may spread from cell to cell by a fus·ion mechanism. virus-induced cell proliferation, as occurs with poxviruses, papovaviruses, and retroviruses, is another mechanism of lesion extension. dissemination of the virus from the initial focus of infection occurs by several mechanisms. · • • • • the first step in generalized dissemination of a virus is the spread from the local lesion to the regional lymph node, either free in lymph or, more frequently, within carrier cells such as lymphocytes or phagocytic cells. virus concentration may be amplified by replication in the regional lymph node; secondary spread of the virus in blood vessels, either free or, more likely, within carrier cells, to other tissues and organs in the body may occur. this secondary spread of the virus may be detected clinically as the second part of a biphasic fever. in many instances, the critical event in viral dissemination is the successful infection of phagocytes. • the macrophage provides an important resistance mechanism of viral infections, and virulent viruses overcome their antiviral action. impaired macrophage function markedly enhances the susceptibility of animals to viruses. neonatal and corticosteroid-treated animals may be more susceptible for this reason. it is likely that several mechanisms of antiviral action exist in macrophages. • often virus penetration occurs in both susceptible and resistant macrophages, but uncoating of the virus is blocked in resistant macrophages, thereby terminating virus replication. various relatively sequestered organs may become infected with certain viruses. the central nervous system (cns) has nonfenestrated vasculature, tightly packed cellular components, and lacks a conventional lymphatic system. • the endothelial cells are joined by tight junctions and are surrounded by dense basement membranes, which in turn are tightly packed against astrocytic footplates. these structural barriers impede virus invasion of the cns from the bloodstream, but transendothelial migration of an infected lymphocyte and endothelial injury (either as a result of the virus infection or some other cause) may result in cns infection. • • the traffic of leukocytes into the cns is very limited under normal circumstances. some viruses are transported across the cells in pinocytotic vesicles and are deposited in the cytoplasm of the adjacent astrocytes. the cns may become infected by virus migration along nerve trunks (herpesviruses, rabies, hemagglutinating encephalomyelitis virus). • • • several mechanisms have been described • : movement within the central axoplasm, along endoneural spaces, and cell-to-cell infection of schwann cells. both centripetal and centrifugal spread along nerve trunks to and from the cns are possible. rabies virus infects salivary glands by the latter route. viral disease of the cns requires that the virus either has the capacity to invade these tissues or that its entry is facilitated by some unrelated event. • virus infection that leads to injury by direct or indirect mechanisms of oligodendrocytes results in demyelinization. • • • neurologic dysfunction may develop in the absence of obvious cell injury. persistent canine distemper virus infections of rat glioma cells cause a reduction of betaadrenergic receptors. some viruses specifically infect neurons of the cns. virus specificity may be so restricted that only certain subpopulations of neurons become infected. in humans, poliomyelitis viral infection is restricted mainly to motor neurons. rabies virus, during the early stages of infection, is confined primarily to neurons of the limbic system. this facilitates transmission by biting because the cortical-neurons are not involved early, and instead of seizures and motor deficits, alertness and aberrant behavior are the predominant clinical signs. virus selectivity determines also the development of hydranencephaly and porencephaly in newborn lambs infected in utero with bluetongue virus. the virus selectively destroys the germinal cells of the subventricular zone, the precursors of the neurons and glial cells of the forebrain. before the development of the cortical mantle in early gestation, necrosis and hydranencephaly occur owing to the destruction of these cells. after the formation of the cortical mantle before midgestation, porencephaly develops because the glial cell precursors are destroyed, resulting in focal white matter necrosis. several parvoviruses, which replicate only in cells in s-phase mitosis, selectively destroy the germinal cells of the cerebellum, resulting in hypoplasia of the cerebellum with abnormal foliation, depleted granular cells, and aberrant synaptic organization. this condition, which occurs in cats after infection as neonates or in late gestation, is known as spontaneous ataxia of kittens and is the most common neurologic disease in cats. for many years, it was thought to be an inherited condition. it has become evident that viruses can injure endocrine tissues specifically with the concomitant deficit in endocrine secretion. • · • • • in some instances, functional impairment develops without obvious cell injury. certain picornaviruses cause selective destruction of beta-pancreatic cells, resulting in diabetes. • evidence suggests that this may occur in humans also. impaired growth hormone production, growth rate, and glucose metabolism result from selective noncytopathic infection of anterior pituitary gland cells with lymphocytic choriomeningitis virus in mice. • immunosuppression vastly increases the potential of a virus to advance from a localized to a generalized infection. • ° complex interactions occur between various microorganisms in natural infections. • • • • a virus that induces immunosuppression in an animal can significantly enhance the severity and change the nature of a concurrent viral disease. one report suggested that canine parvovirus, which is lymphocytolytic, may enhance the neuropathogenicity of modified live canine distemper vaccine virus. bovine viral diarrhea virus, which also affects lymphocyte function, greatly enhances the dissemination of a herpesvirus, infectious bovine rhinotracheitis virus. various host-and virus-related restrictions determine tissue or organ invasion by a virus. cell surface receptors and the virus penetration step have already been discussed. • • • • intracellular hospitality for the particular virus may determine whether virus replication (either partial or complete-with progeny) occurs in a particular cell. • • · defective interfering particles are viruses of subgenomic size that contain most or all of the normal viral proteins. • they lack complete genomes and are unable to replicate in the absence of the parental virus. paradoxically, they interfere with the replication of the parental virus, apparently because the defective particles with their smaller genomes have a replication advantage and are able to sequester the replicase systems. defective interfering particles have been demonstrated in most groups of viruses. they may mediate the cessation of a viral disease or may convert an acute, lytic disease to a persistent or chronic infection. evidence indicates that the generation of defective interfering particles is controlled by host cells. blockage of host dna synthesis blocks generation of these particles when vesicular stomatitis virus free of defective interfering particles is used as the infecting virus. defective interfering particles are produced in such cells if they are introduced with the parenteral virus. this evidence, together with evidence that the defective particles can alter the pathogenesis of viral infections, suggest that cells may have evolved mechanisms to protect themselves against viruses that successfully enter and penetrate. the most rapidly produced defense against viruses is a family of proteins secreted by tissue cells in response to various stimuli such as viruses, bacteria, foreign cells, foreign macromolecules, and several other compounds. • • interferons act indirectly by stimulating surrounding cells to produce protein(s) that, in turn, may regulate virus replication, the immune response, cell growth, and other functions. interferons are unexpressed genetic functions of mature cells and may have a normal role in cell regulation. certain viruses are much more efficient in eliciting interferon production than others, which, of course, affects the course of the virus infection. • generally, the most potent interferon stimulators are also the most susceptible to interferon. · there are many examples of exquisitely sensitive genetically controlled expression of viral disease functional within the host cell. an example of \iral genetic control of pathogenesis is that of reoviruses in newborn mice. reovirus type produces nonfatal infection of ependymal cells after intracerebral inoculation, whereas type results in fatal encephalitis with neuronal destruction. the tropism for ependymal cells or neurons seems to be regulated by a single gene (the s genome segment) that codes for the sigma- capsid protein. another genome segment (m ) determines the ability of these viruses to initiate local or systemic infection in newborn mice after oral inoculation. an example of host genetic control of pathogenesis at the intracellular level is the fv- system in mice, which determines susceptibility to murine leukemia viruses. this mechanism operates after penetration but before integration of viral genetic material in host dna. the cellular gene, known as fv- , is present on chromosome , and the virus determinant with which it interacts to determine this tropism is the p viral protein. this mechanism is illustrated by murine leukemia viruses that preferentially infect cells from ~ih swiss mice (n-tropic) or balb/c mice (b-tropic). cells may be destroyed when they become infected with certain viruses, particularly dna viruses. several mechanisms are responsible for this effect on cells. , , , , , , , , , , , , , , , in some instances, cell metabolism is drastically altered. macromolecular synthesis may be inhibited by a variety of mechanisms and may occur at the level of replication, transcription, or translation. certain viruses 'interfere with host dna replication. vaccinia virus mrna competes with cellular mrna. this virus may also result in disruption of polysomes and impaired rna processing. poliovirus inhibits the initiation factor in mrna translation. increased permeability of virusinfected cells causes increased intracellular sodium ions, which favor viral mrna translation. the rate of formation of viral products may be greater than their release, which has an adverse effect on cellular metabolism. the virus may deplete substrates essential for vital cellular functions, and the physical presence of viral products in a cell has an adverse effect on cell metabolism. complete or partial replication of a virus is necessary for cell injury in many instances, but virus replication does not necessarily result in cell injury, particularly with many rna viruses. several viruses produce a toxin-like substance that is either a direct effect of a virus-induced product or a secondary effect caused by the activation of lysosomal enzymes by a virus. viral cytotoxins have been described for poxviruses and adenoviruses (the penton capsid protein which reversibly modifies cell membranes from the outside). these cytotoxins may be either structural virion components (preformed) or induced during virus replication. vaccinia cytotoxin has been identified as a surface tubular virion protein, whereas adenovirus cytotoxin is contained in the penton capsomere (the hexon capsomeres and fiber antigen inhibit macromolecular synthesis). the cytotoxic effect is markedly amplified in certain virus infections. t lymphocytes infected with dengue virus produce a cytotoxin that induces macrophages to produce a cytotoxic factor. the exact mechanism by which various viruses affect cell metabolism and produce cytopathogenesis is not well known. however, it is known that poxviruses rapidly cut off host macromolecular synthesis, disaggregate host polysomes, interfere with processing of rna, and redistribute lysosomal enzymes. one hypothesis is that the cytopathic and pathophysiologic changes may be due to a common cause-an alteration of the permeability of cell membranes. leakiness of cell membranes occurs during infection with viruses from several virus families and usually precedes cytopathogenesis. certain physiologic and clinical manifestations of virus infections can be attributed to membrane leakiness. these include excess respiratory tract mucus production with rhinovirus infections, loss of vision in corneal keratitis caused by herpesvirus infections, and excessive loss of water and electrolytes from the gastrointestinal tract during rotavirus infections. one of the most important aspects of infectious diseases that is receiving increasing recognition is the interaction and synergism of pathogenic microorganisms in the manifestation of the pathologic state. in many instances of enhanced disease due to microorganism interaction, it is the result of virus-induced immunosuppression. • • - • - a virus-causing immunosuppression can enhance dramatically diseases caused by viral, bacterial, or protozoan infections that normally are relatively innocuous. viral immunosuppression may facilitate dissemination of microorganisms in a host and promote persistent or chronic infections. the cellular basis and consequences of viral-induced immunodeficiency are discussed in detail elsewhere in this issue. a brief summary will be presented here. the most readily recognized types of viral immunosuppression are those infections that result in destruction of immunocompetent cells such as macrophages, neutrophils, and lymphocytes. however, in the majority of instances of viral immunosuppression, cytocidal infection of immunocompetent cells is not recognized, and the phenomenon appears to be the result of impaired function. · · · · - · · · · · ·m virus-induced immune modulation has been recognized in macrophages, neutrophils, various subsets oft lymphocytes, and b lymphocytes. many of the immune responses inhibited by viruses may be attributed to alteration of macrophage function (see the discussion on the antibacterial activity of alveolar macrophages). influenza virus, lymphocytic choriomeningitis virus, bovine viral diarrhea virus, and several other viruses are capable of affecting phagocytic cells. several studies have indicated suppression of phagocytic, chemiluminescence, and chemotactic responses of macrophages and neutrophils. delayed wound healing in mice has also been attributed to viral alteration of macrophage function. paradoxically, impaired function of some macrophages (such as the alveolar macrophage) may be due to immune-mediated injury as a result of virus-specific cytotoxicity directed against the virus-laden macrophage. virus-induced modulation of macrophages conceivably could alter the outcome of numerous cellular interactions, either due to impaired direct participation in lymphocyte functions or because of interference of secretion of regulatory factors. functional impairment of lymphocytes by viruses may be the result of changes in cell surface receptors, competition between virus and immunogen for protein or dna synthetic machinery, generation of suppressor interferon, interruption of the cellular communication network in the immune response, and stimulation of suppressor t cells. • · · · ·m as has been observed for macrophages, viral infection in lymphoid cells may result in the production of a lymphocytotoxic immune response, which may lead to the premature senescence of t cells. another mechanism that may occur in some viral infections is virus-induced alterations in normal lymphocyte traffic and recirculating patterns in the host. this results in redirecting immunocompetent cells away from lymph nodes and spleen and decreasing the immunologic reserve of lymphatic tissues. an active area of investigation is immunosuppression by retroviruses, which seems to be mediated by the pl e envelope protein of some of these viruses (particularly feline leukemia virus). · · · · · impaired immunologic functions associated with exposure to pl e include monocyte chemotaxis, lymphocyte blastogenesis, erythroid colony formation, macrophage accumulation, and tumor immunity. it has been suggested that pl e causes immunosuppression by blocking the production of interleukin by lymphocytes. virus-induced immunosuppression is often accompanied by multiple deficits of the immune response. during certain viral infections, there is both an activation and increase of suppressor t cells, which, in turn, suppress autologous t-cell proliferation to antigens as well as b-cell antibody production. · · ·m certain paramyxoviruses cause silent infections of lymphocytes, which fail to generate natural killer (nk) cell activity (an important antiviral mechanism) or produce antibodies. · several viruses have been identified that inhibit interferon production in a host, resulting in increased susceptibility to other viruses. • host-damaging immune responses in viral infections have been reviewed adequately in the literature. • • the mechanisms and consequences of viral immunopathology will be summarized in this section. generally, host immune responses are responsible for recovery from virus infections but, occasionally, they can initiate or enhance cell injury. certain viruses, such as herpesviruses and picornaviruses, are cytolytic, and disease results from direct destruction of tissue cells. in such instances of viral infection, the immune response can limit infection by destroying infected cells before progeny virus is assembled and released. immune-mediated cytolysis can be deleterious to the host if it occurs late in the replication cycle of the virus, because then it serves only to release infectious virus. noncytolytic viruses often do not cause direct cell injury, but immune responses may injure cells persistently infected with such viruses. a chronic inflammatory response may occur when such cells are not rapidly destroyed by immune processes. such responses frequently are harmful to the host, especially if they interfere with the function of certain tissues and organs. immunopathologic chronic inflammation usually involves antigen-sensitized t lymphocytes and/or antibody immune complexes that also activate complement with inflammatory consequences. viral immunopathology may be divided into two categories: lesions due to antibodies, either directly or with other nonspecific effector mechanisms (for example, antibody-dependent cellular cytotoxity-adcc) or lesions due to specific cellular immune responses. the classical example of virus antibody immunopathology is immune complex disease. • ,· • • · • • • the development of immune complex disease is associated with circulating antibody-antigen complexes in serum, deposition of the smaller complexes (in instances of antigen excess) in tissues with limiting basement membranes (glomeruli, arterioles, choroid plexus, joints, and so on) to produce injury, and the presence of antigen, antibody, and complement components at the site of injury. the initial step in immune complex disease is the release of vasoactive substances, such as histamine and serotonin, and the subsequent increase in vascular permeability. serotonin is released from platelets when they react with immune complexes in the presence of complement. other mechanisms of immune complex-mediated vascular permeability, such as kinins generated from activated hageman factor, may also occur. such platelets may contribute also to immune complex lesions by causing capillary thrombosis. increased vascular permeability facilitates trapping of immune complexes, usually those of small size that develop in moderate antigen excess and in the walls of arterioles and capillaries. the trapped complexes activate complement locally and chemotactically attract polymorphonuclear cells. the circulating and fixed macrophages can degrade immune complexes, but the phlogogenic effect with anaphylotoxins (and the consequent release of vasoactive amines), neutrophil release of cathepsins and proteolytic basic proteins, and subsequent activation of hageman factor (which leads to kinin and plasmin production) cause direct vessel wall injury. arteritis, glomerulonephritis, arthritis, and choriomeningitis are common sequelae of immune complex disease. immune complex disease is particularly prevalent in infections with noncytopathic viruses such as those that cause equine infectious anemia, aleutian disease of mink, and feline infectious peritonitis (fip), but it may also cause some of the lesions of cytocidal viruses (for example, canine distemper virus). evidence indicates that fip is an immune complex disease. • • • humoral immunity is not protective in this disease, but a functional cell-mediated immunity appears to prevent lesions caused by fip virus. impaired cellular immunity is associated with manifestation of the disease (the effusive form with greatly deficient cell-mediated immunity, and the noneffusive form with a partially impaired cellular immunity. • thus, in this disease, we have paradoxical mechanisms of immunopathogenesis. one facet (the humoral response) is enhanced, whereas another (cell-mediated) immunity, may be impaired. this may be the reason fip is associated frequently with feline leukemia virus, a powerful suppressor of lymphocyte function. virus-infected cells may be destroyed by antibody-dependent cellular cytotoxicity (adcc). in this synergistic action of specific antibody and effector cells, the immunologic specificity is provided by the antibody molecules (only minute quantities are required), whereas the effector cells act nonspecifically. the latter must bear fe receptors and include t cells, b cells, killer cells, macrophages, and neutrophils. complement-assisted cytotoxicity occurs when complement enhances adcc. interferon also has been reported to enhance adcc. complement-mediated cytotoxicity involves virus-infected cell destruction by complement following activation by the classical and alternative pathways with specific immunoglobulins. activation of complement by the alternative pathway by virus-infected cells, in the absence of antibody, may also occur. a special case of antibody-induced host injury is the enhancement, by specific antibody, or infectivity of certain viruses of host cells. this has been described for flaviviruses (for example, dengue virus) and for fip virus, for which target cells are macrophages. although mononuclear cells appear to be the only cells that support replication of dengue virus, the latter is not internalized efficiently in the host cell unless complexed with non-neutralizing antibody, which attaches to fe receptors on the cells. some investigators have speculated on the possible allergic immunopathologic responses to viruses. • • the potential for lge-mediated type i hypersensitivity in virus infections exists, but little evidence has been presented to support this hypothesis. an interesting hypothesis has been proposed to explain certain antibodymediated hypersensitivities in virus infections. • it envisages a viral infection (such as dengue virus) in a host with pre-existing parasitic infection leading to depletion of suppressor t lymphocytes. the resultant augmented production of igg and ige could then result in type iii and type i hypersensitivities, respectively. t-lymphocyte cytotoxicity is an immunologically specific and highly effective lysis of cells with viral antigen expression on the plasma membrane. • • ·m in some species (most notably mice), genetic restriction oftcell cytotoxicity occurs in that there is a requirement, not only for specific antigen binding, but also for recognition of certain antigens of the major histocompatibility region. the cytolytic event requires contact between the effector and the target cell. the former can lyse a number of target cells in sequence, but the number of immune t cells at a particular site may not be high; for this reason, indirect t-cell events such as macrophage recruitment may be more important in immunity and immunopathology. cytotoxic lymphokines (lymphotoxins) are soluble products oft cells following reaction with viral antigen. lymphotoxins cause cytolysis by affecting the permeability of the plasma membrane of cells. natural or normal killer (nk) cells are lymphocytes with cell surface fe receptors that nonspecifically destroy certain virus-infected and neoplastically transformed cells. interferon and complement can facilitate cytolysis by t lymphocytes and thus enhance viral immunopathology. macrophages can be directly cytotoxic, but this appears to be a nonspecific event. they may be localized at sites of specific antigen by t cellmediated and phlogogenic recruitment. evidence is accumulating that certain viruses may elicit tissue-reaction antibodies and t lymphocytes, which results in tissue and organ injury. • • • • • in some autoimmune diseases, the organ and tissue specificity is quite broad, whereas in other instances, it is very restricted to specific cell types. a polyendocrine disease affecting several hormones as a result of virus-induced autoimmunity has been described. • several mechanisms of the pathogenesis of viral pathogenesis have been described. one possibility is the virus-induced access of the immune system to sequestered antigens, such as the cns (protected by the blood-brain barrier), which are normally "immunologically privileged" sites and are not normally monitored by recirculating lymphocytes. this may occur during the acute phase of the disease. subacute or chronic demyelinating encephalomyelitis that occurs with some strains of canine distemper may be the result of such a mechanism. another mechanism that is plausible is virus-induced changes in host cell membrane. the expression of new host antigens such as embryonic antigens or alloantigens is induced. a related mechanism may be the result of virus antigens expressed at the cell surface that share antigens with the host cell. the immune response to either the new or cross-reactive antigens is capable of reacting with normal tissues. the final mechanism is one that has been alluded to already-that of loss of immune regulation, such as impaired suppressor cell function. tolerance to self-antigens may be maintained by suppressor cell activity, at least in part. diminished activity of these cells then results in increased responsiveness to foreign antigens and an immune response to self-antigens. a special case of autoimmune disease may occur in mucosal disease and bluetongue virus-associated hemorrhagic disease in cattle that have "tolerant" infections as a result of in utero infections. superinfection with a heterologous strain or an overwhelming dose of a homologous strain may induce an immune response to the "tolerant" viral antigens that are present in most tissue cells and thereby induce cell injury. much of the discussion of viral pathogenesis has focused on structural and functional injury of cells. another virus-induced response of cells is that of immortalization due to oncogenic transformation. the mechanism by which various viruses transform cells depends on the nature of the virus. oncogenic dna viruses belong to several virus families, but oncogenic rna viruses are members of the retroviridae. viral oncogenesis is a complex phenomenon and is an area receiving considerable attention by the scientific community. transformed cells have altered morphology, changed behavior, and altered biochemistry. often new membrane proteins and glycoproteins appear (for example, tumor-and/or virus-specific surface antigens and fetal antigens). co-carcinogens have been identified that appear to promote transformation by certain viruses. immunosuppression, a prominent feature of infection by several viruses, may be an indirect oncogenic mechanism of these viruses. it is still not clear which of the complex of changes induced in cells by viruses cause transformation and which are secondary events. at best, this review can only summarize the information on viral oncogenesis, and in order to achieve this, the subject material is greatly oversimplified. for a more detailed description of viral oncogenesis, please consult the reviews by dues berg, ° fenoglio and lefkowitch, weiss, generally, dna viruses replicate in cells and destroy the cell after progeny is produced. cells that support virus replication in this manner are known as permissive cells. however, in some cells (nonpermissive cells), virus replication is not completed and progeny virions are not produced. on rare occasions, these nonpermissive cells become transformed. defective virus may also, on rare occasions, transform both permissive and nonpermissive cells. an essential event for dna virus-induced transformation appears to be integration of viral dna into host dna. poxviruses seem to be the exception. indeed, poxvirus-induced tumors consist of hyperplastic rather than trans-formed cells. neither the cellular site of integration nor the location on virus dna is unique or even specific, and integration seems to be the result of random "illegitimate" recombination between nonhomologous regions of the virus and the host. there is insufficient evidence that oncogenes are involved in dna virus transformation. although the entire viral dna genome may be integrated in transformed cells, all transformed clones contain at least part of the early region of the viral genome. various early functions seem to initiate and/or maintain the transformed phenotype, whereas late viral functions do not have a role in transformation. thus, many dna viruses mediate transformation through the action of some viral gene products, which generally are required for the virus to complete its normal cytolytic cycle. it is not known exactly how the transformation genes originate, but, unlike the retroviruses, there is little evidence that they are derived from normal host genes. however, there is some evidence for limited transcription from virus dna sequences into flanking host sequences. in addition, recent transfection experiments with burkitt's lymphoma cells resulted in the discovery of an oncogene of cellular origin. this oncogene is activated in burkitt's lymphoma. furthermore, one locus in the human genome has some homology with the gene-enhancer sequences of a human papovavirus, indicating that some of the oncogenic enhancer dna sequences of dna viruses can be evolutionarily related to host cell sequences. it appears also that dna virus oncogenes can interact with retroviral (or cellular) oncogenes. a synergistic interaction of the "ras" oncogene and an adenoviral oncogene has been reported. viral dna can mediate transformation through several possible mechanisms. viral dna integrated at certain sites in host dna could act as a mutagen, thereby destroying the control of cellular genes. perhaps integrated viral genetic material may contain promoters of viral gene expression and also coincidentally affect host gene expression. finally, viral dna may specify protein(s), which when synthesized, may directly cause transformation of the cell. as mentioned earlier, several viral early proteins have been identified that initiate and/or maintain transformation. the large t and small t antigens of sv virus are proteins that function in this manner. co-carcinogens promote oncogenesis by several dna viruses. the flavinoid (quercetin) from bracken fern is associated with progression of upper alimentary tract papillomas to carcinomas in cattle. burkitt's lymphoma, a malignent b-cell lymphoma of humans, is associated with infection by the herpesvirus epstein-barr virus. the most likely co-factor is holoendemic malaria. aflatoxin appears to be a co-factor in primary liver carcinoma associated with hepatitis b virus. the oncovirinae subfamily of the retroviridae, due to its unique intracellular biology, has an exquisitely well-developed mechanism for cell transformation. • • • • the enzyme, reverse transcriptase, constitutes the fundamental basis of the molecular biology of these viruses. the diploid rna genome usually contains four major genes in a specific sequence. the gag gene, which is closest to the ' end, codes for a poly protein, a precursor of four structural proteins of the nucleoid. the next gene, pol, codes for the reverse transcriptase, whereas the env gene codes for the two glycoproteins on the surface of the envelope. the final major gene codes for a phosphoprotein with protein kinase activity and is responsible for neoplastic transformation. the latter gene, known as the virus oncogene, is inconsistently present on oncoviruses. virus replication is initiated when virus binds to specific receptors and penetrates into a cell. in permissive cells, virus progeny is produced and the host cell may or may not become neoplastic. nonpermissive cells may be transformed occasionally, but viral replication is never completed. defective viruses may efficiently transform both permissive and nonpermissive cells. within a few hours, virion reverse transcriptase synthesizes linear double-stranded dna copies of the haploid genome. linear dna molecules migrate to the nucleus and become circularized. the latter becomes integrated into cellular dna, a step essential for retrovirus replication and gene expression. integrated virus dna is known as the provirus, and the number of copies in the haploid host genome varies from to . the integrated provirus retains the topography of the viral genome and is subjected to expression and control by host mechanisms. proviruses can be transmitted vertically and thus inherited in germ cells as genes by mendelian genetics. viral gene expression relies on the hospitality of the host cell. transcription of proviral dna is catalyzed by cellular rna polymerase ii. expression may be governed by genetic determinants of the cell that are closely linked but separable from the provirus. two general types of oncoviruses exist. the sarcoma-type viruses transform fibroblasts in vitro, have an oncogene, and usually are highly oncogenic. often they fail to produce progeny virus because of a deletion of the env function and are known as replication defective (rd). coinfection with a related virus containing intact env activity may provide the deficient envelope glycoproteins to allow full maturation and progeny sarcoma virus but with surface antigen specificity of the helper (or associated) virus. prior infection or occupation of cell surface receptors by an oncovirus with identical envelope characteristics will result in interference and prevent superinfection by the second virus. the initial virus is known as the resistance-inducing factor. leukemia viruses do not seem to have an oncogene and cannot transform fibroblasts in vitro. such viruses are transformation defective (td). thus, the transforming segment, the oncogene, is not essential for virus replication and is not of intrinsic importance to the virus. however, many of these viruses are leukemogenic in animals after a prolonged incubation period and seem to be responsible for many of the leukemias and lymphomas of various animals. the mechanism of leukemogenesis is not known, but it is likely that an indirect mechanism occurs with these viruses. perhaps these viruses recombine with other viral genomes, but more likely, because of their insertion in the vicinity of cellular oncogenes, activate the latter. cellular oncogenes are very similar to those of sarcoma viruses, and it seems plausible that the latter may have acquired oncogenes by transduction during past interactions with their host cells. various hypotheses on oncovirus oncogenesis have been proposed as the information on the biology of these viruses became available. the virogene-oncogene theory was proposed in by heubner. temin introduced in the protovirus concept for viral oncogenesis and the existence of endogenous oncoviruses. however, considerable evidence now exists to support the cellular oncogene theory, which contains some of the concepts of the two hypotheses oftemin and heubner. an oversimplified explanation of this hypothesis is that most cells contain the so-called oncogenes, a highly conserved host gene with a useful function. this gene product seems to be a protein kinase catalyzing the phosphorylation of certain proteins and has been implicated in regulating growth of normal cells by activity on cell surfaces. this unique protein kinase differs from the normal cell kinases in that it phosphorylates tyrosine instead of serine or threonine. a large number of viral oncogenes and cellular equivalents have now been identified. they are distinguished by the prefix "c" for cell oncogenes and "v" for viral oncogenes. viral oncogenes are probably of cellular origin and may be merely a passenger acquired by sarcoma viruses from the host dna. viruses with oncogenes are directly oncogenic, whereas oncoviruses without oncogenes are indirectly tumorogenic because they interfere with a cellular oncogene at or near the site of provirus integration. two hypotheses have been proposed to explain the mechanism of oncogene transformation; the mutational hypothesis and the dosage hypothesis. the former requires that the viral oncogene differs, as a result of mutation, from the parent cellular oncogene and, upon expression of the gene, neoplastic transformation occurs instead of its normal regulatory function. furthermore, in the instance of nononcogene-bearing leukemia viruses, a similar mutation occurs in cellular oncogenes during integration. proto-oncogene (inactive) activation by one-point mutation has been best documented in the family of "ras" oncogenes. the dosage (or amplification) hypothesis requires that excessive production of the gene product occurs after infection with a sarcoma virus and formation of one or multiple copies of the provirus in the cell. nononcogene-bearing leukemia viruses probably stimulate cellular oncogene activity because some are inserted as proviruses in the immediate vicinity of this gene. recent evidence suggests that the latter theory may be accurate. normal cellular control prevents the oncogene's transforming capacity. however, when an oncovirus includes an oncogene in its genome, the oncogene is removed from its controlled environment, resulting in increased production during viral replication. enhanced activity of the oncogene probably is due to the viral promoter. an unexpressed oncogene (proto-oncogene), when introduced with its normal promoter into host cells, does not cause transformation; however, transformation occurs when the viral promoter accompanies the cellular oncogene in transfection experiments. the activation of normal cell oncogenes by leukemia viruses is probably due to the viral promoter inserted in the vicinity of these genes. the delayed tumor induction of leukemia viruses may be due to random integration of the viral genome. thus, the location of the viral promoter at the appropriate site for affecting the cellular oncogene is an inefficient event. the appropriate point of integration of the promoter into the cellular dna varies, and evidence indicates that the site can be some distance from the target oncogene. another mechanism of retroviral neoplasia is related to immunosuppression induced by some of these viruses. immune surveillance may be severely impaired, allowing transformed cells to accumulate unchecked. immunosuppression often precedes tumor development in animals infected by oncoviruses. recent evidence indicates that human t-celllymphotropic virus iii may cause the acquired immunodeficiency syndrome, which, in turn, may 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in relation to pathogenicity the little known determinants of virus pathogenicity. scand immunosuppressive activity of the retroviral envelope protein p e and its possible relationship to neoplasia epithelial and surfactant changes in influenza pulmonary lesions pathogenicity of influenza virus effect of influenza virus infection on phagocytic and cytopeptic capacities of guinea pig macrophages interspecies reactivity of type c and d retrovirus pl e and p c proteins rabies. in topley and wilson's principles of bacteriology respiratory and systemic cellular and humoral immune responses to influenza virus vaccine administered parenterally or by nose drops alterations in macrophage antimicrobial activity associated with viral pneumonia alterations in lung macrophage immune receptor(s) activity associated with viral pneumonia resistance to infectious disease the role of host genetics in the pathogenesis of coxsackievirus infection in the pancreas of mice absolute linkage of virulence and central nervous system cell tropism of reovirus to viral hemagglutinin oncogenes: an overview disseminated intravascular coagulation in experimentally induced feline infectious peritonitis pathogenesis of feline infectious peritonitis: pathologic changes and immunofluorescence vaccinia virus cytotoxin viral myocarditis oncogenic viruses oncogenic viruses virus-induced diabetes mellitus. isolation of a virus from the pancreas of a child with diabetic ketoacidosis key: cord- -gq lp authors: becker, daniel j.; washburne, alex d.; faust, christina l.; pulliam, juliet r. c.; mordecai, erin a.; lloyd-smith, james o.; plowright, raina k. title: dynamic and integrative approaches to understanding pathogen spillover date: - - journal: philosophical transactions of the royal society b: biological sciences doi: . /rstb. . sha: doc_id: cord_uid: gq lp nan pathogen spillover is the process by which a pathogen is transmitted from a reservoir host species to a recipient host species [ , ] . the term is sometimes used more broadly, particularly in public discourse, blending in elements of onward transmission in the novel host species or even pathogen adaptation to the novel host [ , ] . this theme issue focuses on pathogen spillover sensu stricto, except where explicitly noted. many of the examples considered pertain to zoonotic spillover (i.e. from wildlife or domestic animals to humans), given recent epidemics (e.g. ebola virus [ ] ) and pandemics (e.g. h n influenza virus [ ] ); however, we emphasize the general methods and mechanisms involved in understanding spillover between any two species, such as those that threaten wildlife conservation (e.g. mycoplasma ovipneumoniae from domestic sheep to bighorn sheep [ ] ) and the agricultural sector (e.g. brucella abortus from elk to cattle [ ] ). spillover requires the spatial and temporal alignment of several hierarchical factors that must occur for a pathogen to be transmitted from a reservoir or source host to a recipient host of a different species [ ] . these factors include reservoir host distribution and abundance, pathogen prevalence and shedding from reservoir hosts; pathogen survival in the environment or arthropod vector; recipient host contact with the infectious agent, reservoir host or arthropod vector; and susceptibility of the recipient host. following spillover, another suite of factors determines whether a pathogen is transmitted within the recipient host population (e.g. [ ] ). research on pathogen spillover is often focused on a single component of this process through the lens of a particular discipline. for example, the distribution of reservoir hosts is often studied through ecology, contact between reservoirs and humans is often studied via epidemiology or anthropology, and the pathogenesis of zoonoses in humans is often studied with medical microbiology and immunology. while each factor must be studied and quantified, spillover is the emergent property of these collective processes. studying each factor in isolation fails to account for the hierarchical and often nonlinear dynamics of the spillover system [ ] . pathogen surveillance, epidemic preparedness and management interventions would all benefit from integrative approaches that consider multiple components of pathogen spillover [ ] . this theme issue stemmed from a workshop on cross-species transmission of pathogens, where participants from interlinked fields including ecology, mathematical modelling, epidemiology, virology and immunology discussed how to better understand and predict pathogen spillover. here, we bring together a diverse set of perspectives-including empirical research, theory and synthetic reviews-to highlight cutting-edge research and to provide a roadmap for quantifying and integrating hostpathogen dynamics at each step in the spillover process. manuscripts are organized around three approaches. the first set of manuscripts focuses on integrating data streams to understand spillover dynamics and predict risk. the second set of manuscripts focuses on in-depth analysis of each of the factors affecting cross-species transmission: infection dynamics in reservoir hosts, pathogen survival in the environment, recipient host exposure, dose -response relationships and establishment of infection in recipient hosts. the final set of manuscripts focuses on applied perspectives, with an emphasis on surveillance and interventions. here, we summarize these contributions to highlight key insights, methodologies and future directions to improve our understanding of pathogen spillover. because spillover is the outcome of multiple ecological, epidemiological and immunological factors aligning in space and time [ , ] , predictive frameworks aim to integrate data pertinent to these factors to quantify the relative importance of these processes and to estimate risk. cross et al. [ ] review approaches for estimating spatio-temporal variation in spillover risk, focusing on the wildlife -livestock interface. the authors highlight the challenges inherent in either correlating observed spillover events with relevant covariates or integrating data on host density, distribution and pathogen prevalence using mechanistic models. they highlight that mechanistic approaches may be especially useful in systems where spillovers are infrequent, rarely observed or hard to differentiate from within-species transmission; however, linking datasets on different factors in the spillover pathway requires that such datasets be related to a common spatial and temporal resolution. the authors use case studies of brucellosis in the greater yellowstone ecosystem [ ] and of avian influenza virus in china and north america [ ] to emphasize potential solutions to these challenges for estimating spillover risk. emphasizing that statistical modelling efforts may struggle to detect nonlinear and stochastic relationships inherent in pathogen spillover, childs et al. [ ] provide a strong test of theory governing how hierarchical barriers control crossspecies transmission [ ] . the authors focus their case study on yellow fever, a mosquito-borne viral disease of historical importance in south america that persists in the region largely in sylvatic cycles that occasionally spill over to infect humans [ , ] . specifically, they use mechanistic models that incorporate spatial ecological and immunological data from brazil across years to predict yellow fever spillover in humans. the authors show that a mechanistic model of spillover risk, based on the ecology of mosquito vectors and non-human primate reservoirs, best predicts spillover events compared with models that also include human population size and immunity. this result arises because spillover occurs even in areas with low human population density and high vaccination coverage (e.g. parts of the amazon), so population density and vaccination coverage tend to inflate the predicted risk in locations with low ecological suitability. this integrated approach also highlights a key research gap-cyclical dynamics of susceptible primate populations-that could further improve prediction. this work illustrates that mechanistically modelling the interactions among the environment, viruses, vectors, nonhuman primates and humans can predict rare and seemingly stochastic spillover events with high accuracy. washburne et al. [ ] study the general statistical problems that can arise when aiming to forecast spillover risk. the authors highlight that any such statistical efforts will compile a dataset of explanatory variables expected to relate to pre-spillover processes (e.g., infection prevalence in reservoirs, human vaccination coverage) that are aligned with one of two response variables: the presence and absence of spillover or the number of spillover events at some spatial and temporal resolution (e.g., spatio-temporal counts of yellow fever spillovers [ ] ). the authors show how modelling cross-species transmission as a percolation process, in which pathogens move from infected reservoirs to recipient hosts along a graph representing various spillover pathways [ , ] , reveals first principles for how such datasets will behave and how common statistical tools can produce misleading inferences and poor predictions. for example, percolation theory reveals an inherent nonlinearity in modelling spillover counts, in which statistical inferences are driven by the dominant reservoir sources of infection and the most limiting barriers to cross-species transmission; this nonlinearity can mask the influence of alternative reservoir species or barriers, both of which could be modified through interventions but whose sensitivity as a management tool will appear reduced under linear models. percolation models provide a conceptual framework to connect statistical and mechanistic models with applications to limit risk by illuminating unexpected statistical principles governing pathogen spillover and the nonlinear impacts of management actions. the theme issue's second section uses empirical research, theory and synthetic reviews to understand the processes operating at each stage of pathogen spillover, from infection dynamics within the reservoir hosts to susceptibility and establishment of infection in the recipient host. for the former, the distribution and intensity of infection in reservoir hosts over space and time is the first determinant of spillover risk [ ] . data on these spatio-temporal dynamics help elucidate how pathogens circulate in reservoir hosts and when and where to expect pathogen excretion to be greatest [ ] . however, such field data can be expensive and difficult to collect, and researchers inevitably must tradeoff between the extent and intensity of spatial versus temporal sampling. sampling is thus often opportunistic and fails to adequately describe spillover. plowright et al. [ ] review factors that influence spatial and temporal variation in infection in reservoirs and describe sampling designs that can increase the quality and quantity of this information. although the standard prescription from sampling theory is to sample randomly in space and time [ ] , probabilistic sampling designs are rare in the study of wildlife disease, given logistical challenges and non-random distributions of hosts. the royalsocietypublishing.org/journal/rstb phil. trans. r. soc. b : authors highlight how stratified random sampling designs or adaptive sampling designs can help capture spatio-temporal pulses of infection when researchers have little a priori data on concentrations of infection or spillover events in space and time. these sampling designs can be integrated into modelling approaches and used to better quantify pathogen shedding from reservoirs. accordingly, glennon et al. [ ] present a case study for how to use mechanistic models to differentiate among transmission processes for henipaviruses in straw-coloured fruit bats (eidolon helvum). using this virulent zoonosis as a case study, the authors generalize standard frameworks common in epidemiological modelling [ ] . given that henipavirus infection dynamics in bats are poorly understood, the authors study all possible transitions among infection states in bats to produce potential models. using likelihood-based methods, they fit these models to longitudinal data from captive bats to show strong support for reinfection after virus clearance and cycles of recurrent latent infection: key areas for future empirical work. this inclusive approach to confronting epidemiological models with longitudinal data in poorly understood reservoir host systems holds promise for elucidating spatio-temporal risk of pathogen spillover. following pathogen shedding from reservoir hosts, spillover risk is influenced by the duration of pathogen survival and possible reproduction outside the host in the environment [ ] . for pathogens such as avian influenza virus, persistence in the environment (e.g. ponds) can also facilitate viral reassortment when strains co-occur, promoting co-infections during environmental exposure [ , ] . pepin et al. [ ] review and discuss how genomics, experimental ecology and epidemiological modelling can be leveraged to understand viral reassortment in environmental reservoirs. although no gold standard for capturing, isolating and identifying avian influenza virus diversity from the environment exists, environmental metagenomics and field-based viral diagnostics (e.g. field-based nucleic acid extraction, pcr and sequencing) hold promise for characterizing this context of viral reassortment [ , ] . the authors note how standardizing such field protocols and coupling these data streams with quantitative disease models and natural transmission studies should dramatically improve our understanding of viral co-occurrence and reassortment and thus, this additional process in the pathway to spillover. exposure of recipient hosts to pathogens (e.g. those persisting in the environment) can take a variety of forms; however, in a more general sense, exposure often occurs at elevated rates near boundaries between ecosystems [ ] . borremans et al. [ ] review how ecosystem boundaries can promote spillover by applying ecological theory to understand landscape permeability across ecosystems. the authors highlight that the traits of hosts and pathogens are critical for determining effects of ecosystem boundaries on crossspecies transmission. properties of ecosystem boundaries can also promote or inhibit exposure; for example, edge effects can affect species composition, diversity and population size between ecosystems, as can features of landscape configuration such as patch size and perimeter-to-area ratio [ ] . by considering the analogy between parasite flow and resource flow and by applying concepts from movement ecology, borremans et al. [ ] connect contact rates and spillover risk across ecosystem boundaries to generalize between pathogens and integrate into broader ecological theory. following the complex interactions between reservoir hosts, vectors, pathogens, the environment and recipient hosts, a crucial juncture in any potential spillover event is the point when a recipient host is challenged with a given dose of pathogen (through a particular route and sometimes over a particular duration) and a successful infection does or does not ensue [ ] . lunn et al. [ ] describe how the dose -response relationship, which quantifies the probability of successful infection in the recipient host as a function of challenge dose, can act as a filter on the aforementioned upstream dynamics to shape pathogen spillover risk. the authors integrate recent developments in the dose -response literature, as well as re-analysing data from animal challenge experiments with nipah virus and middle east respiratory syndrome coronavirus [ , ] , to highlight challenges and opportunities arising at the intersection of infectious disease ecology, microbial risk assessment and virology. lunn et al. [ ] call for closer interactions between these fields and for a new generation of pathogen transmission models that link dose -response data to epidemiological dynamics. gostic et al. [ ] next provide an example of the epidemiological insights such an approach can yield. they present a modelling analysis of dose -response experiments for leptospira interrogans, a globally important bacterial zoonosis for which environmental exposure to soil or water contaminated by urine of infected reservoir hosts is the primary transmission route [ ] . by conducting well-designed challenge experiments across a range of exposure routes, and then developing a mechanistic model to identify and quantify the key barriers to infection, gostic et al. [ ] show that intact skin is the crucial defence against leptospiral infection and that skin abrasions or wounds can increase recipient host infection risk by at least a million-fold. this close integration of experimental and modelling approaches isolates a potent and well-defined risk factor for infection with leptospira, opening the door to targeted interventions to reduce spillover risk. once a pathogen has crossed these within-host barriers to replicate and disseminate in the recipient host, the outcome of infection may range from subclinical illness to death and from dead-end spillover to sustained onward transmission [ ] . bonneaud et al. [ ] focus on the conditions favouring pathogen emergence, from the initial jump into the recipient host to adaption in the novel host environment [ ] . the authors highlight that our current understanding of host shifts stems primarily from viral infections, limiting generalizations to other pathogen taxa, given substantial differences in ecology and life history [ ] . they propose several non-mutually exclusive hypotheses to explain why novel bacterial pathogens may be less likely to specialize on their novel hosts and then test these with a mathematical model. the authors demonstrate that high levels of phenotypic plasticity, low rates of evolution and the ability to recombine should reduce propensity to specialize, suggesting that novel bacterial infections may be more likely to result in transient spillovers or increased host ranges than in host shifts. wasik et al. [ ] in turn describe the within-host barriers that pathogens, and viruses in particular, must overcome to replicate and spread in new host populations to cause onward transmission. they present three well-documented examples of viruses that have crossed these barriers to cause epidemics or pandemics in the new host species: influenza a viruses [ ] , human immunodeficiency virus [ ] and royalsocietypublishing.org/journal/rstb phil. trans. r. soc. b : canine parvovirus [ ] . the authors emphasize the role of integrated models that consider all the steps required to go from exposure to spillover to epidemic or pandemic. guth et al. [ ] expand upon these ideas through a comparative study of host and viral traits that predict virulence and the capacity for onward transmission in recipient hosts (i.e. humans). by expanding a previous global dataset of viral zoonoses [ ] , the authors show that increasing reservoir host phylogenetic distance from humans positively correlates with human mortality but negatively correlates with humanto-human transmissibility, suggesting that the virulence induced by reservoirs at high phylogenetic distance may limit viral capacity for onward transmission [ ] . in particular, distantly related reservoirs, such as bats, harbour highly virulent zoonotic viruses with a lower capacity for onward transmission in recipient human hosts, building upon prior work describing bats as special reservoirs [ ] . the theme issue's final section focuses on applied perspectives to detect early spillover events (i.e. surveillance) and the role of interventions focused upstream in the spillover pathway. in particular, early detection is critical for minimizing the spread of zoonotic pathogens following an initial spillover event [ ] . a first series of manuscripts emphasize different approaches to the surveillance of zoonoses. schmidt et al. [ ] use machine learning tools (e.g. boosted regression trees [ ] ) to predict which mammal species are more likely to play roles in ebola virus spillover events. the authors show that large-bodied, frugivorous mammals with slow life histories are likely host species, implicating some insectivorous bats, old world monkeys and forest antelopes as possible ebola virus reservoirs. predictions such as these can help prioritize future wildlife surveillance efforts (e.g. [ ] ). kuisma et al. [ ] in turn describe a community-based surveillance effort focused on wildlife mortality reporting and oriented to early detection of ebola virus disease outbreaks. spanning over a decade and covering km of challenging terrain in the congo basin, this programme has reached hundreds of villages and thousands of hunters and forest gatherers. the programme has educated community members in wildlife carcass reporting and behavioral risk reduction as well as built capacity for safe carcass sampling by trained local responders. this region was not confronted with an ebola virus outbreak during the period described here, and all reported carcasses tested negative. nevertheless, given the well-recognized fact that early intervention can avert massive human and economic costs of widespread epidemics, the low-cost and scalable surveillance programme described by the authors could provide key early detection capability more generally. two other contributions focus on zoonotic pathogen surveillance efforts in domestic animals and human populations. mwangi et al. [ ] present a real-time surveillance system that leverages the existing mobile phone network and shows immense potential to improve adaptive management of spillover. this surveillance system has been implemented in households across rural kenya, where participants are asked to report symptom syndromes in their livestock. zoonotic diseases such as rift valley fever present with severe clinical signs in domestic animal populations, but lack of active surveillance can miss these sentinels [ ] . the authors demonstrate that illnesses were more likely to be reported on mobile phones compared with standard routine household animal surveys. they also show that more severe symptoms are likely to be reported, highlighting the utility of this surveillance method for diseases such as rift valley fever. das et al. [ ] similarly describe the implementation of a surveillance system in hospitals in bangladesh that screens symptomatic patients for potential zoonoses. most patients did not have a laboratory diagnosis for their illness, indicating that unidentified pathogens are likely spilling over in human populations. broad-scale, sustainable human surveillance programmes such as outlined by the authors can play a critical role in early detection of zoonotic spillovers. following these approaches to surveillance, interventions can accordingly focus upstream or downstream in the pathway to spillover, given available data and resources, to limit cross-species transmission. at the wildlife-livestock interface, managing pathogen spillover is a main goal for animal husbandry, conservation and food security [ ] . yet, managers are often forced to make control decisions on the basis of limited evidence about intervention efficacy. manlove et al. [ ] develop a spatially explicit, stochastic model of pathogen transmission within and between wildlife reservoirs and livestock recipient hosts to improve evidence-based decisionmaking. by varying host movement patterns and epidemic growth rates, the authors show that biosecurity, containment and retroactive vaccination of the reservoir are the most effective for limiting the spatial spread and magnitude of spillover risk for fast-moving epidemics in mobile hosts. by contrast, prophylactic vaccination and depopulation of the reservoir host were more successful for fast-moving epidemics with low rates of host movement. this framework provides general intuition for how to manage different pathogens at the wildlife-livestock interface, and a flexible platform for more rigorously investigating disease control strategies. ultimately, one of the primary goals of research focused on pathogen spillover is to design interventions that can reduce or eliminate disease burden in recipient hosts. sokolow et al. [ ] explore how ecological interventions, which target the ecological context in which cross-species transmission occurs, can complement more traditional biomedical and veterinary interventions (e.g. vaccination, culling). the authors provide case studies to illustrate the potential for ecological interventions that target the reservoir host (sometimes indirectly, such as through the restoration of natural enemy populations [ ] ), pathogen survival in the environment, contact between reservoir and recipient hosts, or other aspects of risk in the recipient species. the authors also present a simple mechanistic model, parameterized for two example systems, that shows how nonlinear effects can produce counterintuitive results when comparing potential intervention strategies and highlights the importance of a detailed understanding of underlying ecological dynamics when designing and assessing interventions. lastly, the authors draw attention to the importance of social, economic and political considerations to intervention success, as these can derail even the most efficient or cost-effective intervention. in particular, aligning the benefits of an intervention with the costs incurred is crucial to motivate ecological interventions and may require working across sectors for successful implementation. royalsocietypublishing.org/journal/rstb phil. trans. r. soc. b : pathogen spillover is the result of a complex series of events that result in the successful establishment of infection in a recipient host [ ] . as highlighted in the final paper of this theme issue, developing actionable forecasts of risk is further complicated by the various phylogenetic, spatial and temporal scales over which we study and predict spillover [ ] . the authors here contextualize a diverse range of approaches to pathogen spillover within these scales to illustrate critical areas of pragmatic overlap. by focusing on an ecological perspective, the authors outline a research pipeline that connects pathogen discovery and macroecological analyses with spatio-temporal surveillance in reservoir and recipient hosts. through several case studies (e.g. lyme disease [ ] , hendra virus [ ] , plasmodium knowlesi [ ] ), the authors further demonstrate how ecologically focused research has facilitated predicting spillover of particular pathogens in space and time and facilitated design of intervention strategies. this synthesis shows how greater integration of macroecology, pathogen discovery and surveillance could ultimately generate more actionable predictions and interventions to limit spillover risks. recent epidemics, pandemics and disease emergence events all underscore the need to improve approaches to predict and prevent pathogen spillover. this theme issue highlights a range of methods and their commonalities through diverse host -pathogen systems for which researchers are assessing factors driving spillover risk across varying phylogenetic, spatial and temporal scales. contributing manuscripts further emphasize how developing a mechanistic understanding of the hierarchical factors affecting spillover can facilitate quantifying the drivers of crossspecies transmission, deriving generalizable theory and making robust predictions, even for seemingly rare and idiosyncratic spillover events. importantly, such insights can improve our ability to deploy surveillance efforts, design interventions at early stages of the pathway to spillover and manage disease cases in recipient hosts, thereby limiting or preventing further outbreaks. continued study of pathogen spillover as a repeated and hierarchical phenomenon will only improve our ability to predict, prevent and manage cross-species transmission risks. data accessibility. this manuscript has no additional data. authors' contributions. all authors contributed to the development of ideas and to the writing of this manuscript. competing interests. we declare no competing interests. funding. this work was 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heightened vigilance and precautions being urged this winter predictive analysis across spatial scales links zoonotic malaria to deforestation ranch (emigrant, montana) for assistance organizing the working group that led to this theme issue. we also thank helen eaton for all her assistance in preparing this theme issue for publication.disclaimer. the views, opinions and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the department of defense or the us government. key: cord- - giy fow authors: martinez-martin, nadia title: technologies for proteome-wide discovery of extracellular host-pathogen interactions date: - - journal: j immunol res doi: . / / sha: doc_id: cord_uid: giy fow pathogens have evolved unique mechanisms to breach the cell surface barrier and manipulate the host immune response to establish a productive infection. proteins exposed to the extracellular environment, both cell surface-expressed receptors and secreted proteins, are essential targets for initial invasion and play key roles in pathogen recognition and subsequent immunoregulatory processes. the identification of the host and pathogen extracellular molecules and their interaction networks is fundamental to understanding tissue tropism and pathogenesis and to inform the development of therapeutic strategies. nevertheless, the characterization of the proteins that function in the host-pathogen interface has been challenging, largely due to the technical challenges associated with detection of extracellular protein interactions. this review discusses available technologies for the high throughput study of extracellular protein interactions between pathogens and their hosts, with a focus on mammalian viruses and bacteria. emerging work illustrates a rich landscape for extracellular host-pathogen interaction and points towards the evolution of multifunctional pathogen-encoded proteins. further development and application of technologies for genome-wide identification of extracellular protein interactions will be important in deciphering functional host-pathogen interaction networks, laying the foundation for development of novel therapeutics. the plasma membrane constitutes a critical biological interface between the cytosol and the extracellular environment of the cell, and consequently membrane-tethered proteins and secreted molecules (collectively termed extracellular proteins) are essential regulators of cellular communication. from high affinity cytokine-receptor interactions to low affinity cell-cell adhesion contacts, extracellular protein-protein interactions (eppis) are key for information processing and coordination of virtually all processes in a living organism. furthermore, given their fundamental functions and their accessibility to systemically delivered drugs, extracellular proteins are particularly suitable targets for therapeutic intervention. in fact, despite these proteins being encoded by approximately one-fourth of the human genes, at least two-thirds of the existing drugs target either secreted or membrane-bound proteins [ ] . thus, the elucidation of the eppi networks on a global scale has become crucial for the biomedical research. however, in spite of their relevance and abundance, eppis are remarkably underrepresented in available large-scale datasets. this discrepancy is due to the low sensitivity and limited compatibility of current high throughput technologies to detect extracellular interactions because of the unusual biochemical nature of the membrane proteins and the intractability of their binding partners [ ] [ ] [ ] . in particular, transmembrane domain-containing proteins are amphipathic, making it difficult to solubilize them in their native conformation, and often contain posttranslational modifications such as glycans and disulfide bonds, which are not properly added in common heterologous expression systems [ ] . in addition, interactions between cell surface proteins are often characterized by fast dissociation rates and therefore weak binding affinities, and in consequence well-established ppi methods such as yeast-two-hybrid or affinity purification-mass spectrometry (ap/ms) largely fail to detect these interactions. over the last decade, several innovative technologies have been developed to overcome the aforementioned technical challenges and allow for sensitive journal of immunology research detection of eppis [ , [ ] [ ] [ ] [ ] [ ] . nevertheless, the mapping of eppis remains a major challenge in biology. infectious diseases result in millions of deaths each year and therefore identifying new candidate targets for improved therapeutic development remains a pressing health concern. pathogens have evolved a myriad of elegant and often complex strategies to invade the host and commandeer host immune responses to allow pathogen replication, spread, and persistence in the infected organism. many cell surface molecules serve as entry receptors for initial host cell invasion, and concerted responses to the pathogenic challenge critically rely on cell functions mediated by receptors and secreted proteins. to allow host colonization, pathogens encode highly optimized protein modulators, in the form of secreted molecules or receptors expressed on the plasma membrane of the infected cells or the surface of the pathogen [ , ] . interactions between these proteins and extracellular host molecules form the foundation of communication between a host and a pathogen and play a vital role in the initiation and outcome of the infection [ , ] . characterizing host-pathogen eppi networks is therefore of utmost importance to gain a better understanding of the infection process and to inform the development of novel or improved therapeutic strategies. excellent studies on mapping hostpathogen interactions, particularly ms-based analysis of viral infection, have provided a wealth of insight into infectious diseases [ ] [ ] [ ] [ ] [ ] . nevertheless, similarly to host eppis, a significant hurdle to the elucidation of host-pathogen biology has been the shortage of datasets of extracellular interactions between host and pathogen proteins, partly due to the technical challenges that these proteins present. moreover, an additional consideration when studying pathogen-encoded molecules is that these proteins often lack any recognizable homology with any host molecules, thus precluding prediction of their functions [ , ] . robust methodologies that permit unbiased characterization of eppi in the absence of preexisting hypotheses are thus needed to elucidate the binding partners and molecular functions of most pathogenencoded molecules. excellent reviews have recently revisited the currently available technologies for proteome-wide eppi discovery [ , , [ ] [ ] [ ] . here we discuss the application of some of these technologies to the study of host-pathogen interaction and describe some of the major findings that have recently impacted research in the field of extracellular host-pathogen recognition. protein microarrays and functional genomics approaches are highlighted here as emerging techniques with unique potential for the elucidation of host-pathogen eppi networks at a genome-wide scale. classical biochemical and biophysical approaches are particularly suitable for detection of high affinity host-pathogen interactions, such as those mediated by a viral capsid protein and a host cell surface receptor, or a pathogen-encoded glycoprotein and a secreted host factor. typically, these approaches have relied on the utilization of recombinant pathogen proteins as baits to probe for host binding partners, followed by immunoprecipitation and ms, or biophysical techniques for analysis of ppi such as surface plasmon resonance (spr). spr requires prior knowledge of the possible interacting partners and is therefore unsuitable for unbiased ppi discovery, whereas immunoprecipitation and ms approaches usually fail to detect weak interactions, which often characterize eppis, particularly those that take place on the cell surface. notwithstanding, the identification of the receptors for some of the most prominent pathogens, such as the severe acute respiratory syndrome coronavirus (sars-cov) or the new world arenaviruses, was made utilizing standard immunoprecipitation techniques [ , ] . despite their inherent limitations, biochemical approaches continue to provide relevant insights into host-pathogen interactions, such as the discovery of the dipeptidyl peptidase (dpp ) as the receptor for the mers-cov just within a few months after the emergence of this virus [ ] . in addition, more recently kabanova and colleagues identified the cell surface receptor for the trimeric entry complex ghglgo encoded by human cytomegalovirus (hcmv) [ ] . several studies have shown that the ghglgo trimer is involved in the infection of fibroblasts, whereas the ghglul l pentameric complex is required for entry into endothelial, epithelial, and myeloid cells [ ] [ ] [ ] . in this work, both the trimeric and the pentameric cmv protein complexes were generated as recombinant products and used as baits to perform binding experiments on biotinylated cell surfaces, followed by immunoprecipitation and ms identification of bait-cell receptor complexes. using this approach, the authors identified the cell surface protein pdgfr as a high affinity receptor for the ghglgo trimer and demonstrated that this interaction was required for infection of fibroblasts. interestingly, in the case of the pentameric cmv complex, multiple bands were detected upon protein immunoprecipitation from epithelial cells, suggesting the existence of multiple receptors on these cells, which so far remain unknown [ ] (table ) . different biophysical techniques for detection of ppi, in particular spr, have also proven valuable in the field of host-pathogen interactions. spr offers the advantage of label-free, sensitive detection of interactions between a diversity of ligands in real time, thus allowing calculation of kinetic parameters. spr has been widely utilized to monitor antibody binding to a variety of pathogen antigens, information that has informed vaccine development [ ] [ ] [ ] . the spr technology has also been exploited for discovery of eppis. for example, viejo-borbolla and coworkers utilized spr to screen several secreted and membrane-expressed glycoproteins encoded by herpes simplex viruses (hsvs) for binding to chemoattractant cytokines (the chemokine family) and were able to identify a subset of human chemokines that bound to hsv glycoprotein g with high affinity [ ] . recently, day and colleagues utilized a combination of glycan arrays and spr and identified over host-bacterial glycan pairs characterized by a wide range of binding affinities, some of which participated in bacterial adherence to host cells in vitro, leading to the hypothesis that bacteria-host surface glycan interactions may mediate initial attachment to the target cell during infection [ ] . despite spr and related methods offering higher sensitivity for detection of transient biochemical and ms pdgfr identified as a high affinity cell surface receptor for the cmv ghglgo protein complex [ ] herpes simplex viruses (hsvs) biophysical secreted and plasma membrane-expressed glycoprotein g targets a specific set of human chemokines with high affinity [ ] human immunodeficiency virus type (hiv) monoclonal antibodies cd identified as the receptor for hiv infection of t cells [ , ] rhinovirus monoclonal antibodies icam- as the common entry receptor for most rhinovirus serotypes [ , ] plasmodium falciparum avexis basigin identified as the cell-surface receptor that mediates erythrocyte infection [ ] human adenoviruses extracellular human protein microarrays elucidation of the extracellular interactome of adenovirus-encoded immunomodulatory proteins [ ] pseudomonas aeruginosa extracellular pathogen protein microarrays (nappa) screening of patient sera against all p. aeruginosa extracellular proteins. proteins identified as potent antigens [ ] varicella zoster virus (vzv) extracellular pathogen protein microarrays (nappa) identification of extracellular viral proteins that promote humoral responses upon screening of the entire vzv proteome [ ] streptococcus extracellular pathogen protein microarrays identification of new streptococcal proteins that interact with fibronectin, fibrinogen, and c bp factors [ ] hepatitis delta virus lhdag antigen plasma membrane microarrays (mpa) candidate interactions identified between viral antigen and plasma membrane proteins [ ] simian virus (sv ) plasma membrane microarrays (mpa) candidate interactions between whole particles and plasma membrane proteins identified [ ] vaccinia virus (vacv) triceps (ms) candidate cell surface binding partners identified for vacv [ ] viral pathogens computational studies insights into global principles of virus-host ppi networks [ ] [ ] [ ] [ ] [ ] [ ] [ ] hepatitis c virus (hcv) cdna libraries cd , claudin- , and occludin as cell surface receptors and some of the players involved in hsv internalization [ ] [ ] [ ] adenovirus and coxsackievirus b monoclonal antibodies and cdna libraries car identified as a common entry receptor for adenovirus / and coxsackievirus b [ ] sindbis virus sirna screens nramp as cell surface receptor for entry into drosophila cells [ ] murine norovirus crispr/cas cd lf identified as a cell surface receptor that determines virus tropism [ ] bacterial distending toxins (e. coli) haploid cell screens sphingomyelin synthase and the putative g protein-coupled receptor tmem identified as toxin receptors [ , ] clostridium difficile and perfringens toxins haploid cell screens lipolysis-stimulated lipoprotein and the low-density lipoprotein receptor-related protein identified as the receptors for the bacterial toxins, respectively [ , ] ebola virus haploid cell screens hops proteins and the niemann-pick c (ncp ) transporter identified as endosomal receptors that mediate cytosol access [ ] lassa virus haploid cell screens lamp , lysosomal-associated membrane protein identified as an essential host factor mediating virus release to cytosol [ ] adeno-associated virus (aav) serotype haploid cell screens cell host factors identified, including heparin sulfate proteoglycan biosynthesis and intracellular transport genes. the immunoglobulin domain-containing transmembrane protein kiaa l identified as aav receptor [ ] journal of immunology research population genomics analysis p. falciparum ebl- protein binds to the erythrocyte receptor glycophorin b, a highly polymorphic gene in malaria-endemic regions [ ] streptococcus phage display bacterial proteins identified as potential fibronectin-binding proteins [ ] fusobacterium nucleatum transposon-based mutant libraries the bacterial protein fap binds to the receptor tigit and downregulates nk-mediated killing of tumor cells [ ] avexis, avidity-based extracellular interaction screen; nappa, nucleic-acid programmable protein array; mpa, microfluidic-based comprehensive protein array; crispr, clustered regularly interspaced short palindromic repeat; ms, mass spectrometry; ppi, protein-protein interaction. ppis than most biochemical approaches, these biophysical techniques have not been exploited for large-scale eppi discovery, possibly due to the low throughput of the available instrumentation and the overall difficulties for generation of the relevant protein libraries. these studies, among many others, have demonstrated the power of the classical biochemical and biophysical techniques for detection of host-pathogen interactions. nevertheless, these approaches require previous knowledge of the pathogen-encoded proteins responsible for binding and the ability to produce such proteins as recombinant reagents, which may be challenging, as exemplified by the production of hcmv entry complexes [ , ] . alternative methods have been utilized in those cases where there is no previous knowledge of the pathogen proteins required for interaction with the host cells. in this regard, the screening of large collections of monoclonal antibodies raised against membrane proteins has proven particularly useful to identify receptors that mediate viral entry. back in the early s, the discovery of cd as an entry receptor for the human immunodeficiency virus type (hiv) significantly impacted our understanding of viral pathogenesis and subsequent development of therapeutics [ , ] . in this case, the well-defined tropism of the virus determined the choice of over antibodies directed against human leukocyte differentiation antigens, of which only antibodies that recognized the surface receptor cd blocked viral infection [ ] . it is worth noting that similar monoclonal antibody screens have also been utilized for unbiased characterization of viral blockers. for example, colonno and colleagues performed a screen of more than , hybridomas from mice immunized with preparations of plasma membranes from human cells and were able to find one antibody that blocked rhinovirus binding to its cell surface receptor [ ] , identified as the intercellular cell adhesion molecule (icam- ) in subsequent studies [ ] . despite the undoubted importance of the biochemical and biophysical approaches to the study of host-pathogen interactions, the aforementioned limitations have motivated the development of alternative technologies for large-scale analysis of eppis. from the initial utilization of microarrays for detection of ppi over a decade ago, human proteome chips containing thousands of recombinant proteins have been generated, some of which are now commercially available. protein microarrays offer the unique advantage of requiring minimal consumption of protein reagents, fast readouts, and relatively more affordable instrumentation. typically, a fluorescently labeled or tagged protein of interest (the bait) is generated as a recombinant product and screened against all proteins in the array [ , ] . despite the increased availability of highcoverage protein arrays, very few are focused on extracellular proteins and therefore are not suitable for study of hostpathogen eppis. most existing microarray-based methodologies rely on multimerization of the bait protein for increased avidity and detection of weak eppis, mimicking the way these interactions occur in vivo, where proteins are arrayed in the crowded molecular environment of apposing plasma membranes. different multimerization strategies and protein microarray libraries have been developed and utilized for host-pathogen interaction discovery, some of which are described in more detail below. the wright lab developed a novel method for detection of low affinity eppis, termed avidity-based extracellular interaction screen (avexis) [ ] . in brief, avexis consists of the expression of the extracellular domain (ecd) of the bait of interest as a recombinant protein, which retains its binding properties while removing the insoluble transmembrane region of the protein. these ecds are tagged with a coiled-coil sequence from the rat cartilage oligomeric matrix protein to allow for pentamerization of the bait and therefore increased binding avidity, alongside a -lactamase tag for detection of baitprey interactions upon incubation with the colorimetric substrate nitrocefin. this multivalent strategy has been used for medium-scale screens, allowing detection of weak interactions between human receptors with low false-positive rates [ ] . notably, crosnier and colleagues applied avexis to search for the plasma membrane receptor responsible for plasmodium falciparum infection of erythrocytes [ ] . the authors compiled a library consisting of most secreted or cell surface-expressed proteins in erythrocytes and systematically assayed more than red blood cell proteins for binding to p. falciparum protein pfrh , a parasite protein essential for blood stage growth, expressed as an avexis pentameric bait. notably, the ok blood group antigen basigin was identified as a unique receptor for pfrh , and inhibition of this interaction was shown to be sufficient to block parasite invasion of the erythrocyte, findings that may importantly inform antimalarial therapies [ ] . in later studies, avexis was miniaturized making this approach compatible with the protein microarray format, thus permitting more comprehensive and lower resource-intensive screenings [ ] . although this technique should allow for high throughout and sensitive determination of eppis, this approach has not yet been applied to elucidation of pathogen-host interactions. over a decade ago, fueled by the recent completion of the human genome, genentech pioneered a significant effort to identify novel secreted or transmembrane domain-containing proteins, upon careful bioinformatics assessment and high throughput protein purification [ ] . these efforts resulted in the generation of a comprehensive human protein library, which was subsequently utilized to develop an extracellular protein microarray platform, consisting of over , secreted or single-transmembrane domain containing proteins [ ] . for the generation of this human protein library, secreted proteins or the ecd of single-transmembrane receptors were fused to different affinity tags and subsequently purified from cell culture supernatants by size-exclusion chromatography. mammalian cells or baculovirus-insect cells were preferentially used as expression systems, to maximize the likelihood of proper folding and glycosylation of the extracellular protein collection [ , ] . sds-page and multiangle laser light scattering were used to analyze noncovalent aggregation and ensure high-quality protein production. subsequently, the purified proteins were spotted on epoxysilane slides using a nanoprint arrayer, and protein immobilization on the microarray was determined by probing the slides with the relevant anti-tag antibodies [ ] . to enhance detection of low affinity interactions, a rapid method to assemble bait proteins (whose ecd was expressed as a fc tag-fusion protein) into multivalent complexes using fluorescently labeled protein a microbeads was developed. proof-of-concept assays showed high sensitivity for detection of weak eppis characterized by micromolar , a minimal off-target binding, and more than % true-positive to false-positive detection ratio [ , ] . over the years, this extracellular protein microarray has successfully identified counterreceptors for a number of human molecules, providing relevant insights into novel pathways that coordinate a multitude of cell functions [ ] [ ] [ ] . receptor discovery. recently, we applied this eppi discovery platform to the study of extracellular viral proteins ( figure ), with a focus on human adenovirus-(hadv-) encoded immunomodulatory proteins [ ] . despite the increasing relevance of hadv as both pathogens and therapeutic vectors, information on the interaction of these viruses with the host immune system remains scarce [ , ] . interestingly, the immunomodulatory proteins encoded by these viruses, termed e proteins, show substantial diversity in their ecds across and within viral species and constitute one of the most divergent regions of the hadv genome [ , ] . given this striking variability, the e proteins have been suggested to play a role in viral tropism and pathogenesis, yet the functions of virtually all e proteins have remained unknown [ ] . in our study, we took advantage of such unique variability to evaluate the effect of viral immunomodulatory protein diversity in extracellular host targeting. screening of a substantial number of e proteins encoded by different hadv species using the extracellular protein microarray platform allowed identification of over novel virus-host interactions encompassing viral species, which were fully validated by orthogonal methods [ ] . these findings revealed significant diversity in extracellular host targeting and, moreover, allowed identification of semiconserved host targets, pointing towards specific human receptors that may represent previously unrecognized hubs for viral perturbation. furthermore, most of the e immunomodulators were identified as multifunctional proteins, suggesting that viruses have evolved proteins capable of interfering with several cellular functions, a strategy consistent with the optimization of limited genomic resources. such economic targeting has been often observed in intracellular targeting [ ] [ ] [ ] , but so far few examples of widespread targeting in the extracellular environment have been reported [ , [ ] [ ] [ ] [ ] , let alone a global elucidation of eppi networks, in part due to the technical challenges associated with eppi detection. remarkably, many of the hadv e proteins preferentially interacted with host receptors that exert known or predicted inhibitory functions during the immune response (as defined by the presence of intracellular immunoreceptor tyrosinebased inhibitory motif, itim), including lilrb [ , ] , lair [ ] , and mpzl [ ] , suggesting previously unrecognized strategies of immunosuppression that may be utilized by other human pathogens [ ] . moreover, several of the receptors identified as targets for the viral proteins in this study (including the prominent cell surface molecule cd ) do not have known counterreceptors in the host, supporting the longstanding hypothesis that pathogen molecules drive the evolution of immune receptors and in many instances may represent the most relevant modulators of host receptor function [ ] [ ] [ ] . in summary, such unbiased, microarraybased study of immunomodulatory proteins represented the first large-scale analysis of the ppi landscape of a collection of extracellular immunomodulators encoded by viruses. future investigation of other pathogen-encoded molecules using similar extracellular protein microarrays will likely shape our understanding of the pathogen imprint in our immune system. microarrays. one of the main limitations of any protein microarray platform is the lower protein coverage relative to other genome-wide methods for ppi identification, due to the costs and difficulties for generation of comprehensive protein libraries to be deposited onto the microarrays. in an attempt to address this caveat, ramachandran and colleagues developed a method called nucleic-acid programmable protein array (nappa), in which dnas are directly deposited onto the array followed by protein synthesis in situ using an in vitro transcription and translation (ivtt) system, thus avoiding the need for protein purification [ ] . although this promising approach has proven superior in generating transmembrane-containing molecules as soluble proteins, it still remains to be systematically addressed if the extracellular human proteins produced in this manner present the folding and posttranslational modifications necessary for protein functionality. nevertheless, emerging data support the utility of nappa as a useful tool for the study of bacterial proteins. for example, montor and colleagues used a bioinformatics approach to predict the pseudomonas aeruginosa proteins that reside in the outer membrane of the bacteria or are secreted to the extracellular environment of the infected cell [ ] . in this work, the authors utilized the nappa approach to screen all predicted extracellular gene products for interaction with sera from cystic fibrosis patients, where p. aeruginosa establish a life-threatening lung infection. from bacterial proteins initially selected, proteins were recognized by antibodies in the sera, indicating that these bacterial proteins represent major antigens that trigger adaptive immune responses in humans. interestingly, robust antibody responses against three previously uncharacterized proteins were detected, suggesting this approach could help identify new extracellular proteins that exert unknown functions during the infection [ ] . these results confirmed the utility of the microarrays to detect immune responses against membrane proteins encoded by pathogens, and supported the use of this methodology for diagnosis applications. in this regard, several groups have developed microarrays composed of pathogen-encoded proteins [ , [ ] [ ] [ ] [ ] [ ] . such pathogen protein arrays have so far being exploited mainly for diagnosis purposes, to allow screening of antibodies present in patient sera for binding to extracellular bacterial or viral antigens on the array. nevertheless, their inherent high throughput and compatibility with multivalent bait approaches makes them a powerful tool for eppi discovery. for example, margarit et al. developed a streptococcus microarray to find novel microbial proteins capable of binding to the human proteins fibronectin, fibrinogen, and c bp and were able to identify a set of streptococcal proteins that interacted with these factors [ ] . nevertheless, despite such pathogen protein-based arrays offering great promise, this methodology remains to be systematically analyzed for eppi discovery. more recently, yu and colleagues applied nappa technology in combination with the halotag-halo ligand detection system to elucidate the interaction network of two effector proteins (sidm and lida) encoded by legionella pneumophila, a highly pathogenic bacteria that is the causative agent of legionnaire's pneumonia [ ] . similarly to many journal of immunology research pathogen proteins, these virulence factors lack significant homology to host molecules therefore complicating the assessment of their host targets and biological functions. in this work, the bacterial proteins of interest were tagged with a halotag, a modified haloalkane dehalogenase that covalently binds to synthetic halo-ligands (haloalkanes) that can be fluorescently labeled, thus allowing more robust detection of bait protein binding to interactors present on the array. in this study, more than , human proteins were expressed on the nappa array using different ivtt techniques, leading to identification of and binding partners for the lida and sidm effectors, respectively, most of them experimentally verified by pull-down [ ] . although this study focused on identification of intracellular ppi, the applicability of the nappa-halotag technology for eppi determination should be explored in the future. moreover, bait multimerization strategies should be implemented in order to make this approach more suitable for detection of transient ppis. related to the nappa technology, glick and colleagues recently built a miniaturized platform focused on human membrane proteins. by integrating the microfluidics technology, protein microarrays, and an ivtt system, this group built a new device named microfluidic-based comprehensive human membrane protein array (mpa) [ ] . a notable improvement introduced by these investigators was the addition of microsomal membranes to the ivtt system to allow for improved folding and posttranslational modifications in plasma membrane proteins, both common limitations of ivtt systems. in this work, a library of , human genes encoding for membrane proteins was built and subsequently utilized to screen the large-form delta antigen (l-hdag) encoded by the hepatitis delta virus (hda) and whole viral particles of the simian virus (sv ), a nonenveloped human pathogen. proof-of-concept assays showed encouraging results, with over % true-positive rate within a small set of proteins with known interactors and, more importantly, indicated the feasibility of this approach for expression of multitransmembrane-containing proteins, a protein type that has proven challenging given their high hydrophobicity. the mpa screens identified and over interactions for sv particles and l-hdag viral protein, respectively, and around interactions were validated by coimmunoprecipitation or protein-fragment complementation assays [ ] . to our knowledge, this is the first study to assess eppis using a comprehensive human protein library and whole viral particles (sv ) as baits, a valuable approach that may provide important insights into pathogen tropism, alongside a molecular explanation for the cell surface receptors engaged by the pathogen. further utilization of this platform followed by a more systematic analysis of the candidate hits, including nonspecific binder determination, will be needed to assess the overall performance of the mpa technology. regardless, this platform provides an extended version of the nappa approach that focuses on mammalian eppis and may therefore provide relevant insights into extracellular hostpathogen interactions. the protein microarrays have represented one of the most fruitful approaches for unbiased determination of eppis, including host-pathogen interactions. nevertheless, one of the main limitations of this technology is the need to generate comprehensive libraries, a process that is resource consuming and often not available to many researchers [ , ] . consequently, although some of the available arrays were designed to cover a significant fraction of the human proteome, any discoveries made using these platforms are limited to the proteins present in each array. the current libraries are likely to continue expanding alongside innovative approaches to facilitate sensitive detection of eppi using protein microarray formats. over the last decade, ms-based technologies have emerged as a versatile, powerful approach to decipher many aspects of the human proteome, including the characterization of protein complexes. excellent reviews on current ms-based technologies, recent improvements, and future prospects for elucidation of ppi networks are available [ ] [ ] [ ] [ ] . in this review, we briefly discuss the applications of some of these techniques to the study of extracellular host-pathogen interactions. proteins and interacting partners. the proteins expressed on the surface of pathogens mediate functions necessary for survival, replication, immunoevasion, and transmission and therefore are logical candidates for therapeutic and vaccine design. however, the study of the surface proteome in pathogens, particularly in bacteria is constrained by the fact that commonly used prediction algorithms fail to correctly predict the location of several proteins [ , [ ] [ ] [ ] . despite the characterization of the extracellular proteins and their interactions still representing the achilles heel of most proteomics methods, ms has emerged as an invaluable approach to characterize the protein composition of plasma membranes [ ] . to date, several studies have exploited ms-based techniques to gain insights into the extracellular protein composition of bacterial pathogens [ ] [ ] [ ] [ ] . for example, palmer and colleagues studied the surface proteome of the tick-borne intracellular pathogen anaplasma marginale (rickettsiales: anaplasmataceae) using liquid chromatography and tandem ms [ ] . interestingly, the authors found that the surface proteome of a. marginale isolated from tick cells, despite being less complex than that of bacteria isolated from human erythrocytes, contained a novel protein, which the authors hypothesized to play a function in human cell invasion in spite of its human counterreceptor remaining uncharacterized. this interesting observation suggests a remodeling of the bacteria surface proteome during the transition between mammalian and arthropod hosts, an aspect of the infection that could be targeted to block transmission. similarly, several studies have pursued the identification of the proteins present in viral particles utilizing ms. although these analyses suffer from several drawbacks associated with membrane protein characterization, particularly the poor solubility of these proteins and the low abundance of many plasma membrane proteins, these studies have revealed a complex composition for most of the viruses studied, alongside incorporation of many host proteins in the virions, in most cases with undetermined functions [ ] [ ] [ ] . an interesting observation from some of the studies referred above is the fact that certain bacterial proteins, predicted cytoplasmic by consensus, can be found in the extracellular environment of the cell, where they may play alternative functions. in fact, the number of proteins that are secreted through noncanonical signal sequence pathways is increasingly appreciated [ , , , ] . little is known about these bacterial proteins originally described as cytosolic proteins but capable of exerting functions on the cell surface, which some authors have named moonlight proteins, in reference to their potential to exert multiple functions [ ] . there is emerging evidence that protein moonlighting contributes to virulence of important bacterial pathogens including staphylococcus aureus or mycobacterium tuberculosis, sometimes in fascinating ways. for example, m. tuberculosis is known to encode two molecular chaperones, cpn . and cpn . , which function as modulators of myeloid cells among other regulatory functions [ ] . despite these chaperones being by definition cytosolic, cnp . has been detected in significant amounts on the bacterial surface, and either recombinant cnp . or antibodies against this protein efficiently block binding of m. tuberculosis to macrophages [ ] , through a potential interaction with the receptor cd [ ] . in addition, the protein dnak, a hsp related protein encoded by m. tuberculosis, can locate to the bacterial surface and functionally interact with cd [ ] and with the hiv coreceptor ccr [ ] . notably, dnak appears to block hiv binding to ccr in vitro, an interesting observation given the co-occurrence of m. tuberculosis and hiv infection [ ] . although a more detailed revision is out of the scope of this review, bacterial protein moonlighting, excellently revisited by henderson and martin [ ] , is a thought-provoking phenomenon that suggests a much more complex extracellular landscape than anticipated. moreover, such protein moonlighting is in line with the hypothesis that pathogens have evolved multifunctional proteins as a prominent strategy for efficient use of limited genomic resources [ , ] . another ms-based approach that holds great promise for host-pathogen eppi detection is the recently developed triceps [ ] . triceps is a chemoproteomic reagent that consists of three moieties, one that binds the ligand of interest through its amino groups, a second one that binds glycosylated receptors on the cell surface, and a biotin tag for purifying the receptor peptides for subsequent identification by ms. notably, in the initial description of the method, triceps was successfully applied to the identification of receptors for extracellular ligands of diverse nature, such as secreted glycoproteins, small peptide ligands for g proteincoupled receptors, and therapeutic antibodies. importantly, this approach has also been utilized to study cell surface molecules targeted by vaccinia virus (vacv). interestingly, the analysis of vacv binding to hela cells revealed seven candidate binding partners, including the previously identified receptors axl, chondroitin sulfate proteoglycan , and laminin binding protein dystroglycan . further, downregulation of five out of the seven candidates using short interfering rna reduced vacv infection by - %, supporting the functionality of the interactions identified, at least in vitro [ ] . although this technology is still developing and no studies on other pathogens have been published yet, future triceps-based studies promise relevant insights into pathogen interaction with distinct components of the cell surface. as an addendum to the vast amount of knowledge acquired using ms approaches and some of the additional methodologies discussed in this review, bioinformatics offers an in silico systems biology approach that reveals a global perspective on host-pathogen interactions. advances in computation have been fundamental to dissect the complex datasets generated in many genome-wide msbased studies and have enabled the reconstruction of largescale host-pathogens ppi networks, providing fundamental insights into viral disease and hence host biology [ - , - , ] . although the computational tools available for analysis of large datasets, in some cases developed in association with some of the high throughput screens mentioned in this review, certainly deserve a focused chapter, a couple of observations are specially notable. commonly observed in these studies is that the intracellular viral effectors preferentially target host proteins that act as hubs (proteins with many interacting partners) or bottlenecks (proteins central to many pathways in the network) [ , , ] . for example, dyer and colleagues built a network of host-pathogen ppi by integrating published information from pathogens [ ] . supporting previous findings, this analysis indicated that pathogen-encoded proteins preferentially interfere with host molecules that control critical cellular processes, such as cell death or nuclear transportation, possibly as a strategy to maximize control of the host machinery given limited genomic resources. interestingly, this study highlighted a small set of extracellular host proteins recurrently targeted by several of the viral and bacterial pathogens analyzed, including cell surface receptors such as vegfr /kdr and collagen, possibly indicating previously unrecognized roles in the immune response against pathogens. although informative, the analysis performed by dyer and collaborators was skewed towards viruses, with a prominent enrichment in hiv strains [ ] . more extensive analyses encompassing other human viruses and bacterial pathogens may reveal general strategies of immunomodulation and potential human targets suitable to therapeutic intervention. interestingly, increasing evidence suggests that virus-host interactions are governed by principles distinct to those that dictate within-host interactions [ , , , , ] . notably, detailed analyses carried out by the xia group highlighted significant differences between virushost and within-host (also called endogenous) interactions, such as the tendency of viral proteins to compete with host proteins for binding to a given receptor in the absence of sequence similarity with the host counterpart or the observation that viral molecules have evolved multiple short linear motifs capable of mediating a number of diverse interactions [ , ] , features that are consistent with the multifunctional capabilities of some pathogen-encoded proteins [ , , , [ ] [ ] [ ] [ ] ] . altogether, bioinformatics analysis of virushost interactions suggest that virus-mediated targeting of host proteins is characterized by signatures of pleiotropy, economy, and convergent evolution, conclusions that are supported by emerging experimental data. followed by thorough biological experimentation such computationalbased systems biology approaches will provide a unique tool to help decipher basic global principles of pathogenhost interaction and may reveal novel eppis amenable to therapeutic intervention. alongside protein microarrays-based technologies, ms, and computational analysis, the explosion of the functional genomics field in the last years has revolved the avenues to study pathogen interactions with their hosts, often in high throughput. in brief, genetic screens comprise gain-of-function and loss-of-function strategies, represented by complementary dna (cdna) libraries and rna-interference-(rnai-) based approaches, respectively. these methods were developed more than two decades ago and have been widely utilized by the scientific community, providing fundamental insights into the infection process. in particular, the cdna libraries have proven extremely successful in identifying viral receptors through a gainof-function approach, upon transduction of the cdna library from a susceptible cell line into nonpermissive cell lines. the use of cdna libraries is not reviewed in detail here in the interest of a more comprehensive revision of relative newer genomics-based approaches, such as the clustered regularly interspaced short palindromic repeat/ crispr-associated protein (crispr/cas ) or the haploid cell screens. nevertheless, these libraries have represented one of the most significant technologies to further our understanding of the pathogen-host interaction. for example, early studies made use of cdna libraries to shed light on the complex mechanism exploited by hepatitis c virus for initial invasion of the cell [ ] [ ] [ ] , identified car as a common receptor for adenovirus and coxsackievirus b [ ] , and were instrumental to identify slam and pvr as a receptors for measles and poliovirus, respectively [ , ] . in turn, the rnai technology has yielded significant insights into virus-host interactions, such as the identification of the ion transporter nramp as the receptor for the mosquito-borne sindbis virus colonization of drosophila cells [ ] . the main power of the rnai technology is that it allows high throughput genome-wide screens and therefore potential identification of essential factors that play roles in different aspects of the pathogen life cycle, including initial interaction with the host cell. although rnai screens have provided tremendous insights into host-pathogen interactions and remain widely utilized [ ] , inefficient gene depletion and off-target effects are important limitations of this methodology [ ] . technology. the increasingly popular crispr/cas technology overcomes some of the caveats often associated with genetic manipulation and holds enormous promise for genome editing and downstream applications, including host-pathogen interaction discovery [ ] . although still early days, high throughput crispr/cas screens for genome-wide studies have already displayed remarkable results, with high levels of genomic modification, hit confirmation, and strong phenotypic effects [ ] . the development of the crispr/cas technology has undoubtedly transformed the functional genetic analysis in mammals. recent studies have applied the crispr/cas technology to ablate expression of previously identified receptors for viral entry, such as the hiv coreceptors cxcr and ccr , leading to resistance to infection in primary cells [ , ] . an interesting additional application of crispr/cas is the direct editing of viral genes important for viral fitness. this approach has recently been used to target hsv- , cmv, and epstein-barr virus (ebv) essential genes, leading to a significant decrease of viral replication [ ] . these studies suggest the potential use of crispr/cas as an innovative therapeutic strategy, as aspect that will surely be further explored in the near future. another prominent example published recently is the identification of host factors that confer susceptibility to the evolutionary related type iii secretion systems, t ss and t ss , encoded by vibrio parahaemolyticus [ ] . the t sss are highly complex nanomachines utilized by gramnegative pathogens to inject a variable repertoire of virulence factors into the cytosol of the eukaryotic cells, enabling pathogen adhesion and internalization of modulation of host processes. interestingly, using genome-wide crispr/cas screens, sulfation and fucosylation of cell surface components were identified as host determinants of t ss -and t ss mediated cytotoxicity, respectively. the authors hypothesized that interactions between sulfated cell surface molecules such as host proteoglycans and bacterial adhesins act as facilitators of t ss activity, whereas fucosylated glycans on the surface may serve as receptors for t ss components necessary for insertion of the complex in the host membrane [ ] . the crispr/cas approach has just started to reveal its power as a tool for unbiased identification of novel eppis, elegantly exemplified by the identification of cd lf as the cell surface receptor for noroviruses, which, strikingly, was identified as the main determinant for the tropism of the murine norovirus [ ] . further optimization of this technology will unequivocally signify a tremendous advance for the discovery of extracellular host-pathogen ppis, the processes underlying host-pathogen interactions and its possible therapeutic applications. haploid cells, in turn, allow the study of recessive phenotypes that can be masked in diploid cells, due to the difficulties of creating true genetic knockouts in mammalian cells. despite yeast being a useful tool due to the simplicity of obtaining relevant mutants at its haploid life stage, the majority of human pathogens do not replicate in yeast therefore limiting the applicability of this approach [ ] . in recent years, human haploid cells have been increasingly utilized for genome-wide loss-of-function genetic screens using insertional mutagenesis [ , , ] . in initial studies, carette and colleagues took advantage of the kbm cell line, a derivative of the chronic myeloid leukemia cell line (cml) with a haploid karyotype except for chromosome [ ] . using gene-trap retroviruses for efficient insertional mutagenesis, the authors generated a genomewide collection of null mutants for most nonessential genes [ ] . this approach was successfully utilized to identify host factors essential for the functions of the distending toxins or cdts, potent virulence factors secreted by a number of pathogenic bacteria. in particular, mutagenized kbm cells were treated with escherichia coli-derived cdts and resistant clones were isolated, leading to identification of insertions in the sphingomyelin synthase and the putative g protein-coupled receptor tmem , suggesting that this molecule may serve as a surface receptor for the toxin [ , ] . similar haploid screens have identified novel receptors for a number of bacterial toxins, including the lipolysis-stimulated lipoprotein receptor for the clostridium difficile transferase [ ] , or the low-density lipoprotein receptor-related protein as a host receptor of the clostridium perfringens tpel toxin [ ] . in a later study, carette et al. generated a kbm -derived cell line named hap , haploid for all chromosomes [ ] . similarly to previous studies, the authors used the retroviral gene-trap approach to mutagenize hap cells followed by deep sequencing to map more than . insertions. in this study, using a replication competent vesicular stomatitis virus (vsv) carrying the ebola virus glycoprotein, a previously unknown entry receptor for ebola virus was identified. notably, these haploid cell screens identified six members of the hops complex, proteins known to play functions in endosomal/lysosomal trafficking, as well as the niemann-pick c (npc ) transporter as the most prominent hit of the assay. it is worth noting that npc is not a surface molecule but rather an endosomal receptor. these findings led the authors to propose a novel mechanism of entry by which ebola virus is internalized into the endocytic pathway, followed by endosome maturation and cleavage of the surface glycoprotein of the virus. endosome fusion, mediated by the hops complex, would allow interaction with ncp containing endosomes, triggering fusion and release of the viral genome into the cytosol. multiple cell surface receptors can lead to internalization of the ebola virus into the endocytic pathway [ ] ; such redundancy in receptor usage likely explains why these receptors were not identified in the haploid cell screen [ ] . notably, independent studies have confirmed that ncp acts an intracellular receptor for ebola, including a chemical screen approach, a study showing ncp dependence for infection of otherwise nonsusceptible cells, and more recently the elucidation of the crystal structure of this receptor bound to the ebola virus glycoprotein [ ] [ ] [ ] . interestingly, after the aforementioned haploid genetic screens identified ncp as a noncanonical entry receptor (given its intracellular localization), other filoviruses have been shown to take advantage of this receptor [ ] . the relevance of this intriguing mechanism of viral entry is further reinforced by recent work on lassa virus, an old world arenavirus that, similarly to ebola virus, causes severe to fatal hemorrhagic disease in humans [ , ] . a genome-wide haploid screen using vsv pseudotyped with lassa glycoprotein was performed in order to identify host factors essential for viral entry. although -dystroglycan (dag ) was long recognized as the cell surface receptor for lassa virus, additional factors were suspected, given the observation that certain dag -expressing cells are resistant to infection. the authors elegantly demonstrated that at a neutral ph, the lassa virus glycoprotein was bound to dag , whereas upon exposure to lower ph (resembling the lysosome environment), a receptor switch occurred leading to strong association with the lysosomal-associated membrane protein (lamp ) [ ] . thus, similarly to ebola virus, in the model suggested the virus would be incorporated into the endocytic pathway after interaction with its surface receptor dag , followed by increasingly acidic conditions that would result in interaction with lamp in the lysosomal membrane, triggering membrane fusion and release of the virus in the cytosol [ ] . more recently, pillay and colleagues applied the haploid cell screening approach to the identification of host factors essential for the adeno-associated virus (aav) serotype infection, one of the leading vectors for virus-based genes therapies [ ] . notably, the most significantly enriched gene in these screens was kiaa l, a poorly characterized type i immunoglobulin domain-containing transmembrane protein named hereafter as the aav receptor. among the host factors identified as hits, many were implicated in heparin sulfate proteoglycan biosynthesis as well as a number of proteins that participate in intracellular transport processes. aav is known to attach to the cells using heparin sulfate proteoglycans and hijacks endosomal trafficking to travel to the nucleus upon invasion of the cell; thus the authors hypothesized that these additional factors may influence virus tropism [ ] . altogether, these studies elegantly demonstrate the power of genome-wide screens in human haploid cells and the power of this approach to study virus-host interactions. future studies should further assess the applicability of this method for general detection of interactions that take place at the pathogen-plasma membrane interface. it will also be important to generate additional haploid cell lines, in order to broaden the range of pathogens and pathogen-derived molecules that can be studied using these genetic tools. in this regard, a number of haploid cell lines have been generated in mammals [ ] , unique tools to elucidate the basic aspects of human genetics. discovery. pathogens are among the most intriguing and prominent drivers of human evolution. humans have adapted to the pressure imposed by microorganisms through genomic diversification, particularly through variation of genes involved in immune system function, constantly challenged by the rapidly coevolving pathogen genomes. the advent of new technologies such as next-generation sequencing and the computational tools associated have opened new avenues for the study of human genetics, making it possible to evaluate the contribution of genetic diversity to susceptibility to infection at the genomic level. the emergence of datasets of genomic variation in multiple human populations, as well as pathogen genomes, allows detection of signatures of selection, which can be exploited to identify genes with major roles in immunity (for an excellent review see [ ] ). remarkably, the cell surface-expressed receptors are among the most polymorphic gene families in mammals, subjected to strong positive selection and rapid evolution, in many instances possibly driven by pathogen molecules that remain unknown [ , [ ] [ ] [ ] . polymorphisms in receptors and immunomodulatory genes contribute to the natural susceptibility of different individuals to infection [ ] [ ] [ ] , as illustrated by protection against hiv infection in individuals carrying homozygous polymorphisms in the viral coreceptor ccr [ ] . the identification and further study of genes under positive selection may represent a mainstream approach to dissect novel genes involved in disease and hostpathogen interaction. a notable example is the identification of glycophorin b as the erythrocyte receptor for p. falciparum protein ebl- through examination of highly polymorphic genes in populations from malaria-endemic regions [ ] . further population genetics studies promise key insights into novel immunological mechanisms and have the potential to provide molecular details that will ultimately help design effective therapies. in addition to these encouraging technologies, the generation of phagemic libraries has also represented an important tool for deciphering ppis, in this case between particular binding partners and the whole genome of specific pathogens [ ] [ ] [ ] . typically, pathogen-encoded molecules are expressed as fusions with phage envelope proteins, a method known as phage display that has been widely exploited to identify peptides with specific binding properties. for example, beckmann and colleagues built a phage display library to identify novel group b streptococci proteins capable of mediating adherence to fibronectin, a major component of the extracellular matrix often exploited for colonization of the host [ ] . from this analysis, the authors identified genes with homology to known bacterial adhesin proteins, genes involved in virulence, transport, or metabolic processes, along with genes with uncharacterized functions. interestingly, one of these genes showed significant homology with the scpb protein, a peptidase found in other streptococci that inactivates the member of the human complement system c a, suggesting that this bacterial molecule acts as a bifunctional protein, similarly to other examples of multifunctional proteins discussed above [ ] . more recently, a transposon-based insertion-inactivation mutant library was elegantly utilized to identify a bacterial protein capable of targeting the surface receptor tigit, an inhibitory molecule present in natural killer (nk) cells and t cells [ ] . fusobacterium nucleatum is a common oral bacterium that has been associated with colon adenocarcinoma and rheumatoid arthritis among other malignancies. in this study, gur and colleagues showed that different strains of f. nucleatum blocked nk-mediated killing of human tumors. using a library of f. nucleatum mutants, the authors identified fap as the bacterial protein that directly interacted with tigit, leading to inhibition of nk cytotoxicity and downregulation tumor-infiltrating t lymphocytes activation. immunoevasion is a hallmark of cancer; however whether members of the microbiome found within the tumor provide cancer cells with immunoregulatory properties has remained a major matter of debate [ ] . these interesting findings suggest that f. nucleatum present in the tumor niche may enhance tumor escape by inactivating nk-mediated killing upon interaction of the fusobacterial fap with the inhibitory receptor tigit. of note, transposon-based mutant libraries are readily available for other pathogenic bacteria and have been successfully applied to identification of bacterial genes implicated in bacterial physiology [ ] [ ] [ ] . it would be of interest to employ these libraries for unbiased identification of eppis. notably, as mentioned above, we found that hadv immunomodulators preferentially target other immunoreceptors that, similarly to tigit, also play inhibitory functions [ ] , suggesting this might represent a common immunosuppressive tactic evolved by pathogens. in fact, there is emerging evidence suggesting that may be the case, as a number of extracellular proteins from unrelated human pathogens have already been shown to target diverse immune receptors with inhibitory functions [ , , , , , ] . further exploration of inhibitory receptor targeting by other pathogens warrants exciting biological discoveries. deciphering the human genome made possible the categorization of genes that encode for the human secretome; now, the challenge of the postgenomic era is to annotate the functions of those genes and their expression patterns during health and disease. a lot has been learnt from painstaking, highly focused experiments using classical biochemistry. in recent years, the impressive technological advances in proteomics, functional genomics, and computation have revolved our understanding of cell communication and function and have collectively created a versatile platform to enable biological discoveries, from mechanistic explorations to big data and systems biology analysis. nevertheless our understanding of the molecules and mechanisms of extracellular immunomodulation and pathogen invasion remains remarkably limited. extracellular ppis between host-and pathogen-encoded molecules orchestrate an enormous diversity of cellular processes, from initial colonization of the target cell to subsequent immune responses. the elucidation of these extracellular interactomes is integral to understanding the molecular basis of infection and will guide the development of more efficient or innovative therapeutics. improvements in proteomics and genomics approaches have exponentially increased our understanding of how pathogens, particularly viruses, modulate the intracellular environment of the cell. concomitantly, we and several other groups have implemented technologies directed towards elucidation of extracellular interactomes [ , , , , , ] , which have begun to reveal fundamental principles of extracellular journal of immunology research host-pathogen interactions. notably, recent studies have revealed extensive eppi networks in model organisms such as drosophila or zebrafish [ , ] . these undertakings predict that, similarly to intracellular ppis, extracellular networks will be highly connected, with secreted and plasma membrane-expressed proteins having multiple binding partners. however, as discussed in this review, the identification of the host factors and in many cases the pathogen molecules that mediate eppis have largely defied molecular identification, in part due to the technical difficulties inherent to the study of these extracellular proteins. the elucidation of the global principles dictating extracellular pathogen-host ppis will require a coordinated effort to bring together the areas of biology and technology. there are now considerable opportunities for integrating multiple disciplines for eppi discovery, particularly proteomics and crispr/cas genome-wide screens, which should be powered by commensurable advances in bioinformatics and computation for big data analysis. the integration of orthogonal datasets coming from multiple "omics" approaches will be advantageous for elucidating the intricacies of the host-pathogen extracellular interactomes and will further enhance the rational identification of novel therapeutic targets by uncovering fundamental principles of biology. the journey from classical biochemical studies towards a systems biology approach has just begun and promises major technological breakthroughs and surprising biological findings. the development of powerful technologies for eppi discovery has already illuminated sophisticated and sometimes unexpected molecular mechanisms by which pathogens 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cells to assign genes to phenotypes by deep sequencing haploid genetic screens in human cells identify host factors used by pathogens lipolysisstimulated lipoprotein receptor (lsr) is the host receptor for the binary toxin clostridium difficile transferase (cdt) lrp is a receptor for clostridium perfringens tpel toxin indicating a tworeceptor model of clostridial glycosylating toxins ebola virus entry requires the cholesterol transporter niemann-pick c lassa virus entry requires a trigger-induced receptor switch an essential receptor for adeno-associated virus infection glycophorin b is the erythrocyte receptor of plasmodium falciparum erythrocytebinding ligand, ebl- identification of novel adhesins from group b streptococci by use of phage display reveals that c a peptidase mediates fibronectin binding binding of the fap protein of fusobacterium nucleatum to human inhibitory receptor tigit protects tumors from immune cell attack protein microarrays, biosensors, and cellbased methods 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ligand nell trem binds to apolipoproteins, including apoe and clu/ apoj, and thereby facilitates uptake of amyloid-beta by microglia subversion of host defense mechanisms by adenoviruses human adenovirus: viral pathogen with increasing importance molecular evolution of human adenoviruses homologous recombination in e genes of human adenovirus species d direct recognition of cytomegalovirus by activating and inhibitory nk cell receptors crystal structure and ligand binding properties of the d d region of the inhibitory receptor lir- (ilt ) secreted herpes simplex virus- glycoprotein g modifies ngf-trka signaling to attract free nerve endings to the site of infection structural basis for nkg a/cd recognition of hla-e the lilr family: modulators of innate and adaptive immune pathways in health and disease the inhibitory receptor lir- uses a common binding interaction to recognize class i mhc molecules and the viral homolog ul crystal structure and collagen-binding site of immune 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of ehrlichia chaffeensis composition of the surface proteome of anaplasma marginale and its role in protective immunity induced by outer membrane immunization identification of proteins associated with murine cytomegalovirus virions proteins of purified epstein-barr virus identification of proteins in human cytomegalovirus (hcmv) particles: the hcmv proteome fibroblast growth factor secretion is mediated by a non-cleaved aminoterminal signal sequence secretion of fgf- requires an uncleaved bipartite signal sequence bacterial virulence in the moonlight: multitasking bacterial moonlighting proteins are virulence determinants in infectious disease mycobacterium tuberculosis cpn . and dnak are located on the bacterial surface, where cpn . facilitates efficient bacterial association with macrophages mycobacterium tuberculosis employs cpn . as an adhesin that binds cd on the macrophage surface cd is a cellular receptor mediating mycobacterial heat shock protein stimulation of cc-chemokines dendritic cell stimulation by mycobacterial hsp is mediated through ccr inhibition of human immunodeficiency virus type infection of human cd + t cells by microbial hsp and the peptide epitope - epstein-barr virus and virus human protein interaction maps structural principles within the human-virus protein-protein interaction network slam (cdw ) is a cellular receptor for measles virus cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily what have rnai screens taught us about viral-host interactions? expression profiling reveals off-target gene regulation by rnai the crispr/cas bacterial immune system cleaves bacteriophage and plasmid dna high-throughput functional genomics using crispr-cas genome editing of cxcr by crispr/cas confers cells resistant to hiv- infection a cas ribonucleoprotein platform for functional genetic studies of hiv-host interactions in primary human t cells crispr/cas -mediated genome editing of herpesviruses limits productive and latent infections crispr/cas screens reveal requirements for host cell sulfation and fucosylation in bacterial type iii secretion system-mediated cytotoxicity how yeast can be used as a genetic platform to explore virus-host interactions: from "omics" to functional studies isolation and characterization of a near-haploid human cell line ebola virus entry: a curious and complex series of events small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection ebola virus entry requires the host-programmed recognition of an intracellular receptor ebola viral glycoprotein bound to its endosomal receptor niemann-pick c cell entry by a novel european filovirus requires host endosomal cysteine proteases and niemann-pick c molecular mechanism for lamp recognition by lassa virus derivation and differentiation of haploid human embryonic stem cells population genetic tools for dissecting innate immunity in humans the natural selection of herpesviruses and virus-specific nk cell receptors pathogen-driven selection in the human genome positive selection on the human genome genome-wide scans provide evidence journal of immunology research for positive selection of genes implicated in lassa fever toll-like receptor cascade and gene polymorphism in host-pathogen interaction in lyme disease single nucleotide polymorphisms in pathogen recognition receptor genes are associated with susceptibility to meningococcal meningitis in a pediatric cohort homozygous defect in hiv- coreceptor accounts for resistance of some multiply-exposed individuals to hiv- infection cloning of ligand-binding domains of bacterial receptors by phage display m-like proteins of streptococcus dysgalactiae a fibrinogen-binding protein of staphylococcus epidermidis cancer and the microbiota the neglected intrinsic resistome of bacterial pathogens sequence-defined transposon mutant library of burkholderia thailandensis comprehensive transposon mutant library of pseudomonas aeruginosa leukocyte immunoglobulin-like receptor b is critical for antibodydependent dengue hsv- infection through inhibitory receptor, pilralpha the mammalianmembrane two-hybrid assay (mamth) for probing membrane-protein interactions in human cells the author is grateful to lino gonzalez, jennie lill, erik verschueren, and yvonne franke for critical reading of the manuscript and insightful comments and suggestions. avidity-based extracellular interaction screen aav:adeno-associated virus car:coxsackievirus receptor cmv:cytomegalovirus crispr/cas : clustered regularly interspaced short palindromic repeat/crispr-associated protein ebv:epstein-barr virus ecd: extracellular domain eppi:extracellular protein-protein interaction hadv:human adenoviruses hcv:hepatitis c virus hiv:human immunodeficiency virus hsv:herpes simplex virus itim:immunoreceptor tyrosine-based inhibitory motif ivtt:in vitro transcription/translation lair :leukocyte-associated immunoreceptor like lilrb : leukocyte immunoglobulin-like receptor mpa:microfluidic-based comprehensive human membrane protein array mpzl :myelin protein zero-like protein ms:mass spectrometry nappa:nucleic-acid programmable protein array ncp :niemann-pick c pdgfra: platelet-derived growth factor receptor alpha ppi:protein-protein interaction pvr:poliovirus receptor sars-cov: severe acute respiratory syndrome coronavirus sv :simian virus slam:signaling-lymphocytic activation molecule spr:surface plasmon resonance t ss: type iii secretion system vavc:vaccinia virus. the author declares that they have no competing interests. key: cord- -du rqc i authors: louz, derrick; bergmans, hans e.; loos, birgit p.; hoeben, rob c. title: cross‐species transfer of viruses: implications for the use of viral vectors in biomedical research, gene therapy and as live‐virus vaccines date: - - journal: j gene med doi: . /jgm. sha: doc_id: cord_uid: du rqc i summary all living organisms are continuously exposed to a plethora of viruses. in general, viruses tend to be restricted to the natural host species which they infect. from time to time viruses cross the host‐range barrier expanding their host range. however, in very rare cases cross‐species transfer is followed by the establishment and persistence of a virus in the new host species, which may result in disease. recent examples of viruses that have crossed the species barrier from animal reservoirs to humans are hantavirus, haemorrhagic fever viruses, arboviruses, nipah and hendra viruses, avian influenza virus (ai), monkeypox virus, and the sars‐associated coronavirus (sars‐cov). the opportunities for cross‐species transfer of mammalian viruses have increased in recent years due to increased contact between humans and animal reservoirs. however, it is difficult to predict when such events will take place since the viral adaptation that is needed to accomplish this is multifactorial and stochastic. against this background the intensified use of viruses and their genetically modified variants as viral gene transfer vectors for biomedical research, experimental gene therapy and for live‐vector vaccines is a cause for concern. this review addresses a number of potential risk factors and their implications for activities with viral vectors from the perspective of cross‐species transfer of viruses in nature, with emphasis on the occurrence of host‐range mutants resulting from either cell culture or tropism engineering. the issues are raised with the intention to assist in risk assessments for activities with vector viruses. copyright © john wiley & sons, ltd. is known as a zoonosis in the case where the virus is transmitted from non-human hosts to humans and causes disease [ ] . crossing the species barrier is an unpredictable event that involves complex interactions between the virus and the newly adopted host [ ] . the hiv virus and contemporary human influenza viruses are prominent examples of viruses that have crossed the species barrier and established themselves permanently in the human population without further dependence on the presence of the original animal host reservoir. fortunately, natural adaptation of a new virus leading to permanent establishment and dissemination within the human population is a rare event [ ] . often, the virus is not readily adapted to infect and spread efficiently from human to human. the emergence of new viral infections often follows environmental, ecological and technological changes caused by human activities [ ] . these activities may lead to an increased contact between humans and animal hosts acting as reservoirs of zoonotic viruses. agricultural development, an increased exploitation of environmental resources, growth and increase in the mobility of the human population and trade and transportation of food and livestock, have been identified as important factors contributing to the introduction and spread of a number of new viruses in the human population [ ] [ ] [ ] [ ] . the road map for cross-species transfer may differ for individual viruses. however, some common underlying factors that affect the probability of zoonotic events can be identified ( table ). the potential of viruses to adapt to new or changing cellular environments or ecological niches via genetic variation appears to be a key feature [ , ] . the advent of novel technologies for genetic modification of viruses offers new opportunities for biomedical research. often, these activities involve handling of viruses and their genetically modified variants in large quantities. the use of viral vectors in experimental gene therapy or as live vaccines may be also a cause for concern. indeed, such activities meet the primary requirements for cross-species mutations that facilitate the use of alternative receptors in the newly adopted host species feline parvovirus [ ] transmission to humans after occupational exposure hendravirus [ ] intensified contact between natural and new host species due to climatic changes hantavirus [ ] immune evasion by genetic variation (e.g. antigenic drift and shift) influenza virus [ ] introduction in new geographic areas by migrating birds west nile virus [ ] initial close contact between natural and newly adopted host due to changes in natural infrastructures nipah virus [ ] transfer, i.e. contact between infectious viruses and a potential new host species. therefore, it is important to identify those activities that have a finite risk of leading to new viral infections and to practice appropriate biosafety measurements. in this review risk factors associated with activities with viral vectors will be addressed from the perspective of emerging viruses that have crossed the host barrier in nature. the processes that underlie cross-species transfer through host-range expansion and establishment of viruses in new host species depend on the accumulation of genetic changes [ , ] . these are likely to differ for various viruses and may affect virtually every aspect of the viral life cycle. this process of adaptation can occur by a variety of mechanisms including mutation, recombination and reassortment. mutations occur in the genomes of dna as well as rna viruses. in general, mutations occur slower in dna viruses than in rna viruses because of the proofreading function of many dna polymerases. this corrects mistakes made by the polymerase during replication. rna genomes are replicated by rna polymerases that lack proofreading. therefore, mutations in rna viruses can occur up to a million-fold more frequently than in dna viruses [ , ] . as a consequence, rna viruses generally evolve more rapidly, and lead to genetic heterogeneity and the presence of so-called viral quasispecies [ ] . the concept of quasispecies states that in an infected host, the virus exists as a population of genetically related but divergent variants defined by a master sequence and a complex and dynamic series of mutant sequences. while the master sequence remains the predominant sequence present within the population, the spectrum of mutants may shift in response to selective pressures. from the quasispecies population a variant may be selectively expanded [ ] [ ] [ ] . although the major mechanism that drives adaptation is based on accumulation of point mutations, evolution of viruses also occurs through recombination. recombination occurs in both dna and rna viruses leading to the exchange of parts of genomes. this may result in the emergence of new virus variants. for recombination to take place, at least co-infection of a cell by two different virus variants is required. recombination plays an important role in evolutionary changes of dna viruses. in some cases, recombination between viruses and cellular nucleic acid can lead to the capture of cellular coding sequences [ ] . reassortment is another important evolutionary mechanism in rna viruses with a segmented genome, such as influenza viruses and reoviruses. after co-infection of a cell with different strains or subtypes, genomic segments may be shuffled and rearranged in progeny virus particles resulting in the generation of new viruses with different biological properties. this offers viruses a large adaptive potential by facilitating evolutionary leaps in response to changing cellular environments without the need for gradual accumulation of favourable mutations [ ] . in this section a number of examples of animal viruses that have crossed the species barrier are chosen to illustrate how they have evolved in nature through genetic changes. the examples illustrate that under the right environmental conditions host-range variants evolve that may establish themselves in the newly recruited host as new viruses causing disease. after the initial cross-transfer to the new host species, a period of further adaptation may be required. the examples also show how the evolutionary processes continue while an epidemic evolves. in the late s, a new syndrome of viral enteritis and myocarditis emerged in dogs and subsequently swept rapidly across the world, killing thousands of dogs within a few years after its initial appearance. the virus was named canine parvovirus type (cpv- ). phylogenetic analysis revealed that this virus was remarkably similar to feline parvovirus-like viruses such as feline panleukemia virus (fpv) which infects cats, mink, and raccoons, but not dogs [ ] . therefore, cpv- presumably emerged as a natural host-range mutant of a feline parvovirus [ , ] . host-range properties are determined by the capsid protein for both cpv- and fpv. although cpv- is capable of infecting feline cells in culture, the virus does not replicate in cats. fpv, on the other hand, is able to replicate in dogs in a restricted fashion, i.e. in bone marrow and thymus, but not in cultured dog cells [ ] . cpv- differs from fpv by only two nucleotide substitutions within the capsid gene resulting in two amino acid substitutions [ ] . these changes are associated with the ability of cpv- to bind to the canine transferrin receptor with high affinity [ ] . as a result, cpv- acquired the capacity to infect canine intestinal tissue. it is thought that subsequently this virus acquired transmissibility between dogs and further adapted to replicate more efficiently in dogs as it became pandemic. interestingly, within three years after its initial appearance in , cpv- was replaced worldwide by an antigenically and genetically variant virus cpv- a [ ] . this indicates that cpv- a had a strong selective advantage over cpv- . another antigenic variant derived from cpv- a, cpv- b which differs by only two amino acids, arose in . this implies that variants of cpv gradually arose due to further adaptation and selection in dogs, in reaction to selective, most likely immunological, pressure. the two new variants (types a and b) differ at or amino acid from cpv- isolates [ ] . at present, the two variants are still endemic in the canine population. these two variant viruses have an expanded host range compared to the original cpv- since they replicate in cats in both experimental settings and in the wild although with no or relatively low pathogenicity [ , ] . remarkably, the prevalence of cpv- a and cpv- b and new antigenic variants (cpv- c) has now been demonstrated in a wide range of feline populations worldwide (reviewed in [ ] ). the emergence of cpv- serves as an example of rapid global distribution and establishment of host-range mutants in an immunologically naï ve new host. whereas cpv- most likely arose as a natural host-range mutant derived from cats, some other scenarios on its emergence have been suggested [ ] . it was suggested that cpv- may have emerged after cross-transfer from a yet unidentified animal host to dogs, or cpv- may have arisen under selective growth conditions during fpv livevirus vaccine production in canine cells and subsequently spread via vaccination. the high titers of the virus shed in faeces and its resistance to inactivation may explain its initial rapid dissemination, also into countries with strict quarantine regulations for dogs. human activity may have stimulated the spread through mechanical transport, presumably aided by long-distance air travel [ , ] . the aids pandemic is now generally accepted to stem from a viral zoonosis. human immunodeficiency viruses and (hiv- and hiv- ) emerged separately around the same time in distinct geographically areas as the result of multiple zoonotic transmissions from simian immunodeficiency virus (siv)-infected non-human primates to humans [ ] . based on their genomic organization and phylogenetic analyses it is clear that hiv- and hiv- fall into two different siv lineages [ ] . this implicates that both viruses must have had distinct origins. both phylogenetic and epidemiologic evidence indicate that hiv- evolved as a consequence of sivcpz transmission from chimpanzees to humans in central africa [ , ] . however, to date, no serological or genetic evidence of widespread prevalence of hiv- -related strains exists in chimpanzees in the wild in africa. transmission of siv from sooty mangabeys in west africa most probably caused the emergence of hiv- since siv strains derived from sooty mangabeys are phylogenetically closely related to hiv- . hiv- is found at a high prevalence in sooty mangabeys [ , ] . both hiv- and hiv- show enormous genetic diversity. hiv- comprises three genetically distinct virus groups (m, n, and o) of which the predominant group m consists of subtypes or clades of which all but two have spread throughout the world. in contrast, hiv- is mainly confined to the african continent and comprises seven distinct phylogenetic lineages, subtype groups a through g, which can be categorized in epidemic subtypes a and b and non-epidemic subtypes c through g [ , ] . it has been estimated from phylogenetic and epidemiological data that initial cross-species transfers of both the m group of strains of hiv- , and the progenitor group of subtypes of hiv- , may have taken place around in west africa [ , ] . hiv- may have initially started to spread in africa at the beginning of the s [ , ] . therefore, more than two decades of 'silent' human-tohuman transmission may have occurred in africa before aids became apparent and hiv was identified as its causative agent in the early s. in the advent of the hiv epidemic early siv strains initially may have crossed the species barrier as a result of an increase of contact between humans and siv-infected simian species. apparently, activities such as hunting, handling and consumption of contaminated uncooked simian meat led to direct exposure to animal blood and body fluids [ ] . the following years passaging between infected humans of partially adapted siv strains may have resulted in series of cumulative mutations and genetic changes. the large genetic differences that exist between siv and hiv indicate that the initial siv strains that crossed the species barrier must have undergone adaptation in humans in a relatively short period of time. this suggests the involvement of some modern iatrogenic event. massive vaccination programs carried out at that time using non-sterile injection needles may have provided opportunities for transmission and further adaptation of the virus to humans in africa [ , ] . the genetic and phenotypic evolution of the hiv virus still proceeds at a high pace not only between individuals worldwide, but also within infected individuals. during the time-course of infection the extensive genetic diversity originates from the rapid viral turnover and replication errors caused by reverse transcriptase [ , ] . in addition, among the globally pandemic hiv- m group, several circulating recombinant forms (crfs) exist. these crfs result from recombination events between two different strains within the same individual and now constitute - % of newly characterized circulating strains [ ] . this diversity allows the hiv virus to rapidly adapt under selective pressure generated by antiretroviral drugs and host immune responses. selection of hiv variants has been implicated in the use of different co-receptor molecules and selection for different cell types and tissues and body compartments such as lymph nodes and the brain during late stages of infection and manifestation of different disease patterns [ , ] . the emergence of hiv exemplifies how multiple independent cross-species transmissions of simian viruses that are not associated with disease in their natural hosts eventually resulted in the establishment of two types of hiv in the human population. while adapting to its new host the virus underwent a myriad of molecular changes. changes in social behaviour of humans may well have offered opportunities for newly evolved hiv strains to become pandemic. pandemic influenza a is a zoonotic disease caused by cross-species transfer of influenza a viruses from animal reservoirs. the twentieth century has witnessed three influenza pandemics, spanish influenza ( ), asian influenza ( ), and hong kong influenza ( ), that killed millions of people worldwide. although influenza a viruses have been isolated from a variety of vertebrates, including pigs, horses, seals, and whales, birds serve as the main reservoir and are a potential source for new pandemic strains [ ] . influenza a viruses contain eight negative-sense rna segments that code for at least ten polypeptides of which eight are structural viral proteins and two nonstructural proteins. influenza a viruses are divided into subtypes based on both serological and genetic differences between the surface proteins and their encoding genes, respectively. to date hemagglutinin (ha) subtypes (h -h ) and nine subtypes (n -n ) of the neuraminidase (na) proteins have been identified. influenza a viruses containing all different combinations of the ha and na subtypes have been identified in aquatic birds. in humans only influenza a viruses of hemagglutinin subtypes h through and neuramidase subtypes n and n have established permanent lineages. these viruses are considered human influenza a viruses [ ] . in nature new influenza a viruses emerge via two mechanisms of antigenic variation. the first, antigenic drift, is caused by accumulation of point mutations in both the na and ha surface proteins enabling new antigenic variant viruses to evade the human immune system and emerge via selection. influenza a viruses that emerge via antigenic drift are responsible for the yearly epidemics in the human population. antigenic shift occurs when a variant influenza a virus arises that is antigenitically completely distinct from former circulating influenza a viruses. the new virus is a reassortant that is characterized by the presence of a novel hemagglutinin gene segment alone or in combination with a complete novel neuraminidase gene segment. influenza a variants that emerge through antigenic shift are potentially capable of causing novel pandemics in an immunologically naï ve human population [ ] . the avirulent nature of avian influenza virus infections in ducks and waterfowl results from adequate adaptation to their hosts [ ] . avian influenza viruses do not replicate efficiently in humans. similarly, human influenza a viruses do not replicate efficiently in birds [ , ] . the trachea tissue of the pig contains receptors for both avian and human influenza a viruses. therefore, they are permissive to both human and avian viruses and thought to function as a 'mixing vessel' for reassortment of not only human and avian, but also swine influenza a viruses [ ] . pigs are therefore also considered to be ecological niches important for the emergence of new influenza a viruses in humans. in pigs, newly reassorted viruses may further evolve by accumulation of additional mutations, further adapt to a mammalian host and eventually be transmitted to humans. ample data indicate that further adaptation to the human cellular environment is necessary for replication and efficient transmission in humans. adaptation has a polygenic basis and may involve multiple viral gene segments. it should be noted, however, that only in rare cases will cross-species transmission lead to permanent establishment of new lineages of influenza a viruses in humans [ , ] . analyses of the viruses that caused the asian and hong kong pandemics revealed that these were caused by reassortants that contained a mixture of avian and human genome segments [ , ] . genetic analysis of the spanish influenza virus initially suggested that the epidemic originated from a whole avian influenza virus that had been transmitted from infected pigs to humans [ ] . however, the origin of the pandemic strain still remains an enigma since the presence of its ha molecule did not originate from any known avian strain. in addition, there was no evidence of adaptation to a mammalian host [ ] . it was not until the hong kong epidemic that direct transmission of whole avian viruses to humans was observed. analysis of the virus that caused this epidemic revealed that a reassorted influenza a virus (h n ) of entirely avian origin had crossed the species barrier, apparently without adaptation to a mammalian host. interestingly, the virus was able to replicate in humans but had not acquired human-tohuman transmissibility, preventing efficient spread and, potentially, a global epidemic (reviewed in [ ] ). receptor specificity is considered to be a major determinant of the host range of influenza a viruses. the ha protein plays a pivotal role in host-cell receptor recognition and attachment. it binds sialic acid (sa) on the host cells. avian influenza a viruses preferentially bind to terminal sa which is joined by an alpha , -linkage to the sugar chain of the glycoprotein or glycolipid in the gut. however, human influenza a strains bind to terminal sa through an alpha , -bond to cells in the respiratory tract as a result of acquired mutations [ ] [ ] [ ] . the hong kong avian h n strain, however, possessed avian binding properties [ ] . this indicates that receptor specificity alone is not an absolute requirement for birdto-human transmission. the host range of influenza a viruses is determined by a complex interplay of multiple factors [ ] . the influenza a virus illustrates the unpredictability of virus variation as well as the virus's great potential for adaptation. regular close contact between birds, pigs and humans offers opportunities for reassortment and crossspecies transfer. hence, the live-bird markets in south-east asia are considered a risk [ ] . a variety of experimental conditions are applied in the laboratory for propagation and isolation of viruses and their genetically modified derivatives. as a result, these viruses are subject to selective forces that are likely to differ from those experienced in nature. although conditions may be well defined and controlled, various selective pressures are generated in culture due to, e.g., changes of concentrations of nucleotide substrates, the addition of mutagenic substances, the use of different incubation temperatures, incubation with antibodies, or a change of host cells. these different selective forces have unpredictable influences on the virus. since cell culture conditions can have profound effects on the composition of viral populations, viral stocks consist of genetically heterogeneous populations (reviewed in [ , ] the following examples demonstrate that upon persistent infection and passage in cell culture, cross-species transmissibility may be promoted by selection of virus variants with an altered host range. adaptation in cell culture may result in changes in receptor specificity and tropism, and leads to the emergence of host-range mutant viruses. the mouse hepatitis virus (mhv) is characterized by a narrow host-range and tissue specificity, both in vivo and in vitro. this specificity is primarily determined by the virus's surface spike (s) glycoprotein, which is responsible for attachment to specific host-cell receptors [ ] . mhv virus variants with both an altered receptor specificity and a broadened host range were selected during continued passaging in murine or mixed cultures consisting of murine and non-permissive hamster cells. here mhv acquired the ability to infect human, hamster and monkey cells. mhv host-range expansion has been attributed to the presence of virus variants recognizing homologues of the normal receptor. adaptation required mutations in the surface s protein and selection of host-range mutants for the use of the alternative cellular receptor [ ] [ ] [ ] [ ] . selection of variants with a changed receptor specificity resulting from passage in cell culture has also been demonstrated for foot-and-mouth disease virus (fmdv). host-cell specificity of the parental virus is based on an rgd motif-dependent integrin-mediated entry pathway [ ] . the rgd motif is an arginine-glycine-aspartic protein sequence within the virus capsid that recognizes and binds to some integrins on the cell surface of the host cell [ ] . yet, upon multiple passages in bhk- cells, fmdv variants emerged that acquired the ability to infect several initially non-permissive human and animal cell lines via an alternative entry pathway. analysis of these variants revealed that adaptation of fmdv in cell culture led to an enhanced affinity for heparan sulfate as a receptor, independent of the rgd motif. interestingly, these host-range variants were able to maintain infectivity in cell culture not only independent of an rgd motif, but also without the requirement to bind to heparin sulfate [ , ] . this implies the use of alternative receptors. selection of the fmdv host-range mutants was associated with amino acid substitutions in or near the capsid rgd motif [ ] [ ] [ ] . recently it was demonstrated that passaging of fmdv in bhk- cells led to an expansion of the host-cell tropism to non-human primate and human cell lines. selection of these host-range variants was also associated with amino acid substitutions in the viral capsid proteins [ ] . there is less data on mutation frequencies and adaptation of dna viruses in cell cultures compared to rna viruses. however, there are a number of illustrative examples. adaptation of sv and polyomavirus to different cellular environments resulted in the emergence of host-range mutants in cell cultures (reviewed in [ ] ). random mutagenesis of human adenovirus followed by repeated passaging in certain cell lines allowed isolation of adenovirus host-range mutants [ , ] . human adenovirus (hadv ) mutants with altered specificity resulting from reduced binding affinity of the adenovirus penton-base protein for the integrins on the cells were selected in persistently infected cell lines [ ] . more recently, upon passaging in cell culture, adaptation led to the emergence of herpesvirus host-range mutants. these viruses use alternative receptors and replicate in different cell types in the natural host, and in cells from different species that were previously nonpermissive. several glycoproteins are essential for the entry of alphaherpesviruses such as pseudorabies virus (prv), herpes simplex virus (hsv) and bovine herpesvirus (bhv- ) (reviewed in [ ] ). interaction between, e.g., the viral glycoprotein c (gc) and heparan sulfate mediates primary attachment. for infection, however, a secondary interaction between glycoprotein d (gd) and one of several entry receptors is required. single amino acid substitutions in the gd glycoprotein of herpes simplex (hsv- ) as well as complete ablation of this glycoprotein in the swine pseudorabies virus (prv) led to a gd glycoprotein-independent entry mode using alternate receptors [ ] [ ] [ ] : at least three classes of cell-surface proteins are now thought to be involved in alphaherpesvirus entry [ ] . also, cell culture adaptation of human cytomegalovirus (hcmv), a betaherpesvirus, resulted in the selection of phenotypic variants that had lost their endothelial tropism [ ] . in cell culture viruses may readily adapt by mutation, selection and competition. these processes are stochastic in nature. adaptation is therefore an unpredictable process, strongly influenced by the experimental setting, e.g. the multiplicity of infection used, the number of passages employed, and the type of selection employed. one should be aware of the potential adaptation when working with virus-infected cell cultures. recombinant dna technology, including 'reverse genetics' and the availability of complete (infectious) clones for a large number of rna and dna viruses, allows genetic modification of viral genomes and generation of recombinant [ , ] viruses in vitro. these technologies offer the possibility to deliberately change the tropism or host range of the viruses. some of the latest technologies are discussed in the context of two important virus groups, i.e. influenza a viruses and coronaviruses. the advent of reverse genetics systems now allows the generation of recombinant infectious influenza a viruses entirely from cloned cdnas in cell culture. these systems are based on transfection of at least eight plasmids, each containing a copy of one of the eight influenza a virus genomic segments [ , ] . the technology permits the generation of custom-made recombinant influenza a viruses (reassortants) containing specific (heterologous) gene segments of interest and offers the possibility to study their biological properties in cellular and animal model systems. in addition, reverse genetics can be used in the development of vaccine strategies. the use of reverse genetics allows the deliberate introduction of specific mutations in viral genes allowing selective evaluation of the contribution of individual genes or segments to, e.g., the virus's virulence/pathogenicity, transmissibility and host range. this approach has already been shown to be pivotal for the generation and characterization of reassortants containing heterologous influenza a segments from, e.g., highly pathogenic avian influenza (hpai) h n strains [ ] or the pandemic strain [ , ] . in addition, this technology allowed the generation from cloned segments of the hpai h n strain that caused the deadly hong kong outbreak [ ] . reverse genetics systems can also be used as an alternative for the production of both live attenuated and inactivated vaccines in preparing for pandemic influenza a virus threats. conventional annual (inactivated) vaccine production is based on simultaneous infection of chicken eggs with two different influenza a strains followed by selection of the desired vaccine virus. this reassorted virus then contains the na and ha segments of the relevant circulating influenza a virus against the background of six complementary segments derived from an attenuated reference strain (e.g. a/puerto rico/ / h n ), which is safe for humans [ ] . to overcome the difficulties of selecting such reassortants and subsequent laborious time-consuming passaging of these viruses, plasmidbased reverse genetics can be used for fast and directed generation of vaccine strains [ ] . for vaccines based on hpai viruses the use of reverse genetics has another important advantage. such highly pathogenic viruses are lethal to chicken embryos and cannot be grown in large quantities in this way. the virus's high pathogenicity is associated with the presence of basic amino acids adjacent to the cleavage site within the ha molecule [ ] . by using recombinant dna technology this sequence can be eliminated. plasmid-based reverse genetics can then be used to generate the desired vaccine strain containing the attenuated ha molecule [ , ] . in conclusion, plasmid-based reverse genetics enables the generation of defined reassorted influenza a viruses consisting of, e.g., human and avian viral gene segments of interest. however, it is usually not possible to predict the biological properties from the gene constellation of such variant viruses. such activities therefore pose potential risks, in particular when the gene constellation is not based on characterized isolates. recombinant dna technology has allowed the construction of infectious cdna clones of large rna viruses such as coronaviruses, including the sars-associated coronavirus (sars-cov) [ ] [ ] [ ] . these reverse genetics systems can now be used as tools for the production of defined genetically modified coronaviruses [ ] . this allows the introduction of specific mutations into the genome of coronaviruses, and, e.g., the exchange of specific viral genes between different coronaviruses to study their pathogenesis, replication strategy, and cross-species transmissibility. the possibility to engineer tissue and host tropism using these technologies makes coronaviruses potential vectors for vaccine development and possibly for gene therapy [ ] [ ] [ ] . host-range specificity of coronaviruses is primarily determined at the virus entry level. several studies have demonstrated that sequence changes in the gene encoding the coronavirus surface spike (s) glycoprotein can lead to a change of tropism and host-range specificity [ ] . this is illustrated by the generation of a chimeric coronavirus by targeted recombination, in which the ectodomain of the s glycoprotein of mouse hepatitis virus (mhv) was replaced by the ectodomain of the s glycoprotein of feline infectious peritonitis virus (fipv). this substitution conferred specific tropism for feline cells, while the ability to infect murine cells was lost [ ] . vice versa, a reverse genetics strategy for fipv was developed conferring the ability to infect murine cells [ ] . similar techniques may aid the studies on the pathogenicity of the sars-cov. the examples mentioned above indicate that the use of recombinant dna technology now provides for powerful systems to generate and modify, e.g., highly pathogenic viruses such as pandemic influenza a viruses and sars-cov. live attenuated virus vaccines are among the most successful viral vaccines known to date. traditionally, attenuation is achieved by the 'jennerian approach', i.e. serial passaging in cell culture [ ] . by this method a number of useful vaccines currently in use have been generated. still, the mechanism by which the attenuated phenotype evolves is largely unknown. for instance, the nature and degree of genetic variation present at different stages of the attenuation process is usually not known. the presumed mechanism of attenuation is based on host-range restriction due to accumulation of changes in surface (glyco)proteins [ ] . thus selection of variants seems inherent to the process of generating the desired level of attenuation and genetic stability to prevent reversion to the wild-type virus therefore, dependent on the passage history, diversity within the virus population is likely to represent adaptations to growth in cell culture. as a result, genetic variants with different host-range phenotypes may be present in the vaccine strain of the virus. examination of substrains of live attenuated vaccine lots based on the yellow virus d strain and measles virus edmonston strain demonstrate that these virus stocks indeed consist of a heterogeneous population of variants [ ] [ ] [ ] [ ] . a number of adverse consequences of the use of such virus stocks have been reported and associated with possible selective growth advantage of host-range variants in the recipient [ , ] . therefore, caution should be taken before releasing live attenuated viral vaccines based on non-human animal viruses. significant progress has been made in approaches to genetically modify the tropism of vector viruses. such strategies have been used in the development of cancer gene therapy. initially, replication-deficient vectors were used for this purpose. however, to improve efficacy, tumor-targeted replication-competent viruses have been developed for the use of viral therapy of cancer (virotherapy) [ , ] . here we discuss some of the developments with human adenovirus type (hadv ). hadv has been widely used for a number of vector applications [ ] . however, the use of genetically modified adenoviral vectors has some limitations [ ] . their efficacy relies on the presence of its receptor, car (coxackievirus and adenovirus receptor for hadv ), on target cells. primary binding of the virus to car is mediated by the knob domain of the adenoviral fiber protein. subsequent internalization is mediated by the interaction between the rgd motif in the penton base of the virus and secondary host-cell integrin receptor molecules [ , ] . to achieve cell-type specificity and a high efficacy in the absence of the car receptor different strategies have been developed. these include redirecting adenoviral binding to alternative cellular receptors by genetic modification of genes coding for the capsid proteins fiber, hexon and penton base [ ] . this may result in either an expanded tropism or in abolishment of the adenoviral native tropism. table summarizes a number of properties contributing to the relative risk for the use of vector viruses. for this purpose a numerical hazard score was assigned to each property. table summarizes a number of adenoviral vectors with altered properties and their relative risks. such modified viruses are now being evaluated in a clinical setting for experimental gene therapy. table illustrates that a change of cell tropism, tissue tropism, or host range of a viral vector should be considered as factors in risk assessment for activities with genetically altered vector viruses. in general, the use of replicationcompetent viral vectors poses special concerns with regard to unintended spread to new and undesired cell types, as well as horizontal transmission of the vector [ , ] . a replication-competent vector virus with an altered tropism or host range virtually constitutes a new viral pathogen with the potential of a new disease manifestation. in nature many factors may contribute to the emergence of a new zoonotic viral disease. these factors consist of viral evolutionary processes such as mutation, natural selection and competition, host determinants, e.g., immune status and physiological factors, and environmental determinants such as ecological and climatological circumstances. as highlighted by the emergence of new viral diseases in the last two decades, the process of adaptation often involves the acquisition of an altered cell tropism or host range. against this background the intensified use of viruses and their genetically modified variants as viral gene transfer vectors for biomedical research, experimental gene therapy and for live-vector vaccines is a cause for concern. this review highlights the importance of identifying and evaluating the risks and consequences of activities that may generate host-range mutants with the capacity of cross-species transmission. the use of such viruses may lead to inadvertent introduction of vector viruses with a changed cell or tissue tropism and/or host range through an immunologically naï ve and non-adapted hosts. interactions between the virus and the cellular receptor often determine the host range of the virus and therefore constitute a species barrier [ ] . minor mutations in the viral capsid or surface glycoproteins may already result in profound changes in cell tropism or host range of a virus. in this review we have therefore focused on the level of virus entry to address some implications for activities with viral vectors, and in particular with hostrange mutants. this could contribute to a rational and reasoned inventory of factors that should be considered in risk assessments of activities with viral vectors. in considering possible risks involved in handling replication-competent vector viruses in the laboratory, [ ] . this implies that concepts such as host-range barrier and hostcell specificity may be rather flexible than rigid. if the host range and completion of the viral life cycle is exclusively restricted to the level of entry, forced entry may bypass important discriminatory host-cell restriction steps. this may result in distinct pathological phenotypes and new disease manifestations. therefore, precaution should be taken to avoid inadvertent release and spread of such potential harmful vector viruses. it goes without saying that live-virus vaccines are among the most effective modalities to control viral pathogens. it is evident that for such vaccines the benefit is higher than the possible adverse effects. nevertheless, the risks associated with handling vaccine viruses, viral vectors, and exotic viruses are small but finite. we are just beginning to understand the mechanisms that drive the emergence of new viruses and viral diseases in nature. there are parallels between 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receptor-independent cell entry mechanism an adenovirus mutant that replicates selectively in p -deficient human tumor cells a novel threepronged approach to kill cancer cells selectively: concomitant viral, double suicide gene, and radiotherapy infectivity enhanced, cyclooxygenase- promoter-based conditionally replicative adenovirus for pancreatic cancer the authors wish to thank prof. alex j van der eb and dr. huub schellekens for critical review of the manuscript. in addition the authors wish to acknowledge the useful commentaries of the anonymous reviewers. key: cord- -fhmhpobi authors: qi, dan; guan, jitian; wu, erxi title: virus infection-induced host mrna degradation and potential application of live cell imaging date: - - journal: radiol infect dis doi: . /j.jrid. . . sha: doc_id: cord_uid: fhmhpobi viruses exist wherever there is life. they can cause allergy, immune response, inflammation, and even fatal diseases directly or indirectly. accumulating evidence shows that host rna undergoes rapid degradation during virus infection. herein, we focus on several possible mechanisms of infection-induced host rna turnover, which seems to be a common strategy for both prokaryotic and eukaryotic viruses during the very early stage of infection and a potential application of live cell imaging on its visualization. when a virus/bacteriophage infects a host cell, it utilizes its host's molecular machinery to replicate, efficiently generating more than hundredfold of its progeny. in particular, the virus relies on the ribosomes in the host cell to translate viral messenger rna (mrna) into polypeptides. many viruses also impair the translation of cellular mrna [ e ], one of the mechanisms during the shift of gene expression from host to virus, a process termed "host shutoff", in order to prevent the production of anti-viral, host protecting proteins [ ] . both eukaryotic and prokaryotic viruses are reported to have numerous mechanisms during 'host shutoff' that are either essential or can facilitate their propagation under changing environment, such as host interferon antagonist, host dna damage, host transcriptional and translational apparatuses modification [ e ]. among them, virus infection-induced host rna degradation has not been paid enough attention to, which might be the very early step after viral infection in most cases. strict control of their gene expression regulates the timing of host factor suppression to maximize their replication. mrna decay occurs in various pathways and is highly regulated in both eukaryotes and prokaryotes. here we will mainly summarize the common pathways. in mammalian cells, a majority of mrnas are capped by a -methyl-guanosine (m g) and tailed by poly adenine (a) at end. these features physically protect the mrna ends from exonucleolytic decay that remove nucleotides from -end (xrn mainly in the cytoplasm and xrn in the nucleus) or end (the exosome complex) [ ] . these structures of mrna also serve to recruit translation initiation machinery [ ] . cellular deadenylase enzyme complexes, ccr -not, pan -pan , and parn are in charge of the deadenylation, which is the first and often the rate-limiting step of mrna decay, resulting in the shortening of the poly (a) tail. decapping enzymes are required in some cases for their mrna turnover such as dcp , dcp , etc. [ ] . in bacterial cells, the -end and -end of mrna are likewise protected from exonucleolytic decay. mrnas are end triphosphated instead of a cap and transcriptionally terminated at -end with stem-loop [ ] , which is sometimes followed by a poly (u) or poly (a) cluster. endonucleolytic cleavage via endoribonuclease is one of the basal pathways for bacterial mrnas such as that in escherichia coli (e. coli) endoribonuclease e plays an essential role in its mrna turnover process [ ] . another common route for bacteria mrna decay is that a removal of two of the triphosphates via rna pyrophosphohydrolase (rpph) [ ] leaving a monophosphate, causes the message to be destroyed by the exonuclease rnase j, which degrades to , such as the mrna turnover in bacillus subtilis [ ] . promoting global mrna degradation is reported to be one of the host shutoff mechanisms to block host gene expression among at least three different viral subfamilies, alphaherpesviruses, gammaherpesviruses and betacoronaviruses [ , ] . moreover, gaglia et al.'s work showed that viral encoded proteins trigger host mrna degradation by a primary endonucleolytic cleavage causing shutoff of host gene expression and a host exonuclease such as xrn , an important to exonuclease in human cells, were required in subsequent completion of host mrna turnover [ ] . as shown in fig. a and b, nearly all viruses known drive widespread mrna degradation triggered by either internal endonucleolytic cleavage or removal of cap structure of the mrna. one typical example of alphaherpesviruses is herpes simplex virus (hsv- ). virion host shutoff protein (vhs, previously ul ) having an rnase activity [ , ] , is packaged in virions and released after infection [ , ] . vhs is directed to mrnas through interactions with the translation initiation factors eif h and eif ai/ii [ ] and thus induces host mrna decline. several examples of gammaherpesviruses were also characterized: sox in kaposi's sarcoma-associated herpesvirus (kshv) [ ] is the homolog of the alkaline exonuclease of other herpesviruses, which has been shown to function as a dnase involved in processing and packaging the viral genome [ ] as an endonuclease. during kshv infection, sox initiates host mrna degradation by its endonucleolytic cleavage followed by rapid exonucleolytic degradation of host xrn [ ] . recently, mendez et al. demonstrated that sox cleaves its target mrna without other factors by in vitro cleavage assay [ ] . moreover, muller et al. revealed that some mrnas that 'overrided' sox were also degraded, suggesting multiple viral endonucleases' existence [ ] . bglf in epsteinebarr virus (ebv) plays a role in processing non-linear or branched viral dna intermediates in order to promote the production of mature packaged unitlength linear progeny viral dna molecules [ ] , serves as an endoribonuclease of host mrna after infection [ ] . similar to bglf , musox in murine herpesvirus (mhv ), which is also alkaline exonuclease homolog, a member of the pd(d/e)xk restriction endonuclease superfamily have been demonstrated to induce host mrna decay after infection via endonucleolytic attack [ ] . nsp , a protein with no known similarity to cellular or viral nucleases from severe acute respiratory syndrome (sars) coronavirus (scov) belonging to betacoronavirus family, is reported to induce host mrna degradation by binding to s ribosome [ , ] . nsp also prevents sendai virus-induced endogenous ifn-b mrna accumulation. later kamitani et al. found that nsp bound to host s ribosomal subunit and inactivated the translational activity of the s subunits. meanwhile, nsp - s ribosome complex induces the modification of the region of capped mrna template and renders the template rna translationally incompetent [ ] . rna endoribonuclease designated as pa-x, which is encoded in the genome of influenza a virus (iav), stimulates the decay of cellular mrna [ ] . host rna degradation was also reported in iav-infected cells, though the sensitivity of host mrnas to iav-induced host shutoff varies. for example, host proteins that facilitate viral propagation (such as the proteins that maintain oxidative phosphorylation) are less attacked [ , ] . this suggests that like iav, virus infection in fact effectively and strictly control the total pool of mrnas in virus-infected cells, allowing selection of host mrnas that are important for virus replication to persist and be translated [ , ] . as illustrated in fig. b , poxviruses use virally encoded decapping proteins to remove the -cap of the mrna resulting in destabilization of the mrna. a typical poxvirus, vaccinia virus (vacv) encodes two decapping proteins, d and d , and these proteins likely maintain viral stage-specific protein synthesis as well as host cellular mrna turnover [ , ] . other viruses including bunyaviruses and orthomyxoviruses use cap-snatching mechanisms to remove the -cap and use the removed cap to protect viral rnas [ ] . intriguingly, prokaryotic viruses such as bacteriophages also trigger rapid host mrna degradation. as shown in fig. c , bacteriophage t srd is demonstrated to stimulate the activity of e. coli essential endoribonuclease e via binding to its n-terminal in order to induce host mrna degradation immediately after infection [ ] . though there is no homolog of srd found yet in bacteriophage t and t , host bulk mrna degradation was also observed in t -and t -infected cells [ ] . little is known in the case of other bacteria such as b. subtilis yet, but host shutoff was observed in ar phage infected b. subtilis cells [ ] . further research is expected on their mrna elimination after phage infection. influence of functional deficiency of viral factors that raises host mrna turnover on viral replication has been studied for years. functional loss of viral factors mentioned above usually is not fatal for viral growth, but several pieces of evidence have been also obtained that global host mrna turnover should be required for rapid transition of gene expression from host to viruses and efficient viral replication [ , , , ] . for instance, vhs-deficient mutant of hsv- results in low virulence and vhs plays a role in evasion from non-specific host defense mechanisms during primary infection [ , ] . in addition, matsuura's group showed that rna replication of scov was low when nsp was mutated in an in vitro rna replica system [ ] . furthermore, from our own previous work, bacteriophage t srd could stimulate the activity of host e. coli essential endonuclease e inducing bulk mrna degradation and a deletion of srd causes inefficient propagation [ ] . therefore, it is likely to be a common mechanism in both prokaryote and eukaryote that infection with viral organisms activates the host mrna degradation machinery for the shift of gene expression from host to virus and thus facilitates efficient viral propagation. how this contributes to offense immune response in mammalian cells is also an unanswered question so far. in this concise review, we mainly focus on virus-induced degradation of host rnas during host shutoff, which helps to maximize viral gene expression and thus their replication. mammalian viruses as well as bacteriophages evolved unique mechanisms to propagate efficiently during infection. host cells respond in order to create a hostile environment for viral replication, leading to the shutoff of mrna translation (protein synthesis) and the assembly of rna granules [ ] . recently, the hiv- unspliced mrna has been shown to contain n -methyladenosine (m a), allowing the recruitment of yth n methyladenosine rna binding protein (ythdf ), which is involved in mrna decay. this indicates that hiv- interacts with host mrna decay components to accomplish viral replication successfully [ ] . this finding implicates a novel mechanism involved in infection-induced host rna degradation. in addition, viruses also employ numerous strategies to regulate the gene expression of both host and themselves despite of host shutoff. hepatitis b virus (hbv) mrna interacting with host microrna- (mir ) was reported to have an indispensable role for viral replication [ , ] . the interaction between viral mrna and host microrna is also known to be able to facilitate hepatitis c virus (hcv) replication [ ] . under development of imaging technologies, converting virus-host interaction signals to imaging visualization will help decipher the underlying cellular mechanisms. singlemolecule fluorescence imaging has been reported to monitor the entry, transport and virus-host cell interaction for some viruses like polio virus (pv) and adeno-associated virus (aav) [ ] . moreover, live cell imaging technology has also been reported for mrna-protein interaction, which makes it possible to study cellular and viral gene expression directly. yin et al. has constructed red color mcherry-based trimolecular fluorescence complementation (trifc) systems, visualized influenza a viral mrna-protein interaction in living cells and helped understand more about iav mrna nuclear export mechanism [ ] . they show that adapter proteins aly and uap are able to interact with three kinds of viral mrnas while splicing factor g only interacts with one mrna [ ] . therefore, it is expectable that application of advanced live cell imaging technology will facilitate in-depth research on virus-induced host mrna degradation. in summary, comprehensive understanding of virusinduced host shutoff and its connection in the whole process of viral replication as well as its interaction with host response through multiple technologies (e.g. molecular cell biological techniques and advanced imaging methods) is crucial to struggle with viruses. this could facilitate decoding virusraised diseases and may provide new insights for both novel research tool and therapeutic strategy design for disease. a two-pronged strategy to suppress host protein synthesis by sars coronavirus nsp protein inhibition of host protein synthesis during infection of escherichia coli by bacteriophage t . ii. induction of host messenger ribonucleic acid and its exclusion from polysomes viruses: overturning rna turnover closing in on the causes of host shutoff. elife a common strategy for host rna degradation by divergent viruses bacteriophage t -induced shut-off of hostspecific translation phage-induced change in the stability of mrnas a tale of two rnas during viral infection: how viruses antagonize mrnas and small non-coding rnas in the host cell emerging roles for rna degradation in viral replication and antiviral defense interplay between viruses and host mrna degradation messenger rna degradation in bacterial cells rnase e: at the interface of bacterial rna processing and decay the bacterial enzyme rpph triggers messenger rna degradation by ' pyrophosphate removal herpes simplex virus virion host shutoff function herpes simplex virus mutants defective in the virion-associated shutoff of host polypeptide synthesis and exhibiting abnormal synthesis of alpha (immediate early) viral polypeptides the role of the ul gene of herpes simplex virus type in evasion of non-specific host defence mechanisms during primary infection the herpes simplex virus host shutoff rnase degrades cellular and viral mrnas made before infection but not viral mrna made after infection the virion host shutoff endonuclease (ul ) of herpes simplex virus interacts with the cellular cap-binding complex eif f lytic kshv infection inhibits host gene expression by accelerating global mrna turnover the exonuclease and host shutoff functions of the sox protein of kaposi's sarcoma-associated herpesvirus are genetically separable viral nucleases induce an mrna degradation-transcription feedback loop in mammalian cells site specific target binding controls rna cleavage efficiency by the kaposi's sarcomaassociated herpesvirus endonuclease sox nuclease escape elements protect messenger rna against cleavage by multiple viral endonucleases the epstein-barr virus alkaline exonuclease bglf serves pleiotropic functions in virus replication host shutoff during productive epstein-barr virus infection is mediated by bglf and may contribute to immune evasion host shutoff is a conserved phenotype of gammaherpesvirus infection and is orchestrated exclusively from the cytoplasm sars coronavirus nsp protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp -induced rna cleavage severe acute respiratory syndrome coronavirus nsp protein suppresses host gene expression by promoting host mrna degradation selective degradation of host rna polymerase ii transcripts by influenza a virus pa-x host shutoff protein a systematic view on influenza induced host shutoff. elife a cap-to-tail guide to mrna translation strategies in virus-infected cells poxvirus decapping enzymes enhance virulence by preventing the accumulation of dsrna and the induction of innate antiviral responses down regulation of gene expression by the vaccinia virus d protein rapid degradation of host mrnas by stimulation of rnase e activity by srd of bacteriophage t the genome of ar , a giant transducing bacillus phage encoding two multisubunit rna polymerases role of the virion host shutoff (vhs) of herpes simplex virus type in latency and pathogenesis severe acute respiratory syndrome coronavirus nsp facilitates efficient propagation in cells through a specific translational shutoff of host mrna who regulates whom? an overview of rna granules and viral infections interactions between the hiv- unspliced mrna and host mrna decay machineries mir- in hepatic function and liver diseases hepatitis b virus mrna-mediated mir- inhibition upregulates pttg -binding protein, which promotes hepatocellular carcinoma tumor growth and cell invasion modulation of hepatitis c virus rna abundance by a liver-specific microrna single-molecule fluorescence imaging: generating insights into molecular interactions in virology imaging of mrnaprotein interactions in live cells using novel mcherry trimolecular fluorescence complementation systems we thank dr. tetsuro yonesaki, professor emeritus of osaka university, for his valuable comments and suggestions. we thank the grant support from corbett estate fund for cancer research. key: cord- - bhiattk authors: farmaki, anna; miguel, cristina; drotarova, maria hadjielia; aleksić, ana; Časni, anita Čeh; efthymiadou, fani title: impacts of covid- on peer-to-peer accommodation platforms: host perceptions and responses date: - - journal: int j hosp manag doi: . /j.ijhm. . sha: doc_id: cord_uid: bhiattk the covid- pandemic has brought international tourism at a standstill. peer-to-peer (p p) accommodation, in particular, has been greatly affected with platforms being heavily criticised for lacking a strategic response to users’ needs. drawing from semi-structured interviews with p p accommodation hosts, this study aims to explore: a) their perceptions of the short-term impacts of the pandemic on their hosting practice, b) their responses to the pandemic and c) their perceptions of the long-term impacts of the pandemic on the p p accommodation sector. the study offers a continuum of host pandemic responses which illustrates different types of hosts in relation to their market perspective and intention to continue hosting on p p platforms. the continuum carries theoretical implications as it offers insights to academics exploring crisis impacts on p p accommodation. it is also of practical value to platforms and practitioners as it may lead to improved crisis management strategies. Τhe tourism and hospitality industry is no stranger to pandemics, having been impacted by sars in the early s and mers more recently (jamal and budke, ) . nonetheless, the emergence and rapid spread of a new coronavirus has had unprecedented effects on the global tourism and hospitality market. initially detected in the chinese city of wuhan in early december (yang et al., ) , covid- spread rapidly and by may there were over . million reported cases of infected people and reported deaths worldwide (who, ) . as a result, governments around the world imposed strict restrictions prohibiting travel while closing their borders, bringing international travel at a standstill. specifically, airlines have grounded their fleet and suspended operations (sahin, ) . likewise, the international hospitality and leisure industry has been experiencing tremendous economic problems as hotels in most countries were forced to shut down due to governments' lock-down response to the pandemic. for example, % of hotel employees have been laid off or furloughed, leading to a $ . billion loss in weekly wages and . million hospitalitysupported jobs being lost (ahla, ) . unsurprisingly, with a % reduction in revenues (oxford economics, ) , was named the worst year for hospitality in terms of occupancy rates since the great depression (cbre, ) . although crises in general have long-lasting adverse effects on travel patterns, tourist demand and destination image (chew and jahari, ; chien and law, ; corbet et al., ) , the tourism and hospitality industry has proven to be resilient in the past. indeed, in most cases destinations recover (seabra et al., ) particularly when crisis management strategies are in place (alonso-almeida and bremser, ) . for instance, after sars in china, travel bounced back relatively quickly to normal levels (dombey, ) . nonetheless, the unparalleled situation brought about by covid- has led to concerns over the future of global tourism and hospitality industry, which is one of the worst-affect industries by the pandemic (tidey, ) . specifically, scholars estimate that the effects of covid- on tourist risk perceptions and destination marketing will be long-lasting even after the pandemic is controlled (e.g. ying et al., ) , especially on the operational aspects of the industry. for instance, hospitality companies have issued announcements that their cleaning protocols will be revised. in this context, a range of practices such as the use of germ-zapping robots, the removal of breakfast buffets, a -hour gap between check-out and check-in time and even the issuance of a 'clean and safe' certificate has been suggested by hospitality associations and international hotel chains (bagnera and steward, ) . the peer-to-peer (p p) accommodation sector has attempted to follow suit, with platforms such as airbnb and booking.com responding to the effects of covid- in numerous ways. for example, the platforms have sought to establish a new, optional cleaning protocol for properties which requires a -hour waiting time between bookings as well as the use of specific cleaning products to eliminate possible coronavirus transmissions (wood, ) . in addition, the platforms have issued a refund policy which, nonetheless, angered a large majority of guests as they discovered that refunds are partial, dependent on the host and/or given in the form of travel credit (schuk, ; webster, ) . concerns from p p accommodation hosts have also been voiced as many struggled financially during the pandemic due to the loss of reservations (johnson and davis, ) . in relation to this point, hosts on p p accommodation platforms expressed that they felt largely unsupported as many local governments deemed p p accommodation as non-essential business (evans, ) , granting them no financial support and leaving the companies to foot the bill. airbnb, for instance, announced its intentions to provide more than $ million to support its host community. likewise, booking.com has asked financial aid from the dutch government to pay the salaries of its netherlands based staff (sharma, ) . even so, the platforms have been criticised heavily for demonstrating a lack of strategic thinking in response to covid- (carpenter, ) . with airbnb being forced to lay off % of its staff while observing its value depreciate from $ billion in march to $ billion in may (evans, ) and booking.com informing employees that lay-offs are probable (stevens, ) , it is not surprising that some media reports are suggesting that the pandemic might signal the end of p p accommodation. drawing from the perspectives of p p accommodation hosts, this study aims to explore indepth the perceived impacts of the covid- pandemic on host practice specifically and p p accommodation in general. more precisely, we examine p p accommodation hosts' perceptions of the short-term and long-term impacts of the pandemic as well as their associated responses. overall, the study offers several contributions. first, it advances existing knowledge on the pandemic-tourism nexus which has mostly concentrated on destination-level and sectoral-based analyses . while there are past studies examining the impacts of and responses to pandemics in hospitality (e.g. alan et al., ; henderson and ng, ) , these tend to overlook the perspectives of micro-level stakeholders. by drawing from accommodation service providers, this study thus responds to this gap in the literature. second, to the best of our knowledge, this study is the first to examine the associated impacts of the covid- pandemic on the increasingly popular p p accommodation sector (farmaki and kladou, ) . given that health, safety and cleanliness are considered key elements in hospitality decision-making (zemke et al., ) , findings from this study will therefore shed light on the ongoing discourse on p p hosts' practices which have been argued to shift to more institutionalised hospitality services (farmaki and kaniadakis, ) . third, the increase in infectious diseases across the world (jamal and budke, ) will likely preoccupy hospitality practitioners in the foreseeable future. to this end, this study contributes insights that may lead to improved health crisis management strategies in hospitality. the rest of the paper is organised as follows. first, the literature on the effects of pandemics on hospitality is reviewed before considering the impacts of the covid- pandemic on the p p accommodation sector. then, the methodology guiding this study is explained and justified before the findings are presented and discussed. last, the theoretical and practical implications emerging from this study are drawn as conclusions. the threat of pandemic emergence has increased in the st century as a result of various reasons including: the growing mobility of the population, urbanisation, the industrialisation of food production processes and the expansion of global transport networks which contributes to the transmission of pathogens (connolly et al., ; hall, ) . the outbreak of diseases like sars, mers, the ebola and zika viruses and more recently covid- stands as evidence of the growing pandemic threat. in this context, the global tourism industry has been identified as a contributor to the spread of diseases (nicolaides et al., ) as the more people travel, the more likely it is for a disease to spread internationally. on the other hand, the industry was also recognised as being highly susceptible to pandemics, incurring significant economic costs (kuo et al., ) as pandemics negatively influence j o u r n a l p r e -p r o o f tourist demand and destination perceptions (novelli et al., ) . one sector of the tourism industry that is regarded as highly impacted by pandemics is hospitality, given its vulnerability to health-related crises (mckinsey and company, c) . numerous studies examining the effects of pandemics on hospitality may be found in the literature (e.g. alan et al. ; chen et al., ; chien and law, ; kim et al., ; wu et al., ) . this pool of work illustrates the adverse effect of a pandemic on hotel occupancy rates, revenues and stock performance, reminding us of the importance of sanitation and hygiene within the sector (naumov et al., ) . evidently, pertinent studies highlight the significance of response mechanisms of hospitality businesses to epidemics (henderson and ng, ; kim et al., ) whilst identifying customers' self-protective behaviours as equally important (chuo, ) . in this context, hospitality businesses' reactions to pandemics through risk assessment, formal planning and integrated, contingency plans have been noted as particularly critical for the recovery of the sector (jayawardena et al., ) . likewise, support from national governments through assistance programmes was identified as a key factor contributing to sector recovery (chien and law, ) . equally, human resource strategies were acknowledged as important in crisis management efforts during pandemics, with leung and lam ( ) suggesting unpaid leave and involuntary separation as common immediate solutions by hotels. in this context, pandemic-related studies indicate that travel behaviour tends to return to normal as soon as the situation is controlled by authorities. drawing from the sars experience, several studies point out to the risk-taking behaviours of travellers (e.g. lau et al., ) whilst reporting that travel resumes to normalcy as soon as the situation allows it (dombey, ; zeng et al., ) . while much of the previously noted pandemics were shortlived , the newly emergent covid- virus though is anticipated to have long-lasting effects on the tourism and hospitality industry (ying et al., ) . although there are instances of tourists demonstrating irresponsible behaviours during the covid- pandemic (e.g. guy, ) , in most cases travel behaviour has been greatly affected as many individuals will opt for domestic holidays in (euronews, ) . as a result of increasing fears among the general public, industry stakeholders have expressed plans to improve hygiene standards. for instance, hotel companies have announced revisions in their cleaning protocols and food and beverage offerings (bagnera and steward, ) with robotics adoption emerging as a preferred option to minimise human contact (zeng et al., ) . whilst the traditional hospitality industry seems willing and capable of adjusting its operations, concerns have been raised over the future of the p p accommodation sector and, specifically, the ability of hosts to follow suit. p p accommodation has emerged as a disruptor on the traditional hospitality sector due to their growing popularity (sigala, ) . referring to online networking platforms through which individuals can rent out for a short period of time their under-utilised property space (belk, ) , p p accommodation has grown immensely as a result of the numerous benefits it may offers to travellers and property owners. for travellers (guests), it offers a convenient, valuefor-money accommodation option (stors and kagermaier, ; tussyadiah, ) that is generally regarded as more authentic and localised than hotel stays paulauskaite et al., ) offering a 'home away from home' feeling (liang et al., ) . for property owners (hosts), p p accommodation offers opportunities for entrepreneurship (zhang et al., ) and additional income (farmaki and kaniadakis, ; guttentag, ) that improves individuals' standard of living (lutz and newlands, ) . similarly, through hosting individuals have the opportunity to engage in social interaction (moon et al., ) and share experiences, particularly when hosts rent rooms in their homes . in addition, it has been argued that through p p exchanges hosts receive gratification for providing a good hospitality service (lampinen and cheshire, ) . these economic and social benefits drive p p accommodation users to engage with platforms either as hosts or guests. in recent years changes have been observed in the p p accommodation sector as the growth of certain platforms (i.e. airbnb) and the competition among hosts has led to the adoption of professional hospitality standards (farmaki and kaniadakis, ; . the professionalisation turn in p p accommodation was also encouraged by the decision of platforms like airbnb to open up their space to professional accommodation providers such as boutique hotels and attract people who would never thought of staying in a p p accommodation before like business travellers (guttentag and smith, ) . for example, airbnb introduced new search tools for business travellers allowing more customised search results. likewise, the platform launched a 'superhost' and 'superguest' badge that resemble hotel loyalty schemes and award benefits (i.e. discounts) to dedicated users (liang et al, ) . another recent addition of the platform was 'airbnb plus' which refers to an elite selection of properties that have "exceptional hosts" and 'airbnb luxe' that includes luxury properties that come with the services of a dedicated concierge (farmaki and kaniadakis, ) . in this context, p p accommodation hosts started to offer services such as airport pick-ups, meals and concierge service that resemble those of hotels (farmaki and kaniadakis, ) in a bid to reach superhost status and improve their search results and profitability. evidently, while the provision of a clean, functionable property and quality tangibles moon et al., ) continues to be a key prerequisite for guest satisfaction (priporas et al., ) , the recent changes in the p p accommodation sector point towards increasing guest expectations (farmaki and kaniadakis, ) . several studies identify cleanliness and tidiness as key factors for p p accommodation guest satisfaction (lyu et al., ; tussyadiah and zach, ) alongside other elements such as location, amenities and facilities (cheng and jin, ) . nonetheless, there is a growing number of studies that highlight the increasing demand for host friendliness, responsiveness and hospitableness (chen and jin, ; gunter, ; xie and mao, ) in addition to hotel-like products and services. hence, what influences the guest experience and drives demand for p p accommodation is a combination of tangible elements as well as intangible practices, reflecting host attitudes and personality (sthapit and jimenez-barreto, ; zhu et al., ) that denote the quality of the overall service offered (ju et al., ) . as such, it is not surprising that a hybrid form of service is emerging in the hospitality industry combining home feeling with professional hospitality service provisions the outbreak of the covid- pandemic, nonetheless, has challenged p p accommodation host practices, revealing the sector's vulnerability to pandemics. according to industry analysts, hotels will have an advantage over p p accommodation rentals in the post-covid era primarily due to the lack of standardisation in p p accommodation host practices which, in turn, may make the public wary regarding the hygiene of their properties and the fairness of their terms (glusac, ) . although the morality of hosts in maintaining responsible behaviours and hosting practices was previously noted , it was especially highlighted during the covid- pandemic and in relation to practices such as cancellation of bookings. also, while many hosts offered their properties for free to covid- responders and health workers (gtp, ), others driven by economic motives defied lockdown laws and advertised their properties as 'covid- retreats' (criddle, ) . another hosting aspect that is impacted by the pandemic is the interaction with the guest which represents an essential element of the experience ), yet is compromised by the social distancing guidelines imposed by national governments as a measure to combat the spread of j o u r n a l p r e -p r o o f the covid- virus. indeed, the pandemic led individuals and accommodation providers to engage in protective behaviours. although hosts were urged to follow prevention measures (jamal and budke, ) , cost-benefit evaluations tamper hosts' decision to engage in protective behaviour (cahyanto et al., ) . in this context, the perceptions of hosts and their responses to the covid- pandemic emerge as significant in determining the future of the sector. given the purpose of the research, a qualitative approach to research was regarded as more appropriate. qualitative research may provide thick descriptions of people's perceptions and, hence, reveal new understandings of a phenomenon (ezzy, ) . in particular, from may to june semi-structured interviews were performed with p p accommodation hosts from the following countries: croatia, cyprus, greece and spain. located in the mediterranean basin that has been greatly affected by the covid- pandemic, these countries represent suitable contexts for examining hospitality issues as they are popular tourist destinations with an abundance of p p accommodation properties. nonetheless, each country has witnessed different experiences during the covid- pandemic and showed varying responses. for example, greece has been commended for its timely measures against the pandemic which led to a relatively low number of infected cases and deaths whereas spain witnessed a national tragedy, with covid- related deaths exceeding cases (pappas, ) . such differences in experience are likely to shape host perceptions of and reactions to the impacts of the pandemic. purposive sampling was used to select participants. purposive sampling is a sampling technique where participants are selected based on specific pre-selected criteria (etikan et al., ) . therefore, at the first instance, we selected p p accommodation hosts purposively according to the following criteria: a) participants had to be active hosts on p p accommodation platforms and b) participants had to be available and willing to participate in the study as well as be able to describe their perceptions (bernard, ) . the rationale of purposive sampling rests on the fact that the researchers, based on their a-priori theoretical understanding of the topic, assume that certain individuals may have important perspectives on the phenomenon in question (robinson, ) . in so doing, the researcher(s) from each country posted an open call on various social media platforms and p p accommodation forums inviting members to participate in the research whilst ensuring their anonymity and data confidentiality. in order to minimise self-selection bias (bethlehem, ) , the researchers tried to ensure that the sample from each country was diverse enough (ritchie et al., ) in terms of gender, age and host type. data collection came to an end when no new information was observed in the data (fusch and ness, ) and the researchers were confident that data saturation was reached (glaser and strauss, ) in accordance to the leading questions and, thus, ensuring that adequate evidence for each theme was obtained to reach conclusions (saunders et al., ) . overall, interviews were conducted with p p accommodation hosts. the profile of the participants may be seen in table . the type of property of each participant was categorised according to the p p accommodation host typology of farmaki and kaniadakis ( ) which includes: hosts sharing a room in their house, hosts with or listings renting the entire property and hosts renting an entire property that manage multiple listings (their own and of others). due to the lockdown measures imposed across europe at the time of the data collection, the interviews were conducted over skype or zoom, in accordance to the participants' date and time preference. before each interview, the researcher(s) explained to the participants the purpose of the study and the ethical implications involved and obtained their signed consent to being recorded. the interviews lasted approximately to minutes each and were conducted in the local language of the researcher(s) before being translated into english. each interview proceeded from a number of 'grand tour' questions (mccracken, ) seeking to establish the hosting profile of the participants before moving into the topic of: a) hosting motives in order to understand the drivers for engaging in p p accommodation, b) impacts of the pandemic on host practices and their related responses and c) host perceptions of the longterm impacts of the pandemic on their hosting and p p accommodation in general. table illustrates the questions asked. what are the short-term economic impacts of the pandemic on your hosting activity? how has your hosting practice and operational activity changed as a result of the pandemic? have you received petitions of cancellations of bookings asking for % refund pushed by the platforms? have you received phone calls from 'customers service' to encourage you to cancel bookings? which measures are you planning to take to ensure a good level of hygiene is provided in your property(ies)? have you received any requests or enquiries from guests asking about hygiene? have the platforms (e.g. airbnb) been supportive in terms of the impacts hosts have incurred as a result of the pandemic? did you receive any support from the government as a result of the pandemic? what do you think the long-term economic impacts of covid- will be? how do you foresee your hosting practice evolving in the near future as a result of the pandemic? do you plan to move to a long-term rental mode? in your opinion, how has the pandemic changed the p p accommodation sector? any long-term threats or even opportunities emerging from this situation? the translated transcripts were checked by each researcher for accuracy and then imported to the nvivo software to be analysed using thematic analysis (braun and clarke, ) . data analysis was conducted by two researchers who employed the three coding rounds prescribed by gioia et al. ( ) . in more detail, during the first coding round the researchers 'adhered faithfully to informant terms' (gioia et al., : ) by reading the transcripts line by line without imposing restrictions on the text to be analyzed (strauss and corbin, ) . then, the transcripts were analysed more closely with the researchers identifying key topics in a "theorydriven" manner (braun and clarke, : ) . to maximise analytical integrity and ensure the robustness of findings, each researcher undertook an initial round of open coding separately before converging the first set of findings in a process called triangulation. flick ( ) posited that investigator triangulation is an effective method to balance subjective research interpretations due to the collective comparison of coding schemes. hence, in this study researcher triangulation ensured that interviewees' perceptions of the pandemic as pertaining to their hosting practice were objectively interpreted. subsequently, a second round of coding was undertaken whereby emerging topics were grouped into interrelated themes by copying, re-organising and comparing thematic categories whilst refining the data under each theme to identify sub-categories (goulding, ) . in this way, thematic categories were expanded and clarified. last, a third round of coding was used to combine sub-categories with the themes initially identified. in this way, relationships were validated and thematic categories were refined and further developed (strauss and corbin, ) to enhance elaboration on key issues (hennink et al., ) . the following steps were undertaken to ensure reliability in data analysis. first, we ensured interpretative and evaluative rigor (kitto et al., ) was maintained. for instance, in addition to participant validation of the data collected, the transcripts were read and compared by both researchers involved in the analytical process through investigator triangulation (golafshani, ) ; thus, minimising researcher bias. second, in completing the abstraction process, we grouped concepts and/or overlapping categories according to similarities and differences between categories which were discussed between the key researchers following the separate round of coding as proposed by bryman and burgess ( ) . taking thomas and harden's approach ( ) , as part of thematic synthesis the independent researcher identification of themes was followed by discussion among the researchers of overlaps and divergencies until agreement was reached. in so doing, the researchers followed a thorough process of recordkeeping to maximise the consistency of the data interpretation and to clearly demonstrate the decision trail of grouped concepts (noble and smith, ) ; thus maximising transparency. also, we ensured the coding process reflected the richness of the data collected (moretti et al., ) ; as such, we included thick verbatim descriptions of the interviewees' accounts to support key findings. at the first instance, we asked participants to describe the reasons that led them to host on p p accommodation platforms. understanding host motives was important not only to set the background and identify differences between hosts but also to gain a better insight of their perceptions of the pandemic impacts and associated responses. indeed, posited that p p accommodation host behaviours are likely to be influenced by hosting motives. some participants argued that they host for the social benefits (farmaki and stergiou, ) emerging from the interaction with the guest and the enjoyment of hosting people in their house. for these hosts, continuous bookings are not necessarily a concern as they view hosting more as a hobby or a temporary arrangement as a result of personal life changes (i.e. children moving away from home) from which they get personal gratification. generally, though, financial gains emerged as the primary motive for hosting in line with extant literature (e.g. guttentag, ) . around a third of the participants claimed that p p accommodation was their first source of income. as the discussion progressed, it became obvious that many of those hosts emerged as 'professionals' in the p p accommodation domain (farmaki and kaniadakis, ) , often managing multiple listings that are not just their own but also the properties of others. for these hosts, p p accommodation has proven to be a lucrative employment option (zhang et al., ) that is simultaneously flexible. for example, a participant argued that managing p p accommodation properties offers her a good work-life balance, stating that "this job does not distract me from my baby" [p , cyprus] . in their majority, 'professional' hosts seem to have started hosting using a or personal properties and then used their profits to acquire additional ones to expand their business. within this type of hosts, we also identified participants that were previously involved in long-term renting; yet, they decided to switch to short-term rentals via p p accommodation platforms as their popularity grew, allowing them to earn more money. a few participants, though, stated that they continue to manage long-term rentals alongside p p accommodation properties "depending on the type and location of the property and demand" [p , greece]. in addition, for some 'professional' hosts, the transition to p p accommodation came about as a result of negative experiences with tenants. in the words of a participant: "[(the tenant) left without telling me anything…left the apartment in very bad conditions…the deposit didn't cover all the damage. so, i thought i had to control the apartment closer" [p , spain]. further on, we asked the participants to state which platforms they use to advertise their properties, explaining which is their favourite. although several participants, especially professional hosts, seem to use various platforms (e.g. expedia, lastminute, splendia, homeaway, mrbnb, wimdu and other local ones) for greater exposure, airbnb and booking.com emerge as the most popular. some participants seemed to prefer booking.com as they claim it offers more reservations or a higher quality clientele; nonetheless, the majority identified airbnb as their favourite, citing numerous reasons for their choice. for instance, participants stated that airbnb is more user friendly, popular and flexible to use whilst commands lower commission fees than booking.com. participants also added that airbnb is more "pro-people" [p , spain] and "host-oriented" [p , croatia] as it allows hosts to review guests as well while laying down their own house rules. regardless, hosts on both airbnb and booking.com commented on the ease of using the features of platforms to synchronise calendars across platforms, which also explains why most hosts do not use a channel management app. following, we asked participants to elaborate on the impacts of the covid- pandemic on their hosting practice. unsurprisingly, all the participants stated that they have been directly and negatively affected by the measures undertaken by governments to control the spread of the pandemic. indeed, many participants are based in tourist destinations, with a large proportion of their bookings coming from foreigners who at the time of the pandemic were unable to fly (sahin, ) . specifically, hosts argued that they not only saw their bookings cancelled but also requests to book their properties ceased, illustrating similar pandemic effects as in mainstream hospitality (e.g. chien and law, ; kim et al., ; wu et al., ) . with the exception of a few participants who said that they had booking requests from locals who wished to stay in their properties (i.e. healthcare workers distancing from their families, people stranded in the country), hosting for most was basically put on hold. in fact, some participants claimed to have closed their property on the platforms to ensure it is not available for bookings during the pandemic, fearing contagion or platform collapse. the following extracts reflect such sentiments: "if i host someone in my apartment who is not very careful, i can get the virus from this person. the host is always risking to get the virus by hosting" [p , spain] . "my parents live downstairs, they are old and vulnerable. also, i see there are problems with airbnb as it has fired employees…so i changed the limit of the money that airbnb can transfer as i was afraid the platform would collapse" [p , greece]. even so, 'professional' hosts seemed to have experienced the greatest impact as hosting represents their main source of income. while hosts in general expressed difficulty in paying their mortgages/rents and covering maintenance costs as a result of the loss of bookings, 'professional' hosts in particular found themselves in a dire economic situation. for example, participants expressed difficulty in "paying rent to property owners" [p , cyprus] or even covering the salaries of employees such as cleaners, indicating that the pandemic has had an effect on other actors as well. as a participant put it: "i have no job and i cannot pay the lady who is cleaning our properties. i feel very sad about this situation…the unemployment office pays only about % of your original salary…if my husband doesn't work, we are near to bankruptcy…" [p , cyprus] . in order to try to adapt their hosting activity to the pandemic, participants reported different strategies. for example, to boost demand they claimed that they will "lower prices...to attract new guests this year" [p , croatia] although a few participants said they might "raise prices…to recover the lost profit" [p , spain] . other practices participants are contemplating on adopting include: targeting domestic tourists, introducing self-check in to minimise human context, offering antiseptic gels to guests, disinfect the apartments, invest in buying ozone machines for better cleaning and allow hours between bookings. these practices seem to be in line with those suggested within the mainstream hospitality industry (bagnera and steward, ) . in this context, some participants commented on how they previously maintained "high levels of hygiene" [p , croatia] with some, though, acknowledging that such practices may not have been adopted by everyone and even suggesting "some kind of certification of cleaning so that it is not done by individuals or the hosts themselves" [p , greece]. when asked if they thought the platforms were supportive of hosts during the pandemic, the majority of participants answered negatively. as a participant put it, "airbnb returned the full amount to all users of its services without asking the host" [p , croatia] . other participants agreed, commenting how platforms "reacted spontaneously" [p , croatia] as the situation was new to them, leading to host resentment (johnson and davis, ) . more critical participants argued that "the platforms are always pro-guests" [p , spain], with some participants highlighting that airbnb especially was never supportive to hosts as their main strategy is "to grow its clientele" [p , greece] . this argument is in line with past studies claiming that the airbnb is becoming guest-oriented (farmaki and kaniadakis, ) . the major controversy was related to the fact that hosts, who had a strict cancellation policy on airbnb or non-refundable in other platforms like booking and usually receive % of the amount of the booking in case of cancellation, were forced to provide a % refund. as some hosts explained, this was perceived as a betrayal of the platform since they had compromised with less bookings due to that policy to secure some funds. for example: "airbnb cancelled the bookings on my behalf. airbnb didn't respect the hosts cancellation policies. there was a kind of contract between the platforms and the hosts where each host could choose the type of cancellation policy: strict, moderated or flexible. obviously each cancellation policy has pros and cons. for instance, if you choose a strict cancellation policy, you may not have so much demand, compared to a more open cancellation policy, but you secure part of the payment in case of cancellation" [p , spain]. nonetheless, some participants felt that the platforms were communicative and did their best given the unprecedented situation. demonstrating a more empathetic stance, a participant stated that the platforms "informed us in a timely manner of the situation affecting us, but neither could perform miracles" [p , croatia] . others claimed they cancelled the bookings because they put themselves in guests' shoes. "i would do the same [as a guest]. i'd ask for a refund, it is a superior force [pandemic] i did not expect what airbnb would say, it is an issue of being humane" said a participant [p , greece]. another host explained how he appreciates that airbnb launched a $ million fund aimed to help hosts who had strict cancellation policies while pointing that booking did not offer any help: "airbnb has prepared $ million in support for paying all people % of what they should have received as the cancellation fee. to put it simply, if you had a strict policy that pays you % of the total price in case of cancellation, you get % of that %, which is ultimately . % of the total price. although it is only a tenth of the total price, airbnb's consideration for the hosts was appreciated. i point out that airbnb takes only % commission from the hosts and booking.com that did not help the hosts in any way takes %" [p , croatia] . in relation to this point, participants highlighted the positive outcome of receiving at least part of the booking with some going further and suggesting amendments in platform policies such as the issuance of "a travel insurance in case of cancelling bookings" [p , cyprus]. one j o u r n a l p r e -p r o o f host explained how he reacted very quickly and cancelled all his bookings, which then prevented him for getting the support: "i did a small mistake because i cancelled my bookings and return all money back to my guests at the very beginning. so, i am not allowed to get any refund out of these. because, if my guests would cancel their stay and not me, then i would get some money back, but i did not know about this" [p , cyprus]. the way airbnb announced the 'help scheme for hosts' was deemed misleading by some hosts. the information was not very clear and many hosts believed the help was for all hosts and not only for the hosts with strict cancellation policies. also, hosts complained about the lack of information about the 'superhost help scheme' aimed at superhosts who only have one property and depend % on the earnings from that property. some superhosts who fit the requirements called airbnb to request information and they were told they would be contacted by airbnb but never received any communication. as a host put it, "airbnb lied to us, they told us they were going to help us but they didn't" [p , spain]. in addition, some participants pointed to platforms' indirect 'manipulation' of the situation commenting that they "contacted the guest to suggest to cancel the booking under the covid- protocol where the set cancellation policy is not applicable and the guest could receive % refund" [p , spain] whilst "keeping their fee" [p , spain] . such arguments confirm p p accommodation platforms' pro-guest mentality, as previously highlighted by farmaki and kaniadakis ( ) . unsurprisingly, many hosts expressed feeling marginalised from the platforms, suggesting that the platforms "don't treat hosts as partners" [p , cyprus]. as such, hosts commented on how there was poor communication with the platforms as in some cases hosts had made their own arrangements with guests; thus, confirming reports of p p accommodation platforms' lack of strategic thinking in the pandemic (carpenter, ) and support past studies that emphasise contingency plans of hospitality companies as key for their recovery (jayawardena et al., ) . in addition to refunds, participants stated that platforms promised to offer guests a change of dates in bookings or travel vouchers, which came after hosts expressed dissatisfaction with the refund policy (schuk, ; webster, ): "booking.com is starting to offer a change of dates or a voucher to travel in the future. but at the beginning they were just refunding everyone % because of force majeure" [p , spain]. nonetheless, as participants explained, in many cases these alternatives were not preferred by guests, who usually preferred the % refund. participants were also equally critical of governments, citing lack of support to hosts despite their important role in assisting the hospitality sector to recover (chien and law, ) . although 'professional' hosts claim to have received some money from government as part of their subsidy to companies, many participants highlighted the unregulated context in which p p accommodation operates as limiting the potentiality of compensation from governments. in the words of a participant, "i do not think that the government would like to give to hosts any help, since some policy makers believe that they are cheating and not paying their taxes to the government" [p , cyprus] . in fact, several participants said that they don't expect monetary assistance from the government. consequently, we diverted our attention to hosts' long-term perspectives and intentions following the pandemic. in relation to this point, participants expressed varying views leading j o u r n a l p r e -p r o o f us to categorise them accordingly. specifically, as analysis progressed, we identified two main types of hosts in terms of their perspectives: the 'optimistic hosts' and 'pessimistic hosts' which we were able to further categorise according to their decision to exit the platforms or not. overall, five types of hosts were identified and categorised on a continuum (figure ) according to their long-term perspective (i.e. decision to continue hosting on p p accommodation platforms) and level of practice adjustment. <insert figure about here> . . pessimistic hosts on the right-hand side of the continuum, 'pessimistic hosts' stated that they intent to either "sell the property given the situation" [p , spain], "give up renting altogether" [p , croatia] or "switch to long-term renting" [p , cyprus] . for these hosts, the pandemic has exposed the vulnerable aspects of the p p accommodation sector, threatening its existence as "the system has become weak" [p , greece] . such arguments support previous research findings which postulate that the power dynamics in p p accommodation, fostered by platforms' favouring approach to guests, is driving hosts out of the platforms (farmaki and kaniadakis, ; . pessimistic hosts also claimed that tourists would choose hotels over p p accommodation in the current situation, for example: "tourists will probably choose hotels rather than private accommodation because hotels have some standards and for private accommodation there is always a surprise factor" [p , cyprus]. we also identified 'cautious hosts' who appear to contemplate on shifting or returning to longterm rental, albeit temporarily as a reaction to the current situation and until things improve. "(will move to long-term rental) at least for or years. we need to make some money, so the best solution is long-term accommodation" said a participant [p , cyprus] . likewise, some 'cautious hosts' argued that to maximise their feeling of security, they'll opt to rent both shortterm and long-term type of properties. in the words of a participant, "we have created a website where we are going to mix long-term and short-term rental" [p , spain]. in the middle of the continuum, we placed hosts who claimed that it is not possible to foresee the future of the sector due to the unknown outcome of the pandemic. as such, these hosts which we labelled 'ambivalent hosts' stated that they will wait to see "how the situation unfolds" [p , cyprus] before making any decisions to continue hosting on p p accommodation platforms or exit. as a participant explained, "i am waiting for the airports to open first and see what type of people will come…" [p , greece]. moving towards the left-hand side of the continuum, there were 'indolent' hosts who stated that they will continue renting through p p accommodation platforms due to a number of reasons. for example, several participants argued that they cannot switch to long-term renting as their properties are in touristic locations and thus "not suitable for long-term renting" [p , croatia] . other participants explained that renting through p p accommodation platforms is preferable as they can achieve higher profits than long-term renting or, in many cases, longterm renting is not possible as the rented property is attached or within the ground of the host's house. another key factor that seems to deter hosts from renting long-term is that "long-term renting has many problems" [p , greece] including damages to the property that the owner is not being compensated for, unpaid rents and in some legal contexts inability to evict unruly tenants. even so, these hosts seemed unwilling to change their hosting practices as a result of increased hygiene and safety risks. in the words of a participant, "i don't intent to change anything. i prefer to have it closed than go into the mentality of being a labourer of the property" [p , greece]. on the other end of the continuum, there were participants who emerged as 'optimistic hosts' as they believed that the pandemic has brought opportunities that will positively transform the p p accommodation sector. specifically, participants claimed that the pandemic will reinforce demand for p p accommodation as hotels run greater risk of infection and, thus, people will prefer to stay in more isolating types of accommodation with less personal contact. such statements counteract initial estimations by media reports that the p p accommodation sector will be negatively affected by the pandemic due to the lack of standardisation in host practices that reinforces concerns over health and safety criteria (e.g. glusac, ) . in the words of a participant, "there is going to be a complete shift in the way we travel. massive tourist trip, trips for just a weekend, low cost travel, all of that is going to change" [p , spain] . within this context, participants argued that "there will be a cleaning up" [p , spain] in the sector as the pandemic is removing the opportunists. as a participant summed it up "the image that 'anyone rents a property in airbnb' will change" [p , greece] . in relation to this point, some participants commented on how the transition of some hosts to long-term renting will be good for the society as long-term rent will decrease. generally, 'optimistic hosts' seem to plan their future hosting practices accordingly and expressed their intentions of making adjustments. for example: "i have always left one day in between bookings. now i may even leave days" [p , spain]. overall, three conclusions are derived from this study. first, the pandemic's effects have been equally great on p p accommodation as on mainstream hospitality providers (e.g. wu et al., ) , with hosts experiencing both losses of revenue and future booking requests. in the case of professional hosts, the effects of the pandemic extended into inability to pay for salaried staff (i.e. cleaners) and other company-related expenses. second, with economic benefits driving individuals to host on p p accommodation platforms (guttentag, ) , it is not surprising that hosts are contemplating to continue hosting on the platforms in hope that the situation will improve and they will resume making profits. nonetheless, our study identified hosts that have decided to exit the platforms and recover by turning to long-term renting. the decision to exit the platforms seems to have been encouraged by hosts' disappointment over the minimal support received from platforms which, according to our findings, are exhibiting a "pro-guest" mentality that victimises hosts. for instance, hosts expressed frustration over the way the platforms handled the pandemic by encouraging guests to ask for full refunds. this leads to the third finding of our study which reveals a variety in host responses with regard to the pandemic's impacts. specifically, we depict the different types of hosts on a continuum (figure ) in accordance to their long-term market perspective and hosting practice adjustment depending on their decision to stay or exit p p accommodation platforms. in light of these conclusions, this study carries both theoretical and practical implications. although the relationship between pandemics and hospitality has been previously investigated (e.g. chen et al., ; henderson and ng, ) , this study represents the first attempt to examine the impacts and associated responses of pandemics in a p p accommodation context. as such, the study sheds light on p p host practices during a pandemic by exposing the factors driving host decision-making, which does not revolve around economic benefit solely but encompasses personal aspects including ability to respond to health and safety expectations. specifically, the study advances theoretical understanding of the pandemic-hospitality nexus which has insofar focused on destination-level and sectoral-based analyses by investigating micro-level stakeholders' perspectives. in so doing, the study reveals a variance in perceptions and responses of hosts to pandemics which led us to categorise them along a continuum in terms of their market perspective and intention to continue hosting on p p platforms. on the one end of the continuum, there are 'optimistic hosts' that will continue hosting on platforms whilst altering their practices to comply to the emerging need for better health and safety standards. these hosts seem to understand that health and safety are regarded as key in hospitality provision (zemke et al., ) and are willing to adapt their strategies, contrary to 'indolent hosts' who plan to continue their hosting activities without adaptation of their practices. the sentiments of 'indolent hosts' seem to emanate from their belief that their practices are adequately responsive to health standards or their decision to withstand the additional pressures of the platforms and guests on their practice (buhalis et al., ; . on the other end of the continuum, there are 'pessimistic hosts' who intend to cease p p hosting altogether and turn to long-term renting as well as 'cautious hosts' who prefer to maintain both short-term and long-term rentals for greater safety. additionally, we identified hosts that were ambivalent towards their future responses to the pandemic, preferring to see how the situation will unfold before making a decision. the figure can serve as the basis for further investigation into the effects of pandemics on p p accommodation users, primarily by illustrating the need to acknowledge existing variance in service providers' perceptions and responses to crises. correspondingly, the figure may enable researchers to identify specific behaviours and, thus, understand influencing factors and relationships between actors in p p accommodation in order to articulate more targeted questions and designs within their research. the study also offers practical implications. for instance, our typology of host pandemic responses (figure ) can be useful to p p accommodation platforms as it may offer some indications related to the improvement of their governance. as the figure illustrates, there are several types of hosts that depict varying responses in the midst of pandemics. as such, platforms need to adopt a more targeted approach in the development of their crisis management policies and strategies as well as their overall support measures to hosts. otherwise platforms run the risk of losing members, especially individual hosts who tend to share their space and are often unable to meet the increasing needs of guests and/or even platform themselves (farmaki and kaniadakis, ) . considering that many users of p p platforms seek a sharing type of property for social reasons , such a risk might prove to be unprofitable for platforms. in this context, platforms may consider establishing travel insurance features on bookings that will safeguard hosts and/or providing a range of support measures depending on varying types of hosts. given the unregulated environment of p p accommodation which fosters the lack of governmental support towards hosts during the pandemic, it is important that platforms step up to ensure responsibility towards all of their members. in this sense, this study could also inform policymakers in order to help them design appropriate policies to regulate the p p accommodation market sector. although questions have been raised over the future of the p p accommodation sector as a result of the pandemic, the unprecedented situation revealed underlying opportunities which platforms may exploit (glusac, ) . for example, our study found that 'optimistic hosts' anticipate demand for p p accommodation to grow as they are more isolated than hotels. thus, platforms need to promote the related benefits of staying in p p accommodation opposed to traditional accommodation whilst, simultaneously, ensuring that hosts adhere to the required health and safety standards. as such, platforms need to promote a proactive evaluation process before booking in addition to post-stay reviews by, perhaps, offering specific health certifications to hosts who fulfil a set of required criteria. even though health and safety are core to the hospitality product (naumov et al., ) , cleanliness and tidiness are key factors for guest satisfaction in p p accommodation (lyu et al., ; tussyadiah and zach, ) . the covid- pandemic has highlighted the importance of such factors further, heightening them into a prerequisite determining the future of the sector. the supportive stance of platforms is of particular importance for 'ambivalent hosts' who are still indecisive of their future responses. in this context, out typology may be of use to practitioners of p p accommodation allowing them to self-identify with a specific category of host pandemic response and adopt the tactics that are most suited to their needs, preferences and capabilities. this study drew from a european context only; hence, it is advisable that future research examines hosts' perceptions and responses to pandemics within other cultural contexts. similarly, as this study focused on host perspectives it may be worth if future research considers guest views in order to identify potential gaps between guest health and safety expectations and host practices. likewise, researchers may also examine the social impacts of the pandemic on hosts sharing a room in their house as these are more likely to engage in hosting due to social motivations. researchers may also embark on a comparative investigation in terms of the crisis management strategies adopted in p p accommodation and mainstream hospitality to observe areas of convergence and divergence as well as best practices. furthermore, the views of policymakers on the impacts of the pandemic, especially in terms of long-term renting, and their related responses is another area of investigation worth considering. generally speaking, the outbreak of the covid- pandemic has had a profound effect on the global tourism and hospitality market ; nonetheless, the pandemic has opened pandora's box for p p accommodation platforms exposing the vulnerable aspects of the sector. as such, the future of the sector remains to be seen. crisis management and recovery: how restaurants in hong kong responded to sars strategic responses of the spanish hospitality sector to the financial crisis covid- 's impact on the hotel industry navigating hotel operations in times of covid- you are what you can access: sharing and collaborative consumption online qualitative data analysis i: text analysis selection bias in web surveys using thematic analysis in psychology analyzing qualitative data predicting information 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unfold to make decision key: cord- -u wjgw authors: guven-maiorov, emine; tsai, chung-jung; nussinov, ruth title: structural host-microbiota interaction networks date: - - journal: plos comput biol doi: . /journal.pcbi. sha: doc_id: cord_uid: u wjgw hundreds of different species colonize multicellular organisms making them “metaorganisms”. a growing body of data supports the role of microbiota in health and in disease. grasping the principles of host-microbiota interactions (hmis) at the molecular level is important since it may provide insights into the mechanisms of infections. the crosstalk between the host and the microbiota may help resolve puzzling questions such as how a microorganism can contribute to both health and disease. integrated superorganism networks that consider host and microbiota as a whole–may uncover their code, clarifying perhaps the most fundamental question: how they modulate immune surveillance. within this framework, structural hmi networks can uniquely identify potential microbial effectors that target distinct host nodes or interfere with endogenous host interactions, as well as how mutations on either host or microbial proteins affect the interaction. furthermore, structural hmis can help identify master host cell regulator nodes and modules whose tweaking by the microbes promote aberrant activity. collectively, these data can delineate pathogenic mechanisms and thereby help maximize beneficial therapeutics. to date, challenges in experimental techniques limit large-scale characterization of hmis. here we highlight an area in its infancy which we believe will increasingly engage the computational community: predicting interactions across kingdoms, and mapping these on the host cellular networks to figure out how commensal and pathogenic microbiota modulate the host signaling and broadly cross-species consequences. rather than existing as independent organisms, multi-cellular hosts together with their inhabiting microbial cells have been viewed as "metaorganisms" (also termed superorganisms or holobionts) [ ] . millions of commensals, symbiotic, and pathogenic microorganisms colonize our body. together, they comprise the "microbiota". microbiota are indispensable for the host, as they contribute to the functioning of essential physiological processes including immunity and metabolism. hosts co-evolved with the microbiota. while some commensals are beneficial (symbionts), others may become harmful (pathobionts) [ , ] . microbiota immune system development. the immune system recognizes antigens of microorganisms e.g. dna, rna, cell wall components, and many others, through pattern recognition receptors, such as toll-like receptors (tlrs) and downstream intracellular signaling circuitries are activated to generate immune responses [ ] . however, like self-antigens, antigens from commensal microbiota are tolerated with no consequent inflammatory responses. this makes gut microbiota accepted as "extended-self" [ ] . still, under some circumstances, commensals may act as pathogens. for example, staphylococcus aureus [ ] or candida albicans [ ] are commensals of human, but in "susceptible" hosts, they can undergo commensal-to-pathogen transition. thus, identifying microorganisms that reside in the host, and within these, those that are responsible for distinct host phenotypes, and the host pathways through which they act are significant goals in host-microbiota research. microbiota survival strategies within the host are likely to be limited. analysis of their repertoire may reveal core modules, thereby helping in classification, mechanistic elucidation and profile prediction. here we provide an overview of structural host-microbiota interaction networks from this standpoint. the host interacts with microbiota through proteins, metabolites, small molecules and nucleic acids [ , ] . the microbiota employs a range of effectors to modulate host cellular functions and immune responses. they have sophisticated relationships with the host, and network representation enables an effective visualization of these relationships [ ] . most proteins of bacterial and eukaryotic pathogens are not accessible to bind to host proteins; but some of their proteins either bind to host surface receptors [ ] or enter the host cell and interact with host cytoplasmic proteins. various bacterial species have a secretion system-a syringe-like apparatus-through which they inject the bacterial effectors directly into the host cell cytoplasm [ ] . via hmis, they specifically hone in on key pathways, alter host physiological signaling, evade the host immune system, modify the cytoskeletal organization [ , ] , alter membrane and vesicular trafficking [ , , ] , promote pathogen entry into the host, shift the cell cycle [ , ] , and modulate apoptosis [ ] . all are aimed to ensure their survival and replication within the host. host signaling pathways that are targeted by microbiota and turned on or off may change the cell fate. unraveling the hmis for both commensals and pathogens can elucidate how they repurpose the host signaling pathways and help develop new therapeutic approaches. hmis have complex and dynamic profiles. studies often focus on individual protein interactions and try to explain the pathogenicity of a microorganism with a single interaction. however, considering host-microbiota interactions one-at-a-time may not reflect the virulence scheme [ ] . for instance, replication of vaccinia virus necessitates the establishment of a complex protein interaction network [ ] and hence focusing on only one hmi is incomplete and may be misleading. at any given time, hundreds of different species reside in the gut. different microbial compositions and hence effector protein combinations from these microbial species may have additive (cross-activation) or subtractive (cross-inhibition) [ ] impacts on the host pathways, which lead to signal amplification or inhibition, respectively (fig ) . since numerous bacteria will be sensed by the host immune system at any given time, more than one signaling cascade will be active in a cell. communication and crosstalk among active, or active and inhibited, pathways determine the ultimate cellular outcome [ ] : to survive, die, or elicit immune responses. the combinatorial ramifications of all active (or suppressed) host pathways and hmis will be integrated to shape the type and magnitude of the response, and thus the cell state. to tackle the pathogenicity challenge, it is reasonable to concomitantly consider all host pathways and hmis. the transkingdom (metaorganism) network analysis is a robust research framework that considers host and microbiota as a whole [ ] . systems biology approaches that integrate the hmis with host endogenous protein interaction networks reveal the systematic trends in virulence strategies of pathogens. here we ask how interspecies (superorganism) networks can facilitate the understanding of the role of microbiota in disease and health. we focus on host-microbiota protein interaction networks since many bacteria or virus-induced pathological processes require physical interactions of host and microbial proteins [ ] . the availability of genome-wide high throughput omics data makes it possible to associate microbiota with certain host phenotypes at multiple levels and construct host-pathogen interaction networks at the transcriptome [ ], proteome combinatorial effects of microbial effectors and the active host pathways determine the cell response. (a) composition has certain microorganisms that secrete effector protein combination . these effectors activate pathway in the host, which produces pro-inflammatory cytokines. (b) composition secretes effector combination and activates pathway in addition to pathway . additive effects of these two pathways amplifies the signal and promotes inflammation (cross-activation). (c) microbial composition utilize effector combination to activate both pathway and , which have opposing outcomes. subtractive effects of these pathways result in no inflammation (cross-inhibition). https://doi.org/ . /journal.pcbi. .g [ ], and metabolome levels [ ] . steps toward the construction of host-microbiota networks of gene [ ] , mrna [ ], protein-protein interaction (ppi) [ ] [ ] [ ] [ ] , and metabolic networks [ ] have already been taken. within this framework we highlight molecular mimicry, a common strategy that microorganisms exploit to bind to host proteins and perturb its physiological signaling. mimicry of interactions of critical regulatory nodes in core network modules in the immune system, may be a major way through which pathogens adversely subvert-and commensal microbiota may beneficially modulate-the host cell. microbiota developed several strategies to interact with host proteins and modulate its pathways. one efficient way is molecular mimicry, which has been extensively reviewed in our recent study [ ] . molecular mimicry can take place at four levels: mimicking (i) both sequence and d structure of a protein, (ii) only structure without sequence similarity, (iii) sequence of a short motif-motif mimicry, and (iv) structure of a binding surface without sequence similarity-interface mimicry. interface mimicry (protein binding surface similarity) seems to be the most common type of molecular mimicry. global structural similarity is much rarer than interface similarity both within and across species. thus, employing interface mimicry instead of full-length sequence or structural homology allows microbes to target more host proteins. molecular mimicry follows the principle suggested over two decades ago that proteins with different global structures can interact in similar ways [ ] [ ] [ ] . interface mimicry is frequently observed within intra-[ - ] and inter-species [ , ] (fig ) (intra-species interface mimicry: distinct proteins from the same species having the same/similar interfaces; inter-species interface mimicry: proteins from different species hijack the same interface architectures). interface similarity allows proteins to compete to bind to a shared target. if an interface is formed between proteins from the same species, it is an 'endogenous interface'. if it is formed by proteins from two different species, it is an 'exogenous interface' [ , ] . endogenous (intra-species) interfaces mimic each other [ ] [ ] [ ] , and exogenous (inter-species) interfaces mimic endogenous interfaces (fig ) [ , ]. by mimicking endogenous interfaces, exogenous interfaces enable pathogenic proteins to compete with their host counterparts and hence rewire host signaling pathways for their own advantage [ ] . they can either inhibit or activate a host pathway. for example, the helicobacter pylori secreted protein caga interacts with human tumor suppressor tp bp , inhibits apoptosis and allows survival of infected host cells [ ] . however, map protein of e. coli and sope protein of salmonella bacteria bind and activate human cdc , a rho gtpase, and trigger actin reorganization in the host cell, facilitating bacterial entry into the host [ ]. one of the most significant pattern recognition receptor families in the innate immune system is the tlr family. its members detect diverse bacterial compounds, like peptidoglycan, lipopolysaccharide, and nucleic acids of bacteria and viruses. they induce pro-inflammatory or anti-viral responses. once activated, they recruit other tir-containing proteins such as mal and myd or tram and trif through their cytoplasmic tir domains, forming the myd -and trif-dependent tir domain signalosomes, respectively [ ]. myd also assembles into a myddosome structure through its death domain together with irak and irak / death domains. the myddosome then recruits e ubiquitin ligases-either traf or traf -to catalyze the addition of k -linked ubiquitin chains to themselves, which serve as a docking platform for other proteins to bind, such as tak . subsequently, nf-κb and mapk pathways are activated. in the nf-κb pathway, tak phosphorylates and activates ikk. activated ikk in turn phosphorylates iκb, which is the inhibitor of nf-κb. phosphorylated iκb is then ubiquitylated by other e ubiquitin ligases (k -linked ubiquitin chain) and targeted for proteosomal degradation. this liberates the p subunit of nf-κb to translocate to nucleus and initiate transcription. in the mapk pathway, tak serves as a map k that activates erk / , p and jnk pathways. the trif-dependent downstream path of tlrs recruits traf and leads to activation of interferon regulatory factors (irfs) and production of key antiviral cytokines, interferons (ifns). the tlr pathway is regulated by several endogenous negative regulators to prevent excess inflammation [ ] . since this is one of the major immune pathways, its signaling is targeted by diverse microorganisms at various steps (fig ) , compete with endogenous tir-containing proteins and interfere with the assembly of the tir-domain signalosome and prevent downstream signaling. since these microbial proteins do not enzymatically modify the endogenous proteins, elucidation of their inhibition mechanism requires structural information. the availability of the structures of their complexes with the orchestrators of the tlr pathway can clarify how they inhibit downstream signaling. microbial proteases prevent both tlr-induced mapk and nf-κb signaling and lead to proteosomal degradation of the key orchestrators in these pathways: nled of e. coli cleaves jnk and p , inhibiting mapk pathway; and nlec cleaves p , inhibiting nf-κb [ ] . there are also bacterial acetyltransferases ( [ , ] inhibit this protein to limit ifn production [ ] . here, we listed only a couple of microbial proteins targeting tlr pathway as examples. there are many others. the tlr pathway does not constitute the whole innate immune system; other immune pathways also need to be considered as well as how these microbial proteins affect them as a whole. this can help foreseeing what kind of responses the coordinated actions of these pathways together with tlrs would generate. most cellular processes are elicited by proteins and their interactions. graph representations of ppi networks, where proteins are the nodes and their interactions are edges, are helpful for delineating the global behavior of the network. topological features of networks, such as degree (number of edges), betweenness-centrality (how a node affects the communication between two nodes), lethality-centrality, hubs (proteins with high node-degree, i.e. several a, b, c, d are host proteins and p is pathogenic protein. protein a has two interfaces: through blue interface it binds to b and through grey interface it binds to c and d. c and d proteins employ similar interfaces to bind to a. so, endogenous interfaces mimic each other. pathogenic protein p has similar interface as b and competes to bind to the blue interface on a. in this case, an exogenous interface mimics an endogenous interface. (b) the f l protein of variola virus interacts with human bid protein ( ajj:ab.pdb) and inhibits apoptosis in the host cell by hijacking the interface between human bid-bclxl ( qve:ab.pdb): an exogenous interface mimicking an endogenous one. human mcl protein binds to human bid ( c f:ab.pdb) in a very similar fashion that bclxl does: endogenous interfaces mimicking each other. https://doi.org/ . /journal.pcbi. .g interaction partners), non-hubs (with only a few partners), and bottlenecks (nodes with high betweenness-centrality) help characterization of the importance of the nodes, i.e. the contribution of the node to network integrity [ , ] . early on, hubs were classified as either party or date hubs. while party hubs interact with many partners at the same time since they use distinct interfaces, date hubs interact with their partners one at a time due to their overlapping interfaces. to infer whether a hub is party or date hub, structural information (interface residues) [ ] or gene expression data (co-expressed proteins have higher chances of interacting with each other) [ ] were used. later on, this definition was questioned. among the reasons were the many examples where a protein node can serve concomitantly as a party and date hub. large assemblies typically fall into this category. biological networks are often scale-free, with many non-hubs and fewer hubs [ , ] . not all nodes have the same effect on the network: random node attacks do not harm the network as much as removing hubs from scale-free networks [ ] . degree and betweenness-centrality are measures of the contribution of nodes to network integrity. there are also "essential" nodes, knock-out of which leads to lethality: a feature also known as "lethality-centrality". attack of a hub by microbiota is likely to influence the cell, either resulting in lethality, or in beneficial modulation. thus, integrated superorganism interaction networks may suggest candidate host and microbial node targets. structural interspecies networks and their topological features can shed light on how microbiota alter the host signaling and what will the outcome in different settings be. available hmi networks demonstrate that different bacteria often hijack the same host pathway in distinct ways [ ] , like the tlr pathway subversion by numerous microbial species (fig ) . however, importantly, the same host pathway is often targeted at several nodes, which was suggested to guarantee modulation of cellular function [ ] . although there are a number of examples of constructed networks of host-pathogen superorganism interactions [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , there are many fewer attempts of integrating d structural data with the hmi networks [ ] . traditional network representation has low resolution, missing important details. however, structural interaction networks provide a higher resolution with mechanistic insights. they can decipher and resolve those that are not obvious in binary interaction networks [ ] . the potential of structural networks in unraveling signaling pathways was demonstrated earlier [ , , , ] . they are essential to fully grasp the mechanisms exerted by pathogens to divert the host cell signaling and attenuate immune responses. fig displays an example of a structural hmi network, showing how host ppis can be affected by hmis. structures can detail which endogenous host ppis are disrupted by the hmis, possible consequences of mutations on either host proteins or pathogenic proteins, and whether variants of a virulence factor in different strains of the same species have distinct hmis. for instance, the pro- residue on hiv accessory protein vpr is at the interface with human cypa and its mutation to alanine abrogates the interaction [ ] . the structure of the cypa-vpr complex shows that pro- is at the interface. if the structure of the vpr-cypa complex was unknown, it would have been difficult to understand why, or how, this mutation disrupts the ppi. previously built structural hmi networks demonstrated that endogenous interfaces that are hijacked by pathogens are involved in multiple transient interactions [ , ] . these endogenous interfaces exhibit 'date-like' features, i.e. they are involved in interactions with several endogenous proteins at different times [ , ] . hub and bottleneck proteins at the crossroads of several host pathways were suggested to be the major targets of viral and bacterial proteins [ , ] and interface mimics allow transient interactions with the hub [ ] . this allows them to interfere with multiple endogenous ppis. it was proposed that microorganisms causing acute infections, which are dramatic for the host, are likely to interfere with the hubs, whereas others that lead to persistent infections tend to target non-hubs [ ] . during acute infection, pathogens replicate very quickly and are transmitted to new hosts. however, during chronic infections, they adapt to the host environment, which allows them to reside there for a long period of time. thus, how microbiota target certain proteins and pathways at the molecular level is of paramount importance. detecting the hmis, mapping them onto networks and determining their d structures as a complex are the major steps to construct structural hmi networks. despite the progress in experimental techniques, it is still challenging to determine structures of ppi complexes, particularly hmis. since large-scale experimental characterization of host-pathogen ppis is difficult, time consuming, and costly, experimentally verified hmi data is scarce. it is important to note that available endogenous protein structures are biased towards permanent, rather than transient interactions. if majority of the hmis are transient, this presents another hurdle since they will be under-represented in the structural space. several hmi databases have been developed, such as phisto [ ] , hpidb [ ] , proteopathogen [ ] , patric [ ] , phi-base [ ] , phidias [ ] , hopaci-db [ ] , virhostnet [ ] , virbase [ ] , virusmentha [ ] , hcvpro [ ] , and likely some others as well. however, these databases cover only a limited number of pathogens and their interactions. given that thousands of species residing in the host, thousands of hmis are yet to be identified. computational approaches are becoming increasingly important in prioritizing putative hmis and complementing experiments. hence, construction of comprehensive metaorganism networks and increasing the coverage of the host-microbiota interactome will still mostly rely on computational models in the near future [ ] . computational modeling of intra-species interactions is a well-established area; detection of inter-species interactions is relatively new. available computational tools to predict host-pathogen interactions have been recently reviewed by nourani et al. [ ] . current methods mostly depend on global sequence and structure homology. sequence-based methods focus only on orthologs of host proteins. however, sequence by itself is insufficient to detect the targets of pathogenic proteins because several virulence factors do not have any sequence homologs in human. for instance, the vaca protein of helicobacter pylori, the most dominant species in gastric microbiota, has a unique sequence that does not resemble any human protein [ ] . still, it alters several host pathways [ ] . with sequence-based methods, it is impossible to find hmis for vaca. as noted above, global structural mimicry is much rarer than interface mimicry. hence, utilizing interface similarity, rather than global structural similarity in a computational approach would generate a more enriched set of hmi data together with atomic details [ ] . several studies suggested that the available interface structures are diverse enough to cover most human ppis [ ] [ ] [ ] [ ] . therefore, success of template-based methods for prediction of human ppis is very high [ ] . since exogenous interfaces mimic endogenous ones, both available endogenous and exogenous interface structures can be used as templates to detect novel hmis. thanks to the rapid increase in the number of resolved d structures of human-pathogen ppis in recent years [ ] and advances in structural and computational biology, the performance of interface-based methods is expected to increase. both experimental and computational approaches have false-positives and false-negatives with varying rates depending on the approach. although the coverage of interface-based methods is higher, their false-positive rate is also higher. despite this, attempts to complete the host-microbiota interactome will improve our knowledge of microbiota and their roles in health and disease. advances in host-microbiota research will revolutionize the understanding of the connection between health and a broad range of diseases. building the rewired host-microbiota multiorganism interaction network, along with its structural details, is vital for figuring out the molecular mechanisms underlying host immune modulation by microbiota. topological features of such networks can reveal the selection of host targets by the microbiota. structural details are essential to fully grasp the mechanisms exerted by microbiota to subvert the host immunity. identification of the hmis will also help drug discovery and integrated superorganism networks would suggest how inhibition of an hmi can influence the whole system. here we highlighted the importance of building structural hmi networks. however, not only hmis are important; although to date data are scant, crosstalk among microorganisms is also emerging as critical. alterations in their population dynamics may lead to dysbiosis. signals from gut microbiota resulting from population shifts can affect profoundly several tissues, including the central nervous system. dysbiosis of microbiota is involved in several diseases, such as inflammatory bowel disease [ ] , autoimmune diseases (e.g. multiple sclerosis) [ ] , neurodegenerative diseases (e.g. parkinson's) [ ] , and cancer [ , ] . identifying bacterial effectors, or effector combinations, which are responsible for specific phenotypes, is challenging. in line with this, recently, parkinson's disease (pd) patients are found to have altered gut microbiota composition [ , ] . transplanted microbiota from pd patients, but not from healthy controls, induce motor dysfunction and trigger pd in mice. it is not clear however whether dysbiosis triggers pd or it arises as a consequence of the disease [ ] . the role of microbiota in host health and disease might be even more complex than thought: commensals once being benign can convert to disease-causing pathogens; different compositions of microbial communities trigger different phenotypes; more than one host pathway is targeted by more than one 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gut microbiota, probiotics and prebiotics colonic bacterial composition in parkinson's disease gut microbiota are related to parkinson's disease and clinical phenotype key: cord- -no mbg d authors: vandegrift, kurt j.; wale, nina; epstein, jonathan h. title: an ecological and conservation perspective on advances in the applied virology of zoonoses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: no mbg d the aim of this manuscript is to describe how modern advances in our knowledge of viruses and viral evolution can be applied to the fields of disease ecology and conservation. we review recent progress in virology and provide examples of how it is informing both empirical research in field ecology and applied conservation. we include a discussion of needed breakthroughs and ways to bridge communication gaps between the field and the lab. in an effort to foster this interdisciplinary effort, we have also included a table that lists the definitions of key terms. the importance of understanding the dynamics of zoonotic pathogens in their reservoir hosts is emphasized as a tool to both assess risk factors for spillover and to test hypotheses related to treatment and/or intervention strategies. in conclusion, we highlight the need for smart surveillance, viral discovery efforts and predictive modeling. a shift towards a predictive approach is necessary in today’s globalized society because, as the h n pandemic demonstrated, identification post-emergence is often too late to prevent global spread. integrating molecular virology and ecological techniques will allow for earlier recognition of potentially dangerous pathogens, ideally before they jump from wildlife reservoirs into human or livestock populations and cause serious public health or conservation issues. generated concern for the environment and thrust ecologists into a new political field where preserving the integrity of our global ecosystems was the priority [ ] . even so, the society for conservation biology was not established until [ ] . as a part of this transition, ecology shifted from a descriptive science to one of prediction, reflecting the hope that ecologists might mitigate changes which can have negative impacts upon the ecosystem. ecologists have branched out into the study of parasites and disease as it has become increasingly apparent that parasites are inextricably linked to the ecology of their hosts and environments, to the point where they have been a driving force in the evolution of sexual reproduction and in the shaping of biodiversity [ , ] . over the past years, disease ecologists have developed the study of parasites and pathogens in the wild. this knowledge has been synthesized into mathematical models which describe the dynamic properties of ecosystems and predict how parasites and pathogens flow through them. [ , ] . these models are becoming more commonly integrated into epidemiological studies that seek to predict outbreaks or periods of time when cross-species spillover risk is highest. parallel to this progress, the field of virology, particularly the subfields of molecular virology and viral evolution, have also been burgeoning, largely due to advances in technology that have made molecular assays and genetic sequencing more accessible to a greater number of scientists. the development of high-throughput sequencing has greatly increased our ability to efficiently detect known viruses as well as to discover new types of viruses, thereby improving our understanding of viral diversity, pathology and evolution. this increased capacity has spawned the development of new fields of study. for example, phylodynamics allows researchers to determine the origin of circulating viruses in space and time. mutations among viral strains can be used to investigate interactions among host species as well as long-range host movement via corridors and flyways. phylodynamic analyses can also inform livestock management practices, as was the case with foot and mouth disease in the united kingdom [ ] . conducting viral surveillance in animal reservoirs and invertebrate vectors can help explain circulation within host species; observed patterns of zoonotic transmission; and even allow for the prediction of periods of increased risk of zoonotic transmission (e.g., rift valley fever and rainfall [ ] ; west nile virus (wnv) and american robin (turdus turdus) migration [ ] ; as well as hantavirus in mice [ , ] ). understanding viral ecology in wildlife reservoirs and identifying high-risk human-wildlife interfaces is especially critical in the context of ever increasing globalization, whereby transportation networks facilitate rapid spread of pathogens well beyond bounds where traditional epidemiological methods can be effective [ ] [ ] [ ] . the influenza a (h n ) pandemic spread from the presumptive point of emergence in la gloria mexico to new zealand in just under a month [ ] while sars radiated from guangdong, china to different countries within several months [ ] . the negative impacts of emerging infectious diseases are not limited to humans. indeed, wildlife conservationists have documented several mass mortality events in other animal species. western lowland gorillas (gorilla gorilla gorilla) have been decimated by ebola virus [ ] and an especially virulent calicivirus, rabbit hemorrhagic disease virus, spread through both domestic and wild rabbit populations, resulting in tens of millions of deaths [ ] . in some instances the viruses have attenuated, while in others the animal populations have been brought to the brink of extinction. importantly, the risk from disease to humans and animals should not be separated. the global transportation network facilitated the introduction of infected vectors (e.g., mosquitoes) into new york and wnv caused both avian and human mortality, and this virus has subsequently spread across the united states [ ] . table . definitions of key terms used in the text. the formation of a hybrid population through the mixing of two ancestral, or long-separated, populations. a method for estimating historical population dynamics from a sample of sequences without assuming a predefined demographic model. coalescent theory a mathematical framework which describes the distribution of gene trees in populations. it provides mathematical methods for connecting demographic or ecological models with a phylogenetic tree. demography is the statistical study of populations. in the field of ecology, demography encompasses the study of the size, structure and distribution of populations, and spatial and/or temporal changes in them in response to birth, migration, aging and death. however, here we use a more rigid definition of demography-as the pattern and rate of population growth. the number of breeding individuals in an idealized population that would show the same amount of dispersion of allele frequencies under random genetic drift, or the same amount of inbreeding, as the natural population under consideration. the analogue of ne for viruses, the ‗effective number of infections' is related to the number of infected host individuals and to the number of new transmission events [ ] . a discipline which uses next-generation sequencing technologies to characterize the entirety of genomic material found in environmental samples. the molecular clock is derived from the hypothesis that sequence evolution, while random, occurs at stable rate such that the time since the divergence of two or more sequences can be estimated. recent ‗relaxed molecular clock' analysis can account for variation in the rate of sequence evolution through time or between lineages. the relations among a set of sequences showing which shares a most recent common ancestor with other sequences. the study of the principles and processes governing the geographical distribution of genealogical lineages. in the field of population genetics, population structure is defined as the absence of random mating within a population. this is the definition used here. in ecology, population structure is defined by several key parameters including number of individuals in a population, age distribution of individuals, probabilities of survival (or mortality), and rates of fecundity. a process that occurs in segmented viruses by which one or more segments ‗swap' to create a new viral genome. this drives the process of antigenic shift in influenza a viruses. the process by which new genotypes are created by the combination of distinct lineages. in sexual organisms it occurs during meiotic division, by the exchange of dna between different chromosomes or ‗crossing-over'. viral recombination occurs during viral replication and is an important factor in viral evolution (for more details see worobey and holmes, [ ] ). mutation of a virus such that it changes ‗back' to its wild-type state. the timing of an organism's schedule of reproduction and death. species with long life histories, also known as ‗k' strategists, tend to have low reproductive rates, stable populations, long generation times and long lifespans. where the parasite population is not randomly distributed among hosts, such that the variance is greater than the mean. the macroparasites in a host population are often best described by the negative binomial distribution such that a minority of hosts possess the majority of the parasites. r (basic reproduction number) in the case of viruses and other microparasites, r is the average number of secondary infections which an infection produces. as such it is a measure of parasite fitness. a host species that can independently maintain a disease and act as a source of infection to other host species. infection in reservoirs is usually more persistent and less harmful than that of other hosts. zoonotic disease a disease transmissible from animals to humans or vice versa. globalization, host ecology, host-virus dynamics, climate change, and anthropogenic landscape changes all contribute to the complexity of zoonotic viral emergence and disease, and create significant conservation and public health challenges. comprehensive and collaborative scientific approaches that transcend disciplinary boundaries are necessary to address these challenges. it is the goal of this paper to review new methods for understanding viral dynamics and illustrate how and when these techniques can be used by not only public health officials, but also disease ecologists and conservation biologists. the phylodynamic paradigm, established in [ ] , exemplifies the power of a multidisciplinary approach. it unites the ecological and evolutionary study of viruses and builds upon advances in sequencing technologies and coalescent theory, by which gene genealogies are reconstructed backward in time [ ] . the analysis of phylogenetic trees enables researchers to address many of the primary questions posed by disease ecologists (figure ). in some cases this approach can provide an estimate of a virus's basic reproductive number (r ), which is a measure of parasite fitness [ ] . phylodynamics has far-reaching applications for the control of viruses in both human and animal populations, in addition to being vital to our understanding of the interconnectedness between them. phylodynamic studies can be used to identify reservoir species as well as defining the spatial and temporal origin of emerging infectious diseases [ ] . they can also help to elucidate how these viruses spread following their emergence [ ] . firstly, chains of viral transmission can be extrapolated from the branching topology of phylogenetic trees. one example of the utility of this approach is with rabies virus. rabies causes thousands of human deaths a year in africa and has been implicated in the decline and local extinction of several populations of african wild dog (lycaon pictus) [ , ] . lembo et al. analyzed sequences of rabies virus from the serengeti, revealing that domestic dogs were the reservoir of the virus and that they had transmitted it to other resident carnivore populations on repeated occasions [ ] . this work has applicability in that it can be used to design efficient and effective vaccination strategies, both to alleviate current distress and prevent future outbreaks [ ] . secondly, by mapping the geographical origin of each sequence onto the nodes of phylogenetic trees, the geographical origin of a virus might be identified. wallace et al. ( ) [ ] used this phylogeographic approach to identify guangdong province, china as the most parsimonious origin of highly pathogenic h n strain of avian influenza and to delineate the most likely pathways of viral spread [ ] . however, a recent bayesian analysis of this data did not support the conclusion that h n had dispersed from guangdong to indonesia [ ] . instead, the bayesian analysis suggested it had spread to indonesia from guangxi or hunan in china. this example demonstrates that different statistical techniques may yield different conclusions. as yet, neither the bayesian nor the frequentist method is universally considered to be superior and there is much room for improvement as statistical phylogeography develops as a field. phylogeographic tools have also been applied by walsh et al. ( ) [ ] to locate the putative origin of the zaire strain of ebola virus. in an attempt to resolve the controversy over the time of emergence and spreading trajectory of ebola in the congo basin, they then used spatial data and two different tests for the impact of selection on the virus genome [ ] . where a virus is expanding in range, as the zaire strain of ebola virus appears to be, using a ‗landscape genetics' approach may help identify geographical barriers to viral spread and help identify vulnerable human or wildlife populations lying in the path of infection [ , ] . these phylogenies may reflect transmission chains, however sampling must be sufficient for them to do so, while recombination may obscure ‗true' relationships between viral sequences. (b) simple molecular clock theory, predicated on the neutral theory of molecular evolution [ ] assumes that mutation occurs at a constant rate over time, thus the time that has elapsed since a pair of virus strains diverged from a common ancestor may be quantified. methods that account for differences in the evolutionary rates of different strains, and for variation in these rates through time, have been recently developed [ ] . here variants represented by thick lines evolve much faster than those represented by thin lines. (c) using a phylogeographic approach, the location at which a sequence was sampled may be mapped onto the viral phylogeny and the likely spreading trajectory of the virus inferred. while parsimony approaches have been popular, powerful bayesian methods that account for uncertainty of dispersal process and historical phylogeny have been developed to reconstruct viral dispersal events [ ] . crosses on the phylogeny represent such viral dispersal events, in this example. (d) coalescent theory provides the basis for many phylodynamic approaches. here, circles on the same row represent temporally simultaneous infections. working back from sampled infections (red circles), lineages can be traced back to the most recent common ancestor (black circle) via hypothetical, unsampled ancestors (grey circles). the time it takes for sampled lineages to coalesce is dependent on a variety of variables (i.e., viral effective population size, population structure, selection, stochastic infection die-out and recombination). a variety of methods are available to test for selection and recombination. phylodynamic analyses are not without limitations. dense and representative sampling at a scale equivalent to epidemiological surveys is required to fulfill the potential of phylodynamics for understanding epidemics of rapidly-evolving viruses [ ] . the construction of phylogenetic trees from these viral sequences may be complicated by recombination and, in the case of segmented viruses, reassortment of viral genomes [ ] . as a result of these processes, the genes on a single viral sequence may have very different origins (see the discussion of the different origins of the hemagglutinin and neuraminidase segments of h n avian influenza in lemey et al. [ ] ). therefore, concatenated analysis of multiple genes may be confounded. the ability to construct phylogenies is further limited by the total viral genetic information available. genbank is a vast public database that contains records of genetic sequences; however, its usefulness is dependent upon the willingness and/or ability of individuals and organizations to submit viral sequences. governments and industrial institutions may be reluctant to report sequences of economically important viruses (i.e., avian influenza) due to the potential negative economic impacts that may ensue. although phylodynamics is currently encumbered by the aforementioned factors, there is hope for progress. advancements in coalescent theory will help us to deal with the phylogeny construction problems caused by recombination and reassortment. they will also facilitate better utilization of genomic and spatial data, provided these advancements are also accompanied by a simultaneous increase in computing power, which is also currently limiting. the utility of phylodynamics is not limited to questions of interest to virologists and disease ecologists. this approach may also inform investigations of host population biology and, in so doing, aid in the development of conservation policy. host molecular markers (e.g., microsatellites, mitochondrial dna) are used by conservation biologists and ecologists to infer population structure, historical demography and other critical features of wildlife populations [ ] and have proved particularly powerful when analyzed in combination (i.e., [ , ] ). recently, it has been demonstrated that the pathogens of host populations might also be useful to this end. research using helminths and bacteria has revealed patterns of ancient human migration and dispersal [ , ] identified ancient refuges of rodent and bird taxa [ , ] and shown that there was past contact between contemporary non-sympatric bat species [ ] . however, there have been few attempts to utilize viruses to this end (save [ , ] ). it is surprising that viruses have not been used more for the inference of host population biology since some of their characteristics make them ideal for doing so. most viruses have large population sizes and short generation times, and many replicate using a highly error-prone rna-dependent rna polymerase, causing them to accumulate many more mutations (nucleotide changes) per unit time than the host genomes [ , ] . consequently, viruses may provide information about host demographics on a shorter timescale than molecular markers of the host. one of the signature tools of phylodynamics, the bayesian skyline plot, might also be utilized to infer changes in historical population size of the host. these plots incorporate the use of a molecular clock and coalescent theory to infer historical changes in virus population sizes without assuming a predefined demographic model [ ] . it is important, however, that the timescale over which the evolutionary dynamics of the virus population can be reliably reconstructed is appropriate for the parameters of interest in the host population. unfortunately, the very characteristics that make viruses useful for estimating host population structure and demography may also impede the analyses. multiple substitutions can occur quickly in the viral genome and this will obscure the host population's actual evolutionary history. meanwhile, variations in the transmission mechanisms of viruses (vertical vs. horizontal) can alter the ability to accurately infer a virus' relationship to a host population. cross-species transmission is also problematic in that it can cause pathogen phylogenies to inaccurately reflect the history of their hosts [ ] . therefore, before the genetic information contained within a virus population can be used to infer the population structure and demography of the host, it is critical to test for congruence in the evolutionary history of the host and virus populations. this is accomplished by statistically comparing the respective phylogenies within the relevant timescale. viruses with high host specificity have a greater likelihood of exhibiting such congruence. feline immunodeficiency virus (fiv) is known for high host specificity and is, thus far, the only virus to have been used to elucidate changes in host population structure and size. from a phylogenetic analysis of fivpco, the fiv type specific to the cougar (puma concolor), biek, drummond, and poss ( ) [ ] inferred that the north american population of cougars became subdivided during the last century but subsequently expanded in both size and range. subsequently, antunes et al. ( ) [ ] used the distribution of fiv ple subtypes in the serengeti to infer that recent admixture has occurred between the region's lion (panthera leo) populations. these recent changes in felid population size and structure could not have been inferred from host genetic data. there is great potential for the further use of this technique by conservation biologists and ecologists, and for it to complement existing methods which utilize host genetic data. host genetic markers are used to define management units for conservation purposes [ ] . many ‗flagship' endangered species have long life histories [ ] , a feature that correlates with both extinction risk in certain regions [ ] and difficulty in reconstructing recent demographic history from molecular markers. because of the latter, the use of viral genetics to define management units may be an important avenue of exploration. in addition to aiding the definition of management units, viral data could be used to analyze the consequences of management activities and other environmental changes on target species. where viral genetic diversity exists in a spatially heterogeneous distribution, viral movement patterns could be used to study the migratory behavior of animals (as macroparasites have been [ , ] ). as such, researchers could monitor the use of wildlife corridors and the efficacy of control measures aimed at limiting the range of a host species [ , ] . where viruses can be readily amplified from non-invasively collected samples (see [ ] ), the above objectives could be achieved in a cost effective manner with minimal disturbance of the study species. viruses with specific transmission routes may also serve as proxies for behaviors related to transmission (i.e., sexually transmitted diseases). similarly, where cross-species transmission occurs, viruses might be indicative of types of sustained, direct contact between different, sympatric taxa which facilitate such transmission, for example predator-prey interactions [ ] . at the broader ecosystem level, inferences about long-term evolutionary processes might also be made by examining the phylogeographic structure of numerous host and virus populations of a region (see [ ] ). a major hurdle for both virologists and ecologists is defining the biodiversity of life. at present, scientists do not know the actual number of mammal species, much less the diversity of viruses they harbor [ ] . indeed, the diversity of viruses known to infect the house mouse (mus musculus), a staple in biomedical research, is not yet completely known. recent advances in genetic sequencing, including high-throughput sequencing and other -next-generation sequencing‖ techniques, as well as masstag pcr and microarray multiplex assays [ , ] have made the study of microbial diversity feasible [ ] . these technologies have facilitated a movement from classical virology, where the focus was on disease etiology, toward a broader discipline that considers the rest of the viral diversity or -the virosphere‖. metagenomic studies have used next-generation sequencing to study biodiversity in substrates such as ocean water and soil [ , ] . metagenomics has also been used to screen human and animal clinical samples in order to determine etiologic agents of disease or to describe the microbial flora normally present in a vertebrate host-in many cases the result has been the discovery both of novel pathogens and novel associations between clinical disease and agent [ ] [ ] [ ] [ ] . as technology becomes more affordable, and thus accessible, there will be increasing opportunities to ask large-scale questions such as: how does the virosphere vary across space and time? how does it vary across species? can we use this to define risk of cross-species transmission or to inform conservation efforts? and how might co-infections with these undiscovered viruses influence the dynamics of the more well known viruses? the paucity of information about viral diversity within a host poses problems for research progress. it is difficult to understand viral pathogenesis and transmission without completely understanding the dynamics of co-infections. indeed, it is currently difficult to ascribe a host's symptoms to an individual virus with any certainty because a virus' actions, and even its ability to infect the host, could be a function of another (possibly undetected) co-habitant of the host. evidence of interactions between co-infecting species has been clearly demonstrated [ , ] and it will be critically important to elucidate the interactions that occur between multiple pathogens as well as the combined effects they may have on a host's immune system. broadening our understanding of the diversity of pathogens that exist in human and animal hosts through wildlife and domestic animal surveillance will significantly improve our ability to recognize novel zoonotic agents in the context of a disease outbreak. phylogenetic information obtained from comparative sequence analyses can improve our understanding of the impact of sequence mutation on virulence, as well as inform decisions about vaccine development. a final noteworthy benefit of viral discovery efforts is that these techniques should be important for identifying candidates for future vaccines as a virus's most worthy competitor is often another virus. from a health perspective, vaccination is arguably the most important technology that has arisen from the study of viruses. vaccination offers a direct means of intervening in a host-pathogen system and it has become routine in many parts of the world. efforts to this end have resulted in the eradication and/or control of smallpox, polio, mumps, measles, rubella and most recently, rinderpest. vaccines take several forms including live-attenuated viruses; inactivated whole viruses; inactivated toxins and viral protein subunits, and these are often delivered in combination. while live-attenuated vaccines have been predominant, a new generation of techniques including gene delivery and nano-technologies are being used to develop highly-efficacious and safer vaccines, that have less risk of reversion [ ] . new types of administration methods are also being developed with oral, aerosolized and nasal vaccines currently on the market. these less invasive administration techniques decrease labor costs associated with administration and offer increased capacity for mass-dispersal of vaccines to both humans and free-ranging wildlife [ ] . vaccination campaigns aimed at both protecting threatened species and decreasing public health risks via animal vaccination have taken place. swiss health officials were pioneers in this field, using oral vaccines to control rabies in wild red foxes (vulpes vulpes) [ , ] . these vaccines were inserted into chicken heads which were distributed in the wild beginning in [ ] . as can be observed from the supplemental movie (video s ), their initial barrier approach evolved into a large-scale treatment of infected areas and resulted in rabies being successfully pushed back to and then eliminated from the swiss alps [ , ] . following the success of these trials, campaigns were conducted in western europe [ ] and canada [ ] with similar results, though the situation in the united states has proven more challenging. other successful vaccination examples include canine distemper virus in black-footed ferrets (mustela nigripes; [ ] ) and ethiopian wolves (canis simensis; [ ] ), as well as rabies in florida panthers (felis concolor coryi; [ ] ) and african wild dogs [ , ] . a caveat to this success is that there is growing evidence that vaccination against a specific strain of pathogen can result in inadvertent selection for related co-infecting strains. thus vaccination can influence the dynamics of a pathogen [ ] . the possibility of inadvertent viral strain selection highlights the importance of understanding the long-term evolutionary and ecological consequences of vaccination. indeed, where threatened or endangered animals are concerned, mishaps may prove disastrous. attenuated canine distemper vaccines did not provide immunity to critically endangered black-footed ferrets, while the use of a live canine distemper virus vaccine resulted in clinical distemper arising in one of the few remaining populations [ ] . ideally, long-term clinical trials with suitable animal models might avert these problems. these trials should also be used to provide an a priori understanding of how vaccination might shape future evolutionary processes. in contrast to the ferret experience, efforts with the endangered ethiopian wolf serve as an example of a successful vaccination program. wolf populations were suffering severe mortality due to rabies and distemper acquired from the wild dogs that shared their home range [ ] . on the basis of a spatially explicit individual-based model, which indicated rabies could be controlled in dogs given just over % coverage [ ] , knobel et al. executed an intensive vaccination plan [ ] . both the extent and duration of outbreaks in the treated areas were limited and, although monitoring and continued vaccination are required, the situation appeared to be under control in [ ] . wildlife vaccination campaigns are also being investigated as tools to limit public health risks. tsao et al. vaccinated mice in an effort to break the cycle of lyme disease and reduce the risk of emergence in human populations, in which it causes tens of thousands of deaths per year in the us [ , ] . in the same vein, griffing et al. [ ] have tested the efficacy of vaccinating american robins to interrupt the wnv transmission cycle. this species can absorb up to % of the potentially infective mosquito bites in early spring and is thus a key host in the wnv system [ ] . targeted vaccination of this single species could potentially result in herd immunity and reduce the risk of human infection as well as decreasing wildlife mortality. these works exemplify how ecological knowledge can be used to identify and exploit some of the heterogeneities which so often dominate the dynamics of pathogens. while the lasting efficacy of wildlife vaccination efforts has yet to be demonstrated with either endangered species or in breaking the transmission cycle of human pathogens, an increasing number of researchers are drawing attention to systems where it seems feasible [ , ] ; demonstrating that intricate knowledge of host and virus ecology can greatly reduce the amount of vaccine coverage that is necessary to control these viruses. the problems entailed by the sheer number of viruses, viral resistance, the explosive potential for spread, and the economic burden, make it clear that currently available vaccination methods do not provide a sustainable solution for either human or animal disease. the unambiguous indication is that researchers need to work towards the goal of developing a predictive framework where risk can be defined for different scenarios and not only to rank pathogens, and species, but also, places and times of year that can be identified as more or less precarious for global health. pending questions include: which geographic areas will experience more disease and conservation problems? which areas pose the highest risk for pandemic spread of pathogens? what characteristics of hosts and viruses make them more or less likely to be involved in cross-species transmission events? and what are the relative roles of genetic relatedness and contact rate for transmission? some modeling work and reviews of historic data have been informative [ , ] , but novel uses of phylogenies of both viruses and hosts (as discussed above) provide promise for progress to this end, especially when coupled with high quality surveillance data. once we have this information, scientists will be able to design -smart surveillance‖ strategies whereby valuable vaccine resources can be efficiently targeted and efficiently distributed. ecological studies can effectively inform conservation as well as public health policy. gaining knowledge of reservoir host ecology can be critical for the development of eradication strategies. most viral disease systems are dominated by heterogeneities and identifying and understanding these can be crucially important when trying to interrupt the chain of events that leads to persistence. ecological studies of wnv have shown how forest fragmentation and decreased biodiversity can alter transmission among avian hosts as well as to humans [ ] . likewise, researchers have used satellite imagery to identify habitat characteristics that accurately predict the prevalence of sin nombre virus [ , ] , a hantavirus that uses the deer mouse (peromyscus maniculatus) as a reservoir host and it occasionally infects and kills humans [ ] . these studies epitomize the type of effort scientists will need to successfully fight viral pathogens in the future. however, piecing together emerging disease and conservation problems ex posto facto is only of limited value. increased pathogen surveillance and ecosystem process monitoring may provide the insight necessary to mitigate problems before they become serious human health or conservation concerns. this is especially the case for zoonotic viral pathogens where the reservoir hosts are known and a targeted approach is feasible. rodents rank as the number one reservoir of emerging and re-emerging zoonotic viruses [ ] . conveniently, these small mammals also present a manageable system for studying disease dynamics [ ] [ ] [ ] . individuals can be marked and sampled individually through time. their locations as well as their contacts with other individuals can be measured. as such, wild populations of rodents can be valuable as model disease systems to address relevant questions like: are there key hosts for transmission? how does prevalence vary seasonally or over time? what is the contact rate between the reservoir and humans? how do these pathogens flow through populations? the answers are of critical importance because they provide an indication of when and where there is increased risk of a zoonotic event whereby a human becomes infected, or when a species becomes at genuine risk of extinction. by monitoring and manipulating wild populations, one might also be able to identify factors that may increase a pathogens chance of emerging. for instance, what characteristics of hosts and viruses make them more or less likely to be involved in cross-species transmission? and what are the relative roles of genetic relatedness and contact rate for transmission? long-term monitoring and surveillance in reservoirs will also enlighten us to the kind of aggregations and other heterogeneities that exist through time and that and can be exploited with efficient vaccination campaigns. we are experiencing a global increase in the rate of emerging viral zoonoses, which are primarily driven by anthropogenic activities such as land-use change, agricultural intensification, and driven by global travel and trade [ ] . in order to adequately understand, predict and ultimately interrupt the processes by which zoonoses cross the species barrier from their natural reservoirs to humans, and then become established as human pathogens, comprehensive scientific studies that use the tools of ecology, virology, microbiology, and epidemiology are needed [ ] . the study of disease ecology has become an established discipline with advances in both the formulation of new theory as well as the integration of molecular virological techniques that provide important information about epidemiology, ecology and viral evolution, all of which has been applied to both health and conservation [ ] [ ] [ ] . because ecological systems are rife with heterogeneities and often have non-intuitive processes underlying their dynamics, it is critically important for scientists to use a comprehensive approach to understanding the population processes of an ecosystem before successful intervention strategies can be developed or implemented. admittedly this is a daunting task and it is often the case that scientists need to operate with less than complete information. where this is the case, a modeling approach is necessary to identify key processes that allow successful interventions. technological advances in molecular virology and genetics, as well as the expanded use of mathematical models in epidemiology and disease ecology have dramatically changed our ability to manage both conservation and health. finally, it is only with this type of interdisciplinary approach that considers free-ranging wildlife, domestic animals and humans as inextricable components of a single disease system, that progress can be made that will satisfy both conservation and public health needs. this is the essence of conservation medicine [ ] and indeed we believe a -one health‖ approach to infectious disease is necessarily the way forward. supplemental movie (video s ). are we in the midst of the sixth mass extinction? 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the potential role of pterygodermatites peromysci in the population dynamics of free-living mice, peromyscus leucopus predicting the emergence of human hantavirus disease using a combination of viral dynamics and rodent demographic patterns anthropogenic environmental change and the emergence of infectious diseases in wildlife collaborative research approaches to the role of wildlife in zoonotic disease emergence ecology of infectious diseases in natural populations the ecology of wildlife diseases conservation medicine and a new agenda for emerging diseases this work was supported in part by nih/nsf -ecology of infectious diseases‖ awards from the john e. fogarty international center (grant r -tw - s ) and an nih award (k ai ) from the national institute of allergy and infectious diseases. we thank c. j. wale, b. wale and h. ewing for their help in proofing the manuscript. key: cord- -u dh authors: cuthill, jennifer hoyal; charleston, michael a. title: a simple model explains the dynamics of preferential host switching among mammal rna viruses date: - - journal: evolution doi: . /evo. sha: doc_id: cord_uid: u dh a growing number of studies support a tendency toward preferential host switching, by parasites and pathogens, over relatively short phylogenetic distances. this suggests that a host switch is more probable if a potential host is closely related to the original host than if it is a more distant relative. however, despite its importance for the health of humans, livestock, and wildlife, the detailed dynamics of preferential host switching have, so far, been little studied. we present an empirical test of two theoretical models of preferential host switching, using observed phylogenetic distributions of host species for rna viruses of three mammal orders (primates, carnivores, and ungulates). the analysis focuses on multihost rna virus species, because their presence on multiple hosts and their estimated ages of origin indicate recent host switching. approximate bayesian computation was used to compare observed phylogenetic distances between hosts with those simulated under the theoretical models. the results support a decreasing sigmoidal model of preferential host switching, with a strong effect from increasing phylogenetic distance, on all three studied host phylogenies. this suggests that the dynamics of host switching are fundamentally similar for rna viruses of different mammal orders and, potentially, a wider range of coevolutionary systems. viruses with rna genomes are major agents of infectious disease in humans holmes and rambaut ) , livestock, and wildlife (cleaveland et al. ). therefore, the study of their infection dynamics is important for human health (jones et al. ) , livestock economics, and environmental conservation (cleaveland et al. ). however, we still know relatively little about the evolutionary factors affecting host identity and range (the type and number of host species exploited) among viruses (longdon et al. ) , and pathogens and parasites in general (perlman and jaenike ) . rna viruses are widely distributed among mammal species, suggesting an ancient origin (holmes ) . furthermore, it has recently been shown that rna viruses have left "fossils" dating to millions of years ago, in the form of endogenous viral elements integrated into host genomes (reviewed by holmes ) . previously, matching phylogenetic branching patterns for some extant rna viruses and their hosts had been thought to suggest a similarly ancient history for living viral strains, characterized by cospeciation with their hosts over hundreds of thousands, to millions, of years (reviewed by holmes ) . however, the majority of rna viruses appear to have high rates of mutation, indicating an age in the order of hundreds of years for most extant virus taxa below the family level (holmes (holmes , . this apparent discrepancy can be explained by a model of "preferential host switching" (charleston and robertson ) , in which a virus is more likely to successfully colonize a new host species if this is a close relative of the original host than if it is a more distant relative (for similar concepts in the earlier literature, see jermy ; menken , e.g.) . in this model, switches to phylogenetically distant hosts are possible, but are considered less probable than switches between more closely related host species. such preferential host switching is expected because barriers to infection will depend on the physiological similarity between original and potential host species (poulin ) ; factors that can depend strongly on host phylogeny. this is, in turn, because phylogenetic distance represents a general proxy for physiological and ecological similarity, due to the evolutionary inheritance of traits such as suitability for parasite replication (ohishi et al. ) , immune defenses (longdon et al. ), nutrient quality (poulin and mouillot ; poulin ) , and body size (clayton et al. a; bush and clayton ) . ( we acknowledge that species-specific differences in pathogen susceptibility are likely to lead to natural variation around this overall trend, e.g., harder and osterhaus ; poulin and mouillot ; poulin .) in some cases preferential host switching may also be promoted by phylogenetic covariance in the opportunity for infection, for example, if recently diverged host species tend to have overlapping geographic ranges (poulin and mouillot ; davies and pedersen ; but see also daszak et al. ) . since preferential host switching was proposed for primate lentiviruses (charleston and robertson ) , it has been suggested as a general characteristic of viral infection dynamics (holmes ) and as a wider evolutionary phenomenon: documented in phylogenetic studies of fleas (siphonaptera, insecta) (krasnov et al. ) , jumping plant-lice (psylloidea, insecta) (percy et al. ) , and brood-parasitic indigobirds (vidua, passeriformes) (sorenson et al. ) ; and in experimental studies of sigma viruses (longdon et al. ) , spiroplasma bacteria (tinsley and majerus ), lice (phthiraptera, insecta) (clayton et al. a) , and nematodes (allantonematidae, tylenchida) (perlman and jaenike ) . however, the detailed dynamics of preferential host switching have been relatively little studied. in particular, theoretical models describing the probability of host switching with genetic distance between potential hosts (charleston and robertson ; engelstädter and hurst ) have not previously been tested empirically, and the extent to which similar parameters of preferential host switching hold across different host phylogenies has not, until now, been investigated. two possible models of preferential host switching will be considered here. both models have been proposed before now (charleston and robertson ; engelstädter and hurst ) , but it has not previously been possible to test or compare them using real host-pathogen association data. the first, exponential, model is based on the simple notion that a parasite or pathogen colonizing a new host will have reduced fitness on that host if it represents a dissimilar physiological environment to that of its original host (as introduced above). the second, sigmoidal, model derives from the idea that there will be a certain tolerance of pathogens to new environments, within which they may remain optimally or near-optimally fit, but beyond which the combination of multiple factors' deviation from the environment to which the parasite has adapted will become too much and fitness will rapidly decline. these two models can neatly be described by an exponential and half of a gaussian (sigmoidal) function, respectively (see eq. , below). a potential example of a pathogen with a phylogenetically restricted, tolerable host range and infrequent host switches beyond this range (as predicted by the sigmoidal host-switching model) is influenza a virus. this is apparently more easily transmitted between bird species (the reservoir hosts) than to other animals, due to adaptation of virus subtypes to hostspecific surface receptors (webster et al. ; dugan et al. ; bandín and dopazo ; gambaryan et al. ). on the other hand, a pathogen that appears to show an immediate reduction in infectivity with increasing phylogenetic distance from the original host (as predicted by the exponential host-switching model) is measles virus, mv. for this virus, frequency of wild transmission appears considerably higher among humans, the natural hosts, than to nonhuman primates (de swart ) and, even more so, to other mammals such as rodents (dörig et al. ; wyde et al. ) . evolutionary histories of host-pathogen association have often been investigated using cophylogenetic analysis (comparisons of host and pathogen phylogenies, e.g., charleston and robertson ) . however, potential reconstruction of historical host switches using this method is complicated by the finding that both cospeciation and preferential host switching predict similar branching patterns between host and pathogen phylogenies (charleston and robertson ) . current methods of cophylogenetic analysis (implemented in programs such as treemap (charleston and robertson ) and jane (conow et al. ) ) are designed to test hypotheses of cospeciation, and the reconstructions they produce are expected to decrease in optimality as the actual incidence of cospeciation declines (percy et al. ) . to overcome the above complications, this study takes an alternative approach, and reconstructs the dynamics of preferential host switching among recorded "multihost" rna viruses of mammals, on phylogenies of their primate, carnivore, and ungulate hosts. multihost pathogens use multiple host species, often as definitive hosts on which they are able to complete their life cycle and reproduce (banks and paterson ) (here, the term "pathogen" is used for simplicity, though the arguments apply to both pathogens and parasites). although most pathogens are likely to experience some level of species barrier (giving reduced infectivity among individuals of an unusual host species), the majority of pathogens may have the potential to infect multiple host species . two alternative evolutionary processes have generally been suggested to explain the presence of a pathogen species on multiple hosts (banks and paterson ) . one process involves association by evolutionary descent, in which divergent host species inherit a pathogen that does not, itself, speciate (referred to, e.g., as failure to speciate (johnson et al. ; banks and paterson ; clayton et al. b) or to diverge (conow et al. ) ). the other process involves association by colonization, in which one parasite population switches host, but does not speciate from a population maintained on the original host (which we refer to here as incomplete host switching, after clayton et al. b ). failure to speciate and incomplete host switching are comparable to the cophylogenetic events for singlehost parasites of, respectively, cospeciation (which involves association by descent; but with parasite speciation) and host switching (which involves association by colonization; but with either parasite speciation or extinction of the parasite population on the original host (clayton et al. b) ). however, we note that currently available cophylogenetic methods are able to fully reconstruct only one of these theoretical events for multihost parasites (failure to speciate, which can be reconstructed by the software package jane (conow et al. ) ). for extant rna viruses, failure to speciate is ruled out by their young age relative to their host species (as implied by current estimates of substitution rates for the majority of rna viruses (holmes ) and discussed below). this leaves host switching-in this case incomplete, that is, without virus speciation-as the most probable evolutionary mechanism for the spread of a given rna virus species to multiple host species. the distribution of rna viruses on the phylogenies of their mammal hosts, therefore, provides a record of host-switching dynamics that allows us to test alternative theoretical models of preferential host switching. to achieve this, approximate bayesian computation (abc) is used to test the fit of the two models of preferential host switching to the observed distributions of multihost rna viruses on the phylogenies of their mammal hosts (primates, carnivores, and terrestrial ungulates). this method is also used to estimate the value of a parameter describing the decay in the probability of hostswitching success with increasing phylogenetic distance between current and potential hosts (details below). the results support a decreasing sigmoidal (gaussian) function for the probability of a successful host switch given phylogenetic distance, with a strong effect from phylogenetic distance between species on the host phylogeny. records of host species exploited by rna viruses were taken from the global mammal parasite database (gmpd) (nunn and alitzer ) , a large compilation of wild host-parasite/pathogen associations documented in the scientific literature. this database covers three major mammal groups: nonhuman primates, carnivores, and terrestrial ungulates. corresponding host phylogenies, of primates, carnivora and cetartiodactyla, were downloaded from the ktrees project (arnold et al. ) , which provides up-to-date bayesian phylogenies for these mammal orders. for each order, the % majority rule consensus of , phylogenies sampled from the bayesian markov chain monte carlo (mcmc) tree search (excluding burn-in), was used in our main analyses (for details of phylogenetic reconstruction methods see arnold et al. ) . a second analysis, which used phylogenies sampled from the mcmc chain (again excluding burn-in), was conducted to test whether the results were robust to phylogenetic uncertainty (details below). the dataset of host associations was then restricted to those rna virus species recorded on more than one host species represented in a ktrees phylogeny. some extant rna viruses have been suggested to have unusually low substitution rates and old evolutionary origins, making cospeciation with their host species a possibility (reviewed by holmes ) and potentially introducing failure to speciate as an explanation for the host distribution of widespread parasites. of these, one (simian foamy virus, see switzer et al. ) appeared in the primary dataset. because this study is concerned with the dynamics of host switching (rather than "failure to speciate" events), this virus was excluded from the final dataset. had there been any unidentified failures to speciate, among the multihost viruses that were included in our analyses, they could potentially bias our conclusions. however, host-virus associations over the time scales of mammal species divergence (generally, in the order of millions of years, e.g., dos reis et al. ) have been rejected or considered less probable than recent host switches for the remaining viruses in our dataset. these comprised: the caliciviruses reptilian crotalus- , feline calicivirus, and san miguel sea lion virus (smith et al. ; etherington et al. ) ; the coronaviruses canine coronavirus, and feline infectious peritonitis (gorbalenya ) ; the retroviruses feline leukemia virus, feline immunodeficiency virus, and simian t-lymphotropic virus- and -l (van brussel et al. ; switzer et al. ; lópez et al. ; roelke et al. ); rabies virus (jackson and charleston ) ; the morbilliviruses measles virus, canine distemper virus, phocine distemper virus, and dolphin morbillivirus (harder and osterhaus ; haffar et al. ; furuse et al. ; kosakovsky pond , though see ohishi et al. ) ; vaccinia virus (tryland et al. ); footand-mouth disease virus (tully and fares ) ; the orbiviruses bluetongue virus and epizootic hemorrhagic disease virus (biek ; carpi and holmes ) ; chikungunya virus (volk et al. ) ; the alphaviruses mayaro virus and semliki forest virus (gould et al. ; forrester et al. worobey , ) , the orthobunyaviruses bakau and bunyamwera, although these have been relatively little studied (yakondo et al. ; lambert and lanciotti ) ; human respiratory syncytial virus (köndgen et al. ; gaunt et al. ); human parainfluenza virus- (collins ); rotavirus a (ghosh et al. ) ; and the rubulaviruses human parainfluenza virus- and - , although the age and evoloutionary origins of such paramyxoviruses require further study (drexler et al. ) . the final dataset of host-virus associations is shown in the supporting information (table s ). numbers of multihost rna virus species recorded for the three mammal orders were for the primates, for carnivores, and for terrestrial ungulates. the effectiveness of the abc method given these different virus sample sizes was tested by cross-validation, as detailed below. we considered a number of potential sources of sampling bias in the recorded distributions of multihost rna viruses on their host phylogeny. one potential problem for the identification of multihost viruses, and multihost parasites in general, is the accurate designation of species (e.g., van regenmortel ). for example, lumping of distinct parasite populations with different hosts into a single species might lead to overestimation of the number of host species exploited. to minimize this possible effect, the dataset was screened to ensure all included viruses were classified to the species level. we also note that the recent age of origin inferred for the majority of extant rna viruses (holmes ) suggests that any distinct strains, incorrectly lumped within the same species, would still represent very closely related lineages that achieved their current host range by host switches (the subject of this study). another potential sampling issue is that relevant host-parasite associations, present in the wild, might not be recorded in the database (nunn and alitzer ) . to test for such sampling bias, we measured sampling effort for each species of virus, or host, included in the dataset and checked for a relationship with the number of recorded hosts. as in previous studies of host range (e.g., nunn et al. ), we estimated sampling effort using the number of citations for a given species in a comprehensive database of published articles, in this case the web of science (web of knowledge ). the number of citations for each virus was non-normally distributed for the viruses of primates and carnivores (primates: shapiro-wilk w = . , p < . ; carnivores: w = . , p = . ; ungulates: w = . , p = . ). similarly, the average number of citations per host (calculated for each multihost virus) was non-normally distributed for primates (primates: w = . , p = . ; carnivores: w = . , p = . ; ungulates: w = . , p = . ). therefore, nonparametric spearman's rank correlations were performed. neither the number of citations for each virus, nor the average number of citations per host, was significantly correlated with the number of host species recorded for a given virus (virus citations, primates: p = . ; carnivores: p = . ; ungulates p = . , average host citations, primates: p = . ; carnivores: p = . ; ungulates: p = . ). finally, any phylogenetic bias in sampling effort for different species of parasite and host might affect the phylogenetic distribution of recorded hosts for a multihost rna virus. two tests were performed to assess whether measures of sampling effort for virus or host species were significantly correlated with the average phylogenetic distance between hosts (as measured by the hsd "host switch distance" statistic, detailed below). spearman's rank correlations indicated that the sampling effort for each virus was not significantly correlated with hsd, for viruses of primates (p = . ), carnivores (p = . ) or ungulates (p = . ). nor was the average number of citations per host species significantly correlated with hsd (primates: p = . ; carnivores: p = . ; ungulates: p = . ). furthermore, hsd was found to be normally distributed for all viruses of all three mammal orders (primates: w = . , p = . ; carnivores: w = . , p = . ; ungulates: w = . , p = . ). consequently, the main analyses were performed without any transformation or correction of hsd. a measure of phylogenetic distance between the host species exploited by a given multihost rna virus was used to estimate host switch distances. specifically, host switch distance (hsd) was estimated, for each virus, as the average phylogenetic distance between each pair (i, j) of host species of that virus: where d ij is the midbranch distance between two host species i and j, and n is the number of host species. the midbranch distance between two hosts is calculated as d ij = d ij -(a i + a j )/ , where d ij is the patristic distance between host leaves i and j, and a i (resp. a j ) is the length of the branch incident to host leaf i (resp. j). hsd is similar to previously proposed measures of phylogenetic host specificity (poulin and mouillot ; poulin, krasnov, and mouillot ) , but considers distances between branch midpoints rather than distances between leaves. branch midpoints were used to model host switch events from a "take-off" site, at an unknown point along the branch to one of the host leaves, to a "landing" site, at an unknown point along the branch to another host leaf. although this was the biologically motivated method of calculation, simulated phylogenetic distances between leaves were similarly distributed to those calculated between midbranch points (data not shown). hsd was not found to be significantly correlated with the number of hosts for viruses of primates (linear correlation p = . ), carnivores (p = . ), or ungulates (p = . ), suggesting that this measure of interhost phylogenetic distance is comparable for multihost viruses with different numbers of hosts (two hosts, three hosts, etc.). hsd values for each virus were then summarized by the mean, variance, skewness, and kurtosis, calculated separately among the viruses of primates, carnivores, and ungulates. this provided four summary statistics for each host phylogeny, which could then be compared with similar statistics calculated for the data simulated under the theoretical models of preferential host switching (as described below). we tested two theoretical models for the probability of a successful host switch at a given distance between species on the host phylogeny. these represent exponential (charleston and robertson ; engelstädter and hurst ) , versus sigmoidal (engelstädter and hurst ), functions for the decline in host switch probability with phylogenetic distance, respectively: where p e (i j) and p s (i j) give the probability of a successful host switch between species i and j on the host phylogeny (which may have occurred either from i to j, or from j to i ), γ ( ≤ γ ≤ ) is a constant for the host phylogeny (which can be viewed as the probability of successful infection where d ij = (engelstädter and hurst ) ), d ij is the phylogenetic distance between species i and j (as described above), and β is a positive parameter that scales the decay in host switch probability as d ij increases. under both models (fig. ) , the maximum possible probability of a host switch is given by the value of γ (achieved if d ij = ). with a given value of β, the probability of a successful host switch decreases toward zero as phylogenetic distance increases, reaching γ/ when d ij = β (engelstädter and hurst ) . as the decay parameter β decreases, the probability of a host switch at a given phylogenetic distance declines. overall, both models describe a decrease in the probability of a successful host switch as phylogenetic distance increases. however, with a given β value, the sigmoidal function (fig. b) shows a smaller initial decline in host switch probability (when phylogenetic distances are small) followed by a steeper decline toward a host switch probability of zero (as the distance between host species becomes relatively large), compared with the exponential function ( fig. ) (as discussed below). we use as the base of the exponent for consistency with previously proposed forms (engelstädter and hurst ) , noting that changing the base is equivalent to scaling the β value. host switches were simulated, under the two theoretical models of preferential host switching, using a matlab (version . . . , the mathworks inc. ) program written for the purpose (text s ). simulations were performed separately for the three consensus phylogenies (primates, carnivora, and cetartiodactyla). the patristic distance matrix corresponding to each phylogeny was used to derive a matrix of the midbranch distances (d ij ) between each possible pair of hosts (as described above). the probability of host switch success under each of the two models (probability p e (i j) for the exponential model or p s (i j) for the sigmoidal model) was then calculated for each possible pair of host species, with values of the decay parameter β ranging from . to in steps of . . for sampling purposes, host switch probabilities (for a given phylogeny and with a given β value) were then normalized by dividing the probability calculated for each possible host pair by the total probability of host switching (the sum probability over all possible pairs of species). a random sampling procedure was then used to sample interhost phylogenetic distances, according to the normalized probability of each possible host switch. sampling from the normalized probabilities (rather than non-normalized values) removes the effect of the phylogenetic constant γ (the probability of infection at a phylogenetic distance, d ij , of zero), and ensures that a phylogenetic distance is sampled at every iteration. this is suitable for comparison with our observed data, which records only the presence of a pathogen (rather than recording absence vs. presence of each pathogen across all potential hosts, e.g.). each sampled distance d ij provides an estimate of host switch distance, hsd, where n = (eq. ). the sample size was set to match the recorded number of viruses for each host order ( for primates, for carnivora, and for cetartiodactyla). four summary statistics were then calculated for each sample. these were the mean value of the hsd statistic (fig. ) , and its variance, skewness, and kurtosis (figs. s -s ). the use of these different sample sizes allowed us to test whether abc could distinguish between simulations, with different models and parameter values, given sample sizes comparable to the numbers of viruses actually recorded in databases of host-pathogen associations, such as that used in our study. the sampling procedure was repeated times for each unique combination of host phylogeny, model, and β value, and the calculated summary statistics (hsd mean, variance, skewness, and kurtosis) were output for abc. this procedure was also repeated using the average pairwise phylogenetic distance calculated on phylogenies sampled from the mcmc chain (rather than the consensus phylogeny, which was used in the main analyses). taking the average pairwise distances, across the sampled phylogenies, approximately weights them by the relevant clade posterior probabilities. this allows us to test whether the main results (based on the consensus phylogeny) were robust to phylogenetic uncertainty, by comparison against the results based on average phylogenetic distance (across the range of topologies present in the mcmc sample). data simulated under the two models of preferential host switching were compared with the observed phylogenetic distributions of multihost rna viruses using abc, as implemented in the r package abc (csilléry ). abc allows model and parameter selection, and estimation of support for a selected model or parameter, under complex biological models for which computation of the likelihood function may be impracticable (csilléry et al. ). this is achieved by comparing summary statistics for observed data with similar statistics calculated for the simulated data. these comparisons are based on a rejection method, which repeatedly samples from the simulated statistics, accepting parameter values for a given simulation if the distance from the observed statistic is below a set threshold (which determines the proportion of accepted simulations). accepted parameter values can be viewed as a sample from an approximate posterior probability distribution for that parameter. regression techniques may then be applied to improve the estimates of distance between statistic values, and to produce a weighted and corrected approximation of the parameter posterior. average posterior probabilities can then be calculated for different models, over the range of simulated parameter values, and compared using bayes factors (csilléry ) . bayesian confidence (or, credible) intervals were calculated from each estimated posterior probability distribution, giving the interval within which there is a % probability that the parameter value lies. for each rna virus in the dataset, the observed phylogenetic distribution of its hosts was summarized using the mean phylogenetic distance hsd (as described above), and three further summary statistics for hsd: its variance, skewness, and kurtosis. these statistics (mean, variance, skewness, and kurtosis) are widely used to summarize biological data (e.g., dowd ) , and their effectiveness in model and parameter selection was tested using an abc cross-validation procedure, also implemented in the abc package. cross-validation for model and parameter selection was also performed to select the best performing abc method and tolerance level, and to evaluate performance. cross-validation for "model" selection repeatedly samples from the simulated data and records the percentage of samples for which the simulation model is correctly identified, based on the simulated summary statistics. cross-validation for "parameter" selection repeatedly samples from the simulated data and calculates the prediction error for a given parameter (the sum of squared differences between true and estimated values divided by the variance (csilléry ) ). based on cross-validation for parameter selection, the main analyses were performed using a linear regression method to improve posterior probability estimation (csilléry ) and reduce estimation error (relative to the simpler rejection method). based on cross-validation for parameter selection, we used a tolerance level of . (rather than the tested alternatives of . or . ), which gave the greatest model selection accuracy with the abc method and simulation set chosen for the main analyses (parameter prediction error was found to be relatively independent of tolerance level, as illustrated in fig. s ). the abc procedure was performed first on the full simulation set (across all β values) and then on the subset of simulations with β ≤ . , over which the models can be clearly distinguished while covering a relatively wide range of model selection with abc (csilléry ) supported the sigmoidal model of preferential host switching, over the exponential model, for all three studied host phylogenies. mean posterior probabilities for the sigmoidal model were . for primates, . for carnivores, and . for ungulates; whereas posterior probabilities for the exponential model were . for primates, . for carnivores, and . for ungulates. bayes factor comparisons of these posterior probabilities (kass and raftery ) indicate positive support for the sigmoidal model, over the exponential model, among the multihost rna viruses of primates (bayes factor = . ), but only weak support among those of ungulates (bayes factor = . ) and carnivores (bayes factor = . ). for all three host phylogenies, abc selected a relatively low value for the parameter β, indicating a strong effect of host phylogenetic distance (hsd) on the probability of a successful host switch. the modal β value with % bayesian confidence intervals on the posterior probability distribution for this parameter was . [ . , . ] for the primates, . [ . , . ] for carnivores, and . [ . , . ] for ungulates. a second set of analyses, conducted using the average pairwise distance between species (across phylogenies sampled from the bayesian mcmc chain), gave similar results to those based on the majority rule consensus phylogenies (above). these supported the sigmoidal model (posterior probabilities: primates . , carnivores . , ungulates . ) over the exponential model (posterior probabilities: primates . , carnivores . , ungulates . ) and returned relatively low estimates for parameter β (primates . [ . , . ], carnivores . [ . , . ], ungulates . [ . , . ] ). this suggests that the main results, based on the consensus phylogenies, are fairly robust to phylogenetic uncertainty. figure illustrates modeled relationships between phylogenetic distance and the probability of a successful host switch. given levels of β between . and . (as selected for the three host phylogenies), the best supported sigmoidal model indicates, first, that very low phylogenetic distances (below approximately . substitutions per site) present very little barrier to host switching. then, with increasing phylogenetic distance, there is a relatively steep decline in the probability of a successful host switch, nearing zero as phylogenetic distances increase above approximately . - . substitutions per site. figure illustrates the average host switch distance (hsd) returned by computer simulation on each host phylogeny. with the β parameter value selected using abc (for the given host phylogeny), the average phylogenetic distance of a simulated host switch under the best supported sigmoidal model is markedly lower than that expected under the exponential model (given the same value of β). cross-validation for model selection, using the simulated host switch data, confirmed that the simulation model and β parameter value could be effectively estimated from the summary statistics for each host phylogeny. this indicates that abc model selection was effective with each of the three sample sizes used for calculation of the hsd summary statistics (which corresponded to the number of observed host-virus associations, of for primates, for carnivores, and for ungulates). the sigmoidal model was correctly selected in % of the sampled simulations on the primate phylogeny, % on the carnivore phylogeny, and % on the ungulate phylogeny. the exponential model was correctly selected in % of sampled simulations for primates, % for carnivores, and % for ungulates. cross-validation for parameter selection indicated that the prediction error (csilléry ) for the β parameter was very low for the simulations on the primate phylogeny (error = . ), for which the sample of virus species was largest. prediction error was higher, for carnivores (error = . ) and ungulates (error = . ), which had lower sample sizes. however, error plots illustrate that estimated parameter values (within the relevant bayesian confidence intervals) are approximately linearly related to true parameter values, showing no evidence for prediction bias (fig. s ). this indicates that relatively large samples of host-virus associations allow for more effective model and parameter testing, but that even relatively small samples can provide information on host-switching dynamics. our survey of the rna viruses of mammals recorded in the gmpd (nunn and alitzer ) found a total of multihost rna virus species, collectively infecting wild hosts among the primates, carnivores, or terrestrial ungulates. of the rna viruses recorded in the gmpd, multihost viruses represented % of the total among primates, % among carnivores, and % among ungulates. of these recorded multihost viruses, % had just two hosts and % had fewer than seven hosts, although a few virus species infected relatively large numbers of host species. (harder and osterhaus ; purse et al. ; chambers ; maclachlan and guthrie ; de almeida et al. ) . comparison of two theoretical models of preferential host switching (eq. ) using abc supported a sigmoidal model, over an exponential model, with a strong effect from phylogenetic distance for the three studied host phylogenies. this suggests that the decay in host switch probability is initially shallow, before proceeding steeply as phylogenetic distance increases. it is noteworthy that our tests across three mammal phylogenies (primates, carnivores and ungulates) all supported the same theoretical model and similarly strong effects from phylogenetic distance. these results suggest that strong preferential host switching, with a sigmoidal decay in the probability of a successful host switch as phylogenetic distance increases, may be a general characteristic of infection dynamics among mammal rna viruses. this result feeds into our understanding of the coevolutionary dynamics of host switching by parasites and pathogens, potentially generalizing across many different coevolutionary systems (see below). although it has long been held that the success of host switching diminishes with phylogenetic distance between current and nascent hosts, it has not previously been possible to test particulars of this decay. our work suggests that ecological arguments of a certain "tolerable range" of host environment are indeed reflected in a sigmoidal reduction in host-switching success with increasing phylogenetic distance. this agrees with previous experimental observations from a wide range of host and parasite taxa. first, it has been observed that small phylogenetic distances (such as those between sister species, or within a subgenus (perlman and jaenike ) or genus (tinsley and majerus ) ) can present very low barriers to host switching. second, a number of recent studies suggest that the distribution of pathogens and parasites on the phylogeny of their hosts is affected by the phylogenetic distance between the host species (charleston and robertson ; clayton et al. a; perlman and jaenike ; poulin and mouillot ; krasnov et al. ; percy et al. ; sorenson et al. ; tinsley and majerus ; longdon et al. ) . third, it has been suggested that host switches over very large phylogenetic distances are comparatively rare. for example, few pathogens or parasites have hosts in difference orders or classes (gifford et al. ; woolhouse and gowtage-sequeria ) and very few infect hosts in different phyla (poulin and mouillot ) . although the sigmoidal host-switching model had the highest posterior probability (compared with the exponential model) for all three studied orders, and is consistent with previous suggestions (as described above), bayes factor support was weaker for the smaller carnivore and ungulate datasets than for the larger primate dataset. this suggests that the generality of the sigmoidal host-switching model should be corroborated as more host-virus association data becomes available for nonprimate groups. our abc parameter selection supported a relatively low value of the model parameter β, which scales the decay in host switch probability with increasing phylogenetic distance between current and potential hosts. increasing values of β indicate a declining effect of phylogenetic distance. however, the average host switch distance modeled with a particular β value depends on the distribution of branch lengths on the host phylogeny (fig. ) . thus, the significant point is not the selected value of the β parameter per se, but that the value supported for each phylogeny falls in the range where phylogenetic distance strongly affects the distribution of simulated host switches. this suggests that the dynamics of host switching by mammal rna viruses have been strongly affected by host phylogeny. although our analysis focused on the decay parameter β describing the probability of a successful host switch at a given phylogenetic distance, the models of host switching also contain a parameter γ, which represents the probability of infection within the original host species (engelstädter and hurst ) . our abc procedure could perhaps be extended to incorporate estimates of γ, based on statistics describing intrahost pathogen infectivity (e.g., see tinsley and majerus ) , though it is not yet clear how informative this would be. here we have tested two simple models that could be compared with the available data. of course, simply finding that one model is better supported than another does not mean that that model is the correct one in all cases: however, we regard this as an important first step in understanding the general dynamics of host switching by rna viruses. furthermore, the abc methods used here could be applied to other models as they arise. in summary, we present a novel empirical test of two theoretical models of preferential host switching and the model parameter that scales the predicted decay in host switch probability. comparison of observed and computer simulated host phylogenetic distributions for mammal rna viruses, using abc, supports a strong sigmoidal decay in the probability of a successful host switch as phylogenetic distance on the host phylogeny increases. the same model, with similar decay parameter values, is supported for all three studied host phylogenies (primates, carnivores, and ungulates) . this suggests that the supported model is applicable across mammal rna viruses, and may generalize to a wider range of coevolutionary systems. these findings provide an insight into the dynamics of host switching, an evolutionary factor thought be responsible for a majority (holmes and rambaut ) of emerging and re-emerging infectious disease. this work was supported by an australian research council grant, dp , which we gratefully acknowledge. we also thank the curators of the ktrees project for granting us early access to their phylogenies of carnivora and cetartiodactyla. associate editor: s. remold additional supporting information may be found in the online version of this article at the publisher's website: table s . recorded mammal hosts of rna viruses, as used in this study. text s . matlab program for data simulation. figure s . variance among the simulated hsd statistics, otherwise annotated as for figure . figure s . skewness among the simulated hsd statistics, otherwise annotated as for figure . figure s . kurtosis among the simulated hsd statistics, otherwise annotated as for figure . figure s . prediction error for model parameter β (eq. ) determined using an approximate bayesian computation cross-validation procedure (csilléry ) for simulated host switches on the phylogenies of primates (a), carnivores (b), and ungulates (c). the ktrees website: a new online resource for primate phylogeny evolution and dispersal of st. louis encephalitis virus in the americas host range, host specificity and hypothesized host shift events 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contributions of phylogenetics to understanding the history and epidemiology of the preeminent arboviral disease evolution and ecology of influenza a virus purifying selection can obscure the ancient age of viral lineages a challenge to the ancient origin of sivagm based on african green monkey mitochondrial genomes host range and emerging and reemerging pathogens population biology of multihost pathogens use of cotton rats for preclinical evaluation of measles vaccines evolutionary history and phylodynamics of influenza a and b neuroaminidase (na) genes inferred from large-scale sequence analysis molecular characterization of african orthobunyaviruses key: cord- - hiss j authors: kong, qingming; xue, chunyi; ren, xiangpeng; zhang, chengwen; li, linlin; shu, dingming; bi, yingzuo; cao, yongchang title: proteomic analysis of purified coronavirus infectious bronchitis virus particles date: - - journal: proteome sci doi: . / - - - sha: doc_id: cord_uid: hiss j background: infectious bronchitis virus (ibv) is the coronavirus of domestic chickens causing major economic losses to the poultry industry. because of the complexity of the ibv life cycle and the small number of viral structural proteins, important virus-host relationships likely remain to be discovered. toward this goal, we performed two-dimensional gel electrophoresis fractionation coupled to mass spectrometry identification approaches to perform a comprehensive proteomic analysis of purified ibv particles. results: apart from the virus-encoded structural proteins, we detected host proteins in the purified virions which can be grouped into several functional categories including intracellular trafficking proteins ( %), molecular chaperone ( %), macromolcular biosynthesis proteins ( %), cytoskeletal proteins ( %), signal transport proteins ( %), protein degradation ( %), chromosome associated proteins ( %), ribosomal proteins ( %), and other function proteins ( %). interestingly, of the total host proteins have not been reported to be present in virions of other virus families, such as major vault protein, tenp protein, ovalbumin, and scavenger receptor protein. following identification of the host proteins by proteomic methods, the presence of proteins in the purified ibv preparation was verified by western blotting and immunogold labeling detection. conclusions: the results present the first standard proteomic profile of ibv and may facilitate the understanding of the pathogenic mechanisms. infectious bronchitis virus (ibv), the coronavirus of domestic chickens that causes acute, highly contagious respiratory disease, is one of the most important causes of economic loss in the poultry industry. ibv is an enveloped virus with continuous, positive and single-stranded rna genome, which is the largest of any rna virus characterized [ ] and encodes four types of structural proteins. the spike (s) glycoprotein, together with small envelope (e) protein and matrix (m) glycoprotein, consists of the viral envelope, whereas the nucleocapsid (n) protein interacts with genomic rna of the virus to form the viral nucleocapsid, in the invariable order '-s-e-m-n- '. proteins s, e, and m have been studied for their important roles in receptor binding and virus budding. s mediates attachment to cellular receptors and entry by fusion with cell membranes, whereas m interacting with s and n proteins is an essential component of virion and plays pivotal roles in virion assembly, budding and maturation [ , ] . in addition, s protein can inhibit host cell translation by interacting with eif f [ ] and the interaction between m and actin facilitates virion assembly and budding [ ] . e is a poorly characterized small envelope protein present in low levels in the virions. the significance of the e protein appears to be critical for viral budding. another role for protein e is that it can promote apoptosis [ , ] . viruses constantly adapt to and modulate the host environment during replication and propagation. to govern egress from the host cell and initiation of replication in the target cell, viruses will carry some of the host proteins when released from infected cells. enveloped viruses particularly encoding only small proteins have the capability of incorporating numerous host proteins into or onto the newly formed viruses. it is an important prerequisite for the functional studies to know the protein composition of the purified viral particles, as it allows the analysis of specific proteins and their roles during the virus life cycle, resulting in better understanding of the infection process and the pathogenesis of viruses. as a large number of virus complete genomes have been sequenced since s, more and more host proteins in different enveloped viruses have been studied using viral proteomic approaches. herpesviruses have been the most extensively studied in this respect, such as kaposi's sarcoma-associated herpesvirus (kshv) [ , ] , marek's disease virus (mdv) [ ] , epstein-barr virus (ebv) [ ] , human cyotomegalovirus (hcmv) [ ] and murine cyotomegalovirus (mcmv) [ ] . other doublestranded dna (dsdna) viruses including vacciniavirus have also contributed to a better understanding of this intriguing phenomenon [ ] [ ] [ ] . furthermore, recent studies on identification of the incorporated host proteins in rna viruses have also been undertaken. for retrovirus, various studies in this research area have been performed on human immunodeficiency virus type (hiv- ) [ ] [ ] [ ] [ ] and moloney murine leukemia virus (mmlv) [ ] . for paramyxovirus, numerous host proteins have been found incorporated into avian influenza virus (aiv) particles and respiratory syncytial virus (rsv) particles [ ] [ ] [ ] . to date, no study of the host proteins in the virions of coronavirus has been performed yet. in this study, we performed two-dimensional gel electrophoresis fractionation coupled to mass spectrometry identification approaches to perform a comprehensive proteomic analysis of purified ibv particles. our analysis resulted in the identification of virus-encoded structural proteins and incorporated host proteins. in addition, we also discussed the functional implications of some host proteins in ibv infection and pathogenesis. viral proteomic analysis requires a highly purified preparation of virions. there was no available permissive cell line capable of supporting productive replication of ibv. although primary chick embryo kidney cell (cek) and chick kidney cell (ck) are capable of supporting productive replication of ibv, their poor yields prohibit them from being used for producing large quantity of ibv. in order to obtain large quantity of ibv virions, this study selected -day-old spf embryonated chicken eggs for the growth of ibv strain h . the af with enrichment of h was clarified by differential centrifugation in order to remove the contamination of nuclei, mitochondria, lysosomes, peroxisomes and so on from the chicken embryo. the virus was concentrated through a % (wt/vol) sucrose cushion before purified over a non-linear %- % sucrose-tne (tris-buffered saline including mm tris, mm nacl, mm edta, ph . ) gradient. two distinct types of ibv particles were isolated by sucrose density gradients. the higher density particles banded at %- % sucrose-tne gradients while the less density particles banded at %- % sucrose-tne gradients. the purity of ibv was confirmed by electron microscopy analysis following negative staining to ensure that the virions have normal viral morphology and to exclude the possible inclusions of vesicles, other cellular organelles and debris (fig. a ). an abundance of intact virions were observed without obvious contamination from host cellular materia. proteins in purified virions were separated on % sodium dodecylsulfate polyacrylamide gel electrophoresis (sds-page) and stained withc coomassie brilliant blue (fig. b ). there were also some lighter bands visible that may represent cellular proteins besides the conjectured major virus-encoded structural proteins. furthermore, the four virus-encoded structural proteins were confirmed by immunoblotting test (fig. c) . taken together, the best purification of the ibv was obtained after differential centrifugation to remove the cellular contamination and condensation through a % (wt/vol) sucrose cushion with a non-linear sucrose gradient. to obtain a detailed protein composition profile associated with the ibv particles, the proteins in purified ibv particles were extracted for -de experiments. to authenticate the results and to compensate the variability figure analysis of purified avian infectious bronchitis virus preparations. a: specific pathogen free (spf) chick embryo-grown major particles h from %- % sucrose density gradients, negatively stained with % potassium phosphotungstate, ph . . b: sds-page separation of proteins in a purified h preparation. μg of proteins were separated on an - . % polyacrylamide gel and stained with coomassie blue. c: western blotting of the purified h virions. viral proteins were separated on % polyacrylamide gel and analyzed by western blot with chicken polyclonal antibody against infectious bronchitis virus (massachusetts). the identified viral proteins are indicated. s: spike, n: nuclecapsid, m: mebrane, e: envelope. of gel electrophoresis, three independent experiments were performed with three replicate gels for each experiment. the viral protein profiles were analyzed by -d with μg of protein. after the electrophoresis separation, gels were stained with silver and processed for image analysis. for ibv particle-associated proteins separated in the ph - range, protein spots were detected (fig. ) . to identify the proteins associated with ibv particles, all protein spots detected in the gels were excised and in-gel digested with trypsin followed by maldi-tof/tof (matrix-assisted laser desorption/ionizationtime of flight mass spectrometry) analysis. database search analysis revealed that virus-encoded structural proteins and host proteins were successfully identified. detailed information of the full set of the identified proteins is listed in table ; additional file . to better understand the host proteins incorporated with ibv virion and their roles played in ibv infection, these proteins were categorized with biological processes according to uniprot knowledge database (swiss-prof/ trembl) and gene ontology database. the identified host proteins were comprised of cytoskeleton proteins, molecular chaperone, macromolecular biosynthesis proteins, signal transport proteins and glycolytic enzymes (table s ; additional file ). these host proteins were located mainly in the cytoplasm, including cytoskeleton, cytosol and mitochondrion (fig. ) . to confirm the presence of host proteins in the purified ibv particles after the identification of them by pro-teomic method, we performed immunoblotting experiments. ibv preparation purified from af was analyzed for the presence of n protein, actin, hsp , annexin a and tubulin (fig. ) . extracts from -day-old specific pathogen free (spf) embryonated chicken eggs were included as a positive control. when analyzing the results of virion proteomic studies, the challenge is to prove that the host proteins are really an part of the virion and that they are not just attached non-specifically to the outside of the virus. to address this question, the af from uninfected -day-old spf embryonated chicken eggs were parallelly subjected to our standard density centrifugation procedure and the protein extracts from %- % sucrose gradient was used as negative control. gradientpurified virions and the control were separated on % sds-page gels, transferred to pvdf membranes, and probed with the appropriate antibodies. as shown in fig. , actin, tubulin and annexin a were both found in the purified virions and positive control but not in the af extracts from uninfected -day-old spf embryonated chicken eggs. hsp is a member of the heat shock protein family which is upregulated in response to stress and has low abundance in unstressed cells. in present study, we detected it only in the purified virions but not in normal cells. it is an expectable result that we also detected actin and tubulin in the af extracts from uninfected day-old spf embryonated chicken eggs which resulted from their high concentrations in all eukaryotic cells and subcellular fractions. to provide additional evidence that the host proteins are not just derived from a microvesicle or exosome that co-purified with the virus, we used the bromelain protease protection assay which has been shown to efficiently remove microvesicles from ibv virion preparations [ ] . protease treatment of the purified virus preparation strips proteins off any contaminating microvesicles and off the outside of virus particles, such as s protein. in doing so, the microvesicles become lighter than the virions and therefore the virions can be isolated by density centrifugation. proteins that are inside the virion are protected by the lipid envelope and therefore will remain after the protease treatment. and then immunogold labeling of the purified virions was performed ( fig. ). virions were either mock treated or subjected to digestion with bromelain and then were incubated with antibodies against actin, annexin a , hsp , ibv massachusetts strain and secondary gold antibodies followed by negative staining. one or two gold particles located on the surface of a virion could be seen for hsp . this was significantly less compared with the degree of other labelings which is consistent with the fact that there is most likely far more actin, annexin a present on the virions than hsp . in addition, the abundance of actin detected in the -de gels is much higher than that of hsp . virus exploits multiple host proteins during infection for successful entry, replication, egress, and evasion. this is especially true for rna viruses because they encode only little proteins. learning the protein composition profile of the infectious viral particle is prerequisite for studying the role of host proteins during infection. to our knowledge, incorporation of host proteins in the envelopedvirus family coronaviridae has not been investigated so far. in this study, we revealed the presence of virusencoded proteins in infectious bronchitis particles and for the first time confirmed the incorporation of host proteins. a total of viral and host proteins associated with purified ibv particles were identified. in the present study, we failed to obtain m and e protein while other two structural proteins n and s were identified successfully. n protein is easy to identify because it is the most abundant virus-derived protein produced throughout the process of the virus infection, whereas s protein as a major structural protein of ibv located on the surface of viral particles is also easy for identification because it is large (about kda) and has many tryptic cleavage peak in the ms analysis. the identification of m and e protein of coronavirus by ms has been thought to be a difficult task due to their properties, especially in the case of e protein for the following reasons [ ] . first, e protein is a very hydrophobic protein. second, it is low-abundant in the virions. third, e is a low molecular weight protein with the mass about kda. fourth, e protein contains two cysteines, which may form disulfide bonds within itself or with other proteins and make e protein difficult to be reduced and subsequently digested. of the total host proteins, have also been described to be present in virions of quite diverse virus families, such as herpesviruses, poxviruses, paramyxovirus and retroviruses [ , ] . there are some explanations that the incorporated host proteins are common to other virus families. first, they are all enveloped viruses. enveloped viruses contain the viral genome and core proteins wrapped within one or more membranes which are acquired from the host cell during virus assembly and budding. these viruses all share some fundamental feature in the particular stage of their life-cycle and these host proteins are involved in the common process. second, these host proteins would be either highly abundant cytosolic proteins or enriched at the virus budding sites. several of the highly abundant cytosolic proteins found within both ibv and other viral particles are beta actin, tubulin, annexins and enolase. proteins enriched at the virus budding sites including hsp , hsp and gapdh are also identified in ibv and other viruses [ , , , , , ] . some host proteins may be specially incorporated into the virions. in this study, of the total host proteins are reported for the first time. the identified host protein functions in diverse biological processes and some functional groups are analyzed. these proteins participate in a broad array of cellular functions and are involved in many processes in the viral life cycle. the potential roles of some of these proteins are discussed below in relation with ibv infection, pathogenesis and early host antiviral response. numerous viral proteins interact with cytoskeletal elements. many viruses, such as retroviruses, herpesviruses and picornaviruses, even contain the main cytoskeletal element actins in their infectious particles. the transport machinery of actins are proven to be critical at almost every step along the infectious cycle [ ] . actin has been found in preparations from several types of retroviruses and paramyxoviruses. for coronavirus, an association of m with cytoskeletal elements has been reported [ ] , which indicates an essential function of actin in the replication cycle of coronavirus ibv. in our studies, actin and tubulin were all present in the interior of infectious bronchitis particles and this observation most likely reflects their active participation in moving the viral components to assembly sites. actin and tubulin have been characterized as the major folding substrates for cct (chaperonin containing tcp- , also termed tric). both cytoskeletal proteins require in vivo and in vitro the interaction with cct to fold to their native states [ ] . cct is the most different and complicated protein of all group ii chaperonins in eukaryotic cytosolic chaperonins, which might be involved in the assistance of the folding of a small set of proteins. in addition to the already mentioned actin and tubulin, cct has been found to interact either in vitro or in vivo with other cytoskeletal proteins, cell division control protein , protein phosphatase type a, and guanine nucleotide-binding protein (g protein) beta subunit [ ] , which are all found to be associated with infectious bronchitis particles in present study. it's pleasantly surprising to find that certain viral proteins such as the epstein barr virus-encoded nuclear protein (ebna- ), the hepatitis b virus capsid and the type d retrovirus gag polyprotein are also folded by cct [ ] [ ] [ ] . thus, cct may have an important role in infectious bronchitis viral proteins assembly. other cytoskeletal proteins found to be associated with infectious bronchitis particles are actin-related proteins, wd repeat containing protein, destrin and annexin. several annexin family members (a , a and a ) were identified in purified infectious bronchitis particles. annexins are a well-known multigene family of ca + regulated phospholipid-binding and membrane binding proteins with diverse functions. the presence of annexin a is thought to support viral binding, fusion and replication [ ] [ ] [ ] [ ] [ ] . annexin a , which interacts with annexin a , has the opposite effect by preventing fusion, which possibly indicates a potential regulatory role [ ] . annexin a tightly binds to a member of the s family of calciumbinding proteins, s a (p ). upon binding, annexin a and p form a heterotetramer which is capable of binding two membrane surfaces simultaneously, which potentially promotes fusion events and also plays a role in exocytosis [ ] . the p protein was also detected by our analysis, suggesting that ibv is also incorporated this complex. other s family members such as s a and s a were also detected in viral samples and could play various roles in fusion and membrane organization [ , ] . heat-shock proteins (hsps) have been known as multifunctional proteins. they facilitate the folding and unfolding of proteins, participate in vesicular transport processes, prevent protein aggregation in the densely packed cytosol and are involved in signaling processes. most, but not all, hsps are molecular chaperones. several viruses require host molecular chaperones for entry, replication, and assembly, as well as other steps in viral production [ , ] . hsp and hsp have been found incorporated into ibv. hsp interacts with various viral proteins and may be involved in the assembly of adenovirus [ ] , enterovirus [ ] , vaccinia virus [ ] and hantaan virus [ ] . alternatively, upon entry into susceptible target cells, virion-associated hsp might participate in early events of infection. for example, hsp might actively uncoat the viral capsid in a manner similar to its role in the uncoating of clathrin cages [ ] . hsp and hsp have been shown to interact with hepatitis b virus reverse transcriptase and to facilitate the initiation of viral dna synthesis from hepatitis b virus pregenomic rna [ , ] . for sendai virus (sv), the viral proteins synethsis will be inhibited as long as hsp synthesis occurs [ ] . thus, hsp in ibv virions might serve a similar function in the virus life cycle. the chaperone hsp has been identified as an essential factor in the folding and maturation of picornavirus capsid proteins [ ] . the involvement of hsp in viral replication has also been reported for many viruses and it has been demonstrated that hsp inhibition blocks viral replication [ ] . recently, a role for hsp in the control of hepatitis c, flock house and influenza virus polymerase function has been shown [ ] [ ] [ ] [ ] [ ] [ ] [ ] and it has been proposed that hsp is a major host factor that is of central importance for viral replication for a wide spectrum of rna viruses [ ] , which implies the crucial roles of hsp in ibv replication. the importance of hsp for the replication of multiple viruses opens up an interesting possibility for developing new antiviral therapies which have not yielded drug-resistant viruses [ ] . some proteins involved in the glycolytic pathway were identified, such as aldehyde dehydrogenase family, member a (aldh a ), glyceraldehyde- -phosphate dehydrogenase (gapdh), alpha-enolase, which were identified in other viral particles, like hiv- , mmlv, hcmv, kshv and aiv [ , , , , ] . some studies have suggested that several glycolytic enzymes interact with microtubules and tubulin [ ] [ ] [ ] and may also contribute to transcription of rna virus genomes. in higher eukaryotes, enolase is found as a dimer of subunits, α, β, or γ. all enolase isoforms from mammalian have been reported that are capable of stimulating transcription of svgenome [ ] . gapdh is a well-characterized key enzyme in glycolysis, but recent evidence suggests it also has rna binding properties and binds to the untranslated rna sequences of several different viruses, including human parainfluenza virus type (hpiv ), japanese encephalitis virus (jev), hepatitis a virus (hav) and hepatitis b virus (hbv) as well as hepatitis c virus (hcv) [ ] [ ] [ ] [ ] [ ] . in the case of hpiv , gapdh has been reported to inhibit actin-dependent in vitro transcription and is also present in purified virions [ , ] . in vitro data indicates that gapdh serves a negative regulatory role in hpiv transcription and in a phosphorylation-dependent on manner [ ] . in addition to these host proteins associated with enveloped viruses, the roles of which in the virus life cycles have been studied well, we also identified host proteins in purified infectious bronchitis particles, which have not been described to be present in other virions of quite diverse virus families, such as apolipoprotein a-i (apoa-i), fatty acid-bingding protein , ovalbumin, tenp protein, tumor protein translationally controlled- , transthyretin and so on. apoa-i, a major constituent of highdensity lipoproteins, alters plasma membrane morphol-ogy by participating in the reverse transport of cholesterol binding with atp-binding cassette transporter a [ ] , and activates the small gtp-binding protein cdc associated signaling including apoa-i induced cholesterol efflux, protein kinases, and actin polymerization [ ] . what important is that apo a-i can inhibit herpes simplex virus (hsv)-induced cell fusion at physiological concentrations. this function may be related to the structure of apoa-i and before long its amphipathic peptide analogue was also found to inhibit cell fusion, both in hiv- infected t cells and in recombinant vaccinia-virusinfected cd + hela cells expressing hiv envelope protein on their surfaces [ ] . the results indicate that amphipathic helices may be useful in designing novel antiviral agents that inhibit penetration and spreading of enveloped viruses. ovalbumin is the main protein found in egg white, making up - % of the total protein. the chicken ovalbumin upstream promoter transcription factors (coup-tfs), members of the steroid/thyroid hormone receptor superfamily, binds to a negative regulatory region in the human immunodeficiency virus type long terminal repeat (ltr). ltr contains a negative regulatory element which downregulates the rate of ltr-directed transcription and hiv- replication [ ] . the interaction between ovalbumin and np from influenza a virus as well as glycoprotein c from the herpes simplex type i virus was reported long time ago [ ] . the tenp protein from g. gallus, however, was isolated as a transiently expressed gene in neural precursor cells in retina and brain, and has been proposed to function in the transition to cell differentiation in neurogenesis. after expressed in chicken embryonic fibroblast cells, tenp was immunodetected in membrane fractions, implying that tenp might be a membrane protein as predicted by a computer analysis of its primary sequence [ ] . to date, there have been no reports about tenp associated with virus, but it's an enriched and abundant protein identified in purified infectious bronchitis particles which suggests to us that it may be a requisite host protein in ibv life cycles. the present study ) provides the first proteomic analysis of infectious bronchitis particles, ) establishes the most comprehensive proteomic index of ibv and ) shows that most of the virion incorporated host proteins have central roles in virus life cycle. although some proteins may be associated with virus biology, further investigation of the function of these host proteins may facilitate the understanding of the pathogenic mechanisms. the ibv strain h was obtained from qianyuanhao biological corporation limited (beijing, china). virus was propagated in -day-old specific pathogen free (spf) embryonated chicken eggs (beijing merial vital laboratory animal technology co, ltd, beijing, china) for h at °c. the allantoic fluid (af) with enrichment of ibv h was clarified by differential centrifugation. af was first centrifugated at , × g for min and then the supernatant was centrifugated at , × g for min. clarification and all subsequent centrifugations were performed at °c. the virus was sedimented through . ml of % (wt/vol) sucrose in tne buffer ( mm tris, mm nacl, mm edta, ph . ) by centrifugation in a ti rotor (beckman coulter, optima™ l- xp preparative ultracentrifuge) at , × g for . h. condensed virions were then diluted with . ml tne buffer and centrifuged to equilibrium in . ml non-linear %- % sucrose-tne gradients at , × g for . h in a sw rotor (beckman coulter, optima™ l- xp preparative ultracentrifuge). purified virions were diluted with tne buffer and pelleted by sedimentation at , × g for . h in a sw rotor to remove the sucrose. the purified ibv pellets were stored at - °c until use. the purified ibv particles were dissolved in about μl lysis buffer ( m urea, m thiourea, % triton x- , mm dtt, % biolyte ph - ) and incubated for min at °c. then the lysis solution was sonicated for min (pulse durations of s on and s off ) in an ice bath sonicator. the viral protein samples were prepared when the indiscerptible sediments were wiped off by centrifugation at , × g for min. the supernatant was collected and the concentration of the prepared protein samples was determined by the bio-rad protein assay kit ii according to the manufacturer's instructions. the samples were then aliquoted and stored at - °c until used for further analysis. purified virus particles treated with bromelain (bb , bbi) at . mg/ml in mm dtt (ph . ) in dulbecco's phosphate buffered saline (pbs) at °c for min. after incubation, the treated virus was directly centrifuged to equilibrium in . ml non-linear %- % sucrose-tne gradients at , × g for . h in a sw rotor (beckman coulter, optima™ l- xp preparative ultracentrifuge). purified virions were diluted with tne buffer and pelleted by sedimentation at , × g for . h in a sw rotor to remove the sucrose and then subjected to immunogold labeling and electron microscopy analysis. two-dimentional gel electrophoresis analysis was performed using cm immobile drystrip (ipg strips, ph - non-linear, ge healthcare). first, μl samples containing μg protein were added into μl sample rehydration buffer ( m urea, m thiourea, % (w/v) chaps, mm dtt, . % bio-lyte ph - ) and incubated for min at °c prior to their separation by isoelectric focusing (ief) in the first dimension. the ipg strips were rehydrated at °c for h by a passive rehydration method. ief was carried out for a total of kvh at °c on an ettan ipgphor iii electrophoresis unit (ge healthcare). second, ipg strips were further transferred onto the second dimension of gel electrophoresis. before this step the ipg strips were reduced and alkylated in a equilibration buffer containing mm tris-hcl, ph . , m urea, % sds and % glycerol supplemented with % (w/v) dl-dithiothreitol (dtt) or . % iodoacetamide (iaa) instead of dtt for min. subsequently, the viral protein samples were separated at v on linear %- . % sodium dodecyl sulfate gradient polyacrylamide gel (sds-page) in tris: glycine buffer ( mm glycine, mm tris, . % sds, ph . ) for about h. third, proteins in the gel were stained by the modified silver staining method compatible with ms [ ] and the gels were scanned at a resolution of dpi using imagescanner™ iii (ge healthcare). gel pieces ( . mm ) containing the whole protein spots from the d gel were cut and washed three times with mm carbonic acid, monoammonium salt (nh hco , amresco). these gel pieces were destained with mm potassium ferricyanide (k fe(cn) , amresco) and mm sodium thiosulfate (nas o , amresco) in mm nh hco and dehydrated in % acetonitrile (acn, wako) until gel pieces turn to white. after dring in speedvac concentrator (thermo savant, usa) for about min, gel pieces were incubated with . ng/μl trypsin (sequenceing grade, promega) to cover dry gel pieces completely at °c overnight. the gel pieces were then extracted three times in % acn water solution containing % trifluoroacetic acid (tfa, wako). the supernatant was pooled and dried thoroughly in speed-vac. protein digestion extracts were resuspended with μl of . % tfa and then the peptide samples were mixed ( : ) with a matrix consisting of a saturated solution of αcyano- -hydroxycinnamic acid (α-cca, sigma) in % acn containing . % tfa. . μl aliquot was spotted onto stainless steel target plates. peptide mass spectra were obtained on an applied biosystem/mds sciex maldi tof/tof plus mass spectrometer. data were acquired in positive ms reflector using a calmix standard to calibrate the instrument (abi calibration mixture). mass spectra were obtained from each sample spot by accumulation of laser shots in an - mass range. for ms/ms spectra, the - most abundant precursor ions per sample were selected for subsequent fragmentation and laser shots were accumulated per precursor ion. both the ms and ms/ms data were interpreted and processed by gps explorer software (v . , applied biosystems), then those obtained ms and ms/ms spectra per spot were combined and submitted to mascot search engine (v . , matrix science, london, u.k.) by gps explorer software and searched with the following parameters: trypsin as the digestion enzyme, one missed cleavage site, partial modification of cysteine carboamidomethylated and methionine oxidized, none fixed modifications, ms tolerance of ppm, ms/ms tolerance of . da. mascot protein score in ipi_chicken (v . ) database (based on combined ms and ms/ms spectra) of greater than (p ≤ . ) or in ncbinr database of greater than (p ≤ . ) was accepted. mouse monoclonal antibodies against actin (mab ) and hsp ( - ) were purchased from millipore. rabbit polyclonal antibodies against annexin a (ab ) and tubulin alpha- (ab ), and chicken polyclonal antibody against ibv (massachusetts) (ab ) were purchased from abcam. mouse monoclonal antibody against nucleoprotein of ibv ( bn ) was purchased from hytest ltd. for control, the af from -day-old spf embryonated chicken egg performed with the same protocol as the purification of ibv particles and the protein extracted from the normal -day-old spf embryonated chicken eggs included for western blot analysis. samples were separated at v on linear %- . % sds-page with % stacking gels in tris: glycine buffer for about h. for purified virus, μg of total proteins were used per lane. for the control, a total of μg proteins were loaded. after separated by sds-page, the proteins were transferred to a polyvinylidene fluoride membrane (pvdf, p/n , biotrace, pall corporation). the membrane was blocked in freshly prepared % bovine serum albumin (bsa) with . % tween- for h at room temperature with constant agitation. the pvdf membrane was washed three times with tris buffered saline plus . % tween (tbst) and then incubated with properly diluted primary antibodies for h at room temperature or overnight with agitation at °c. anti-rabbit or anti-mouse immunoglobulin g antibody conjugated to horseradish peroxidase (hrp) ( - , proteintech group, inc) was used as the secondary antibody and the pvdf membrane was incubated in it for h at room temperature. the chemiluminescence system (ar , boster bio-technology co. ltd) was used for detection of antibody-antigen complexes. rabbit polyclonal antibody against chicken igg ( nm gold) (ab ), goat polyclonal against rabbit igg ( nm gold) (ab ) and goat polyclonal against mouse igg ( nm gold) (ab ) were purchased from abcam. purified ibv particles were suspended in pbs (ph . ) and then were collected onto -mesh formwar-coated nickel grids and adsorbed on the grids for min. the viruses were fixed in % paraformaldehyde for min at rt and treated with triton x- ( . %) in pbs (ph . ) for min and then blocked with % bsa in pbs-tween (ph . ) for min at rt. all grids were then blocked with blocking buffer ( % bsa, % normal serum, . % cold water skin gelatin, mm phosphate buffer, mm nacl, ph . ) for min. after washing with pbs, immobilized virions were incubated for . h with μg/ml primary antibody (in % bsa), and washed three times for min in pbs/ % bsa. anti-rabbit or anti-mouse immunoglobulin g coupled to nm colloidal gold particles was used as the secondary antibody and virions were incubated in it for min at room temperature. the grids were then washed extensively with pbs, washed twice more with distilled water to remove excess salt and negatively stained with % sodium phosphotungstate for min. negatively stained virions were examined on a scan and transmission electron microscope. abbreviations d: two-dimensional; -de: two-dimensional electrophoresis; sds-page: sodium dodecylsulfate polyacrylamide gel electrophoresis; ms: mass spectrometry; maldi-tof: matrix-assisted laser desorption/ionization time of flight mass spectrometry; spf: specific pathogen free; af: allantoic fluid; bsa: bovine serum albumin; dtt: dithiothreitol; iaa: iodoacetamide; acn: acetonitrile; tfa: trifluoroacetic acid; α-cca: α-cyano- -hydroxycinnamic acid; tne: tris-buffered saline including mm tris; mm nacl; mm edta: ph . ; pbs: phosphatebuffered saline; tbs: tris-buffered saline; tbst: tris buffered saline plus . % tween ; hrp: horseradish peroxidase; pi: isoelectric point; mw: molecular weight. coronavirus avian infectious bronchitis virus envelope glycoprotein interactions in coronavirus assembly characterization of the coronavirus m protein and nucleocapsid interaction in infected cells coronavirus spike protein inhibits host cell translation by interaction with eif f interaction of the coronavirus 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ribonucleoprotein of avian infectious bronchitis virus proteomic analysis of sars associated coronavirus using two-dimensional liquid chromatography mass spectrometry and onedimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by mass spectroemtric analysis plunder and stowaways: incorporation of cellular proteins by enveloped viruses viral proteomics interaction of epithelial ion channels with the actin-based cytoskeleton the tcomplex polypeptide complex is a chaperonin for tubulin and actin in vivo systematic identification of protein complexes in saccharomyces cerevisiae by mass spectrometry epstein-barr virus-encoded nuclear protein ebna- interacts with the epsilon-subunit of the tcomplex protein chaperonin complex a eukaryotic cytosolic chaperonin is associated with a high molecular weight intermediate in the assembly of hepatitis b virus capsid, a multimeric particle type d retrovirus gag polyprotein interacts with the cytosolic chaperonin tric annexin ii enhances cytomegalovirus binding and fusion to phospholipid membranes secretory leukocyte protease inhibitor binds to annexin ii, a cofactor for macrophage hiv- infection annexin ii incorporated into influenza virus particles supports virus replication by converting plasminogen into plasmin annexin -mediated enhancement of cytomegalovirus infection opposes inhibition by annexin or annexin annexin : a novel human immunodeficiency virus type gag binding protein involved in replication in monocyte-derived macrophages annexins: linking ca + signalling to membrane dynamics intracellular and extracellular roles of s proteins s -annexin complexes: some insights from structural studies recruitment of hsp chaperones: a crucial part of viral survival strategies synthesis and quality control of viral membrane proteins association of hsp with the adenovirus type fiber protein in infected hep- cells association of heat shock protein with enterovirus capsid precursor p in infected human cells vaccinia virus infection induces a stress response that leads to association of hsp with viral proteins increased expression of hsp and co-localization with nuclear protein in cells infected with the hantaan virus uncoating atpase is a member of the kilodalton family of stress proteins hsp is required for the activity of a hepatitis b virus reverse transcriptase two-dimensional blue native/sds-page analysis reveals heat shock protein chaperone machinery involved in hepatitis b virus production in hepg . . cells selective inhibition of virus protein synthesis by prostaglandin a : a translational block associated with hsp synthesis evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance molecular chaperone hsp is important for vaccinia virus growth in cells hsp inhibitors suppress hcv replication in replicon cells and humanized liver mice hepatitis c virus rna replication is regulated by fkbp and hsp the cellular chaperone heat shock protein facilitates flock house virus rna replication in drosophila cells identification of hsp as a stimulatory host factor involved in influenza virus rna synthesis involvement of hsp in assembly and nuclear import of influenza virus rna polymerase subunits antiviral activity and rna polymerase degradation following hsp inhibition in a range of negative strand viruses herpes simplex virus type dna polymerase requires the mammalian chaperone hsp for proper localization to the nucleus development and application of hsp inhibitors enhanced association of mutant triosephosphate isomerase to red cell membranes and to brain microtubules a glycolytic enzyme binding domain on tubulin glycolytic enzyme-tubulin interactions: role of tubulin carboxy terminals enolase, a cellular glycolytic enzyme, is required for efficient transcription of sendai virus genome specific interaction in vitro and in vivo of glyceraldehyde- -phosphate dehydrogenase and la protein with cis-acting rnas of human parainfluenza virus type human hepatic glyceraldehyde- -phosphate dehydrogenase binds to the poly(u) tract of the ' noncoding region of hepatitis c virus genomic rna identification of glyceraldehyde- -phosphate dehydrogenase as a cellular protein that binds to the hepatitis b virus posttranscriptional regulatory element glyceraldehyde- -phosphate dehydrogenase (gapdh) interaction with ' ends of japanese encephalitis virus rna and colocalization with the viral ns protein functional significance of the interaction of hepatitis a virus rna with glyceraldehyde -phosphate dehydrogenase (gapdh): opposing effects of gapdh and polypyrimidine tract binding protein on internal ribosome entry site function specific phosphorylated forms of glyceraldehyde -phosphate dehydrogenase associate with human parainfluenza virus type and inhibit viral transcription in vitro specific binding of apoa-i, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing abc apolipoprotein a-i activates cdc signaling through the abca transporter apolipoprotein a-i and its amphipathic helix peptide analogues inhibit human immunodeficiency virus-induced syncytium formation immunoprecipitation, with an antiserum to ovalbumin, of protein np from influenza a virus and of glycoprotein c from the herpes simplex type i virus identification and characterization of tenp, a gene transiently expressed before overt cell differentiation during neurogenesis a modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry proteomic analysis of purified coronavirus infectious bronchitis virus particles proteome science the authors declare that they have no competing interests. qk performed the main proteomic experiments and data analysis and drafted the manuscript. cx created the detailed experimental design. xr and cz contributed to the initial phase of the proteomic experiments. ll and ds assisted in the propagation and purification of ibv. yb and yc helped conceive the research. all authors read and approved the final manuscript. key: cord- -hayhbs u authors: gonzalez, jean-paul; souris, marc; valdivia-granda, willy title: global spread of hemorrhagic fever viruses: predicting pandemics date: - - journal: hemorrhagic fever viruses doi: . / - - - - _ sha: doc_id: cord_uid: hayhbs u as successive epidemics have swept the world, the scientific community has quickly learned from them about the emergence and transmission of communicable diseases. epidemics usually occur when health systems are unprepared. during an unexpected epidemic, health authorities engage in damage control, fear drives action, and the desire to understand the threat is greatest. as humanity recovers, policy-makers seek scientific expertise to improve their “preparedness” to face future events. global spread of disease is exemplified by the spread of yellow fever from africa to the americas, by the spread of dengue fever through transcontinental migration of mosquitos, by the relentless influenza virus pandemics, and, most recently, by the unexpected emergence of ebola virus, spread by motorbike and long haul carriers. other pathogens that are remarkable for their epidemic expansions include the arenavirus hemorrhagic fevers and hantavirus diseases carried by rodents over great geographic distances and the arthropod-borne viruses (west nile, chikungunya and zika) enabled by ecology and vector adaptations. did we learn from the past epidemics? are we prepared for the worst? the ultimate goal is to develop a resilient global health infrastructure. besides acquiring treatments, vaccines, and other preventive medicine, bio-surveillance is critical to preventing disease emergence and to counteracting its spread. so far, only the western hemisphere has a large and established monitoring system; however, diseases continue to emerge sporadically, in particular in southeast asia and south america, illuminating the imperfections of our surveillance. epidemics destabilize fragile governments, ravage the most vulnerable populations, and threaten the global community. pandemic risk calculations employ new technologies like computerized maintenance of geographical and historical datasets, geographic information systems (gis), next generation sequencing, and metagenomics to trace the molecular changes in pathogens during their emergence, and mathematical models to assess risk. predictions help to pinpoint the hot spots of emergence, the populations at risk, and the pathogens under genetic evolution. preparedness anticipates the risks, the needs of the population, the capacities of infrastructure, the sources of emergency funding, and finally, the international partnerships needed to manage a disaster before it occurs. at present, the world is in an intermediate phase of trying to reduce health disparities despite exponential population growth, political conflicts, migration, global trade, urbanization, and major environmental changes due to global warming. for the sake of humanity, we must focus on developing the necessary capacities for health surveillance, epidemic preparedness, and pandemic response. infectious diseases have swept the world, taking the lives of millions of people, causing considerable upheaval, and transforming the future of entire populations. every year pathogens cause nearly million deaths worldwide, mostly in developing countries. more than infectious diseases have emerged between the s and [ ] . also among the known arboviruses, only are known to be human pathogens, while the others only infect wild animals and/or arthropods. to anticipate an epidemic one must identify the risk, prepare an appropriate response, and control the disease spread by first identifying the vulnerabilities of the population and circumscribing the potential space into which a disease will extend. when the epidemic expansion risk is identified, adequate information must be communicated to decision makers. ultimately, an appropriate response will depend on biosurveillance, prevention, sustained data processing, communication, strategic immunization campaigns, resilience, and mitigation strategies. the viral hemorrhagic fevers (vhfs) are a diverse group of human illnesses caused by rna viruses including approximately species of the arenaviridae, filoviridae, bunyavirales, flavi viridae, and rhabdoviridae (table ) . despite the efforts placed on early detection, viruses like dengue, ebola, lassa, crimean-congo hemorrhagic fevers continue to threaten the health of millions of people, mostly in areas where demographic changes, and political and socio-economic instability interrupt vaccination campaigns [ ] . however, the threat of vhf to global health is increased by intercontinental travel and global trade. moreover, because of the high case fatality rate of some of these pathogens, such concerns extend to the potential use of these viruses by bio-terrorists [ ] . global expansion of several diseases is exemplified by the spread of yellow fever from africa to the americas, the spread of dengue fever across continents, and recently, the spread of ebola virus from the democratic republic of the congo to western africa. the concept of an epidemic, as a disease affecting many persons at the same time and spreading from person to person in a locality where the disease was not previously prevalent, was not enunciated until when john snow produced his admirable demonstration of the emergence of an infectious disease in an urban area: the emergence of a cholera epidemic in london. at that time, none could clearly comprehend the mechanisms of emergence and spread since the existence of microbes had just been demonstrated by louis pasteur in the late s and microbe transmission modes were more speculative than based on medical or scientific facts, until when robert koch demonstrated that bacteria can be transmitted and responsible for diseases. nowadays, it is extremely difficult to make a retrospective diagnosis of historical pandemics, there are currently species in the orthohantavirus genus. the pathogeny of most of them is unknown during times when clinical descriptions were rare or lacking accuracy, and the extent of an epidemic was extremely subjective. thus, it is common to note that the first outbreak described in the western world was that of the plague of athens for which thucydides rather precisely reported the symptoms; today this epidemic has often been attributed to typhus through its clinical picture and epidemic profile [ ] . the first historically recorded outbreaks due to viral agents date to antiquity when the roman armies were returning from distant countries bringing with them "exotic" diseases. indeed, the rise of a "new" virus is an extremely rare event. most often, in terms of pathogen emergence, a virus adapts through mutation and selection pressure to a human host causing disease. presumably, smallpox, measles, and influenza were among the plagues that struck the ancient latins in gusts of epidemics more or less severe. the antonin plague that extended from to ad in much of western europe, when the troops of emperor lucius verus returned from war against the parthians, is often attributed to a smallpox pandemic by historians. in the middle ages, it seems that smallpox made a return around ad to france, germany, belgium, and the british islands [ ] . the acute respiratory infections reported during the winter of - ad accompanying the return of the carolingian armies from italy have been attributed by historians to a flu epidemic. many soldiers of charlemagne died then. the disease returned regularly and fiercely in and ad to the western european peninsula [ , ] (table ) . from the plague (sensu lato, including all transmissible diseases) of antiquity, to the severe acute respiratory syndrome that emerged on the eve of the third millennium, pandemics have followed in the history of mankind. as noted by mirko grmek, a historian of medicine, it seems that one pandemic will drive in another. if several diseases circulate concomitantly, one of them will take precedence over the other, an epidemic over the previous, and it is more likely that a pandemic will prevail [ ] . plague temporarily replaced the leprosy that appeared in eurasia for over , years; during the first millennium, plague was manifested by successive pandemics that crossed continents. during the first half of the past millennium, syphilis started its expansions, crossed oceans, and became global. tuberculosis originated in europe more than , years ago, but it was only at the turn of the seventeenth century that it was considered a pandemic; smallpox was also manifest as epidemics and then was pandemic at its peak in the late nineteenth century, then smallpox persisted until the jenner area. although early medical records of smallpox are available (egypt, china, india), large and devastating epidemics were only identified in the late fifteenth century of the millennium. smallpox was introduced into the americas by spanish settlers in the caribbean island of hispaniola in and arrived in mexico in . on hispaniola island, one third of a million of the inhabitants died of smallpox in the following years. smallpox devastated the native amerindian population and was an important factor in the conquest of the aztecs and the incas by the spaniards [ ] . in , children died in goa, india, from a smallpox epidemic. in europe, smallpox was a leading cause of death in the eighteenth century, killing an estimated , europeans each year [ ] . during the twentieth century, it is estimated that smallpox was responsible for - million deaths. the last known natural case of smallpox occurred in somalia in [ ] . it is only at the end of the first millennium that all these pathologies were better understood and their infectious origins elucidated. the first pandemic of the twentieth century was attributed to the h n spanish flu that emerged in kansas in . however, this "flu pandemic" is now thought to have had subepidemic circulation earlier in france or germany or even prior emergence in china in or [ ] , and to be exascerbated by concurrent bacterial infections. although it burned out quickly by , it has been estimated that one third of the world's population was afflicted; million people died, half of them in the first weeks of the outbreak. since the s, the frequency and magnitude of dengue fever epidemics increased dramatically as the viruses and the mosquito vectors have both expanded geographically in pandemic proportions [ ] largely extending the pandemic to all the intertropical zone. in the early s, human immunodeficiency viruses (hiv- and hiv- ) spread as an acquired immunodeficiency syndrome (aids), a pandemic that continues to take its terrible toll at the global level. since the emergence of aids, organization updates, as of june only million people were accessing antiretroviral treatment and among them, seven of ten pregnant women received treatment. in , a severe acute respiratory syndrome, sars, inaugurated the twenty-first century as a first pandemic of the millennium, involving more than countries with secondary epidemic chains in asia, europe, north america, south america, and a total of cases [ ] . ultimately, one of the major characteristics that defines today's pandemics, apart from the introduction of the disease within several continents or the rapid expansion across the administrative borders of countries, is the initiation of locally active transmission of the pathogen. although, the first ebola virus disease outbreak of western africa was considered a pandemic and witnessed several exported cases with secondary epidemic chains in distant countries of the african continent (i.e., nigeria, mali), outside of africa, exported cases rarely sparked local transmission. emergence from a sporadic case to an outbreak, to an epidemic, and ultimately to a pandemic depends upon effective transmission among nonimmune hosts, host availability (density), characteristics of the vector (natural or human made) that would enable it to circumvent distances, and the pathogen infectiousness. all these dynamics are essential for an effective disease transmission and spread. an outbreak is a sudden increase in occurrences of a disease in a particular time and place, more localized than an epidemic. an epidemic occurs as the disease spreads to a large number of people in a given population within a short period of time. to spark an epidemic chain of transmission depends on factors like immune population density, virus infectiousness, promiscuity, vulnerability, etc., while the efficiency of such transmission depends on how many persons will be infected by one person (i.e., the reproductive ratio or r ). an epidemic event will therefore expand in space (beyond the first cluster of cases) and time (rapid spread). a pandemic is essentially spatial, and represents an epidemic of infectious disease that has spread through human populations across a large region, extensively across two or more continents, to worldwide. however, all these typologies harbor the same fundamentals: emergence from one index case, transmission from one host to another, and spatial expansion. altogether, an epidemic and a pandemic are respectively a local and a global network of inter connected infectious disease outbreaks (i.e., epidemic chains). ultimately, understanding how disease (i.e., pathogens) spread in the social system is fundamental in order to prevent and control outbreaks, with broad implications for a functioning health system and its associated costs [ ] . also, after the last case occurs at the end of an epidemic, the goal is to control the risk of transmission for a -day time period. this three-week period represents an incubation when the infected subject does not transmit the virus and remains asymptomatic. the " days" is based on experimental methods use in virology to detect virus replication: influenza virus infected eggs should hatch in days, there is a -day limit for an arbovirus to infect a living model (suckling mice, mice, rats, guinea pigs, cell lines). moreover, most viral infectious diseases have a maximal incubation period of days, with few exceptions (e.g., hiv, and rabies). ultimately, such -day periods multiplied by the potential of a carrier to travel will produce the risk area for the emergence of secondary cases (from a walking distance to the long distances covered by commercial jets). however, it is important to clarify that many vhf including ebola virus can be carried by an asymptomatic host for several months [ , ] . the mode of transmission profiles the epidemic pattern of a transmissible disease. it is extremely helpful when a disease emerges to rapidly surmise the mode of transmission and how to respond (e.g., water-borne disease, arthropod-borne disease, human-tohuman transmission). pathogen transmission can be interspecific or hetero-specific, direct or indirect. direct transmission occurs by close contact with infected biological products (e.g., blood, urine, saliva). indirect transmission occurs with intermediate hosts such as arthropod vectors (e.g., mosquito, tick) or mammalian vector/reservoir (e.g., rodent, chiropteran) or from infected environmental means (e.g., soil, water, etc.). mobility and transportation are the main factors for diseases dispersion, as an emblematic example, one can simply show how the - evd outbreak of western africa expanded due to the transportation of patients during their -day incubation periods, first by foot-paths, then by motorbike, then taxis and public transportation, finally becoming a global concern with patients traveling by boat or commercial airline [ , ] . host population density and promiscuity, crowded places (like schools, markets, mass transportation system) also play an important role in the efficiency of transmission as well as the level of herd immunity (e.g., annual pandemic flu), altogether this gives us the level of population susceptibility (i.e., vulnerability). environmental factors can also be major drivers of pathogen expansion, for example the emergence of nipah encephalitis. the nipah virus, when it emerged for the first time in malaysia in , was transported by its natural host, a frugivorous chiropteran. a year earlier, an immense forest fire affecting several indonesian islands had forced the escape of disease-carrying bats that took refuge in malaysian orchards, planted to nurture newly developed pig farms. both pigs and farmers became infected and nipah virus was discovered for the first time. another classical example, more associated with human environment and behavior, is the old story of the spread of dengue virus via the used tires carrying infected aedes aegypti eggs and transporting dengue across oceans and continents [ ] . understanding the mechanisms of transmission and expansion of disease vectors with respect to the typology (epidemic pattern) of a disease is the ultimate challenge for controlling and preventing disease. typologies from human-to-human transmission, zoonotic diseases, arboviruses, water-borne diseases, and others play different roles in the rate of disease spread and need to be clearly understood. finally, while an epidemic pattern is driven intrinsically by the virus and its vector, the host population, the mode of transmission, and even the human environment (e.g., population density, urbanization, agricultural practices, health system, public health policies) as well as physical environment (season, meteorology, climate changing, latitude, altitude) factor into the rate of disease spread. with respect to pandemic risk (the rapidity and area covered by disease), the main characteristics of a virus are found in its environmental persistence while remaining infectious. environmental persistence depends on: virus structure, enveloped viruses are more sensitive than the naked viruses; its mode of entry into the body of the susceptible subject (transdermal, oral, respiratory); its ability to diffuse out of the body for a sufficient period of time which will, in turn, enable transmission to a greater number of subjects (r ). altogether these intrinsic factors link to the infectivity of the virus, indeed, viruses transmitted by aerosol possess certainly the most efficient way to spark an epidemic that increases with population density and vulnerability as well as with the resistance of the virus to environmental factors outside the host cells. the cycle of transmission shapes the epidemic in time and spatial dispersion. for example, animal to human zoonoses are dictated by chance encounters between host (population density, animal farming, pets, hunting) and, eventually transmission such as that observed between human and nonhuman primates [ ] . vectortransmitted diseases (i.e., arthropod-borne diseases) depend on the vector ecology (ability to transmit, length of the intrinsic cycle of the virus, trophic preferences, vector density, seasonality, reproduction, breeding sites, food abundance for hematophagous arthropods). mobility of hosts/vectors that are part of the natural cycle will also play a role in the potential for disease expansion (e.g., mosquito-flying distance, cattle transhumance, human migration). also, other factors associated with the hosts will render a more efficient transmission: human behaviors like fear/social responses, nosocomial infections, super-spreaders); viruses having multiple natural hosts (vicariates) or vectors; vectors with multiple trophic preferences (e.g., biting cattle, birds, and primates); the incubation period in the vertebrate hosts as well as the intrinsic replication in the arthropod vectors will also intervene; ultimately subclinical infection is also an underestimated factor of virus dispersion and transmission that modifies the epidemiological pattern of disease. one can distinguish also a typology of communicable diseases that reflects the spatial and temporal mode of transmission including arthropod-borne transmission, human-to-human transmission, human-to-animal (and vice versa) transmitted diseases (i.e., zoo- predicting hfv pandemics noses) including vector and nonvector transmitted diseases, and some other types of environmentally transmitted diseases. all of them represent unique types of transmission and risk of spread with a variable path of time, and also dependent on multiple factors (environment, climate, behavior, etc.). we have to consider territories as spaces where disease can potentially expand and that can be characterized by the fundamental factors of emergence and spread: the vulnerability of the population, the level of favorable transmission factors, and the probability for the population to be exposed to the virus. vhf are exemplary for their epidemic patterns of expansion dependent on the above reviewed factors (i.e., fundamentals of emergence) and their epidemiological characteristics (i.e., virus, host, environments). for example, let us consider the control of arenavirus spread by their strong host-species association. on a geological time scale, arenaviruses such as the agent for argentinian hemorrhagic fever (ahf) coevolved with their natural rodent host and then spread according to the expansion of the rodent host. one host-one virus ultimately produces a localized endemic cycle, the distribution of the disease overlaps the distribution of the rodent host while enzootic patterns appear naturally limited to an ecosystem (e.g., local rodent populations, behaviors, and environmental factors). hantaviruses also appear as a global complex, resulting from the coevolution of virus and rodent hosts and a global dispersion of generally localized enzootic diseases [ ] [ ] [ ] . as for the pandemic risk associated with a natural virus reservoir, chiropterans are unique flying and migratory mammals that have been associated with filoviruses and other viruses of major public health importance [ ] , their potential as vectors will eventually favor the spread of these viruses into new territories. also because there is potential for a long coevolution, epidemiological patterns are also dependent on virus-host spillover, host vicariate, and other environmental factors (e.g., climate change and man-made changes in land use). other arboviruses such as yellow fever virus, dengue virus, as well as west nile, chikungunya, or zika viruses show a pandemic risk associated with the existing distribution of their respective arthropod vector, vector density, and ability to transmit virus. investigating the fundamental factors of transmission and favorable territories for disease emergence are necessary to evaluate the risk, respond to the epidemic, and control its expansion from an index case to a pandemic. ultimately, when the fundamentals are understood and epidemic/pandemic risk identified, suitable emergency funding needs to be identified and made available in endemic areas to insure political willingness and community participation. ultimately, a suitable response will improve biosurveillance, data processing, communication, strategic immunization campaigns, and research for future risk prevention. several emblematic vhf and their original "epidemiological engineering" are presented in herein. vhf such as ebola virus disease, lassa fever, rift valley fever, or marburg virus disease are highly contagious and deadly diseases, with potential to become pandemics. remarkably, vhf are essentially caused by viruses of eight families; arenaviridae, filoviridae, hantaviridae, nairoviridae, peribunyaviridae, phenuiviridae, flaviviridae, and more recently rhabdoviridae [ ] (table ) . hemorrhagic fever viruses (hfv) have been classified as "select agents" because they are considered to pose a severe threat to both human and animal health due to high mortality rate, human-to-human transmission, and, in some cases, the potential to be aerosolized and used as bioweapons [ ] . each of these hfv shares some common features that define the nosology of the vhf group, from virus structures to the clinical and epidemiological characteristics of their diseases. -hfv spread person-to-person through direct contact with symptomatic or asymptomatic patients, body fluids, or cadavers. -vhf can have a zoonotic origin, as when humans have contact with infected livestock via slaughter or consumption of raw meat, unpasteurized milk, bushmeat, inhalation or contact with materials contaminated with excreta from rodents or bats. -hfv can be vector-borne, i.e., transmitted via rodents, mosquitos, and ticks. -vhf are zoonotic diseases. accidental transmission from the natural host to humans can eventually lead to human-to-human transmission, human infection, and sporadic outbreaks. -with a few noteworthy exceptions (i.e., ribavirin), there is no cure or established drug treatment for vhf, while limited vaccines could be available, including yf, ahf, and rvf (the latter is for animals only). -vhf have common features: they affect many organs, they damage blood vessels, and they affect the body's ability to regulate itself. clinical case definitions describe vhf with at least two of the following clinical signs: hemorrhagic or purpuric rash; epistaxis, hematemesis, hemoptysis, melena, among other hemorrhagic symptoms without known predisposing host factors for hemorrhagic manifestations. in fact, during an epidemic, all infected patients do not show these signs and a specific case definition needs to be defined in accordance with the suspected or proven viral etiology of the disease [ ]. also, vhf pathogenesis encompasses a variety of mechanisms including: ( ) alteration of hepatic synthesis of coagulation factors, cytokine storm, increased vascular permeability, complement activation, disseminated intravascular coagulation. moreover, severe pathogenic syndrome is often supported by an ineffective immunity, high viral loads, and severe plasma leakage and co-infection with other pathogens [ ] . the present chapter will mainly focus on the factors that can specifically and eventually contribute to a pandemic risk and how did we learn from historical spread of the vhf. the yellow fever disease pandemic is thought to have originated in africa, where the virus emerged in east or central africa and spread to western africa. in the seventeenth century, it spread to south america through the "triangular" slave trade, after which several major outbreaks occurred in the americas, africa, and europe [ , ] . the yellow fever vaccine is a fantastic gift from pioneering vac- cinology; it is efficient, affordable for developing countries, and protects for at least a decade or even life-long. however, yf remains a particular concern at the global level and the number of cases has unexpectedly increased this past decade. nowadays, yfv causes , infections and , deaths every year, with nearly % occurring in africa. nearly a billion people live in an endemic area [ ] . although yfv is common in tropical areas of south america and africa, it has never been isolated in asia [ ] . ultimately, the pandemic risk is there, from the uncontrolled epidemic as for example in the inland remote area of the brazilian mato grosso state, to the recent burst of epidemics in west and central africa including angola, drc, as well as imported cases in kenya and china [ , ] . indeed, the risk of a pandemic exists if any imported case goes to an area where the fundamentals of emergence are present (i.e., aedes aegypti and a nonimmune human population). for years it has been stressed that yf coverage needs to be exhaustive in the endemic area, and the who international health regulations (ihr) need to be strictly respected when peoples are crossing frontiers to or from an endemic area [ ] . even though the virus was known to actively circulate in asia, north america, and africa years ago, a global pandemic of dengue fever began in southeast asia in the s [ , ] . dengue virus (denv) expansion was followed by the emergence of a dhf pandemic that occurred in the late twentieth century (see above, the "tire-mosquito larvae connection"). by the end of the century, dhf emerged in the pacific and the americas, and extended to all asian continents [ ] . lately, in the s, epidemic dengue fever occurs in africa, with a predominant activity in east africa, while sylvatic denv circulation was described in western africa [ ] . the different dengue virus serotypes spread also independently to all continents. while it is remarkable that infection with one serotype does not provide cross-protective immunity against the others, epidemics caused by multiple serotypes became more frequent, and highly pathogenic denv were identified [ ] . dengue fever to date has a global distribution with an estimated . billion people at risk. yearly, hundreds of thousands of dhf cases occur [ ] . altogether, the requirements for a dhf pandemic are globally present [ ] : the highly competent aedes aegypti and aedes albopictus denv vectors, the globally distributed denv serotypes and highly pathogenic strains, and finally, climate change that opens new breeding opportunities for these mosquitoes to expand and eventually transmit imported denv into new populations and territories [ ] . mankind will have to live with this pandemic until the new denv vaccines can be implemented. in , an unknown disease was reported by a group of laboratory workers in west germany and former yugoslavia [ ] . over the course of months, cases and seven deaths occurred. conclusions made by treating physicians at the time (and published shortly thereafter) highlighted the following: high fatality rate, risk of relapse; risk of sexual transmission [ ] . a connection was made to infected african primates, chlorocebus aethiops, when laboratory workers were exposed to their imported tissues. it took years to effectively connect marburg virus, marv, to a bat, rousettus aegyptiacus, as a natural marv reservoir in central africa [ ] . marv is considered to be extremely dangerous for humans, is classified as a risk group pathogen, and also is listed as a select agent; however, the pandemic risk cannot be assessed because only four epidemics have occurred. although marv expansion appears to be limited to a few countries in africa, the recent emergence (estimated at a few decades ago) of a second human pathogenic marburgvirus known as ravn virus, and the widely distributed old world rousette fruit bats (rousettus spp.) serving as reservoir for both viruses [ ] , are two factors that favor pandemic risk. although more than years after its emergence from a remote area on the ebola river in the central african rain forest, ebola virus (ebov) remained hidden in a cryptic natural cycle. then a series of outbreaks occurred in the large congolese rain forest of central africa [ ] . the epidemic risk was always considered to be localized and circumscribed [ ] . then, suddenly without warning, in the late months of , ebov emerged for the first time in a remote area of western africa and sparked an outbreak more massive than ever witnessed before. more than , people were infected, ten countries recorded cases (transmitted or imported), the pandemic risk raised fear, and who declared it as an inter national health emergency that requires a coordinated global approach [ ] . besides the lack of preparedness of national and international public health systems, the other major factor that played an immense role for the dispersion of evd in western africa was the extreme mobility of village populations. they followed the kissidougou forest foot-paths to the towns in guinea using motorbikes, cars, and other public transportation, then later evd traveled by plane to the global level. the evd epidemic went from outbreak to pandemic risk. like marburg virus, another member of the filoviridae, ebola virus, shares bats as a potential virus reservoir, human and nonhuman primates are highly sensitive to the virus, and inter-epidemic periods play an important role since the epidemic silences tend to diminish the attention of health services and increase epidemic risk. in this way, the first western african evd epidemic is exemplary for showing the hidden risks contained in the natural cycle of a virus, and the sudden emergence followed by an unprecedented velocity of spreading. in the absence of biosurveillance, a pandemic risk remains. hemorrhagic fever with renal syndrome, hfrs, appears first as a global concern of one virus family, several human pathogenic viruses of the genus orthohantavirus, multiple clinical presentations, and different epidemiological patterns [ ] . hantaviruses and hfrs were first described in asia [ ] ; nowadays, hantaviruses are the cause of zoonoses that are expanding worldwide. indeed, since when a previously unknown hantavirus was implicated in the first hantavirus pulmonary syndrome (hps) outbreak in the united states, several other hantavirus infections were reported in western europe, and then hantaviruses were described in south america. ultimately, after an early suspicion of the presence of the hanta viruses in africa [ ] , a novel hantavirus, sangassou virus, was isolated in in guinea [ ] . altogether we observed the emergence of the hantaviridae in the western hemisphere, from the old world to the new world, and recently discovered its first tentative steps on the african continent. with respect to the orthohantavirus genus, a real pandemic exists even when multiple viruses are involved. ultimately, as for the arenaviridae, hosts are specific and certainly the major vectors of virus dispersion. the arenaviridae includes different viral species grouped as old or new world arenaviruses [ ] , each is maintained by rodents of individual species as natural reservoir host and as vector for the viruses that are human pathogens. the rodent hosts are chronically infected without obvious illness and they pass virus vertically to their offspring. de facto, the distribution of the virus covers that of its natural hosts but is isolated in an ecosystem generally limited by natural barriers, e.g., mountains, river. a phenomenon in which rodent lineages are naturally infected by a virus and remain in such a limited environment is called "nidality" [ ] . this is what it is observed for argentinian hf, venezuelan hf, bolivian hf, and lassa hf. regarding the pandemic risk of any of these hf, arenaviruses because of their strict association with their natural hosts, like the hantaviruses, have their expansion potential limited by their natural hosts even though the latter are widely spread and could certainly be infected. such risk lies in an unexpected encounter between infected and noninfected populations under the pressures of (as yet unknown) factors that favor their migration from enzootic to non-enzootic areas. in that matter, lymphocytic choriomeningitis virus, another member of the arenaviridae, has a worldwide distribution through its domesticated natural host, the ubiquitous house mouse, mus musculus. although crimean-congo hemorrhagic fever, cchf, is a widespread disease endemic to africa, the balkans, western asia, and asian countries south of the th parallel north, it is generally transmitted by ticks to livestock or humans and therefore geographically limited to regions where tick vectors feed on humans. although the competent ixodid vector is limited, as is the abundance of their natural hosts, climate change modifies the distribution and abundance of tick hosts (i.e., tick abundance) [ ] . additionally the cchfv pandemic risk is limited by low mobility, geographical repartition, and seasonal activity, although its main natural hosts are widely dispersed from africa, to asia and europe [ ] . ultimately, human-to-human transmission occurs from close contact with the blood, secretions, or other biological fluids of infected persons but these remain rare events with a r < . altogether, a cchf pandemic risk remains hypothetical but underlined by the risk of human-to-human transmission [ ] . as for cchf, rift valley fever, rvf, is first a disease of cattle and illustrates a unique subcontinental zoonotic spread along the path of traditional herders. rvf became a transcontinental risk with trade and transportation when the virus spread from north east africa to western africa, and even to madagascar [ ] . if one considers its pandemic risk, with respect to rvf epidemiology as a mosquito-transmitted disease, two factors have to play concomitantly: the presence of infected cattle (i.e., nonimmune) and competent mosquito abundance, both considered hazards, while concretizing the risks from human vulnerability (nonimmune; mosquito bite; direct exposure to infected blood). in order to streamline the prevention and the actions to reduce epidemic risk, the various elements involved in an outbreak are here considered from a systemic point of view, considering the risk as the convergence of a hazard and vulnerability: -the presence of the threat (or "hazard" pathogen, i.e., vector, virus reservoir) is considered to be a necessary-but not sufficient-condition for the development of a disease. it is often known only in terms of probabilities, sometimes very low and therefore often subject to significant random variability in time and space. we often seek to evaluate the spatial and temporal differences of this probability, trying to measure its significance. sometimes, it only uses one character necessary to the presence of the pathogen or vector (e.g., the presence of water, a minimum temperature, a type of vegetation). -the susceptibility of the host (which is essentially linked to individual characters, genetic, biological, such as immune status or age) is individual, and often given by a probability. -direct exposure of the host to the hazard is an element of active vulnerability, depending on the behavior of the host that increases the likelihood of contact between host and hazard by exposing it to an environment conducive to his presence (e.g., travel and contacts, professional activities). it also includes all the known "risk" behaviors that increase the likelihood of direct exposure to the hazard. -passive vulnerability of the host, which is not directly dependent on the pathology, is not even necessary nor sufficient for pathology, but influences the exposure of the host to the hazard or to protection from the pathology. this protection consists of prophylaxis, access to care, availability of care. it is independent of the real presence of the hazard; the host can be vulnerable without being exposed to the threat. the vulnerability is often defined by several levels (individual, context). it is very often "spatial" as linked to phenomena of segregation or spatial concentration. this is an area primarily studied by geography. ultimately, this vision can differentiate what is active, often subject to high variability, random in time and space (the emergence or the presence of hazards is often difficult if not impossible to control) from what is passive, generally situated among more stable population levels (sensitivities, exhibitions, behaviors, and vulnerabilities). this allows for better public health preventive actions, and also to understand rationally crisis situations by preemptively targeting the most important elements of the system in terms of vulnerability, and secondly by optimizing risk reduction (elimination of vectors, vaccinations, quarantine, etc.). in all cases, these actions must be adapted to social contexts to have a real impact on risk behaviors and vulnerabilities that they generate, hence the increasing role of anthropology in the field of health. to prevent or reduce the epidemic risk, it is necessary to act on each component of this system: -reducing the susceptibility of the host (e.g., immunization, vaccination, prophylaxis). -reducing host exposure to the pathogen (e.g., vector control, quarantine, exclusion zone). -eliminating the pathogen directly (e.g., animal slaughter, disinfection, hygiene), or indirectly (e.g., suppress transmission). -reducing host vulnerability (e.g., socio-economic, behavioral, access to health care system). -reducing host exposure to emergency condition (e.g., realtime data collection, warning systems for emergency, crisis management, implementation of treatment). the rapid detection of emergence is the key to controlling the spread of an epidemic. it requires comprehensive monitoring to trigger alerts and all other risk-reducing actions, in particular, reducing the exposure of the host to the pathogen and, if possible, the elimination of the pathogen. in parallel to the monitoring and warning systems, protocols must always take into account local characteristics of political power and decision-making bodies that could otherwise render ineffective year-long action plans or warning systems (for example, the management of the chikungunya epidemic in reunion island was largely impacted by bottlenecks related to local political system) [ ] . biosurveillance and efficiency in data collection and management will be the technical keys for prevention (early detection of epidemic risk) and forecasting epidemic emergence and spread (i.e., analyzing the data in near real time taking into account the vulnerability of a given population). also, this can be achieved only by exhaustive capacity building (human and technical) mostly in the more vulnerable developing countries but also where the most advanced technology needs to be developed. networking biosurveillance systems are a major undertaking from regional to global, involving politics and diplomacy. taking in account the local characteristics of political structures and decision systems is fundamental. despite our current recognition of the risks posed by emerging and re-emerging infectious diseases to global public health and stability, reliable structured data remains a major gap in our ability to measure (and therefore manage) globally infectious diseases. who has long served as an information hub for infectious disease events worldwide; however, extracting quantitative data from who information bulletins (weekly epidemiological record and the more recent disease outbreak news alerts) proves to be a time-consuming effort with limited results in terms of operability, and exists more for the record and future analysis. the current proliferation of geospatial information tools (i.e., geographical information system, gis) and stepwise advances in data extraction capabilities have made it possible to develop robust, systematic databases facilitating anomaly detection (like clusters), infectious disease models (and model evaluation), and apples-to-apples comparisons of historic infectious disease events worldwide. however, biosurveillance capabilities-the key to global prevention and health securityremain inadequate to support true early detection and response. increased access to technology, rapidly developing communications infrastructures, smartphone usage for suspected-case reporting, and global networks of (formal and informal) disease surveillance practitioners provide an explosive opportunity to patch and improve surveillance networks. the challenge is to leverage all these developments, implement technical and capacity building where needed, before the next epidemic with global impact emerges. several organizations have developed systems to collect epidemic information and facilitate rapid response: who has the department of pandemic and epidemic diseases (ped) that develops mechanisms to address epidemic diseases, thereby reducing their impact on affected populations and limiting their international spread. among them some have self-explanatory titles: the battle against respiratory viruses (brave); early warning and response systems for epidemics in emergency (eware); emerging and dangerous pathogens laboratory network (edpln); international coordinating group for access to vaccines for epidemics (icg); global infection prevention and control network; (gipcn ); global influenza surveillance and response system (gisrs); global leptospirosis environmental action network (glean); meningitis environmental risk information technologies (merit); weekly epidemiological record (wer); emerging diseases clinical assessment and response network (edcarn). global commitment to these efforts will insure their readiness in times of need. most certainly and most importantly, any preparedness and response requires emergency funding [ ] . it has been estimated that if the ebola virus disease response started months earlier, it could have reduced the total number of deaths by % in liberia and sierra leone [ ] . we learned from this last evd epidemic that in march , the african union's minister of finance requested the african risk capacity (arc) agency to help member states to better plan, prepare, and respond to devastating outbreaks by developing new applications for financial tools, like insurance, that can significantly improve the speed of funds to affected countries and shorten the time between event and response. the agency is now developing an outbreak and epidemic insurance product primarily based on responsibly and timely budget reallocation; however, viruses do not wait. moreover, the world bank's pandemic emergency facility is designed to finance surge capacity and support international government partners to actively participate to the response. ultimately, epidemics are not one-off events, but rather demonstrate financial patterns similar to other natural catastrophes. as natural catastrophes, large epidemics can be insured by creating financial mechanisms to facilitate the movement of critical resources within affected countries and ultimately manage the spread of disease and minimizing macroeconomic impact [ ] . classical tools and strategies for predicting epidemics encompass human disease surveillance (e.g., public health and hospital statistics) and, sometimes, environmental surveys (e.g., climate, el niño, earthquake, tsunami); also more recently complying with one health concept, human and veterinary health as well environmental risk factors have been reunited in a comprehensive approach of public health risk (i.e., outbreak, epidemic risks). however, this heuristic approach of health remains limited to specific diseases and territories and does not apply as a global predictor of pandemics. first, historical data is the only available objective view of past epidemics and pandemics, needs to be collected, formatted, corrected, and analyzed. this will be the foundation of the different tools and strategies described below. in that matter, with respect to the depth of the past data available, time series of disease observation, modern tools such as internet search data have actually led to the development of several specific sites (e.g., google flu and dengue) [ ], whose search-term reports have correlated strongly with incidence estimates in several public health reports in europe, asia, and the u.s. however, even though such tools can complement classical disease surveillance, most of these sites are geographically limited and cannot be used for live monitoring of epidemic risk and for neglected tropical disease surveillance [ , ] . however, from such historical and live-collected data, health alert systems can be implemented, and prediction models can be developed. moreover, thanks to the spatial analyses, combining multiple data sources will provide the ultimate tools for livemapping an outbreak, which will lead to an efficient response when tools and strategy have been specifically identified (i.e., sufficient and available in-country heath system resources and funding; identifying variations in pathogen sequences that contribute to ro and pathogenicity; monitoring population movement; etc.). the amount of data being digitally collected and stored is exponentially accumulating. it is estimated that, as of september of , the world wide web reached . billion pages containing eight zettabytes of accessible data, and the accumulation of information is growing around % every year [ ] . this situation has generated much discussion about how to use the unprecedented availability of information and computational resources and the sophistication of new analytic and visualization algorithms for decision-making to reduce the impact of infectious diseases. in fact, it is argued that the paradigm of "big data" will change not only the way business and research is done, but significantly improve the understanding of factors leading to the emergence of infectious diseases. big data could lead to the implementation of a decentralized biosurveillance enterprise allowing organizations and individuals to take full advantage of a large collection of disparate, unstructured qualitative, and quantitative datasets. with the proper integration and the right analytics, big data could find unusual data trends leading to better pathogen detection systems, as well as therapeutic and prophylactic countermeasures. however, the impact of these analyses and forecasts depends not only on how the data is collected, ingested, disambiguated and processed, but also on how it is relayed in different operational contexts to users with different backgrounds and understandings of technology. while impressive in data mining capabilities, real-time content analysis of social media data misses much of the factual complexity. quality issues within freeform user-provided hashtags and biased referencing can significantly undermine our confidence in the information obtained to make critical decisions about the natural versus intentional emergence of a pathogen. risk factors associated with a health event in a population are often linked to environmental factors (fig. ) . they are also linked to spatial relationships between individuals, especially for infectious diseases. the geographical distribution of these phenomena reflects spatial relationships. beyond "classic" epidemiology mainly based on statistical analysis, using the location and spatial distribution is essential in the understanding of health events and analysis of their mechanisms. spatial analysis in epidemiology is a method to help determine the location (georeferenced) of risk factors. it allows one to identify the spatial and temporal differentiation in the distribution of events, using their location in time and space. when the location is available, with precision for each studied object (i.e., individuals or geographical units), it is possible to: -characterize the overall spatial distribution, using synthetic indices on the absolute position of an object, on the average spatial arrangement of objects or their values (grouping/ fig. mapping environmental factors that have a major impact on insect vector population (i.e., mosquitoes and ticks). this map of laos constitutes the basis of a risk map showing part of the hazards contributing to virus vector density that could be matched with human density and pathogen prevalence leading to a risk map (spatial risk) and eventually extended through seasonality (temporal risk). mean temperature and mean rainfalls are interpolated as climatic conditions, as environmental factors influencing the presence of mosquitoes dispersion, spatial dependence, variogram measure of autocorrelation space). -look for characteristics of the overall shape of the phenomenon (tendency, shape), and search for a theoretical spatial distribution, or for a process to model the observed spatial distribution. -look for unusual places (geographical centers and source sites; aggregates; exclusions; hot spots, cold spots), and to study the spatial relationships at the individual level. -conduct spatiotemporal analysis: search index cases, reconstruction of paths, diffusion models, models of extinction, etc. -spatial analysis allows the development of applications for modeling epidemics, preparing warning systems, as well as crisis management systems, risk prevention and analysis systems, and vaccination campaigns. many tools for biomonitoring and prevention of epidemic risk have been developed (fig. ) , as well as software tools to: (a) visualize spatial distributions. (b) synthesize and analyze position and spatial relationships between events (continuity, consolidation, attractionrepulsion, shape, centrality, displacement, diffusion processes). (c) to analyze the relationship between spatial distribution of attributed values and environmental characteristics of the phenomenon (environmental correlations). (d) to model the phenomena of emergence, dissemination, extinguishment of an epidemic. cluster detection, space-time analysis, and spatial integration with environmental and demographic data are widely used in such warning systems. multiple and complex factors are associated with the emergence and impact of pathogens in a given geographical area. therefore, public health analysts are confronted with the task to identify the likely, and unlikely, consequences and alternative critical outcomes of a given vhf outbreak. this requires the ability to monitor in near real time the dynamics of the geographical dissemination of these viruses in villages, cities, countries, continents, or the globe using new analytical techniques within the emerging field of genomicbased biosurveillance. this concept integrates microbial genotyping, next generation sequencing, metagenomics, big data and database analytics, and contextualized visualization to identify, characterize, and attribute known and unknown pathogens and generate estimates of how different contingencies will affect their impact [ ] . a genomic-based biosurveillance system includes powerful microbial genomic characterization to rapidly identify a pathogen [ ] . this characteristic makes a genomic-based biosurveillance a useful approach not only for public health but serves as a deterrence tool for intentional biological weapon development and deployment. the initial step consists of integration of signals generated by molecular-based assays and next generation dna sequencing and unbiased microbial characterization for pathogen source tracing, attribution and forensics. while each of these techniques has been discussed in the literature in detail [ ] , the integration of this information can yield a more extended view of the scale of a pathogen outbreak. the development of high-throughput the exemplary case of the highly pathogenic avian influenza virus h n in thailand. from the emergence of one imported case (red-filled circle), the pathway direction (arrowed green lines) of h n infection in farms (yellow points) is reconstituted, using dates of infection and distance between farms. results show local spread with time-to-time medium distance jumps dna sequencing technologies (i.e., dna and cdna forms of rna viral genomes) is allowing the genomic characterization of previously unknown pathogens without relying on prior reference molecular information [ , ] . this information is available within days, and even hours, of sample collection, and well before the development of animal infection models. because of their portability, this technology will become widely used in the next years in routine clinical settings. however, to be clinically and epidemiologically relevant, dna sequences must be rapidly and effectively translated into actionable information defining pathogen characteristics (i.e., virulence or drug resistance), it must point to a source of origin, and discriminate a natural event from a manmade release [ ] . while some government agencies are considering use of genomic information to develop next generation level- and level- detection/surveillance devices [ , ] , there is no reference database where researchers can retrieve standardized genomic signatures and motif fingerprints to develop primer-, probe-, and antibody-based detection technology using reference moieties. the impact of genomic-based biosurveillance in public health and biodefense will not be fully realized until addressing the current impracticality of transferring the terabytes of genomic data generated by dna sequencing devices to a centralized architecture performing analysis operations, as that might take hours or even days. therefore, a new paradigm could emerge from encouraging the development of decentralized algorithms that first determine in situ the presence of pathogen-specific genomic signatures or motif fingerprints, summarize and relay the results into an operational biosurveillance metadata format for contextualized decision support. the localized data management, time, and space required for spatial analysis is performed by geographic information systems (gis). these are computer systems that manage large volumes of data and easily use the location to perform spatial analysis. most gis are not limited to data management functions, but also integrate multiple analysis tools, data transformation, and cartographic representation. these are for the most part complex applications with enormous features. the "gis" designation covers a wide variety of software projects built according to different technical options, functionality, and diverse performances. a gis is essentially a management tool (structure, organization, entry, storage), an analytical tool (statistical and geographical treatment, spatial analysis), and a communication tool (data visualization, descriptive mapping, thematic mapping, atlas). it is also a tool that allows the use of a spatial model for the simulation of a process, such as the development of an epidemic. gis facilitates the interface between modeling and simulation program, and the geographic database, and can ultimately take over the whole of access to spatial information needed by the modeling program. the gis should thus be at the heart of organizing the collection and processing of monitoring data. to ensure the management of this system, it is important to set up a body specifying all the collection, validation, processing and dissemination of information and results (alerts, risk modeling, near real-time dissemination of results). this body must be proposed and validated by political authorities, preemptively, to avoid further blockage and to ensure effectiveness in situations of epidemic crisis. mathematical modeling is a mathematical formulation of a parameter or risk; it depends on identified or hypothesized risk factors whose coefficients are determined by a statistical or heuristic analysis from historical or observed data with the use of r , as a basic reproduction rate, to timely and spatially predict the spread-speed of an emerging outbreak. spatial-temporal modeling of health events can be seen as the final stage of the analysis. it is different from statistical modeling. despite using risk factors, it considers the epidemic phenomenon as a whole, taking into account the spatial relationships between agents (hosts, vectors, reservoirs, and pathogens), between individuals, and relationships between individuals and their environment. this model is thus useful for understanding and anticipating the epidemics, and can be generally used to classify individuals in different states (susceptible, infected, sick healed, immune) and to model the major phenomena that can change the state of an individual. however, when a model takes into account many phenomena, it can quickly become very complex. the vast majority of models are simplifications of assumed reality. two broad categories of methods are usually developed in modeling: -a deterministic approach, based on differential equations whose coefficients are adjusted from observed data, or monitoring data from epidemics. in this model, one can introduce stochastic types of components in the coefficients, studying the variability of observed data. taking no account of spatial relationships is difficult in these models, which deal in general populations, not individuals. -a nondeterministic approach, which is based on agents whose behavior is described by expertly determined rules (multiagent models). the status of each agent is calculated at each time step, from its behavior, environment, and relations between the agent and all other agents. these models take into account a more realistic description of the phenomenon, near the complex system finely describing reality. they allow us to consider spatial relationships in each time step. these models require intensive calculation, and their use is made possible by development of the power of computer calculations. let us first honestly address the fundamental questions about epdimeics and preparedness: what did we learn from all the past epidemics, what will we remember in times of need? are we prepared for the worst of these hypothetic pandemics abundantly illustrated in the cinema and unfortunately sometimes overwhelmed when reality goes beyond fiction? certainly, we are not "globally" prepared, unfortunately, at that scale, the immense natural and human disparities do not permit it, but we do our best in our own societies. the concept of disease emergence, born only at the end of the twentieth century, is a societal marker, our desire to be on alert, understand and predict epidemics. ultimately, there are a few, but necessary and difficult goals to reach for the prevention and control of any epidemic, also these goals are part of the development of our societies, as well as for education, they become part of the wellbeing for all: first, beyond understanding transmission, is needed a clear understanding of the epidemiological pattern and the spread of a given disease, before it is too late; then, which is certainly one of the more complex and costly things to achieve, is having an efficient health system to respond to an epidemic and an operational network to respond at the regional and global levels; and last but certainly not a least, having identified funding for any public health emergency will be crucial to changing our world. perhaps, in a shrinking global community, after too many ebola virus disease outbreaks, we will learn and be prepared for future epidemic challenges? the progress made, mostly by computer sciences in the overall analysis of health data, should serve as a tool in the prevention of major epidemics. let us ultimately use our predictions of pandemic risk to meet and unite beyond the current frontiers of political and social wills. epidemic predictions in an imperfect world: modelling 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in sierra leone: stalking the virus in the threatening wake of emergence perspectives on west africa ebola virus disease outbreak aedes alpobictus and the world trade in used tires, - : the shape of things to come? men, primates, and germs: an ongoing affair seewis virus: phylogeography of a shrew-borne hantavirus in siberia coevolution of rodent and viruses: arenaviruses and hantaviruses the arenavirus and rodent coevolution process: a global view of a theory bats worldwide carry hepatitis e virus-related viruses that form a putative novel genus within the family hepeviridae a novel rhabdovirus associated with acute hemorrhagic fever in central africa federal select agent program: select agent and toxins list pathogenesis of the viral hemorrhagic fevers viruses, plagues, and history: past, present and future yellow fever fact sheet n° . world health organization yellow fever in africa: estimating the burden of disease and impact of mass vaccination from outbreak and serological data the changing epidemiology of yellow fever and dengue resurgence of yellow fever in angola international health regulations in practice: focus on yellow fever and poliomyelitis yellow fever in africa: a disaster waiting to happen dengue/dengue hemorrhagic fever: the emergence of a global health problem delaporte ( ) rapp. au ministre de la marine the global epidemiology of infectious diseases, global burden of disease and injury series epidemiology of arthropod-borne viral disease the xxth century dengue pandemic: need for surveillance and research emergence of epidemic dengue/dengue hemorrhagic fever as a public health problem in the americas zur Ätiologie einer unbekannten, von affen ausgegangenen menschlichen infektionskrankheit isolation of genetically diverse marburg viruses from egyptian fruit bats virological and serological findings in rousettus aegyptia cus experimentally inoculated with vero cellsadapted hogan strain of marburg virus ebola virus circulation in. africa: a 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epidemiology - the chikungunya epidemic on la réunion island in - : a cost-of-illness study global health risk framework: pandemic financing: workshop summary african risk capacity. executive perspective: outbreak and epidemic insurance, new solution to an old problem. the rockefeller foundation from the ebola river to the ebola virus disease pandemic: what have we learned? using web search query data to monitor dengue epidemics: a new model for neglected tropical disease surveillance evaluation of internetbased dengue query data: google dengue trends biosurveillance enterprise for operational awareness, a genomic-based approach for tracking pathogen virulence bioinformatics for biodefense: challenges and opportunities biosurveillance of emerging biothreats using scalable genotype clustering optimizing biosurveillance systems that use threshold-based event detection methods biodefense oriented genomic-based pathogen classification systems: challenges and opportunities biosurveillance observations on biowatch generation- and other federal efforts: testimony before the subcommittees on emergency preparedness, response, and communications and cybersecurity, infrastructure protection, and security technologies, committee on house homeland security, house of representatives congress. house ( ) committee on homeland security. subcommittee on emergency preparedness response and communications., united states. congress. house. committee on homeland security. subcommittee on cybersecurity infrastructure protection and security technologies., united states. government accountability office: biosurveillance observations on biowatch generation- and other federal efforts: testimony before the subcommittees on emergency preparedness, response, and communications and cybersecurity, infra structure protection, and security technologies, committee on house homeland security, house of representatives acknowledgments w.a. valdivia-granda has been funded by the department of homeland security and the department of defense. we are greatfull to sarah cheeseman barthel, director, data acquisition & management metabiota, inc., for her review and input of the section on "global surveillance and data collection." key: cord- -ctass hz authors: bull, james j.; nuismer, scott l.; antia, rustom title: recombinant vector vaccine evolution date: - - journal: plos comput biol doi: . /journal.pcbi. sha: doc_id: cord_uid: ctass hz replicating recombinant vector vaccines consist of a fully competent viral vector backbone engineered to express an antigen from a foreign transgene. from the perspective of viral replication, the transgene is not only dispensable but may even be detrimental. thus vaccine revertants that delete or inactivate the transgene may evolve to dominate the vaccine virus population both during the process of manufacture of the vaccine as well as during the course of host infection. a particular concern is that this vaccine evolution could reduce its antigenicity—the immunity elicited to the transgene. we use mathematical and computational models to study vaccine evolution and immunity. these models include evolution arising during the process of manufacture, the dynamics of vaccine and revertant growth, plus innate and adaptive immunity elicited during the course of infection. although the selective basis of vaccine evolution is easy to comprehend, the immunological consequences are not. one complication is that the opportunity for vaccine evolution is limited by the short period of within-host growth before the viral population is cleared. even less obvious, revertant growth may only weakly interfere with vaccine growth in the host and thus have a limited effect on immunity to vaccine. overall, we find that within-host vaccine evolution can sometimes compromise vaccine immunity, but only when the extent of evolution during vaccine manufacture is severe, and this evolution can be easily avoided or mitigated. recombinant vector vaccines are live replicating viruses that are engineered to carry extra genes derived from a pathogen-and these extra genes produce proteins against which we want to generate immunity. these vaccine genomes may evolve to lose the extra genes during the process of manufacture of the vaccine or during replication within an individual, and there is a concern that this evolution might severely limit the vaccine's efficacy. the dynamics of this process are studied here with mathematical models. the potential for vaccine evolution within the host is somewhat limited by the short-term growth of the vaccine population before it is suppressed by the immune response. we find that evolution is a problem only when the process of manufacture results in the majority of the vaccine virus being revertant. we show that increasing the vaccine inoculum size or reducing a a a a a live vaccines replicate within the host. as true of any reproducing population, these withinhost vaccine populations may evolve. for live vaccines that do not transmit, any within-host evolution is a dead end and might thus seem to be irrelevant to vaccine function. but if the process is fast enough, or the vaccine population replicates long enough, the vaccine population may evolve to a state where it is ineffective or virulent-either change would be bad. the two main types of live viral vaccines are attenuated and recombinant-vectored. most live virus vaccines in use today are attenuated, their reduced virulence typically achieved by adapting the wild-type virus to a new environment (e.g. replication in a novel cell line or low temperature), with a consequent reduced replication rate in humans. the use of attenuated vaccines is too risky for pathogens such as hiv, and a safer alternative is to develop a live, recombinant vector vaccine where one or a few pathogen genes with immunogenic activity (proteins that elicit protective immunity) are expressed from a benign virus vector. the expected consequences of within-host evolution differ between these two types of vaccines ( table ) . evolution of an attenuated vaccine is likely to be a reversion toward the wildtype state, the rate of this process depending heavily on vaccine design and the duration of vaccine virus replication in the host (reviewed in [ ] ). to a first approximation, reversion toward the wild-type state should lead to the vaccination more closely resembling natural infection [ ] , such as higher virus densities, side-effects and disease, and possibly an increased immune response. within-host evolution of an attenuated vaccine might also predispose the virus to better transmission-also reflecting the wild-type state-but this outcome is not assured: viral adaptation to different tissues within the host may hamper growth in and dissemination from tissues important in transmission (e.g., [ ] ). the expected consequences for evolution of a recombinant-vectored vaccine are fundamentally different [ ] . in most cases, the antigen against which immunity is sought comes from a foreign transgene inserted into a competent viral vector without replacing any vector genes. vectors in development include adenovirus, vsv (vesicular stomatitis virus) and cmv (cytomegalovirus). the vector genome carries out all viral amplification and transmission functions, and the transgene does not contribute to any process benefiting vector reproduction. from an evolutionary perspective, the transgene is both dispensable and potentially costly: selection may favor loss of the transgene and thus loss of vaccine's ability to elicit immunity against the antigen encoded by the transgene. this evolution therefore generates something akin to infection by the wild-type vector. as vectors are typically chosen to be avirulent for immune competent hosts, vaccine evolution will result in no more than a harmless infection that does not generate immunity to the antigen encoded by the transgene. considerable attention has recently been given to the evolution of attenuated vaccines and designs that retard their evolution. evolutionary stability of attenuated vaccines seems attainable by engineering designs, including the introduction of hundreds of silent codon changes, genome rearrangements, and some types of deletions (comparisons and reviews are provided by [ , , ] ). far less thought has gone into the consequences of evolution for recombinant vector vaccines or of strategies to minimize this evolution. although recombinant vector vaccines are not yet in widespread use, many are under development [ , ] , and their success may rest on understanding within-host evolution. here we explore how the combination of evolution during the process of vaccine manufacture and during its within-host dynamics following vaccination could affect the immune responses elicited by a recombinant vector vaccine and reduce its efficacy-the specific interaction between evolution and immunity. we consider viral vaccines and focus on vaccines that cause shortduration (acute) infections. the ideas we discuss also apply to live vaccines of bacteria and other pathogens. our overall message is that while vaccine evolution may occur, it is either unlikely to be a problem (i.e., compromise the generation of immunity), or it is easily mitigated. when vaccine evolution does limit the adaptive immune response, we identify ways of escaping such outcomes. our analysis rests on mathematical models, but most results can be explained intuitively (perhaps only in hindsight), with the main results illustrated graphically; many analyses are relegated to supporting information. our analysis assumes that vaccines replicate within the host untill cleared by host immunity; we exclude vaccines that reproduce for just a single infection cycle (e.g., modified vaccinia virus ankara), as they have no significant opportunity for evolution. our models are numerical analyses of ordinary differential equations. the equations are given in supporting information (s appendix). the were numerically evaluated and graphed in r (s file, a markdown file), sometimes also evaluated in mathematica (s file). the key question is whether evolution of the vaccine virus (henceforth just 'vaccine') meaningfully affects immunity to the antigen encoded by the foreign transgene (henceforth just 'antigen'). the potential for vaccine evolution is easy to understand. through mutation, any large vaccine population will contain mutants that inactivate or delete the foreign transgene, and those revertants will then grow amidst the vaccine. vaccine inferiority may accrue in two different ways: the transgenic insert and its expression may intrinsically impair vaccine growth, and adaptive immunity to the foreign antigen may impair the vaccine's growth but not the revertant's during an infection. it is easy to appreciate how and why the vaccine may be inferior to the revertant, and this can result in an increase in frequency of the revertant. however, the relationship between this evolution and the extent of immunity to the vaccine antigen is more complex. we thus explain some of the factors that affect how this evolution translates into a reduction in immunity to the antigen, and why in some circumstances, substantial evolution can result in little change in immunity to the antigen, while in different situations it can result in a substantial reduction. two realms of vaccine evolution. vaccine evolution can be inimical to immunization by limiting vaccine antigen levels in the host. as noted above, one realm in which evolution may occur is within the host, starting with the inoculum and ensuing until vaccine clearance. a second realm of evolution affecting antigen levels is that occuring during manufacturingduring growth of the virus to prepare the stock used for inoculation. both realms are part of a continuum, any evolution during manufacturing advancing evolution within the host (fig ) . the inoculum sets the starting point for within-host evolution, and indeed, an inoculum that is mostly revertant will limit vaccine efficacy even if no further evolution occurs within the host. from population genetics principles [ ] , even if the inoculum has a seemingly low frequency of revertant, the evolution that has occurred prior to inoculation may greatly accelerate within-host evolution (fig ) . paradoxically, when considerable evolution occurs in manufacture, such that the inoculum is primarily revertant, there is little opportunity for further evolution within the host-the damage is already done. the effect of any 'pre-host' evolution on the host immune response is potentially as important as the effect of within-host evolution. an important difference between the two realms is that pre-host evolution may be more easily mitigated than is within-host evolution. that is, controlling pre-host evolution may be a feasible way to limit within-host evolution and to limit the loss of immunity from vaccine evolution. since pre-host and within-host evolution represent different realms on a continuum, we adopt a language that attempts to distinguish them and avoid confusion. we use the following: • 'pre-host evolution' refers to evolution during manufacture that affects inoculum composition • 'within-host evolution' refers to evolution that occurs within the host after inoculation • 'evolution' refers to either or both of the above. it is easily appreciated that the specifics of evolution in the two realms may differ-vaccine growth and fitness in an in vitro environment (pre-host) will often differ from that within the host. regardless, however, any pre-host evolution will advance subsequent within-host evolution, unless the revertant is selected in opposite directions pre-host and within-host. there may even be a common molecular basis to vaccine inferiority in both the pre-host and withinhost environments that will render the two processes somewhat similar (see below). a short duration of infection limits within-host evolution. any fitness advantage of revertant means that its frequency-its abundance relative to the vaccine-will increase during active viral growth (fig ) . however, when the infection caused by the vaccine is acute, as we consider here, the magnitude of possible within-host evolution is limited by the short duration of viral growth before clearance. if the inoculum is largely free of revertant, even a moderate fitness cost of the vaccine may have little effect on vaccine evolution, such that vaccine fitness effects and evolution can be ignored. evolution versus immunity. surprisingly, vaccine evolution per se need not reduce the immune response, even when its magnitude is large. if overgrowth by revertant does not interfere with vaccine growth, then vaccine growth and antigen production are not affected (fig ) . evolution affects antigen production only to the extent that revertant superiority suppresses vaccine growth and thereby suppresses antigen production. numbers versus frequencies. models of evolution often address relative frequencies, on a scale of to . immunity develops in response to vaccine density. in addressing immunity, it is thus necessary for the model to track densities, whereas any evolution is more easily described with frequencies (the two approaches can be compared between figs and ). the challenges are thus to understand (i) when and how much vaccine evolution occurs; (ii) whether and to what extent evolution affects the abundance of vaccine virus in the host over time; and (iii) how changes in vaccine abundance affect the generation of adaptive immunity against the antigen. the arguments presented above are qualitative and only superficially identify the scope of the problem. quantitative understanding ultimately rests on analysis of mathematical models. however, as the models have many interacting processes-minimally innate immunity, adaptive immunity and intrinsic growth differences between vaccine versus revertant-we first verbally explain the biology underlying the processes that go into those models. intrinsic fitness differences. intrinsic fitness effects are considered here to be those that stem from the intracellular processes of viral gene expression and assembly, independent of host immune responses. intrinsic fitness differences between the vaccine and the revertant (wild-type vector) are plausible because the transgene is non-essential and has no evolutionary history with the vector genome. thus, the insertion may be disruptive, and the resulting antigen expression may interfere with vector functions. intrinsic fitness effects are expected to affect evolution during vaccine manufacture as well as within-host evolution, but it has largely been investigated in vitro, as would apply to manufacture and the pre-host phase. indeed, intrinsic fitness differences may be the sole or at least the most important bases of vaccine inferiority. because recombinant viruses have often been observed to evolve loss or down-regulation of engineered inserts, they are now commonly observed during in vitro growth for their 'genetic stability' (e.g., [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). some recombinant viral genomes are stable over short term transfers in culture, others not, indicating that intrinsic fitness effects of the engineering are not universal. thus, the possibility of vaccine inferiority should not be ignored, and furthermore, even when a vaccine appears to be stable over a few transfers, the short term population where the revertant has a % fitness advantage and starts at a frequency of − . the curve shows the well-known population genetic principle that, while the favored type (revertant) is rare, its absolute frequency changes very little. but the frequency eventually reaches a level at which evolution is rapid. the yellow box represents a possible period of pre-host evolution, the green box representing the period of within-host evolution. the periods of within-host evolution are drawn to be the same length in right and left panels, as if the vaccine has the same within-host duration in both cases. the arrow represents a possible point at which manufacture would end and an inoculum be created, thus defining the boundary between pre-host and within-host evolution. the left panel depicts a short period of vaccine manufacture, the right a longer period of vaccine manufacture and one in which more pre-host evolution has occurred. it is thus easy to see the potential importance of pre-host vaccine evolution on within-host evolution, for even when the revertant is not a large component of the inoculum, it can be poised for rapid evolution within the host (right panel). the curve obeys p t ¼ p w t p w t þ À p , in which p t represents the revertant frequency in generation t and w the fitness of revertant relative to vaccine. the curve is drawn for a common evolutionary process across pre-host and within-host evolution, but evolution in the within-host phase will typically experience different parameters than evolution in the pre-host phase. retention of antigen expression may mask an underlying long term instability. thus most observations of stability merely set limits on the possible magnitudes of inferiority. yet even if vaccine selective 'neutrality' turns out to be fleeting, merely a mistaken impression from shortterm observations, we will find that the phenomenon of short-term stability mirrors a solution to minimize vaccine evolution within the host-the solution of limiting vaccine growth. the revertant virus has the superior growth rate, but in the absence of interference between the two, vaccine growth is unimpeded and immunity is triggered. https://doi.org/ . /journal.pcbi. .g three mechanisms of vaccine-revertant interference. fig presented a hypothetical case in which evolutionary superiority of revertant did not suppress vaccine growth, hence evolution had little effect on antigen production. that process was one in which there was no interference between vaccine and revertant growth. evolution does become important to antigen levels if vaccine and revertant interfere so that vaccine growth is depressed by the revertant, or if the duration of infection by the vaccine strain is reduced. in either case the revertant will then suppress antigen levels. again, the problem is complicated by the limited duration of the infection: reduced antigen production due to vaccine evolution depends not only on interference between the two genomes but also on overall growth and the extent to which it affects the level of immunity to vaccine and vector. a mechanism that forces interference between vaccine and revertant can also limit the total amount of viral growth, thereby limiting evolution. evolution of vaccine versus revertant thus depends on details, in particular, the specific mechanism by which revertant interferes with vaccine growth. we describe three different mechanisms that have been proposed that may be relevant to vaccine competing with revertant: innate immunity, resource limitation, and adaptive immunity to the vector backbone shared by the vaccine and revertant virus. (these are not the only possible mechanisms of within-host parasite competition [ ] , but they capture the relevant immune processes.) for many vaccines, each mechanism will impede revertant and vaccine equally as a collective population, thus ensuring interference. it was initially believed, implicitly if not explicitly, that the adaptive immune response played the dominant role in the control of viruses and other infections. in the 's, janeway and medzhitov identified shared pathways for the control of pathogens between vertebrates and drosophila, even though drosophila lacks an adaptive immune response (reviewed in [ ] ). this led to a resurgence of interest in the role of innate immunity in the initial control of infections. later modeling studies of influenza infections suggested yet another mechanism, that the initial control of these infections could be largely described by simple resource limitation models, of the type used in ecology for population growth [ , ] . the realization that all three different processes might suppress viral infection led to more careful examination of the roles of different factors in the early control of acute infections [ ] [ ] [ ] [ ] . the relative role of each mechanism in clearing infections is the basis of ongoing discussion, but it is widely accepted that the roles differ among infections by different viruses and that each mechanism is potentially important for some viruses. • innate immunity. there are two broad arms of immunity for suppressing vaccine growth within the host, the innate and the adaptive immune responses. innate immunity is triggered by conserved molecules associated with pathogens (pathogen associated molecular patterns, [ ] ). conserved structures of pathogens targeted by innate immunity include dsrna, frequently accompanying viral replication, plus lipopolysaccharides and endotoxins of bacteria [ ] . because innate immunity involves the activation of a standing population of immune cells such as macrophages and dendritic cells, or triggering of the complement pathway, it can be elicited much more rapidly than the adaptive response; the latter requires many rounds of clonal expansion of rare antigen-specific cells to generate a population large enough to control the infection [ ] . furthermore, recent studies have shown that the innate response is required for the initial stimulation of the adaptive response [ ] . thus, innate immunity has a major role in early control of the viral population. innate immunity can control both vector and vaccine, and it is not likely to discriminate between two genomes that differ by a single, non-essential gene (the transgene). • resource limitation another mechanism by which revertant levels can suppress vaccine levels is resource limitation. both the vaccine and revertant virus use the same resource (susceptible host cells). resource limitation can control the infection if the virus depletes this resource, whereby the rate of virus output falls below its intrinsic death rate [ ] . like innate immunity, resource limitation is expected to affect vaccine and revertant similarly. resource limitation has been considered an important mechanism for competing malarial strains within the host [ , ] . • adaptive immunity adaptive immunity can be induced by the revertant and the vaccine virus. adaptive immune responses to antigens expressed by the revertant will presumably affect the vaccine and revertant equally-because the vaccine encodes a complete vector genome, and the revertant is also a complete vector/virus. as with the preceding pair of mechanisms, adaptive immunity elicited by the revertant will also depress the abundance of the vaccine virus. adaptive immunity to the vaccine antigen will be considered shortly. all three interference mechanisms will potentially operate in any vaccinated host. with all three operating, one mechanism may take precedence over the others, simply because it is activated earlier or enforces a lower limit on viral density than the others. however, there are different stages or degrees of vaccine suppression, so an early mechanism may act to control the infection without clearing it, and another mechanism may act later to clear. because of the delay in developing an adaptive response, viral suppression by adaptive immunity typically occurs later than effects of innate immunity or resource limitation and so might seem to be unimportant in vaccine evolution. yet adaptive immunity may be important in clearing the vaccine following control by other mechanisms, in which case it could have an important role in vaccine evolution. adaptive immunity to the vaccine antigen may also contribute to vaccine inferiorityand feed back to inhibit itself. the preceding paragraphs omitted adaptive immunity to the antigen. by its very nature, adaptive immunity suppresses vaccine growth. but adaptive immunity to the antigen is specific to the vaccine and is thus another reason-besides intrinsic fitness effects-that the vaccine may have lower fitness than revertant. the evolutionary consequences should be the same for both types of inferiority, reducing the long term generation of antigen levels within the host, but adaptive immunity would be irrelevant to vaccine evolution during manufacturing and during early growth within the host. an interesting twist is that adaptive immunity to the antigen might eventually feed back negatively on itself to limit its own growth-immunity against a virus is intrinsically inhibitory, so adaptive immunity against the vaccine will limit vaccine growth and thus limit antigen build-up that would fuel further immunity. one question is whether this self-inhibition is worsened with vaccine evolution. the effect is biologically complicated because adaptive immunity to the antigen does not necessarily translate into selection against the vaccine. selection against the vaccine per se operates only when adaptive immunity specifically targets the vaccine genome over the revertant genome, and this selection need not occur-either because adaptive immunity is so delayed that it is never manifest during vaccine growth, or because the antigen is physically decoupled from its genome when attacked by the adaptive response. without imposing selection on the vaccine, antigen-directed immunity will not affect vaccine evolution. we now employ quantitative models to evaluate the intuitive ideas presented above. given the high dimensionality of the problem, we are especially interested in how well intuition works and whether generalities are observed across large regions of parameter space. a flow diagram of the elements and interactions within the host reveals the complexity of the model (fig ) and facilitates understanding the dynamical equations. v and w are the respective vaccine and revertant densities, with intrinsic growth and death rates governed by four parameters (not illustrated). the model also includes variables for resources (r), innate immunity (z), adaptive immunity to vector (y), and adaptive immunity to antigen (x) that are both influenced by and influence v and w. in the following sections, we explore the dynamics of these interactions with simulations and present results graphically (the results presented do not allow resource limitation to influence dynamics; trials where resource limitation matters were conducted but are not shown). equations and parameter values are provided in s appendix. resource limitation and innate immunity yield qualitatively similar results, so trials with resource limitation are not illustrated in the main text. the equations apply only to within-host processes; any pre-host evolution is subsumed into inoculum composition. the models assist us by forcing us to specify assumptions for how the viruses and immunity interact, and by allowing us to rigorously explore outcomes in different scenarios. however, there is uncertainty in the model structure, many parameter values are unknown, and different viruses will behave somewhat differently. consequently, we focus on broad generalities that arise from many simulations and illustrate these for a few specific cases, reserving supporting information files for further details. the presentation below briefly discusses the dynamics of individual trials for illustration but then moves to contour plots that reveal differences in outcomes as the key parameters are changed. the model used here incorporates the structure of earlier models that described immune responses [ ] [ ] [ ] ; parameter values used here were chosen as described in some of these earlier studies. evolution can matter. in the trials used for illustration, we allow innate immunity to control the infection and adaptive immunity to cause final clearance. such a scenario might correspond to the dynamics of listeria or influenza infections of mice [ ] , or the early dynamics of siv infections [ ] . to get a sense of the full dynamics in the model, we show the time course of dynamics for the different variables (fig ) under conditions of no evolution (top left), just pre-host evolution-revertant abundant in the inoculum but with no fitness advantage (top right), mostly within-host evolution (bottom left), and both (bottom right). the effect of different types of evolution is seen from a comparison of the panels. our chief interest is in final immunity to vaccine, the blue curves. the most visible effect of evolution on immunity to the antigen is evident in the lower right panel, which combines pre-host evolution with a fitness advantage of the revertant. however, the log scale diminishes the visual impact of substantial evolution in other cases. when the revertant is half the inoculum but has no fitness advantage, the immune response is diminished by nearly -fold (top right). overall, the impression is that one must at least suppress either pre-host or within-host evolution to avoid a large loss in immunity (lower right versus the others). illustrations of dynamics from individual trials convey many details. however, without a specific empirical basis for the parameter values chosen, the details have little assured relevance. we therefore provide contour plots that allow easy comparison of many different trials in which parameters of specific interest are varied (fig ) . these graphs show the cumulative vaccine load (left panel) and final level of immunity to vaccine antigen (right) as a function of initial revertant frequencies and selective advantage of the revertant (c). a strong correspondence exists between vaccine load and the level of immunity generated, as is observed empirically following infection [ ] . subsequent figures therefore illustrate the level of immunity. the initial composition of the inoculum matters somewhat more to the adaptive response than does the intrinsic cost of the vaccine (as evident by the contours being closer to vertical rather than horizontal), but this pattern rests heavily on the parameter ranges chosen. indeed, unrealistically large values of initial revertant levels (w( )) are illustrated to offer contrast, as the outcomes are otherwise moderately insensitive to vaccine composition in these graphs. the good news is that, when the inoculum is mostly vaccine and revertant fitness is not high, evolution has little effect on viral load or final level of immunity (i.e., the lower left of each panel has a broad area of one color). this occurs because of the short duration of infection. over longer periods of vaccine growth, the selective advantage of the revertant would undoubtedly play an increasing role in evolution. the large number of parameters ( ) limits the degree to which we can conduct comprehensive sensitivity tests, so the trials are confined to variations in those parameters of greatest interest. vaccine evolution driven by adaptive immunity. we focus on infections of short duration-that are cleared and do not rebound once suppressed. factors that limit the duration of infection include resource limitation, and innate and adaptive immunity. for the most part these factors act equally against vaccine and revertant virus. only one factor, adaptive immunity to the vaccine antigen (x), acts specifically on the vaccine virus and not the revertant. intuition suggests that this adaptive immunity to the antigen can potentially suppress the vaccine's growth and give an advantage to the revertant. as with intrinsic fitness costs, this selection might feed back to limit vaccine growth and thus limit the development of further immunity to antigen by allowing revertant to grow and interfere with vaccine. this section considers whether these arguments are supported by the model. any real vaccine that elicits immunity against the antigen may also experience an intrinsic fitness cost. the effect of immunity on evolution would then be confounded with the effect of intrinsic fitness effects on evolution, making it difficult to isolate one from the other. the models do not face this problem, however. they can be parameterized so that the only possible selection against the vaccine comes from immunity (by setting c = ). vaccine populations can also be freed of revertant by omitting revertant from the inoculum and setting the mutation rate to . thus, we can measure the effect of adaptive immunity on vaccine growth from trials that lack revertant and then compare those results with trials that include revertant. the revertant is included at half the inoculum (representing pre-host evolution); it has no intrinsic fitness advantage over vaccine. immunity (to vaccine) is reduced to just over a third of the level with no evolution. (lower left): the revertant is a small fraction of the inoculum ( . ) but it has a % fitness advantage over vaccine. the level of immunity is % that with no evolution. (lower right): the revertant is half the inoculum and has a % fitness advantage over vaccine. the level of immunity is now less than . % that with no evolution-a depression of almost orders of magnitude. the trials are parameterized so that virus is controlled by innate immunity with final clearance due to adaptive immunity; the mutation rate is in all cases. equations, initial conditions and parameter values not shown here are given in s appendix; r code is included in s file. there are several background points to note about the model structure. first, adaptive immunity specific to vaccine (x) develops at a rate proportional to the vaccine abundance (v) and parameters s (rate of clonal expansion of adaptive immunity) and ϕ x (antigen concentration yielding half the maximum growth rate of adaptive immunity x). in contrast, the impairment specific to vaccine is due to the level of immunity (x) and the vaccine impairment parameter k x . thus, immunity can develop while imposing little or no impairment, i.e., when k x ! . second, adaptive immunity to the vector (y) develops according to its own growth rate parameter (ϕ y ) in response to vaccine plus revertant abundance (v + w), and it impairs both vaccine and revertant growth equally by impairment parameter k y . when revertant is present, it increases the level of immunity to vector backbone/revertant but does not directly affect immunity specific to the vaccine. this immunity will result in faster clearance of both revertant and vaccine, and this results in decreased immunity to the antigen; this is the 'interference' that causes a problem from vaccine evolution. trials were run that contrasted revertant absence versus revertant introduced at % of the inoculum-no evolution versus primairly pre-host evolution, respectively (fig ) . absence of the revertant is the baseline against which the effect of evolution can be compared. the horizontal axis varies k x , the parameter for impairment/killing specific to vaccine, and the vertical axis varies k y , impairment to vector, which affects vaccine and revertant equally. in both panels, increasing impairment against vaccine leads to lower levels of immunity to the vaccinethis is the self-limiting effect of adaptive immunity, which exists even in the absence of evolution. as expected, impairment of immunity to vaccine by immunity to vector is also found. a large effect of inoculum composition on vaccine immunogenicity is evident by comparing the left panel (no mutation, no evolution) and right panel (chiefly prehost evolution): introduction of revertant reduces the level of immunity against vaccine up to -fold. for the -host evolution) . the final vaccine load and immunity against the vaccine antigen depends heavily on two parameters, the inoculum composition (plotted on the x-axis as initial abundance of the revertant virus, w( )) and the growth advantage of the revertant within the host (c, plotted on the y-axis). the heat maps show how, as the composition shifts toward revertant or as vector superiority increases (as we move to the right or up), there is a reduction in the viral load of the vaccine (defined as r v dt, left panel) and in the magnitude of immunity to the vaccine antigen (x, right panel). the initial amount of vaccine virus is always v( ) = (i.e. logv( ) = ). note that the graphs span high frequencies of revertant in the inoculum that should be easily avoided (log w( ) = , i.e. w( ) = v( ))-if the researcher is alert to the possibility. we include such extremes merely to show that the outcome is relatively insensitive to small changes in vaccine composition. equations, initial conditions and parameter values not shown here are given in s appendix; r code is included in s file. https://doi.org/ . /journal.pcbi. .g within-host vaccine evolution right panel, the revertant is / the inoculum and has no intrinsic advantage over vaccine; total inoculum size is unchanged. all reduction of immunity against vaccine is thus due to revertant in the inoculum and any within-host evolution from the selective effect that stems from immunity against vaccine. of the two parameters, k y has a much larger effect than does k x : compared to the left panel, the right panel, with revertant present, has vaccine-specific immunity suppressed more than an order of magnitude along the k y axis, much less so on the k x axis. we attribute this effect of k y to interference by the revertant: the revertant elicits high levels of immunity (y) that indiscriminately also suppress vaccine, thereby suppressing vaccine-specific immunity x. the magnitude of interference depends not only on revertant abundance but also on the values of k x , k y , and the innate immune response (k z ), so interference can appear more or less important in other trials using the same revertant abundance. a question motivating this analysis was one step deeper in the complexity of these effects: does the self-limiting effect of adaptive immunity worsen when revertant is present? this question can be answered by comparing the self-inhibitory effect between left and right panels as k x is increased. by inspection of colors along the horizontal axes, it is seen that the self-inhibitory effect is actually somewhat reduced by the revertant. the revertant lowers the overall response to vaccine, but when correcting for that difference, the effect of increasing k x is slightly weaker in the right panel than in the left. we attribute this weakening of self-limitation as due to vaccine levels being increasingly controlled by immunity against revertant. in sum, therefore, immunity to the vaccine (x) is reduced by itself (depending on the immunity parameter, k x ) and by revertant. the two effects do not interact to make the problem worse than from their separate effects. the results above suggest that vaccine evolution is only likely to compromise immunity if there is substantial pre-host or within-host evolution and if this evolution depresses vaccine effect of evolution on the suppression of immunity by impairment parameters. the final level of immunity to the vaccine antigen (x) depends heavily on the inhibitory parameters k x and k y -which respectively describe how strongly immunity to the vaccine (x) and revertant (y) suppress the viral populations. the left plot considers the absence of revertant, hence no evolution. the right panel introduces revertant at / virus in the host. as the short duration of infection limits within-host evolution, one means of achieving vaccine efficacy is to control the inoculum. two ways of controlling the inoculum are to control its composition and to control its size. pre-host evolution can be reversed by purifying the inoculum after the fact or by taking care to start with a pure isolate and limiting growth (e.g., fig ) . the benefit of suppressing revertant frequency in the inoculum is evident in fig : the magnitude of immunity to the vaccine increases by orders of magnitude as the initial frequency of the revertant is decreased. the effect is strongest at low inoculum levels, pointing to the other solution-increase inoculum size. intuition also suggests that the deleterious effects of evolution can be reduced by increasing the inoculum size, provided the composition does not change: to achieve a threshold antigen level, a large inoculum requires less growth than a small one. less growth reduces the potential for evolution-in the extreme, a large enough inoculum requires no vaccine growth, as with killed vaccines. these conjectures are supported by fig : when the revertant frequency in the inoculum is high, increasing the inoculum size appreciably increases the magnitude of immunity; a much reduced benefit is seen when revertant frequency is low, likely because there is less evolutionary interference from the revertant. these results suggest parallel benefits from reducing the frequency of the revertant in the inoculum and increasing the dose. consideration of the gains from each could help choose an economically feasible strategy, since both purifying the inoculum and increasing its dose are likely to incur financial costs. whether and how well controlling the inoculum will work in practice will depend on details. solutions may be quantitative rather than absolute. intuition is useful for guidance but needs to be confirmed by formal analyses, guided by data from the specific implementation. any live viral vaccine may evolve within the host. the potential for attenuated viruses to revert to wild-type virulence is well appreciated [ , ] , even if it presents a problem for relatively few vaccines (e.g., attenuated polio, [ ] ). there is also a potential for live, recombinant vector vaccines to evolve-our focus in this paper-with the main concern being loss or reduced expression of the transgenic insert [ , ] . if such a vaccine were to evolve fast enough or long enough that it lost the insert, vaccine efficacy might well suffer. we find that evolution during manufacture (pre-host evolution) can play a more important role than within-host evolution in reducing vaccine efficacy, and furthermore that it may be the more easily mitigated. we developed and analyzed models to explore ways in which vaccine evolution could lead to a reduction in vaccine efficacy. an intrinsic fitness advantage of the revertant virus, expected because transgene expression is likely to have metabolic and other costs, will lead to vaccine being gradually overgrown by revertant. this is only likely to cause a reduction in the immunity to the vaccine antigen if it leads to a reduction in the absolute amount (as opposed to merely a reduction in relative frequency) of the vaccine virus. there are in fact several mechanisms by which an ascending revertant population may suppress vaccine: revertant can reduce the amount of the vaccine virus in the host if the revertant uses resources required for virus replication or if the vaccine virus is cleared by the innate or adaptive responses elicited by the revertant. the clear and positive message from our study is that vaccine evolution, if it proves to be a problem for immunization, should be easily mitigated by manipulating the vaccine inoculum. critical to understanding and addressing this problem is recognizing that the vaccine may evolve both within the host and also during manufacture, whereby the inoculum already carries modest to high levels of revertant. the composition of the inoculum can have a large effect on within-host evolution and immunity. by limiting the amount of revertant in the inoculum, and also by boosting the inoculum level, it should usually be possible to limit the amount of within-host vaccine evolution and ensure that immunization is effective. we emphasize, however, that this solution will typically not work for transmissible vaccines and vaccines that establish long term infections within the host. furthermore, using a large inoculum may seem to defeat the purpose of using a live vaccine. there may be cases in which vaccine evolution is so rapid that controlling the inoculum is not sufficient. the solution in this case is to develop or engineer the vaccine with less of a disadvantage. the timing and tissues of antigen expression, location of the transgene in the vector genome, and the size of the transgene may all influence intrinsic fitness effects [ , , , , ] . directed evolution approaches might also improve vaccine efficacy: one simple approach in reducing an intrinsic cost might be to adapt the vector in vitro to host cells expressing the effects of manipulating the inoculum on immunity to the vaccine. small inocula that contain vaccine plus revertant are more prone to reduced immunity levels than are large inocula with little revertant. composition of the vaccine has the larger effect for the inoculm sizes and initial revertant fractions shown, as indicated by the contours being more horizontal than vertical. an intrinsic fitness cost of c = . was set for these trials. smaller c values would lead to higher vaccine and immunity levels across the graphs. equations, initial conditions and parameter values not shown here are given in s appendix; r code is included in s file. https://doi.org/ . /journal.pcbi. .g antigen in trans, allowing compensatory mutations to evolve in response to the antigen before the transgene is cloned into the genome. this adapted vector would then be used as the vaccine backbone. another simple approach would be to compete several different vaccine designs in vitro and pick the design with highest retention of the transgene. any approach using in vitro adaptation needs to avoid adapting the vector to the extent that it compromises ability to grow in vivo. most of these possibilities are ways to reduce pre-host evolution and reduce revertant concentration in the inoculum. one may hope that vaccine designs which reduce pre-host evolution also reduce within-host evolution. measuring the intrinsic fitness effect of the transgene is likely to be an important step in vaccine design. for assessing vaccine evolution, the relevant biological realms are within the host and in vitro. in vitro growth environments are the more easily studied and may reveal much about a vaccine's intrinsic propensity to evolve loss of antigen expression. there are various ways intrinsic fitness effects and their evolutionary consequences might be studied. vaccine growth in tissue culture may reveal some aspects of intrinsic fitness effects and should be relatively easy to study. deletion of the transgene per se would be detectable by pcr, and the fitness advantage of revertant over vaccine could be measured from changes in revertant frequency. the quantitative relevance of an in vitro estimate to in vivo growth would be unknown, but the measure should allow qualitatively comparing engineering designs that improve intrinsic vaccine fitness. if vaccine reversion were due to down regulation of the transgene instead of deletion, fitness estimation would require knowing the mutations responsible and monitoring their frequencies. use of culture-wide antigen levels to measure fitness might provide a sense of whether vaccine evolution would lead to reduced antigen levels in vivo, but it would be less sensitive in measuring evolution than is measuring mutation frequencies. evolution is not the only consideration in designing a recombinant vector vaccine, and the model helps us identify vaccine properties that promote efficacy. first the vaccine should elicit an immune response that rapidly clears the pathogen (i.e. the rate constant for clearance of the pathogen, call it k p , is high). second, the vaccine should elicit a large response to this antigen. this requires that the antigen rapidly elicits immunity (i.e. has low ϕ x , and in terms of immunology it should be an immunogenic antigen), and also requires a high vaccine viral load to generate a large response. engineering this requires tackling a trade-off between avoiding vaccine clearance (i.e. having a low k x ) but allowing for rapid clearance of the pathogen (having a high k p ). vaccines designed to express the antigen in a form that is different from that in the pathogen might help solve this problem. thus, to elicit immunity to influenza, one might design secreted forms of the hemagglutinin or neuraminidase proteins. a recombinant hemagglutinin protein that is secreted rather than on the virion surface would prevent the antibody response to this protein from clearing the recombinant vector vaccine (have low k x ) without compromising the clearance of the influenza virus pathogen which has hemagglutinin on its surface (i.e. has high k p ). in this manner our model allows the identification and tuning of parameters that affect vaccine efficacy, and a comprehensive search of parameter space would identify ideal combinations of vaccine properties. in vitro assays may be useful in measuring intrinsic fitness effects, but in vivo-in the patient-is the ultimate environment for studying within-host evolution and its effects. not only are the dynamics of viral spread different between in vitro and in vivo environments, but most immune components will be in play only in vivo. furthermore, those components may vary across tissues within the host. sampling across this heterogeneity in vivo will be challenging but may be necessary to know whether, when, and where vaccine evolution is a problem. if revertant remains a minority of the population, we expect that vaccine evolution can be ignored. perhaps in vitro studies of vaccine evolution will provide most of the information relevant to in vivo evolution, but it is too early to know. we have focused on recombinant vector vaccines that cause acute infections. necessarily, our recommendations are based on simple models that are caricatures of the complex withinhost dynamics of acute infections. simple models are appropriate at this stage because of uncertainties at many biological levels, and under these circumstances simple models frequently generate more robust results than do complex models [ , ] . the generation of innate and adaptive responses can be modeled with different assumptions than used here, and those alternative processes may affect the conclusions. for example, time-lags in the activation of cells may dominate the time for the generation of an innate immune response, with virus density having a consequently smaller role than assumed here (as can be seen in [ ] and modeled in [ ] ). we have modeled that responses to different antigens are generated independently of each other and do not compete. we have done so because vaccines are likely to cause relatively mild infections during which the densities of pathogen and immune cells do not reach sufficiently high levels required for competitive interactions to be important. the adaptive immune response may be more influenced by recruitment which is followed by a period of proliferation even in the absence of antigen [ ] [ ] [ ] . both these scenarios would minimize the impact of evolutionary changes in the vaccine on the amount of immunity generated to the transgene. finally, it is easily appreciated that there are realms we do not consider, such as within-host spatial structure [ ] and recombinant vector vaccines based on viruses such as cytomegalovirus that cause persistent infections [ ] or that are transmissible. spatial structure may limit the impact of vaccine evolution on immunity (e.g., prevent mutants from taking over the entire population). in contrast, vaccines that cause persistent infections or are transmissible are likely to be more severely affected by evolution than are vaccines causing acute infections, as there is a longer timeframe for evolution to operate. with so little experience from recombinant vector vaccines, we can merely guess how commonly the neglect of within-host evolution will compromise vaccine efficacy. given that simple steps can be taken to reduce vaccine evolution, vaccine development programs should at least entertain the possibility that evolution can underlie failure. avoiding vaccine evolution may be easier than developing an entirely new vaccine. supporting information s appendix. equations and parameters. 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disease, what form that disease takes, and how severe the disease will be. though the pathogenesis of each virus is unique, there are several common points in the virus life cycle that are shared between all pathogenic viruses, and by considering these common aspects of the virus-induced disease process, we can explore some general concepts in viral pathogenesis while illustrating some of the virus specific processes that shape disease outcomes. viral pathogenesis is a term that generally describes the processes by which viral infection results in a disease. however, viruses can range from small rna viruses (e.g., flaviviruses such as dengue virus) to large dna viruses (e.g., herpesviruses and poxviruses), all of which interact with the host in unique ways to drive the virus-induced disease process. these virus specific disease outcomes are driven by fundamental differences in viral replication cycles, modes of transmission, tissue tropism, interactions with the host immune response, as well as a multitude of other variables. furthermore, due to differences in a wide range of factors, including elements such as host genetic variation, host immune status, viral dose, or route of inoculation, infection with the same virus often results in varied disease outcomes in different individuals, where some individuals may develop no disease at all, while others are symptomatic and may develop serious or life threatening disease. therefore, it is difficult to provide a general overview of viral pathogenesis that accurately encompasses the full range of virus-induced disease processes. however, while the pathogenesis of each virus and its associated disease(s) has unique aspects, it is also true that there are several common stages in the viral life cycle/disease process that are shared between all pathogenic viruses, and consideration of these common processes can be used to illustrate several key concepts in viral pathogenesis. for example, since viruses are obligate intracellular pathogens that are not capable of reproducing themselves outside of a permissive host cell, a virus must successfully gain entry to a target cell and propagate itself to cause a disease. whether a virus can accomplish this task depends on interactions with key host molecules, such as cell surface receptors, which determine whether the virus can successfully infect and reproduce itself within its target cells. therefore, for the purposes of this overview, we will consider several common virus/host interactions, including: ( ) factors that affect viral tissue tropism and host range, ( ) viral immune evasion, and ( ) viral effects on target cells or tissues. by focusing on these key steps in the viral life cycle/disease process, we can discuss some general concepts that illustrate how these interactions impact viral pathogenesis, while also emphasizing the virus specific aspects of these interactions that result in each virusinduced disease having unique attributes. as noted above, viruses cannot replicate outside of a permissive host cell, and gaining access to and being able to replicate within these cells represents a key part of any virus's life cycle. furthermore, the induction of disease is usually dependent upon the effects of the virus on cells or organ systems that it infects, such as direct killing of essential host cells (e.g., neurons) by the virus (see below). therefore, viruses that cannot gain access to and replicate within permissive host cells are generally not able to cause disease. furthermore, viruses that cannot gain access to the specific tissue that is associated with a disease, such as the central nervous system for viruses that cause encephalitis, are also less likely to cause severe diseases. though there are multiple virus-host interactions that determine whether a virus can successfully replicate within a target cell, for the purposes of brevity, we will consider specific examples of stages in the viral life cycle where interactions with the host determine whether the virus can successfully replicate in target cells, and how these interactions promote the virusinduced disease process figure . viral entry into host cells usually involves interactions between molecules on the surface of the virus and specific cell surface receptors, which allow the virus to bind to the host cell and initiate the viral entry process. in some cases, viruses interact with a single cell surface molecule, which mediates viral binding to the cell and also facilitates viral cell entry. for example, several rhinoviruses bind to icam- (cd ), and these interactions promote viral infection of the cell (reviewed in rossmann et al., ) . in contrast other viruses, such as herpes simplex virus, interact with cell surface molecules such as heparin sulfate to facilitate viral attachment to the cell, and then engage specific host receptor proteins on the cell surface that mediate viral entry into the cell (reviewed in spear, ) . viral interactions with these receptors can have a significant impact upon several aspects of viral pathogenesis, including determining the cell or tissue tropism of a virus or even whether a virus can efficiently infect and cause disease in a specific host species. the importance of virus-receptor interactions in disease pathogenesis is nicely illustrated by interactions between human immunodeficiency virus (hiv) and two host molecules, cd and ccr , which mediate hiv binding and entry. hiv infection requires interactions between the viral gp protein and cd (sweet et al., ) , a host protein expressed on a subset of t cells (cd positive helper t cells) and a limited number of other cell types, such as macrophages. since hiv infection is dependent on interactions with cd , cd positive helper t cells are the major target of hiv replication, and viral replication in these cells results in the progressive loss of cd t cells over the course of the hiv infection. cd t cells play an essential role in regulating the immune response, and hiv-mediated killing of these cells ultimately leads to immune suppression that leaves hiv infected individuals susceptible to lethal opportunistic infections that would normally be controlled by persons with fully functional immune systems. therefore, viral interactions with cd and subsequent viral tropism for these cells, directly contributes to disease pathogenesis during hiv infection. in addition to the importance of cd in determining hiv tropism, a second hiv/receptor interaction further illustrates the importance of receptors in driving viral pathogenesis. following hiv gp binding to the cd molecule, the gp molecule undergoes a conformational change, which allows gp to interact with one of two coreceptor molecules, ccr or cxcr , that then leads to viral fusion (alkhatib et al., ; feng et al., ) . the importance of these interactions is illustrated by the fact that a small subset of humans has a nonfunctional form of ccr , and these individuals are highly resistant to hiv infection (liu et al., ; dean et al., ; huang et al., ) . in other words, if the virus cannot get into its appropriate target cells through interactions with ccr , it cannot efficiently infect these individuals and cause disease. in addition to affecting disease pathogenesis by determining viral cell tropism, virus-receptor interactions can also broadly impact disease pathogenesis by determining whether a virus will efficiently infect a new host. this is particularly important for zoonotic viruses, which are viruses that naturally reside in animals, but which jump to humans and cause diseases. to successfully make the transition from its natural animal host to humans, a zoonotic virus must either interact with receptors that are highly conserved between species, or the virus must change (mutate) in a way that allows it to adapt to efficiently interact with receptors in the new host. an example of the first situation is provided by sindbis virus, a mosquitoborne virus that must efficiently infect both mosquitos and vertebrate hosts. recent studies have identified natural resistance-associated macrophage protein (nramp), as a receptor for sindbis virus in both mosquitoes and vertebrate cells (e.g., humans) (rose et al., ) . nramp is highly conserved between species, and it is likely that by interacting with this highly conserved receptor protein, sindbis virus is able to readily replicate in both mosquitoes and vertebrates. in contrast to situations where a virus interacts with a highly conserved receptor, there are also situations where the virus receptor is significantly different between species. therefore, for the virus to successfully make the transition between the original animal host and humans, it is likely that the virus will have to adapt to more efficient use of the receptor in the new species. an example of this type of interaction is provided by the sars coronavirus (sars-cov), which caused an outbreak of severe acute respiratory disease in - . sars-cov infects cells through interactions between the viral spike (s) protein and angiotensin converting enzyme- (ace ) (li et al., ) . however, sars-cov is thought to normally reside in bats (lau et al., ) and the bat-derived virus does not efficiently interact with human ace . however, studies suggest that mutations within the receptor binding domain of sars led to more efficient interactions with the human ace molecule, and that viruses with these adaptive mutations were better able to infect human cells (reviewed in bolles et al., ; graham and baric, ) . this is supported by the finding that introducing the region of the s protein that binds to human ace into the bat sars virus allows that virus to bind to human ace and efficiently infect human cells (becker et al., ) . these results further reinforce the idea that virus receptor interactions play a crucial role in determining whether the virus can efficiently infect the host and ultimately cause disease. though receptor interactions represent a crucial component of virus-host interactions and viral pathogenesis, a number of other factors can also determine which tissues a virus infects. most viruses encode their own replication machinery, however they are still dependent upon the host cell for a number of functions, including processes that promote viral entry or the translation and assembly of viral proteins. recently, the field of virology has become very interested in identifying 'proviral' factors, which are host molecules that promote efficient viral replication. identification of these proviral factors not only enhances our knowledge of how viruses interact with the host cell, but also may identify host pathways that could be targeted to inhibit viral replication and develop new therapies. many proviral factors are components of generally important cellular processes, such as the host translation machinery or cellular protein transport pathways, which are likely to be important for broad classes of viral pathogens. however, there are instances where host factors interact with specific viruses to enhance viral replication in specific cell types, thereby affecting both viral cell tropism and disease pathogenesis. in the most extreme examples, viral replication, and hence the ability to cause disease, would be severely compromised by the absence of a specific proviral factor. an excellent example of a virusspecific proviral factor is provided by hepatitis c virus (hcv) interactions with mir- . hcv is a positive stranded rna virus that infects the liver and causes chronic disease in a significant fraction of infected individuals, putting these individuals at risk for the development of chronic liver failure and the development of hepatocellular carcinoma (reviewed in cabibbo et al., ) . though hcv may be able to replicate in cell types such as b and t cells, the major site of hcv replication are hepatocytes within the liver. a key determinant of hcv's tropism for hepatocytes is the host microrna mir- . micrornas are small ( - ) nucleotide host rnas, which regulate a number of biological processes within the host (reviewed in szabo et al., ) . in the context of hcv infection, mir- is expressed specifically within the liver and its expression enhances hcv rna levels in hepatocyte cell lines (jopling et al., ) . though the mechanisms of mir- 's actions on hcv are not completely understood, mir- interacts with rna structures in the end of the viral genome, and these interactions are essential for mir- 's effects on hcv (jopling et al., ) . the importance of these interactions for hcv replication and disease pathogenesis is illustrated by recent studies in a chimpanzee model of hcv where administration of a mir- specific antagonist resulted in decreased viral loads within the liver and a reduction in hcv associated disease signs within the liver (lanford et al., ) . therefore, hcv interactions with mir- affect viral replication and disease pathogenesis within the liver, illustrating the potential importance of viral interactions with proviral host factors in promoting viral replication and driving the pathogenesis of virus-induced diseases. it is important to note that in addition to proviral factors, there are also classes of host proteins that can act as restriction factors that actively inhibit a virus's ability to replicate and cause disease. these types of host restriction factors have been extensively studied in the context of retro/lentivirus infection and an excellent example of this type of factor is provided by trim a. trim a is part of a class of tripartite motif containing host proteins, a large number of which have been shown to exhibit antiviral or immune regulatory functions (reviewed in mcnab et al., ) . studies looking at restriction factors that limit the ability of hiv to replicate in nonhuman primate cells found that the rhesus macaque trim a molecule strongly interacts with the hiv capsid to block viral infection at an early stage in the viral replication process (stremlau et al., ) . in contrast, the human trim a molecule, which interferes with retroviruses and lentiviruses such as equine infectious anemia virus, interacts less efficiently with hiv (stremlau et al., ) . efficient interactions between trim and hiv appear to protect macaques from hiv replication and disease, while less efficient interactions between human trim a and hiv in part explain the enhanced susceptibility of humans to hiv infection. therefore, the presence or absence of appropriate restriction factors can have a major impact on host susceptibility to virusinduced disease, where the presence of a strong restriction factor would inhibit viral replication and prevent or limit virusinduced disease. in addition to trim a, other factors that mediate host range restriction during lentivirus/retrovirus infection include apobec and tetherin (reviewed in luban, ) . the host innate and adaptive immune systems play a major role in protecting from virus-induced disease by limiting or preventing viral replication. the type i interferon system and other components of the innate immune response are rapidly activated in response to viral infection and play a crucial role in limiting viral replication and spread within the host. likewise, components of the adaptive immune system, such as cytotoxic cd positive t cells and antibody, are involved in clearing virus from infected tissues and can provide long-term immunity to prevent reinfection. the complexity of the immune systems and its interactions with each viral pathogen is such that a comprehensive overview of virus-host immune interactions is beyond the scope of this article. however, though there are aspects of the virus-host immune interaction that are unique to each pathogenic virus, every viral pathogen must successfully avoid or actively antagonize the host immune response to infect, replicate, and disseminate within the host. in fact, viruses with defects in blocking the host immune response are often attenuated in their ability to cause disease. therefore, immune evasion represents a common theme in viral pathogenesis that we will explore further. while different viruses employ a wide range of strategies to avoid/antagonize aspects of the innate and adaptive immune system, we will focus specifically on viral interactions with the innate immune system and the interferon response in particular to illustrate the importance of these interactions on viral pathogenesis. though multiple arms of the innate immune system contribute to viral control, including components such as the complement cascade, natural killer cells, and proinflammatory cytokines, several lines of evidence suggest that the type i interferon system plays an essential role in the pathogenesis of most viral pathogens. most, if not all of the pathogenic viruses affecting humans either avoid or actively antagonize some aspect of the type i interferon response, and viruses that are defective for avoiding/antagonizing the type i interferon system are often attenuated for replication and disease in immunocompetent animals (garcia-sastre et al., ; talon et al., ; leib et al., ; bouloy et al., ) . furthermore, animals lacking a functional type i interferon system exhibit enhanced sensitivity to a wide array of viral pathogens (leib et al., ; ryman et al., ; white et al., ; bouloy et al., ; schilte et al., ) , which suggests that type i interferon (ifn) plays an essential role in limiting viral replication and protecting from disease. the type i interferons are a group of cytokines consisting of a several related alpha interferon molecules and a single interferon beta protein, that are induced in response to stimuli associated with viral infections, such as double stranded rna, and which bind to a common type i interferon receptor complex. signaling via the type i interferon receptor leads to the induction of hundreds of interferon-stimulated genes (isgs). though the function of many of these isgs still needs to be elucidated, it is clear that many of these molecules have antiviral activity against one or more viral pathogens (schoggins and rice, ) , while some isg molecules modulate other aspects of the host immune response, including antigen presentation (schoggins and rice, ) . therefore, the type i interferon system limits viral replication and dissemination by inducing an antiviral state within host cells, and then promotes viral clearance by modulating other aspects of the host immune response, including host antibody and t cell responses. for the purposes of this overview, we will briefly summarize several key aspects of the type i interferon response to illustrate how viruses can avoid or antagonize this response. the production of type i interferon can be induced by signaling through several different pattern recognition molecules, including certain toll-like receptors and cytoplasmic nucleic acid sensors, such as rig-i. these pattern recognition molecules recognize pathogen associated molecular patterns (pamps), which are molecules that have signatures commonly associated with viral infection, such as double stranded rna. readers who are interested in more detailed discussion of the pattern recognition molecules that regulate the type i ifn response are directed to the following reviews (kawai and akira, ; nakhaei et al., ) . though simplified for this overview, following interactions with a viral pamp, each pattern recognition receptor will activate a signaling cascade that ultimately converges on a set of transcription factors (irf or irf ), which upon activation, transit to the nucleus to induce type i ifn transcription and subsequent production of type i interferon. type i interferons are secreted from the cell, and can then act in an autocrine (on the cell that produced the interferon) or a paracrine (affecting the surrounding cells) manner. the type i interferon receptor consists of two subunits ifnar and ifnar , which upon interferon binding, dimerize at the cell surface. the receptors are associated with two protein tyrosine kinases, janus activated kinase (jak ) and tyrosine kinase (tyk ), that are brought together during receptor dimerization. jak and tyk then undergo auto-and/ or transphosphorylation (de weerd et al., ; de weerd and nguyen, ) , which leads to their activation, and they in turn phosphorylate tyrosine residues present on the receptor tails that serve as docking sites for signal transducers and activators of transcription (stat) factors. jak and tyk phosphorylate stat and stat , which then form heterodimers and interact with interferon regulatory factor (irf ), where this complex localizes to the nucleus and binds promoters containing ifn-stimulated response elements to drive expression of isgs, which mediate direct antiviral effector functions and modulate the host immune response (kawai and akira, ; nakhaei et al., ; deweerd, ) . importantly, though there are instances of viruses avoiding or antagonizing specific antiviral effector molecules (daffis et al., ) , the majority of known viral interferon evasion strategies are directed at either avoiding recognition by host pattern recognition molecules or targeting the signaling pathways associated with either type i interferon induction or interferon receptor signaling. therefore, we will briefly discuss a few examples of interferon antagonism to illustrate the importance of these interactions in viral pathogenesis. a number of viruses antagonize type i interferon induction either by targeting specific components of the interferon induction pathways or by broadly inhibiting de novo rna synthesis/protein translation in the infected cell, which effectively blocks the production of type i interferon. one strategy for avoiding type i interferon induction is to shield viral pamps (e.g., viral rna) from recognition by host rna sensors, such as rig-i and mda . a number of viruses encode rna binding proteins that have been shown to inhibit type i ifn induction. for example, the ns protein of influenza a virus, which interferes with type i ifn induction at several stages in the induction process, exhibits potent antagonist activity against type i interferon induction, and this antagonism is in part due to rna binding activity by ns (donelan et al., ) . another example of this type of strategy is provided by the nucleocapsid protein (n) of the sars coronavirus, which also inhibits type i interferon induction at an early stage in the rna recognition process, and this inhibitory activity is dependent upon the rna binding activity of the n protein (lu et al., ) . in addition to blocking host recognition, a wide array of viruses encode proteins that block specific components of the type i interferon signaling cascade. for example, the influenza a virus ns protein, in addition to shielding the viral rna from recognition, prevents rig-i activation and interferon induction in response to influenza infection by blocking the function of trim , a ubiquitin ligase that regulates the activation of the rna sensor, rig-i (gack et al., ) , while a second viral protein, pb -f interferes with type i interferon induction through interactions with mavs, an adaptor molecule that is essential for rig-i-mediated type i interferon induction (conenello et al., ; varga et al., ) . the fact that influenza virus employs multiple strategies to block type i interferon induction argues that viral interactions with the type i interferon system are particularly important in regulating viral replication, and this is further supported by the fact that viruses with mutations in either ns or pb -f induce higher levels of type i interferon and are subsequently attenuated in their ability to cause disease (conenello et al., ; donelan et al., ) . therefore, viruses that are defective in their ability to antagonize the host type i interferon system are often unable to replicate and spread efficiently within the host, illustrating the importance of viral immune evasion strategies in determining whether a virus will be pathogenic ( figure ) . in addition to blocking type i interferon induction, a number of viruses also interfere with type i interferon receptor signaling, since this effectively blocks the induction of antiviral isgs. for example, the type i interferon signaling pathway is targeted at multiple stages by members of the flavivirus family, with multiple proteins from the same virus often targeting different steps of the pathway (reviewed in diamond, ) , again illustrating the importance of interferon antagonism for viral success. as is the case with antagonism of type i interferon induction, viral interactions with the type i interferon signaling pathways are likely to have a major impact on virus-induced disease. studies with reovirus have shown the induction of virus-induced myocarditis is associated with suppression of type i interferon receptor signaling, where the viral m gene from a myocarditic virus exhibits enhanced ability to interfere with the irf component of the isgf signaling complex (zurney et al., ) . these results suggest that viral antagonism on interferon receptor signaling can have a major impact on the pathogenesis of virus-induced disease, and that differences in the efficiency of interferon antagonism between related viruses can have significant impacts on what types of disease those viruses cause. as noted above, viruses are obligate intracellular pathogens that cannot reproduce themselves outside of host cells. therefore, the types of cells that a virus targets and what effect the virus infection has on a target cell plays a major role in determining whether a virus will induce disease and if so, what type of disease the virus causes. most simply, some viruses are cytopathic, in that the virus infection results in direct killing of the host cell, while other viruses are noncytopathic and do not directly kill the infected cell. however, while some viruses cause disease due to their targeting and killing of essential cell types, such as neurons, the mechanisms leading to virusinduced disease are often complex and we will discuss some examples of different interactions between viruses and host cells or the host immune response that affects disease outcomes (figure ). immune killing of host cell transformation tumor development in the presence of a robust type i interferon response, and the absence of effective viral interferon antagonism, type i interferon will induce an antiviral state that limits viral replication and spread, thereby limiting virusinduced disease. (b) if the virus effectively interferes with the type i interferon response, interferon will be prevented from inducing a robust antiviral state within the host, and the virus is able to replicate to higher levels, will spread more efficiently, and may cause more severe disease. a number of viruses are directly cytotoxic and productive viral replication leads to direct cell killing. therefore, if the cell type that these viruses replicate in is essential, such as neurons, virus-induced killing of these cells can directly lead to disease. a number of viruses, including alphaviruses such as sindbis virus and venezuelan equine encephalitis virus (vee), cause direct killing of host cells. while the mechanisms leading to cell death are not completely worked out, it is generally accepted that these viruses induce apoptotic cell death in infected cells griffin et al., ) . since these viruses exhibit strong tropism for neurons, viral replication within the central nervous system leads to neuron death and degradation of neurologic function. though there is evidence that the host immune response exacerbates virus induced disease during both sindbis virus and vee infection (rowell and griffin, ; kimura and griffin, ; charles et al., ) , in the case of vee, mice lacking a functional adaptive immune system still succumb to virus-induced disease (charles et al., ) , suggesting the direct cell killing by the virus contributes to disease pathogenesis. the host immune systems plays a crucial role by protecting from virus-induced disease, however there are clear instances where an overactive or inappropriate host immune response contributes to the pathogenesis of virus-induced disease. this is perhaps most clearly illustrated in situations where a virus is noncytopathic and does not cause the direct death of infected host cells, yet viral infection still results in tissue destruction and disease. hepatitis b virus (hbv), which causes serious acute and chronic hepatitis in infected humans, nicely represents this situation and also illustrated both the importance of the host immune system in promoting viral clearance and the potential for ineffective or overly active immune responses to potentiate virus-induced disease. in most immunocompetent individuals, hbv infection causes acute hepatitis, where the host immune response plays an essential role in viral clearance, but in the process, also causes some liver injury (chisari et al., ) . hbv is noncytopathic for hepatocytes and during the early stages of hbv infection there are no signs of liver disease, even though the virus is replicating to high levels within the liver (chisari et al., ) . it is only after the host adaptive immune response has started to clear the virus from the liver that signs of liver injury are evidence, which suggests that though beneficial in clearing the virus, aspects of this immune response are pathologic. through the use of transgenic mouse models and hbv infection of chimpanzees, it has been shown the virus specific cd t cells are responsible for both the clearance of the virus from the liver and are the mediators of the acute liver injury associated with hbv infection (thimme et al., ) . immune interactions also play a major role in the pathogenesis of chronic hbv infection, where chronic disease is associated with a weak hbv specific cd t cell response that is unable to clear the infection (chisari et al., ) . furthermore, evidence from hbv transgenic mouse models suggests that suboptimal cd t cells responses that fail to clear hbv infection may be associated with chronic liver inflammation and damage that ultimately leads to the development of hepatocellular carcinoma (chisari et al., ) . immune pathology is not restricted to infection by noncytopathic viruses, and overactive or inappropriate immune responses are thought to contribute to disease even when the virus itself is capable of causing direct cell killing. as noted above, even though sindbis virus and vee are thought to directly contribute to virus-induced neurologic disease by killing neurons, it is also clear that components of the host adaptive immune response can exacerbate the disease process with these viruses (charles et al., ; rowell and griffin, ; kimura and griffin, ) . this concept is also illustrated by ross river virus (rrv), another alphavirus that is associated with severe arthralgia and myalgia in infected humans. studies in humans and mouse models have shown that rrv replicates to high levels in joint and muscle tissues and that viral replication within these tissues leads to the development of arthritis and myositis hazelton et al., ; morrison et al., ) . further analysis found that even though rrv is cytopathic, depletion of macrophages significantly reduced the severity of virus-induced disease, suggesting that components of the inflammatory response, rather than direct virus-induced killing, are responsible for virus-induced disease (lidbury et al., ) . furthermore, activation of specific components of the host complement cascade, including mannose binding lectin and the c component of complement, are associated with severe rrv-induced disease in humans and mice lacking either of these factors are highly resistant to virus-induced disease (morrison et al., ; gunn et al., ) . it is also important to remember that while the above examples focus on instances where components of the immune response directly contribute to cell killing in virally infected tissues, immune-mediated pathology may not always be restricted to direct effects on virally infected cells or within virally infected tissues. a number of chronic viral infections, including hepatitis c virus (hcv) infection, are associated with the development of immune complexes, where aggregates of virus and antibodies precipitate within small blood vessels and lead to the development of inflammation (vasculitis) (reviewed in lauletta et al., ) . therefore, while generally beneficial, there are instances where overactive or inappropriately activated components of the antiviral host immune response directly contribute to the pathogenesis of virusinduced disease. since there is significant person to person variation in immune function, variation in the magnitude and composition of the host immune response may play a major role in determining whether an individual mounts an immunopathologic immune response and thereby develops disease. not all pathologic interactions between viruses and their target cells/tissues involve direct cell killing and tissue damage, and virus-induced cancer is a serious consequence of some viral infections. while cancers associated with some viral infections are due to indirect effects such as immune suppression associated with hiv infection or chronic inflammation associated with hbv or hcv infection (see above), there are also examples of situations where viral infection directly promotes tumor development. several gamma herpes viruses, including the human gamma herpes viruses, epstein barr virus (ebv) and kaposi sarcoma-associated herpesvirus (kshv), are associated with lymphoproliferative disorders and cancer in humans. both of these viruses encode a number of proteins, such as ebv ebna and lmp or kshv lana- and v-cyclin that are associated with cellular transformation and cancer (reviewed in cesarman, ) . likewise, human papilloma viruses (hpv), which can cause several types of cancer, including cervical cancer, encode several proteins, including viral e and e proteins, that interfere with cell cycle progression checkpoints and promote cellular transformation (korzeniewski et al., ) . though infection with any of these viruses has the potential to cause cancer, as is the case with most aspects of viral pathogenesis, there are multiple additional factors that determine whether an individual is at risk for development of disease. in the case of ebv, since the viral proteins are recognized by the host immune response, individuals with healthy immune systems are at much lower risk of developing ebv associated cancers (reviewed in cesarman, ) . likewise, in the case of hpv infection, there are multiple hpv genotypes and the risk of developing hpv associated cervical carcinoma is much greater with certain high risk hpvs, such as hpv or (korzeniewski et al., ) . therefore, like other aspects of viral pathogenesis, a complex series of virus-host interactions determines whether infection with cancer associated viruses ultimately results in disease development. viral pathogenesis represents a complex series of interactions between viruses and the host that determine whether the virus will successfully establish infection within the host and if so, whether this infection will result in virus-induced disease. as discussed above, though the pathogenesis of each virus is unique, there are several stages in the viral life cycle that are shared by all pathogenic viruses which illustrate common themes 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factor key: cord- - k w authors: leitão, jorge h. title: microbial virulence factors date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: k w microbial virulence factors encompass a wide range of molecules produced by pathogenic microorganisms, enhancing their ability to evade their host defenses and cause disease [...]. most represented, followed by enterobacter and citrobacter [ ] . the isolates were further characterized concerning the presence in their genomes of genes encoding selected virulence factors, as well as their phenotypes related to biofilm formation and resistance to a selection of antibiotics. results point out that the isolates do not encompass particularly virulent strains and in most of the cases were susceptible to antibiotics [ ] . yang et al. investigated the role of the salmonella enterica serovar typhimurium (st) pdxb-usg-trua-deda operon on intracellular survival using deletion mutants constructed with the λ-red recombination technology [ ] . the salmonella genus comprises several facultative intracellular pathogens capable of infecting both human and animal hosts. the st deletion mutants was investigated in j a. macrophage cells. the deletion mutants ∆pdxb, ∆usg, and ∆trua exhibited reduced replication abilities compared to st and the deletion mutant ∆deda. the pdxb-usg-trua-deda operon is shown to contribute to st virulence in mice, and to resistance to oxidative stress [ ] . aeromonas hydrophila is an aquatic gram-negative bacterium, capable of causing serious and lethal infections to a wide range of hosts, including fish, birds, amphibians, reptiles, and mammals [ ] . dong et al. described the identification and functional characterization of the lahs global regulator of a. hydrophila [ ] . lahs was identified after the screening of a tn -derived library of a. hydrophila mutants for reduced hemolytic activity. the lysr family transcriptional regulator family member lahs was found to play a role in biofilm formation, motility, antibacterial activity, resistance to oxidative stress, and proteolytic activity, as well as essential for a. hydrophila virulence to zebrafish [ ] . the comparative proteomics analysis performed by the authors confirmed the role of the protein as a global regulator in a. hydrophila [ ] . bacteria of the dickeya genus comprise plant pathogens that affect crops such as potatoes. in order to succeed when infecting their hosts, dickeya secrete several proteins with plant cell wall degrading activities, including pectinases, cellulases, and proteases [ ] . to investigate the role played by the protease lon on d. solani pathogenicity towards potato, figaj et al. used a λ-red-derived protocol to construct a lon deletion mutant [ ] . results presented indicate that the lon protein plays a role in protecting the bacterium to high ionic and temperature stresses, affecting the activity of pectate lyases, the organism motility, and delaying the onset of infection symptoms in the potato host [ ] . the plant pathogen candidatus phytoplasma mali is the causal agent of apple proliferation disease, that affects apple production in northern italy [ ] . phytoplasma are biotrophic, obligate plant and insect bacterial symbionts, with a biphasic life cycle comprising reproduction in phloem-feeding insects and in plants [ ] . the paper of mittelberger et al. focused on the effector protein pme (protein in malus expressed ), expressed by p. mali when infecting apples [ ] . the in silico analysis of the pme protein sequence performed revealed that the protein has features of effector proteins of gram-positive bacteria, with a predicted final localization at the cytoplasm or nucleus of the host [ ] . two main protein variants, pme st pme at, were found associated in infected apple trees from italy and germany. using protein variants tagged with gfp, both variants were found to translocate to the nucleus of nicotiana spp. protoplasts. a better understanding of the molecular mechanisms used by p. mali to manipulate its host will rely on genomics analysis, since no genetic manipulation is presently available for these organisms [ ] . the necrotrophic fungal pathogen sclerotinia sclerotiorum (lib.) de bary infects a wide range of plants causing devastating agricultural losses. the organism forms a typical structure named sclerotia when vegetative hyphae gather to form a hardened multicellular structure important in its development and pathogenesis, and that under favorable conditions germinate leading to vegetative hyphae or apothecia that will initiate novel disease cycles by producing ascospores [ ] . li et al. used a proteomics approach based on d gels followed by spot isolation and protein identification by maldi-tof to identify proteins differentially expressed between a wild-type strain and a deletion mutant on the gene ssnsd encoding a type ivb gata zinc finger transcription factor [ ] . although the gene encoding ssnsd was found as expressed at low levels during the hyphae stage, the mutant is unable to form the compound appressoria. the authors were able to identify a total of proteins as differentially expressed, with predicted functions and as hypothetical proteins [ ] . the authors emphasize the utility of the approach used to identify important proteins involved in the ssnsd -mediated formation of appressorium. in addition to other factors, the success of pathogens rely on cell-cell communication. bacterial outer membrane vesicles (omv) are recognized as an efficient means of bacteria-bacteria and bacteria-host communication, not only intra-species, but also interspecies [ ] . despite the lack of data on a possible role played by omvs in bacterial-yeast communication, roszkowiak et al. investigated the role played by moraxella catarrhalis omvs on the susceptibility of selected bacterial and fungal pathogens to the cationic peptide polymyxin b, and to the serum complement [ ] . using omvs from m. catarrhalis strain , the authors found that these omvs conferred protection against the cationic peptide polymyxin b to the non-typeable haemophilus influenzae, pseudomonas aeruginosa, and acinetobacter baumannii. furthermore, omvs also protected serum-sensitive non-typeable h. influenza and promoted the growth of the serum-resistant p. aeruginosa and a. baumannii against the complement [ ] . in addition, the results presented also show that omvs facilitate the formation of hyphae by the pathogenic yeast candida albicans, promoting its virulence [ ] . as stated by the authors, this work might pave the way to uncover additional roles played by omvs-dependent interactions in multispecies communities [ ] . the rna chaperone hfq is a master regulator of gene expression in bacteria, mediating the interaction of small noncoding rnas with their mrna targets, including those related to virulence in gram-negative bacteria [ ] . dienstbier et al. performed an integrated omics comparative analysis of the hfq regulon in the bordetella pertussis human pathogen, responsible for respiratory tract infections, in particular of a whooping cough [ ] . based on the use of rnaseq, and gene ontology analysis, genes significantly upregulated in the hfq mutant fall into categories including "translation", "regulation of transcription", and "transmembrane transport", while genes downregulated fall in the categories "transmembrane transport", "iron-sulfur cluster assembly", "oxido-reduction process", "pathogenesis", and "protein secretion by the type iii secretion system" [ ] . correlations of transcriptome, proteome, and secretome datasets are also presented [ ] . results presented corroborate the central role played by hfq on the physiology and pathogenicity of b. pertussis [ ] . in their brief report, maisetta et al. performed the ex vivo evaluation of the bactericidal activity of combinations of the semi synthetic antimicrobial peptide lin-sb - in combination with edta (ethylenediaminetetraacetic acid) against endogenous p. aeruginosa present in the sputum from patients suffering from primary ciliary dyskinesia (pcd) [ ] . the authors observed that the peptide and edta were almost inactive against pcd sputum endogenous p. aeruginosa when used alone, but exhibited a significant synergistic killing effect with a sputum sample-dependent efficacy [ ] . edta, but not lin-sb - , was found to inhibit biofilm formation and the production of virulence factors including alginate, pyocyanin, and the metalloprotease lasa [ ] . various bacterial species have evolved various strategies to invade, survive, and multiply intracellularly in host cells. the paper of denzer et al. presents an updated review of the mechanisms used by bacteria to invade the host cell, to manipulate their biochemical and gene expression machinery, and to multiply and escape from the host cell [ ] . the authors present a thorough review of mechanisms used by intracellular pathogens, including the highjack of host immune defenses to enter into the host cell. central attention is given to the various mechanism used to manipulate gene expression, including histone modification, control of host dna methylation patterns, sabotage of host long non-coding rnas, interfering with the host rna transcription and translation, as well as with host protein stability [ ] . the importance of the detailed molecular knowledge of pathogenesis mechanisms to the development of strategies to combat bacterial infections is highlighted [ ] . the functions of grimelysin of serratia grimesii and protealysin of serratia proteamaculans that use actin as a substrate and promote bacterial invasion was reviewed by khaitlina et al. [ ] . the serratia genus comprises facultative pathogens able to cause nosocomial infections or infections in immunocompromised patients, but nosocomial infections by s. grimesii or s. proteamaculans are low [ ] . the paper focused on the discovery, properties and substrate specificity of the two proteases, their high specificity towards actin, and discussed their contribution to the invasiveness of serratia, although further knowledge of the bacterium virulence factors and the cellular response mechanisms is required to fully understand the mechanism of serratia invasion of the host cell [ ] . the virulence factors that the bacteria use to cross the blood-brain barrier and cause meningitis is reviewed by herold et al. [ ] . meningitis remains a worldwide problem often associated with fatalities and severe sequelae. after reviewing important traits of the central nervous system barriers to bacterial entrance, the authors review the various stages of the virulence processes of bacterial meningitis, including the processes of attachment and invasion, the routes used to enter the central nervous system, and the general mechanisms used to survive intracellularly [ ] . the roles played by virulence factors produced by bacteria when crossing the central nervous system is also addressed, followed by the review of the specific traits of bacterial species more commonly associated with meningitis [ ] . coagulase-negative staphylococci are a broad group of skin commensals that emerged as major nosocomial pathogens, with the species s. epidermidis, s. haemolyticus, s. saprophyticus, s. capitis, and s. lugdunensis as the most frequent pathogens [ ] . in their paper, argemi et al. reviewed the recent progress achieved in the pathogenomics of these species, based on published work supported by whole-genome data deposited in public databases [ ] . as stated by the authors, the ever increasing amount of data available at the genomic, molecular, and clinical levels is expected to enhance the development of innovative approaches to characterize the pathogenicity of this bacterial group of pathogens [ ] . bacteria of the trueperella pyogenes species are considered as belonging to the microbiota of animals skin and mucous membranes of the upper respiratory and urogenital tracts, but it is also an important opportunistic pathogen to animals, leading to important economic losses [ ] . in their paper, rzewuska et al. reviewed the taxonomy of the species, their pathogenicity to animals, and the various diseases associated, as well as their possible involvement in zoonotic infections, as well as the reservoirs and routes of transmission and infections [ ] . the authors also present a thorough review of the main virulence factors used by the organism, including pyolysin, fimbriae, extracellular matrix-binding proteins, neuraminidases, and ability to form biofilms [ ] . the availability of complete genome sequences and a better knowledge of t. pyogenes virulence factors, transmission routes, and epidemiology of infections is expected to lead to the development of effective vaccines, with particular hope deposited on dna vaccines [ ] . candidiasis are on the rise worldwide, with candida albicans and candida glabrata as the more prevalent etiologic agents of these fungal diseases [ ] . the paper by galocha et al. thoroughly reviewed the distinct strategies used by the two candida species to successfully cause human infections, starting by the adhesion and ability to form biofilms [ ] . while c. albicans is dimorphic, growing as yeast or pseudohyphae, c. glabrata cannot undergo hyphal differentiation. as a consequence, c. albicans relies on the production of proteolytic enzymes and hyphal penetration to invade the host cell, while c. glabrata is thought to invade host cells by inducing endocytosis [ ] . the authors extensively review the distinct mechanisms used by the two pathogenic to evade the host immune system, and succeed as pathogens. the detailed knowledge of the virulence mechanisms is critical to develop therapies that specifically target virulence traits of these two pathogenic yeasts [ ] . bacterial small non-coding regulatory rnas (srnas) have emerged over the last decade as key regulators of post-transcriptional regulators of gene expression, being involved in a wide range of cellular processes, including bacterial virulence [ ] . in their review, pita et al. updated knowledge on srnas from two pathogens associated with respiratory infections and lung function decline of patients suffering from cystic fibrosis, p. aeruginosa and bacteria of the so-called burkholderia cepacia complex (bcc) [ ] . as stated by the authors, the knowledge on p. aeruginosa srnas is far more extensive than from bacteria of the bcc. after reviewing the main molecular characteristics of bacterial rnas and their modes of action, including the role played by hfq as a mediator of rna-rna interactions, the authors detail the description of the roles played by p. aeruginosa srnas known for their involvement in virulence traits of the bacterium. despite the shorter information on bcc srnas, the authors make a brief description of known srnas from bcc [ ] . the identification and functional characterization of additional srnas from these two pathogens will certainly enlighten our knowledge on their virulence traits. the development of new tools to investigate microbial pathogenesis, at the molecular and cellular level, is of keen importance to comprehend how the microorganism can invade the host and cause infection. the paper from hatlem et al. reviewed the basic molecular traits and applications of the spycatcher-spytag system, originally developed as a method for protein ligation [ ] . the system consists of a modified domain of the spycatcher surface protein from streptococcus pyogenes that recognizes the cognate spytag peptidic sequence composed of amino acid residues [ ] . upon recognition, a covalent isopeptide bond is formed between a lysine side chain of the spycatcher and an aspartate of the spytag [ ] . the authors describe in detail the fundamentals of the system and of related variants, emphasizing their uses in molecular studies of microbial virulence factors, surface proteins, membrane dynamics, as well as in the development of vaccines [ ] . microorganisms employ a wide array of virulence factors to successfully thrive and flourish with their hosts, leading this interaction to the development of infections that can often be fatal. the molecular knowledge of the virulence traits, associated with the recent availability of genomics data and bioinformatics tools for the more frequent human pathogens, is expected to lead in the near future of novel molecules and strategies to battle infectious diseases. funding: this research was funded by fundação para a ciência e a tecnologia, through project uidb/ / from ibb-institute for bioengineering and biosciences. the serine protease autotransporters tagb, tagc, and sha from extraintestinal pathogenic escherichia coli are internalized by human bladder epithelial cells and cause actin cytoskeletal disruption antibiotic resistance, virulence factors, phenotyping, and genotyping of non-escherichia coli enterobacterales from the gut microbiota of healthy subjects relation of the pdxb-usg-trua-deda operon and the trua gene to the intracellular survival of salmonella enterica serovar typhimurium discovery of lahs as a global regulator of environmental adaptation and virulence in aeromonas hydrophila lon protease is important for growth under stressful conditions and pathogenicity of the phytopathogen, bacterium dickeya solani a novel effector protein of apple proliferation phytoplasma disrupts cell integrity of nicotiana spp proteomics analysis of ssnsd -mediated compound appressoria formation in sclerotinia sclerotiorum types and origins of bacterial membrane vesicles interspecies outer membrane vesicles (omvs) modulate the sensitivity of pathogenic bacteria and pathogenic yeasts to cationic peptides and serum complement hfq: a multifaceted rna chaperone involved in virulence comparative integrated omics analysis of the hfq regulon in bordetella pertussis targeting pseudomonas aeruginosa in the sputum of primary ciliary dyskinesia patients with a combinatorial strategy having antibacterial and anti-virulence potential from gene to protein-how bacterial virulence factors manipulate host gene expression during infection bacterial actin-specific endoproteases grimelysin and protealysin as virulence factors contributing to the invasive activities of serratia virulence factors of meningitis-causing bacteria: enabling brain entry across the blood-brain barrier coagulase-negative staphylococci pathogenomics pathogenicity and virulence of trueperella pyogenes: a review divergent approaches to virulence in c. albicans and c. glabrata: two sides of the same coin small noncoding regulatory rnas from pseudomonas aeruginosa and burkholderia cepacia complex catching a spy: using the spycatcher-spytag and related systems for labeling and localizing bacterial proteins acknowledgments: ibb-institute for bioengineering and biosciences is acknowledged for funding. the author declares no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -rlvk bcx authors: balloux, francois; van dorp, lucy title: q&a: what are pathogens, and what have they done to and for us? date: - - journal: bmc biol doi: . /s - - -z sha: doc_id: cord_uid: rlvk bcx microbes are found on us, within us and around us. they inhabit virtually every environment on the planet and the bacteria carried by an average human, mostly in their gut, outnumber human cells. the vast majority of microbes are harmless to us, and many play essential roles in plant, animal and human health. others, however, are either obligate or facultative pathogens exerting a spectrum of deleterious effects on their hosts. infectious diseases have historically represented the most common cause of death in humans until recently, exceeding by far the toll taken by wars or famines. from the dawn of humanity and throughout history, infectious diseases have shaped human evolution, demography, migrations and history. a pathogen is defined as an organism causing disease to its host, with the severity of the disease symptoms referred to as virulence. pathogens are taxonomically widely diverse and comprise viruses and bacteria as well as unicellular and multicellular eukaryotes. every living organism is affected by pathogens, including bacteria, which are targeted by specialized viruses called phages. the number of viruses and bacteria on earth is staggering and they occupy essentially every environment. a liter of surface seawater typically contains in excess of ten billion bacteria and billion viruses. the number of viruses on earth is estimated to be around , which corresponds to roughly ten billion times the number of stars in the universe [ ] . an average human is made up of about trillion cells but carries a similar number of bacteria, mostly in the gut [ ] . the vast majority of viruses and bacteria we are exposed to have no negative effect and some can even * correspondence: f.balloux@ucl.ac.uk ucl genetics institute (ugi), darwin building, gower street, london wc e bt, uk be beneficial, though a tiny fraction of these can severely affect our health. specifically, about one in a billion microbial species is a human pathogen. indeed, approximately human pathogens have been described, whereas it has been estimated that there are one trillion microbial species on earth, the vast majority of which remain uncharacterized [ ] . pathogens can be divided into two main categories, namely facultative and obligate pathogens, reflecting how intimately their life cycle is tied to their host. facultative pathogens are organisms for which the host is only one of the niches they can exploit to reproduce. facultative pathogens are primarily environmental bacteria and fungi that can occasionally cause infection. they include many of the most problematic hospital-acquired bacteria involved in the antimicrobial resistance pandemic. a distinction is sometimes made between facultative and accidental pathogens, with the latter representing those which only occasionally infect weakened or immunocompromised hosts. typical examples of 'accidental' pathogens include neisseria meningitidis or escherichia coli. obligate pathogens require a host to fulfil their life cycle. all viruses are obligate pathogens as they are dependent on the cellular machinery of their host for their reproduction. obligate pathogens are found among bacteria, including the agents of tuberculosis and syphilis, as well as protozoans (such as those causing malaria) and macroparasites. some obligate pathogens require multiple different hosts to fulfil their life cycle. the definite host, which supports the adult form of the pathogen, is often a vertebrate and the intermediate host (referred to as a vector) is generally an arthropod or a mollusc. this alternation of vertebrate and invertebrate hosts is found in viruses (for example the zika virus), bacteria (for example lyme disease) and protozoa (malaria). trematodes (parasitic flatworms) go even further and some exhibit among the most baroque life cycles. digenetic trematodes have a basic three-host life cycle, and for some species a fourhost life cycle. for instance, halipegus occidualis sequentially has to infect a freshwater snail, an ostracod, a dragonfly nymph and ends its cycle after the dragonfly is eaten by the green frog rana clamitans, where it resides under its tongue [ ] . what is the host range of pathogens? some pathogens are limited to infecting a single host species, whereas others can infect a multitude of host species. host ranges can feel highly idiosyncratic if not outright puzzling. for example, leprosy in humans is caused by two related intracellular bacteria mycobacterium leprae and mycobacterium lepromatosis, which are essentially restricted in the wild to humans, as well as armadillos in the americas and red squirrels in scotland [ ] . conversely, yersinia pestis, another intracellular obligate bacterium and the agent of plague, has a natural life cycle involving alternating infections of rodents and fleas, but can infect essentially any mammalian host. an interesting twist in the case of plague is that y. pestis is not well adapted to the human host. with the exception of uncommon occurrences of human-to-human transmissions, referred to as pneumonic plague, plague epidemics (bubonic plague) are caused by plagueinfected fleas biting humans. somewhat ironically for a pathogen that is possibly the biggest killer in human history, bubonic plague is a complete evolutionary disaster. the human host is at a very high risk of dying, the flea cannot reproduce on a meal of human blood and the bacterium is stuck in an evolutionary dead-end as it cannot transmit to another host. there is no obvious predictor for the host range of different pathogens. intuitively, it may be tempting to predict that pathogens with a more intimate relationship with their host are more closely adapted to their host, and thus have a more restricted host range. however, there is no obvious pattern suggesting that viruses (that rely on the host cells' machinery for reproduction) have a narrower host range than bacteria. also, intracellular bacteria do not seem to have a markedly narrower host range than extracellular ones, despite being more intimately tied to their host. we know relatively little about the underlying genetic changes required for a pathogen to infect a new host, though, interestingly, only a few mutations can be required for a host jump. for example, avian influenza is only around five mutations away from being able to transmit in mammals [ ] , and a single amino acid change was sufficient for the humanadapted bacterium staphylococcus aureus to become a pathogen of rabbits [ ] . obligate pathogens tend to be highly adapted to their hosts, with sophisticated mechanisms to synchronise their life cycles with that of the host, and the ability to manipulate the host's immune system, metabolism and sometimes even behaviour. genes encoding proteins specific to pathogenicity are referred to as virulence factors, which include a variety of molecules required for colonization of the host, immunoevasion and immunosuppression, scavenging nutrients within the host, and entry into and exit out of cells for intracellular pathogens. in bacteria, virulence factors are often found in groups of genes on pathogenicity islands, which can be transferred horizontally by plasmids or other transposable elements. for example, one of the defining features of the plague bacterium y. pestis from its less virulent closest relative yersinia pseudotuberculosis, is the inclusion, early in its evolution, of two plasmids carrying genes involved in pathogenicity [ ] . while acquisition of novel genes and repurposing of existing ones is essential in the evolution towards pathogenicity, a general feature during the evolution towards pathogenicity is genome reduction through the inactivation and loss of genes. this can be primarily explained by the fact that a host represents a fairly stable and resource-rich environment where some metabolic pathways required in the environment are not necessary. genome reduction is a general trend accompanying the evolution towards pathogenicity and is observed in mycobacterium tuberculosis, pathogenic e. coli strains and in the ongoing adaptation of klebsiella pneumoniae lineages to cystic fibrosis patients. the most extreme example is leprosy (m. leprae and m. lepromatosis), which has shed nearly half the genes found in their environmental relatives [ ] . another interesting tendency of many bacterial pathogens is the secondary loss of the ability to undergo genetic recombination [ ] . pathogens cause illness to their hosts through a variety of ways. the most obvious means is through direct damage of tissues or cells during replication, generally through the production of toxins, which allows the pathogen to reach new tissues or exit the cells inside which it replicated. bacterial toxins are among the deadliest poisons known and include famous examples such as tetanus, anthrax or botulinum toxin, known as botox in its commercial application. however, the damage to the host is often self-inflicted through a strong or sometimes excessive immune response that indiscriminately kills infected and uninfected cells and damages host tissues. typical examples of maladaptive over-reaction of the immune system include cirrhosis and liver cancer in hepatitis b [ ] , or the - influenza epidemic, where the toll was highest amongst the young and healthy possibly because they mounted the strongest immune response and as such died from a 'cytokine storm' in the lungs leaving patients literally drowning in their own body fluids [ ] . some pathogens benefit from the hosts' immune reaction to spread within an infected host or increase their transmission to uninfected hosts. influenza transmits mainly through aerosols created through the sneezing and coughing it causes. vibrio cholerae triggers a strong inflammatory response in the gut mucosa, leading to watery diarrhoea and ensuring its release in the environment and thus infection of further hosts. pathogens greatly vary in the severity of their symptoms from a mild inconvenience to assured death. it is sometimes assumed that the deadliest pathogens represent recent host jumps where the pathogens' virulence is maladapted to the new host, and that co-evolution between host and pathogen will lead to more benign symptoms over time. however, this is only true in the case of strict vertical transmission (such as from mother to child), where survival and transmission of host and pathogen are intimately linked. in the case of horizontal transmission, the situation is more complex and there is no straightforward way to predict the evolution of future virulence, as it will depend on a variety of factors, including the population structure of the host and the correlation between virulence and transmission [ ] . a textbook example for reduction in virulence is the introduction of myxomatosis into the european rabbit population in australia and france in and , respectively. upon introduction, the virus initially killed about % of infected rabbits but over a few years mortality went down to %, following the emergence of attenuated virus strains and genetic resistance in the rabbit population. while the virulence went down, which translated into higher transmission rates, the current % mortality rate remains exceedingly high and there is no evidence for further reduction in the short term [ ] . bdgpl, the global lineage of the amphibian fungal pathogen batrachochytrium dendrobatidis (bd), is the only known pathogen to have extirpated entire host species. yet, over the three decades since its discovery, it shows no sign of evolving lower virulence. the primary reason there is little selective pressure on bd for virulence attenuation is that it can infect a very vast host range so that the extinction of any particular host species has limited impact on its fitness. even worse, some host species, such as the widely introduced african clawed frog, xenopus laevis, and the american bullfrog, rana catesbeiana, carry the disease asymptomatically, fuelling the global bd pandemics and limiting any shortterm prospect for significant decrease in virulence [ ] . these are just examples of the evolution of virulence but both illustrate that there is no simple pattern of decrease in pathogenicity with time. how old are the major human pathogens? apart from a few putative ancestral pathogens, including helicobacter pylori [ ] , that might have co-speciated with their human host, the infectious diseases afflicting us were acquired through host jumps from other wild or domesticated animal hosts or sometimes from the wider environment. the timing of these events and the original source remains unclear in many cases. the traditional view has been that many human pathogens emerged during the neolithic revolution. the main arguments for an origin of human pathogens linked to agriculture are based on the proximity between traditional farmers with their livestock and the emergence of higher human population densities in stable settlements enabled by agricultural subsistence. high population density is indeed required by some epidemic diseases which could not have maintained themselves on scattered groups of hunter-gatherers [ ] . this argument, however, neglects the fact that pathogens can evolve fast. also, while the proximity of humans and livestock is conducive to host jumps, humans transmitted more diseases to domestic animals than they acquired, with tuberculosis in particular having probably jumped from humans to cattle rather than the other way around [ ] . finally, this argument also neglects the high burden of pathogens in wild populations, including in the great apes. ancient direct evidence is scant for pathogens, and historical records rarely allow unambiguous attribution of described symptoms to a disease. that being said, recent progress in sequencing technology and in particular the ability to generate sequences, if not complete genomes, from ancient samples has greatly improved our understanding of the age of the major human pathogens, often leading to unexpected results. figure summarises the current knowledge on the age of the seven current 'major killers' , as well as plague, which was included due to its major impact in the past. while some of these estimates may need to be updated in the future after the emergence of new evidence, it is unlikely the general pattern will change much. some human diseases are old (for example plasmodium falciparum malaria) and others recent, such as hiv or, more surprisingly, measles. there is also no obvious pattern pointing to the neolithic revolution as a strong driver for the emergence of human pathogens. where are the resistance-conferring genes in the human genome? infectious diseases have killed well over half of all humans who have ever lived on earth. pathogens, such as childhood diseases, that affect their host prior to reproduction, through death or reduced fertility, have exerted enormous selective pressures. yet, scans of the genome for signatures of pathogen-driven selection have identified only a few variants with clear effects. similarly, genome-wide association studies (gwas) for infectious disease resistance/susceptibility have identified only few loci impacting on infectious disease susceptibility [ ] , despite their success in identifying thousands of variants involved in chronic diseases and phenotypic traits such as height. even for diseases that have affected us for a long time, for example tuberculosis, we know of no obvious protective genetic variant. given the high selective pressure pathogens must have exerted, it is reasonable to ask where all the resistance genes are. strongly protective variants may have reached fixation, rendering them undetectable unless the pathogen has a highly heterogeneous distribution range. an interesting case for regional selective pressure is the duffy negative antigen mutation protective against plasmodium vivax that is found at close to % in sub-saharan africa but virtually absent anywhere else. another situation where a resistance gene does not reach fixation arises when the protective variant is deleterious when homozygous, as in sickle cell anaemia. we might also speculate that the evolutionary potential and high genetic diversity of most pathogens limits our ability to detect protective variants in the human genome, particularly so if these were only effective against a subset of lineages within a pathogenic species. in addition to the few variants protective against specific pathogens, we also know of genomic regions involved in immunity against a wide spectrum of pathogens, such as interleukin genes or the major histocompatibility complex (mhc) system. the very high genetic diversity of the mhc is believed to have been shaped by exposure to different pathogen species [ ] . also, following the recent development of techniques to sequence ancient dna, it has been suggested that immunity genes such as those encoding toll-like receptors have been acquired following hybridization with archaic humans and are over-represented in the current gene pool of anatomically modern humans relative to genes not involved in immunity [ , ] . infectious diseases had a massive impact on our history, leading to the rise and fall of civilizations, both through the toll they took on human life but also through economic and societal collapse following epidemics. more military campaigns have probably been lost or won due to infectious diseases than due to the tactical acumen of the armies' commanders. thucydides reports in his history of the peloponnesian war, written in the th century bc, how the plague of athens devastated the citystate of athens in ancient greece during the second year [ ] ; influenza [ ] ; hiv [ ] ; tuberculosis [ ] [ ] [ ] [ ] ; p. falciparum malaria [ , ] ; hepatitis b [ ] ; measles [ ] ; plague [ , ] of the peloponnesian war ( bc) when it was on the cusp of victory against sparta, ending the golden age of pericles and athenian predominance in the ancient world. the eventual fall of the roman empire was also largely down to another epidemic, the justinian plague in - ce, which precluded the emperor justinian recovering lost territories in the western part of the empire [ ] . infectious disease played an equally important role in past human migrations. the conquistadores' sweeping conquests of large swaths of the americas in the th century was greatly aided by the diseases they brought with them, such as measles and smallpox, to which the indigenous populations had limited immunity [ ] . conversely, one of the possible reasons europeans managed to colonize africa was that they used quinine, an antimalarial drug derived from the bark of the cinchona tree [ ] . history has been shaped not only by pathogens infecting humans, but also those affecting domestic animals and crops. for example, it has been suggested that the islamic conquest of the th and th centuries did not extend to sub-saharan africa because the horses and camels of the islamic armies were dying from trypanosma spread by tsetse flies [ ] . conversely, pathogens were at other times the drivers of large migration. around one million irish people died and another million migrated to the us to escape the famine caused by phytophthora infestans destroying potato harvests between and [ ] . at least in the developed world, the leading causes of human mortality are no longer infectious diseases but instead age-associated disorders such as cancer, heart disease and diabetes. numerous countries have undergone an epidemiological transition, starting some years ago in some developed countries and less than years ago for developing countries. diseases that once devastated human populations, such as smallpox, are now eradicated. others, such as the plague or leprosy, are largely under control with the exception of a few hotspots. the current situation is, however, one of new challenges. globalization and increased mobility, particularly air travel, have facilitated the transmission of diseases not just locally but between continents. the recent outbreak of zika in the americas, for example, has been attributed in part to an increase in air travel from infected areas into brazilian airports, extending both the incidence and geographic range of the virus [ ] . the outbreak of severe acute respiratory syndrome (sars) and recurrent ebola crises in central africa highlight the ability of new and existing diseases rapidly to become significant international health threats. in addition, our ability to combat infectious diseases is also challenged by the widespread emergence of pathogen drug resistance. the global antimicrobial resistance (amr) crisis is increasingly limiting our resources to combat disease through antimicrobial therapy. thus, in spite of the global health narrative supporting a decline in the number of deaths caused by infectious disease, the complexity of our interactions with diseasecausing agents are as significant now as through history. infectious diseases continue to be a major cause of mortality globally, responsible for between a quarter to a third of all deaths and nearly half of all deaths in people under the age of , with most of these in principle avoidable. microbiology by numbers revised estimates for the number of human and bacteria cells in the body the role of damselflies (odonata: ztgoptera) as paratenic hosts in the transmission of halipegus eccentricus (digenea: hemioridae) to anurans red squirrels in the british isles are infected with leprosy bacilli airborne transmission of influenza a/h n virus between ferrets a single natural nucleotide mutation alters bacterial pathogen host tropism yersinia pestis, the cause of plague, is a recently emerged clone of yersinia pseudotuberculosis insight into the evolution and origin of leprosy bacilli from the genome sequence of mycobacterium lepromatosis microbial diversity and the genetic nature of microbial species immunobiology and pathogenesis of viral hepatitis the influenza pandemic: insights for the st century infectious diseases of humans myxoma virus and the leporipox viruses: an evolutionary paradigm emerging fungal threats to animal, plant and ecosystem health age of the association between helicobacter pylori and man myths and misconceptions: the origin and evolution of mycobacterium tuberculosis evolution, revolution and heresy in the genetics of infectious disease susceptibility pathogendriven selection and worldwide hla class i diversity genomic signatures of selective pressures and introgression from archaic hominins at human innate immunity genes introgression of neandertal-and denisovan-like haplotypes contributes to adaptive variation in human tolllike receptors cooling and societal change during the late antique little ice age from to around ad insights from paleomicrobiology into the indigenous peoples of pre-colonial america -a review selected papers from the first international conference 'camel cultures: historical traditions, present threats and future prospects'. london: rn books after the famine: plant pathology, phytophthora infestans, and the late blight of potatoes zika virus in the americas: early epidemiological and genetic findings th century variola virus reveals the recent history of smallpox pandemic influenza -including a risk assessment of h n the early spread and epidemic ignition of hiv- in human populations detection and molecular characterization of -year-old mycobacterium tuberculosis from a neolithic settlement in the eastern mediterranean armed conflict and population displacement as drivers of the evolution and dispersal of mycobacterium tuberculosis pre-columbian mycobacterial genomes reveal seals as a source of new world human tuberculosis eighteenth-century genomes show that mixed infections were common at time of peak tuberculosis in europe plasmodium falciparum accompanied the human expansion out of africa genomes of cryptic chimpanzee plasmodium species reveal key evolutionary events leading to human malaria dating the origin and dispersal of hepatitis b virus infection in humans and primates origin of measles virus: divergence from rinderpest virus between the (th) and (th) centuries early divergent strains of yersinia pestis in eurasia , years ago historical variations in mutation rate in an epidemic pathogen, yersinia pestis the authors are grateful to liam shaw for discussions on the manuscript and for financial support from the mrc, bbsrc, nerc and esrc (grants ne/m / and mr/p / ). lvd and fb wrote the manuscript. both authors read and approve the final version. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - pj fmv authors: perdue, michael l.; seal, bruce s. title: impact of avian viruses date: - - journal: viral ecology doi: . /b - - / - sha: doc_id: cord_uid: pj fmv nan in considering the ecology of the avian viruses and their impact on life on earth, it may be useful to first consider the host itself. the class aves first diverged from the reptiles between and million years ago, depending on which current paleontological interpretations one accepts. of the vertebrate classes, they are most often compared with the reptiles from which they evolved and with mammals because of their common warm-blooded nature. this shared feature with mammals is probably the most influential with respect to ecology, since a virus adapted to warm-blooded physiology would not fare well in the cold-blooded world, and vice versa. along these lines, our recent experience is that some virologists think that a bird is a bird, and that if a given virus replicates in one, it will replicate in them all. this is, of course, far from the truth and perhaps should be a starting point for discussing avian virus ecology. according to fossil records, the class aves emerged from the extinctions of the late cretaceous period , , years ago, somewhat bottlenecked, as did the class mammalia, but since that time they have undergone parallel evolution with mammals and are equally diverse in their own right. certainly some viruses, such as avian paramyxovirus (of which newcastle disease virus is the prototype), are infectious for numerous orders of birds. there are also other important avian virus strains such as the gallid herpesvirus (infectious laryngotracheitis) and the avihepadnaviridae, which appear to be exclusively confined to a single bird family or even genus. it is clear that birds, because of their close association with powered flight, do present a more homogeneous anatomy and physiology than do the class mammalia (feduccia, ) . whether this affects or specifies the molecular nature of viruses that infect birds is not really known. this feature, however, almost certainly uniquely impacts the natural distribution and ecology of the viruses that inhabit the flying birds. a virus infecting or persisting in an arctic tern could potentially be translocated up to , miles in a few weeks. viruses such as some avian orthomyxovirus or avian paramyxovirus strains, which may exhibit subclinical infections, might be shared among a migrating flock of waterfowl and persist for indefinite periods as the flocks move from lake to lake. along the way the virus might be shared with other birds crossing flight paths. so birds do present unique environments for transmission of viruses. the commercial practices of humans have further provided unique opportunities for transmission not normally seen for birds. the order galliformes in particular, which includes domestic and wild fowl; pheasants, quail, and turkeys, has been unquestionably affected. it would be quite safe to say that humans have both determined and upset the ecological balance among members of this order and the viruses that affect them. due to continuous breeding practices, live-virus vaccination regimes, and by housing tens of thousands of birds in a single enclosure, situations never encountered in natural settings are created. whether this has affected the virus-host ecological balance in other orders of undomesticated birds as well is not known; but it would seem highly likely. one of the most important features of a host-parasite relationship, of course, is the host's immune defense. while there are similarities shared with respect to the immune system, particularly the dual (humoral and cell-mediated) nature, there are marked differences between cold-and warm-blooded vertebrates and additional significant differences between birds and mammals (eerola et al., ) . the discovery of processing and maturation of immunoglobulin-producing lymphocytes was made as a result of characterization of an avian-specific organ, the bursa of fabricius (ratcliffe, ) . although functional equivalents exist in mammals, the bursa is a distinct and wholly avian-specific organ. since several viruses are known to affect this organ specifically, it should be considered a unique ecological niche. a second significant difference in the immune system of birds is in the apparent genetic content responsible for specifying the avian major histocompatibility complex (mhc). the chicken mhc appears considerably more simple (providing the oxymoron: a simpler complex) than the mammalian mhc (kaufman and wallny, ) . several alleles have arisen and considerable recombination documented in the mammalian mhcs that thus far have been studied. these are responsible for producing a great variety of class i-, ii-, and iii-type proteins used in recognition and presentation of antigen. in the chicken, some mhc haplotypes produce only one type of class i protein and there is no evidence for recombination at all (kaufman and wallny, ) . this has led to speculation that the relationship with the avian pathogens has evolved significantly differently from the mammals. one result of this difference may be the occurrence and frequency of either resistance or sensitivity to specific viral infections encountered in chickens (see below). thus, the class ayes presents several unique features that might ultimately affect the ecology of viruses that infect them. the classification of birds has presented a significant challenge to systematists. the number of species has actually decreased over the years because of the clearer genetic relationships that have emerged. conversely, additional species have been defined as recognition of convergent evolution has become clearer. currently, new species are identified at a rate of about two per year. while there remains some disagreement among specialists, most accept the current classification of orders, families, genera, and + species (gill, ). molecular analysis of avian genes for phylogenetic studies is still in its early stages. restriction fragment length polymorphism (rflp) analysis and sequence analysis of s mitochondria dna has yielded some molecular phylogenetic information but not enough to gain any insight regarding the viruses of birds (hedges et al., ; cooper and penny, ; mindell et al., ) . roughly speaking, the flightless orders represented by ostriches, rheas, cassowaries, and kiwis are thought to be the most ancient, while the bewildering array of members of the order passeriformes (representing % of known species and % of known families) are thought to be the most recent (gill, ) . trying to determine how long viruses have been associated with various avian groups is of course as impossible as it is with virus-host relationships in any other classes. orthomyxoviruses, paramyxoviruses, and coronaviruses have recently been isolated from ostriches, rheas, and emus, indicating no absolute barriers in these more ancient birds. the class aves is of course distributed worldwide. in addition, there are several hundred species that migrate, sometimes in spectacular fashion. biogeographers have divided the earth into six distinct faunal regions, which correspond somewhat with the major continental areas (welty, ) . these include the nearctic (north america and greenland), palearctic (asia, europe, and north africa), ethiopian (central and southern africa), oriental (india, southeast asia), australasian (australia, new guinea), and neotropical (south and central america). each area contains its own characteristic birds. in the northern hemisphere (nearctic and palearctic), most species are migratory, which is not the case in the other areas. the neotropical has the richest and most abundant bird life, and the southern hemisphere has by far the most families represented, as well as the most families peculiar to a given region. each year, billions of land birds and waterfowl in north america and asia head south to south america and africa, respectively, carrying their viruses with them. the size and scale of these geographic relocations are unmatched by any other land vertebrates. the sea mammals are the only other comparable migrating vertebrates, and they surely cross paths with the birds. in the most interesting putative contacts, purely avian-origin type a orthomyxovirus of at least two different subtypes were isolated from dead and dying seals off the coast of new england in (webster et al., b hinshaw et al., ) . this represents viral ecology at its most forceful, being effected between two warm-blooded vertebrate orders during their natural migration and geographic interaction. if one takes a broad look at the virus families associated with avian hosts (table i) , it is actually easier to list the families of viruses that infect vertebrates but that do not yet have a clear avian member. these are the iridoviridae, arenoviridae, african swine fever-like viruses, and filoviridae. a similar comparison of families that do not contain a mammalian member yields only the birnaviridae. so, in one sense, the mammalian host might be considered more virus-friendly, rather than the alternative. still, many investigators think of bird tissues, in particular the embryo, as being an ideal medium for identifying new viruses. if just from an experimental and practical, rather than a natural, point of view, the avian host has played a tremendous role in our understanding of viral ecology. many of the most important findings in virology have been made utilizing the chicken embryo and in chicken cell cultures. the egg also continues to provide an abundant and important substrate for the production of veterinary and human vaccines. if we think of ecology as the study of organisms and their relationship with their environment to include all other organisms, we most certainly encounter a unique relationship with viruses. unlike higher pathogens, viruses really do not mate, communicate, or colonize (except in the very broadest stretches of the imagination); they simply parasitize and replicate. "we try and make them more familiar by defining higher organism-type genetic alterations as "evolution," when in truth the viruses may simply be adapting to the evolutionary pressures encountered by, or within, its host. obviously, the host is central to the viruses' lifestyle and must be considered a major part of virus ecology. in this sense, information gathered on various viruses as they replicate in embryos or embryo cells, in the absence of immune pressure, should only be considered "natural" for those viruses that are transmitted vertically. the embryo or cell culture then can only provide a window on the natural ecology of the organism in which the immune system is not a factor. in ecological terms, only the natural state of the virus-host relationship becomes important. viruses being parasites, these relationships more often than not eventually result in a disease state. it is not possible to distinguish the molecular biology of avian viruses from that of non-avian viruses on the basis of features unique to the avian system. however, there are some important distinctions with which viruses must deal. one dramatic difference between birds and mammals is body temperature. on average, birds operate at -- ~ higher than mammals, and most can readily regulate their temperature, some dropping body temperature as much as ~ during the night (gill, ) . while this certainly could influence such things as rates of viral enzyme activity, polymerase fidelity, or protein stability, there are no situations documented in which a particular virus group is restricted to class aves solely on the basis of body temperature. it is possible to distinguish some important receptor-specific distinctions unique to avian systems. one example is found in the influenza type a viruses. the glycosidic linkages associated with sialic acid residues on the avian versus mammalian cell surface serves to restrict the strains of viruses able to replicate. influenza virus hemagglutinin (ha) proteins bind to sialic acid residues on the surface of the host cell. although there is no absolute barrier, those viruses that replicate well in avian cells have a receptor binding pocket on the surface of the ha that has a preference for the o~- , siallyl-sugar linkages abundant in bird tissues. those viruses that replicate well in mammalian cells have a preference for ~- , linkages more prevalent in mammalian cells (rogers and paulson, ; murphy and webster, ) . there are probably many more receptor specificities associated with other avian virus infections, particularly among the herpesviruses, where host range is narrowly dictated. another example of virus-specified tissue tropism unique to birds would be infectious bursal disease virus (ibdv; genus avibirnavirus), which has "evolved" an affinity for the bursa, a bird-specific organ. the other two known genera of birnaviridae infect fish and invertebrates. since there are as yet no mammalian members, it is tempting to speculate that ibdv is bird specific because of its evolved affinity for the bursa. air sacs are also uniquely avian structures. the bird lung is a fixed tissue incapable of expansion like mammalian lungs. the air sacs are a series of extensions of the respiratory system that expand with the musculature of the body cavity and allow the large-scale rapid oxygen transfer needed for powered flight. the air sac system is extensive throughout the bird, even encroaching into bone tissue; some birds have as many as nine distinct air sacs. many viruses replicate and cause disease in this unique organ system, and there are examples of apparent preferences by some viruses for air sac tissue over other respiratory tissue. finally, feathers, the most notable and prominent distinguishing feature of the class aves, provide a unique niche for at least two three viruses-avipox, marek's disease, and psittacine beak-and-feather disease virus --which can replicate in and are spread from feather follicles (biggs, ; tripathy and reed, ) . as mentioned previously, the immune system of the intact bird is a critical feature in the establishment of ecological relationships between many viruses and hosts. as the immune system plays a major role in the relationships, they will be covered, but there are some extraordinary examples that deserve their own mention. marek's disease, caused by an alphaherpesvirus, is most commonly associated with lymphomas developing relatively early in the life of a chicken. the initial infection in md is a respiratory infection probably initiated in macrophages. a typical field infection would then progress through the following phases in a bird: latency is established primarily in lymphoid cells, and mostly in activated t lymphocytes. in susceptible birds, the latent state progresses to a second round of lytic infections at multiple sites in the bird. interestingly, at this point the feather follicle epithelium is the only site where complete virus replication occurs and becomes a significant source of environmental infectious virus (calnek and witter, ) . concomitant and permanent immunosuppression occurs in the affected bird. the disease then progresses into a lymphoproliferative phase in susceptible birds that can range in severity depending on the virus strain and breed of chickens. some breeds have shown natural resistance to this progression, and, although the mechanism of resistance is not completely understood, it involves primarily lymphoid tissue and is dictated mostly by genes involved in the immune response (venugopal and payne, ) . it should be clear from this scenario that the immune system in this host-parasite relationship plays a critical role in the ecology of marek's disease virus. infectious bursal disease virus, as mentioned earlier, is a virus that replicates exclusively in lymphoid tissue. the virus can be detected replicating in the bursa of fabricius and within circulating lymphocytes as early as hours after infection of a chicken. it causes acute degeneration of various lymphoid tissues within the first day of infection and results in a severe, albeit age-dependent, depression in the humoral immune response, being most dramatic in very young birds. interestingly, the infection does not suppress b-cell responses to the viral antigens themselves; in fact, there is stimulation of proliferation of b cells committed to anti-ibdv antibody production (mcferran, ) . one may only speculate as to what role, if any, this may play in the viral ecology or replicative cycle, but the immune system is once again a major participant in this host-parasite relationship. finally, the effects of vaccination programs on the ecology of avian viruses cannot be overemphasized. in one sense, we have an ongoing experiment where humans control the type of viruses to which certain species of birds are exposed. since live viruses generally yield much better immune responses, they are employed most often. in the case of the single-stranded rna viruses, noted for their ability to rapidly mutate and avoid the immune system, this has the effect of artificially challenging the immune system, creating selection pressure between host and parasite that would not normally occur. this may be effective in the short run, protecting against disease, but the long-term effects are unknown. table i are some of the most important features of relationships of various viruses with their avian hosts as well as the wide variety of relationships that exist. it is not feasible to list all the interesting attributes for each virus-host relationship, but the table presents a variety of relationships that will be covered in more detail in the following sections. as such, the table should not be taken as the final word on each member virus. for example, in the case of turkey meningoencephalitis virus, a flavivirus, reduced egg production is listed under pathogenesis. reduction in egg production is a common feature in many virus infections of poultry, and while listing it under pathogenesis somewhat stretches the meaning of that word, this clinical sign is one of the most important aspects of that disease in turkeys. although there is variation in the economic or ecological impact of various viral groups from year to year and among geographic sites, the "top ten" list of virus groups exhibiting routine significant impact on commercial poultry worldwide (not necessarily in order of impact) are paramyxoviruses (newcastle disease); coronaviruses (infectious bronchitis); herpesviruses (infectious laryngotracheitis; marek's disease; duck enteritis); reoviruses (viral arthritis); picornaviruses (avian encephalomyelitis); adenoviruses (egg drop syndrome); retroviruses (lymphoid leukosis); orthomyxoviruses (avian influenza); poxviruses (fowlpox); and birnaviruses (infectious bursal disease). the circoviruses (chick anemia) could likely be included in the above list, except it is not yet known to what extent the viral infection alone influences morbidity and mortality (see below). what may not be obvious from table i is that if one searches for viruses in a given avian species one will likely find them. in some of the virus families listed, investigators were forced to clearly separate the disease-causing virus in question from accompanying "contaminant" viruses, which may or may not have influenced the original disease manifestation. there appear to be many viruses of birds that in certain ecological conditions and in certain species may be considered "normal flora" and are not associated with disease. these, obviously, are less interesting to any funding agencies and consequently do not receive much research attention. no one really knows to what extent their transmission and persistence in avian populations affect their own ecology or that of their hosts. the office international des epizooties (oie), the principal world organization for animal health, provides listings of the most serious infectious diseases of animals (oie, ) and divides them into two groups: list a diseases, which "includes those diseases that spread rapidly, the scope of which extends beyond national borders" and "have particularly serious socioeconomic or public health consequences"; and list b diseases, which include those "that are considered to be of socioeconomic and/or public health importance within countries." of the avian viral diseases listed above, only highly pathogenic avian influenza and velogenic newcastle disease are in list a; marek's disease, infectious bursal disease, infectious bronchitis, duck enteritis, and infectious laryngotracheitis are in list b. the only avian disease in oie's list and not on our top list is duck hepatitis, which is a complex of diseases caused by at least three virus families, and generally limited to country-specific origins. the ecological impact of viruses of birds ultimately interests us as homo sapiens, perhaps only to the extent that we are affected. in this respect, there is no question that the major impact thus far has been on raising birds as a food source for our species. ecologically speaking, this impact could have very significant consequences when one considers that poultry provide the most widely used protein source in the world. thus, we will consider for the most part how viruses affect this food source. the most significant and widespread infections of wild birds will be discussed as they are encountered relative to commercial and domestic birds. the most imminent and significant human public health concerns with regard to bird viruses appear to be twofold: the potential relationships with type a orthomyxoviruses that have become at least partially adapted in a totally nonpathogenic state to some avian orders. there is compelling evidence that these viruses may also replicate in pigs and re-assort with pig and/or human strains of influ-enza, yielding new variants capable of replicating and causing disease in humans. they also find their way into commercial poultry, sometimes with devastating consequences. with the recent documented transmission of a lethal avian influenza virus from commercial poultry to humans, these ecological relationships take on new significance. . the presence of a large reservoir of arboviruses in wild birds, some of which, when transmitted by invertebrate vectors to mammals, cause disease. beyond these two examples, other relationships are less directly important to human public health. other avian-origin viruses are capable of replicating in and sometimes causing mild disease in humans, but there is obviously not room in one chapter to cover each in detail. infectious laryngotracheitis (ilt) is a respiratory disease almost exclusively of chickens. infections in turkeys and pheasants have been reported, but surveys have yielded no wild bird reservoir or other domestic poultry reservoir (cranshaw and boycott, ) . based on this and the knowledge that ilt apparently exhibits little antigenic heterogeneity, it has been proposed that through proper husbandry practices and appropriate vaccination techniques the disease could be eliminated from commercial poultry (bagust and johnson, ) . the virus is a member of the alphaherpesvirinae subfamily and is identified taxonomically as gallid herpesvirus i. the disease is almost exclusively respiratory, with no systemic involvement. the severity of disease can vary from significant mortality ( %) in young birds to an inapparent infection of adult birds. there are age-dependent effects on the pathognomonic signs, and there do appear to be strain-specific virulence differences. however, different isolates do not exhibit sufficient genetic heterogeneity thus far to identify specific virulence factors (bagust and guy, ) . the most interesting aspect of ilt is its capacity for persistence in infected birds and flocks showing no disease signs. this persistence is most likely due to establishment of the latent state and recrudescence. numerous studies have demonstrated re-isolation of virus many months after initial infection, and one more recent study demonstrated reactivation of latent virus due to stress factors (hughes et al., ) . this latency achieved by herpesviruses could certainly be considered a unique ecological state, and most herpesvirus infections in birds are associated with its establishment. duck plague, also known as duck viral enteritis (dve), is caused by an alphaherpesvirus classified as anatid herpesvirus- , which infects free-living and domestic ducks, geese, and swans (sandhu and leibovitz, ) . the disease is acute and often associated with high morbidity and mortality. the virus has a worldwide distribution and has caused numerous documented outbreaks in free-living anatids. the first documented north american outbreak was in commercial ducks on long island, new york in (leibovitz and hwang, ) , and since that time sporadic reappearance of the virus in commercial and wild populations has occurred. major outbreaks in free-living birds along the mississippi flyway and a large epornitic in south dakota in killed tens of thousands of wild ducks and geese (brand, ) . vertical transmission and recrudescence of latent virus has been established experimentally in mallard ducks but has not been demonstrated in wild waterfowl. species susceptibility may vary among various waterfowl, although more than species have been shown to be naturally or experimentally infected and virulence differences among dve strains have been demonstrated. the other notable avian herpesvirus infection occurs in pigeons. the virus is taxonomically designated as columbid herpesvirus i and is antigenically indistinguishable from natural isolates taken from wild falcons and owls (vindevogel and duchatel, ) . the causative virus is antigenically distinct from ilt, mdv, herpesvirus of turkeys (hvt), and the anatid herpesvirus i. the disease associated with infection by columbid herpesvirus i is a major cause of growth retardation and bad performance in homing pigeons, though mortality is generally low. this virus, like ilt, becomes latent, reappears, and is shed in asymptomatic birds and flocks. other antigenically distinct herpesviruses have also been isolated from cormorants, quail, and storks. kaleta ( ) has proposed the division of these various herpesviruses into eight antigenic groups. the host specificity for each group varies, but in general they appear to be strictly adapted to the host of origin (as exemplified by the gallid herpesvirus i). gallid herpesvirus ii and hvt will be discussed in the following section. the impact of transmissible neoplastic diseases of poultry has been quite variable over the years. prior to vaccination, losses to marek's disease were often devastating, and even in marketable flocks condemnations due to lymphomas at processing plants could reach %. lymphomas caused by mdv and retroviruses are still the most common viral neoplastic diseases of poultry, and a recent increase in mortality and evolution of more virulent mdv strains indicates that the impact of these viruses will continue to be felt (witter, ) . marek's disease is caused by a herpesvirus that has two very similar relatives: a second nononcogenic serotype and the herpesvirus of turkeys (hvt). these are sometimes classified as serotypes - , respectively, of the gallid herpesvirus ii strain. they share common antigens that distinguish them from the nononcogenic herpesviruses but can be distinguished on the basis of antigenic differences (calnek and witter, ) . only the oncogenic mdvs (serotype ) cause significant problems in commercial poultry. the herpesvirus of turkeys is ubiquitous among commercial flocks and quite prevalent in wild turkeys but has not been directly associated with disease. the serotype strains were originally thought to be nononcogenic apathogenic mdv isolates until they were serologically distinguished from the original isolates. the type serotypes are associated with subclinical infections in chickens, although not as prevalent as turkey strains. the hvt isolate is a very interesting and important isolate, as it was used (and is still used) in vaccine formulations against marek's disease in chicken flocks. it has been very effective at protecting flocks against lymphomas and thus correctly billed as the first anticancer vaccine. marek's disease is relatively well controlled by using mixtures of primarily serotype and isolates in vaccine formulations. however, this vaccination program may ultimately extract a price. shown in figure is a diagrammatic representation of the evolution of virulence in mdv strains associated with changing vaccine formulations. the extent to which these formulations have influenced the evolution of virulence is not proven, but certainly the association is undeniable. the s have brought an increase in incidence of marek's disease cases, and the presence of these acutely virulent strains raises concerns for the future. what is clear is the role of humans in the generation of these strains. there is evidence that the commercial housing practices developed in the s and s resulted in generation of strains that were oncogenic and that this rapid evolution of virulence in the last years is probably due to human control of commercial bird populations. the avian retroviruses have one of the most interesting histories of all of the avian viruses. the first transmissible lymphomas were demonstrated in by ellermann and bang ( ) and the first cell-free transmissible solid tumor by peyton rous three years later (rous, ) . the etiologic agents of both of these diseases were later shown to be members of what is now known as the avian leukosis virus-avian sarcoma virus complex (alv-asv) of related retroviruses. scientists investigating this interesting group of viruses have garnered more nobel prizes (six) than with any other group. because of their relative simplicity in genetic content and their close association with the genetic character of their host, they have provided a bountiful model for the study of oncogenesis. these are rna-containing viruses that replicate via an intermediate dna stage that is most often incorporated into the host genome. the integrated viral genomes serve as templates for production of new progeny genomic rna molecules and the mrnas needed to make new viral proteins (coffin, ) . in the process, these viruses transform the host cell into a tumor cell. there is a significant array of viral subtypes and relationships that exist between the host and avian retroviruses and numerous reviews that may be consulted (crittenden, ; swanstrom and vogt, ) . there are several viral subgroups, based on antigenic differences alone, in the surface envelope glycoproteins. viral subtypes have also been grouped on the basis of whether the viruses rapidly induce neoplasia because of the presence of a viral oncogene, or whether they induce slow development of tumors due to their integration into the host genome and subsequent activation of cellular oncogenes. in commercial poultry, the predominant problems are caused by the latter, the slow-inducing lymphoid leukosis viruses (llvs). the sarcoma-, myeloblastosis-, and erythroblastosis-inducing strains cause only sporadic problems. additionally, it has been calculated that losses due to llv because of poultry performance decreases are actually greater than those due to the lymphomas. still, these viruses continue to cause condemnations at slaughter and must be distinguished from the more rapidly forming mdv-induced lymphomas. two other oncogenic avian retroviruses pose significant problems and possess much wider host ranges than the llv-sarcoma complex. the relatively new subgroup "j" has been characterized in europe as causing significant myelocytoma and endothelioma. in contrast to the llvs, subgroup j viruses have a high tropism for cells of the myelomonocytic series but a very low tropism for bursal cells (arshad et al., ) . a related subgroup j virus has been identified in broiler-breeder flocks in the united states as well . additionally, of note are those retroviruses of the reticuloendotheliosis virus (rev) group. infection with members of this group only rarely results in disease, and then disease most commonly occurs only in the hosts from which rev was first isolated: turkeys. still the rev group is predicted to have significant potential for causing problems in poultry (witter and johnson, ) , and the wide host range of the virus group has prompted speculation regarding potential as a public health risk. there are, in fact, data demonstrating antibodies to the virus in human and other mammalian sera . coronaviruses contain a large positive-sense rna genome of approximately kb. members of this virus family infect both mammals and birds (cavanagh et al., ) . infectious bronchitis, caused by a coronavirus, infectious bronchitis virus (ibv), is one of the major poultry viral diseases of worldwide economic importance. it is also one of the most rapidly spreading avian respiratory diseases known (mcmartin, ) . the virus is antigenically variable owing to a high mutation rate in the surface glycoprotein s gene. new antigenic variants of ibv continue to be isolated from various geographic regions. thus far, it has not been possible to determine the number of distinct ibv subtypes, but there are at least six (siddell, ) . strains can differ in their virulence and tissue tropism, but in general the disease is a rapid-onset respiratory distress that can cause significant mortality in young birds. in established flocks, the infection is often associated with growth retardation and reduction in egg production that may be exacerbated by other respiratory pathogens. estimates of a to % loss in market value have been made for a typical outbreak in a flock. vaccination is employed to control the disease, and an interesting related feature is the demonstration that vaccine strains can undergo recombination with wild-type strains. consequently, new ibv antigenic variants of the s gene emerge with characteristics of both viruses (cavanagh et al., ; wang et al., ) . ibv is also an excellent example of a virus exquisitely adapted to its host, as it is not found naturally in any other reservoir. chickens seem to be the only reservoir, and the virus is capable of persisting in some manner within populations and later being transmitted to naive flocks. evidence suggests the mode is primarily airborne transmission. in controlled studies, birds in houses feet away from an experimentally "seeded" house were infected via aerosol transmission (mcmartin, ) , and circumstantial data from natural outbreaks suggest transmission over distances of several hundred yards (cumming, ) . coronaviral enteritis of turkeys, also known as bluecomb disease, may also be species specific since chickens, pheasants, and quail do not exhibit any disease following inoculation with a strain of virus that is virulent for turkeys (hofstad et al., ; larsen, ) . coronaviruses have been isolated from ratites with enteric disease in zoological collections, but the relationship of these isolates with the turkey coronavirus (tcv) has not been investigated (frank and carpenter, ; kennedy and brenneman, ) . transmission of tcv appears to be restricted to the fecal-oral route, although cell-free lysates of the bursa from infected birds can be used to transmit the agent orally. among turkey flocks, the primary mode of transmission of coronaviral enteritis is via contaminated personnel and equipment. however, since tcv is excreted in fecal material and is very stable, it is conceivable that wild birds may serve as vectors for the agent. morbidity is usually as high as %, with mortality becoming as high as % in an infected turkey flock. mortality among poults may be much higher with increased numbers of secondary bacterial gastrointestinal infections contributing to severity of disease. turkey coronavirus also shares features reported for mammalian hemagglutinating coronaviruses and is closely related by sequence and antigenic crossreactivity to bovine coronavirus and a human coronavirus isolate (verbeek and tijssen, ) . this close relationship suggests recent interspecies transmission of the coronaviruses. while no etiologic agent has been pinpointed, a bovine origin coronavirus has been implicated in an emerging enteric disease of turkeys called poult enteritis mortality syndrome (pems) (barnes and guy, ) . the arthropod-bome viruses (arboviruses) present a unique challenge to evaluating ecological virus-host relationships. this grouping includes members from several virus families, six of which have members isolated from birds: togaviridae, flaviviridae, reoviridae, arenoviridae, bunyaviridae, and rhabdoviridae. of these, only the togaviridae and flaviviridae have strains that have caused documented disease in commercial poultry and game birds. although difficult to assess in feral populations, isolates from the remaining virus families as well as several isolates from the togaviridae and flaviviridae are not associated with any pathology in birds. of the togaviridae, the genus alphavirus contains the encephalitic strains causing eastern equine encephalitis (eee), western equine encephalitis (wee), and a closely related wild bird isolate --the highlands j virus (hjv). pheasants have been the primary targets of significant outbreaks of eee. signs of infection are primarily but not exclusively neural, and mortality rates have reached % in some natural outbreaks eleazer and hill, ) . economically significant outbreaks of disease due to eee virus in turkeys have occurred in wisconsin, with severity of the disease decreasing with increasing bird age. significant outbreaks have also been recorded in chukar partridges, ducks, and chickens. wee has been only rarely associated with disease in avian species, and the closely related hj virus appears to be the eastern united states equivalent to wee. hjv has caused severe neuropathogenic outbreaks in chukar partridges and has been associated with infections in turkey flocks resulting in acute reduction in egg production . of course, these encephalitic viruses also cause disease in humans and horses. another interesting alphavirus, ockelbo virus, related to sindbis virus, has been implicated in causing arthralgia and rash in humans following its isolation from mosquitos collected during the outbreak. these viruses are transmitted by mosquitos among bird populations, which may act as the vector for transmission between humans and avian species. ockelbo virus, therefore, is apparently maintained in an enzootic cycle involving birds and mosquitos with transmission to other hosts such as humans. antibodies to ockelbo virus, either experimentally or naturally infected, have been detected in passeriformes, galliformes, and anseriformes (lundstrom et al., ; lundstrom and niklasson, ) . viremia in the absence of disease resulting from infection with ockelbo has also been demonstrated in these bird groups. given the widespread occurrence of antibodies to these encephalitic alphaviruses, it seems logical to conclude that birds in many cases act as "natural" and reservoir hosts. the only flavivirus thus far associated with disease in birds is the israel turkey meningoencephalitis virus. infected birds exhibit neurological dysfunction and occasional significant mortality. this virus has also been identified in turkeys in south africa. double-stranded segmented rna viruses are unique and intriguing. the distinguished virologist dr. wolfgang k. joklik, when asked what in the world he thought they meant in the grand scheme of biology, replied (and i paraphrase), "they represent an evolutionary step forward in the establishment of the ideal genetic material." the reoviruses have fascinated virologists for some time. they have an unusual and complex replication strategy, are able to undergo reassortment of their genes, and are curious in that they retain their infecting subviral cores as their rna replication template. reoviruses are also quite stable outside the host, remaining viable for up to a year at room temperature (nibert et al., ) . the mammalian strains have a wide host range but are found without any associated clinical signs, the origin of the name reo (respiratory and enteric orphan) telling most of the story. in poultry, several antigenic subtypes have been identified, and the virus can be classified based on serotyping and virulence, but there is no unified typing scheme as yet (kawamura and tsubahara, ; robertson and wilcox, ) . the most severe and common pathology associated with reoviruses is arthritis-tenosynovitis, although associations with other clinical syndromes including respiratory and enteric disorders have been described (rosenberger and olson, ) . often, the clinical states are influenced by the presence of other pathogens. arthritis is a significant problem in birds but primarily only in young chicks and turkey poults. other reoviridae in birds include several members of the genera orbivirus (arboviruses; see below) and rotavirus, which have been associated with a runting-stunting syndrome in chickens. the birnaviridae are a relatively recently characterized family of viruses that contain two double-stranded segments of rna and have no mammalian members thus far (ictv, ) . they were difficult to classify for many years because of their cell-associated nature and slow replication in cell culture systems. currently, two serotypes ( and ) are accepted, with significant antigenic variation and numerous proposed subtypes within each serotype (mcferran et al., ; jackwood and saif, ) . chickens are the only animals known to develop disease and lesions when naturally infected by avibirnaviruses. both serotypes are distributed worldwide, but serotype strains are the only ones associated with pathogenicity and immunosuppression. the bursa is the primary target organ, and strains of differing virulence have been identified. the disease is most significantly manifested when young birds are infected and permanent immunosuppression results. this sets the stage for subsequent severe viral and bacterial infections later in life. interestingly, ibd is an example where maternal antibodies derived by either vaccinations or natural infections provide significant immune protection. highly virulent strains of these viruses exist in various countries worldwide, and there are indications that new virulent variants do arise in the face of immune pressure (chettle et al., b) . avian adenoviruses are double-stranded dna viruses containing - genes. these viruses can be loosely divided into three groups based on antigenic relationships of internal proteins (mcferran, ) , although the three groups have not been officially recognized by the ictv. the avian group i adenoviruses include at least serologically distinct types, all of which have been isolated from mildly or asymptomatic poultry. the type species is known as the celo (chick embryo lethal orphan), or phelps strain, or f (fowl) strain. there are also numerous isolates from other avian species plus electron microscopy and immunological evidence that the type i aviadenoviruses are widely distributed in birds. by themselves, these viruses present no particular clinical problems in commercial poultry, but they are thought to cause significant problems in mixed infections with immunosuppressive viruses such as ibdv and chick infectious anemia virus (ciav). a virus virtually indistinguishable from the celo strain known as quail bronchitis virus (qbv) can be devastating in commercial quail operations, causing as high as % mortality (dubose and grumbles, ; montreal, ) . virus isolations and significant antibody levels in wild quail suggest that the virus may present disease problems in nature (king et al., ) , although there are no documented cases or epizootics. the group ii aviadenoviruses include turkey hemorrhagic enteritis virus (hev), pheasant marble spleen disease virus (msdv), and the avian adenovirus splenomegaly (aas) of chickens. these three agents are serologically indistinguishable but induce differing clinical manifestations in the different species. infections caused by this virus group appear to target lymphoid tissue, often resulting in immunosuppression, and there are strain-specific differences in virulence among the various group ii isolates (pierson and domermuth, ) . hev reached epidemic proportions in the s and still causes significant problems in turkeyproducing states in the united states and elsewhere. msdv is a significant pathogen in confinement pheasant operations, causing significant economic losses. aas is virtually ubiquitous among chicken flocks in the united states, in which case mild respiratory signs and splenomegaly are occasionally associated. the syndrome is similar to the disease state observed in pheasants, but in general aas virus causes no significant problems among chickens. evidence indicates that these group ii avian adenoviruses are limited to the order galliformes, and that wild bird populations (even wild turkeys) are unaffected. the more interesting group iii adenoviruses first appeared in associated with a distinct clinical manifestation called egg drop syndrome (eds), in which egg production decreases and thin-shelled or shell-less eggs are produced. the disease has caused significant egg production losses mostly in the eurasian and australian-pacific poultry markets. a virus named eds , which has been found associated with this syndrome, may have been originally introduced via a contaminated vaccine, but it seems clear now that sporadic cases are initiated by introduction of the causative virus from domestic and wild waterfowl, mostly ducks and geese. when endemic in flocks, the virus is transmitted vertically and exhibits a latent phase, which is reactivated in laying hens, usually after egg production begins. the virus initially appears to replicate in lymphoid tissues but rapidly moves to the oviduct, where replication causes inflammation and production of aberrant eggs. unlike other avian adenoviruses, the group iii strains do not replicate in the intestinal mucosa. it is likely that eds virus or very similar strains are present ubiquitously in wild ducks and geese, but rarely appear to be associated with disease (mcferran, ) . the avipoxviruses are widely distributed throughout the class aves, having been isolated from some species representing avian taxonomic families. the avipoxviruses are responsible for economically important disease problems in commercial poultry and aviaries (tripathy, ) . they may cause a slowly developing cutaneous disease with low mortality or, conversely, significant mortality, and generalized infections when in the diphtheritic form on mucosal surfaces of the respiratory tract and associated areas. these large dna viruses replicate in the cytoplasm of the cell, where they form characteristic inclusion bodies within rapidly proliferating nodular lesions. the poxviruses may be transmitted by mechanical means such as introduction from poultry workers into abrasions in the skin of uninfected poultry. there is also real evidence for transmission of the disease by mosquitos and other vectors such as mites in close conditions, where the number of diseased birds is high. the avipoxviruses can also apparently establish a latent state and be naturally or chemically induced to reactivate (kirmse, b) . cutaneous lesions containing infectious virus persistent for more than a year have also been documented in wild birds (kirmse, a) . variant strains of avipoxviruses have since been isolated from previously vaccinated flocks that were experiencing significant mortality from the diphtheritic form of the disease (tripathy and reed, ) . this suggests that, similar to marek's disease, vaccination against poxviruses may ultimately lead to escape of more virulent forms of the viruses. the avipoxviruses are also considered one of the most promising dna virus vectors for delivery of recombinant poultry vaccines, and poxvirus-vectored vaccines against newcastle disease and avian influenza have been licensed. avian encephalomyelitis is primarily a disease of young chicks caused by a picornavirus. the disease was quite economically important prior to initiation of live-virus vaccination. the host range is very limited (order galliformes), and there is only one virus serotype. the natural isolate is enterotropic and is transmitted horizontally (orally) and vertically. there is a gradient of pathology dependent on the age of infection of young chicks. pre-immune chicks from non-immune parents will generally die if infected within to days of hatch. if infected between and days of age, they may live but exhibit significant nervous involvement (encephalomyelitis). if infected beyond that age, they may exhibit enteric pathology but not neural signs. at adulthood or full immunocompetence, they may be infected but refractory to any clinical signs. so the immune status of the host in this case is very important in affecting the course of the viral replication (calnek, ) . another avian picornavirus, distinct from aev, is the avian nephritis virus (shirai et al., ) . this virus is similar to aev in that it is primarily a problem in very young birds and has a very limited host range. as the name implies, a distinct clinical syndrome is associated with the agents, but it is has not yet been determined how extensive infections are in commercial poultry. finally, another picornavirus, duck hepatitis (dhv- ), has caused significant problems in com-mercial duck operations. there are still unknowns with regard to the overall importance of dhv- , and there are additional virus-induced types of hepatitis disease with at least two others often encountered. the most significant cost involved in raising birds for food sources is feeding them. consequently, diseases that affect the feed-protein conversion ratio will directly affect the cost of marketable birds. as such, enteric disease, even when nonlethal or mild in nature, can affect the performance of food-source birds. in recent years, investigators have identified rotaviruses in chickens, astroviruses in turkeys, and enterovirus-like particles in several avian species associated with enteric diseases. many of these have been associated with significant pathogenesis, particularly in mixed infections where immunosuppression occurs, with subsequent decrease in marketability due to weight loss (barnes, ) . the virus designated chicken infectious anemia virus (ciav) is a singlestranded circular dna-containing virus, tentatively classified with two similar agents in a new family: circoviridae. the agent has only recently been recognized, purified, and characterized, and its unequivocal role in economically important disease is not yet fully established. purified virus inoculated into young chicks results in severe anemia and immunosuppression, but inoculation of -to -week old birds results in little if any pathology (yuasa and imai, ) . the virus has been associated with adult anemic conditions, however, in conjunction with other viral and bacterial infections and may thus play a significant role. the agent is widespread in commercial poultry flocks worldwide, and infection with the virus has been statistically related to a decrease in overall growth and performance (chettle et al., a) . the extent to which two other tentative members of the family circoviridae --psittacine beak-and-feather disease virus (bfdv) and porcine circovirus-are related to ciav is under question, and these stated taxonomic relationships may differ in the future. bfdv has proven to be a significant pathogen in aviaries and commercial in psittacine birds to the extent that vaccines are now routinely administered. recent evidence also suggests that circovirus particles can be detected by em and isolated from several other wild and captive bird species. consequently, the circoviridae may emerge as a more significant disease-causing agent in the near future. the caliciviridae is a family of small single-strand positive-sense rna viruses with a polyadenylated genome. virus-like particles resembling caliciviruses have been isolated from a variety of wild-bird and captive-raised species. a chicken calicivirus has been replicated in cell culture and caused apparent gastrointestinal disease in specific pathogen-free day-old chicks (cubitt and barrett, ) . intestinal contents of goldfinches with hemorrhagic enteritis have been found to contain calicivirus-like particles and gastrointestinal disease associated with caliciviruses has been reported in guinea fowl and pheasants (gough et al., ) . caliciviruses were originally isolated from marine mammals and have caused disease in both domestic and feral swine due to consumption of uncooked garbage containing seafood. interestingly, caliciviruses have been detected in pelagic birds, such as the white tern (poet et al., ) . calicivirus isolations such as these lead to the speculation that wild sea birds may be important in the transmission of these agents across large areas or to other animals. parvoviruses are the smallest of the dna-containing viruses, carrying a singlestranded genome of about nucleotides. they cause or are associated with three infections in birds (kisary, ) . in wild and domestic geese and muscovy ducks, derzy's disease refers to a syndrome that had been variously called goose influenza, goose hepatitis, gosling enteritis, and infectious myocarditis in different countries. this collection of names gives some indication of the variety of signs associated with derzy's disease. the virus, which can be transmitted horizontally and vertically, induces differing pathologies depending on the age of the bird and, in the case of hatchlings, the level of maternal antibodies. an interesting pathology often associated with infection of older birds is the virtually complete loss of feathers. the extreme stability of the parvoviruses makes control of this disease difficult in commercial operations worldwide, and the disease can result in % mortality in young hatchlings. in chickens and young turkeys, parvoviruses have been associated with runting stunting syndrome (rss), as have other agents. experimental inoculations have indicated that the associated viruses, which are distinct from the goose viruses, can cause significant pathology, but the extent to which these viruses participate in the rss is not yet established (trampel et al., ; decaesstecker et al., ) . another parvovirus, the avian adeno-associated virus is almost always found associated with adenovirus infections in poultry. the avian adeno-associated viruses are grouped with their mammalian counterparts in the dependovirus genus, although they are serologically unrelated. the avian dependovirus appears to contribute to disease only in the sense that it can affect multiplication of the associated adenoviruses (yates and piela, ) . newcastle disease virus (ndv), classified as avian paramyxovirus- , contains a single-strand negative-sense rna genome of kb containing coding sequences for six genes. the virus infects all bird species tested to date, including flee-living and domestic species. one panzootic outbreak of the disease is thought to have originated in asia, with subsequent spread to europe, with outbreaks of disease first reported in poultry during the s in java, indonesia, and newcastle-upon-tyne, england. worldwide dissemination of the disease, particularly during the s and s, has been attributed to increased international trade of both commercial poultry and pet birds. this led to development of inactivated and live-virus vaccines for commercial poultry. the transmission of infectious ndv may occur by either ingestion or inhalation, and this knowledge is the basis for mass-application vaccination procedures during poultry production. isolates of ndv are grouped into three main pathotypes depending on the severity of disease that they cause (alexander and parsons, ) . mildly virulent "lentogenic" viruses may cause unnoticeable infections in adult chickens or a mild respiratory distress and are used extensively as live-virus vaccines. "mesogenic" ndvs are of intermediate virulence and cause respiratory distress, with mild infections of various organs detectable only by histopathology. highly virulent viruses that cause severe morbidity and mortality are termed "velogenic." velogenic viruses can manifest themselves as neurotropic or viscerotropic forms of newcastle disease with extensive systemic replication throughout a bird. virulent forms of ndv can replicate within cultures of most avian and mammalian cell types without the addition of trypsin, while lentogens require added proteases for replication in cell culture. the presence of dibasic amino acids at the proteolytic cleavage site (pcs) of the viral fusion protein and the ability of cellular proteases to cleave the fusion protein of various pathotypes specify the molecular basis for ndv virulence. fewer basic amino acids are present in the fusion protein cleavage site of lentogenic ndv than is the case for mesogenic or velogenic isolates. the presence of the increased number of dibasic amino acids in the fusion protein pcs sequence of ndv allows for systemic replication of these more pathogenic viruses in the host (nagai et al., ) . although the principal concern with ndv is its effect on poultry production, it may have severe consequences in other flee-living avian species. major outbreaks of newcastle disease have occurred in north american cormorants during the summers of and , and again in . outbreaks during and occurred in the north central united states and south central canada, while in newcastle disease occurred among cormorants of the western united states and canada. mortality in young nestlings in some areas was as high as to %, and the affected birds had characteristic neurotropic lesions (heckert et al., ) . during the outbreak in cormorants, an unvaccinated north dakota turkey flock became infected with ndv, resulting in high mortality. using nucleotide sequence analysis, the virus causing disease in turkeys was proven to be the same virus isolated from afflicted cormorants in the central united states and canada. the cormorant virus was also phylogenetically related to other known ndv isolates of psittacine origin (seal et al., ) that had caused a major outbreak in southern california poultry during the early s that resulted in a depopulation of two million chickens (utterback and schwartz, ) . illegal importation of pet bird species into the united states continues to be a source of highly virulent ndv and certainly may play a role in spread of viruses that threaten commercial poultry worldwide (panigrahy et al., ) . what role free-living birds play in spread of ndv is unclear. although persistent infections of chickens by ndv do not appear to occur, the virus' persistence in poultry flocks may result from virus reintroduced from wild populations. also, different bird species vary in their level of susceptibility to ndv. ducks, geese, and certain psittacine birds do not exhibit signs of disease when infected with highly virulent ndv, while other psittacine species may have high mortality. persistent infections have been demonstrated in various psittacine birds, with virus isolations noted up to a year following experimental infection of parrots with velogenic ndv chicken isolates (erickson et al., ) . although psittacine birds have been directly linked as a source of ndv highly virulent for gallinaceous birds, such as chickens and pheasants, no studies have shown direct isolation of ndv from feral psittacines. newcastle disease occurred in racing and show pigeons during the s in both western europe and north america. the outbreaks in western europe were linked to contaminated feed, and disease was controlled in both areas via vaccination with mildly virulent ndv commonly used for poultry. the virus isolated from these birds varied somewhat from traditionally virulent ndv in having only one set of dibasic amino acids in the fusion protein cleavage site. increased virulence for poultry subsequently occurred only after passage in chickens (collins et al.. ) . these examples demonstrate the fact that variant forms of ndv may arise in different bird species and that free-living birds may be a constant source ot viruses that affect both domestic and wild birds. vaccination programs against ndv for the most part have been effective al controlling the virus in commercial poultry. however, all of the vaccine strains available today, while effective against lethal disease, share less sequence identit with currently circulating disease strains. this is effectively illustrated in figure , which may also be considered a useful example for other rna-containin~ viruses. the phylogenetic tree demonstrates the relationship between currenl . phylogenetic relationships among newcastle disease virus isolates. nucleotide sequences from the fusion protein gene coding for the cleavage site from several newcastle disease virus isolates were aligned and analyzed by parsimony analysis. all the viruses listed are intermediately virulent mesogens or highly virulent velogenic isolates when inoculated into chickens, with the exception of the two vaccine viruses and the chicken/ field isolate. the data demonstrate that field isolates from chickens related to vaccine virus are isolated from poultry. the chicken/ virus caused the last major outbreak of newcastle disease in u.s. poultry and was epidemiologically linked to a pet bird. it is genetically related to a parrot isolate and viruses that caused disease in pigeons during the s. viruses isolated from cormorants in infected an unvaccinated turkey flock. these viruses appear to be circulating among cormorants, since closely related viruses were isolated again in . no viruses related to the virulent chicken viruses from have been isolated since that time, indicating these virus types may no longer be circulating among birds. all the recent virulent newcastle disease viruses were isolated from pet or exotic birds and chickens infected by free-living birds. these viruses are related to virulent viruses originally isolated during the s. avian influenza (ai) presents one of the most interesting ecological relationships between birds and their viruses. its ecology has been reviewed in depth hinshaw et al., ) , but ai presents such a complete picture of a virus with multiple impacts on several species that it must be included in this chapter. the type a orthomyxoviruses are essentially bird viruses. the infections in birds can range from clinically inapparent with minimal serologic response, to a devastating systemic disease that can result in % mortality within a matter of days. there are identified subtypes of ai viruses based on the hemagglutinin (ha) protein antigenic structure and nine subtypes based on the neuraminidase (na) structure. these two genes code for the predominant surface glycoproteins, which are embedded in the lipid bilayer of the viral envelope. these are only of genes coded for by the virus. the virus' genetic material is contained on eight separate negative-sensed single-stranded rna segments ranging from to nucleotides in length. all of the ha and na viral subtypes have been identified in feral waterbirds, and it has been proposed that these birds act as a "natural" reservoir for the virus slemons and easterday, ) . determination of the phylogenetic relationships of several genes from several subtypes of aiv collected from wild birds indicated that the general rate of evolution in the avian reservoir is low compared to the rate of evolution observed in human and other mammalian strains of type a orthomyxoviruses . other recent studies, however, have shown that the mutation rate of the ha gene and the ns gene even in wild birds approaches that seen in human strains . measurement of clinical signs among infected migrating waterbirds is, of course, a difficult task. thus far, only a single instance of severe clinical signs has been associated with free-living birds --a lethal outbreak in terns in south africa in caused by an h n strain (becker, ) . given the recently measured mutation rates, and the appearance of this severe lethality in a wild bird population, it must be considered that aivs do continue to evolve in feral birds (indeed subtypes have arisen already!). there is no reason to believe that the present count of subtypes will be the final tally. while some have proposed that the aiv strain in wild birds causes no disease problems as a result of "evolutionary stasis," without question the virus has dramatic effects when it leaves this proposed reservoir. in addition to the fixed influenza populations in pigs and horses, avian strains can directly infect mammals and have been identified in whales, seals, and mink (lang et al., ; berg et al., ) . the dramatic association with seals in and mentioned earlier clearly points out the potential impact of aivs. this avian reservoir then serves as a sort of base for the viral "biological invasions" that are a part of interspecies transmission. ecologically and economically, the transmission of these aiv subtypes to commercial poultry has perhaps yielded the most important impact. the situation is most clearly played out in minnesota each year. due to the large number of lakes in the state, migratory waterfowl, primarily anseriformes (ducks, geese), frequent the area in large numbers seasonably. nearby commercial turkey operations suffer infections nearly every year, particularly in the cooler fall months, caused by different influenza subtypes of different virulence (halvorson et al., ) . in severe years, the associated costs due to turkey mortality, as well as performance and egg production losses, can reach several millions of dollars (poss and halvorson, ) . in chickens, introduction of influenza viruses from waterfowl has been more devastating. in the northeastern united states, the h subtype caused the first large-scale outbreak of highly pathogenic ai in numerous flocks in . twentythree million birds were destroyed at a cost of more than $ , , in order to contain the infection. clinically, the disease was indistinguishable from the disease originally described as "fowl plague" in in europe (eckroade and silverman, ) . today we know that all of the classical "fowl plague"-type outbreaks have been associated exclusively with only two subtypes: h and h . evolution to virulence in these subtypes has been closely associated with accumulation of basic amino acids at the proteolytic cleavage site of the hemagglutinin protein (bosch et al., ; webster and rott, ) . in order for aiv strains to be infectious, the ha protein must be cleaved into ha and ha subunits, which subsequently allows structural rearrangement and exposure of a protein sequence needed to fuse the viral envelope with the plasma membrane of the target cell (klenk et al., ; skehel et al., ) . isolates of low pathogenicity lack multiple basic amino acids and are only cleaved by trypsin-like extracellular proteases. these proteases are abundant on the mucosal surfaces of the respiratory tract and gut, so the viruses replicate unrestricted at these sites. the highly pathogenic (hp) forms, however, have additional basic amino acids, which are recognized by the intracellular furin-like proteases ubiquitous in bird tissues (rott et al., ) . thus, in birds, these viruses are able to escape the respiratory tract and infect a wider variety of internal tissues and organs. this is one of the most important of the virulence factors (webster and rott, ) . only two subtypes have been shown to accumulate these basic amino acids: h and h . these subtypes appear to do so by either base substitution or by insertion events (wood et al., ; perdue et al., ) . nucleotide insertion events appear to be the most common mechanism, and table ii and figure illustrate what is presently known about occurrence of these hp virus isolates in commercial poultry. the hp isolates, with only two exceptions, have additional inserted basic amino acids within the well-conserved region surrounding the cleavage site. from the data in table ii and figure , it may be easily surmised that both the number of hp isolates and the number of basic amino acids at the cleavage site are increasing. athe amino acids surrounding the hemagglutinin protein cleavage sites of a representative member of each of the subtypes of avian influenza viruses are shown above. subtypes , , and , which cluster together in a phylogenetic tree, all have five amino acids between the conserved proline and arginine; the remaining subtypes have four. highly pathogenic isolates (subtypes h and h ) more often than not, have insertions of arginines and lysines, increasing the length of the cleavage site and making the hemagglutinin accessible to ubiquitous proteases. this has the effect of greatly increasing the tissue distribution and virulence of the virus. table ii for further explanation. the avian influenza viruses are distributed worldwide. in addition to the united states, outbreaks of highly pathogenic ai have occurred in mexico, canada, europe, asia, and australia. in most cases, there is some sort of connection between the affected flocks and nearby waterfowl. the extent to which the reservoir of viruses exists in waterfowl is not really known. surveys have shown that, in addition to the viruses being widespread, the various subtypes increase and decrease in prevalence over successive years. the order charadriiformes (shorebirds and allies), in addition to anseriiformes, has been shown to carry aiv strains (kawaoka et al., ) , and the extent to which aivs are carried in other orders has really only been superficially explored. molecular and phylogenetic evidence have clearly shown that these segmented viruses share various genes back and forth within the populations of circulating virus strains . they are also capable of donating genes through genetic reassortment to viruses in swine and those genes ultimately end up in humans (webster et al., ) . add this capacity to the known (albeit rare) transmission of purely avian influenza strains directly into mammals and one creates a potential ecological bonanza for the virus, and potential nightmares for new hosts. as advanced as our study of viruses has become, there are still a variety of unknowns with regard to the extent to which avian viruses infect other vertebrates. it would not be unreasonable to suggest that, because of their mobility and longevity of existence, that the class aves may have played a critical role in distribution of viruses to other classes. in the case of the avian orthomyxoviruses, phylogenetic evidence points to recent introduction of these viruses from birds to mammals. following introduction, the natural mutation and evolution of the virus produced strains that now appear to be adapted to their new hosts. there are swine, equine, and human strains of type a influenza viruses that do not replicate well outside their respective host species. if birds are indeed responsible for us coming down with the flu today, we know of no reason why they might not have participated over the eons in the establishment of other purely mammalian diseases. of course, one could easily argue the opposite relationship, but, intuitively, birds do present an ideal mobile vector, particularly for the enterically transmitted viruses. in addition to the diseases discussed here, there is a growing group of emerging diseases or diseases of as yet unknown etiology in commercial poultry (saif, ) . many of them have virus particles clearly associated with the syndrome, but only limited research and characterization has been carried out thus far. it is probably safe to assume that the future will continue to bring new avian diseases and bring to light new or undiscovered avian viruses. the relationships existing among birds, insects, and viruses is one area that should be explored in more detail by increased research efforts. in free-living birds, the large number of apparently innocuous infections with bunyaviridae, togaviridae, and flaviviridae suggest that birds may act as an ecological reservoir for maintenance of these virus populations. from a virocentric point of view (if there is such a thing), having a potentially highly mobile population of susceptible hosts on which transmission vectors may feed would be advantageous. it would provide a mechanism by which to transfer viruses over large geographical distances where they could then be transmitted by new related or unrelated vectors, thus increasing host range. of course, one runs the risk here of assuming that the virus would be ecologically improved by extending its replicative capacity in new hosts. this may not be a valid assumption. for example, if variola virus or human poliovirus type had been exploiting this strategy, they made a bad choice in extending their host range into humans! from one human vantage point, birds provide a useful monitor for detection of arboviral diseases in a given area. sentinel chickens are actually in use today along the east coast of the united states to gauge the extent to which some mosquito populations are carrying eastern equine encephalitis and st. louis encephalitis viruses. the future is perhaps the most important aspect to consider. what viruses other than the orthomyxoviruses may someday establish a similar relationship where transmission to a mammalian intermediate or even direct transmission may allow introduction into the human population? one fundamental unanswered question is whether birds, acting as reservoirs for arboviruses, should be considered an important ecological niche for those viruses. there is no evidence of which we are aware to indicate either yes or no. there have been no documented transmissions of arboviruses from birds to mammals without the insect vector in nature, however, so at this point wild birds could be considered dead-end hosts for the arboviruses. in commercial poultry during outbreaks of the alphavirus eeev, there is no evidence of infection of workers in close contact with infected birds. if, however, a bird provides the necessary reservoir of eeev that infected the human, then this bird becomes very important. but there are no data that as yet identify the bird as the required reservoir for disease transmission to human. newcastle disease virus provides an interesting new disease problem in humans that has only arisen as a result of increased vaccination of birds. a live-virus vaccine is available for control of ndv that is administered by aerosol spray. in a small number of cases where precautions have not been taken, vaccinators can contract a conjunctivitis caused by the vaccine. this outcome has been seen in the heavy poultry-producing areas of northern georgia, on a common enough basis now, to be easily recognized by ophthalmologists. there is usually no seroconversion to the virus in these cases, and they are not particularly serious infections. there is also suggestive evidence in the form of measured seroconversion that poultry workers occasionally may be subclinically infected with avian coronaviruses and avian retroviruses. the evidence for direct infection of humans with avian influenza viruses is growing. human strains have classically included only three subtypes (h - ). until recently, the only other strains shown to cause disease in humans were ot the h subtype. conjunctivitis caused by purely avian h subtype viruses has been reported on two occasions (kurtz et al., ; webster et al., a) . the most disturbing set of events, however, was the more recent highly publicized influenza outbreak in hong kong. a purely avian h subtype virus was isolated from some patients, of whom died, from may through december of . the index case, a -year-old child, in may followed a highly pathogenic outbreak of avian influenza that had just previously occurred in chickens in hong kong and its environs. the child had been exposed to ill birds at a day care center, and it was clear that the recovered virus was essentially the same as that recovered during the chicken outbreak. considerable efforts were made to determine whether the virus was a contaminant and whether it was replicating in the child. the evidence strongly suggested that it was indeed replicating (subbarao et al., ) . subsequent to this event, in november-december the remaining confirmed cases were reported and an intense effort was mounted to determine exactly how this virus was transmitted. it is clear now that the h n viruses infecting humans in hong kong were all of avian origin . there is limited serologic suggestion of human-to-human transmission but no genetic evidence of adaptation to humans. this case was touted as a premier test run for the next predicted influenza pandemic. whether this outbreak represents a simple dead-end zoonotic transmission or whether these purely avian influenza viruses can become fixed in the human population is a matter for conjecture. the avian h and h influenza strains have been the only natural influenza strains thus far to unequivocally cause systemic lethal infection in any species owing to the unique structure of their ha protein (see fig. ). a major fear is that by adding such ha subtypes to a population of mammalian viruses a whole new class of virulent strains might be created. our experience in working with numerous highly pathogenic and nonpathogenic strains has been one of very little or no evidence for human infection in the years that scientists and technicians have spent working with hundreds of different stains. but our experience has also been that, when one attempts to confine influenza viruses by setting biological rules for them, they usually find a way to break them. if ecology is the study of the relationship of organisms to their environment, for bird viruses, the active, pertinent, environment is always within the bird and its tissues. while one cannot discount the effects of the environment outside the host, there are no positive effects on an avian virus life cycle outside the host of which we are aware. that is, there are no activation events outside the avian host of which we are aware; only events of inactivation. certainly, avian viruses, as all viruses, have evolved survival strategies to allow passage from host to host, and some are much more refractory to environmental inactivation than others. but without really knowing which, if any, "evolutionary direction" viruses are taking, it is impossible to determine whether a large herpesvirus of genes is ecologically more fit than the enterovirus with five genes! the relationship of birds and insects in the transmission and life cycle of the arboviruses is poorly understood. until this recent transmission of h n influenza strains to humans, this relationship appeared to be the only significant one in which there is interaction among a genetically unaltered virus, an avian host, and a non-avian host. as mentioned earlier, other documented cases of avian-to-mammalian viral transmission are newcastle disease infections of humans and influenza a infections of mammals and humans. there is also serological evidence for avian retrovirus and avian coronavirus infections. in the case of newcastle disease, the documented infections are mostly minor conjunctivitis. in the case of influenza, discussed earlier, the avian viruses most often acquire rna segments from other sources before they are established in a new host. thus, in general, the impact of avian viruses on public health might currently be considered small, but clearly the potential exists for significant future impact. a major interesting question arises when evaluating virulence of avian-origin viruses. conventional wisdom seems to be suggesting that virus entry into naive host populations is more likely to result in a general decrease in virulence as the virus adapts to the new host (morse, ) . this may be true under truly "natural" conditions; but what happens under human-dictated conditions such as vaccination and by defining the host population through breeding and housing? in the case of marek's disease, we see an example of virulence increasing in the face of continued vaccination against the virus (fig. ) . whether this is occurring as a result of the vaccination or a combination of factors, the fact is that the population of marek's disease herpesviruses in nature is becoming more virulent. the vaccination programs for newcastle disease utilize vaccines whose genetic compositions are becoming farther and farther removed from the virulent strains circulating in wild fowl (fig. ) . this may not bode well for future control of virulent strains in commercial poultry. in the case of avian influenza viruses, we have seen the number of highly virulent outbreaks increase in recent years, often following prior circulation of a nonpathogenic precursor, and we have seen concomitant changes in genetic structure related to virulence (fig. ) . again there are unknowns in this system, but the fact remains that we are seeing more virulence as the nonpathogenic aiv subtypes replicate in commercial poultry settings. thus, this naive commercial population, unlike the waterfowl hosts to which the virus has adapted, appears to allow generation of heretofore unencountered virulence phenotypes. similar scenarios appear to be playing out as more virulent infectious bronchitis strains and infectious bursal disease strains appear worldwide. one must be cautious, then, in working strictly under the previously mentioned virulence-decrease paradigms for mammalian viruses. these have been suggested mostly by the experiences with artificial introduction of rabbit papilloma viruses in australia or where new introductions occur as human encroachment ensues (such as in the recent cases of ebola virus or monkey poxvirus infections). in these cases, it was proposed that virus transmission results first in high virulence for the host followed by subsequent adaptation and attenuation. finally, it may be dangerous to attempt to confine avian viruses to some of our more "logical" inferences based on darwinian evolution. one paraphrased definition of evolution is "moving from a prior more primitive (or less fit) state to a current more advanced (or more fit) state." it is difficult, for us at least, to say that any avian viruses are following such a progression. additionally, other than use as molecular biological vectors, viruses in general have not been shown to provide anything positive to the ecology of any other organisms. the future may yet reveal such relationships, but, for now, ridding ourselves of these fascinating parasites by continuing such policies as those resulting in the eradication of smallpox and polio still appears to be our most prudent course. newcastle disease virus pathotypes tissue tropism of the hprs- strain of j subgroup avian leukosis virus and of a derivative acutely transforming virus diseases of poultry avian infectious laryngotracheitis: virus-host interactions in relation to prospects for eradication viral enteric infections poult enteritis syndrome of turkeys --mortality the isolation and classification of tern virus influenza virus a/tern/south africa/ close relationship between mink influenza (h n ) and concomitantly circulating avian influenza viruses spread of marek's disease proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between ha and ha determines proteolytic cleavability and pathogenicity of avian influenza viruses duck plague avian encephalomyelitis diseases of poultry diseases of poultry infectious bronchitis virus: evidence for recombination within the massachusetts serotype revision of the taxonomy of the coronavirus, torovirus and arterivirus genera an outbreak of disease due to chicken anemia agent in broiler 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infections of birds diseases of poultry epizootology of velogenic viscerotropic newcastle disease in southern california molecular pathogenesis of marek's disease --recent developments sequence analysis of the turkey enteric coronavirus nucleocapsid and membrane protein genes: a close genomic relationship with bovine coronaviruses miscellaneous herpesvirus infections egg production drop in turkeys associated with alphaviruses: eastern equine encephalitis virus and highlands j virus evidence of natural recombination within the s gene of infectious bronchitis virus influenza virus a pathogenicity: the pivotal role of hemagglutinin conjunctivitis in human beings caused by influenza a virus of seals b). characterization of an influenza a virus from seals evolution and ecology of influenza a viruses influenza: a model of an emerging virus disease increased virulence of marek's disease virus field isolates epidemiology of reticuloendotheliosis virus in broiler breeder flocks deduced amino acid sequences at the haemagglutinin cleavage site of avian influenza a viruses of h and h subtypes avian adenovirus-associated virus pathogenicity and antigenicity of eleven isolates of chicken anemia agent (caa) key: cord- - lta tbp authors: overstreet, robin m.; lotz, jeffrey m. title: host–symbiont relationships: understanding the change from guest to pest date: - - journal: the rasputin effect: when commensals and symbionts become parasitic doi: . / - - - - _ sha: doc_id: cord_uid: lta tbp the several meanings for the term “symbiosis” create confusion, which can be avoided when the author provides details of the interrelationships between the symbiotic organism and the “host” so that a reader can clearly understand what definition is implied in each case. for example, we, as opposed to many other mentioned readers, consider a symbiont as an organism living in an association with another regardless of whether it causes a pathologic response or not, but from our title, the reader may incorrectly infer that we consider a parasite to be different from a symbiont. a symbiont is an organism that uses another organism as a habitat. this chapter discusses the primary associations and associated conflicts involving the terminology. it also provides both differentiation between and conflicting views regarding the interpretation of the terms “infect” and “infest,” “infection” and “disease,” and other terms. many seemingly harmless symbionts of a wide array of taxonomic groups are triggered to become pathogenic or virulent, and we provide several examples of the provoking (stimulating) triggers, with the understanding that in most cases, the conditions for the triggered activities are much more complex and complicated than presented. examples of triggers follow: environmental ones like temperature, toxic chemicals (dose), chemotherapeutics, dietary changes, and geographic habits; internal ones like host site, host resistance or susceptibility, and host modifications; and combinations of these and other conditions. we provide examples involving multiple triggers for organisms associated with termites, for an endemic virus being affected by multiple factors and having multiple effects on its commercial penaeid shrimp hosts, and for contrasting variables associated with two exotic viruses in wild and cultured commercial penaeid shrimps with an emphasis on hypothesizing how the pathogenicity developed in these two viruses. the chapter ends by trying to answer the question of why would a symbiont become pathogenic in some hosts and not in others from an evolutionary perspective. it uses two hypotheses to explain the increased virulence. when reading an article on symbiosis, most readers assume they know the definition of all the associated words in the document. this is not the case; even the term "symbiosis" is defined differently by different authors in different fields, by those in different geographic areas, and by those taught by different mentors. the term "symbiosis" was originally used by the german de bary ( ) to mean "living together." his meaning referred to all situations where either similar or unlike organisms or species live together in an intimate association. this is typically thought to include "commensalism," "mutualism," and "parasitism." we prefer to include all such associations under the general term "symbiosis" but realize that our title does not comply, and many readers prefer to use a more specific term for symbiosis and other associations. in the preface of his book ( ) , the eminent parasitologist william trager quoted "the conflict in nature between dissimilar kinds of organisms has been popularly expressed in phrases like 'struggle for existence' and 'survival of the fittest.' yet few people realize that mutual cooperation between different kinds of organisms-symbiosis-is just as important, and that the 'fittest' may be the one that most helps another to survive." for purposes of this series, we restrict "symbiont" to organisms that use other organisms as habitat. therefore, symbiont is a division of nature similar to "terrestrial" or "marine." the symbiont is characterized by where it lives rather than the quality of the relationship. for our purposes, we divide up the world into two kinds of organisms, free living and not free living. this accords us the advantage of comparing the quality of the relationship between free-living organisms and their habitat with that between host and symbiont. the symbiont is dependent upon the host as any inhabitant is on its habitat. we consider symbionts to include bacteriophages (viruses that infect bacteria), bacteria, viruses, "protozoans," and metazoans and symbiosis to include commensalism, mutualism, parasitism, and other relationships. moreover, all symbionts, even metazoans, observed best with a microscope will be considered microbes. more important, we will examine in this series a shift in the interaction among a host and some stage in the life cycle of the symbiont, whether it be free living, facultative, or obligate. we will emphasize that some symbiotic relationships can change from harmless to something that becomes far less favorable for the host. symbiosis this term as defined above is usually thought of as an association (mutualism, commensalism, or parasitism) between organisms of different species involving a unilateral or bilateral exchange of material or energy. the symbiont, or symbiote, is any member of a pair of organisms involved in this symbiotic relationship, with the larger member usually designated as the host. barrows ( ) provided a classification of symbiosis separating nonsocial symbiosis from social symbiosis, and others classify various terms differently. most of these terms and classifications will not be treated in this chapter. commensalism a symbiotic relationship in which one of two partner species benefits and the other shows no apparent beneficial or harmful effect. mutualism a symbiotic relationship in which two or more partners gain reciprocal benefits, usually mutual ones. parasitism a symbiotic relationship in which a symbiont lives all or part of its life in or on a living host, usually benefiting while harming the host in some way and usually having a higher reproductive potential than the host. noble et al. ( ) define it as an association between two different species of organisms in which the dependence of the parasite on its host is metabolic and involves mutual exchange of substances; this dependence is the result of a loss of genetic information by the parasite. there are several atypical kinds of parasites. an accidental one infects an unusual or unnatural host; a commensal one derives its substances from the food of its host; and an erratic, or aberrant, one infects an unusual site. a facultative parasite is usually parasitic but is capable of an independent existence, while an obligate one cannot lead an independent, nonparasitic existence. the late gerald schmidt's definitions are listed by roberts and janovy ( ) . he considered a parasite the "raison d'être" for parasitologists, a parasitologist as a quaint person who seeks truth in strange places and one who sits on one stool while staring at another, and parasitology as a study of the most common mode of life on earth. many parasites have complicated life cycles with multiple hosts and with a variety of stages, not all of which harm or depend on the host. one can glean that students find it difficult to define a parasite. this series will treat a variety of facultative and other relationships. predatory association a relationship in which a predator obtains its living by preying on other animals, usually consuming all or part of it, usually killing it. this relationship can be symbiotic when a predator feeds on one or a few species or when it is relatively small and not harming its host. for example, a trematode (all trematodes are considered parasites) may live in the lumen of a host without causing any harm; that trematode is occasionally called a micropredator. other terms that will be causing confusion in this series are infection and infestation. american parasitologists usually define an internal association as an infection, whether harm is caused or not or the organism is small or large. if the association is external, it is referred to as an infestation, and the symbiont is called an ectosymbiont or an ectoparasite. for an internal association to be called an infection, microbiologists usually restrict the agents to viruses, bacteria, and fungi. consequently, some authors restrict an infestation to a metazoan parasite (e.g., maggenti et al. ) . barrows ( ) provided three meanings for an infestation: ( ) a parasite's colonization, utilization, or both of the host; ( ) a host being colonized, utilized, or both by parasites; and ( ) and environment being colonized, utilized, or both by pests. other authors consider an infestation to refer to a population rather than to individuals. still, others use the word "infestation" to suggest an action and the term "infection" to suggest a condition or a state. microorganisms such as oral bacteria that live naturally in the mouth or elsewhere in a body are not considered infections or infectious agents by many microbiologists, but the organisms are symbionts. another important term for this series is disease. in medical cases, this often refers to dose. a well-nourished person infected with the american hookworm (necator americanus) with a normal hemoglobin and five eggs per milligram of feces, perhaps resulting from up to adult worms, has an "infection" but without a corresponding disease caused by the loss of blood or other detrimental signs. an egg count of per mg in a healthy person or in one anemic with iron or protein deficiency usually indicates the threshold of disease. massive infections with disease can produce as many as eggs per mg of feces (beaver et al. ) . lightner and redman ( ) discussed this term when dealing with shrimp infections. they said "in veterinary and human pathology, the terms 'disease' and 'syndrome' have a range of definitions. dorland's medical dictionary ( ) defines disease as 'a definite morbid process, often with a characteristic train of symptoms' and syndrome as 'a combination of symptoms [signs] resulting from a single cause or so commonly occurring together as to constitute a distinct clinical entity.' a simpler definition of disease is 'any alteration from the normal state of health.' the latter definition permits the inclusion of alterations in health that may result in subtle conditions in which poor health or reduced resistance to stress are the only signs of disease, as well as those disease syndromes on the other extreme, that may be accompanied by catastrophic losses." an important, prudent course of action for an author is to carefully define a term or description that is being treated in any manuscript. a reader, on the other hand, may have serious problems understanding what is being said without that definition. terms being used in symbiosis are especially difficult because research conducted to define an entire relationship seldom exists. several glossaries are careful not to include definitions of either infections or infestations, and others are careful not to define a parasite! many terms used for defining ecology of parasites such as "incidence," "prevalence," "intensity," "mean intensity," "component population," and many others are used differently by different people, and we strongly recommend the article by bush et al. ( ) as a standard or at least a source of discussion. usually, a combination of factors triggers a harmful condition, but only one or two of these factors are known in any detail for most cases. furthermore, most of the examples below treat only one or two of these triggering factors. differentiating the triggering factors into those from the external environment and those originating from the host is, for the most part, impossible to characterize because the two are typically entwined. moreover, genetic triggers that involve expression such as upregulation, downregulation, and gene knockdown may each necessitate a variety of triggers for expression. triggers causing a harmless symbiont to transform into a harmful one can also involve the initiation, interruption, or inhibition of biochemical pathways, complicated actions that sometimes incorporate a cascade of reactions. this chapter will not treat the details of mechanisms but rather present examples of what appear like results of cause-and-effect. as indicated above, the headings indicate only some of the triggers in each case, with an emphasis on the primary one. temperature often regulates disease in marine animals to benefit stocks of all parties. for example, the english sole, parophrys vetulus, arrives into its estuarine nursery area in the northern oregon coast from january to april, grows dramatically in the upper estuary during the summer, and then migrates offshore in the autumn, never to return inshore. when inshore, the sole gets infected by the microsporidian glugea stephani that develops in the intestine at temperatures ! c. this agent appears to cause mortalities from september through november, but this period is when most of the fish routinely migrate offshore where the temperature remains less than c, a temperature that was shown experimentally to inhibit the microsporidian agent's development, which ultimately results in uninfective spores within a few months. consequently, the parasite seldom harms the host stock. however, approximately % of the sole stock remained in the upper estuary where an estimated % of those fish had a rapidly developing infection and most probably died and decomposed or were eaten, dispersing the spores. those fish plus the overwintering infected fish and few reservoir starry flounder juveniles (platichthys stellatus) (a reservoir host is a primary host that harbors the pathogen but typically exhibits no ill effect and serves as a source of infection) provided infective spores for next year-class of sole (olson ; overstreet ) . temperature also has a great bearing on many other pathogenic triggers. for example, the coccidian calyptospora funduli commonly infects the liver, pancreas, and occasionally other tissues in the gulf killifish, fundulus grandis, in the coastal gulf of mexico. the fish becomes infected by feeding on either the infected intermediate host palaemonetes pugio (the common daggerblade grass shrimp) or related species. infections are synchronous, meaning that all the developing stages are the same. early developing stages and mature oocyst stages are rarely seen in the same fish. in some cases, over % of the liver can be infected without causing any notable severe harm to the host. when heavily infected and lightly or noninfected killifish were maintained in outdoor raceways and freezing occurred, the heavily infected fish all selectively died. presumably, that was because the liver serves as a storage reservoir for necessary glycogen, vitamins, minerals, and other necessary nutritional resources. those resources found in - % of the liver were presumably not enough to satisfy the needs of the fish during stressful low-temperature conditions. a series of experimental infections conducted at about , , and c for periods between and days in length (solangi et al. ) showed that both low-temperature treatments inhibited all developmental stages. when the fish were returned from the low temperatures to c, development of all the stages resumed except in fish exposed to c for days. in those infections, many of the coccidian organisms were atrophied or disintegrated within their parasitophorous vacuoles, and necrosis also occurred in some of the pancreatic tissue. in any event, decreased parasitism during inhibition was not linked with a leucocytic inflammatory response. when fish remained at a constant temperature of about c, an inflammatory response to the organism commenced at day , intensified at day , and diminished by about day (hawkins et al. , solangi and . in other words, inflammation associated with infections in warm water begins as gamonts developed and ends after formation of the oocyst wall. consequently, infections that occur during cold winters can be either helpful or harmful to the host, depending on the stage of the parasite, the extent of low-temperature exposure, the age of the fish, and other variables. atypical temperatures, such as warm water associated with power plants, can cause infections of a specific parasite during periods when the hosts are more likely to be consumed by predators, more susceptible to disease, or more susceptible to interactions among parasites that can occur and result in unusual pathogenic conditions. as also suggested above, temperature can have an effect on development of a parasite and can occasionally result in a harmful effect. the ascaridoid nematode contracaecum multipapillatum in a coastal lagoon at celestún, state of yucatán, mexico, infects as definitive hosts the olivaceous cormorant and the great egret. the intermediate host for this avian nematode is the mayan cichlid (cichlasoma urophthalmus). the juvenile in the fish host for all members of the genus contracaecum is usually a third stage; however, in the warm area of mexico, many of the juveniles had developed into the fourth stage. when these fourthstage juveniles were fed to a kitten, they developed into adults in the intestine, often associated with hemorrhaging small ulcers (vidal-martínez et al. ); they did not develop or cause a pathogenic response in rats, ducks, or chickens. people commonly eat the cichlid in mexico and, consequently, are potential hosts. when deardorff and overstreet ( b) fed third-stage juveniles, the typical stage occurring in fish, to day-old chicks and ducklings and to mammals, they neither developed nor survived; however, when the third-stage juvenile was surgically inserted within tied off semipermeable dialysis tubing into the abdominal cavity of the animals, the worms did mature. the gnathostomatid nematode echinocephalus sinensis matures in the intestine of the eagle ray, which acquires the third-stage juveniles by eating the infected oyster crassostrea gigas. infections in this commercial oyster were most prevalent and had the highest intensity in hong kong between july and september. kittens, monkeys, and puppies were fed the infective stage from oysters during every month, but those worms collected only from the warm months of august to october infected the mammals (ko ) . the worms penetrated the wall of the stomach and intestine, migrated to and lodged in various tissues, and often killed the hosts. to test this temperature-triggering condition, ko ( ) obtained oysters during the infective season and acclimated them to , , about , , and c. worms from these different temperature groups were fed to kittens, and infections were most abundant in the c group and were less so in the c group; only one kitten became infected from the c group, and no kitten from the or c group became infected. since people eat raw oysters, the nematode constitutes a potential public health risk. under normal environmental conditions, parasites seldom harm their hosts. when the habitat is a series of aquaculture ponds, several conditions are modified. the pond design usually accommodates a heavy density of snail intermediate hosts near the shallow shore where fish fry occur, and the fish stock inhabiting the middle of the ponds creates an abundance of prey for fish-eating birds. for example, primarily in mississippi but also less so in adjacent states, the channel catfish, ictalurus punctatus, is reared, comprising a multimillion dollar industry. in the natural environment, catfishes, including their madtom host relatives, do not occur as juveniles in habitats where the host marsh ramshorn snail, planorbella trivolvis, and american white pelican, pelecanus erythrorhynchos, occur and defecate in any abundance. consequently, infections of the metacercaria (larval stage acquired from a cercaria shed by the ramshorn snail) of the pathogenic diplostomoid trematode bolbophorus damnificus are relatively rare in those natural waters. a total of five different diplostomoid trematode species infecting the catfish were able to kill it in aquaculture environments. two of these developed from cercariae shed from p. trivolvis that ultimately mature in the american white pelican; two were from worms shed from the ash gyro, gyraulus parvus, that mature in the doublecrested cormorant; and the fifth species is one that matures in seagulls. the most harmful species, b. damnificus, can be present in the catfish with infections of less than metacercariae without harming it. heavier infections seem to have an effect on the kidney of the fish aiding in their mortalities. in the shallow water where large numbers of infected snails occur, thousands of cercariae can penetrate the young catfish and kill it quickly, even before developing into metacercariae. this is in contrast to two other trematodes, bursacetabulus pelecanus and austrodiplostomum compactum, which both infected the nerve cord, brain, optic nerve, and eye and at least in experimental infections can occur as several hundred individuals without killing the host (overstreet and curran ) . the trigger for mortality regarding b. damnificus can be more complicated than pure numbers. in experimental infections with fingerling channel catfish exposed to a sublethal infection of trematode cercariae per fish, they died when additionally exposed to the bacterium edwardsiella ictaluri, the agent of enteric septicemia of catfish. controls given either the cercariae or nothing did not die. those given .  colonyforming units per ml of bacteria for min singularly without cercarial exposure started dying at day , and, by day , the percent cumulative mortality leveled at about compared with % for the group given both the cercariae and bacteria. then at day when the metacercariae fully developed, groups consisting of the remaining fish given only the trematode and of the negative controls were exposed to the bacteria. both groups starting dying at day , but, by day , there was no significant difference in the percent cumulative mortality of about % (labrie et al. ) . when contaminating forage fish that occur in the ponds in addition to the commercial catfish became exposed to the cercariae of b. damnificus, they did not become infected. on the other hand, there is an unnamed species of bolbophorus which also infects both the same snail and pelican, concurrently with b. damnificus. however, it does not use the catfish as the second intermediate host, but it uses mosquitofish and sunfish, which it can kill when in high numbers (overstreet et al. ) . also of note is the finding that channel catfish exposed to the e. ictaluri bacterium day prior to being exposed to the pathogenic ciliate ichthyophthirius multifiliis had % mortality compared with , , and % for those respectively given only the bacterium or the ciliate or given neither. the bacterium could be detected by pcr in the gills, brain, liver, and kidney of the fish whether infected with the ciliate or not, but, by day , the bacterium no longer persisted in the bacteria-only group except in the kidney (shoemaker et al. ) . a few free-living amebas are well known because they can infect humans and occasionally cause fatalities (see overstreet ) . best known is naegleria fowleri because it causes fatal "primary amebic meningoencephalitis (pam)," but it is restricted to freshwater, and victims are typically those who swim for extended periods underwater in open bodies with a silty-muddy substratum. the swimmers' nasal mucosa becomes weakened to the point that the ameba can penetrate the membrane and enter the central nervous system (cns) along the olfactory nerve. because that site contains little cellular inflammatory response but provides a good medium for amebic growth, the organism replicates rapidly, usually before the disease can be diagnosed and treated. marine free-living species of the genus acanthamoeba do not grow as rapidly and, depending on the species, invade different sites. they enter the skin, lower respiratory tract, or nasopharynx, and the vegetative trophozoite can reach the cns through the circulatory system. these acanthamebic infections may take weeks or months to cause death, and often infections occur in patients that are immunocompromised (unlike the otherwise healthy hosts that become ill due to pam) but are still difficult to diagnose. six different ameba species have also been associated with painful amebic keratitis, a difficult to treat corneal disease. even though recognized in , amebic keratitis was not common until when contact lenses became popular; these lenses typically are maintained overnight in a saline solution that can become a source of contamination. toxic waste products can trigger a reduction in host resistance, resulting in susceptibility to rapidly reproducing agents that would normally be held in check or not able to produce infections. the myxosporidian henneguya gambusi infects the western mosquitofish, gambusia affinis, and is rare, not previously considered pathogenic, and not known from any other fish (parker et al. ) . we have also seen it in mosquitofish but only from a location in mississippi receiving wastes from a timber treatment facility, involving the wood preservative chromated copper arsenate, a mixture of chromium, copper, and arsenic (as copper(ii) arsenate) (overstreet ) . presumably, the mosquitofish becomes infected from being in close contact with actinospores shed from a tubificid oligochaete. a small percentage of the few fish in the contaminated creek exhibited mass infection (figs. . and . ), and it was typically histozoic with plasmodia throughout the skeletal muscle mixed with tissue debris not associated with an obvious inflammatory response rather than in pseudocysts located in the epidermis, corium, and subdermal connective tissue as originally described. a few of those fish from the wild had a severely pathogenic infection, with mature spores liberated from the plasmodia and spread into adjacent muscles, replacing most tissues throughout the body (e.g., dyková and lom ) . however, when we collected fish from this station and from another station containing tubificids and then maintained them in aquaria in our laboratory and fed them commercial flakes daily, over half the fish from the contaminated location became moribund or died within months. no fish from the other location died or appeared unhealthy. the moribund fish were sectioned or examined fresh and demonstrated the myxosporidian with its by μm spores mass infection of the myxosporidian henneguya gambusi triggered within the skeletal muscle of the western mosquitofish, gambusia affinis, from a location in coastal mississippi that contains heavy metal contamination. vegetative and mature stages in plasmodia located between and within muscle fibers, with mature triggered spores being liberated and destroying muscle and showing no plasmalemma separating spores from fibers invading most tissues throughout the samples. representatives of live fish from both groups did not exhibit infection in section, and none of the remaining fish exhibited infection when critically examined as fresh. the contamination apparently reduced the host's protective immune response which would normally keep a low infection in check. light infections had to be present in about half the fish from the contaminated location when brought into the laboratory, even though not evident in fresh or sectioned material. under normal conditions, spores of all histozoic species occur in small to large pseudocysts, the latter containing millions of spores. when the plasmodium is extremely small or when few vegetative or spore individuals are present, an infection is difficult to see, and polymerase chain reaction (pcr) probes are necessary to detect infections. in fact, the only person we have seen skilled enough to routinely detect minimal infections is iva dyková of the academy of sciences of the czech republic. another example of a pathogenic agent in the western mosquitofish deals with a free-living organism rather than a known parasite. the free-living organism, the ciliate tetrahymena corlissi, has been reported from fish in aquaria and hatcheries, causing pathological alterations and mortality by hoffman et al. ( ) . they were unable to experimentally infect fish with the ciliate and suggested that infection resulted from a wound or stress. we (overstreet et al. ) sampled the mosquitofish from an outlet canal from an integrated pulp and paper mill, . km upriver from the canal where water entering from a dam kept mill effluent from flowing upstream, and three downstream locations. two of fish from upstream, but none from the other locations, had a proliferation of the ciliate in the head and in the musculature, brachial chamber, and pericardial sac; that location also had fish with a significantly higher prevalence of macrophage aggregates in the spleen, a good indicator of stress. after publication, we learned of a long submerged pipe emptying toxic wastes upstream from our locations and originating from a nonrelated facility located several km from the river and apparently promoting the ciliate infection. some ciliate specimens measured larger than those reported by hoffman et al. ( ) , but sequencing of presumed free-living ciliates infecting various aquatic hosts should allow identifications and experimentation to determine details of the mechanisms that shift a free-living organism to become a pathogenic one. for parasites or diseases of a host organism to be used as monitors of environmental health or biological activities, both the host and the symbionts or pathological responses need to fit several criteria (overstreet ) . chemotherapeutics can also serve as toxins. a cdc report (u.s. department of health and human services, centers for disease control and prevention ) estimated that about million people worldwide possess a chronic infection with the nematode strongyloides stercoralis, and prevalence of the infection in refugee populations ranged from to % by serosurveys. this species has the unusual ability to replicate and auto-infect its human host, persisting for decades. strongyloides hyperinfection syndrome may be triggered many years after migration of a prior refugee to a nonendemic locality, with large numbers of the parasite infiltrating internal organs, resulting in fatality rates exceeding %. the syndrome is generally induced when an individual is placed on corticosteroids, although other immunosuppressive conditions such as cancer and transplant chemotherapeutic immunosuppression may also trigger the hyperinfection. a change in diet as well as some chemotherapeutic compounds can serve as a trigger and induce a parasite located in one internal habitat such as the intestine, blood, visceral organ, or muscle to migrate to more sensitive tissue or be released from a cyst or an encapsulation and migrate or replicate. free-living or symbiotic metazoans, protozoans, algae, fungi, and bacteria can get into abnormal sites (locations in host) or habitats and develop into pathological agents. some cases are rare and others are common. a rare case consisted of finding the diatom amphora sp., which normally is a free-living alga, in the white shrimp, litopenaeus setiferus, presumably after the shrimp's carapace had been punctured or abraded. the diatom occurred in abundance as clusters in the hemolymph, plus some individuals were present in the gills associated with a melanistic response. the shrimp died. to test the effect of the related amphora coffeaeformis on shrimp, overstreet and safford ( ) injected cultures into shrimp resulting in melanistic responses, but no extensive replication occurred as had been observed in the shrimp killed with amphora sp. a more common opportunist is the fungus fusarium solani, which also developed in penaeid shrimp. farfantepenaeus californiensis, a species of penaeid initially being considered for aquaculture, is a highly susceptible species to this fungus. when experimentally infected with a cultured conidial suspension of this free-living species (hose et al. ) , all shrimp became infected within days, tissue destruction and a strong but often unsuccessful hemocytic inflammatory response occurred, and over half of the shrimp died within days. biochemical and hemocytic parameters of the hemolymph changed significantly as the infection developed when compared with noninfected shrimp. when farfantepenaeus aztecus or litopenaeus setiferus was held in seawater with macroconidia spores cultured from an infection from farfantepenaeus californiensis or injected with those spores, resistance to infection occurred. a strong hemocytic encapsulation and melanization of the macroconidia and lysis of the conidiospores occurred in the gills by h. if spore dosage was excessive, .  or greater, all brown shrimp died within h from the macroconidia and hyphae blocking the distal portion of the gill lamellae (solangi and lightner ) . in litopenaeus vannamei, the primary cultured penaeid today, naturally infected individuals in culture are moderately susceptible to infection and the spore aggregate, often grossly apparent in the distal portion of the eyestocks; penaeus monodon, which used to be the primary commercially cultured species, is relatively resistant (lightner ) . for pathogenicity by free-living algae and fungi, the triggering factor seems to be a wound or being compromised by other infectious agents or toxicants, but, as pointed out, the host species is also critical. one or more ciliates identified as or presumed to be the scuticociliate orchitophrya stellarum has an extensive host and geographic range in europe, australia, and north america as determined by morphological and sometimes molecular techniques. it is considered a facultative parasite that can live indefinitely outside the host when cultured with bacteria and tissue detritus or yeast but with different size and morphology when compared with material in male sea stars. cultured specimens enter male but not female sea stars, probably through the gonopores, where it enters the testes and feeds on sperm of fully mature individuals only (stickle et al. ). it has been reported from several sea star species and may have been inadvertently introduced into the northeast pacific region in the late s (boom, in bates et al. ) . recently, small et al. ( ) determined using its (internal transcribed spacer) sequences and pcr (polymerase chain reaction) analyses that the histophagous ciliate infecting the blue crab, callinectes sapidus, in research facilities in virginia and previously thought to be mesanophrys chesapeakensis was actually o. stellarum. the same or similar infectious ciliate was probably responsible for histophagous disease in wild and captive blue crabs in mississippi (shields and overstreet ) , penaeid shrimps in mississippi, and the wild lined shore crab, pachygrapsus crassipes, from carpinteria salt marsh, california, that we examined for ryan f. hechinger, university of california, santa barbara. this infection spreads in the partially closed circulatory system of the decapod (mcgaw ) from the hemolymph to muscle, visceral organs, and other tissues (figs. . , . , . , and . ), usually killing the host. it has been assumed to enter into wounds of its hosts and now shown by miller et al. ( ) to cause rapidly developing fatal infections in the blue crab inoculated with the ciliate or exposed to ciliates after experimental autonomy. when exposed to ciliates and not wounded, the crab seldom died. for comparisons, the fiddler crab uca minax was inoculated with doses of either or over ciliates per crab, and crabs with sometimes established infections, but those with the higher doses developed them rapidly. the infections developed at - c, and ciliates were attracted to blue crab serum over other nutrient sources, suggesting the facultative nature of a blue crab parasite. viridans streptococci typically occur harmlessly in the mouth. these bacteria can be differentiated from streptococcus pneumoniae using several tests. moreover, they lack either the polysaccharide-based capsule typical of s. pneumoniae or the lancefield antigens, based on the carbohydrate composition of bacterial antigens found on the cell walls in beta-hemolytic bacteria of the pyogenic, or pus-producing, members of the genus. some may be involved in other mouth or gingival infections as pericoronitis or inflammation of the gums around molar teeth. however, if they are introduced into the bloodstream from surgical or other lesions, they have the potential of causing endocarditis, especially in individuals with damaged heart valves. viridans streptococci have the unique ability to synthesize dextrans from glucose, allowing them to adhere to fibrinplatelet aggregates at damaged heart valves. often there occurs an indirect site-associated triggering mechanism for parasite establishment, with a corresponding associated ability to enhance or decrease pathogenic effects. the bothriocephalid anantrum tortum, a long, up to over cm, cestode, twists either singly or in groups of up to eight, within the intestine of its relatively small fish host, synodus foetens (inshore lizardfish). it can occur near the pyloric ceca, along the intestine, or near the anus of the samples, measuring up to mm long. overstreet ( ) detected a % level of significance over a -year period between the monthly prevalence of worms located near the anus and water temperature. salinity was held constant, and an inverse sine transformation was applied to the percentage data. this anal location favors loss of the worm during warm weather, possibly assisting probability of best completing life cycle, but to be expelled, the lizardfish appears to have needed a several-mm-long packet of prey fish scales with a diameter much greater than that of the intestine. the carnivorous lizardfish can eat fishes and shrimps larger than itself. consequently, fish prey with large scales tends to be advantageous for the lizardfish to rid itself of the parasite. sometimes, a fortuitous site creates a trigger for pathogenicity. for example, a diver accidently punctured his hand while reloading his spear gun. later, unconcerned about his wound, he was filleting a jackfish, and a female of the nematode philometra sp. invaded the wound and attached deeply. after several unsuccessful attempts to remove the worm, the pain-inflicting infection required surgery (deardorff et al. ). not to be overlooked is the obvious matter of dose. this obvious matter is treated in discussions and is important for all agents from viruses to metazoans. in the case of metazoans, a host can come into contact with an especially high, unnatural dose of infective agents such as thousands of cercariae killing an intermediate host well before the corresponding metacercariae develop (e.g., overstreet and curran ) . intermediate hosts under specific conditions can be heavily infected simultaneously with several parasitic species. some parasites such as microphallid trematodes infect a variety of birds and mammals. therefore, a bird could acquire a harmful infection by feeding on individual heavily infected intermediate hosts, or source communities (bush et al. ). the host species is also critical for many protozoans that are acquired by feeding on an intermediate host in which replication or maturation of a stage is necessary. for example, whereas a few killifish species are natural hosts of calyptospora funduli, several related atheriniform fishes can be experimentally infected, but no nonatheriniform could be infected (fournie and overstreet ) . those experimentally infected demonstrated a variety of abnormalities, including asynchronous development, degeneration of early developmental stages, formation of macrophage aggregates, and a granulomatous inflammatory response, especially one exhibiting liver destruction in fundulus olivaceus and rivulus marmoratus. of course, feeding behavior, environmental conditions, and geographic isolation can also serve as barriers to infection in fish in addition to innate immune barriers. when introduced species become established in a system, they can be resistant to harmful factors in the environment, or they can be susceptible to extensive predation, diseases, and parasites. species of the ascaridoid genus goezia typically embed in an encapsulated ulcer in the wall as well as free in the lumen of the stomach of their natural fish hosts. these associations are described by deardorff and overstreet ( a) for the nematode species goezia pelagia in the cobia, rachycentron canadum, and the atlantic spadefish, chaetodipterus faber, as well as for goezia minuta in the gafftopsail catfish, bagre marinus; the hardhead catfish, ariopsis felis; and the inshore lizardfish, synodus foetens. in none of these hosts does there appear to be any significant harm associated with the presence of the nematode (deardorff and overstreet a) . however, when the introduced host species such as the blue tilapia, oreochromis aureus; striped bass, morone saxatilis; and hybrid striped bass become infected with goezia sinamora, the nematode produces massive fibrotic nodules in the fish stomach, and the nematode can also cause mortality. goezia sinamora was implicated in mortality of hatcheryreared striped bass and tilapia introduced into a series of lakes in florida, including lake parker [gaines and rogers , see correction by deardorff and overstreet ( a) ]. in the tilapia, this roundworm has been observed to migrate through the intestinal wall, causing extensive lesions, in addition to forming nodules in the stomach. when the largemouth bass, micropterus salmoides, and other native species of fish were exposed to this nematode, the resulting infections contained relatively few of the roundworms and demonstrated no conspicuous harmful effect (deardorff and overstreet a ). several books and chapters [such as those by overstreet ( ) , barnard and behnke ( ) , lewis et al. ( ) , moore ( ) , lefèvre et al. ( ) , adamo ( ), adamo and webster ( )] have reported on parasite infections resulting in virulence, evolutionary, and behavioral changes in intermediate hosts such that the intermediate host has a better opportunity to be preyed upon by the appropriate definitive host than by chance alone. whereas these changes are not necessarily triggers that make an agent more pathogenic, they often are changes that reflect the point at which the undeveloped, noninfective agent becomes infective to the definitive host and pathogenic to the intermediate host. a good example is the microphallid trematode levinseniella byrdi, which infects the intestinal ceca of a few bird species such as the seaside sparrow, clapper rail, willet, and semipalmated sandpiper. these birds acquire the infection from one of a few talitrid amphipods such as uhlorchestia uhleri from salt marshes of texas to north carolina and uhlorchestia spartinophilia in a similar habitat from cape canaveral, florida, to central maine. in the gulf of mexico, the metacercaria also occurs in the amphipod species orchestia grillus and chelorchestia forceps (bousfield and heard ) . the dramatic modification in the amphipod usually occurs after about a month, once the metacercaria becomes infective. at this point, the host amphipod turns from greenish or grayish to a translucent or bright orange (fig. . ) . the infected amphipod also unusually slows down its movements and, in contrast to its uninfected, negatively phototactic cohorts, does not always hide under wracks of dead, dissociated leaves and stems of spartina grass or other of their dietary debris shelters. apparently, the amphipod carotenoids become unbound from protein of the infected host, allowing the bird host to be preferentially attracted to the now brightly colored amphipods containing infective metacercariae (bousfield and heard ) . johnson et al. ( ) experimentally manipulated a few tidal salt marsh creeks in plum island estuary, massachusetts, by nutrient fertilizer enrichment and exclusion of the killifish fundulus heteroclitus (mummichog), a primary predator in the system. interaction of the two treatments reduced abundance of the common uhlorchestia spartinophilia, and apparently infected amphipods moved from the marsh edge to the adjoining creek-wall habitats during year, resulting in % of the amphipods clinging on the creek walls and exhibiting the bright orange. a subsample of the colored amphipods was confirmed to be infected, and they were seen being fed upon by the semipalmated sandpiper and seaside sparrow. in regard to the above host modification, other microphallid species in those amphipods did not turn their hosts orange or noticeably modify their behavior. a similar situation has been noted to occur where levinseniella tasmaniae but not other microphallids induced an orangish color in the amphipod austrochiltonia australis in tasmania (smith ) . that is not to say all species of levinseniella induce coloration and other modifications in their amphipod hosts since levinseniella tridigitata does not modify gammarus aequicauda (see thomas et al. ) and levinseniella carteretensis (or levinseniella hunteri) does not induce a color change in any of the five talitrids, including u. uhleri, when experimentally infected (bousfield and heard , heard personal communication) . another trigger involves photoreception. the snail host typically sheds large quantities of cercariae of levinseniella tasmaniae infective for the amphipod host when under light conditions, whether natural or experimentally reversed (smith ) . and yet, cercariae of other species are shed during dark or other physical conditions. almost all symbiotic relationships, including those discussed above, involve a combination of triggering factors. those factors and host-symbiont relationships involving termites are particularly well studied. we, ourselves, have examined in fig. . specimens of the amphipod orchestia grillus in coastal mississippi showing on the top an uninfected one and on the bottom a transformed orangish one that is infected with the microphallid levinseniella byrdi, a trematode that infects the intestinal ceca of a few bird species. the transformation triggers phenotypic and behavioral changes specifically attracting infective specimens to predatory birds in which the trematode matures considerable detail the host-symbiont relationships affecting the outcome of pathogenic viruses in populations of commercial penaeid shrimp. we will address in this section some of the symbiotic relationships and interrelationships that can involve a shift from harmless to harmful relative to three host groups. the relationships between termites or related insects and their symbionts are numerous and provide examples of an abundance of triggers, including diet, to control the various relationships. some of these involving termites will be discussed elsewhere by david bignell (volume , chapter of this series). investigations by l. r. cleveland (e.g., ) were designed to better understand symbiosis using termites and their intestinal flagellates. he was well aware that bacteria and yeasts were involved in the relationships and that there was a fine line separating where one symbiotic association ended and the other began. for example, when one agent in a so-called mutualistic relationship could survive without the host, it nevertheless also can become a parasite living off the host. this situation, however, is complicated and difficult to assess. he tried to remove one or more flagellate microorganisms from a host without harm to the host so that the association could be manipulated. he also thought it best to consider all components, which he defined differently than we do (he considered a commensal association to be one when neither party was benefited nor injured), of an association as symbionts. depending on a specific termite host, he could void the flagellates with starvation, a high temperature not lethal to the host, a specific level of moisture, and oxygen under pressure. for example, by oxygenating the large pacific coast termite (zootermopsis nevadensis) for h at . atm, two (leidyopsis sphaerica and trichonympha campanula) of the four flagellates survived, and the host lived "indefinitely." additionally, starving the termite at the above conditions for days left only one flagellate (l. sphaerica), showing that both l. sphaerica and the termite do together constitute a necessary and mutualistically beneficial symbiotic relationship. without any flagellates but with a diet of wood, the termite lived for about weeks. reintroducing either l. sphaerica or t. campanula allowed the termite to experience its normal much longer longevity (usually - days). cleveland realized that the presence of the other two flagellates (trichomonas termopsidis and streblomastix strix, both having a poorly understood symbiosis with epibiotic bacteria) in the termite starved for days would help the termite survive for about weeks but were not necessary symbionts; he realized that bacteria played a role in digesting the diet. the "primitive" termite mastotermes darwiniensis from australia was studied by li et al. ( ) , and it had in its hindgut six flagellates, none of which can yet be cultured. historically, these flagellates were considered to use their digestive enzymes to digest cellulose for the benefit of the termite. however, they determined using pcr technology that the main endoglucanase activity in the flagellates appears to originate from termite cellulases produced in the salivary glands. at least two of the flagellates possess their own endoglucanase genes, which are expressed but without significant enzyme activity in their nutritive vacuole. after millions of years of evolution, these flagellates, suggested by li et al. ( ) , are heading for a secondary loss of their own endoglucanases to exclusive use of the termite cellulases. feeding on the symbionts still seems to be an important nutritional component of the termite diet. some termites, such as those which are soil-feeders, depend entirely on bacterial symbionts (bignell et al. ). this association, often involved with coprophagy, feeding on fecal pellets containing termite bacteria that digest the cellulose, will be treated by d. e. bignell elsewhere in this series (volume , chapter ). other termites, such as members of the macrotermitinae, depend on a relationship with mutualistic fungal symbionts of the genus termitomyces, which form fungal combs in the nests. these and attine ants can produce nests that often are thousands of liters in volume, able to persist for decades, and contain millions of sterile helper individuals usually resulting as offspring from a single queen. those termites are major decomposers of the old world tropics, and the ants are dominant herbivores of the new world tropics. the fungal symbionts of the termites can produce sexual fruiting bodies allowing their horizontal acquisition, but those of the attine ants rarely fruit and are typically propagated clonally and vertically by the dispersing queens. the life cycles of the fungus are again shown to help trigger different symbiotic relationships. aanen et al. ( ) present phylogenies of the termites (about species in genera) and their fungal symbionts which number about and are shared by different termite species. they show significant congruence between the termite and fungal phylogenies because the interactions at higher taxonomic levels show considerable specificity. they also considered the trait of biparental colony founding to constrain evolution of vertical symbiont transmission in termites, where the male survives and mates repeatedly with the female for life but not in ants where males die after a single mating. the formosan subterranean termite pest (coptotermes formosanus) builds a large nest with spongelike networks of intricate feces-lined tunnels (carton material). fungal pathogenic agents are used, usually unsuccessfully, to kill the termite. studies by chouvenc et al. ( ) have shown that environmental conditions within termite nests promote the growth of actinobacteria, whose presence in turn seems to protect the termite colony against fungal entomopathogens, including metarhizium anisopliae. in other words, the actinobacteria, which represented a nonnutritional exosymbiosis in the termite, was a defensive mutualism that increased survival of the termite and was additive to the termite's individual immunity and social defensive capacity, which in turn increased survival of the termite. baculovirus penaei, commonly called bp or bp (pvsnpv), has a widespread distribution in cultured and wild penaeids, and it can cause severe epizootics in larval, post-larval, and juvenile stages. it is an enveloped, polyhedrosis, rod-shaped, double-stranded, intranuclear, dna virus infecting epithelial cells of either the anterior midgut and midgut gland, or the hepatopancreas (hp; r-cells, f-cells, and b-cells, most commonly found near the base where the hp tubules join the anterior midgut). the e-cell populations (stem cells) at the distal portion of the hepatopancreatic tubules produce cells so quickly as to assure noninfected new cells (figs. . , . , and . ) (overstreet et al. ). baculoviral infections of bp can involve nearly % of the shrimp hp during mysis and early postlarvae stages without necessarily causing death. a number of triggering factors can tip these acutely infected shrimp into mortality. the virus is one out of or so that infects penaeid shrimp and one of a few thousand known from invertebrates. because it has a proteinaceous tetrahedral occlusion body, infections can be detected and followed with a light microscope; because of these bodies, it was the first species reported and characterized from a shrimp (summers ) . the virus has a simple direct life cycle, although unknown means allow the agent to remain dormant or in a reservoir host. it can infect an acceptable penaeid either from free or occluded virions (fig. . ) in the surrounding water or through a carrier host. experimental infections are best accomplished by feeding the virus concentrated in a rotifer to protozoeal or early-stage mysis larvae or concentrated in a brine shrimp to infect late-stage mysis or early postlarvae (overstreet et al. ). depending on a number of factors, the virus replicates in the host alimentary cells and is most prevalent and infective at about day after being fed. the nucleus of those cells enlarges, and the polyhedra and associated virions rupture into the lumen of the host midgut. we have observed as many as relatively large tetrahedral bodies (about μm on a side) or small distinct bodies in experimental infections. the infective agent is then passed out through the intestine or it can be released when the infected larva is eaten by an appropriate predator. unlike some insect baculoviruses that are used to kill agricultural pests, the bp virion remains active in the polyhedron for less than weeks rather than for or so years. on the other hand, bp maintained in an ultralow freezer remained active for at least three and a half years. natural infections of bp in the brown shrimp, farfantepenaeus aztecus, were monitored monthly between january and november with seasonal the monitoring took place in the northern gulf of mexico in or off mississippi waters, but shrimp from florida were also examined. during the mid- s, infections occurred in wild populations of the pink shrimp, farfantepenaeus duorarum, but when we monitored all commercial penaeid shrimps for those infections, the virus primarily infected the brown shrimp and only occasionally infected the pink or white shrimp, litopenaeus setiferus, which exhibited the otherwise rare infection. during the monitoring, we tried to use -cm long shrimp because they exhibited the highest prevalence and intensity of infection. infection usually occurred only about or months of the year between about april and june, with the highest prevalence occurring in when we detected values of , , and %. values reaching and % occurred in may and , respectively . typically, the prevalence reached about - % during the remaining years, and infections were almost always absent in fall and winter. rarely was the intensity of infection great, as usually observed in experimental infections with larvae and early postlarvae. the outbreak dynamics probably resulted from a variety of factors, including genetics (primarily brown shrimp), the interaction between the life cycle of the shrimp and the presence of the virus, the age of shrimp when initially infected, and resistance to infection, which occurred primarily in larger shrimp. these factors will be discussed below in more detail such as with infections and problems in aquaculture hatcheries. strains there are at least four strains of bp as differentiated with molecular probes. each seems to be restricted to specific penaeid shrimps. one strain occurs in some commercially important shrimp in the northern gulf of mexico; another occurs in noncommercially important shrimp (trachypenaeus spp.) in the northern gulf of mexico; another occurs in melicertus marginatus in hawaii; and the last occurs in litopenaeus vannamei in ecuador. we have used for our experimental research the latter strain originally obtained and frozen from ecuadorian aquaculture facilities by biologist james brock. there is also a series of strains of mbv (monodon baculovirus), a baculovirus that produces spherical polyhedral bodies but which have many similarities to bp. we attempted cross infections with bp for all but the hawaiian strain, using presumably susceptible penaeid postlarvae without success. free and occluded virions in addition to the virions occurring in the tetrahedron, which occasionally does not contain any virions, free ones lined up along the abnormally-distorted internal surface of the nuclear membrane. the free viral particles were collected from a freeze-thaw preparation either by filtration through a . μm filter or from processing of the preparation by relatively low-speed centrifugation to prepare a cell-free supernatant, and when next exposed to shrimp larvae, they produced infections. these subsequent infections did not appear to be more virulent than those initiated from virions incorporated in the tetrahedron. however, the free virions of some insect baculoviruses produce secondary infections in the hemocoel, rather than gut cells, and those are considerably more virulent (kelly ) . dose dose of bp is difficult to determine because there is no appropriate crustacean cell line available to culture the virus. however, we conducted a replicated relative study using the same standard viral stock suspension and determined a clear dose response involving both prevalence of bp and mortality of shrimp (overstreet ) . environmental factors temperature and probably other factors affect both the production of free virus and the relationship between the virus and its shrimp host. when a homogenate of bp-infected cells is placed in seawater ( ppt), the virus becomes completely inactivated between and days when maintained at c. however, when maintained at c, some virions remained active after days. a low temperature also delayed or inhibited infections in larvae and presumably adults. virions were inactivated by a -min exposure to temperatures of - c. a variety, but not all, of toxicants can probably affect the relationship between the viral agent and shrimp host. couch ( ) reported an increase in prevalence of infection in pink shrimp exposed to low levels of aroclor ® . we tried to duplicate his experiments using brown shrimp and ppb aroclor and also ppm nickel but with unsuccessful outcomes. we later discovered from couch (personal communication) that the aroclor he used was probably unknowingly contaminated with another toxin (overstreet ) . studies were conducted by treating free bp in a variety of ways and then feeding it to larvae of l. vannamei as a bioassay (overstreet et al. ). unlike insect baculoviruses, all of the bp virions that we tested were inactivated when desiccated for h. tests conducted for possible treatments in aquaculture also showed that the virus was completely inactivated by a -min exposure to ph ; ph extended the pre-patency period of infection, but it did not inactivate the virus. ultraviolet irradiation for min at a wavelength of nm when the virus was cm from the light source also inactivated the virus (leblanc and overstreet a). the virus was also completely inactivated by a chlorine (in the form of calcium hypochlorite) concentration of mg/l when treated for h and by mg/l when treated for a time period as short as s (leblanc and overstreet b). other than for sterilizing aquariums, these methods other than desiccation and steam cleaning seemed impractical for an aquaculture facility. research host shrimp to conduct experimental infections, we used mostly litopenaeus vannamei cultured at the gulf coast research laboratory consortium either at oceanic institute (oi) in hawaii or at gcrl in ocean springs. our early studies with larvae required antibiotics to counter bacterial infections, which otherwise often reduced a larval stock by % and would have been considered an acceptable loss in commercial hatcheries at that time. even with antibiotics, we obtained bp infections with associated catastrophic mortalities when the shrimp larvae were exposed to the virus. after hybridization crosses of shrimp and introducing special wild brood stock, the result was creation of shrimp stocks with improved resistance to bp, and we noted as a bonus our having created crosses exhibiting resistance to other microbial agents. the bp prevalence could be variable, but mortality was reduced or eliminated. in a few cases, we obtained shrimp from commercial hatcheries or other research facilities and had to use antibiotics. host resistance the genetic families of litopenaeus vannamei produced at oi gradually became more resistant to bp. in some cases, it was easy to obtain % prevalence but without the associated mortalities observed using other stocks of shrimp. middlebrooks et al. ( ) examined lectin levels in the hemolymph of monthly samples of wild farfantepenaeus aztecus, and the levels were found to vary by season and among individuals, suggesting that levels could serve as indicators of health or immune status. high titers of activity for lectin agglutination occurred in september, and low levels occurred in april. those observations of positive bp infection results when lectin levels were low corresponded with the time when bp first became apparent in the wild shrimp population, and polyhedra subsequently were no longer apparent when lectin levels were high. age and growth the age of litopenaeus vannamei influences bp infections in a variety of ways. stuck and overstreet ( ) exposed pathogen-free shrimp ages mysis - through -day-old postlarvae (pl) from different sources, each with a single viral exposure, and cultured them for - days. all groups pl or younger became heavily infected within - days, some experiencing high mortalities compared with controls. surviving postlarvae had reduced growth as determined by dry weight. one of these groups was cultured for an additional days, and the smaller postlarvae that survived the infection appeared similar to the controls after weeks by which time viral prevalence had decreased. exposure of the virus to older postlarvae produced a high prevalence of infection but with little effect on either survival or growth. when -to -day-old postlarvae in similar size groups of previously infected and noninfected individuals were starved for days, less than % of the infected postlarvae survived the -day period compared with % of the noninfected ones. leblanc and overstreet ( ) exposed infected bp to groups of l. vannamei , , , , , , and days after reaching postlarvae. the postlarvae exposed when -days-old (pl ) exhibited infections in % of those examined at days postinfection (pi) and in % of the same group when examined at day compared with % prevalence in the controls; % of the shrimp that were exposed when postlarvae for days (pl ) had developed an infection at day pi when % of those exposed as pl s exhibited infections; and the prevalence in the latter group increased to % at day . with the exception of one shrimp exposed when a pl , none developed an observable infection at days, and only of of the exposed pl group examined at day exhibited an infection. no control became infected. other experiments also showed this decrease in prevalence as well as intensity of infection and mortality with age. in general, most postlarvae days old or older did not become infected, and those that became infected did not die. this age corresponds with the age that most postlarvae leave the offshore waters and settle on the substratum in the estuarine area where the infective agents occur. energy the nutritional condition of litopenaeus vannamei can be assessed biochemically in terms of available energy reserve. the principal energy storage materials in the penaeid shrimp are lipids and proteins, with carbohydrates considered to be a minor energy reserve. triacylglycerol (tag), an ester derived from glycerol, and three fatty acids serve as the primary classes of lipid used for energy storage. in patent infections of bp conducted by stuck et al. ( ) , the polyhedra first appeared in the hp - -h postexposure with a maximal infection usually occurring at h (also see hammer et al. ) ; however, progression of the infection after the initial appearance of the polyhedra was variable among shrimp potentially corresponding with different levels of body lipid used for energy storage. we measured preinfection and postinfection tag and protein levels during a series of experimental bp exposures using shrimp from a variety of sources with different inherent protein and tag levels. mysis-stage or early post-larval protein levels measured either preexposure or postexposure to bp showed no consistent relationship with bp infections. however, a viral prevalence of - % occurred at h when initial tag levels measured above . μg/mg in contrast to % or less in shrimp when initial tag levels < . μg/mg. tag levels in mysis stage- larva rose rapidly from to about μg/mg in days for both infected and negative control populations, then dipped to about μg/mg at day , and subsequently again rose in both populations but was about μg/mg higher in the infected shrimp at day . most shrimp died before the end of a week. two other experiments starting with -day postlarvae (pl s) were conducted when initial tag levels were either about or μg/mg, and substantial mortalities did not occur in either. all individuals in the infected groups became infected, at about day in the initial low tag group and at day in the initial high group. in the initial low tag group, the tag level of the infected group stayed about the same at day , but that of the control groups increased to about μg/mg. in contrast, in the initial high tag experiment in which the tag level for both infected and control groups dipped from to about . μg/mg at day , the level increased to about μg/mg at day and about μg/mg at day . stress caused by starving postlarvae (pl s) reduced tag but not protein reserves. detection of the bp infection was delayed for h when the group to be infected was starved for h compared with continuously fed controls, but the prevalence of infection in both groups increased rapidly to similar high prevalence values above % between and h pi. genetics offspring of male and female crosses of high-growth and low-growth "families" of a well-defined population at gcrl consortium at oi, hawaii, were fed bp as -day-old postlarvae (pl s) (alcivar-warren et al. ) . the high  high-growth and low  high-growth (female  male) postlarvae, respectively, had a and % survival rate at days postexposure compared with and % survival for the low  low-growth and high  low-growth offspring. all but the high  low-growth offspring, with a % prevalence of infection, had an - % prevalence at day . the low  low-growth cross of -month-old shrimp fed the virus ihhnv (infectious hypodermal and hematopoietic necrosis virus, which has been classified as penaeus stylirostris densovirus) exhibited the highest prevalence ( %) at days compared with the lowest ( %) in the high  low-growth cross. random amplified polymorphic dna polymorphisms for the four crosses showed no clear relationship between the prevalence values of ihhnv and bp. even with similar mtdna haplotypes included in the initial crosses, the offspring of those crosses exhibited major differences in both steadystate levels and patterns of expression of mitochondrial s rrna at various early developmental stages of the resulting offspring of the different crosses. even though specific genetic markers and differences could not be associated with specific differences in susceptibility to different infections, shrimp growth, or regulation of gene expression, the genetics of the shrimp play an important role in triggering infections and mortality from infections. occur concurrently with other disease-causing viruses producing a synergistic effect. we (krol et al. ) found such a virus infecting the anterior midgut epithelium and the r-and f-cells from the hp in larval specimens of litopenaeus vannamei. the occurrence of the reo-like virus was observed only in bp-infected shrimp; however, shrimp with bp seldom had the reo-like virus. other agents often associated with stress also occur in shrimp (overstreet ) . susceptibility to viral infections appears to be enhanced by crowding of host individuals. we determined that the prevalence and intensity of infections in shrimp crowded in commercial bait tanks were higher than in those collected from the wild shrimp population that was used to stock the bait tanks. in summary for the endemic virus bp, there are a variety of susceptibility factors dealing with the environment, virus, and host, including the nutritional and molting state of the host, which can trigger a relatively harmless infection to develop into a severely pathogenic condition. in nature, this condition is seldom recognized because only microscopic-sized larvae and early postlarvae usually die from the infection, dead animals cannot be seen because they are small or readily eaten, and the opportunity for the agent and the larvae to come into close proximity is relatively rare. in aquaculture, the virus and larvae can and do come into contact. hatcheries in the multibillion-dollar shrimp industry have prompted research to solve the previous major threat that collapsed hatcheries in the usa and elsewhere in the western hemisphere during the early days of the industry when brood stock was obtained from the wild. the related mbv in the eastern hemisphere also caused a similar major problem. now that the industry can rear most penaeid species; detect bp and other infectious agents with pcr, gene probes, and other methods in routine monitoring; produce its own brood stock relative to disease resistance and growth potential; and accommodate ambient temperatures, infections of bp in aquaculture are rare. if they do occur, an entire spawn can be destroyed and disposed and the system decontaminated, and there would be a time loss of only a few days before a new spawn could be produced. pathogens of shrimp aquaculture with the exception of baculovirus species, most or all the pathogenic penaeid viruses are not native to the shrimp species being cultured or perhaps to any commercial shrimp. consequently, the cultured species may be highly susceptible to and die from one or more of the viral agents. most of the presently known or so shrimp viruses cause mortality in subadult animals that have been reared for a few months. the high cost of feed and labor plus the relatively long animal growing period results in a high economic cost. therefore, it is necessary to incorporate the costs of an inability to quickly replace the entire infected pond-reared adult or subadult stock as can be done for larvae in hatcheries infected with bp infections. production losses associated with the exotic viruses white spot syndrome virus (wssv) and taura syndrome virus (tsv) provide excellent examples for examining the emergence of pathogenicity in aquaculture on a large scale. the history of the shrimp aquaculture industry is a case study of the emergence of severe pathogens in hosts where none was known previously. for about two decades after the industry began in the s, few viral pathogens were known. beginning in the early s, several severely pathogenic viral diseases emerged. we will consider two of the pathogens in some detail as they inform our understanding of the appearance of highly pathogenic symbionts through host switching. white spot syndrome virus (wssv) was first reported in from taiwan. the virus belongs to the order baculovirales. although once considered a member of the nudiviridae or baculoviridae, wssv is more closely related to the recently erected family hytrosaviridae (wu et al. ; wang and jehle ) . wssv is now considered a member of the genus whispovirus in the family nimaviridae. the virus is a rod-shaped, enveloped virion and among the largest viruses known at - -nm wide by - -nm long. the genome is double-stranded dna of kbp. the host range is among the widest of crustacean viruses and infects all decapod crustaceans that have been tested. taura syndrome virus (tsv) was first observed from ecuador also in . the virus belongs to the order picornavirales and is a member of the family dicistroviridae and the genus aparavirus. the virion is icosahedral, nonenveloped, and nm in diameter. the genome contains positive sense, single-stranded rna of kb. the host range is restricted mostly to members of the genus penaeus sensu lato, although recently overstreet et al. ( ) reported experimental infections with replication in at least some symbiotic barnacles. pathogenicity varies with host species being most virulent in litopenaeus vannamei and less so in other species of penaeid shrimp . although it is possible that the viruses were symbionts of wild shrimp prior to being detected in shrimp aquaculture, we wish to explore the hypothesis that they emerged by host transfer from insects or other terrestrial arthropods during the development and expansion of shrimp aquaculture. roekring et al. ( ) perhaps first suggested this tantalizing hypothesis in their study of three shrimp parvoviruses whose closest known relatives are parasites of insects. host switching is a common mechanism for the emergence of pathogenic pathogens. for example, in humans, % of today's emerging human viral diseases are zoonotic, e.g., hiv- and hiv- , influenza virus, ebola virus, hantaviruses, nipah virus, zika virus, and the sars coronavirus that causes severe acute respiratory syndrome, and many of the rest of human infectious diseases have their closest relatives residing in nonhuman animal hosts (parrish et al. ; pearce-duvet ) . most theories of the emergence of new diseases through host switching recognize several steps: cross-species transmission, establishment, and spread (parrish et al. ; domingo ; antia et al. ). the first step in the process is an increase in the number of contact events between the recipient host and the donor host. the increase in contacts is usually due to an ecological change or disturbance. as the number of contacts between the donor and recipient hosts increases, there may be a concomitant increase in successful transfer of the pathogen. in the case of shrimp viruses, we suggest below that there was an increase in contact between penaeid shrimp and other arthropods during the early s, and that facilitated cross-species transmission and the emergence of pathogens in penaeids. although cross-species transmission may occur, permanent establishment within the new host requires enough within-species transmission to maintain the pathogen in the new host. it is unknown how many times the ancestor of these two symbionts was transmitted to penaeid shrimp only to be eliminated because shrimpto-shrimp transmission was not sufficient. however, at some point, either prior to cross-species transmission or after initial colonization, a mutation of the ancestor resulted in penaeid shrimp being highly susceptible to them, resulting in the maintenance of the infection locally in shrimp aquaculture. a third set of factors allows for the pandemic spread. sharp and hahn ( ) review the successful establishments of hiv from primates to humans. although there may have been as many as seven successful transmissions, most are restricted to africa, and only one of the seven establishments of hiv (hiv- -m) is responsible for the pandemic of hiv in the s (sharp and hahn ; pepin ) . one or more factors other than trans-species transmission allowed for the amplification and spread after a transmission. in the case of hiv, it is thought that the increased use of vaccination in colonial africa, chance, and increased world travel may have been the contributing factors (pepin ) . in the case of shrimp viruses, it may be that the greater number and higher shrimp densities in aquaculture settings coupled with the long-distance transport of live and fresh frozen shrimp has allowed worldwide viral spread after cross-species transmission. the rapid expansion of the shrimp aquaculture industry in the s and s brought with it conditions conducive to increasing the rate of contact between shrimp and terrestrial arthropods, the majority of which are insects. according to fao statistics (fao a, b) in the late s, most of the penaeid shrimp were coming from capture fisheries (fig. . ) . however, between and production of penaeid shrimp from aquaculture surpassed % of all penaeid shrimp production worldwide (fig. . ). this local peak occurred near the time that the two viruses appeared in shrimp aquaculture. subsequently, culture production declined as a percentage of total penaeid production and remained below % throughout the s. however, the first decade of the twenty-first century saw a substantial increase and by the contribution from aquaculture to world's commercial shrimp increased supply by nearly %. clearly, in the early s, when viral pathogens emerged, there were twice as many penaeid shrimp in the market place as there had been a decade before. although this does not mean that the total number of shrimp in the world doubled (not all of the shrimp in the world were captured), it does suggest that aquaculture substantially increased the total number of penaeid shrimp in the world and continues to do so. it is also clear that the increased number of aquacultured shrimp were at higher densities than wild shrimp. this increased population size and density are conducive to the spread of infectious diseases. perhaps more important for the cross-species transmission of the viruses from insect to shrimp is the habitat of those new shrimp. for the most part, they were in earthen ponds in the coastal zone. however, many of the ponds were located a distance from the coastline, and seawater was pumped over a considerable distance to provide the necessary culture medium. the point is that there may be more shrimp that were likely to have contacted terrestrial insects and other terrestrial arthropods, after the expansion of shrimp aquaculture than before it. the second point in building a case for the origin of the two shrimp viruses from insects or other terrestrial arthropods is that the hosts of the viruses most closely related to shrimp viruses are insect or terrestrial arthropod hosts. roekring fig. . total world's supply of penaeid shrimp in metric tonnes. production from the capture fishery is light bars. production from aquaculture is in dark bars. source fao statistics (fao a, b) et al. ( ) indicate that shrimp parvoviruses do not form a single clade but are distributed among clades that include insect parvoviruses. roekring et al. ( ) suggested that viral transfers between crustaceans and insects occurred. most members of the dicistroviridae are found in insects (baker and schroeder ) . guo et al. ( ) noted that the exceptions are tsv and the recentlydescribed mud crab dicistrovirus- (mcdv- ). mcdv- groups with tsv which could indicate that tsv in shrimp aquaculture arose from other crustaceans. it should be noted that the first reports of tsv were from the new world in the s and that mcdv- was described from asia long after tsv was transferred from the new world into asia in , suggesting that mcdv- arose after tsv was introduced into asia. similarly, wssv as a member of the order baculovirales has as its closest relatives symbionts of insects. in particular, jehle et al. ( ) group wssv most closely with two insect viruses causing salivary gland hypertrophy, musca domestica salivary gland hypertrophy virus (mdsghv) and glossina pallidipes salivary gland hypertrophy virus (gpsghv). we have provided a number of examples and triggers of the switch from one quality of a symbiotic relationship to another, specifically from commensal to pathogen. examples of triggers include environmental ones like temperature, toxic chemicals (dose), chemotherapeutics, dietary changes, and geographic habits; internal ones include host physical site, host resistance or susceptibility, host modifications, and host switching; and combinations of these and other conditions. fig. . proportion of world's penaeid shrimp from aquaculture. note that the proportion of penaeid shrimp production saw a local peak in the early s above . and then declined during the emergence of taura syndrome virus (tsv) and white spot syndrome virus (wssv). source fao statistics (fao a, b) the question arises, "is becoming a pathogen an adaptation for a symbiont?" in an evolutionary context, which is where the concept of adaptation lies, why would a symbiont switch to being pathogenic, either at some stage in its life cycle or in some hosts at the same stage but not in others? in some cases, symbionts are pathogenic as a clear adaptation, e.g., the levinseniella byrdi, referenced above causes damage or even death of the intermediate host to complete its life cycle. in particular, l. byrdi causes a phenotypic change in the host that increases the host's chances of becoming a prey. in other cases, it is not so clear. facultative symbionts, such as entomophagous nematodes, are both free living and symbiotic (sudhaus ) . the adaptation of being symbiotic is that it increases the habitat breadth of the symbiont, and inhabitation of a living organism removes the organism from competition with other organisms encountered living freely in the habitat. thereby, the relationship allows the symbiont an escape from competition. so that may be the reason for a life history strategy in which both symbiotic and free-living phases exist. however, we are most interested in answering the question of why would a symbiont become pathogenic in some hosts and not in others from an evolutionary perspective. there is a body of theory that addresses virulence evolution in symbiotic organisms. traditionally, the virulence of a pathogen was thought to be a reflection of a recent host-symbiont association and that over time there would result a diminishment in virulence of the pathogen (may and anderson ; anderson and may ; ewald ) . it was argued that no symbiont would benefit from killing its host (destroying its habitat and thereby destroying itself). however, this thinking fails to recognize that natural selection maximizes reproductive success of the organism. reproductive success is a composite of births (new infections) and survival (loss of infection) not just survival. more recent theoretical studies suggest the evolution of an intermediate level of virulence. the new conclusion results from the recognition that there is often a trade-off between pathogen transmission (births) and virulence (pathogen survival) (anderson and may ; may and anderson ; antia et al. ; frank ; koella and restif ) . one indicator of reproductive success of a symbiont is the basic reproduction number (r ), which is the mean number of new infections produced by a single infection (anderson and may ; diekmann et al. ; lotz et al. ) . it is calculated as the mean life span of an infection times the number of transmissions (equivalent to births) that would occur over that infectious period (mollison ) . it is very much the population growth rate of an infection and is analogous to the net reproductive rate (r ) of free-living organisms. r for both free-living and symbiotic organisms indicates population growth and therefore reproductive success (fitness). and as such, it should be maximized by natural selection. for simplicity, we can represent transmission as β and life span as the reciprocal of the virulence α or pathogen-induced mortality. in this case, r ¼ β α and r increases as the infected hosts live longer (α decreases), and the infection is highly transmissible (β increases). however, there often occurs a trade-off between transmission and virulence. for example, in the case of tsv, the greater the load or intensity of the virus in a shrimp host, the greater the transmission (β increases). however, the greater the viral intensity, the greater the chance of mortality of the host (α increases) (lotz ) . therefore, the trade-off is mediated by viral intensity. as a result, a balance exists between infectivity and virulence, and the maximum r is obtained at intermediate levels of virulence (fig. . ). this much of the theory assumes that the source of new infections is living infected hosts and that the death of the host ends infectivity (ewald ; hochberg ) . what if transmission occurs not so much from living infected hosts but from hosts after they die? this is likely to be the case for several shrimp pathogens. soto and lotz ( ) and lotz et al. ( ) demonstrated for wssv and for tsv that transmission from dead infected shrimp is considerably greater than from living infected shrimp. this indicates that the transmissibility of a shrimp pathogen does not end with the life of the shrimp. if transmission occurs from dead infected hosts, then r is determined by the time to death of an infected host and the length of time that a dead infected host remains infectious. the infectious time of a dead shrimp depends on two factors, the ingestion of dead shrimp by other shrimp and the decay of the carcass (soto and lotz ; lotz et al. ) . the rate at which dead shrimp infectivity declines, whether by cannibalism or by carcass decay, is unrelated to the viral load; however, transmission rate (β) is. in this case, the load does not affect the time of infectivity of a dead infected shrimp, and the trade-off between load and time of infectivity disappears. in fact, increased virulence in live infected hosts causes a high pathogen load in dead animals and therefore increased infectivity of dead hosts without graph of relationship between two sources of infection (or two habitats) or two hosts assessed as pathogen-induced mortality. curve a is the relationship between r virulence when the source is a living host; b is the relationship between r and virulence when the source is a dead host. which curve obtained depends on the relative contribution of both living and dead hosts to the overall r affecting the time of infectivity. so as virulence increases, r also increases, and we expect virulence to increase over time if transmission is from dead shrimp ( fig. . ). lotz ( ) did not consider the outcome if both living and dead hosts contribute; however, overall r is β a α þ β d δ , where β a is the transmission rate from a living host, β d is the transmission rate from dead hosts, α is the virulence of a pathogen to the living hosts, and δ is the infectivity decay of a dead infected host. the relative contributions of β a α and β a δ to r are what will determine the final virulence and whether or not virulence will increase or decrease over evolutionary time. although the above reasoning and fig. . have been applied specifically to tsv and living and dead shrimp, they apply more generally to any two states that contribute to an overall r . the two states instead can be two habitats or two species of host. the state (habitat or species) that contributes the greatest to r will dominate setting the optimal virulence. for species of symbiont that have both free-living and symbiotic relationships, the free-living habitat will contribute to the net reproductive rate (r ), and the symbiotic habitat will contribute to the basic reproduction number (r ). the two r s contribute to the overall growth (and thereby fitness) of the symbiont. if the two states represent different host species, then the host species that is responsible for the greatest contribution to the basic reproduction number will predominate. the conclusion is that what happens in the lesser contributing state will not be as important, and therefore very high virulence could be obtained in a host with little contribution to fitness of the symbiont. the observed virulence is a coincidental by-product of the adaptation to other habitats (brown et al. ; 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characterization of shrimp baculovirus pattern of infection of gammarus aequicauda (amphipoda) with metacercariae of levinseniella tridigitata (trematoda: microphallidae) intestinal parasite guidelines for domestic medical examination for newly arrived refugees experimental infection of contracaecum multipapillatum (nematoda: anisakinae) from mexico in the domestic cat nudiviruses and other large, double-stranded circular dna viruses of invertebrates: new insights on an old topic whole-proteome phylogeny of large dsdna virus families by an alignment-free method acknowledgments we thank janet wright, jean jovonovich, and kim overstreet for assistance. some funding was provided by a grant from bp exploration & production, inc. key: cord- -hobh akq authors: king, k c; lively, c m title: does genetic diversity limit disease spread in natural host populations? date: - - journal: heredity doi: . /hdy. . sha: doc_id: cord_uid: hobh akq it is a commonly held view that genetically homogenous host populations are more vulnerable to infection than genetically diverse populations. the underlying idea, known as the ‘monoculture effect,' is well documented in agricultural studies. low genetic diversity in the wild can result from bottlenecks (that is, founder effects), biparental inbreeding or self-fertilization, any of which might increase the risk of epidemics. host genetic diversity could buffer populations against epidemics in nature, but it is not clear how much diversity is required to prevent disease spread. recent theoretical and empirical studies, particularly in daphnia populations, have helped to establish that genetic diversity can reduce parasite transmission. here, we review the present theoretical work and empirical evidence, and we suggest a new focus on finding ‘diversity thresholds.' it seems to be conventional wisdom that genetically homogeneous populations suffer from more severe pathogen outbreaks than diverse populations (elton, ; sherman et al., ; schmid-hempel, ; altizer et al., ) . infection is more likely to be transmitted between genetically similar hosts (anderson and may, ) , and upon encountering resistant hosts, parasites would likely die, fail to successfully reproduce (anderson and may, ) , or otherwise be removed from the population (keesing et al., ) . as such, the risk of infection, especially by virulent pathogens, may select for outcrossing over uniparental forms reproduction, such as self-fertilization or parthenogenesis king et al., ; morran et al., ) , resulting in an overall increase in genetic diversity (king et al., ) . there may not seem to be an escape from disease, but genetic diversity in host populations may reduce the risk of infection. the empirical link between disease spread and genetic diversity has its origin in agricultural research. agricultural fields represent environments in which plants are selected for high yield, and may therefore exhibit less genetic polymorphism than those in the wild. it is well known that disease epidemics have devastated agricultural monocultures (for example, rice blast, zhu et al., ) , and that monocultures are typically more susceptible to outbreaks than diverse mixtures of crops (mundt, ) . this association between low diversity and high disease incidence is called the 'monoculture effect' (for example, elton, ; leonard, ; garrett and mundt, ; zhu et al., ; pilet et al., ) . here, we examine how genetic diversity affects the spread of disease in natural populations. we also consider the theoretical basis for the monoculture effect in host populations, and suggest that there may be a 'diversity threshold' for disease spread. the most notable examples of the monoculture effect in natural populations come from the dramatic epidemics in small populations of endangered species, particularly mammals (for example, o'brien et al., ; thorne and williams, ) . genetic bottlenecks have reduced the genetic variability in cheetahs, particularly at the major histocompatibility complex, and endangered populations of these animals have been decimated by coronavirus epizootics (o'brien et al., ) . similarly, the endangered black-footed ferret may have been extirpated from its natural habitat, because low genetic diversity aided the spread of a virulent canine distemper epizootic (thorne and williams, ) . range expansion and habitat isolation caused by human activities can also generate small, founder populations with low genetic diversity. newly-colonised, western populations of italian agile frogs (rana latastei) are genetically depauperate, and they have an increased susceptibility to a novel, emerging ranavirus (pearman and garner, ) . similarly, dam construction in the senegal river basin likely permitted the rapid expansion of snails (biomphalaria pfeifferi ), which serve as the first-intermediate host for the human disease schistosomiasis (campbell et al., ) . recently established biomphalaria snail populations are genetically homogenous compared with those in natural habitats in zimbabwe; they are also more susceptible to infection and this increases the opportunity for parasite transmission to humans (campbell et al., ) . by reducing individual-level and population-level genetic heterozygosity, inbreeding can increase host susceptibility to infectious parasites (dwyer et al., ; acevedo-whitehouse et al., ; spielman et al., ; ellison et al., ) . in wild populations, acevedo-whitehouse et al. ( ) found that heavily inbred california sea lions were more infected, and consequently, may act as pathogen reservoirs, spreading parasites in sea lion populations. but, is increased susceptibility directly due to reduced diversity, or does inbreeding depression have an effect by compromising host condition? by manipulating the levels of inbreeding in drosophila melanogaster, in laboratory experiments spielman et al. ( ) confirmed that the increase in disease susceptibility resulted from a lower frequency of resistance alleles in the population, and not by generalized inbreeding effects. inbreeding effects were similarly excluded as a reason for why offspring from inbred populations of daphnia, a freshwater crustacean, were more susceptible to a vertically-transmitted parasite than those from outbred populations (ebert et al., ) . finally, kerstes and wagner ( ) found that inbreeding increased parasite-induced mortality in the red flour beetle (tribolium castaneum) by prolonging development time, but it did not increase susceptibility to infection. founder populations provide the opportunity to examine the effects of inbreeding and small population size on the link between genetic diversity and parasite resistance. for example, in a comparison of founder versus ancestral mainland populations of deer mice, meagher ( ) found that inbred, island populations in lake michigan had higher infection levels and lower genetic diversity. the link between diversity and parasite spread has even been revealed when comparing large and small founder populations. hawks colonizing smaller galápagos islands possessed lower genetic diversity, produced low antibody titer, and had a higher abundance of parasites than more outbred populations on larger islands (whiteman et al., ) . uniparental forms of reproduction, such as self-fertilization or parthenogenesis, should have similar consequences for parasite resistance as biparental inbreeding. along these lines, self-fertilizing populations and inbred sexual populations were both found to have higher infection rates by a trematode parasite compared with outbred sexual populations of topminnows (poeciliopsis monacha) (lively et al., ) . in another species of partially-selfing fish (kryptolebias marmoratus), ellison et al. ( ) found that outcrossing increased the genetic diversity of wild populations and decreased their susceptibility to multiple parasites. mating systems can also directly affect genetic diversity and parasite resistance (busch et al., ; williams et al., ) . for example, in eusocial insect colonies, queens can mate with a single male or with multiple males (polyandry), which determines the level of relatedness among individuals within a colony. high relatedness among individuals in a population can enhance the evolution of cooperation (hamilton, a (hamilton, , b, , but the genetic similarity between individuals may also facilitate the spread of parasites (shykoff and schmid-hempel, ; schmid-hempel, ) . as such, multiple mating has been suggested as an evolutionary response to parasite pressure (hamilton, ; sherman et al., ) , which may counteract the high risk of parasite transmission and increase the overall productivity of the colony (schmid-hempel, ; schmid-hempel and crozier, ; brown and schmid-hempel, ) . numerous studies on ants and bees have indeed found that multiply mated queens form more resistant colonies schmid-hempel, , ; tarpy, ; hughes and boomsma, ; tarpy and seeley, ; seeley and tarpy, ) , and that offspring fathered by different males do vary in susceptibility to infection (baer and schmid-hempel, ) . ultimately, the balance between the costs (for example, within-colony conflict, reduced offspring output) and benefits (for example, resistance to a range of parasites) associated with heterogeneity in colonies may determine the optimal level of polyandry (baer and schmid-hempel, ; van baalen and beekman, ) . polygyny (the presence of multiple queens within a single colony) can also decrease the relatedness among individuals and increase the variety of resistance alleles in the colony. studies in which the number of founding queens was experimentally manipulated have confirmed that colonies founded by multiple queens have lower parasite loads (liersch and schmid-hempel, ; reber et al., ) . in addition, workers from polygynous colonies of an ant (cardiocondyla obscurior) were better at detecting disease and removing infected individuals from the nest than workers from nests having a single queen (ugelvig et al., ) . increases in infection prevalence in the wild is associated with genetic bottlenecking and inbreeding, induced by founder effects or mating systems. thus, host population genetic diversity seems to have an important role in buffering populations against epidemics. but, how exactly does genetic diversity reduce disease spread? how much diversity is necessary? an association between genetic diversity and disease spread might be detected by categorizing populations as being either genetically homogenous or diverse. this comparative method can tell us that diversity matters, but does not indicate the amount of genetic diversity required for a population to be resistant, or the 'diversity threshold' . recently, two insightful empirical studies have quantified the effect of genetic diversity on resistance in host populations. altermatt and ebert ( ) and ganz and ebert ( ) conducted semi-natural mesocosm and lab experiments, respectively, whereby monoclonal and polyclonal daphnia populations were exposed to microparasites. parasites spread significantly faster (altermatt and ebert, ) and infection rates are higher (ganz and ebert, ) in host monocultures compared with 'polycultures' of several genotypes with higher allelic diversity. these studies suggest that the relationship between host diversity and infection may not be complex, and that a 'handful' of host genotypes in the population can be enough to hamper parasite transmission. the benefits of host genetic diversity, however, may also depend on the genetic diversity of the parasite population (boomsma and ratnieks, ; van baalen and beekman, ; ganz and ebert, ) . if the parasite population is genetically homogenous, increases in host population genetic diversity might boost the opportunity for parasites to encounter a susceptible host (boomsma and ratnieks, ; van baalen and beekman, ) . alternatively, in a diverse parasite population, there is a high probability that one of a diverse set of parasite genotypes can infect a homogeneous host population (van baalen and beekman, ) and genetically diverse host populations are at an advantage. consistent with these ideas, ganz and ebert ( ) found no difference in infection levels among experimental daphnia monocultures and polycultures when populations were exposed to a single-parasite genotype; however, polycultures were more resistant when populations were exposed to multiple parasite genotypes. there has been surprisingly little theoretical work on the effect of genetic diversity on disease spread. two models suggest that genetic variation in host susceptibility would not affect infectious disease spread (springbett et al., ; yates et al., ) , but it might reduce the severity of infection (springbett et al., ) . in these models, hosts varied in susceptibility to a single pathogen strain, but no host genotype was completely resistant to infection. in contrast, lively ( a) found that host genetic diversity could reduce the risk of disease spread, assuming that each host genotype was susceptible to a different parasite genotype. this assumption is consistent with the 'matching-alleles' model for infection (frank, ; otto and michalakis, ) . the matching-alleles model is a useful framework for studying self/non-selfrecognition systems in animals, and it is supported by studies on invertebrate immunity (grosberg and hart, ; carius et al., ; dybdahl et al., ; duneau et al., ; luijckx et al., ) . in addition, the framework seems robust to the assumption of singlegenotype specificity (agrawal and lively, ; engelstaedter and bonhoeffer, ) . the more recent model suggests that increases in the genetic diversity of host populations could have a large effect on disease spread and prevalence at equilibrium (lively, a) . the model assumes that there are no co-infections, and that each parasite genotype can only infect one genetically determined resistance phenotype in the host population, which is the standard assumption of the matching-alleles model for infection. the results suggest that r for each parasite genotype i depends on total host density, as well as the frequency of the matching host genotype, where matches between host and parasite genotypes yield an infection. thus, disease transmission is both density and frequency dependent. the effect of host density on r is asymptotic on bg i , where g i is the frequency of the matching host genotype, and b is the number of parasite propagules produced by each infection that make contact with a host (lively, a) . thus, b is equal to the total number of propagules produced by an infection multiplied by the frequency of propagules that contact a host. for large host populations (n ) the effect of further increasing the host population size has little effect; but, increasing the number of host genotypes has a large effect, because increasing the number of host genotypes decreases the frequency (g i ) of each genotype. under parasite-mediated, frequency-dependent selection, the frequency of each host genotype would be expected on average to be approximately /g, where g is the total number of host genotypes in the population. under these conditions, r for large populations is approximately b/g. this result suggests that experimentally doubling the number of host genotypes in the population would reduce r by one half, and that this would be roughly true even if the experiment increased the total number of hosts in the population (assuming the population is already large). the analytical results also suggest that the parasite would die out, following the addition of genetic diversity to the host population, provided that the frequency of each host genotype declines to less than /b. we used computer simulations to examine the gist of these ideas. the simulations assumed a haploid host with two loci coding for resistance. each locus could have up to three alleles for a total of nine different genotypes. each of the host genotypes could be infected by one of nine different parasite genotypes, consistent with the matching-alleles model for infection. in these simulations, birth rates of the host were density dependent, and infection reduced the intrinsic birth rate by % (the parameters where chosen for illustrative purposes). the details of the simulation are given in lively ( b) . we began the simulation with two alleles at one locus and three alleles at the other locus, giving six possible host genotypes. an uninfected host population was initiated at carrying capacity (k ¼ ), where the number of hosts of each genotype was determined by randomly assigning allele frequencies at the two loci. at generation , one host of each genotype was introduced as infected. thereafter, the infected hosts of each genotype were introduced into the population with a probability of . per generation, to simulate immigration of infected individuals. we started by assuming b ¼ , meaning each infection produced nine propagules that contacted a host. from the analytical results we would expect under that the average value for r would be b/g ¼ / ¼ . (where g is the number of genotypes). as such, the pathogen would be expected to spread in the population, as was indeed the case (figure a) . after an initial oscillatory period, the gene frequency dynamics stabilized (figure a) , and the prevalence of infection also stabilized for the parameter values considered here (figure b) . r converged on the predicted value of . (figure c) . we then introduced a third allele at the second locus at generation , which increased the number of possible genotypes from six to nine. the allele quickly spread, as it conferred resistance on three of the nine possible genotypes for which there was no matching parasite genotype (figure a ). average r quickly dropped to below as the allele spread in the population (figure c) , and the frequency of infection declined sharply (figure b) . matching parasite genotypes were introduced into the population by migration (at a rate of . per genotype per generation), which lead to selection against the new allele after it became common, as well as to a slight increase in r (figure c ). this resulted in sufficient selection to equalize allele frequencies so that the frequency of each genotype approximated /g. at this point, r was equal to one (b/g ¼ / ¼ ), and parasite prevalence fell close to zero, but was maintained at a low level by immigration (figure b) . the point is that introducing a single novel allele in the host population increased the number of possible genotypes by %. this lead to virtual elimination of the parasite, as the prevalence of infection plummeted from about % to near zero. a reduction in prevalence occurred despite the fact that matching parasite genotypes were also introduced into the population, and that the host population size remained large (that is, close to individuals; results not shown). clearly, under the assumptions of the present model, small increases in allelic diversity can cause dramatic reductions in parasitism, even in very large host populations. in addition, elimination of the pathogen is not necessarily followed by a loss of genetic diversity in the host population, as the different host genotypes are selectively neutral in the absence of parasite pressure (figure a) . the available data and the model are consistent with the idea that genetic diversity in host populations can reduce the spread of disease. however, a practical question arises: would the beneficial effect of adding hosts with novel genotypes, in order to increase local genetic diversity, outweigh the positive effect of increasing population size on r ? a possible answer is also suggested by the model, which suggests that the effect of increasing population size on r shows diminishing returns with host density, such that r is asymptotic on b/g ( figure ). as such, while increasing host population size does strongly affect r in small host populations, it has a small effect in large host populations (figure ). this suggests that increasing genetic diversity can still reduce parasite prevalence, even though host population size is also increased. the results in figure suggest that boosting genetic diversity could overcome the effect of increasing host population size, even when the latter is increased by fourfold. critically, the diversity threshold does not work by simply reducing the population size of the individual genotypes. the threshold, in fact, was calculated by assuming an infinite host population size. rather it works by reducing the probability of successful infection by reducing the frequency of matching host genotypes for each parasite genotype. consistent with results from agricultural populations, the existing literature suggests that high genetic diversity could buffer host populations against disease spread. although observational studies from natural populations of vertebrates (for example, cheetahs, sea lions, fish and frogs) may have limitations, they strongly suggest that diversity matters, and their results are consistent with experimental studies on freshwater crustaceans and social insects. however, several questions remain: ( ) does a diversity threshold exist? in other words, can parasites be eliminated by increasing host genetic diversity above some threshold value? ( ) what are the relative effects of host density and host genetic diversity on disease spread? ( ) what are the effects of genetic diversity in the parasite population versus that of the host population? ( ) what is the heritability of parasite resistance in natural populations? very little is known about the heritability for resistance in natural populations. it should be high in populations where genetic diversity is maintained by parasite-mediated frequencydependent selection. these issues would be best addressed by data from natural populations. if parasites take hold or die out depending on how much host genetic diversity exists relative to the threshold, determining whether diversity thresholds exist in natural populations may have great value. this may be particularly helpful for conserving endangered species and mediating vector-human-parasite transmission. there were no data to deposit. figure the effect of increasing genetic diversity on r by adding new hosts. suppose we have hosts, with genotypes in equal frequency (point a). now suppose that we experimentally double the host population size, without affecting the genetic diversity (point b). we see an increase in r , but the increase is not large. now suppose we double the host population size, but also increase the number of genotypes from to (point c). we now see a large decrease in r , but it does not go below , so the disease can still spread. finally, suppose that we experimentally double the host population size, but we also increase the number of genotypes by fourfold (point d). now r not only deceases, but it goes below , so the disease will die out. redrawn from lively ( a). disease susceptibility in california sea lions infection genetics: gene-for-gene versus matching-alleles models and all points in between genetic diversity of daphnia magna populations enhances resistance to parasites rapid evolutionary dynamics and disease threats to biodiversity the invasion, persistence and spread of infectiousdiseases within animal and plant communities experimental variation in polyandry affects parasite loads and fitness in a bumble-bee unexpected consequences of polyandry for parasitism and fitness in the bumblebee bumblebee workers from different sire groups vary in susceptibility to parasite infection paternity in eusocial hymenoptera the evolution of female multiple mating in social hymenoptera evidence for maintenance of sex by pathogens in plants low genetic diversity in a snail intermediate host (biomphalaria pfeifferi krass, ) and schistosomiasis transmission in the senegal river basin genetic variation in a host-parasite association: potential for coevolution and frequency-dependent selection resolving the infection process reveals striking differences in the contribution of environment, genetics and phylogeny to host-parasite interactions host heterogeneity in susceptibility and disease dynamics: tests of a mathematical model hybrid fitness in a locally adapted parasite a short term benefit for outcrossing in a daphnia metapopulation in relation to parasitism best of both worlds? 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the contribution of genetic diversity to the spread of infectious diseases in livestock populations genetic diversity within honeybee colonies prevents severe infections and promotes colony growth lower disease infections in honeybee (apis mellifera) colonies headed by polyandrous vs. monandrous queens disease and endangered species: the black-footed ferret as a recent example rapid anti-pathogen response in ant societies relies on high genetic diversity the costs and benefits of genetic heterogeneity in resistance against parasites in social insects disease ecology in the galapagos hawk (buteo galapagoensis): host genetic diversity, parasite load, and natural antibodies dioecy, hermaphrodites and pathogen load in plants how do pathogen evolution and host heterogeneity interact in disease emergence? genetic diversity and disease control in rice we thank c buser, l delph, l morran, and e rynkiewicz for comments on the manuscript. the authors acknowledge financial support from the national science foundation (deb- to cml and j jokela) and a royal society newton international fellowship (kck). cml also gratefully acknowledges a fellowship at the wissenschaftskolleg zu berlin during the construction of this paper. the authors declare no conflict of interest. key: cord- -vg bwed authors: haagmans, bart l.; andeweg, arno c.; osterhaus, albert d. m. e. title: the application of genomics to emerging zoonotic viral diseases date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: vg bwed interspecies transmission of pathogens may result in the emergence of new infectious diseases in humans as well as in domestic and wild animals. genomics tools such as high-throughput sequencing, mrna expression profiling, and microarray-based analysis of single nucleotide polymorphisms are providing unprecedented ways to analyze the diversity of the genomes of emerging pathogens as well as the molecular basis of the host response to them. by comparing and contrasting the outcomes of an emerging infection with those of closely related pathogens in different but related host species, we can further delineate the various host pathways determining the outcome of zoonotic transmission and adaptation to the newly invaded species. the ultimate challenge is to link pathogen and host genomics data with biological outcomes of zoonotic transmission and to translate the integrated data into novel intervention strategies that eventually will allow the effective control of newly emerging infectious diseases. result in the emergence of new infectious diseases in humans as well as in domestic and wild animals. genomics tools such as high-throughput sequencing, mrna expression profiling, and microarray-based analysis of single nucleotide polymorphisms are providing unprecedented ways to analyze the diversity of the genomes of emerging pathogens as well as the molecular basis of the host response to them. by comparing and contrasting the outcomes of an emerging infection with those of closely related pathogens in different but related host species, we can further delineate the various host pathways determining the outcome of zoonotic transmission and adaptation to the newly invaded species. the ultimate challenge is to link pathogen and host genomics data with biological outcomes of zoonotic transmission and to translate the integrated data into novel intervention strategies that eventually will allow the effective control of newly emerging infectious diseases. most of the well-known human viruses persist in the population for a relatively long time, and coevolution of the virus and its human host has resulted in an equilibrium characterized by coexistence, often in the absence of a measurable disease burden. when pathogens cross a species barrier, however, the infection can be devastating, causing a high disease burden and mortality. in recent years, several outbreaks of infectious diseases in humans linked to such an initial zoonotic transmission (from animal to human host) have highlighted this problem. factors related to our increasingly globalized society have contributed to the apparently increased transmission of pathogens from animals to humans over the past decades; these include changes in human factors such as increased mobility, demographic changes, and exploitation of the environment (for a review see osterhaus [ ] and kuiken et al. [ ] ). environmental factors also play a direct role, and many examples exist. the recently increased distribution of the arthropod (mosquito) vector aedes aegypti, for example, has led to massive outbreaks of dengue fever in south america and southeast asia. intense pig farming in areas where frugivorous bats are common is probably the direct cause of the introduction of nipah virus into pig populations in malaysia, with subsequent transmission to humans. bats are an important reservoir for a plethora of zoonotic pathogens: two closely related paramyxoviruses-hendra virus and nipah virus-cause persistent infections in frugivorous bats and have spread to horses and pigs, respectively [ ] . the similarity between human and nonhuman primates permits many viruses to cross the species barrier between different primate species. the introduction into humans of hiv- and hiv- (the lentiviruses that cause aids), as well as other primate viruses, such as monkeypox virus and herpesvirus simiae, provide dramatic examples of this type of transmission. other viruses, such as influenza a viruses and severe acute respiratory syndrome coronavirus (sars-cov), may need multiple genetic changes to adapt successfully to humans as a new host species; these changes might include differential receptor usage, enhanced replication, evasion of innate and adaptive host immune defenses, and/or increased efficiency of transmission. understanding the complex interactions between the invading pathogen on the one hand and the new host on the other as they progress toward a new hostpathogen equilibrium is a major challenge that differs substantially for each successful interspecies transmission and subsequent spread of the virus. new molecular techniques such as high-throughput sequencing, mrna expression profiling, and array-based single nucleotide polymorphism (snp) analysis provide ways to rapidly identify emerging pathogens (nipah virus and sars-cov, for example) and to analyze the diversity of their genomes as well as the host responses against them. essential to the process of identification and characterization of genome sequences is the exploitation of extensive databases that allow the alignment of viral genome sequences and the linkage of these genomics data to those obtained by classical viral culture and serological techniques, and epidemiological, clinical, and pathological studies [ ] . extensive genetic analysis of hiv- , for example, has provided clues to the geography and time scale of the early diversification of hiv- strains when the virus emerged in humans. hiv- strains are divided into multiple clades, each of which has independently evolved from a simian immunodeficiency virus (siv) that naturally infects chimpanzees in west and central africa. current estimates date the common ancestor of hiv- to the beginning of the twentieth century [ ] . because zoonotic pathogens typically may cause variable clinical outcomes in human hosts that differ in age, nutritional status, genetic background, and immunological condition, deciphering the complex interactions between evolving pathogens and their hosts is a great challenge. the genome sequences of many host species have become available the last decade, and with them a range of novel tools are available to study virus-host interactions at the molecular level. this progress, together with advances in high-throughput sequencing technology and, not least, in (bio)informatics and statistics, allows us to analyze the ''genomewide'' networks of gene interactions that control the host response to pathogens. by comparing and contrasting the outcomes of infection with closely related pathogens in different but related host species, we can further delineate the various host pathways involved in the different outcomes. the power of this approach was nicely demonstrated for siv infection of various primate host species. natural reservoir hosts of siv do not develop aids upon infection, whereas non-natural hosts, such as rhesus macaques and pig-tailed macaques, when infected experimentally with siv, develop aids in a similar manner to hiv-infected humans. transcriptional profiling indicates that siv infection of these species produces a distinctive host response [ ] . siv-infected primates with symptoms of aids have a high viral load, immune activation, and loss of certain types of t cells, whereas sivinfected sooty mangabeys (the species from which hiv- is thought to have originated) have substantially lower levels of innate immune activation than the symptomatic primates, partly due to the production of less interferon-a by plasmacytoid dendritic cells in response to siv and other toll-like receptor ligands [ ] . identification of host factors that restrict hiv infection may aid the development of effective intervention strategies. below, we elaborate on two other examples of recent important zoonotic events that led to sustained virus transmission in the human host, and the role that genomics has played in the elucidation of their pathogenesis thus far. influenza is caused by rna viruses of the orthomyxoviridae family. whereas fever and coughs are the most frequent symptoms, in more serious cases a fatal pneumonia can develop, particularly in the young and the elderly. typically, influenza is transmitted through the air by coughs or sneezes, creating aerosols containing the virus; but influenza can also be transmitted by bird droppings, saliva, feces, and blood. birds and pigs play an important role in the emergence of new influenza viruses in humans. fecal sampling of migratory birds has revealed that they harbor a large range of different subtypes of influenza a viruses [ ] . some wild duck species, particularly mallards, are potential long-distance vectors of highly pathogenic avian influenza virus (h n ), whereas others, including diving ducks, are more likely to act as ''sentinel'' species that die upon infection [ ] . following the introduction of a new pandemic influenza a virus subtype from an avian reservoir, either directly or via another mammalian species such as the pig, the virus may continue to circulate in humans in subsequent years as a seasonal influenza virus. in the past century, three major influenza epidemics resulted in the loss of many millions of lives. spanish flu alone caused the deaths of more than million people by the end of world war i in . the outbreak of a new h n virus (causing ''swine flu'') that started in mexico further illustrates the pandemic potential of influenza a viruses. after introduction of a new influenza a virus from an avian or porcine reservoir into the human species, viral genomics studies are essential to identify critical mutations that enable the circulating virus to spread efficiently, interact with different receptors, and cause disease in the new host. for example, the importance of residue of the pb protein of the viral polymerase in determining species restriction has been demonstrated through these kinds of approaches [ ] . furthermore, changes in the hemagglutinin molecules may allow influenza a viruses to switch receptor specificity. the hemagglutinin of avian h n influenza viruses preferentially binds to oligosaccharides that terminate with a sialic acid-a- , -gal disaccharide, whereas the hemagglutinins of mammalian influenza a viruses prefer oligosaccharides that terminate with sialic acid-a- , -gal (figure ) . fatal viral pneumonia in humans infected with avian h n viruses is partly due to the ability of these viruses to attach to and replicate in the cells of the lower respiratory tract, which have oligosaccharides that terminate in sialic acid-a- , -gal disaccharide [ , ] . the sequence of the hemagglutinin protein may also affect its binding affinity for neutralizing antibodies. understanding the relationship between genetic diversity and antigenic properties of these viruses [ ] may help to predict the emergence of influenza viruses and to develop effective vaccines. microarray-assisted mrna expression profiling of emerging zoonotic viral infections, including influenza a virus, is used to phenotype the host response in great detail. by comparing mrna expression in individuals infected with an emerging virus to expression in individuals infected with a related established virus, researchers can generate a ''molecular fingerprint'' of the host response genes or pathways specifically involved in the oftenexuberant host responses to the emerging virus. by using genetically engineered influenza a viruses, a role for the nonstructural ns viral protein in evasion of the innate host response has been demonstrated [ ] . interestingly, the ns protein derived from the spanish h n pandemic influenza virus blocked expression of interferon-regulated genes more efficiently than did the ns protein from established seasonal influenza viruses [ ] . other genomics studies of genetically engineered influenza a viruses containing some or all of the gene segments from either the h n virus or the highly pathogenic avian influenza a virus (h n ), suggest that these highly pathogenic influenza viruses induce severe disease in mice and macaques through aberrant and persistent activation of proinflammatory cytokine and chemokine responses [ ] [ ] [ ] [ ] . application of genomics tools not only supports the elucidation of mechanisms underlying pathogenesis but may also help to identify leads for therapeutic intervention. in ferrets, h n infection induced severe disease that was associated with strong expression of interferon response genes including the interferon-cinduced cytokine cxcl . treatment of h n -infected ferrets with an antagonist of the cxcl receptor (cxcr ) reduced the severity of the flu symptoms and the viral titers compared to the controls [ ] , clearly demonstrating the potential of biological response modifiers for the clinical management of viral infections. the host evasion and evolution of influenza virus is further discussed in [ ] . coronaviruses (covs) primarily infect the upper respiratory and gastrointestinal tract of mammals and birds. five different currently known covs infect humans and are believed to cause a significant percentage of all common colds in human adults. surprisingly, recent studies revealed that approximately % of bats sampled in china were positive for covs [ ] . subsequent phylogenetic studies revealed that bat covs that resembled human sars-cov clustered in a putative group comprising one subgroup of bat covs and another of sars-covs from humans and other mammalian hosts. according to the current hypothesis sars-cov has arisen by recombination between two bat viruses. phylogenetic analysis of sars-cov isolates from animals indicate that the resulting bat virus was transmitted first to palm civets (paguma larvata), a wild cat-like animal hunted for its meat, and subsequently to humans at live animal markets in southern china [ ] . genome analyses have provided evidence that genetic variation in the spike gene of these viruses from civets is associated with increased transmission of the virus [ ] . in addition, species-tospecies variation in the sequence of the gene angiotensin-converting enzyme (ace ), which encodes the sars-cov receptor, also affects the efficiency by which the virus can enter cells [ ] . by a combination of phylogenetic and bioinformatics analyses, chimeric gene design, and reverse genetics-aided generation of viruses that encode spike proteins of diverse isolates, researchers have reconstructed the events that led to the emergence of a virus able to spread efficiently in humans [ ] . structural modeling predicted that the sars-cov that caused the epidemic had an increased affinity for both civet and human ace receptors due to adaptation (figure ) . subsequent functional genomics studies of these viruses in diverse species provided further insight into the role of specific host genes involved in the pathogenic response [ , ] . the pathological changes observed in the lungs are initiated by a disproportionate innate immune response, illustrated by elevated levels of inflammatory cytokines and chemokines, such the hemagglutinin of avian influenza a viruses (blue) preferentially bind to oligosaccharides that terminate in sialic acid-a- , -gal (red), whereas the hemagglutinin on human influenza a viruses (green) prefer oligosaccharides that terminate in sialic acid-a- , -gal (orange). fatal viral pneumonia in humans infected with the h n subtype of avian influenza a viruses is likely due to the ability of these viruses to attach to and replicate in the lower respiratory tract cells, which have sialic acid-a- , -gal terminated saccharides. the horizontal arrows indicate interspecies transmission, including the transmission from an avian or porcine reservoir into the human species. image credit: bart as cxcl (ip- ), ccl (mcp- ), interleukin (il)- , il- , il- , il- b, and interferon-c [ ] . these clinical data were confirmed experimentally by demonstrating that sars-cov infection of diverse cell types induces a range of cytokines and chemokines, thus providing a conceptual framework for sars-cov pathogenesis. host genome expression analyses of various animal hosts and humans with different outcomes of infection indicated differential activation of innate immune genes in, for example, aged subjects compared to young subjects. importantly, treatment of aged macaques with pegylated interferon-a (i.e. interferon-a covalently modified with polyethylene glycol polymer chains, to enhance its bioavailability) reduced sars-cov replication and pathogenic responses [ ] . thus, host genomics analysis may provide markers of pathogenesis and leads for therapeutic intervention, as in this example of sars-cov infection. rapid identification of newly emerging viruses through the use of genomics tools is one of the major challenges for the near future. in addition, the identification of critical mutations that enable viruses to spread efficiently, interact with different receptors, and cause disease in diverse hosts through, for instance, enhanced viral replication or circumvention of the innate and adaptive immune responses, needs to be further expanded. although microarrayassisted transcriptional profiling can provide us with a wealth of information regarding host genes and gene-interacting networks in figure . zoonotic transmission of sars-cov. genomic analyses provided evidence that genetic changes in the spike gene of sars-cov from bats (left) and civet cats (center) are essential for the animal-to-human transmission (horizontal arrows). species-to-species genetic variation in the (thus far unidentified) viral receptor in bats and in the angiotensin converting enzyme (ace ) gene, encoding the sars-cov receptor in civet cats and humans also affects the efficiency with which the virus can enter cells (vertical arrows). the sars-cov that caused the epidemic evolved a high affinity for both civet (center) and human (right) ace receptors (indicated by the single diagonal and the right side vertical arrow). virus-host interactions, future research should focus on combining data obtained in different experimental settings. therefore, the careful design of complementary sets of experiments using different formats of virus-host interactions is absolutely needed for successful genomics studies [ ] . special attention should be addressed to the comparative analysis of the host response in diverse animal species. thus far a limited number of laboratory animal species has been studied, but the recent elucidation of the genome of several other animal species will provide tools to decipher the virus-host interactions in the more relevant natural host. recent developments in the sequencing of the rna transcriptome may aid this development. ultimately, microarray technology may also extend to genotyping of the human host by snp analysis, to identify markers of host susceptibility and severity of disease, that can be used in tailor-made clinical management of disease caused by emerging infections. comparative analysis of host responses to emerging viruses may also point toward a similar dysregulated host response to a range of emerging virus infections, enabling the rational design of multipotent biological response modifiers to combat a variety of emerging viral infections. by focusing on broad-acting intervention strategies rather than on the discovery of a newly emerging pathogen that is not characterized yet, we may be able to protect ourselves from several unexpectedly emerging infections with the same clinical manifestations. this approach may readily reduce the burden of disease and time will be gained to design preventive pathogen specific intervention strategies such as antiviral therapy or vaccination. clearly, for all stages of combating emerging infections, from the early identification of the pathogen to the development and design of vaccines, application of sophisticated genomics tools is fundamental to success. catastrophes after crossing species barriers public health. pathogen surveillance in animals henipaviruses: emerging paramyxoviruses associated with fruit bats viruses and koch's postulates direct evidence of extensive diversity of hiv- in kinshasa by transcriptional profiling in pathogenic and non-pathogenic siv infections reveals significant distinctions in kinetics and tissue compartmentalization divergent tlr and tlr signaling and type i interferon production distinguish pathogenic and nonpathogenic aids virus infections spatial, temporal, and species variation in prevalence of influenza a viruses in wild migratory birds wild ducks as long-distance vectors of highly pathogenic avian influenza virus (h n ) molecular basis for high virulence of hong kong h n influenza a viruses h n virus attachment to lower respiratory tract haemagglutinin mutations responsible for the binding of h n influenza a viruses 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diversity of coronaviruses in bats from china cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human receptor and viral determinants of sars-coronavirus adaptation to human ace mechanisms of zoonotic severe acute respiratory syndrome coronavirus host range expansion in human airway epithelium early upregulation of acute respiratory distress syndrome-associated cytokines promotes lethal disease in an aged-mouse model of severe acute respiratory syndrome coronavirus infection functional genomics highlights differential induction of antiviral pathways in the lungs of sars-cov-infected macaques genomic analysis reveals age-dependent innate immune responses to severe acute respiratory syndrome coronavirus pegylated interferon-alpha protects type pneumocytes against sars coronavirus infection in macaques virogenomics: the virus -host interaction revisited key: cord- -nquov i authors: murphy, f.a. title: epidemiology of human and animal viral diseases date: - - journal: encyclopedia of virology doi: . /b - - . - sha: doc_id: cord_uid: nquov i viral disease epidemiology is the study of the determinants, dynamics, and distribution of viral diseases in populations. the risk of infection or disease in a population is determined by characteristics of the virus, the host, and the host population, as well as behavioral, environmental, and ecological factors that affect virus transmission from one host to another. viral disease epidemiology has come to have a major role in clarifying the etiologic role of particular viruses and viral variants as the cause of specific diseases, in improving our understanding of the overall nature of specific viral diseases, and in determining factors affecting host susceptibility and immunity, in unraveling modes of transmission, in clarifying the interaction of viruses with environmental determinants of disease, in determining the safety, efficacy, and utility of vaccines and antiviral drugs, and especially in alerting and directing disease prevention and control actions. information on incidence, prevalence, and morbidity and mortality rates contributes directly to the establishment of priorities for prevention and control programs, whether this involves vaccine or drug development and delivery, environmental and hygienic improvements, enhancement of nutritional status, personal or community behavior, agricultural and food processing enhancements, reservoir host and vector control, and international cooperation and communication. evs, they encode other types of defense molecules. among these are the inhibitor of apoptosis (iap) genes. the iap of amev has been well characterized and functionally inhibits apoptosis. a related amev gene that functions to inhibit apoptosis is a homolog of the baculovirus pan-caspase inhibitor, p . another novel protein expressed by amev is a cu-zn superoxide dismutase (sod) . although a number of the orthopoxviruses encode genes with homology to this class of sods, neither the vv or myxoma virus proteins are functional in that capacity, although they are present within the virion. the sod expressed by amev is functional as an sod but is not essential for virus growth in culture. the deletion of the sod gene from amev appears to have no effect on the growth of the virus in gypsy moth larvae. it is clear that this large subfamily of the family poxviridae provides a wealth of possible information about the basic mechanisms of the poxvirus lifecycle. there appear to be a number of interesting variations on the molecular details which define this overall family of viruses. there are clear similarities to the vertebrate poxviruses in virion morphology, double-stranded dna genome, cytoplasmic life cycle, and rna expression. yet the differences between the cvs and evs are significant and represent an area of research that has not been fully explored. the data that have been obtained from genomic sequencing has been essential to identifying some of the different proteins that are present in the evs, as well as identifying potentially missing homologs of vv proteins. it is important to note that there are large differences at the dna level between the two sequenced evs, indicating that there is probably a wide variety of unique features within the evs as a group. as more sequence information becomes available, the diversity of this family of viruses may become more evident. direct contact transmission involves actual physical contact between an infected subject and a susceptible subject (e.g., kissing, biting, coitus). epidemic major increase in disease incidence affecting either a large number of humans or animals or spreading over a large area. epidemiology the study of the determinants, dynamics, and distribution of diseases in populations. fomite an inanimate object that may be contaminated with virus and become the vehicle for transmission. herd immunity the immune status of a population that affects viral transmission rates. often used in describing the elimination of a virus from a population when there are too few susceptible hosts remaining to sustain a transmission chain. horizontal transmission the transfer of infectious virus from one human or animal to another by any means other than vertical transmission. iatrogenic transmission transmission via health care procedures, materials, and workers (e.g., physicians, nurses, dentists, veterinarians). incidence rate (or attack rate) a measure of the occurrence of infection or disease in a population over time -it refers to the proportion of a population contracting a particular disease during a specified period. mathematical model (epidemiological) a means to convey quantitative information about a host-virus interaction, such as an epidemic or an emerging disease episode, by the construction of a set of predictive mathematical algorithms. nosocomial transmission pertains to infections acquired while a patient, human or animal, is in hospital. prevalence rate the ratio, at a particular point in time, of the number of cases currently present in the population divided by the number of subjects in the population at risk; it is a snapshot of the occurrence of infection or disease at a given time. species jumping (or host range extension) referring to a virus that derives from an ancient reservoir life cycle in animals, but has subsequently established a new life cycle in humans or a different animal species and no longer uses, the original animal reservoir. transmission the process by which a pathogen is shed from one host and infects the next. vector-borne transmission involves the bites of arthropod vectors (e.g., mosquitoes, ticks, sandflies). vertical or transplacental transmission occurs from mother to fetus prior to or during parturition, either across the placenta, when the fetus passes through the birth canal, or via colostrum and milk. vertical transmission transmission of virus from parent to progeny through the genome, sperm, or ovum or extracellularly (e.g., through colostrum or across the placenta). zoonosis disease which is naturally transmitted to humans from an ongoing reservoir life cycle in animals or arthropods, without the permanent establishment of a new life cycle in humans. viral disease epidemiology is the study of the determinants, dynamics, and distribution of viral diseases in populations. the risk of infection or disease in a population is determined by characteristics of the virus, the host, and the host population, as well as behavioral, environmental, and ecological factors that affect virus transmission from one host to another. epidemiology attempts to meld these factors into a unified whole. the depiction of the interaction of factors favoring the emergence of a viral disease (figure ) , called 'the convergence model', is taken from the us institute of medicine study, microbial threats to health, emergence, detection and response (national academy press, ) . at the center is a box representing the convergence of factors leading to 'the black box', reflecting the reality that many unknown interactions are important virologically and epidemiologically. the foundations of epidemiology predate the microbiological and virological sciences, starting with hippocrates, the greek physician and father of medicine, who in the fourth century bc made important epidemiologic observations on infectious diseases. john snow is called the father of modern epidemiology because he developed excellent quantitative methods while studying the source of a cholera outbreak at the broad street pump in london in . snow was followed by william farr, who in the s advanced the use of vital statistics and clarified many of the principles of risk assessment and retrospective and prospective studies. their vision is reflected in the fast-changing science of epidemiology which is now supported by advanced computer technology, sophisticated statistical methods, and very sensitive and specific diagnostic systems. by introducing quantitative measurements of disease trends, epidemiology has come to have a major role in improving our understanding of the overall nature of disease and in alerting and directing disease control activities. epidemiology is also effective in ( ) clarifying the role of particular viruses and viral variants as the cause of disease, ( ) clarifying the interaction of viruses with environmental determinants of disease, ( ) determining factors affecting host susceptibility, ( ) unraveling modes of transmission, and ( ) field testing of vaccines and antiviral drugs. the comparison of disease experience between populations is expressed in the form of 'rates'. the terms 'incidence rate' and 'prevalence rate' are used to describe quantitatively the frequency of occurrence of infection or disease in populations. 'incidence rate' (also called attack rate) is defined as the ratio of new cases occurring in a population to the size of the population during a specified period of time. prevalence rate is the ratio of the total number of cases occurring in a population to the size of the population during a specified period of time. 'seroprevalence rate' relates to the occurrence of antibody to a particular virus in a population. because viral antibodies, especially neutralizing antibodies, often last a lifetime, seroprevalence rates usually represent cumulative experience with the virus. the term 'case-fatality rate' is used to indicate the percentage of subjects with a particular disease that die from the disease. all these rates may be affected by various attributes that distinguish one individual from another: age, sex, genetic constitution, immune status, pregnancy, nutritional status, and various behavioral and medical care and patient management parameters. the most widely applicable attribute is age, which may encompass immune status as well as various physiological variables. a viral disease is characterized as 'endemic' when there are multiple or continuous chains of transmission resulting in continuous occurrence of disease in a population over a period of time. 'epidemics' are peaks in disease incidence that exceed the endemic baseline or expected rate of disease. the size of the peak required to constitute an epidemic is arbitrary and is related to the background endemic rate and the anxiety that the disease arouses (e.g., a few cases of rabies is regarded as an epidemic, whereas a few cases of influenza is not). a 'pandemic' is a worldwide epidemic. a proper description of an outbreak of disease or an epidemic must include the parameters of 'person (or subjects in the case of animals), place, and time'. such descriptive information is a necessary first step in describing the occurrence, distribution, course, threat, and anticipated action response to the initial recognition of a cluster of cases of disease. much of the initial investigation called for rests in common sense, observational acuity, and an insightful 'index of suspicion'. much of the initial investigation has been termed 'shoe-leather epidemiology'. the trigger for such initial investigation is most often an astute clinician (physician or veterinarian) or an astute pathologist. there are two basic analytic techniques used to investigate relationships between cause and effect and to evaluate risk factors of disease. these are the 'case-control study' and the 'cohort study'. in the case-control study, investigation starts after the disease has occurred -it is a retrospective study, going back in time to determine causative events. although this kind of study does not require the creation of new data or records, it does require careful selection of the control group, matched to the test group so as to avoid bias. the retrospective case-control study lends itself to quick analysis and is relatively inexpensive to carry out. in the cohort study, the prospective study, investigation entails the gathering of new data to identify cause-effect relationships. this kind of study is expensive and does not lend itself to quick analysis as groups must be followed until disease is observed. however, when cohort studies are successful, proof of cause-effect relationship is often incontrovertible. the term 'molecular epidemiology' is used to denote the use of any of a large number of molecular biological methods in support of epidemiologic investigations. for example, with herpesviruses, restriction endonuclease mapping has provided a means of identification of unique viral genotypes -in an epidemiologic study recognized as the first based upon viral molecular characterization, the source of herpes simplex virus causing disease in a hospital newborn nursery was traced to one persistently infected nurse rather than any of several other possible shedders. with rotaviruses and bluetongue viruses, polyacrylamide gel electrophoresis of the segmented viral rna has been used epidemiologically, for example, to unravel outbreaks involving multiple viral variants. panels of monoclonal antibodies have been used to distinguish virus variants for epidemiologic purposes; they have been particularly useful in elucidating host-range and geographic variants of rabies virus. today, partial sequencing has become the most commonly used molecular epidemiologic methodology; partial sequencing of poliovirus isolates recovered from patients indicates whether they are wild type (even local or introduced wild type), attenuated vaccine type, or a vaccine type that has reacquired neurovirulence during human passage. partial sequencing of foot-and-mouth disease viruses can offer the same kind of geographic information of virus movement as has proved so useful in polio control and eradication programs, but because of political sensitivities in some countries a robust international reference laboratory system has not been established that could provide the same kind of practical disease control information as has been the case with polio. thus, with many human and animal viruses molecular epidemiologic studies are flourishing, but more such studies should lead to international reference laboratory systems to guide prevention and control actions. such studies are developing rapidly today to deal with the threat of a human pandemic of avian influenza, but there are many more viral diseases, especially animal diseases, in need of this kind of development. one of the landmarks in the history of infectious diseases was the development of the henle-koch postulates which established the evidence required to prove a causal relationship between a particular infectious agent and a particular disease. these simple postulates were originally drawn up for bacteria, but were revised in by rivers and again in by evans in attempts to accommodate the special problem of proving disease causation by viruses ( table ) . in many cases, virologists have had to rely on indirect evidence, 'guilt by association', with associations based on epidemiologic data and patterns of serologic positivity in populations. today, many aspects of epidemiologic investigation play roles, especially in trying to distinguish an etiological, rather than coincidental or opportunistic relationship between a virus and a given disease. for example, early in the investigation of human acquired immunodeficiency syndrome (aids), before its etiology was established, many kinds of viruses were being isolated from patients and many candidate etiologic agents were being advanced. prediction that the etiologic agent would turn out to be a member of the family retroviridae was based upon years of veterinary research on animal retroviruses and animal retroviral diseases. this prediction was based upon recognition of common biologic and pathogenetic characteristics of aids and animal retroviral diseases. this prediction guided many of the early experiments to find the etiologic agent of aids; later, after human immunodeficiency virus (hiv ) was discovered, its morphological similarity to equine infectious anemia virus, a prototypic member of the genus lentivirus, family retroviridae, was the key to unraveling confusion over the fact that the human virus killed host lymphocytes rather than transforming them as typical oncogenic retroviruses would do. ever since, this essence of comparative medicine has been guiding hiv/ aids research in many areas, including drug design, diagnostics, and vaccine development. hiv/aids epidemiologic research has often been intertwined with research on the several simian immunodeficiency viruses (sivs). seroepidemiology is useful in public health and animal health investigations and in research to determine the prevalence or incidence of particular infections, to evaluate control and immunization programs, and to assess past history when a 'new' virus is discovered. when paired serum specimens are obtained from individuals several weeks apart, the initial appearance of antibody in the second specimen or a rise in antibody titer indicates recent infection. similarly, the presence of specific immunoglobulin m (igm) antibody in single serum samples, indicating recent infection, may be used in seroepidemiologic studies. correlation of serologic tests with clinical observations makes it possible to determine the ratio of clinical to subclinical infections. because of advanced diagnostic/serologic methods, sentinel studies can yield many valuable data in timely fashion about impending disease risks. for example, sentinel chicken flocks are set out for the early detection of the presence of arboviruses such as west nile virus in the united states. these flocks are bled and tested weekly for the presence of virus or antiviral antibody; they provide an early warning of the levels of virus amplification that occur before epidemics. the immunogenicity, potency, safety, and efficacy of vaccines are first studied in laboratory animals, followed by small-scale closed trials, and finally in large-scale open trials. such studies employ epidemiologic methods, rather like those of the cohort (prospective) study. in most cases, there is no alternative way to evaluate new vaccines, and the design of trials has now been developed so that they yield maximum information with minimum risk and acceptable cost. viruses survive in nature only if they are able to be transmitted from one host to another, whether of the same or another species. transmission cycles require virus entry into the body, replication, and shedding with subsequent spread to another host. portals of virus entry into the body include the skin, respiratory tract, intestinal tract, oropharynx, urogenital tract, and conjunctiva. in some cases, viruses use a particular portal of entry because of particular environmental or host-behavior factors and in other cases because of specific viral ligands and host-cell receptors. in many cases, disruption of normal host-defense mechanisms leads to entry that might otherwise be thwarted; for example, papillomaviruses may enter the deep layers of the skin via abrasions, acid-labile coronaviruses may enter the intestine protected by the buffering capacity of milk, and influenza viruses may enter the lower respiratory tract because a drug has dampened cilial action of the respiratory epithelium. the exit of virus from an infected host is just as important as entry in maintaining its transmission cycle. all portals used by viruses to gain entry are used for exit. the table criteria for disease causation: a unified concept appropriate for viruses as causative agents of disease, based on the henle-koch postulates, and modified by a. s. evans important elements in virus shedding are virus yield (from the standpoint of the virus, the more shedding the better) and timeliness of yield (again, the earlier the shedding the better). viruses that cause persistent infections often employ remarkable means to avoid host inflammatory and immune responses so as to continue shedding. for example, the epidemiologically important shedding of herpes simplex viruses and that perpetuates the viruses in populations requires recrudescence of persistent ganglionic infection, centrifugal viral genomic transit to peripheral nerve endings, and productive infection of mucosal epithelium, all in the face of established host immunity. virus transmission may be 'horizontal' or 'vertical'. the vast majority of transmission is horizontal, that is, between individuals within the population at risk. modes of horizontal transmission of viruses can be characterized as direct contact, indirect contact, common vehicle, airborne, vector-borne, iatrogenic, and nosocomial. vertical or transplacental transmission occurs between the mother and her fetus or newborn. some viruses are transmitted in nature via several modes, others exclusively via one mode (see table ). 'direct contact transmission' involves actual physical contact between an infected subject and a susceptible subject (e.g., kissing, epstein-barr virus, the cause of mononucleosis, biting (e.g., rabies); coitus (sexually transmitted viral diseases)). indirect contact transmission occurs via 'fomites', such as shared eating utensils, improperly sterilized surgical equipment, or improperly sterilized non-disposable syringes and needles. 'common vehicle transmission' pertains to fecal contamination of food and water supplies (e.g., norovirus diarrhea). common vehicle transmission commonly results in epidemic disease. 'airborne transmission' typically results in respiratory infections (and less typically in intestinal infections), but these infections may also be transmitted by direct and indirect contact. airborne transmission occurs via large droplets and via very small droplet nuclei (aerosols) emitted from infected persons during coughing or sneezing (e.g., influenza) or from environmental sources. large droplets (> mm in diameter) settle quickly, but droplet nuclei evaporate forming dry particles (< mm in diameter) which remain suspended in the air for extended periods. droplets may travel only a meter or so while droplet nuclei may travel over much longer distances. 'vector-borne transmission' involves the bites of arthropod vectors (e.g., mosquitoes, ticks, and sandflies). 'iatrogenic transmission' involves health care procedures, materials, and workers (e.g., physicians, nurses, dentists, and veterinarians). 'nosocomial transmission' pertains to infections acquired while a patient, human or animal, is in hospital. 'vertical or transplacental transmission' occurs from mother to fetus prior to or during parturition. certain retroviruses are vertically transmitted in animals via the integration of viral dna directly into the dna of the germline of the fertilized egg. other viruses are transmitted to the fetus across the placenta; yet others are transmitted when the fetus passes through the birth canal. another vertical transmission route is via colostrum and milk. vertical transmission of a virus may or may not be associated with 'congenital disease' (i.e., disease that is present at birth) which may be lethal (and the cause of abortion or stillbirth) or the cause of congenital abnormalities. the herpesviruses, especially cytomegaloviruses, and rubella virus cause important congenital diseases in humans, and pestiviruses, such as bovine viral diarrhea virus, in animals. enteric infections are most often transmitted by direct contact and by fomites in a 'fecal-oral cycle' that may include fecal contamination of food and water supplies; diarrheic feces may also splash to give rise to aerosols (droplets and droplet nuclei). respiratory infections are most often transmitted by the airborne route or by indirect contact via fomites in a 'respiratory cycle', that is, virus is shed in respiratory secretions and enters its next host through the nares during inhalation. the respiratory cycle is responsible for the most explosive patterns of epidemic disease in humans and all domestic animal species. perpetuation of a virus in nature depends upon the maintenance of serial infections, that is, a chain of transmission; the occurrence of disease is neither required nor necessarily advantageous. infection without recognizable disease is called 'subclinical' or 'clinically inapparent'. overall, subclinical infections are much more common than those that result in disease. their relative frequency accounts for the difficulty of tracing chains of transmission, even with the help of laboratory diagnostics. although clinical cases may be somewhat more productive sources of virus than subclinical infections, because the latter do not restrict the movement of the infected host, they can be most important as sources of viral dissemination. in most acute infections, whether clinically apparent or not, virus is shed in highest titers during the late stages of the incubation period, before the influence of the host-immune response takes effect. persistent infections, whether or not they are associated with episodes of clinical disease, also play an important role in the perpetuation of many viruses in nature. for example, prolonged virus shedding can reintroduce virus into a population of susceptibles all of which have been born since the last clinically apparent episode of infection. this is important in the survival of rubella virus in some isolated populations. sometimes the persistence of infection, the production of disease, and the transmission of virus are dissociated; for example, togavirus and arenavirus infections may have little adverse effect on their reservoir hosts (arthropods, birds, and rodents), but transmission may be very efficient. on the other hand, the persistence of infection in the central nervous system, as with measles virus in subacute sclerosing panencephalitis (sspe), is of no epidemiological significance, since no infectious virus is shed from this site. the virulence of the infecting virus may directly affect the probability of its transmission. the classic example of this is rabbit myxomatosis. in australia, mosquito-borne transmission of myxoma virus was found to be most effective when infected rabbits maintained highly infectious skin lesions for several days before death. highly virulent strains of the virus were found to kill rabbits so quickly that transmission did not occur, and naturally attenuated strains were found to produce minimal lesions that healed quickly and did not permit transmission. virus strains at either extreme of this virulence spectrum were found not to survive in nature, but virus strains of intermediate virulence have circulated for many years. with most viruses, endemic or epidemic transmission leads to a level of immunity in the host population that affects or even interrupts further transmission. the 'herd immunity' effect is countered in some cases by viral antigenic variation. for example, influenza viruses undergo genetic variations ('shift' and 'drift') such that persons immune to previously circulating virus strains are susceptible to new strains. assessing these genetic changes is the main objective of laboratory-based surveillance programs, which in turn are the basis for decisions on the formulation of each year's influenza vaccine. it is self-evident that the long-term survival of a virus requires that it be continuously transmitted from one host to another. in general, for rapidly and efficiently transmitted viruses such as many respiratory viruses, local survival of the virus requires that the susceptible host population be very large. a virus may disappear from a population because it exhausts its potential supply of susceptible hosts as they acquire immunity to reinfection with the same virus. depending on duration of immunity and the pattern of virus shedding, the 'critical population size' varies considerably with different viruses and with different host species. the most precise data on the importance of population size in acute nonpersistent infections come from studies of measles. persistence of measles virus in a population depends upon a continuous supply of susceptible children. analyses of the incidence of measles in large cities and in island communities have shown that a population of about half a million persons is needed to ensure a large enough annual input of new susceptible hosts, by birth or immigration, to maintain measles virus in the population. because infection depends on respiratory transmission, the duration of epidemics of measles is correlated inversely with population density. if a population is dispersed over a large area, the rate of spread is reduced and the epidemic may last longer, so that the number of susceptible persons needed to maintain transmission chains is reduced. on the other hand, in such a situation a break in the transmission chain is much more likely. when a large proportion of the population is initially susceptible, the intensity of the epidemic builds up very quickly and attack rates are almost % ('virginsoil epidemic'). on the other hand, when measles vaccination programs are implemented properly the virus disappears completely from the regional population. because most viruses are host-restricted, most viral infections are maintained in nature within populations of the same or related species. however, there are a number of viruses that may have multiple hosts and spread naturally between several different species of vertebrate host, for example, rabies and eastern equine encephalitis viruses. the term 'zoonosis' is used to describe multiple-host infections that are transmissible from animals to man. the zoonoses, whether involving domestic or wild animals or arthropods, usually represent important problems only under conditions where humans are engaged in activities involving close contact with animals or exposure to arthropods. many viral zoonoses are caused by arboviruses. arboviruses have two classes of hosts, vertebrate and invertebrate. over arboviruses are known, of which about cause disease in humans and in domestic animals; some of these are transmitted by ticks, some by mosquitoes, and yet others by phlebotomine flies (sandflies) or culicoides spp. (midges). arthropod transmission may be 'mechanical', where the arthropod acts as a 'flying pin', or more commonly, 'biological', involving replication of the virus in the arthropod vector. the arthropod vector acquires virus by feeding on the blood of a viremic person or animal. replication of the ingested virus, initially in the arthropod's gut, and its spread to the salivary glands takes several days; the interval varies with different viruses and is influenced by ambient temperature. virions in the salivary secretions of the vector are injected into human or animal hosts during subsequent blood meals. most arboviruses have localized natural habitats in which specific receptive arthropod and vertebrate hosts are involved in the viral life cycle. vertebrate reservoir hosts are usually wild mammals or birds; humans are rarely involved in primary transmission cycles, although the exceptions to this generalization are important (e.g., venezuelan equine encephalitis, yellow fever, and dengue viruses). humans are in most cases infected incidentally, for example, by the geographic extension of a reservoir vertebrate host and/or a vector arthropod. ecological changes produced by human activities disturb natural arbovirus life cycles and have been incriminated in the geographic spread or increased prevalence of arbovirus diseases. from the time of william farr, who studied epidemic disease problems in the s, mathematicians have been interested in 'epidemic curves' and secular trends in the incidence of infectious diseases. with the development of computer-based mathematical modeling techniques, there has been a resurgence of interest in the population dynamics of infectious diseases. there has also been a resurgence in controversies surrounding the use of models; critics say 'for every model there is an equal and opposite model'. so, the proof of the value of models lies in their practical application, and in recent years there have been more and more successes. for example, when for counterterrorism reasons universal smallpox vaccination was being considered, models that showed that vaccine could be used effectively after rapid detection of a terrorism incident led to a decision to stockpile, but not widely use vaccine. as another example, when a foot-and-mouth disease epidemic raged in the united kingdom in , a model showed that only the most vigorous stamping-out campaign could get ahead of the movement of the virus across the country. the model, seeming eminently logical now, importantly provided the kind of veracity and political will needed to accelerate the stamping-out campaign. models may be used to determine ( ) patterns of disease transmission, ( ) critical population sizes to support the continuous transmission of viruses with short and long incubation periods, ( ) the dynamics of endemicity of viruses that become persistent in their hosts, and ( ) the variables in age-dependent viral pathogenicity. computer modeling also provides useful insights into the effectiveness of disease control programs. much attention has been given to modeling the future of the aids epidemic in the united states and the rest of the world. such models usually start with historical data on the introduction of the etiologic virus, hiv , proceed to the present stage of the epidemic where the disease has become well established in many countries and in fewer countries subject to prevention and treatment strategies, and then proceed to project its course into the future. during the first years of the aids epidemic in the united states, african countries, and then in asian countries, most models underestimated developing trends; more recently models have become more accurately predictive -but in many places more and more sobering. knowledge of the epidemiology and modes of transmission of infectious diseases is critical to the development and implementation of prevention and control strategies. data on incidence, prevalence, and mortality contribute directly to the establishment of priorities for prevention and control programs while knowledge of viral characteristics and modes of transmission are used in deciding prevention strategies focusing on vaccine development and delivery, environmental improvements, enhancement of nutritional status, improvement in personal hygiene, and behavioral changes. see also: disease surveillance; viral pathogenesis; zoonoses. prevalence of the disease is significantly higher in subjects exposed to the putative virus than in those not so exposed. . incidence of the disease is significantly higher in subjects exposed to the putative virus than in those not so exposed temporally, the onset of disease follows exposure to the putative virus, always following an incubation period a regular pattern of clinical signs follows exposure to the putative virus, presenting a graded response, often from mild to severe a measurable host-immune response, such as an antibody response and/or a cell-mediated response, follows exposure to the putative virus experimental reproduction of the disease follows deliberate exposure of animals to the putative virus, but nonexposed control animals remain disease free. deliberate exposure may be in the laboratory or in the field, as with sentinel animals elimination of the putative virus and/or its vector decreases the incidence of the disease prevention or modification of infection, via immunization or drugs, decreases the incidence of the disease control of communicable diseases manual, th edn mandell, douglas, and bennett's principles and practice of infectious diseases the epidemiology of viral infections veterinary virology fields virology evolution of viral diseases virus dynamics: mathematical principles of immunology and virology emerging microbial threats to health in the st century. institute of medicine/ national academy of sciences microbial threads to health, emergence, detection and response veterinary epidemiology general features all rights reserved. the lymphocryptoviruses (lcvs) present in old world nonhuman primates, including ebv-like viruses of chimpanzees and rhesus monkeys. these viruses share homologous sequences and genetic organization, and infect the b lymphocytes of their host species, resulting in the establishment of latent infection in vivo and transformation key: cord- - twm hmc authors: vourc’h, gwenaël; plantard, olivier; morand, serge title: how does biodiversity influence the ecology of infectious disease? date: - - journal: new frontiers of molecular epidemiology of infectious diseases doi: . / - - - - _ sha: doc_id: cord_uid: twm hmc over the past years, biodiversity has been reduced on an unprecedented scale, while new infectious diseases are emerging at an increasing rate. greater overall biodiversity could lead to a greater diversity of hosts and thus of pathogens. yet disease regulation – due to the buffering role of host diversity – is considered to be one of the services provided by biodiversity. in this chapter, we ask how biodiversity is linked to infectious disease. first, we investigate the influence of the biodiversity of pathogens. we highlight that the number of pathogen species is not well known but that new findings are facilitated by the rapid expansion of molecular techniques. we show that, although there is a trend to find higher pathogen richness toward the equator, identifying a global pattern between the richness of all pathogen species and their latitudinal distribution is challenging. we emphasize that pathogen intraspecific diversity is a crucial factor in disease emergence and allows pathogens to adapt to the selective pressures they face. in addition, the selective pressure acting on hosts due to parasite, and reinforced by parasite diversity within hosts seems to be a major evolutionary and ecological force shaping hosts biodiversity. second, we investigate how the diversity of hosts influences infectious disease ecology. for multi-host diseases, a change in host species richness or abundance can modify the dynamics of local infectious diseases by either reducing (“dilution effect”) or increasing (“amplification effect”) the risk of transmission to the targeted host species. the underlying hypothesis is that, the competence of reservoirs varies according to the host species. the dilution effect has been demonstrated mainly through theoretical work and there have been only few case studies. regarding the genetic diversity of host, an important issue is: to what extent does a reduction of this diversity impact the ability of the host population to response to infectious diseases? third, we rapidly examine the role of biodiversity in the treatment of infectious diseases. to conclude, we consider that the consequences of the loss of species biodiversity on infectious diseases is still largely unknown, notably due to the lack of knowledge on the dynamics of host-pathogen relationships, especially at the population and at the community level.. we highlight that work on multi-host/ ulti-pathogen systems should be fostered and that new approaches, such as metagenomic investigations that does not require a priori assumptions, are promising to describe a community of pathogens and their interactions. investigate how the diversity of hosts influences infectious disease ecology. for multi-host diseases, a change in host species richness or abundance can modify the dynamics of local infectious diseases by either reducing ("dilution effect") or increasing ("amplification effect") the risk of transmission to the targeted host species. the underlying hypothesis is that, the competence of reservoirs varies according to the host species. the dilution effect has been demonstrated mainly through theoretical work and there have been only few case studies. regarding the genetic diversity of host, an important issue is: to what extent does a reduction of this diversity impact the ability of the host population to response to infectious diseases? third, we rapidly examine the role of biodiversity in the treatment of infectious diseases. to conclude, we consider that the consequences of the loss of species biodiversity on infectious diseases is still largely unknown, notably due to the lack of knowledge on the dynamics of host-pathogen relationships, especially at the population and at the community level.. we highlight that work on multi-host/ ulti-pathogen systems should be fostered and that new approaches, such as metagenomic investigations that does not require a priori assumptions, are promising to describe a community of pathogens and their interactions. over the past years, human activity has altered habitats and reduced biodiversity on an unprecedented scale that comes close to mass extinction (mea ) . at the same time, new infectious diseases seem to be emerging at an increasing rate (wilcox and gubler ) . during this period, there has been a dramatic spread of highly pathogenic diseases such as aids and multi-drug resistant bacterial infections, and in more recent years sars, west nile in north america, and highly pathogenic influenza viruses (jones et al. ) . habitat loss, largely a result of the conversion of forests and savannas into agricultural land, cities, and industrial sites, is the major cause of change in biodiversity. biodiversity represents the diversity of life at all levels of biological organization, from the genes within populations, the species that compose a community, to the communities that compose ecosystems. intuitively, one might assume that greater overall biodiversity would lead to a greater diversity of pathogens and hosts, and thereby increase the incidence of infectious diseases (dunn et al. ). yet disease regulation is said to be one of the services provided by biodiversity because a high level of species diversity creates a buffer that reduces the risk of transmission (mea ; walpole et al. ). scientific evidence supporting both of these views is beginning to emerge, but the core question remains: how is biodiversity linked to infectious disease? this is the question addressed in this chapter. pathogens are organisms that have a negative impact on the fitness of their host(s), often, if now always, by producing visible symptoms (e.g. a disease). such trophic interaction between two organisms, a host and a parasite, is just one of several interactions that take place within communities and ecosystems, others being those of prey-predator and plant-phytophagous for instance (begon et al. ) . to date, more attention has been paid to these other interactions, and their roles in ecosystem functioning (e.g. steffan and snyder ) , than to pathogen-host interactions, and food web studies only recently have begun to take parasites into consideration (arias-gonzález and morand ; lafferty et al. ) . studies incorporating pathogens are scarce (hudson et al. ) , probably due to the difficulties of surveying pathogens (using intrusive or even destructive sampling methods…). moreover, the systematics and even basic aspects of parasite biology often are unknown. however, although numerous species of pathogens still need to be described (dobson et al. ) , there is no doubt that pathogens represent a large part of biodiversity on earth. given that each free living species is host to numerous pathogens, and that pathogens of pathogens also exist (consider, for example, phages that are virus affecting bacteria), several authors believe that pathogens may be the most diverse living group on earth (windsor ) . the link between biodiversity and the ecology of infectious diseases is not simple. in this chapter, we investigate how biodiversity influences the ecology of infectious diseases at the intraspecific level (genetic variability of pathogens and hosts) and at the level of communities (species composition). although we mainly provide examples from human and animal diseases, we also use some illustrations from plants. we describe patterns of biodiversity and the consequences of changes in biodiversity on the ecology of infectious diseases. lastly, we rapidly examine the role of biodiversity in the treatment of infectious diseases. infectious disease ecology? we shall consider infectious diseases caused by bacteria, virus, fungi, protozoa and endo-parasites, and exclude from our analysis ecto-parasites that are considered here as disease vectors. in the light of the discussion above, the pathogen status of a given living organism clearly is not a straightforward question (consider, for example, the case of some rickettsia species that are considered to be not only blood vertebrate pathogens, but also tick symbionts, perlman et al. ). this status is related to the host, and varies with individual hosts and species as well as in space and in time (different hosts, for example, can have different resistance or susceptibility). when pathogens have complex life-cycles, some stages may have a different biology (such as biotrophic or necrotrophic plant pathogens, morris et al. ). furthermore, horizontal gene transfer is so extensive in bacteria that many microbio logists question the existence of species in bacteria, preferring to consider bacteria as populations that exchange genes. however, the existence of core genes responsible for the maintenance of species-specific phenotypic clusters is an argument supporting the identification of bacterial species (riley and lizotte-waniewski ) . for these reasons, combined with the limited knowledge available of the systematics of many pathogens (brooks and hoberg ) , it is difficult to accurately estimate the number of pathogen species. estimations of pathogen species richness vary from % to % of living beings (de meeûs et al. ; poulin and morand ) . in estuaries, the biomass of macro and micro-parasites has been estimated as exceeding that of top predators . although the existence of pathogens has been known for a long time, lists of species only were compiled recently for human and animals (ashford and crewe ; cleaveland et al. ; taylor et al. ) , with an update on human pathogens completed in (woolhouse and gaunt ) . approximately , human pathogens were reported, livestock pathogens (cattle, sheep, goats, pigs and horses), and domestic carnivore pathogens (dogs and cats). no clear figure was given for wildlife (but see the global mammal parasite database at http://www.mammalparasites.org/). on average, over two new species of human viruses also are discovered each year (woolhouse et al. ) . pathogens affecting humans have received more attention than those affecting other species. if one assumes that other animal species are affected in a proportional manner, huge numbers of pathogens remain to be discovered. altogether, , , , and , of fungi, viruses and bacteria respectively have been described, which represent only %, %, and - % of the total estimated number of species of fungal, viral and bacterial species. it is difficult to know the number of plant pathogens, but a significantly proportion of the fungal, viral and bacterial species are likely to be plant pathogens (ingram ) . until recently, many new pathogen discoveries relied on the investigation of atypical symptoms. today, new findings are facilitated by molecular techniques that render it possible to detect and characterise unculturable pathogens and to investigate the presence of genes and genomes independently of individuals (metagenomics). although multi-host pathogens are more numerous than single hosts, interactions between pathogens and hosts can evolve towards the specialisation of pathogens on a given host species (cleaveland et al. ; huyse et al. ; pedersen et al. ) . such a specialisation can lead to speciation, id est the birth of a new pathogen species. co-cladogenesis, a process of parallel diversification in hosts and pathogens, also can give birth to numerous pathogen species (page ) . until the development of molecular tools, these species were very difficult to distinguish (cryptic species). systematic investigations using molecular tools have made it possible, however, to reveal a high diversity of pathogens. for instance, in a systematic inventory of viruses in various vertebrate hosts conducted over a year period in the central african republic, different viruses were isolated, including new ones (saluzzo et al. ) . two species of plasmodium, p. falciparum, infecting humans, and p. reichenowi, infecting chimpanzees, were long considered to be within the clade that includes humans and the great apes. however, recent studies of apes in their natural habitat have revealed a much higher diversity of species infecting great apes; in addition, it has been found that p. falciparum also infect gorillas (liu et al. ; prugnolle et al. ) and are at the origin of human malaria. metagenomic studies in ecosystems such as human faeces (zhang et al. ) and marine sediments (breitbart et al. ) also have revealed that the majority of viral sequences found did not match in the databanks. finally, new investigations have been launched to monitor people, animals and animal die-offs in areas where people have a high exposure to wildlife. generic, broad screening tools will be used to detect pathogen species (wolfe et al. ). to our knowledge, a similar approach has not yet been implemented for pathogens of animals or plants. in addition to the inventories of pathogen biodiversity, scientists have investigated which part of the world holds the highest diversity of pathogen species. many studies on plants and animals have shown that species richness decreases the further one moves away from the equator. the reasons most likely are linked to the area, energy, time and habitat heterogeneity, and geometric constraints (gaston and blackburn ) . comparative studies exploring pathogen species richness in the tropics compared to temperate zones are scarce and have produced discrepant results. guernier et al ( ) studied the worldwide distribution of human pathogens (bacteria, virus, fungi, protozoa, and helminths) according to environmental, demographic and economical factors. they found that parasite species richness decreased with latitude and had a spatially nested organization; i.e. some species are widely distributed and occur in many communities while others have more restricted distributions and occur only in a subset of locations. such findings were confirmed by the analysis of dunn et al. ( ) , who showed that human pathogen diversity was strongly related to both mammal and bird species richness. diseases that occur in temperate zones also tend to occur in the tropics, while some tropical diseases are restricted only to the tropics. in primates, the number of protozoa species, which primarily are vector-borne transmitted, increase as one approaches the equator, however, the same trend was not found for viruses and helminths (nunn et al. ). lindenfors et al ( ), who examined the parasite richness of parasite species and terrestrial carnivore species, found that helminth parasite species richness increased the further away one moved from the equator. the reason for this finding is unknown and may be related to a bias in sampling because carnivores inhabiting areas of industrialized countries in the northern hemisphere may have been sampled more intensely. poulin ( ) and bordes et al ( ) did not find any correlation between helminth species richness at intra or interspecific levels and latitude. some studies have shown higher tick species richness at lower latitudes (cumming ) . however, this is not the case for flea species, which have been found to have low richness at lower latitudes (krasnov et al. ) . a final example is ichneumonid parasitoid hymenoptera. although a higher specific host diversity is found in the tropics, the number of species of this parasitoid group is similar in both tropical and temperate regions. it has been hypothesised that this is due to habitat fragmentation (leading to a lower density of hosts); lack of seasonality (and thus of a host population dynamics with peaks and high density of hosts), or the higher content in toxic compounds of tropical plants and thus in phytophagous insects (the "nasty hypothesis") (gauld et al. ) . a meta-analysis of parasite-associated host mortality (robar et al. ) revealed that host mortality risk declined as one moves away from the equator, indicating that, in addition to parasitic load, the effect of parasites on host mortality might be determined by some abiotic factors. thus, although there is a trend to find higher pathogen richness as we move toward the equator, it is thus challenging to identify a global pattern between the richness of all pathogen species and their latitudinal distribution. however, it should be noted that of the pathogens that have been discovered since , most have a global distribution (woolhouse and gaunt ) . pathogens generally are characterised as having higher mutation rates and generation times than those of their hosts (hamilton et al. ). genetic variability also results from recombination during sexual reproduction of eukaryotic pathogens, and any other genetic exchange mechanisms such as bacterial conjugation or viral recombination. in addition, many animal and plant pathogens use a vector to increase gene flow among populations and to reach a new individual host. this genetic diversity is a crucial factor in disease emergence (cleaveland et al. ) and allows pathogens to adapt to the main selective pressures they face: hosts' immune systems, the need to be transmitted, and treatments or vaccines used to counter infections. the capacity of some pathogens to genetically diversify facilitates their ability to evade host immune systems. one of the best examples is the human immunodeficiency virus (hiv), which is able to change its appearance faster than the time its takes for the immune system to reply (drosopoulos et al. ). another example is p. falciparum, which generates high levels of variability in genes involved in antigenic variability and virulence (var genes) by producing frequent recombination events between heterologous chromosomes (freitas-junior et al. ) . high genetic variation of pathogens also is involved in the interspecies infection process as it facilitates the infection of a broader range of host species, which is another characteristic of emerging pathogens (cleaveland et al. ; woolhouse and gowtage-sequeria ) . the evolutionary potential of pathogens allows them to respond quickly to the directional selective pressure provided by the massive use of drugs (palumbi ) . in areas where selective pressure is important, such as in hospitals, multiresistant bacteria are very frequent (levy and marshall ) . for bacteria, resistant genes probably originated from environmental organisms with which they shared their ecological niche (aminov and mackie ) . these genes can be transferred between different species of bacteria and even between species that colonize different hosts (nikolich et al. ) . although vaccination is a major advance of modern medicine, it thus far has contributed to the eradication of only one infectious disease in humans (small pox, www.who.int/mediacentre/factsheets/smallpox/en) and one in cattle, buffalo and wildebeest (rinderpest, normile ) . as many vaccines do not totally block transmission, vaccination modifies the selective pressure on pathogens. depending on how vaccines act on the pathogen, the epidemiology consequences can differ (gandon and day ) . for instance, theoretical work has shown that vaccines that reduce the growth rate or toxicity of pathogens also reduce selection pressure against virulent pathogens, leading to higher intrinsic virulence (gandon et al. ). in the poultry industry, an increase in virulence of avian tumour viruses has followed the use of vaccines that reduce virus growth rates (witter ) . although plants lack an adaptive immune system, through evolution they have developed various strategies to stop plant pathogen infections. an induced or acquired systemic resistance occurs following host recognition of a pathogen, which triggers the production of a hypersensitive reaction (jones and dangl ) . through this mechanism, the plant provides itself protection from secondary infection in distal tissues, even if the plant faces a pathogen for which it does not have the resistance gene (durrant and dong ). the immune system of vertebrates acquires its efficiency by being exposed to a diverse array of pathogens. the striking increase in hygiene standards that began in the early twentieth century has considerably lowered humans' exposure to pathogens, at least in developed countries. the immune response triggered by a pathogen can provide some cross protection against other pathogens (e.g. huang et al. ) . a low exposure to a diversity of pathogens has had immediate consequences in decreasing the risk of disease. but could this low exposure also induce evolutionary change in the ability of a host to respond to infection? due to a trade off between investment in disease resistance and other traits linked to fitness, low exposure could decrease the frequency of resistance down through the generations, setting the stage for a potentially devastating outbreak (altizer et al. ; graham et al. ) . domestic species that are bred to be protected from pathogens might be more susceptible to infectious diseases (lyles and dobson ) . furthermore, it has been suggested that on islands, where some pathogens may be absent, hosts may have lower immune response abilities (island syndrome) (lee and klasing ) . however, studies that have tested this hypothesis, both using experimental and theoretical approaches, have had contrasting results (beadell et al. ; hochberg and møller ; matson ) . infections by different species of pathogens or by different strains of the same species within the same individual host or vector are common (abbot et al. ; cox ) . in fact, parasite diversity in hosts seems to be a major evolutionary and ecological force for hosts (bordes and morand ). these concomitant infections can trigger cross-effective immune responses between pathogens that are antigenically similar, having thus an impact on the issue of the infection (lee et al. ). an infection also can enhance susceptibility to subsequent infection (cattadori et al. ). in particular, individuals with already are in poor physical condition may be more susceptible to multiple infections (beldomenico and begon ; telfer et al. ) . furthermore, concomitant infection may allow the exchange of genetic material between strains of a given pathogen species or even between species through horizontal gene transfer (see sect. . . above), allowing the emergence of new virulent strains. an extreme case is one in which a pathogen drives the extinction of a population or species. such scenarios are rare but do occur, generally due to a conjunction of pathogens and other causes. for instance, the decline of amphibian populations around the world is thought to be linked to a fungal pathogen batrachochytrium dendrobatidis causing chytridiomycosis (crawford et al. ). amphibians could have an increased susceptibility to the fungus due to changes in temperature variability (rohr and raffel ) . another example is the dramatic decline of the native red squirrel in the uk that has been attributed to a combination of direct competition with the grey squirrel and disease-mediated competition because the grey squirrel is a reservoir host of the squirrelpox virus that causes disease in the red squirrel (tompkins et al. ) . the local extinction of a host also may have tremendous consequences on an entire ecosystem (see for example the case of the wildebeest /rinderpest interactions in the serengeti, holdo et al. ). disease ecology? a change in species richness or abundance can modify the dynamics of local infectious diseases by either reducing or increasing the risk of transmission to the targeted species. the first outcome has been named, the "dilution effect", the second, the "amplification effect". the term "dilution effect" has conveyed different meanings since its first use in disease ecology literature (see box in the paper keesing et al. ) . the broad definition of the dilution effect refers to "the phenomenon -the net effect -when increased species diversity reduces disease risk" that is produced by a variety of mechanisms ("amplification effect" refers to the opposite phenomenon) (keesing et al. ) . this applies to vector-borne and directly transmitted diseases, although the concept of dilution has been developed most with regards to the tick-borne lyme disease (allan et al. ; logiudice et al. logiudice et al. , . the hypothesis underlying the amplification and dilution effect is that for many diseases, the competence of reservoirs, i.e. the ability to become infected and retransmit the pathogen, varies according to the host species (haydon et al. ) . the composition of the host community thus can influence the transmission dynamic of the disease. similarly, since vectors have different competence to transmit pathogens, the composition of the vector community likely influences transmission dynamics. different mechanisms are thought to be involved, but they are difficult to differentiate (begon ; keesing et al. ) . one is the modification of the encounter rate (when reduced, this corresponds to the "dilution effect" sensu stricto). in the presence of species that are poorly competent, the transmission event that should link an infectious individual to a susceptible individual instead links infectious individuals to non-competent individuals. for vector-borne diseases, the increased diversity of a poorly competent host species on which the vector feeds increases the proportion of vector bites that are wasted. for direct transmission, the addition of non competent hosts can decrease transmission if these hosts remove infectious particles (begon ) . a second mechanism at work is that a high diversity of host species regulates the abundance of the competent host population. this regulation can be mediated by interspecific competition for limiting resources or by predation upon competent hosts. this typically is the idea that underlies biological controls, where organisms prey upon reservoir hosts, vectors or intermediate hosts (straub and snyder ) . a third mechanism is based on the link between species richness and host mortality. this is the case when predators modify the mortality rate of a host and lower pathogen transmission by feeding on a heavily diseased individual (packer et al. ) . two other mechanisms are cited by keesing et al ( ) , but they are difficult to demonstrate: (i) the modification of recovery when species added to a community facilitate the recovery of infected individuals by, for instance, providing resources, and (ii) the modification of transmission once the contact has occurred, for instance, when adding a species modifies the pathogen load within the host. the dilution effect has been demonstrated mainly through theoretical work; there have been few case studies. one of the main examples is lyme disease in the usa that is caused by pathogenic bacteria transmitted by ticks. these ticks feed readily on many species of vertebrates and these species vary in their degrees of reservoir competence. the white-footed mouse (peromyscus leucopus) is thought to be the most competent host and dominates in fragmented forests. in native forests, which harbour a higher diversity of species than fragmented forests, ticks have a higher probability to dilute their bite by feeding on a less competent host (allan et al. ; logiudice et al. logiudice et al. , . however, such a dilution effect has not been demonstrated in europe, probably because of the complexity of the disease ecology which involves numerous reservoir host and bacteria species (halos et al. ) . another example is the west nile virus, where an increased diversity of non passerine birds, which are less competent reservoir hosts compared to passerines, was associated with decreased west nile virus infection in mosquitoes and humans (ezenwa et al. ; swaddle and calos ) . to date, there have been few examples of directly transmitted diseases, but studies on hantaviruses have shown that higher diversity of small mammals appears to regulate reservoir host populations through competition or predation. high small-mammal diversity also might inhibit intraspecific aggressive encounters between reservoir hosts that result in hantavirus transmission (suzán et al. ). in plants, crop diversity can reduce the total burden of disease in agricultural systems. this results from the combined effects of (i) the limitation of pathogen dispersal thanks to the physical barriers provided by the presence of non-host plants (burdon and chilvers ) , (ii) induced systemic resistance, and (iii) competition among pathogens. the efficiency of crop mixtures is linked to the size of the area on which this method is used: a high level of success has been observed in a field trial with susceptible and resistant varieties of rice conducted on a large scale ( , ha) in china (zhu et al. ) . illustrations of amplification effects are typically the consequences of species introduction that radically modifies encounter rates. the added species can introduce new pathogens that infect native hosts (spillover) (bruemmer et al. ) or amplify the circulation of local pathogens (spillback) (kelly et al. ). the introduction of additional species also can provide sources of vector meals and increase vector numbers or activity (saul ) . for instance, the introduction of siberian chipmunks (tamias sibiricus) in suburban forests could increase the risk of lyme disease because this host seems to be more competent than native hosts (vourc'h et al. ). the introduction of the mosquito aedes albopictus in many parts of the world has facilitated the transmission of the chikungunya virus (benedict et al. ; charrel et al. ) . theoretical works based on deterministic modelling have looked at the conditions in disease transmission dynamics that are needed for the amplification or the dilution effect to occur (begon ; dobson ) . when there is a relationship between the risk of a disease, the abundance of the reservoir host, and the abundance of an additional host, the addition of a species does not necessarily decrease the risk. in the case of lyme disease, for example, tick abundance mainly is determined by the abundance of deer, which are in fact a non competent reservoir. an increased abundance of deer may reduce infection prevalence when immature ticks are feeding on the deer. at the same time, however, the overall number of adult ticks increase proportionally with the number of deer (begon ) . further research in this field are relying on the modelling of the global community competence of hosts and vectors (roche ) . scientists and societies are increasingly interested in the dilution effect (mea ) due to the link between habitat disturbance, generalist host characteristics, and their competence in disease transmission. disturbance seems to favour generalist hosts (hosts that use different types of habitats) (devictor et al. ; marvier et al. ) , and these hosts often have a broad geographical distribution (mckinney and lockwood ; smart et al. ). crucially, these species also seem to have a higher competence reservoir or vector reservoir than species that are not favoured by disturbance (mills ; molyneux et al. ; vittor et al. ) . for example, many murid rodents that are recognized hosts of hemorrhagic fever viruses are opportunistic species that seem to be favoured in disturbed environments. the question is whether there is a causal link between a species' generalist and opportunist character and its disease competence. why are murid species associated with hemorrhagic fever more generalist than those which are not? could it be possible that specialist species also carry hemorrhagic fever viruses, only these viruses have not yet been identified? or is there something intrinsic in opportunistic species that makes them more likely to evolve and maintain hemorrhagic fever viruses (mills )? only a very small subset of plant and animal species have been domesticated (diamond ) . many species of that small subset, for example, cattle (in animals) and maize (in plants), have seen their genetic diversity considerably reduced for the purpose of intensive production (the bovine hapmap consortium, matsuoka et al. ) . in the wild, small populations of endangered species often have a very reduced genetic diversity (keller and waller ) . this then raises the following question: to what extent does a reduction of the genetic diversity in a host species impact the ability of the host population to response to infectious diseases (may ) ? genetic loci associated with the major histocompatibility complex (mhc) plays a key role in the acquired immune response of vertebrates (altizer et al. ) . mhc genes code for molecules that recognize foreign peptides (antigens) and display them on the cell surface. when the mhc-protein is displayed, it can be presented to immune cells, such as t lymphocytes or natural killer cells, which subsequently can trigger an adaptive immune response. because mhc genes are faced with an important diversity of antigens, they must themselves be diverse. the measure of the genetic diversity of mhc based on an analysis of polymorphism sequences of mhc among individuals in populations has been widely used in conservation biology as a proxy to estimate the fitness of populations confronted by pathogens (alcaide et al. ; bernatchez and landry ; sommer ) . however, the level of genetic variation at mhc loci results from different evolutionary forces (selection, gene flow, mutation) varying both in space and time in co-evolutionary systems involving both hosts and pathogens, making conservation genetics of non-model organism a challenging task (stockwell et al. ) . we already have many examples where low genetic diversity of species has favoured the diffusion of, and/or susceptibility to, pathogens. for example, the low genetic diversity of the tasmanian devil could be involved in its susceptibility to facial tumor disease (mccallum ) . the low genetic diversity found in commercially traded bee queens has been hypothesised as being one of the factors explaining colony collapse disorder (le conte and navajas ). the problem is even more critical in intensive crops in which disease resistance has relied on the use of a very small number of genes. this selection strategy has proven to be ineffective as pathogens manage to overcome the resistance. for example, the resistance of brassica napus (canola, oilseed rape and colza) to leptosphaeria maculans (causing the blackleg disease) due to a major resistance gene was overcome in an area covering approximately , ha in south australia in a period of years (sprague et al. ). even with advances in synthetic chemistry, which provides many biologically active molecules, pharmaceuticals derived from nature remain an important part of pharmaceutical practice today (newman et al. ). all organisms have developed compounds to protect themselves against infectious diseases and to interact with individuals of their own species or other species (e.g. rogerio et al. ). these molecules, coming from all organisms (bacteria, fungus, animals, plants) in terrestrial, marine and extreme ecosystems, represent an amazing diversity that has been tested in the field for millions of years by involving millions of individuals. however, only a very small subset of plants and marine organisms has been investigated for novel bioactive compounds. furthermore, it is estimated that less that % of bacterial species and only % of fungal species are known. those which have not yet been identified could be sources of novel molecules (cragg and newman ) . observations of natural medicine practices used by indigenous people have led to the discovery of many drugs. the most well known and widely used pharmaceuticals are quinine, used as a model to synthesize anti-malarial drugs (chloroquine and mefloquine), and artemisinin, indentified as a potent anti-malarial drug by chinese scientists (newman et al. ) . animals also are a source of inspiration for drugs against infectious diseases. for instance, compounds of the sponge cryptotethya crypta inspired the elaboration of antiviral medication such as azt used in the treatment of hiv/aids (cragg and newman ) . observing great apes medicate themselves through the plants they eat also could help to reveal new active compounds (krief et al. ). pathogens constitute a large part of biodiversity on earth and are present in all ecosystems and at all trophic levels, where they have a large impact on ecosystem functioning and on the population dynamics and evolution of their hosts. the recent acceleration of biodiversity loss due to human activities deeply impacts host-pathogen dynamics. pathogens and hosts form co-evolving systems exercising major selective pressures on each other. furthermore, the virulence or pathogenicity of a given species depends on its environment -which includes the hosts -that is highly variable in space and time. in such a context, human beings will never be able to completely control or eradicate every pathogen species; rather, we should accept that we must coexist with pathogens. to better understand and predict the evolution of pathogens and the impact of human activities on them, more in-depth studies are needed on how pathogens interact with host communities within different ecosystems. to understand human, animal, and plant epidemics, two-species systems involving only a single host and a single pathogen species are no longer appropriate. the approach considering multi-host/multi-pathogen systems in their environment is the framework that now needs to be used to deepen our understanding of disease dynamics woolhouse et al. ) (fig. . ) . however, these dynamics are very complex and difficult to study because precise knowledge regarding the diversity of pathogens and of interactions taking place on several scales is lacking (lloyd-smith et al. ). in addition to intensive fieldwork to collect adequate data and modeling to understand the main processes, the use of molecular tools in a multi-host/multi-pathogen framework will facilitate investigations into pathogen-host community interactions. in particular, new generation sequencing techniques render it easier to characterize the genetic diversity of pathogens and hosts. for instance, metagenomic investigations allow an approach that does not require a priori assumptions that is useful to describe a community of pathogens and their interactions. molecular techniques also may be used to clarify the taxonomic status of pathogens, revealing cryptic species or host races. with suitable molecular fig. . schematic representation of the link between biodiversity and the ecology of infectious diseases. diseases results from the complex interactions between the three compartments corresponding to pathogens, hosts and vectors (in the case of vector-borne diseases). the biodiversity of these three compartments can be considered at the community level (each circle corresponding to a species) or at the intraspecific level (each circle corresponding to a population or an individual within a population). the gray shading off of each unit considered (species, population or individual) illustrates its genetic or phenotypic variability in space and time, while variations in size illustrate frequency or density differences within the ecosystem. interactions within each compartment can be direct (competition) or indirect (apparent competition…), synergic or antagonistic as illustrated by the different arrows markers (producing a high level of polymorphism), the analysis of genetic variability within a spatially explicit framework renders it possible to identify the routes followed by a given pathogen. moreover, molecular techniques can be used to identify genes involved in important life history traits of a pathogen such as virulence and transmission. better knowledge of the mechanisms involved in host-pathogen interactions, and the extent of their variability, will significantly advance our understanding of outbreaks. although our knowledge of the number and variety of pathogens is not complete, it appears that their diversity, like that of their hosts and vectors, is greater in tropical areas than in temperate ones, and in undisturbed habitats than disturbed ones (chaisiri et al. ; friggens and beier ) . the reason we are so concerned by the loss of species biodiversity is because a reduction of biodiversity seems to favour opportunistic species that are highly competent as pathogen reservoirs and vectors. however, this observation was derived from only a few studies and theoretical works, mainly undertaken in the temperate zones. further investigations should be launched to verify the link between, and understand the process involved in, biodiversity loss and disease dynamics. this especially should be done in the tropics to understand whether high levels of biodiversity create a buffer reducing the risk of disease 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biology of multihost pathogens temporal trends in the discovery of human viruses rna viral community in human feces: prevalence of plant pathogenic viruses genetic diversity and disease control in rice key: cord- - x j iqg authors: bösl, korbinian; ianevski, aleksandr; than, thoa t.; andersen, petter i.; kuivanen, suvi; teppor, mona; zusinaite, eva; dumpis, uga; vitkauskiene, astra; cox, rebecca j.; kallio-kokko, hannimari; bergqvist, anders; tenson, tanel; merits, andres; oksenych, valentyn; bjørås, magnar; anthonsen, marit w.; shum, david; kaarbø, mari; vapalahti, olli; windisch, marc p.; superti-furga, giulio; snijder, berend; kainov, denis; kandasamy, richard k. title: common nodes of virus–host interaction revealed through an integrated network analysis date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: x j iqg viruses are one of the major causes of acute and chronic infectious diseases and thus a major contributor to the global burden of disease. several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signaling pathways. a collective view of these multiple studies could advance our understanding of virus-host interactions and provide new therapeutic perspectives for the treatment of viral diseases. here, we performed an integrative meta-analysis to elucidate the different host-virus interactomes. network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. we also identified the core cellular process subnetworks that are targeted by all the viruses. integration with functional rna interference (rnai) datasets showed that a large proportion of the targets are required for viral replication. furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis c virus and human metapneumovirus. altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape. viruses are one of the major causes of acute and chronic infectious diseases and thus a major contributor to the global burden of disease. several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signaling pathways. a collective view of these multiple studies could advance our understanding of virus-host interactions and provide new therapeutic perspectives for the treatment of viral diseases. here, we performed an integrative meta-analysis to elucidate the different host-virus interactomes. network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. we also identified the core cellular process subnetworks that are targeted by all the viruses. integration with functional rna interference (rnai) datasets showed that a large proportion of the targets are required for viral replication. furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis c virus and human metapneumovirus. altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape. viruses continue to be a major contributor to the global burden of disease through acute and chronic infections that cause substantial economic impact in addition to increased mortality and morbidity ( ) . despite the tremendous improvement in the understanding of the antiviral immune response and the availability of therapeutics, existing and emerging viral diseases are an ever-growing problem, particularly in developing countries. development of antiviral resistance of hepatitis c virus (hcv), influenza a virus (iav), herpes simplex virus (hsv), human cytomegalovirus (hcmv), human immunodeficiency virus (hiv), and other viruses is a major concern ( - ). one of the main reasons for increasing resistances is the accumulation of mutations in the viral genome caused by multiple factors including the polymerase infidelity ( , ) . therefore, the world health organization (who) and the united nations have urged for better control of viral diseases. this has led to turning the focus on the host for therapeutic intervention. targeting the host factors has been proven to be useful for restricting viral infections ( , ) . the small molecule ccr inhibitor maraviroc and the anti-cd monoclonal antibody ibalizumab are examples of successful use of host-directed therapies for combating hiv in clinic ( ) ( ) ( ) . viruses have evolved to evade the host antiviral response at various stages starting from viral sensing to antiviral proinflammatory responses ( ) ( ) ( ) . multiple studies attempted to understand global principles of the viral evasion employed by various viruses, including dengue virus (denv), ebola virus (ebov), iav, and hiv ( ) ( ) ( ) ( ) ( ) ( ) . global systems-level approaches including functional rnai screens, interactome mapping technologies such as affinity-purification mass spectrometry (ap-ms), quantitative proteomics, and crispr/cas -based screens have provided unparalleled details and insights into the dynamics of host proteome in immune cells ( ) ( ) ( ) ( ) , host-virus interactome ( - , , ) , and also identified important host dependency factors of various viruses ( , , ) . meta-analyses of such high-dimensional datasets have been crucial for identifying novel host factors as drug targets such as ubr in iav infection ( ) . moreover, some of these factors represent drug targets for multiple viruses ( ) . we hypothesized that combining a meta-analysis of host-virus protein-protein interactions of multiple viruses and functional rnai screens would provide novel insights for developing broadspectrum antiviral strategies. for this, we assembled a host-virus protein-protein interactome of , host-virus interactions (hereafter referred to as "hvppi") covering viral proteins from different viruses and , host proteins. we performed extensive bioinformatics and network analysis and integrated this dataset with genome-wide or druggable-genome rnai screen data from published studies. this resulted in the assembly of critical nodes of viral evasion and identification of core cellular processes and druggable nodes that were verified by a drug re-purposing screen using broad-spectrum antivirals. host-virus protein-protein interactions were downloaded from published studies ( - , , - ) we performed gene-set enrichment analysis using david bioinformatics resources [version . , ( ) ]. for all enrichment analysis, a p-value cutoff of ≤ . was used as significant. protein disorder analysis was performed using iupred a software. we used the offline version with protein sequences downloaded from uniprot. statistical analysis of disordered region distribution was performed by kolmogorov-smirnov test in r statistical environment. annotation of human proteins was mapped from uniprot id to ensembl using ensdb.hsapiens.v . the index of subcellular localization of interaction partners of single viral proteins was calculated for all viral proteins with ≥ five host targets. localization of host targets was mapped using compartments ( ) , filtered for a minimum evidence score of in the knowledge channel, excluding non-experimental based localization predictions. evidence for all protein was subsequently divided by the absolute number of host-targets per viral protein. multiple sequence alignment was performed using clustal x [version . , ( )]. genome-wide rnai screen data for hcv ( ) and hpv ( ) , through genomernai database [( )-gr ], as well as druggable rnai screen data for hpv ( ) , vacv ( ) , and sv ( ) were integrated in the existing network as z-scores. drug-gene interaction data was downloaded from dgidb and drugbank. the identifiers were mapped to uniprot ids and then compared with hvppi. for the hmpv nl/ / -gfp screen, approximately × human retinal pigment epithelial (rpe) cells were seeded per well in -well plates. human non-malignant rpe cell line represents excellent model system for studying replication of many viruses including respiratory ( , , ) . the cells were grown for h in dmem-f medium supplemented with % fbs, . % nahco , and µg/ml streptomicine and iu/ml penicillin. the medium was replaced with virus growth medium (vgm) containing . % bovine serum albumin (bsa), mm l-glutamine, . % nahco , and µg/ml l- -tosylamido- -phenylethyl chloromethyl ketone-trypsin in dmem-f . hcv screen-associated cell culture conditions are described in kim et al. ( ) . the compounds were added to the cells in fold dilutions at seven different concentrations starting from µm. no compounds were added to the control wells. the cells were mock-or virus-infected at a multiplicity of infection (moi) of one. after h of infection, the medium was removed from the cells. to monitor cell viability, celltiter-glo reagent was added ( µl per well). this assay quantifies atp, an indicator of metabolically active living cells. the luminescence was measured with a plate reader. to determine compound efficacy, hmpv nl/ / -mediated gfp expression was measured. the half-maximal cytotoxic concentration (cc ) and the half-maximal effective concentration (ec ) for each compound were calculated after non-linear regression analysis with a variable slope using graphpad prism software version . a. the relative effectiveness of the drug was quantified as the selectivity index (si = cc ec ). cytotoxicity and antiviral activity of the compounds against gfp-expressing hcv in huh- . cells was determined as previously described ( ). to provide new and critical insights into viral evasion mechanisms we performed a comprehensive meta-analysis of the host-virus interaction landscape. we assembled the hostvirus protein-protein interaction data ("hvppi") from published studies ( figure a ) ( - , , - ) . this dataset comprised of protein-protein interactions from two different types of experimental methods-affinity purification mass spectrometry (ap-ms) and yeast two-hybrid screens (y h). altogether, this combined dataset includes viral proteins, , host proteins, and , protein-protein interactions ( figure b and figure s ). many interactome networks including yeast and human are scale-free networks, where a large portion of the nodes (e.g., a protein in the network) have few interactions and only a few nodes have large number of interactions. the latter are often referred to as "hubs" which are crucial in keeping the network intact ( ) . we performed network topology analysis to infer the properties of the host proteins targeted by the viral proteins in the context of the human protein interactome. we considered two important parameters-relative betweenness centrality (which reflects the amount of information that passes through this protein in the human interactome) and degree (number of binding partners in the human interactome) of the host proteins targeted by each virus. the targets of all the viruses showed higher betweenness centrality and degree as compared to an average protein in the human interactome (figures c,d) . this shows that viruses, by targeting "hubs" and proteins that serves as key communication nodes, have evolved the best way to disrupt the scale-free human interactome. this topological property thereby shows how viruses having small genomes achieve the maximal effect in rewiring the human interactome to benefit viral survival and replication. our analysis is in agreement with several previous studies, which have highlighted this property ( , , , , ) . we propose that this could be a general principle for all viruses. proteins typically fold into stable three-dimensional structures that mediate specific functions. in addition, there are substructures in proteins termed "intrinsically disordered regions (idrs)" which lack stable structures under normal physiological conditions. idrs are required for multiple cellular functions even though they lack these defined structures ( ) . many studies have highlighted the presence of such idrs in viral proteins ( ) ( ) ( ) , such as e from human papilloma virus, that are crucial for hijacking the cellular machinery. we analyzed the host proteins from the hvppi for presence of idrs using the prediction software iupred ( ) . it is estimated that the human proteome more than , short linear binding motifs in idrs ( , ) . proteins with idrs are often signaling hubs and might form dynamic complexes with other proteins through specific motif based interactions ( ) . we found a statistically significant enrichment (p-value < . × − ) of idrs in the host proteins targeted by viruses (figure a and figure s ). we then identified the subnetwork in the hvppi which contained the top host targets with high disorderness score ( figure b) . the top five proteins with large idrs include cd antigen (cd ), serine/arginine repetitive matrix protein (srrm ), myristoylated alaninerich c-kinase substrate (marcks), bag family molecular chaperone regulator (bag ), and mitochondrial antiviralsignaling protein (mavs; figure c ). cd is a marker of exhausted cd + t cells ( ) and replication of hcv in t cells was shown to decrease cell proliferation by inhibiting cd expression and signaling ( ) . srrm is a serine/arginine-rich protein involved in rna splicing ( ) . srrm is differentially phosphorylated in hiv- infected cells and absence of srrm lead to increased hiv- gene expression, since it regulates the splicing of hiv- ( ) . in the hvppi, srrm is targeted by multiple viral proteins including the tat protein from hiv- . tat protein has an important role in the stimulation of the transcription of the long terminal repeat (ltr) ( ) . in addition, ns protein from influenza b virus has also been reported to interact with srrm ( ) . proteins of the marcks family are involved in a range of cellular processes including cell adhesion and migration ( ) . marcks is a negative regulator of lipopolysaccharide (lps)-induced toll-like receptor (tlr ) signaling in mouse macrophages ( ) . mavs is an adaptor protein in the rig-i signaling pathway involved in the sensing of rna. ablasser et al. ( ) reported that double-stranded dna serves as a template for rna polymerase ii and is transcribed into a ′ triphosphate containing double-stranded rna, which activates the rig-i signaling pathway. in the hvppi, mavs is targeted by several proteins from dsdna viruses such as ebv and hpv. altogether, our analysis shows that the idr-high part of the human proteome is an essential part of the viral evasion strategy and some of the selected targets highlighted here could show novel insights into the viral evasion mechanisms. however, the very flexible protein structure of disordered proteins also makes them also difficult to target with drugs. to assess the signaling pathways and cellular processes within the hvppi, we identified highly connected subnetworks within hvppi network. we constructed a host-host interaction network based on the host targets in the hvppi and identified a number of highly connected subnetworks/clusters (figure ) . we then performed a gene-set enrichment analysis of significantly enriched biological processes. we found one or more enriched processes for each of this subnetwork including core cellular processes such as proteasome, spliceosome, protein translation, protein/rna transport, and cell cycle. next we listed the viruses that target one or more of these processes, and found that almost all the core pathways and processes are targeted by all the viruses that are part of the hvppi (figure s ). this analysis highlights the core components of the cellular process subnetworks which are targeted as part of the viral evasion strategies and thus could be broad-spectrum antiviral hot-spots from a therapeutic point of view. given that the viral proteins were interacting with a large number of host proteins, we analyzed the sub-cellular location of the host proteins. we performed gene-set enrichment analysis of sub-cellular localization information provided by uniprot database. we binned the localization into compartments and estimated the percent of host proteins in a given compartment as compared to the total number of host proteins targeted by a given figure | clusters of hvppi involved in core cellular processes. network view of the "clusters" or highly-connected sub-networks and their associated cellular processes. each cluster is marked in a unique color. virus. we found that the viral targets were distributed across multiple subcellular compartments with cytoplasm being the most common ( figure s a ). the hvppi includes two different strains of iav-pr (h n ) and udorn (h n ). the subcellular localization analysis showed that both strains were enriched for nuclear proteins. non-structural protein (ns ) from both the strains had the highest number of nuclear targets but their targets were very different ( figure a) . ns of udorn was enriched for a large number of histones as compared to ns of pr that had large number of heterogeneous nuclear ribonucleoproteins (hnrnps), such as hnrnpu−a known restriction factor for many viruses. this corroborates with the observation that ns protein has short linear histone mimicry motifs that can suppress the host antiviral response ( ) . in our analysis, we found that it is ns of udorn that has a histone mimicry motif "arsk" (figure s b) . similarly, hpv and hpv e proteins interact more often with host proteins located in the endoplasmic reticulum (er). we found both common and specific subsets of er proteins targeted by the e protein ( figure b ). hpv e protein er targets were enriched for phospholipid biosynthesis as well as gpi anchor related proteins, such as phosphatidylinositol glycan anchor biosynthesis class s/t/u (pigs, pigt, and pigu), glycosylphosphatidylinositol anchor attachment (gpaa ) and phosphatidylserine synthase (ptdss ). hpv e protein er targets were enriched for er-associated ubiquitindependent protein catabolism involving host proteins such as er degradation enhancing alpha-mannosidase-like protein (edem ) and er lipid raft associated (erlin ). er targets common to hpv and hpv e protein were enriched for unfolded protein response, n-linked glycosylation and protein folding involving host proteins such as srp receptor alpha/beta subunit (srpra/srprb) and catalytic subunits of the oligosaccharyltransferase complex (stt a and stt b). two independent crispr/cas screening studies identified multiple er associated components including stt a and stt b as host factors for denv, zika virus (zikv) and japanese encephalitis virus (jev) ( , ) . the non-canonical function of stt a and stt b is required for denv replication and that ns protein of denv interacts with these proteins ( ) . our orthogonal approach can lead to the identification of critical host factors, and similar functions of er components, such as stt a and stt b, are used by hpv and hpv as well. thus, targeting the non-canonical function of stt a and stt b could be a broad antiviral strategy. overall, the enrichment analysis clearly shows that there is commonality and specificity in the subcellular targets of the viral proteins and that detailed interrogation of these targets can give vital clues into the viral evasion mechanisms. rnai screens have been a powerful high-throughput method to identify various cellular functions, including for identification of host restriction factors of viruses ( ) . in order to explore the functional relevance of the host targets in the hvppi, we integrated it with five published rnai screens that performed genome-wide or druggable-genome-wide rnai screens for identifying host factors of hcv ( ), hpv ( ), hpv ( ), sv ( ) , and vacv ( ) . we found that host targets from the hvppi were spread across the spectrum of genes with proviral as well as antiviral phenotype (figure s ) , thus showing that targeting of the host protein by the virus could lead into any direction that favors the virus. we then investigated the top proviral genes that are also targeted by the viral proteins as seen in the hvppi. we identified host proteins ( figure a ) that were significantly enriched for coatomer protein complex (copi), protein translation/transport and proteasome ( figure b) . this further substantiates the findings from the earlier section on the core cellular processes targeted by the viruses. network analysis of these top hits showed high level on connectivity and crosstalk-for example between the translation and proteasome machinery (figure c ). vesicle carriers are involved in the transport of membranes and proteins. copi system is one of the three vesicular carrier systems that is involved in the early secretory pathway ( ) ( ) ( ) . moreover, it has been pointed out that there is a strong similarity between vesicular transport and viral transport [viral entry to budding process, ( ) ]; thus making copi system important for the viral life cycle. in addition to the findings of the present study, sirna-based silencing of copi lead to a decrease in entry and subsequent gene expression of iav, vsv, lcmv, and hpiv and disruption of the copi complex inhibited the production of infectious progeny virus ( , ) . copi coatomer inactivation results in a direct decrease of vsv attachment and uptake, but not for membrane fusion or rnp release; however the direct mechanism remains unclear ( ) . altogether, these top hits including the copi system could serve as targets for developing therapeutic antiviral intervention strategies for a broad group of viruses. our analyses pointed out that viral evasion mechanism observed in one virus could also be relevant for other viruses. to test this, we obtained known drug-gene interactions from dgidb ( ). we selected investigational/experimental/approved antivirals compounds ( ) which had dgidb annotated targets that are part of the hvppi. we added agents as controls (table s and figure s ) . we tested broad-spectrumantivirals against gfp-expressing human metapneumovirus (hmpv) nl/ / strain ( ) . seven different concentrations of the compounds were added to hmpv or mock-infected cells. hmpv-induced gfp expression and cell viability was measured and after h to determine compound efficiency and toxicity. after the initial screening, we identified five compounds, which lowered gfp-expression without detectable cytotoxicity (with si > ). we repeated the experiment with these compounds. the experiments revealed novel activity of azacytidine, lopinavir, nitazoxanide, itraconazole, and oritavancin against hmpv ( figure s a and table , table s ). similarly, we examined toxicity and antiviral activity of broad-spectrum-antivirals against gfp-expressing hcv in huh- . cells using previously described procedures ( ) . our test identified azithromycin, cidofovir, oritavancin, dibucaine, gefitinib, minocycline, and pirlindole mesylate as novel anti-hcv agents with si > ( figure s b and table , table s ). in summary, our metaanalysis approach of the hvppi could provide novel and faster approaches for the re-purposing of existing drugs as antiviral agents. using integrative analysis of orthogonal datasets our study provides a comprehensive view of viral evasion mechanisms. in particular, our analysis of the hvppi network revealed that all the viruses have evolved to target proteins that are central and have strong control over the human interactome. host proteins targeted by viruses contain a high proportion of intrinsically disordered regions. we identified the core cellular processes and associated proteins that are targeted by all viruses. detailed comparative analysis of the subcellular localization of the host proteins showed commonality and specificity both between viral proteins from different strains of the same virus; and between viruses. integrating hvppi with functional rnai screens showed that % of the hvppi are host factors of one or more virus. hvppi data-based drug re-purposing screen identified novel activities for various broad-spectrum antivirals against hmpv and hcv. this unique dataset can be used for further detailed interrogation of the mechanisms behind viral evasion. this could serve as a starting point for identifying novel host targets and generating hypothesis in the context of viral evasion and development of pan-viral therapeutic intervention strategies. the methods described here also provide unique ways of dissecting the orthogonal datasets. various analyses from this study have highlighted the existence on pan-viral evasion points that could be utilized for the development of host-directed antiviral therapies. it is also intriguing to see that there is commonality and specificity at the level of sub cellular localization of the viral targets. our analyses have underlined some salient features in the context of iav, hpv, denv, and hcv. further detailed analysis in this context along with protein sequence features, such as short linear motifs [slims; ( ) ] would provide novel insights as well as deeper understanding of how small sequence features are involved in the hijacking of the host machinery. integration of such data with known drug-gene interactions provides a clear estimate of the druggable proportion in the hvppi. our meta-analysis approach of the hvppi could provide novel avenues of re-purposing existing drugs for antiviral targeting strategies. our meta-analysis approach of the hvppi could provide novel avenues of re-purposing existing drugs for antiviral targeting strategies. to prove the concept, we tested bsas against hmpv, hcv, sindbis virus (sinv), cytomegalovirus (cmv), and hepatitis b virus (hbv). importantly, bsas have dgidb annotated targets that are part of the hvppi, whereas were used as controls. these safein-man drugs have already been used as investigational agents or experimental drugs in different virus infections (table s ) . we demonstrated novel antiviral effects of azacytidine, itraconazole, lopinavir, nitazoxanide, and oritavancin against hmpv, as well as cidofovir, dibucaine, azithromycin, gefitinib, minocycline, oritavancin, and pirlindole against hcv. azithromycin, is an fda-approved antibiotic of the macrolide family. it is also an investigational agent against rsv and experimental agent against ebov, hrv-a, zikv, and rsv. cidofovir is an fda-approved anti-cmv drug. it is also investigational agent against adv, bkv, hpv, hsv- , hsv- , and experimental drug against b v. dibucaine is an fdaapproved amide local anesthetic. in addition, it is experimental anti-hev-a, hev-b, hev-d, and ebov agent. gefitinib is an fda approved anticancer drug. it has also antiviral activity against bkv, cmv, and vacv. minocycline is a broad-spectrum antibiotic and experimental anti-denv, hiv- , and wnv agent. oritavancin is a semisynthetic glycopeptide antibiotic used for the treatment of gram-positive bacterial skin infections. it also inhibits ebov, mers-cov, and sars-cov infections. pirlindole is an antidepressant, which is also experimental anti-hev-a, hev-b, and hev-d agent. azacitidine is a chemical analog of cytidine, which is used in the treatment of myelodysplastic syndrome. it is also an experimental anti-adv, the measurements were repeated three times (p < . ). fluav, rvfv, hiv- , and hiv- agent. itraconazole is an antifungal medication. it is also used as experimental anti-hev-b, hrv-b, hrv-a, par-a , and safv agent. nitazoxanide is a broad-spectrum antiparasitic drug, which is also investigational agent against fluav and hcv and experimental anti-chikv, rsv, hbv, hiv- , vacv, rv, jev, mers-cov, nov, ruv, and zikv agent. lopinavir is an fda-approved antiretroviral of the protease inhibitor class. it is also investigational anti-mers-cov and experimental anti-zikv agent (table s ). in addition to inhibition of viral proteases (table s ) , lopinavir was reported to induce host rnasel production in infected and non-infected cells ( ) . rnasel is endoribonuclease that is a part of interferon (ifn) antiviral response, which is the most critical node of virus-host interactions. although, the antiviral mechanisms of action of other compounds are still unknown, these agents could inhibit steps of viral infections, which precede reporter protein expression from viral rna. in summary, our results indicate that existing bsas could be re-purposed to other viral infections. to further expand a spectrum of their activities, these bsas could be tested against other viruses. re-purposing these and other safe-inman antiviral therapeutics could save resources and time needed for development of novel drugs to quickly address unmet medical needs, because safety profiles of these agents in humans are available. effective treatment with broad-spectrum-antivirals may shortly become available, pending the results of further pre-clinical studies and clinical trials. this, in turn, means that some broad-spectrum-antivirals could be used for rapid management of new or emerging drug-resistant strains, as well as for first-line treatment or for prophylaxis of acute virus infections or for viral co-infections. the most effective and tolerable compounds could expand the available therapeutics for the treatment of viral diseases, improving preparedness and the protection of the general population from viral epidemics and pandemics. all datasets used for this study are accessible as stated in the materials and methods section . . we thank christian sinzger and group for the egfp-expressing tb e cmv strain. we thank vironovative and erasmus mc for the gfp-expressing hmpv nl/ / strain. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material redefining chronic viral infection antiviral drug resistance as an adaptive process emerging virus diseases: can we ever expect the unexpected? emerg microbes infect herpes simplex virus resistance to acyclovir and penciclovir after two decades of antiviral therapy mechanisms of viral mutation complexities of viral mutation rates host-directed therapies 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proteins hijack the cell? iupred a: context-dependent prediction of protein disorder as a function of redox state and protein binding a million peptide motifs for the molecular biologist intrinsically disordered proteins in cellular signaling and regulation molecular and cellular insights into t cell exhaustion hepatitis c virus infection of t cells inhibits proliferation and enhances fas-mediated apoptosis by down-regulating the expression of cd splicing variant the srm / splicing coactivator subunits quantitative phosphoproteomics reveals extensive cellular reprogramming during hiv- entry the hiv- tat protein has a versatile role in activating viral transcription human interactome of the influenza b virus ns protein cross-talk unfolded: marcks proteins marcks as a negative regulator of lipopolysaccharide signaling rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate suppression of the antiviral response by an influenza histone mimic the use of rnai-based screens to identify host proteins involved in viral replication the copi system: molecular mechanisms and function bi-directional protein transport between the er and golgi copi-mediated transport viruses as vesicular carriers of the viral genome: a functional module perspective dissecting the role of copi complexes in influenza virus infection rnai screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand rna viruses genetic inactivation of copi coatomer separately inhibits vesicular stomatitis virus entry and gene expression dgidb . : a redesign and expansion of the drug-gene interaction database an improved plaque reduction virus neutralization assay for human metapneumovirus how viruses hijack cell regulation lopinavir up-regulates expression of the antiviral protein ribonuclease l in human papillomavirus-positive cervical carcinoma cells key: cord- -quw gyw authors: martin, lynn b.; addison, brianne; bean, andrew g.d.; buchanan, katherine l.; crino, ondi l.; eastwood, justin r.; flies, andrew s.; hamede, rodrigo; hill, geoffrey e.; klaassen, marcel; koch, rebecca e.; martens, johanne m.; napolitano, constanza; narayan, edward j.; peacock, lee; peel, alison j.; peters, anne; raven, nynke; risely, alice; roast, michael j.; rollins, lee a.; ruiz-aravena, manuel; selechnik, dan; stokes, helena s.; ujvari, beata; grogan, laura f. title: extreme competence: keystone hosts of infections date: - - journal: trends ecol evol doi: . /j.tree. . . sha: doc_id: cord_uid: quw gyw individual hosts differ extensively in their competence for parasites, but traditional research has discounted this variation, partly because modeling such heterogeneity is difficult. this discounting has diminished as tools have improved and recognition has grown that some hosts, the extremely competent, can have exceptional impacts on disease dynamics. most prominent among these hosts are the superspreaders, but other forms of extreme competence (ec) exist and others await discovery; each with potentially strong but distinct implications for disease emergence and spread. here, we propose a framework for the study and discovery of ec, suitable for different host–parasite systems, which we hope enhances our understanding of how parasites circulate and evolve in host communities. individual hosts differ extensively in their competence for parasites, but traditional research has discounted this variation, partly because modeling such heterogeneity is difficult. this discounting has diminished as tools have improved and recognition has grown that some hosts, the extremely competent, can have exceptional impacts on disease dynamics. most prominent among these hosts are the superspreaders, but other forms of extreme competence (ec) exist and others await discovery; each with potentially strong but distinct implications for disease emergence and spread. here, we propose a framework for the study and discovery of ec, suitable for different host-parasite systems, which we hope enhances our understanding of how parasites circulate and evolve in host communities. most epidemiological theory has tended to discount intraspecific variation in host competence (see glossary); the capacity for a host to cause an infection in another susceptible host or vector [ ] . only relatively circumscribed forms, such as variation among age classes or individuals with different levels of prior parasite exposure, have been well studied [ ] . heterogeneities in host competence have recently been recognized as integral to understanding and control of many outbreaks and epidemics [ , ] . in many systems, a small number of host individuals drive disease emergence and spread [ , ] . the best-known human example is mary mallon, a cook responsible for many cases of typhoid fever. although superspreaders such as mallon exist for many host-parasite combinations (table ) , we still know little about what imbues some hosts with such ec [ ] . although extreme often connotes high in colloquial use, here we use its statistical form; both very high and very low values of competence constitute extreme in this paper. presently, many forms of ec probably remain unknown, largely because of biases in the way we study many infections. this dearth of information potentiates a missed opportunity, as many diseases are most effectively controlled when these key hosts are targeted [ ] . here, we have three goals. first, we describe the ecological importance of individual host-level ec and summarize some known examples from the literature (table ) . superspreading is by far the most common form, but others exist and yet others seem to await discovery. second, we propose a framework for studying and revealing new forms of ec (figure ), which we apply to two distinct infections: malaria and lung nematode infection. finally, we highlight a few a few members of host populations, so-called superspreaders, have disproportionate impacts on the risk of infectious disease emergence and spread. several other forms of ec exist; some of which might be exceptionally protective. to discover and understand forms of ec, it is imperative to describe the distribution of, and covariation among, traits of individual hosts that mediate the many stages of host-parasite interactions. here, we provide a framework to do so, emphasizing how interplay among host traits related to parasite exposure behavior, susceptibility, replicability of parasites on/in hosts, and transmissibility, comprise host competence. we hope this framework helps reveal new forms of ec and informs and improves management of disease risk. promising research paths (boxes and ) to reveal how hosts become so competent; a complement to other recent efforts [ , ] . we anticipate that this framework will be of value because it links disease processes within and among individual hosts for most host-parasite systems [ ] . central to most mathematical models for infectious diseases are estimates of r , which quantify the number of new infections generated by the average infected host in a wholly susceptible population [ ] . typically, the mean values of host traits within species are used to estimate r and individual heterogeneity is ignored. this lack of inclusion of heterogeneity might explain why simple epidemiological models have been unable to explain or predict the behavior of some outbreaks [ ] . recently, researchers have demonstrated the value of using individuallevel variants of r [ ] , such as n [ ] , to model disease dynamics. one particular advantage of n is that it can be decomposed into three elements: infectiousness, contact rate, and infectious period [ ] . this empirical tractability facilitates linkage of within-and between-host processes in traditional mathematical frameworks. when the distribution of n is described in a population, it is often non-normal with only a few individuals having very high n [ , ] . this distribution (k) is not altogether surprising; in many host-parasite systems, we have long known that % of hosts are responsible for % of new infections [ ] . what remains obscure, though, is whether n (or other proxies of r ) capture the true extent of heterogeneity in host competence that resides in populations; in particular, functionally exceptional forms. superspreaders are often conspicuous behaviorally or in terms of their high parasite burdens or shedding rates [ ] , but more cryptic forms of ec might not be revealed by typical approaches to estimating n. for instance, n does not explicitly take into account that host behavior sometimes changes, often dramatically, in the presence and absence of infection. parasites commonly alter host behavior for their own interests. host sickness behaviors too are highly variable among individuals; some hosts become lethargic and seek refugia when infected but others behave almost as if uninfected, and still others become more active [ ] . consider that asymptomatic carriers of human dengue virus are responsible for up to % of new infections [ ] ; behaviorally, these individuals are not conspicuous. also, a key aspect of n, infectiousness, is often inferred from the parasite burdens of hosts. this proxy is sometimes representative of true infectiousness, but oftentimes it is not. many hosts are much less competent than their parasite burdens would suggest [ ] . some hosts tolerate infections well and shed many parasites in the right places and times to infect other hosts [ , ] , but others, even those with high burdens, generate few infections because of particular behaviors or high mortality postinfection [ ] . in table , we list several published examples of ec, which represent the most distinct and best-supported examples we could find. superspreading, unsurprisingly, is the most commonly documented form. most likely, its apparent commonness is related to sampling bias; its importance in some high-profile cases and its relative conspicuousness facilitate its discovery. notably, though, what comprises superspreading can vary depending on parasite taxonomy, mode of transmission, and several other factors ( resistance: the ability of a host to limit parasite burden after exposure to a parasite. suitability: the propensity of a host to permit parasite survival long enough for the parasite to produce viable offspring, including the number of such offspring. superattractor: a more conspicuous and/or less behaviorally defended host (to vectors). superblocker: a host with high connectivity, but low suitability, in a social network context. superbreeder: a host that makes strong terminal investments upon parasite exposure or infection. superconsumer: a host that consumes and digests many parasites or vectors. superdiluter: a host that greatly reduces risk of infection for other hosts. superevolver: a host that facilitates transmissible evolutionarily change in a parasite. supermover: a host that moves a parasite across a large physical distance. superrecaller: an individual host that remains protected against reinfection for a long period postparasite exposure and recovery. superreceiver: a host having a high number of contacts with infected hosts, vectors, or sites in an environment; functionally, resembles a superattractor but is agnostic in regard to mode of transmission. supershedder: a host that passes a large parasite burden into the environment. taurus) deposit more escherichia coli into feedlots than others, making them putative supershedders [ ] . supershedding, unlike superspreading, could make some terrestrial habitats enduring hotspots for infection [ ] . in aquatic systems, supershedding near currents could enhance parasite dispersal opportunities, making some supershedders supermovers too. superattractors and superreceivers are often cast as variants of superspreaders, but their functional roles will depend on covariation between traits affecting (at least) suitability and transmission. in one series of experiments, physiologically stressed, west nile virus-infected zebra finches (taeniopygia guttata) attracted twice as many vectors as controls and were infectious to vectors, whereas controls were not [ , ] . there, superattractors were also superspreaders, but in other species low wnv suitability coupled with high vector attractiveness could produce superdiluters. for contact-transmitted infections, forms of network centrality can drive superspreading or superblocking (table ) , depending on the traits of hosts at these key nodes and how such traits affect betweenness versus degree [ ] . a particular goal of this paper is to advocate for research on forms of ec that would make some hosts exceptionally protective against infection risk in communities [ ] . such superdiluters might be just as pivotal to understanding and managing disease dynamics as superspreaders, but tailored investigations will be required to identify them. unlike superspreaders, one cannot identify superdiluters by measuring parasite burden or even obvious behaviors, which might be why we found no examples of them in the literature. the existence of such hosts, however, is plausible. for water-borne infections, as in the daphnia-metschnikowia (host-fungal parasite) system [ ] , hosts can act as superdiluters by (i) consuming parasites (as food; superconsumers) or (ii) competing with hosts with higher suitability or transmissibility (reducing contact rates with susceptible hosts; superblockers) [ ] . there is already indirect evidence for superdiluting with regards to human malaria: a single human genetic mutation is associated with increased phagocytosis of malaria parasites, which could make some individual hosts highly susceptible but unlikely to reach parasitemia sufficient for transmission [ ] . some individual hosts might also dilute local infection risk by maintaining protective levels of antibodies or other forms of immunity for exceptionally long periods (i.e., superrecallers). a final plausible example of nonobvious ec, worthy of study, are superevolvers. this type of ec might be particularly applicable to some viruses, such as influenza virus, which is transmitted directly through respiratory fluids and/or fecal matter and is capable of circulating in a wide range of mammalian and avian species [ ] . most human influenza infections tend to resolve over the course of days, but sometimes they are chronic. such chronically infected hosts might sometimes be able to generate genetically unique forms of viruses [ ] . only recently have the tools become precise enough to reveal genetic differentiation of viruses within single hosts, and for influenza virus, as many as ten variants per individual human infection have been observed [ ] . the high mutation rate of influenza virus (and perhaps other rna viruses), as well as their capacity to share nucleic acids horizontally (when the same host cells are coinfected by different strains; [ ] ), could enable some hosts to facilitate parasite evolution. although most such variants will be eliminated before transmission because of strong purifying selection, genetic drift, bottleneck effects, and elimination by the host immune system, some few could manifest new forms of virulence or propensities to infect novel host cell types (via sialic acid receptors). it is too soon to know whether superevolvers are common, much less if they impact global infection dynamics [ ] . however, the propensities of many viruses (e.g., hiv and hepatitis c virus) to hide out in tissues [ ] , as well as the heterogeneities among hosts in their abilities to cope with viral infections [ ] , should instigate efforts to identify them. in figure , we provide a framework for decomposing host competence into four componentsexposure, susceptibility, suitability and transmissibilitythat should be amenable to studying ec, including revealing novel forms of ec if they exist. although these aspects of competence are coarse and multifaceted, just as with terms that comprise n, they are amenable to description at the individual level and even decomposable to their physiological bases [ , ] . so as to ground our framework in familiar territory, we collected data and plotted frequency distributions of all four aspects of host competence for two different infections: malaria parasites and lung nematodes ( figure ). in one system (lung nematodes) we used data from one host species (cane toads); for malaria, we produced a composite of available data from humans and wild birds. a complete set of information was unavailable for either host group alone. for both examples, we encourage caution in overinterpreting apparent patterns, as our intention is simply to demonstrate the promise of the framework. the critical next step will be to collect data on the four dimensions of competence for the same host individuals in several host populations. malaria figure a depicts the distribution of traits from human and avian malaria hosts based on published and unpublished data. although humans and birds harbor distinct malaria species, the biology of both systems is similar. the rate of bites from infected vectors per host is a good proxy for malaria exposure risk [ ] ; some hosts are more conspicuous to or favored by vectors than others are (figure ai ). susceptibility, defined in this system as successful new infections relative to the number of vector bites per individual per unit time, ranges between % and % in humans [ ] , indicating that susceptibility is generally high (figure aii ). experimental inoculations reveal that, although the likelihood of an infection establishing is dose dependent, only extremely low doses are uninfective [ ] . with regard to host suitability, a survey of a community of tropical birds (l. peacock and r.h. clarke, unpublished data) revealed a preponderance of low parasitemia representing chronic infections (figure aiii ). under these conditions, only the small proportion of individuals with high parasitemia are likely to infect biting vectors. we know that suitability varies depending on host genetic and resistance factors, including immune defenses [ ] and other aspects of host physiology and life history [ ] . there is also growing evidence of the importance of host tolerance (box ) [ ] ; host genotypes that affect blood cell turnover (e.g., sickle cell anemia and b-thalassemia) can extend host lifespan postinfection [ ] . in terms of transmissibility (figure aiv ), although humans with low parasitemia (based on visual analysis of blood smears) are thought to sustain infections, the relationship between parasitemia and transmissibility is complex. for instance, high parasitemia often increases transmission probability, but exceptionally high parasitemia can negatively impact vector lifespans [ ] , meaning that intermediate parasite burden might be most transmissible. malaria parasites can increase host co and volatile compound output [ ] , making them more conspicuous to vectors. altogether for malaria, then, the most competent host would be one that is attractive to infected vectors when uninfected, attractive to uninfected vectors when infectious, and able to generate and tolerate a high enough parasite burden that parasites are taken up in vector bites. in figure b , we emphasize how one form of ec, superattracting, could lead to superspreading or superdiluting, depending on how attractiveness to vectors relates to suitability and transmissibility. lung nematode infection nematodes either have a free-living life stage before infecting a definitive host species, or they require both intermediate and definitive hosts to complete their life cycles. nematodes (and macroparasites generally) do not replicate on or in their hosts; burden is increased by exposure and decreased immunologically or through grooming. in figure c , we plotted the distributions superspreader: a host that greatly increases risk of infection for other hosts. susceptibility: the propensity of a host to become infected upon exposure to a parasite. tolerance: the relationship between parasite burden and host fitness, performance or health (box ). transmissibility: the propensity of a host to transfer parasites to another susceptible host or vector, including the sensitivity of said host to manipulative effects of parasites. n: individual reproductive number, as opposed to the average reproductive number in r , for an infection. of aspects of host competence among introduced australian cane toads and the lung nematode, rhabdias pseudosphaerocephala [ ] . parasite exposure is high for most individual hosts (figure ci ), especially when toads congregate seasonally around dwindling water supplies [ ] . most toads can also be readily infected (experimentally) with lung nematodes [ ] , suggesting that susceptibility is generally high (figure cii ). in the case of suitability (figure ciii) , toads differ in their capacity to maintain infections [ ] . finally, transmission is highly dependent on host density [ ] ; hosts from low-density areas (i.e., range edges) have low infection rates but hosts in the range core have high prevalence (figure civ ) (ii) susceptibility to parasites; (iii) suitability of a host for a parasite; and (iv) transmissibility of parasites once infected. the composite of these traits is host competence. panel a depicts existing information on competence for human and avian malaria (plasmodium and haemoproteus). exposure and transmissibility both depend on vector biting rates and are strongly right skewed in humans. by contrast, susceptibility is universally high. data from a wild tropical avian community suggest that most infections are chronic with most individuals maintaining parasite burdens insufficient for transmission to vectors (i.e., low suitability). in panel a, a malaria (vector) superattractor has high exposure risk, but it is unknown whether such hosts tend to have high or low suitability and transmissibility and thus act as superspreaders or superdiluters. red and blue circles denote traits of two different individuals in all four stages of the host-vector-parasite interaction. panel b depicts that superattracting could have either superdiluting or superspreading consequences depending on relationships between traits within hosts. white-filled symbols depict uninfected hosts, black-filled symbols depict infected hosts, blue and red symbols reflect alternate forms of competence, and green-shaded circles reflect host impacts on local transmission. in panel c, frequency distributions reflect data from invasive populations of cane toads (rhinella marina) and their lung nematodes (rhabdias pseudosphaerocephala). exposure rates are high, except at the leading edge of the geographic range of this host. susceptibility is also high ( % success rates in experimental infections), yet suitability is variable with some hosts capable of clearing worms and others less so. transmissibility is high for most hosts. whether a host with a high burden has high transmissibility depends on parasite-mediated effects on factors determining the behavior during and duration of the period over which hosts shed parasites. red and blue triangles denote traits of the two different individuals in all four stages of the host-parasite interaction. panel d depicts the two possible outcomes of different trait combinations. white-filled symbols depict uninfected hosts, black-filled symbols depict infected hosts, blue and red symbols reflect alternate forms of competence, and green-shaded circles reflect local risk. also see the supplemental information online regarding the supporting material for this figure. increase host body temperature and moisture content of feces as well as choice of defecation sites [ ] . this toad-nematode system presents an opportunity to discern whether high parasite burden equates to supershedding or superdiluting. many occurrences of supershedding are known for other macroparasites and parasites generally (table ). in figure d , we highlight how the within-individual relationship between suitability and transmissibility might lead to different forms of ec in toad hosts. parasite tolerance is a neglected aspect of host competence [ , ] , probably because it is such a new concept to disease ecology. tolerance is typically quantified by plotting a reaction norm between host fitness, health or performance (y axis) and parasite burden (x axis). in this construct, a perfectly tolerant host will have zero slope (e.g., a reaction norm parallel to the x axis), whereas a completely intolerant host will have an infinite slope (i.e., its performance will be zero at any parasite burden; figure i ). tolerance in a host population thus intervenes two extremes. the reaction norm framework produces at least three estimable parameters for quantification of competence: the intercept, slope, and area under the curve (auc). the intercept captures host vigor in the absence of infection, the slope equates to host tolerance, and auc represents the aggregate of host performance across the course of the infection, which could serve as a composite of suitability and transmissibility ( figure i ). reaction norms are estimable for genotypes, individuals, or populations, but only with repeated measures of individuals across different parasite burdens can one definitively estimate the contribution of tolerance to competence as emphasized here. for instance, superspreaders should have the largest aucs ( figure ia, host ) . in contrast, supersensitive hosts should have aucs near zero; their performance declines abruptly at very low burden ( figure ia, host ) . figure ib -d depicts other ways that tolerance could affect competence in a host population. in figure ib , the reaction norm of dispersal is plotted for two host genotypes; host has higher competence than host because of higher tolerance and concomitantly greater auc; it represents a supermover. in figure ic , two hosts differ in tolerance and vigor. host has higher performance than host in the absence of infection, exposing it to greater parasite risk. however, the auc for host is also lower than that of host , making it a superattractor. finally, in figure id , host has higher performance than host at the beginning of infection, with a positive reaction norm at early stages of infection followed by a sharp decline as parasite burden increases. in contrast, host follows an average trajectory in the population. in this case, host represents a superbreeder (terminal investor); its competence operates indirectly on a system by contributing new susceptible hosts, which could be more or less competent depending on inheritance of suitability or behavioral differences. the literature is replete with examples of superspreading and their ecological and evolutionary ramifications [ ] . however, there are data to implicate other forms of ec; many of which will have distinct consequences for epidemic risk and dynamics [ ] . going forward, perhaps the greatest need is to describe trait distributions for host competence in populations, including description of what covariation structure exists among traits within individuals [ ] . we might aspire to refine our lexicon as well, asking whether it is effective to recognize both individuals and species as ec when the former is a nested element of the latter. as the components and consequences of ec within species vary, we think it sensible to study ec at the level of individuals, scaling up to species-level composites when appropriate (see outstanding questions). we might also resolve whether the prefix 'super' connotes the form of ec that individuals manifest or the effects that such hosts have on their communities. finally, for many human diseases (including zoonoses), we have artificial mechanisms (e.g., education, prophylaxis, and institutional and social forms of hygiene) to control some infections. these conditions could make efforts to exploit knowledge about ecs difficult but often worthwhile to implement. supplemental information associated with this article can be found online at https://doi.org/ . /j.tree. . . . a few papers have summarized how ec, and particularly superspreading, might manifest physiologically and behaviorally [ ] , but several factors have yet to receive much attention. behaviorally, for vertically transmitted infections, higher rates of host reproduction, quicker maturation, or long breeding seasons could foster superspreading [ ] . migratory disposition [ ] through selection of stopover and breeding sites by infected individuals as well as local movements could affect ec depending on habitat quality [ ] . more nomadic individuals can be better spreaders of feline influenza virus [ ] and distemper [ ] , but particularly far-ranging individuals could dilute risk if their movements take them away from conspecifics until they become noninfectious or die. physiological drivers of ec are also numerous, particularly those involving the endocrine, nervous, and immune systems [ ] . one of the best known involves responses to stressors [ ] . variation in stress responses affect multiple aspects of competence in most taxa, but few studies have yet considered whether ec can arise via stress [ , , ] . symbionts that live on and within hosts might also affect ec [ ] , as these organisms sculpt host immune functions and behavior [ , ] . a final understudied but putative driver of ec is mitochondrial function. among other activities, mitochondria regulate innate immune activation, adaptive immune cell differentiation [ ] , and detection of viruses through pathogen recognition receptors [ ] . some viruses and toxoplasma gondii can hijack mitochondria to modulate host apoptosis [ ] or meet parasite nutrient needs [ ] . collectively, we expect that major advances in understanding ec will occur when host defenses are studied in an organismal manner [ , ] . syndromes between host physiology and behavior are well known, but multiple aspects of host competence (figure ) are rarely quantified in the same individuals. descriptions of trait distributions in host populations (figure ) , and covariance thereof, would resolve whether 'super' refers to the magnitude of effects of a particular individual on a system, its relative rarity, or both. we lack viable proxies of ec, but the use of experimental, laboratory organisms could provide some tools for difficult-to-study wild-animal and human systems (e.g., infection duration, exposure risk, and transmission success). some forms of ec (e.g., superdiluters) warrant attention in laboratory studies, as field methods tend not to be amenable to their discovery. laboratory studies are also more likely to reveal how different exposure doses and route of transmissions impinge on ec. dose dependency of susceptibility and parasitemia transmission thresholds warrant more experimental attention, as they will modify multiple aspects of host competence. ec variants affect communities differently depending upon niche and spatiotemporal overlap among hosts, parasite and vectors, and the quality of environments in which interactions occur. studies on the context dependency of ec will be critical. the advent of tools such as therapeutic interfering particles, which inhibit the growth of pathogens [ ] , have shown promising signs for lowering hiv/aids prevalence more effectively than vaccines or drugs alone. these tools might be impactful, as they directly target traits of ec hosts. this paper emphasizes hosts, but ec in vectors and parasites also warrants investigation. clearly, parasite and vector strains differ in virulence, infectivity, and even mutability, but how much individual heterogeneity in these traits exists is little known. the context of host competence: a role for plasticity in host-parasite dynamics 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transmission potential of tick-borne parasites variation in natural exposure to anopheles mosquitoes and its effects on malaria transmission feeder use predicts both acquisition and transmission of a contagious pathogen in a north american songbird key: cord- -v lahyw authors: van seventer, jean maguire; hochberg, natasha s. title: principles of infectious diseases: transmission, diagnosis, prevention, and control date: - - journal: international encyclopedia of public health doi: . /b - - - - . - sha: doc_id: cord_uid: v lahyw infectious disease control and prevention relies on a thorough understanding of the factors determining transmission. this article summarizes the fundamental principles of infectious disease transmission while highlighting many of the agent, host, and environmental determinants of these diseases that are of particular import to public health professionals. basic principles of infectious disease diagnosis, control, and prevention are also reviewed. an infectious disease can be defined as an illness due to a pathogen or its toxic product, which arises through transmission from an infected person, an infected animal, or a contaminated inanimate object to a susceptible host. infectious diseases are responsible for an immense global burden of disease that impacts public health systems and economies worldwide, disproportionately affecting vulnerable populations. in , infectious diseases resulted in over million years lost due to disability and over million deaths (naghavi et al., ) . lower respiratory tract infections, diarrheal diseases, hiv/ aids, malaria, and tuberculosis (tb) are among the top causes of overall global mortality (vos et al., ) . infectious diseases also include emerging infectious diseases; diseases that have newly appeared (e.g., middle east respiratory syndrome) or have existed but are rapidly increasing in incidence or geographic range (e.g., extensively drug-resistant tuberculosis (xdr tb) and zika virus (morse, ) . infectious disease control and prevention relies on a thorough understanding of the factors determining transmission. this article summarizes some of the fundamental principles of infectious disease transmission while highlighting many of the agent, host, and environmental determinants of these diseases that are of particular import to public health professionals. a classic model of infectious disease causation, the epidemiological triad (snieszko, ) , envisions that an infectious disease results from a combination of agent (pathogen), host, and environmental factors ( figure ). infectious agents may be living parasites (helminths or protozoa), fungi, or bacteria, or nonliving viruses or prions. environmental factors determine if a host will become exposed to one of these agents, and subsequent interactions between the agent and host will determine the exposure outcome. agent and host interactions occur in a cascade of stages that include infection, disease, and recovery or death (figure (a) ). following exposure, the first step is often colonization, the adherence and initial multiplication of a disease agent at a portal of entry such as the skin or the mucous membranes of the respiratory, digestive, or urogenital tract. colonization, for example, with methicillin-resistant staphylococcus aureus in the nasal mucosa, does not cause disease in itself. for disease to occur, a pathogen must infect (invade and establish within) host tissues. infection will always cause some disruption within a host, but it does not always result in disease. disease indicates a level of disruption and damage to a host that results in subjective symptoms and objective signs of illness. for example, latent tb infection is only infectionevidenced by a positive tuberculin skin test or interferon gamma release assaybut with a lack of symptoms (e.g., cough or night sweats) or signs (e.g., rales on auscultation of the chest) of disease. this is in contrast to active pulmonary tb (disease), which is accompanied by disease symptoms and signs. recovery from infection can be either complete (elimination of the agent) or incomplete. incomplete recovery can result in both chronic infections and latent infections. chronic infections are characterized by the continued detectable presence of an infectious agent. in contrast, latent infections are distinguished by an agent which can remain quiescent in host cells and can later undergo reactivation. for example, varicella zoster virus, the agent causing chicken pox, may reactivate many years after a primary infection to cause shingles. from a public health standpoint, latent infections are significant in that they represent silent reservoirs of infectious agent for future transmission. when a potential host is exposed to an infectious agent, the outcome of that exposure is dependent upon the dynamic relationship between agent determinants of infectivity, pathogenicity, and virulence, and intrinsic host determinants of susceptibility to infection and to disease (figure (b) ). environmental factors, both physical and social behavioral, are extrinsic determinants of host vulnerability to exposure. environment disease figure the epidemiological triad model of infectious disease causation. the triad consists of an agent (pathogen), a susceptible host, and an environment (physical, social, behavioral, cultural, political, and economic factors) that brings the agent and host together, causing infection and disease to occur in the host. infectivity is the likelihood that an agent will infect a host, given that the host is exposed to the agent. pathogenicity refers to the ability of an agent to cause disease, given infection, and virulence is the likelihood of causing severe disease among those with disease. virulence reflects structural and/or biochemical properties of an infectious agent. notably, the virulence of some infectious agents is due to the production of toxins (endotoxins and/or exotoxins) such as the cholera toxin that induces a profuse watery diarrhea. some exotoxins cause disease independent of infection, as for example, the staphylococcal enterotoxins that can cause foodborne diseases. agent characteristics can be measured in various ways. infectivity is often quantified in terms of the infectious dose (id ), the amount of agent required to infect % of a specified host population. id varies widely, from organisms for shigella dysenteriae to - for vibrio cholerae (gama et al., ; fda, ) . infectivity and pathogenicity can be measured by the attack rate, the number of exposed individuals who develop disease (as it may be difficult to determine if someone has been infected if they do not have outward manifestations of disease). virulence is often measured by the case fatality rate or proportion of diseased individuals who die from the disease. the outcome of exposure to an infectious agent depends, in part, upon multiple host factors that determine individual susceptibility to infection and disease. susceptibility refers to the ability of an exposed individual (or group of individuals) to resist infection or limit disease as a result of their biological makeup. factors influencing susceptibility include both innate, genetic factors and acquired factors such as the specific immunity that develops following exposure or vaccination. the malaria resistance afforded carriers of the sickle cell trait exemplifies how genetics can influence susceptibility to infectious disease (aidoo et al., ) . susceptibility is also affected by extremes of age, stress, pregnancy, nutritional status, and underlying diseases. these latter factors can impact immunity to infection, as illustrated by immunologically naïve infant populations, aging populations experiencing immune senescence, and immunocompromised hiv/aids patients. mechanical and chemical surface barriers such as the skin, the flushing action of tears, and the trapping action of mucus are the first host obstacles to infection. for example, wound infection and secondary sepsis are serious complications of severe burns which remove the skin barrier to microbial entry. lysozyme, secreted in saliva, tears, milk, sweat, and mucus, and gastric acid have bactericidal properties, and vaginal acid is microbicidal for many agents of sexually transmitted infections (stis). microbiome-resident bacteria (a.k.a. commensal bacteria, normal flora) can also confer host protection by using available nutrients and space to prevent pathogenic bacteria from taking up residence. the innate and adaptive immune responses are critical components of the host response to infectious agents ( table ) . each of these responses is carried out by cells of a distinct hematopoietic stem cell lineage: the myeloid lineage gives rise to innate immune cells (e.g., neutrophils, macrophages, dendritic cells) and the lymphoid lineage gives rise to adaptive immune cells (e.g., t cells, b cells). the innate immune response is an immediate, nonspecific response to broad groups of pathogens. by contrast, the adaptive immune response is initially generated over a period of - days, it recognizes specific pathogens, and it consists of two main branches: ( ) t cell-mediated immunity (a.k.a. cell-mediated immunity) and ( ) b cellmediated immunity (a.k.a. humoral or antibody-mediated immunity). the innate and adaptive responses also differ in table comparison of innate and adaptive immunity innate immune response adaptive immune response immediate response; initiated within seconds gradual response; initially generated over - days (primary response) targets groups of pathogens targets-specific pathogens no memory memory progression from one stage to the next is dependent upon both agent properties of infectivity, pathogenicity, and virulence, and host susceptibility to infection and disease, which is in large part due to both protective and adverse effects of the host immune response. credit: modification of original by barbara mahon, md, mph. that the latter has memory, whereas the former does not. as a consequence of adaptive immune memory, if an infectious agent makes a second attempt to infect a host, pathogenspecific memory t cells, memory b cells, and antibodies will mount a secondary immune response that is much more rapid and intense than the initial, primary response and, thus, better able to inhibit infection and disease. immune memory is the basis for the use of vaccines that are given in an attempt to stimulate an individual's adaptive immune system to generate pathogen-specific immune memory. of note, in some cases the response of the immune system to an infectious agent can contribute to disease progress. for example, immunopathology is thought to be responsible for the severe acute disease that can occur following infection with a dengue virus that is serotypically distinct from that causing initial dengue infection (screaton et al., ) . an immune host is someone protected against a specific pathogen (because of previous infection or vaccination) such that subsequent infection will not take place or, if infection does occur, the severity of disease is diminished. the duration and efficacy of immunity following immunization by natural infection or vaccination varies depending upon the infecting agent, quality of the vaccine, type of vaccine (i.e., live or inactivated virus, subunit, etc.), and ability of the host to generate an immune response. for example, a single yellow fever vaccination appears to confer lifelong immunity, whereas immune protection against tetanus requires repeat vaccination every years (staples et al., ; broder et al., ) . in malariaendemic areas, natural immunity to malaria usually develops by years of age and, while protective from severe disease and death, it is incomplete and short-lived (langhorne et al., ) . functionally, there are two basic types of immunization, active and passive. active immunization refers to the generation of immune protection by a host's own immune response. in contrast, passive immunization is conferred by transfer of immune effectors, most commonly antibody (a.k.a. immunoglobulin, antisera), from a donor animal or human. for example, after exposure to a dog bite, an individual who seeks medical care will receive both active and passive postexposure immune prophylaxis consisting of rabies vaccine (to induce the host immune response) and rabies immune globulin (to provide immediate passive protection against rabies). an example of natural passive immunization is the transfer of immunity from mother to infant during breastfeeding. vaccination does not always result in active immunization; failure of vaccination can be due to either host or vaccine issues. individuals who are immunosuppressed as, for example, a result of hiv infection, malnutrition, immunosuppressive therapy, or immune senescence might not mount a sufficient response after vaccination so as to be adequately immunized (protected). similarly, vaccination with an inadequate amount of vaccine or a vaccine of poor quality (e.g., due to break in cold chain delivery) might prevent even a healthy individual from becoming immunized. environmental determinants of vulnerability to infectious diseases include physical, social, behavioral, cultural, political, and economic factors. in some cases, environmental influences increase risk of exposure to an infectious agent. for example, following an earthquake, environmental disruption can increase the risk of exposure to clostridium tetani and result in host traumatic injuries that provide portals of entry for the bacterium. environmental factors promoting vulnerability can also lead to an increase in susceptibility to infection by inducing physiological changes in an individual. for example, a child living in a resource-poor setting and vulnerable to malnutrition may be at increased risk of infection due to malnutritioninduced immunosuppression. table provides examples of some of the many environmental factors that can facilitate the emergence and/or spread of specific infectious diseases. a unique characteristic of many infectious diseases is that exposure to certain infectious agents can have consequences for other individuals, because an infected person can act as a source of exposure. some pathogens (e.g., sti agents) are directly transmitted to other people, while others (e.g., vectorborne disease (vbd) agents) are transmitted indirectly. from a public health standpoint, it is useful to define stages of an infectious disease with respect to both clinical disease and potential for transmission ( figure ). with respect to disease, the incubation period is defined as the time from exposure to an infectious agent until the time of first signs or symptoms of disease. the incubation period is followed by the period of clinical illness which is the duration between first and last disease signs or symptoms. with respect to transmission of an infectious agent, the latent (preinfectious) period is the duration of time between exposure to an agent and the onset of infectiousness. it is followed by the infectious period (a.k.a. period of communicability) which is the time period when an infected person can transmit an infectious agent to other individuals. in parasitic infections, the latent and infectious periods are commonly referred to as the prepatent period and patent period, respectively. the duration of disease stages is unique for each type of infection and it can vary widely for a given type of infection depending upon agent, host, and environmental factors that affect, for example, dose of the inoculated agent, route of exposure, host susceptibility, and agent infectivity and virulence. knowledge of the timing of disease stages is of key importance in the design of appropriate control and prevention strategies to prevent the spread of an infectious disease. for example, efforts to control the recent ebola west africa outbreak through contact tracing and quarantine were based on knowledge that the infectious period for ebola does not begin until the start of the period of clinical illness, which occurs up to days following exposure (figure (a) ; pandey et al., ) . a carrier is, by definition, an infectious individual who is not showing clinical evidence of disease and, thus, might unknowingly facilitate the spread of an infectious agent through a population. incubatory carriers exist when the incubation period overlaps with the infectious period, as can occur in some cases of chicken pox (figure (b) ). convalescent carriers occur when the period of infectiousness extends beyond the period of clinical illness (figure (c) ). carriers of this type can be a significant issue in promoting the spread of certain enteric infections, such as those caused by the bacterium, v. cholerae. healthy carriers, infected individuals that remain asymptomatic but are capable of transmitting an infectious agent, occur commonly with many infectious diseases (e.g., meningococcal meningitis and typhoid fever) and are also significant challenges to disease control ( figure (d)). a variety of terms are used to describe the occurrence of an infectious disease within a specific geographic area or population. sporadic diseases occur occasionally and unpredictably, while endemic diseases occur with predictable regularity. levels of endemicity can be classified as holoendemic, hyperendemic, mesoendemic, or hypoendemic depending upon whether a disease occurs with, respectively, extreme, high, moderate, or low frequency. for some infectious diseases, such as malaria, levels of endemicity are well defined and used as parameters for identifying disease risk and implementing control activities. malaria endemicity is quantified based upon rates of palpable enlarged spleens in a defined (usually pediatric) age group: holoendemic > %, hyperendemic - %, mesoendemic - %, and hypoendemic < % (hay et al., ). an epidemic refers to an, often acute, increase in disease cases above the baseline level. an epidemic may reflect an escalation in the occurrence of an endemic disease or the appearance of a disease that did not previously exist in a population. the term outbreak is often used synonymously with epidemic but can occasionally refer to an epidemic occurring in a more limited geographical area; for example, a foodborne illness associated with a group gathering. by contrast, a pandemic is an epidemic that has spread over a large geographic region, encompassing multiple countries or continents, or extending worldwide. influenza commonly occurs as a seasonal epidemic, but periodically it gives rise to a global pandemic, as was the case with h n influenza. two fundamental measures of disease frequency are prevalence and incidence. prevalence is an indicator of the number of existing cases in a population as it describes the proportion of individuals who have a particular disease, measured either at a given point in time (point prevalence) or during a specified time period (period prevalence). in contrast, incidence (a.k.a. incidence rate) is a measurement of the rate at which new cases of a disease occur (or are detected) in a population over a given time period. usually measured as a proportion (number infected/number exposed), attack rates are often calculated during an outbreak. in some circumstances, a secondary attack rate is calculated to quantify the spread of disease to susceptible exposed persons from an index case (the case first introducing an agent into a setting) in a circumscribed population, such as in a household or hospital. during the sars epidemic, secondary attack rates in toronto hospitals were high but varied from % to % depending upon the hospital ward (cdc, b) . the basic reproductive number (basic reproductive ratio; r ) is a measure of the potential for an infectious disease to spread through an immunologically naïve population. it is defined as the average number of secondary cases generated by a single, infectious case in a completely susceptible population. in reality, for most infectious diseases entering into a community, some proportion of the population is usually immune (and nonsusceptible) due to previous infection and/or immunization. thus, a more accurate reflection of the potential for community disease spread is the effective reproductive number (r) which measures the average number of new infections due to a single infection. in general, for an epidemic to occur in a population, r must be > so that the number of cases continues to increase. herd immunity (a.k.a. community immunity) refers to population-level resistance to an infectious disease that occurs when there are enough immune individuals to block the chain of infection/transmission. as a result of herd immunity, susceptible individuals who are not immune themselves are indirectly protected from infection ( figure ). vaccine hesitancy, the choice of individuals or their caregivers to delay or decline vaccination, can lead to overall lower levels of herd immunity. outbreaks of measles in the united states, including a large measles outbreak at an amusement park in california, highlight the phenomena of vaccine refusal and associated increased risk for vaccine-preventable diseases among both nonvaccinated and fully vaccinated (but not fully protected) individuals (phadke et al., ) . an important public health consequence of herd immunity is that immunization coverage does not need to be % for immunization programs to be successful. the equation r ¼ r ( À x) (where x equals the immune portion of the population) indicates the level of immunization required to prevent the spread of an infectious disease through a population. the proportion that needs to be immunized depends on the pathogen (table ) . when the proportion immunized (x) reaches a level such that r < , a chain of infection cannot be sustained. thus, ro and r can be used to calculate the target immunization coverage needed for the success of vaccination programs. proper diagnosis of infectious illnesses is essential for both appropriate treatment of patients and carrying out prevention and control surveillance activities. two important properties that should be considered for any diagnostic test utilized are sensitivity and specificity. sensitivity refers to the ability of the test to correctly identify individuals infected with an agent ('positive in disease'). a test that is very sensitive is more likely to pick up individuals with the disease (and possibly some without the disease); a very sensitive test will have few false negatives. specificity is the ability of the test to correctly identify individuals not infected by a particular agent ('negative in health'); high specificity implies few false positives. often, screening tests are highly sensitive (to capture any possible cases), and confirmatory tests are more specific (to rule out false-positive screening tests). broadly, laboratory diagnosis of infectious diseases is based on tests that either directly identify an infectious agent or provide evidence that infection has occurred by documenting agent-specific immunity in the host ( figure ). identification of an infecting agent involves either direct examination of host specimens (e.g., blood, tissue, urine) or environmental specimens, or examination following agent culture and isolation from such specimens. the main categories of analyses used in pathogen identification can be classified as phenotypic, revealing properties of the intact agent, nucleic acid-based, determining agent nucleic acid (dna or rna) characteristics and composition, and immunologic, detecting microbial antigen or evidence of immune response to an agent ( figure ). direct phenotypic analyses include both macroscopic and/or microscopic examination of specimens to determine agent morphology and staining properties. cultured material containing large quantities of agent can undergo analyses to determine characteristics, such as biochemical enzymatic activity (enzymatic profile) and antimicrobial sensitivity, and to perform phage typing, a technique which differentiates bacterial strains according to the infectivity of strain-specific bacterial viruses (a.k.a. bacteriophages). nucleic acid-based tests often make use of the polymerase chain reaction (pcr) to amplify agent dna or complementary dna (cdna) synthesized from messenger rna (mrna). the ability of pathogen-specific pcr primers to generate an amplification product can confirm or rule out involvement of a specific pathogen. sequencing of amplified dna fragments can also assist with pathogen identification. restriction fragment analysis, as by pulse-field gel electrophoresis of restriction enzyme-digested genomic dna isolated from cultured material, can yield distinct 'dna fingerprints' that can be used for comparing the identities of bacteria. the cdc pulsenet surveillance program uses dna fingerprinting as the basis for detecting and defining foodborne disease outbreaks that can sometimes be quite widely dispersed (cdc, ) . most recently, next-generation sequencing technologies have made whole-genome sequencing a realistic subtyping method for use in foodborne outbreak investigation and surveillance (deng et al., ) . the objective of immunologic analysis of specimens is to reveal evidence of an agent through detection of its antigenic components with agent-specific antibodies. serotyping refers to the grouping of variants of species of bacteria or viruses based on shared surface antigens that are figure herd immunity occurs when one is protected from infection by immunization occurring in the community. using influenza as an example, the top box shows a population with a few infected individuals (shown in red) and the rest healthy but unimmunized (shown in blue); influenza spreads easily through the population. the middle box depicts the same population but with a small number who are immunized (shown in yellow); those who are immunized do not become infected, but a large proportion of the population becomes infected. in the bottom box, a large proportion of the population is immunized; this prevents significant transmission, including to those who are unimmunized. source: national institute of allergy and infectious diseases (niaid). identified using immunologic methodologies such as enzymelinked immunosorbent assay (elisa) and western blotting. immunologic assays are also used to look for evidence that an agent-specific immune response has occurred in an exposed or potentially exposed individual. serologic tests detect pathogen-specific b cell-secreted antibodies in serum or other body fluids. some serologic assays simply detect the ability of host antibodies to bind to killed pathogen or components of pathogen (e.g., elisa). others rely on the ability of antibodies to actually neutralize the activity of live microbes; as, for example, the plaque reduction neutralization test which determines the ability of serum antibodies to neutralize virus. antibody titer measures the amount of a specific antibody present in serum or other fluid, expressed as the greatest dilution of serum that still gives a positive test in whatever assay is being employed. intradermal tests for identification of t cell-mediated immediate type (type i) hypersensitivity or delayed type (type iv) hypersensitivity responses to microbial antigen can be used to diagnose or support the diagnosis of some bacterial, fungal, and parasitic infections, such as, the mantoux (tuberculin) test for tb. based on the classic model of leavell and clark ( ) , infectious disease prevention activities can be categorized as primary, secondary, or tertiary. primary prevention occurs at the predisease phase and aims to protect populations, so that infection and disease never occur. for example, measles immunization campaigns aim to decrease susceptibility following exposure. the goal of secondary prevention is to halt the progress of an infection during its early, often asymptomatic stages so as to prevent disease development or limit its severity; steps important for not only improving the prognosis of individual cases but also preventing infectious agent transmission. for example, interventions for secondary prevention of hepatitis c in injection drug user populations include early diagnosis and treatment by active surveillance and screening (miller and dillon, ) . tertiary prevention focuses on diseased individuals with the objective of limiting impact through, for example, interventions that decrease disease progression, increase functionality, and maximize quality of life. broadly, public health efforts to control infectious diseases focus on primary and secondary prevention activities that reduce the potential for exposure to an infectious agent and increase host resistance to infection. the objective of these activities can extend beyond disease control, as defined by the dahlem workshop on the eradication of infectious diseases, to reach objectives of elimination and eradication (dowdle, ; box ). as noted earlier, the causation and spread of an infectious disease is determined by the interplay between agent, host, and environmental factors. for any infectious disease, this interplay requires a specific linked sequence of events termed the chain of infection or chain of transmission ( figure ). the chain starts with the infectious agent residing and multiplying in some natural reservoir; a human, animal, or part of the environment such as soil or water that supports the existence of the infectious agent in nature. the infectious agent leaves the reservoir via a portal of exit and, using some mode of transmission, moves to reach a portal of entry into a susceptible host. a thorough understanding of the chain of infection is crucial for the prevention and control of any infectious disease, as breaking a link anywhere along the chain will stop transmission of the infectious agent. often more than one intervention can be effective in controlling a disease, and the approach selected will depend on multiple factors such as economics and ease with which an intervention can be executed in a given setting. it is important to realize that the potential for rapid and far-reaching movement of infectious agents that has accompanied globalization means that coordination of intervention activities within and between nations is required for optimal prevention and control of certain diseases. the cause of any infectious disease is the infectious agent. as discussed earlier, many types of agents exist, and each can be characterized by its traits of infectivity, pathogenicity, and virulence. a reservoir is often, but not always, the source from which the agent is transferred to a susceptible host. for example, bats are both the reservoir for marburg virus and a source of infection for humans and bush animals including african gorillas. however, because morbidity and mortality due to marburg infection is significant among these bush animals, they cannot act as a reservoir to sustain the virus in nature (they die too quickly), although they can act as a source to transmit marburg to humans. infectious agents can exist in more than one type of reservoir. the number and types of reservoirs are important determinants of how easily an infectious disease can be prevented, controlled, and, in some cases, eliminated or eradicated. animal, particularly wild animal, reservoirs, and environmental reservoirs in nature can be difficult to manage and, thus, can pose significant challenges to public health control efforts. in contrast, infectious agents that only occur in human reservoirs are among the dahlem workshop on the eradication of infectious diseases defined a continuum of outcomes due to public health interventions targeting infectious diseases: " ) control, the reduction of disease incidence, prevalence, morbidity or mortality to a locally acceptable level as a result of deliberate efforts; continued intervention measures are required to maintain the reduction (e.g. diarrheal diseases), ) elimination of disease, reduction to zero of the incidence of a specified disease in a defined geographical area as a result of deliberate efforts; continued intervention measures are required (e.g. neonatal tetanus), ) elimination of infections, reduction to zero of the incidence of infection caused by a specific agent in a defined geographical area as a result of deliberate efforts; continued measures to prevent re-establishment of transmission are required (e.g. measles, poliomyelitis), ) eradication, permanent reduction to zero of the worldwide incidence of infection caused by a specific agent as a result of deliberate efforts; intervention measures are no longer needed (e.g. smallpox), and ) extinction, the specific infectious agent no longer exists in nature or in the laboratory (e.g. none)" (dowdle, ) . the chain of infection (a.k.a. chain of transmission). one way to visualize the transmission of an infectious agent though a population is through the interconnectedness of six elements linked in a chain. public health control and prevention efforts focus on breaking one or more links of the chain in order to stop disease spread. those most easily targeted, as illustrated by the success of smallpox eradication. humans are the reservoir for many common infectious diseases including stis (e.g., hiv, syphilis) and respiratory diseases (e.g., influenza). humans also serve as a reservoir, although not always a primary reservoir, for many neglected tropical diseases (ntds) as, for example, dracunculiasis (a.k.a. guinea worm). from a public health standpoint, an important feature of human reservoirs is that they might not show signs of illness and, thus, can potentially act as unrecognized carriers of disease within communities. the classic example of a human reservoir is the cook mary mallon (typhoid mary); an asymptomatic chronic carrier of salmonella enterica serovar typhi who was linked to at least cases of typhoid fever (soper, ) . animals are a reservoir for many human infectious diseases. zoonosis is the term used to describe any infectious disease that is naturally transmissible from animals to humans. these diseases make up approximately % of all infectious diseases, and an estimated % of recently emerging infectious diseases (burke et al., ) . zoonotic reservoirs and sources of human disease agents include both domestic (companion and production) animals (e.g., dogs and cows) and wildlife. control and prevention of zoonotic diseases requires the concerted efforts of professionals of multiple disciplines and is the basis for what has become known as the one health approach (gibbs, ) . this approach emphasizes the interconnectedness of human health, animal health, and the environment and recognizes the necessity of multidisciplinary collaboration in order to prevent and respond to public health threats. inanimate matter in the environment, such as soil and water, can also act as a reservoir of human infectious disease agents. the causative agents of tetanus and botulism (clostridium tetani and c. botulinum) are examples of environmental pathogens that can survive for years within soil and still remain infectious to humans. legionella pneumophila, the etiologic agent of legionnaires' disease, is part of the natural flora of freshwater rivers, streams, and other bodies. however, the pathogen particularly thrives in engineered aquatic reservoirs such as cooling towers, fountains, and central air conditioning systems, which provide conditions that promote bacterial multiplication and are frequently linked to outbreaks. soil and water are also sources of infection for several protozoa and helminth species which, when excreted by a human reservoir host, can often survive for weeks to months. outbreaks of both cryptosporidiosis and giardiasis commonly occur during summer months as a result of contact with contaminated recreational water. soil containing roundworm (ascaris lumbricoides) eggs is an important source of soil-transmitted helminth infections in children. early steps in preventing exposure to an infectious agent include interventions to control or eliminate the agent within its reservoir, to neutralize or destroy the reservoir, and/or to stop the agent from exiting its reservoir. central to these interventions are surveillance activities that routinely identify disease agents within reservoirs. when humans are the reservoir, or source, of an infectious agent, early and rapid diagnosis and treatment are key to decreasing the duration of infection and risk of transmission. both active surveillance and passive surveillance are used to detect infected cases and carriers. some readily communicable diseases, such as ebola, can require isolation of infected individuals to minimize the risk of transmission. as part of the global effort to eradicate dracunculiasis, several endemic countries have established case containment centers to provide treatment and support to patients with emerging guinea worms to keep them from contaminating water sources and, thereby, exposing others (hochberg et al., ) . contact tracing and quarantine are other activities employed in the control of infections originating from a human reservoir or source. during the west africa ebola outbreak, key control efforts focused on the tracing and daily follow-up of healthy individuals who had come in contact with ebola patients and were potentially infected with the virus (pandey et al., ) . one health emphasizes the importance of surveillance and monitoring for zoonotic pathogens in animal populations. for some diseases (e.g., rift valley fever) epizootics (analogous to epidemics, but in animal populations) can actually serve as sentinel events for forecasting impending human epidemics. once animal reservoirs (and sources) of infection are identified, approaches to prevention and control include reservoir elimination and prevention of reservoir infection. zoonotic diseases exist in nature in predictably regular, enzootic cycles and/or epizootic cycles and are transmitted to humans via distinct pathways. the focus of prevention and control activities for these diseases reflects the extent to which a zoonotic pathogen has evolved to become established in human populations (wolfe et al., ) . for some zoonotic diseases (e.g. anthrax, nipah, rabies), primary transmission always occurs from animals, with humans acting as incidental (dead end) hosts; control of these diseases thus concentrates on preventing animal-to-animal and, ultimately, animal-to-human transmission. currently, most human cases of avian influenza are the result of human infection from birds; human-to-human transmission is extremely rare. thus, reservoir elimination by culling infected poultry flocks is a recommended measure for controlling avian influenza in birds and preventing sporadic infection of humans (cdc, ) . other zoonotic diseases demonstrate varying degrees of secondary human-to-human transmission following primary transmission (a.k.a. spillover) from animals. both rates of spillover and the ability to sustain human-tohuman transmission can vary widely between zoonoses and, in consequence, control strategies can also be quite different. for example, outbreaks of ebola arise following an initial bush animal-to-human transmission event, and subsequent human-to-human transmission is often limited (feldmann and geisbert, ) . in contrast, the four dengue viruses originally emerged from a sylvatic cycle between non-human primates and mosquitoes, and are now sustained by a continuous human-mosquito-human cycle of transmission with outbreaks occurring as a result of infected individuals entering into naïve populations (vasilakis et al., ) . thus, while ebola outbreak prevention efforts would include limiting contact with bush animals, such efforts would not be useful for prevention of dengue outbreaks. hiv is an example of a virus that emerged from an ancestral animal virus, simian immunodeficiency virus, but has evolved so that it is now hiv is an example of a virus that emerged from an ancestral animal virus, simian immunodeficiency virus, but has evolved so that it is now only transmitted human to human (faria et al., ) . infectious agents exit human and animal reservoirs and sources via one of several routes which often reflect the primary location of disease; respiratory disease agents (e.g., influenza virus) usually exit within respiratory secretions, whereas gastrointestinal disease agents (e.g., rotavirus, cryptosporidium spp.) commonly exit in feces. other portals of exits include sites from which urine, blood, breast milk, and semen leave the host. for some infectious diseases, infection can naturally occur as a result of contact with more than one type of bodily fluid, each of which uses a different portal of exit. while infection with the sars virus most frequently occurred via contact with respiratory secretions, a large community outbreak was caused by the spread of virus in a plume of diarrhea (yu et al., ) . control interventions targeting portals of exit and entry are discussed below. there are a variety of ways in which infectious agents move from a natural reservoir to a susceptible host, and several different classification schemes are used. the scheme below categorizes transmission as direct transmission, if the infective form of the agent is transferred directly from a reservoir to an infected host, and indirect transmission, if transfer takes place via a live or inanimate intermediary (box ). direct physical contact between the skin or mucosa of an infected person and that of a susceptible individual allows direct transfer of infectious agents. this is a mode of transmission for most stis and many other infectious agents, such as bacterial and viral conjunctivitis (a.k.a. pink eye) and ebola virus disease. direct droplet transmission occurs after sneezing, coughing, or talking projects a spray of agent-containing droplets that are too large to remain airborne over large distances or for prolonged periods of time. the infectious droplets traverse a space of generally less than m to come in contact with the skin or mucosa of a susceptible host. many febrile childhood diseases, including the common cold, are transferred this way. diseases spread by direct contact and droplet transmission require close proximity of infected and susceptible individuals and, thus, commonly occur in settings such as households, schools, institutions of incarceration, and refugee/displaced person camps. infectious agents spread exclusively in this manner are often unable to survive for long periods outside of a host; direct transmission helps to ensure transfer of a large infective dose. direct contact to an agent in the environment is a means of exposure to infectious agents maintained in environmental reservoirs. diseases commonly transmitted in this manner include those in which the infectious agent enters a susceptible host via inhalation (e.g., histoplasmosis) or through breaks in the skin following a traumatic event (e.g., tetanus). animal bites are another way in which some infectious agents are directly transferred, through broken skin. this is the most common means of infection with rabies virus. transplacental (a.k.a. congenital, vertical) and perinatal transmissions occur during pregnancy and delivery or breastfeeding, respectively. classic examples include mother-to-child transmission of the protozoa toxoplasma gondii during pregnancy, hiv during pregnancy, delivery, or breastfeeding, and zika virus during pregnancy (rasmussen et al., ) . case finding and contact tracing are public health prevention and control activities aimed at stopping the spread of infectious diseases transmitted by either direct contact or direct spread of droplets. once identified, further activities to limit transmission to susceptible individuals can involve definitive diagnosis, treatment, and, possibly, isolation of active cases and carriers, and observation, possible quarantine, or prophylactic vaccination or treatment of contacts. patient education is an important feature of any communicable infectious disease control effort. environmental changes, such as decreasing overcrowded areas and increasing ventilation, can also contribute to limiting the spread of some infectious diseases, particularly respiratory diseases. central to prevention of transplacental and perinatal infectious disease transmission is avoidance of maternal infection and provision of early diagnosis and treatment of infected women prior to or during pregnancy. for example, public health efforts targeting congenital toxoplasmosis focus on preventing pregnant women from consuming undercooked meat or contacting cat feces that may be contaminated. current who guidelines for prevention of mother-to-child hiv transmission recommend that hiv-infected pregnant and breastfeeding women should be maintained on antiretrovirals (who, ) . there are three main categories of indirect transmission: biological, mechanical, and airborne. box provides definitions of the different types of hosts, vectors, and vehicles involved in the life cycle of agents that are transmitted indirectly. biological transmission occurs when multiplication and/or development of a pathogenic agent within a vector (e.g., biological vector or intermediate host) is required for the agent to become infectious to humans. the time that is necessary for these events to occur is known as the extrinsic incubation period; in contrast to the intrinsic incubation period which is the time required for an exposed human host to become infectious. indirect transmission by mosquito vectors is the primary mode of transmission of a large number of viruses (arthropod-borne viruses or arboviruses) of public health concern (e.g., west nile, zika). a number of ntds are also transmitted by biological vectors including lymphatic filariasis (a.k.a. elephantiasis) by mosquitoes. ticks are biological vectors for many bacterial etiological agents (e.g., lyme disease and ehrlichiosis), and the parasitic agent causing babesiosis. the infectious agent of the helminthic ntds, schistosomiasis, and dracunculiasis are transmitted indirectly via intermediate freshwater snail and copepod hosts, respectively. mechanical transmission does not require pathogen multiplication or development within a living organism. it occurs when an infectious agent is physically transferred by a live entity (mechanical vector) or inanimate object (vehicle) to a susceptible host. classic examples of diseases spread by mechanical vector transmission are shigellosis (transmission of shigella spp. on the appendages of flies) and plague (transmission of yersinia pestis by fleas). many diarrheal diseases are transmitted by the fecal-oral route with food and water often acting as vehicles (figure ) . other types of vehicles for infectious disease agents are biologic products (e.g., blood, organs for transplant) and fomites (inanimate objects such as needles, surgical instruments, door handles, and bedding). transfusion-related protozoal infection resulting in chagas disease has been of increasing concern to the us blood banks that have instituted screening measures (cdc, ) . airborne transmission involves aerosolized suspensions of residue (less than five microns in size, from evaporated aerosol droplets) or particles containing agents that can be transported over time and long distance and still remain infective. tb is a classic example of an infectious disease often spread by airborne transmission. vbds comprise approximately % of the global burden of infectious diseases (townson et al., ) . for some diseases (e.g., dengue, zika, chagas), chemoprophylaxis and immunoprophylaxis are not prevention and control options, leaving vector control as the primary means of preventing disease transmission. integrated vector management is defined by the who as, "a rational decision-making process to optimize the use of resources for vector control" (who, ) . there are four major categories of ivm vector control strategies: biological, chemical, environmental, and mechanical. ivm interventions are chosen from these categories based upon available resources, local patterns of disease transmission, and ecology of local disease vectors. two key elements of ivm are collaboration within the health sector and with other sectors (e.g., agriculture, engineering) to plan and carry out vector control activities, and community engagement to promote program sustainability. another core element is the integrated approach which often permits concurrent targeting of multiple vbds, as some diseases are transmitted by a single, common vector, and some vector control interventions can target several different vectors. in addition, combining interventions serves not only to reduce reliance on any single intervention, but also to reduce the selection pressure for insecticide and drug resistance. table , adapted from the who handbook for ivm, illustrates some of the many types of ivm activities and provides examples of vbds that might be controlled by such interventions (who, ) . diarrheal diseases primarily result from oral contact with water, food, or other vehicles contaminated with pathogenic agents originating from human or animal feces. most ($ %) of diarrhea-associated deaths are attributable to unsafe drinking water, inadequate sanitation, and insufficient hygiene (black et al., ; prüss-Üstün et al., ) . interruption of fecaloral transmission through provision of safe water and adequate sanitation, and promotion of personal and domestic hygiene are fundamental to diarrhea prevention and control. fecaloral transmission of a diarrheal agent can occur via one of several routes. in , wagner and lanoix developed a model of major transmission depicted in what has become known as the 'f-diagram,' based on steps within the fecal-oral flow of transmission starting with the letter 'f': fluids (drinking water), definitive host: a host in which a parasite reproduces sexually. humans are definitive hosts for roundworms. by strict definition, mosquitoes are the definitive host of malaria as they are the organism in which sexual reproduction of the agent protozoa, plasmodium spp., occurs. reservoir host: a host that serves to sustain an infectious pathogen as a potential source of infection for transmission to humans. note that a reservoir host will not succumb to infection. lowland gorillas and chimpanzees can be infected by ebola virus, but they are not a reservoir host as they suffer devastating losses from infection. bats are a suspected reservoir for ebola virus. intermediate dead-end host: a host from which infectious agents cannot be transmitted to other susceptible hosts. humans are a dead-end host for west nile virus which normally circulates between mosquitoes and certain avian species. vector: a generic term for a living organism (e.g., biological vector or intermediate host) involved in the indirect transmission of an infectious agent from a reservoir or infected host to a susceptible host. biological vector: a vector (often arthropod) in which an infectious organism must develop or multiply before the vector can transmit the organism to a susceptible host. aedes spp. mosquitoes are a biological vector for dengue, chikungunya, and zika. mechanical vector: a vector (often arthropod) that transmits an infectious organism from one host to another but is not essential to the life cycle of the organism. the house fly is a mechanical vector in the diarrheal disease shigellosis as it carries feces contaminated with the shigella spp. bacterium to a susceptible person. vehicles: inanimate objects that serve as an intermediate in the indirect transmission of a pathogen from a reservoir or infected host to a susceptible host. these include food, water, and fomites such as doorknobs, surgical instruments, and used needles. fingers, flies, fields (crops and soil), floods (representative of surface water in general), and food (wagner and lanoix, ; figure ). other f's that can be considered include facilities (e.g., settings where transmission is likely to occur such as daycare centers) and fornication. the f-diagram is useful for depicting where water, sanitation, and hygiene (wash) interventions act as barriers in the fecal-oral flow of diarrheal pathogens. safe excreta disposal and handling act as primary barriers to transmission by preventing fecal pathogens from entering the environment. once the environment has become contaminated with pathogen-containing feces, secondary and tertiary barriers to transmission include water treatment, safe transport and storage of water, provision of sewage systems to control flooding, fly control, and good figure the 'f-diagram' illustrates major direct and indirect pathways of fecal-oral pathogen transmission and depicts the roles of water, sanitation, and hygiene interventions in providing barriers to transmission. primary barriers prevent contact with feces, and secondary barriers prevent ingestion of feces. source: water, engineering and development centre (wedc), loughborough university. personal and domestic hygiene (e.g., food hygiene) practices (requiring adequate water quantity) ( figure ) . as with ivm, the control of diarrheal diseases increases with integration of control measures to achieve multiple barriers to fecal-oral transmission. the basic approach to preventing transmission of an infectious agent from a contaminated vehicle is to prevent contamination of, decontaminate, or eliminate the vehicle. food is a common vehicle for infectious agents, and it can potentially become contaminated at any step along the food production chain of production, processing, distribution, and preparation. production refers to the growing of plants for harvest and raising animals for food. an example of contamination at this step includes the use of fecally contaminated water for crop irrigation. processing refers to steps such as the chopping, grinding, or pasteurizing of food to convert it into a consumer product; if the external surface of a melon is contaminated, chopping it into pieces for sale can result in contamination of the fruit. distribution, in which food is transferred from the place where it was produced and/or processed to the consumer, can result in contamination if, for example, the transportation vehicle is not clean. finally, preparation is the step in which food is made ready to eat; not cleaning a cutting board after cutting raw chicken can result in microbial pathogen crosscontamination of other food items. food hygiene is the term used to describe the conditions and activities employed to prevent or limit microbial contamination of food in order to ensure food safety. decontamination includes sterilization, the destruction of all microbial agents, and disinfection, the destruction of specific agents. control of airborne diseases focuses on regulating environmental airflow and air quality to minimize contact with infectious droplet nuclei. in health-care settings, negative pressure isolation rooms and exhaust vents can be used to manipulate airflow. recirculating, potentially infectious air can undergo high-efficiency particulate air (hepa) filtration and/or be mixed with 'clean' (noncontaminated) air to remove or dilute the concentration of infectious particle to below the infectious dose. health-care workers should use n masks. on commercial aircraft, airborne pathogen transmission is minimized by methods including regulating airflow to prevent widespread dispersal of airborne microbes throughout the cabin, hepa filtering recirculating air, and mixing recirculating air with fresh air (considered sterile) (dowdall et al., ) . the portal of entry refers to the site at which the infectious agent enters a susceptible host and gains access to host tissues. many portals of entry are the same as portals of exit and include the gastrointestinal, genitourinary, and respiratory tracts, as well as compromised skin and mucous membrane surfaces. some infectious agents can naturally enter a susceptible host by more than one portal. for example, the three forms of human anthrax can be distinguished according to the route of agent entry: cutaneous anthrax due to entry through the skin, gastrointestinal anthrax resulting from ingestion of spores, and pulmonary anthrax following inhalation of spores. standard infection control practices target portals of exit (and entry) of infectious agents from human reservoirs and sources. cdc guidelines suggest two levels of precautions to stop transmission of infectious agents: standard precautions and transmission-based precautions (siegel et al., ) . standard precautions prevent transmission of infectious agents that can be acquired by contact with blood, body fluids, nonintact skin, and mucous membranes. they can be used to prevent transmission in both health-care and non-health-care settings, regardless of whether infection is suspected or confirmed. hand hygiene is a major component of these precautions, along with use of personal protective equipment (ppe). common ppe include gloves, gowns, face protection (e.g., eye-protecting face shields), and respiratory protection using n masks to prevent inhalation of airborne infectious particles (e.g., from mycobacterium tuberculosis). of note, depending on the circumstance, ppe can be used to prevent dispersal of infectious agents from their source by providing a barrier to the portal of exit, or to protect a susceptible individual by placing a barrier to a portal of entry. respiratory hygiene/cough etiquette is used to prevent spread of infection by respiratory droplets. main elements of respiratory hygiene/cough etiquette include covering the nose and mouth area with one's elbow during coughing or sneezing or using a surgical mask to limit dissemination of infectious respiratory secretions, and hand hygiene after contact with respiratory secretions. other components of standard precautions include needle stick and sharp injury prevention, safe injection practices, cleaning and disinfection of potentially contaminated equipment and other objects, and safe waste management. a susceptible host is an individual who is at risk of infection and disease following exposure to an infectious agent. as discussed previously, there are many determinants of host susceptibility, including both innate factors determined by the genetic makeup of the host and, acquired factors such as agent-specific immunity and malnutrition. important prevention and control interventions that target the susceptible host include both those that address determinants of susceptibility in the host (e.g., immunoprophylaxis, provision of adequate nutrition, treatment of underlying diseases) and those that target an infecting agent (e.g., chemoprophylaxis). immunoprophylaxis encompasses both active immunization by vaccination and passive immunization through provision of pathogen-specific immunoglobulin. malnutrition is a strong risk factor for morbidity and mortality due to diarrheal disease, and a vicious cycle exists between infectious diarrheal disease leading to malnutrition and impaired immune function which, in turn, promotes increased susceptibility to infection (keusch et al., ) . consequently, breastfeeding and safe complementary feeding play crucial roles in protecting infants and young children from infectious diseases, particularly in resource-poor settings. micronutrients are required for normal immune function, and vitamin a and zinc supplementations have been shown to decrease some types of infections in children deficient in these micronutrients (mayo-wilson et al., ; imdad et al., ) . in certain circumstances, chemoprophylaxis is employed to protect a susceptible host in anticipation of, or following exposure to an infectious agent. antimalarial drugs are routinely used in combination with personal protective measures to prevent malaria in travelers and established guidelines exist for antibiotic prophylaxis prior to surgery. another important element in the prevention and control of infections is the recognition and management of patients with underlying diseases and conditions that can weaken host barriers to infection. for example, tb is the leading opportunistic infection in hivinfected individuals, and antiretroviral therapy reduces risk of developing tb and mortality due to tb disease. infectious complications are a major cause of morbidity and mortality in cancer and transplant patients, often resulting from immunosuppression that can be primary or related to drug and/or radiation therapy. infectious disease control is also critical in individuals with compromised physical barriers to microbes as, for example, burn patients and patients with cystic fibrosis. dr william h stewart, the one-time surgeon general of the united states, has been quoted (perhaps mistakenly) as saying in the s "it is time to close the book on infectious diseases, and declare the war against pestilence won (spellberg, ) ." these words clearly do not hold true today, and public health practitioners wage an ever-growing fight against emerging pathogens, drug-resistant organisms, and vaccine-preventable diseases. in this light, it is all the more important that we have the tools needed to understand transmission dynamics and implement effective prevention and control programs. clear definitions of terminology and elucidation of fundamental principles lay the foundation for effective public health interventions. hopefully, this article helps strengthen the armamentarium of the public health 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communities. who consolidated guidelines on the use of antiretroviral drugs for treating and preventing hiv infection large outbreak of cryptosporidium hominis infection transmitted through the public water supply origins of major human infectious diseases impact of deforestation and agricultural development on anopheline ecology and malaria epidemiology evidence of airborne transmission of the severe acute respiratory syndrome virus key: cord- - ebmq t authors: duplantier, allen j.; shurtleff, amy c.; miller, cheryl; chiang, chih-yuan; panchal, rekha g.; sunay, melek title: combating biothreat pathogens: ongoing efforts for countermeasure development and unique challenges date: - - journal: drug discovery targeting drug-resistant bacteria doi: . /b - - - - . - sha: doc_id: cord_uid: ebmq t research to discover and develop antibacterial and antiviral drugs with potent activity against pathogens of biothreat concern presents unique methodological and process-driven challenges. herein, we review laboratory approaches for finding new antibodies, antibiotics, and antiviral molecules for pathogens of biothreat concern. using high-throughput screening techniques, molecules that directly inhibit a pathogen’s entry, replication, or growth can be identified. alternatively, molecules that target host proteins can be interesting targets for development when countering biothreat pathogens, due to the modulation of the host immune response or targeting proteins that interfere with the pathways required by the pathogen for replication. monoclonal and cocktail antibody therapies approved by the food and drug administration for countering anthrax and under development for treatment of ebola virus infection are discussed. a comprehensive tabular review of current in vitro, in vivo, pharmacokinetic and efficacy datasets has been presented for biothreat pathogens of greatest concern. finally, clinical trials and animal rule or traditional drug approval pathways are also reviewed. opinions; interpretations; conclusions; and recommendations are those of the authors and are not necessarily endorsed by the us army. the concept of bioterrorism and the intentional release of biothreat agents for purposes of harm to human and agricultural interests stimulates discussion of some unanswerable questions. questions ranging from protection of a nation's security to military defense tactics, all point to the gravity of the problem for which scientists are working together in many areas of study such as the development of novel medical countermeasures to combat lethal infections, the prevention of the spread of disease in the general populace, and design of field-worthy diagnostic tools. a biothreat organism is generally thought to be one causing severe or lethal disease or has potential to induce panic over the prospect of infection therewith; one with high pathogenicity and/or contagious infectivity; one with strong environmental stability or probable transmission as an aerosol; one with ease of large-scale production for far-reaching dissemination; and one that can be controlled for directing the release to only the intended target rather than accidental harm to the perpetrator [ ] . improved preparedness for intentional release of bacteria, viruses, and toxins will not only protect military positions and strategies but will also increase ability to combat disease in naturally occurring epidemics of diseases caused by some of these organisms. the priorities for the development of medical countermeasures against these organisms have been defined through international discussions [ ] [ ] [ ] . currently classified as tier select agents are those pathogens of grave concern, whereas other useful classification categories are in use by us government entities such as national institute of allergy and infectious diseases and the us centers for disease control and prevention (cdc) list, denoting the pathogens as category a, b, and c agents [ ] . various biothreat pathogens addressed in this chapter are grouped by general category and disease associated therewith (table . ). there are many organisms on the cdc list; consequently, not all of them are addressed in this chapter [ ] . the authors of this chapter have endeavored to provide a comprehensive survey of the literature and described the development hemorrhagic fever a crimean-congo hemorrhagic fever virus hemorrhagic fever a, b, c denote additional categorization into category a, b, and c pathogens, per niaid [ ] . a tier agents of human pathogenicity are presented [ ] . two more tier agents are rinderpest virus and foot-and-mouth disease virus, which are of agricultural concern (not covered in this chapter). b important non-tier agents for which countermeasures are described in this chapter [ , ] . see www.selectagents.gov for a comprehensive list of non-tier select agents and toxins.sars, severe acute respiratory syndrome; mers, middle east respiratory syndrome-related coronavirus. of medical countermeasures against high-priority bacterial and viral biothreat agents where the most progress has been made, and/or the most novel ground has been broken. bacteria cause disease in humans by invading tissue, altering the host immune response, and/or producing toxins or virulence factors. many of the bacteria described here are difficult to treat clinically. the potential bacterial threat agents that pose the greatest risk to national security are ones that can be easily disseminated and result in high morbidity and mortality rates. the former soviet union is known to have weaponized at least viral and bacterial agents, including several vaccine or drug-resistant strains [ ] . each agent has unique properties that present both a distinct threat and challenge for detection, prevention, and control. bacillus anthracis and clostridium botulinum are gram-positive bacterial agents of grave biothreat concern. b. anthracis is a spore-forming bacterium that causes cutaneous, respiratory, or intestinal forms of anthrax disease, which is an acute, rapidly progressing infection in any form. the b. anthracis spores are highly stable both in the environment and in the exposed individuals and can be easily disseminated via the aerosol route, thus making it a dangerous bacterium [ ] . the anthrax attacks in caused widespread panic, damage, disease, and death, which increased national awareness to the threat of bioterrorism. the bacterium produces a lethal toxin that disrupts the host innate responses during the early stages of infection and ultimately leads to septicemia and death of the host (fig. . a) . antibiotic treatment requires a lengthy dosing regimen and is effective only if it is initiated during the early stage of the infection. two monoclonal antibody (mab)-based anthrax antitoxin therapeutics [abthrax (raxibacumab) and anthim (obiltoxaximab)] have been approved by the us food and drug administration (fda) and included in the strategic national stockpile for treating inhalational anthrax [ ] . biothrax, the only licensed anthrax vaccine, is indicated for preexposure prophylaxis of disease in persons at high risk of exposure and postexposure prophylaxis of disease following suspected or confirmed b. anthracis exposure [ ] . botulinum neurotoxin (bont), produced by c. botulinum, is extremely potent, lethal, and easy to produce, transport, and misuse. the toxin itself is the select agent, but the clostridium organism, as an isolate capable of producing the toxin, is also classified as a tier select agent. there are seven serotypically distinct bonts (serotypes a-g) and they act by blocking neurotransmitter release and thereby preventing transmission of nerve impulses, which can lead to botulism, hallmarks of which are paralysis and respiratory arrest [ ] (fig. . b) . current treatment is limited to botulism immune globulin intravenous, human-derived antibotulism toxin antibodies for the treatment of infant botulism types a and b, and botulism antitoxin heptavalent (a-g), a mixture of immune globulin fragments developed from equine plasma for the symptomatic treatment of adult and pediatric botulism. the us army has developed a similar antitoxin based on equine neutralizing antibodies that is effective against a number of serotypes, but there is a limited supply and risk of horse serum sensitivity. an investigational vaccine also exists, but it offers limited protection and painful side effects [ ] . many of the bacterial agents of biothreat concern are intracellular gram-negative organisms. intracellular bacteria are particularly difficult to treat because the intracellular niche protects bacteria from the innate or adaptive immune surveillance. these bacteria can enter host cells through phagocytosis, and to prevent their destruction in the endocytic pathways, intracellular bacteria have adapted to survive in a host lysosome and replicate within the acidic endolysosomal compartment (e.g., coxiella burnetii). another intracellular bacteria brucella spp. can traffic from a mature lysosome to endoplasmic reticulum-derived compartments, while bacteria such as burkholderia mallei, burkholderia pseudomallei, francisella tularensis, and yersinia pestis can prevent acidification and maturation of the phagosome and escape to the cytosol, where they can replicate and then disseminate to neighboring cells [ ] [ ] [ ] . one characteristic feature of the b. mallei and b. pseudomallei intracellular life cycle is the fusion of infected mononuclear cells, forming multinucleated giant cells (mngcs). although the role of b. pseudomallei-induced mngcs is unclear, it is believed that cell fusion facilitates localized dissemination of the bacteria [ , ] (fig. . c) . brucella spp. are nonmotile bacteria that cause brucellosis, a world-wide chronic debilitating disease in both humans and animals. although not typically fatal, brucella spp. are stable and infectious as aerosols and can lead to sterility and abortions [ ] . the nonmotile bacillus b. mallei is the causative agent of glanders that usually infects equids but is highly infectious to humans at low doses, producing septicemia, severe pulmonary infection, and chronic inflammation of the skin and eyes. b. mallei can be easily aerosolized, and even with antibiotic treatment there are high mortality rates [ ] . the motile bacterium b. pseudomallei, the causative agent of melioidosis, is a close relative to b. mallei and can lead to severe illness in humans, such as pulmonary infection and septic shock. b. pseudomallei is an environmental saprophyte that is naturally resistant to many antibiotics [ ] . q-fever is caused by direct contact with the nonmotile bacterium c. burnetii that was previously weaponized because of its ease of aerosolization, its environmental stability, and its ability to infect animals or humans with a single bacterium [ ] . q-fever is not typically lethal but can be incapacitating, causing fever and difficulty breathing, and antibiotic therapy is not always effective, thus leading to persistent infections. f. tularensis is the causative agent of tularemia and is highly infectious, resulting in an acute, rapidly progressing local or systemic infection [ ] . y. pestis, the causative agent of plague, is a nonmotile bacterium that can be disseminated by aerosol, transmitted from person-to-person, and is characterized by a severe clinical disease course with potentially high case-fatality rates. there is a limited window for effective treatment against plague, since the resulting respiratory and circulatory collapse from septic shock is usually fatal [ ] . there are a great number of viruses on the list of select agents and toxins, and some of these can only be handled in the laboratory at the highest biocontainment level (biosafety level ). the causative agents of some of the most lethal hemorrhagic fever infections are filoviruses, paramyxoviruses, and arenaviruses. filoviruses such as ebola virus (ebov) and marburg virus infect humans and nonhuman primates (nhps) and have caused large outbreaks in recent years. these viruses are likely transmitted in nature by fruit bats and are spread from person to person via contact with body fluids or fomites [ ] . marburg virus was reportedly weaponized through activities carried out by the former soviet union [ ] . because of the large scale of recent ebov outbreaks, this virus may have become available to nefarious people through access to corpses and contaminated clinical waste. two more viruses transmitted in nature at least in part by fruit bats are the nipah and hendra paramyxoviruses that belong to the henipaviridae family and cause severe neurological and/or respiratory diseases in humans [ ] . these viruses can infect many domestic and agricultural animal species and are frequently transmitted between humans via droplets or fomites, leading to concerns of new human outbreaks in areas of malaysia, bangladesh, india, and australia where case-fatality rates range from % to % [ ] . arenaviruses, specifically lassa virus (lasv) and junin virus (junv), machupo, and other south american viruses are transmitted not by bats but by peridomestic rodent species [ ] . none of the filoviruses or henipaviruses has any fda-approved therapeutics or vaccines available for prevention or treatment of human disease, and while ribavirin is sometimes used to treat lassa fever, it is not a terribly effective drug against this viral infection [ ] . variola virus (varv) is the causative agent of smallpox, a human viral disease for which tecovirimat was recently approved as a therapeutic by the fda, a successful therapeutic development story [ ] . this pathogen has been eradicated since through successful vaccine campaigns [ ] , but because the vaccine is no longer administered in most countries, populations may be susceptible in the event that varv or an intentionally modified or related poxvirus with similar virulence factors and similar human lethality is resurrected [ ] . arthropod-transmitted alphaviruses and bunyaviruses are also biothreat concerns. for the alphaviruses specifically, the venezuelan encephalitis viruses (veev), eastern encephalitis viruses, and western equine encephalitis viruses belonging to the family togaviridae are found in the americas and cause equine disease [ , ] . these new world alphaviruses cause encephalitis-like symptoms, are stable in the environment, grow to high titers easily in cell culture, are highly infectious by aerosol, and affect humans with incapacitating neurological disease, sometimes with high morbidity rates [ ] . bunyaviruses such as rift valley fever virus and the related tick-transmitted nairovirus causing crimean-congo hemorrhagic fever also cause human diseases with high morbidity and mortality rates. some investigational new drug vaccines exist for these agents. these viruses replicate quickly in humans and cause rapid disease; therefore the timing for therapeutic intervention is short, making treatment postinfection very challenging. the lack of approved therapeutics available to combat biothreats may be in part attributed to the unique challenges for the discovery and development process of evaluating drugs that target select agents. foremost is the implementation of high-throughput screening (hts) efforts for the discovery of new compounds against authentic or wildtype biothreat bacterial and viral pathogens [ ] . specifically, the requirement of work to be performed in high-level biocontainment laboratories (bsl or bsl ) is a major limiting factor since laboratories with these capabilities are not widely available. in addition, highly trained personnel that can handle infectious agents use robotic instruments and adhere to operational, engineering, and government regulations are a critical requirement for working with biothreat agents [ ] . in the united states, strict guidelines have been instated for generating government-approved methods and processes for inactivation of pathogens before plates/samples can be brought out of biocontainment suites for further experimentation, and to track the inactivated material [ ] . other challenges that need to be considered include the prevention of pathogen aerosolization while handling screening plates in biocontainment laboratories and ensuring that inactivation chemicals and methods are compatible with downstream procedures. new therapeutics effective against both natural and engineered resistant forms of bacterium are vital to the biodefense armory. screening for novel antimicrobials is traditionally done by scoring for growth inhibition in vitro, using the standard clinical & laboratory standards institute guidelines. this generally involves performing a dose-response assay in a multiwell plate format and monitoring growth in the absence or presence of the test compounds. the compound concentration that shows no visible growth is considered the minimum inhibitory concentration (mic). over the years, this approach has led to the discovery of only a limited number of novel antimicrobial compounds and resistance has already been generated against most of the antibiotics used in the clinic. one disadvantage to this approach is the inability to identify potent immunomodulatory compounds against intracellular pathogens that require the host for replication. new approaches to understanding bacterial pathogenesis have enabled researchers to elucidate mechanisms that could be targeted to control and clear infection in lieu of simply targeting in vitro bacterial viability. targeting the host under in vivo-like conditions (e.g., in cell culture or animal models) will be a key feature of study design to combatting intracellular pathogens that require the host for invasion and replication and will likely identify new host-directed therapeutics. the development of host-directed therapeutic (hdt) strategy relies on an understanding of the interactions between pathogens and their hosts and appropriate tools and hts assays to screen and identify therapeutics. technological progress in assay miniaturization has emerged from a combination of advanced robotic systems, high-throughput microscopy, automated image analysis, and data analysis using powerful bioinformatics tools, and this has led to the development of high-content imaging (hci), allowing for large-scale quantification of multiple cellular phenotypes at the system level. such phenotypic screening platforms rely on physiologically relevant host cell types that are permissive to pathogen infection and have the potential to identify compounds that modulate relevant biological processes in an unbiased, target, and mechanism-agnostic fashion. this cell-based approach has the added advantage that compounds that have greater mammalian cell membrane permeability, reduced cellular toxicity, and target the host proteins will be readily identified in the context of their desirable function in cells. pharmacologically active compounds can be selected that inhibit the uptake or intracellular replication of the bacterium or disrupt the host-pathogen interactions. the general workflow for high-throughput, imagebased phenotypic screening approach to identify hdts is outlined in fig. . [ , ] . using bacterial antigen-specific antibody to detect bacteria, this method can quantitate the number of intracellular or cell-associated bacteria and the effect of the compounds in reducing the bacterial number (% inhibition of bacterial infection), and cellular toxicity (based on loss in cell number). alternatively, one can use hci to quantitate the morphological changes of mngcs based on nuclei number and mngc size/area and use this phenotype to screen and identify compounds that prevent bacterial spread [ ] . to overcome the problem of multidrug resistant bacteria, there is a growing focus on identifying small molecules that target drug resistant mechanisms or virulence factors, or agents that prevent/disrupt biofilm formation. virulence factors, such as secretion systems in gram negative bacterial pathogens, are promising therapeutic targets. specifically, the type secretion system (t ss) present in y. pestis is responsible for injecting effectors that target the cytoskeleton and proinflammatory signaling pathways. a number of techniques have been used to screen and identify potential t ss inhibitors that can be adapted for biothreat pathogens. these include an enzyme-linked immunosorbent assay (elisa)-based detection of proteins secreted from enteropathogenic escherichia coli (epec), inhibition of sheep erythrocyte lysis by epec, inhibition of induction of a yope luciferase fusion in yersinia pseudotuberculosis, and a pseudomonas aeruginosa cell-based bioluminescent reporter screen [ ] [ ] [ ] [ ] . using a high-throughput luminescence screening assay, three compounds were identified that inhibit y. pestis t ss-mediated cytotoxicity that relieves the growth inhibition associated with in vitro activation of t ss [ ] . another promising approach to disarm the bacteria is to prevent/disrupt biofilms, a barrier produced by bacteria to protect itself from the aggressive host environment. small molecule therapeutics that specifically disrupt or prevent the biofilm formation could be used in combination with antibiotics. the common method to quantitate biofilms is a colorimetric-based assay that utilizes a crystal violet dye to stain the biofilms and subsequent extraction of the dye using organic solvents or detergents [ ] followed by absorbance measurement. to improve sensitivity, robustness, and throughput, a fluorescent-dye-based assay was developed, wherein the biofilms are stained with fm - fluorescent dye and fluorescence signal is measured following organic extraction of the dye [ ] . screening of a small molecule library in this assay identified rifabutin and ethavarine, as potential inhibitors of b. pseudomallei (bp ) and acinetobacter baumannii biofilm production, respectively, without directly affecting the bacterial growth. phenotypic screening using high-throughput hci. cells susceptible to the pathogen of interest are seeded in hci plates. next day, cells are pretreated with appropriate concentration of the compounds and then infected with the pathogen of interest for optimal time wherein %- % infection results. the infected plates are then submerged in % formalin for h to inactivate the pathogen and to fix the cells. immunofluorescence staining is then performed, using a primary antibody specific to a pathogen antigen, and an appropriate fluorescence-labeled secondary antibody. dyes such as cell mask red and hoechst are added to detect the cell cytoplasm and nuclei, respectively. automated image acquisition and analysis is performed and data are analyzed using columbus software to quantitate the percentage of inhibition of pathogen infection and loss in cell number that represents cellular toxicity, in the presence of the compound [ ] . hci, high-content imaging. there is a possibility that therapeutics targeting the virulence factors or other drug resistance mechanisms may not be effective by themselves and will need to be evaluated in combination with antibiotics to treat multiple drug resistance (mdr) infections. thus screening experiments designed to find combination therapies are warranted. to determine the synergy of two drugs (antibiotic and nonantibiotic), conventional checkerboard assays are set up wherein the two drugs are tested in combination at varying concentrations and the mic of each drug either alone or in combination is then used to calculate the fractional inhibitory concentration [ ] . similarly, in the case of the biofilm assay, testing a biofilm disruptor and an antibiotic together at varying concentrations will help one to assess the effectiveness of combination therapies. unlike most bacteria, viruses require the mammalian host for replication. the virus life cycle can be divided into distinct stages that include the entry, uncoating, replication, genome packaging, assembly, maturation, and budding. various cell-based and in vitro biochemical assays have been developed to study virus life cycles as well as to screen and identify antivirals [ ] . the conventional plaque-forming assay used to evaluate antivirals is time-consuming, not amenable for hts, and not very robust. alternatively, in the absence of more sophisticated instruments or technologies, a virus-induced cytopathic effect can be used as an endpoint to test antivirals. with advances in imaging instruments and informatics, a cell-based hci platform ( fig. . ) that uses viral antigen-specific antibodies to detect and quantitate the viral infection is now a general approach to identify compounds that inhibit viral infection [ , , ] . however, this approach will not provide information on which steps in the viral life cycle the inhibitors are disrupting. to help one to deconvolute the mechanism of action of identified hits ( fig. . ), cells pretreated with an inhibitor prior to virus exposure can potentially identify compounds that inhibit viral entry, while treatment of cells after exposure (i.e., after the entry step) would identify compounds that inhibit intracellular replication and/or viral spread. assays utilizing recombinant noninfectious viruses have been generated to screen and identify inhibitors that target different stages of the viral life cycle [ ] (fig. . ). pseudotyped virion assays are well suited as safe alternatives for hts, since bsl and bsl wild-type pathogens are not required to complete the screens. these assays are based on viral vectors that harbor glycoproteins (gps) of different enveloped viruses and a reporter gene such as green fluorescent protein (gfp) or luciferase flanked by packaging signals, are used to generate chimeric replication-deficient viruses, and then used to screen and identify entry inhibitors. this approach has successfully identified entry inhibitors for lassa, ebola, and nipah viruses [ ] [ ] [ ] [ ] . cell fusion assays, including cell-cell or cell-virus fusions, have been developed to screen and identify hiv- fusion inhibitors, but to date no such assays have been developed for biothreat viruses [ ] . reverse genetic systems or minigenome assays have proven to be valuable models to study rna virus replication and transcription. this model system is used to screen and identify antivirals [ , ] . replication competent minigenome systems wherein some of the viral open reading frame is replaced with a reporter gene (gfp or luciferase) and the cdna copy cloned into a plasmid is cotransfected into mammalian cells with individual plasmids each containing a viral ribonucleoprotein (rnp). the target genes in the expression vectors are under the control of either a mammalian rna polymerase i or ii or t rna polymerase (which will require transfection of a plasmid containing the t rna polymerase) promoters. following transcription, the resulting viral rna is complexed with the rnp components and there is subsequent replication of the virus genome and expression of the reporter protein. minigenome systems have been developed for several biothreat pathogens, including filoviruses, arenaviruses, and bunyaviruses, and have been used to screen and identify small molecule inhibitors of filovirus and arenavirus replication [ ] [ ] [ ] [ ] [ ] . to study the viral assembly and budding, another surrogate model that can be used is based on virus-like particles (vlps) that are mimics of viral protein assemblies made by reconstituting the viral recombinant structural proteins. vlps are noninfectious as they do not contain any viral genome, but are intrinsically immunogenic, and hence are being extensively investigated as potential vaccine candidates [ ] . in the case of the ebov vlp-based assay, cotransfection of plasmids encoding the viral gp and the matrix protein (vp ) results in spontaneous formation of filamentous vlps that are released into the medium and can be quantitated by elisa [ ] ; thus this model can be useful in drug discovery research [ ] . to identify inhibitors of viral genome replication, in vitro biochemical assays targeting viral enzymes such as polymerases, methyltransferases, helicases, as well as viral and host proteases such as cathepsins or kinases have been developed. a number of antivirals that have been approved by the fda target either the dna or rna polymerases. incorporation of radioactive nucleotide either to a dna oligonucleotide by dna polymerase [ ] or to a homopolymeric rna as a template by rna polymerase are common methods to determine polymerase activity [ ] . a recent study reported the use of fluorescent dye to detect the double-stranded rna and the feasibility of developing this assay to screen and identify inhibitors of zika virus polymerase activity [ ] . the host lysosomal protease cathepsin l (cat l) is necessary for the processing and cleavage of the gp of enveloped viruses, so that the virus can fuse with the host cell membrane and gain entry into the host. thus cat l has been regarded as an ideal target for drug discovery. a fluorescence resonance energy transfer (fret)-based cat l enzymatic assay was developed, wherein peptides derived from gps of viruses such as ebola, nipah, hendra, and severe acute respiratory syndrome and middle east respiratory syndrome coronavirus and containing cat l cleavage site were chemically conjugated with a quencher -carboxytetramethylrhodamine at the n-terminus and -carboxyfluorescein fluorophore at the c-terminus [ ] . the intact peptides exhibited minimal to no fluorescence, but following cleavage of the peptide by cat l, there was an increase in fluorescence intensity. screening of a chemical library in this assay identified small molecules that selectively inhibited cat l-mediated cleavage of multiple viral peptides over host proneuropeptide y [ ] . viral proteases are also good drug targets as they play a vital role in viral replication. for example, the ns b-ns protease is highly conserved among the flaviviruses and a fret-based enzymatic assay using a synthetic peptide substrate [ ] was developed to identify west nile virus protease inhibitors [ ] . functional genomic screening using gene-trapping, crispr's gene editing, or rna interference (rnai) technologies has been applied to identify host factors that are required for replication or involved in pathogenesis of several biothreat viral and bacterial agents and are summarized in table . . the activities of several identified host factors can be perturbed by small molecules and thus serve as potential therapeutic platforms. for example, it was demonstrated that the novel host factor inositol-requiring enzyme α is required for brucella infection in mammalian cells [ ] . reducing the levels of either the retromer cargo-adapter complex or retromer-associated sorting nexins abrogated c. burnetii replication [ ] . multiple host kinases such as camp-dependent protein kinase, protein kinase b, and protein kinase c all play a role during c. burnetii infections [ , ] . zhou et al. [ ] identified tnfrsf and serpini that may promote activated macrophages in controlling f. tularensis replication. akimana et al. [ ] showed that f. tularensis utilizes host ubiquitin turnover in distinct mechanisms during the phagosomal and cytosolic phases and that phosphoinositide metabolism is essential for cytosolic proliferation of f. tularensis. connor et al. [ ] revealed that host proteins are required for intracellular survival of y. pestis. of particular, interest was the enrichment for genes involved in endosome recycling. using the gene trapping approach, carette et al. [ ] first identified several host factors that are required for ebov infection. these include a cholesterol transporter niemann-pick c factor involved in the fusion of endosomes and lysosomes (homotypic fusion and protein sorting complex), biogenesis of endosomes (pikfyve), lysosomes (bloc s , bloc s ), and targeting of luminal cargo to the endocytic pathway [ ] . many of these hits reoccurred in several crispr and small interfering rna (sirna)/shrna screenings [ , , ] . in addition, lysosomal protein (bri ) and a gtpase involved in the regulation of vesicle trafficking (rab b), pi k, calcium/ calmodulin kinase-related network, and de novo pyrimidine synthesis pathway are essential for ebov replication and transcription [ ] [ ] [ ] . the application of rnai screening has been utilized for other viral pathogens such as henipavirus, junv, poxvirus, vaccinia virus, and veev [ , [ ] [ ] [ ] [ ] [ ] . it is demonstrated that catalytic activity of fibrillarin, the enzymatic subunit of the snornp complex that is responsible for catalyzing the transfer of a methyl donor from a bound cofactor s-adenosyl methionine to ribose sugars of the target pre-rrna, is required for henipavirus infection [ ] . voltage-gated calcium channel (vgcc) subunits were shown to be important in junv-cell fusion and entry into cells. gabapentin, an fda approved anticonvulsant drug against α δ subunitcontaining vgccs, inhibited replication of the vaccine strain of junv in mice [ ] . other sirna-based screens against v. virus identified that amp-activated protein kinase (ampk) promotes viral entry through the control of actin dynamics, and knockdown of nuclear pore protein (nup ) arrests virion morphogenesis [ ] [ ] [ ] . lastly, an sirna screen identified trafficking host factors that modulate veev infection [ ] . an alternative approach to gain an in-depth understanding of host-pathogen interactions during infection is to construct a protein-protein interaction network between host protein and bacterial virulence factors. using a yeast two-hybrid (y h) library, memiševic´ et al. [ ] identified a molecular network that governs b. mallei infection. similarly, a y h study conducted by yang et al. [ ] showed the involvement of focal adhesion, regulation of cytoskeleton, leukocyte transendoepithelial migration, toll-like receptor (tlr), and mapk signaling pathways during y. pestis infection. to complement the y h study, reverse-phase protein microarray analysis was used to interrogate changes in protein expression and posttranslational modification. this further revealed the roles of ampk-α , src, and gsk β in regulating b. mallei and b. pseudomallei infection [ ] , and thus, as viable host targets for countermeasure development. prior to the initiation of medical countermeasure development against specific pathogens, a target product profile (tpp) is needed to define the required features of potential drug candidates (e.g., route of administration, prophylactic vs therapeutic, trigger to treat, and onset of action requirements). once a tpp is in place, a screening funnel is drafted that sets laboratory criteria and defines clear go/no-go decision points that are needed to progress countermeasures from discovery through preclinical development and into human clinical trials. irrespective of the types of assays used for countermeasure screening, compounds identified as having significant inhibition in primary screens are validated in subsequent dose-response experiments to determine the half maximal effective concentration (ec ) and cytotoxic concentration (cc ). potent compounds that have an adequate selectivity index (e.g., > ) that is defined as a ratio of cc / ec , are then often tested in orthogonal assays in appropriate cells/tissues to better understand or validate the antipathogen activity. ideally, compounds are further optimized for potency, selectivity, physicochemical, and pharmacokinetic (pk) properties and safety prior to in vivo evaluation to assess efficacy in appropriate animal models of infection ( fig. . ) . many of the therapeutics that are in different stages of either preclinical or clinical development for select biothreat pathogens include small molecule antivirals (tables . and . ), antibody (or antibody cocktails) against viruses or bacteria/virulence factors (table . ), and combination drug therapy (table . ). the increased use of antivirals and antibiotics has set the stage for rapid adaptation mechanisms that microbes can use to counteract them. the development of antimicrobial resistance is one of the biggest public health threats and hence alternative approaches to treat infectious diseases are urgently needed. table . lists the resistance mechanisms identified in each biothreat bacterial pathogen and provides references for targets of resistance. since stand-alone antibiotics may not be sufficient to overcome resistance and/or completely clear some biothreat bacterial infections, we have also included encouraging data on host directed therapeutics, and combination therapy. hdt is an emerging approach in the field of antiinfectives discovery. the strategies behind hdt can include modulation of host immune responses, or interference/manipulation/targeting of host-cell factors that are required for pathogen replication [ ] . for example, in a potential bioterror scenario, where the identity of the etiological agent causing the disease is unknown, stimulation of innate immunity may be particularly useful as induced immune responses are often capable of providing protection against a broad range of pathogens. although no fda-approved hdt therapies are yet available for treating infectious diseases, we have summarized in this section the antimicrobial primary screening of small molecule chemical libraries in the phenotypic hci assay will identify compounds that inhibit pathogen infection as well as those that may contribute to cellular toxicity. generally, hits that show ≥ % infection inhibition and ≤ % loss in cell number are then subjected to hit triage or in silico filtering wherein compounds with optimal physicochemical properties such as solubility, lipinski's rule of , metabolism are selected for potency testing in the phenotypic screening assays. compounds that exhibit an ec ≤ µm and si, which is a ratio of cc /ec > , are then further optimized through iterative cycles of synthesis, testing in cell-based and orthogonal assays and in in vitro admet studies to improve potency and physicochemical properties. if the target of the hit molecule is identified then a target-based screen is performed and the hits identified are optimized through the iterative structureactivity relationship cycle. the lead series candidates are then evaluated in vivo for their pharmacokinetic properties and then for efficacy in appropriate challenge models of infection. admet, absorption, distribution, metabolism, excretion, and toxicity; hci, high-content imaging; si, selectivity index. antibiotics aigiv combination therapy with antibiotics and aigiv is more effective than antibiotics alone in a rabbit model of inhalational anthrax and improved survival compared to the antibiotic treatment alone [ ] ciprofloxacin clindamycin treatment of rabbits with systemic anthrax with clindamycin and ciprofloxacin had improved efficacy compared to monotherapy and could be used to prevent relapse of infection [ ] ciprofloxacin combination therapy for anthrax, including antiprotective antigen (pa igg) antibodies and ciprofloxacin in a rodent anthrax model increased survival significantly compared to ciprofloxacin treatment alone [ ] levofloxacin raxibacumab combination therapy with raxibacumab, an igg monoclonal antibody that binds protective antigen, and the antibiotic levofloxacin provides protection in rabbits late in the disease course [ ] oligochlorophen antibiotics targeting cytoskeletal proteins such as ftsz with oligochlorophen analogs is a promising new treatment method that has a -fold lower development of resistance compared to antibiotics used for anthrax treatment in humans [ ] penicillin, meropenem, or rifampin linezolid treatment of antibiotic-resistant inhalation anthrax with linezolid and penicillin, meropenem, or rifampin had the inhibitory effect on mean lethal factor levels compared to the control groups and successfully treated fluoroquinolone-resistant b. anthracis infection [ ] rifampin clindamycin combination therapy for anthrax with rifampin and clindamycin was shown to be synergistic in vitro [ ] brucella spp. rifampin successful combination therapies used to treat pulmonary brucellosis in humans is doxycycline and rifampin for weeks [ ] burkholderia mallei antibiotic heat-killed vaccine combination of an antibiotic moxifloxacin, azithromycin, or sulfamethoxazole-trimethoprim, and vaccination using heat-killed b. mallei can protect balb/c mice from lethal glanders infection, potentially by stimulating immune responses, such as gamma interferon, which acts synergistically with antibiotic therapy to inhibit bacterial growth [ ] enrofloxacin, trimethoprim, and sulfadiazine doxycycline successful -week combination treatment of parenteral administration of enrofloxacin and trimethoprim with sulfadiazine followed by oral administration of doxycycline eliminated b. mallei from glanderous horses during an outbreak [ ] burkholderia pseudomallei combination therapy with farnesol a sesquiterpene alcohol that damages biofilm matrix and interferes with cell wall and peptidoglycan biosynthesis, facilitates antimicrobial penetration, and reduces the minimum biofilm eradication concentration for ceftazidime, amoxicillin, doxycycline, and sulfamethoxazole-trimethoprim in vitro [ , ] ceftazidime avibactam avibactam restores susceptibility to ceftazidime for genetically diverse extremely drug resistant isolates of burkholderia from cystic fibrosis patients by binding pena and the combination treatment significantly improved survival of larvae infected with the drug resistant isolates [ ] ceftazidime ifn-γ interferon gamma-induced reactive oxygen species with ceftazidime leads to synergistic killing of intracellular b. pseudomallei and markedly increases the effectiveness of antimicrobial therapy for the treatment of b. pseudomallei infection in mice [ , ] clostridium botulinum bont serotypes and subtypes differences present a significant challenge for creating monoclonal antibody treatments for neutralization, by diversifying the v-regions of mabs and selecting cross reactivity, a combination treatment of three antibodies neutralized bont/f , f , f , and f in mice and was times more potent than equine antitoxin [ ] coxiella burnetii doxycycline chloroquine combination therapy of doxycycline and hydroxychloroquine combination shortened the duration of therapy and reduced the number of relapses in patients with q fever endocarditis. and a case of q fever endocarditis with biological prosthetic aortic valve and aortic homograft was successfully treated with doxycycline and chloroquine combination therapy [ , ] francisella tularensis cytochalasin b, ly , wortmannin, nocodazole, mg , and xva inhibitors reduce f. tularensis update and reduce inflammatory cytokine production and can be used in combination with antibiotics to improve survival of infected mice [ ] gentamicin membrane antigen immunization postexposure immunization with membrane protein fraction antigens and treatment with low-dose gentamicin increased survival of mice and significantly reduced bacterial burdens in the liver and spleen [ ] potential of several small molecule immunomodulators and host cell factors that have been investigated to date. immunomodulators directly target the host rather than the pathogen (fig. . ). this is accomplished by targeting pattern recognition receptors, such as tlrs that are present on innate immune cells in the host to detect features of microbes known as pathogenassociated molecular patterns. since immunomodulators target host immune cells, they are an attractive candidate for use against bacterial agents as they are unlikely to result in the development of antibiotic resistance even after repeated use. in particular, the threat of an intentional release of a highly virulent bacterial pathogen that is either intrinsically resistant to antibiotics, or has been weaponized via the introduction of antibiotic resistance, makes immunomodulation an attractive complementary or alternative strategy to directly targeting bacterial biothreat agents. for example, a synthetic tlr agonist, '-c-phosphate-g- ' oligodeoxynucleotide (cpg odn), appears to be able to stimulate protective immunity against intracellular bacterial infection and/or eliminate chronic infections. indeed, studies in mice have demonstrated that the innate immune defenses activated by cpg odns protect against lethal challenge with b. anthracis, b. mallei, and combination therapy, including antibiotics with an efflux pump inhibitor, would be a novel mechanism to restore the efficacy of the antibiotic in resistant strains of y. pestis [ ] antibody therapy corticosteroid the addition of antiinflammatory methylprednisolone, a corticosteroid, in combination with antibody therapy correlates with improved mouse survival, with reduction in neutrophil and matrix metalloproteinase in the tissue, and the mitigation of tissue damage [ ] ciprofloxacin l- - a novel postexposure medical countermeasure l- - , an a adenosine receptor antagonist blocks lps-induced activation of immunomodulatory cytotoxic substance accumulation to prevent acute lung injury, and in combination with ciprofloxacin improves survival of rats following infection with y. pestis [ ] aigiv, anthrax immune globulin intravenous; ifn, interferon; lps, lipopolysaccharide; pa, protective antigen. aminoglycosides rnd-type efflux pumps, s-adenosyl-l-methioninedependent methyltransferase, amrr [ , ] β lactams pena a , nlpd , dacc, flgn, sch, tr _ , tr _ , ftsi, amrr, bper, bpet, spot, trna, rrna, proteins with unknown function, sers seryl-trna synthetase, and rnd efflux pump amrab-opra, and bpeef-oprc [ , [ ] [ ] [ ] [ ] [ ] macrolides amrab-opra efflux pump [ ] quinolones amrab-opra efflux pump, bpeab-oprb efflux pump [ ] sulfamethoxazole/ trimethoprim rnd bpeef-oprc efflux pump, lysr-type regulator bpet bpes, ptr , fola, amrr tetr-type regulator, amrab-opra, metf [ , ] quinolones gyra a [ ] tetracyclines putative protein secretion targets, biosynthesis of pantothenate and coenzyme a, aspartate biosynthesis, dna replication [ ] francisella tularensis β lactams blab [ ] chloramphenicol s rrna, the l and l ribosomal proteins, and overexpression of efflux pumps [ ] quinolones gyra a and gyrb [ ] f. tularensis or their surrogates [ ] . similarly, human monocyte-derived macrophages treated with poly(i:c), a synthetic tlr agonist, showed significantly reduced intracellular f. tularensis [both schu and lvs (live vaccine strains)] replication. mice administered with poly(i:c) before or after schu or lvs infection showed reduced bacterial burden in the lungs and prolonged survival. mice treated with poly(i:c), challenged with f. tularensis, and then treated with levofloxacin showed % survival relative to no survival in animals receiving levofloxacin alone [ ] . in addition to targeting innate immune cell receptors, there is a growing interest in modulating autophagy as an immunotherapeutic intervention. autophagy is a dynamic process that targets cellular cytoplasmic contents for lysosomal degradation. more specifically, xenophagy is a type of selective autophagy that specifically targets intracellular pathogens to lysosomes, retracing their replication and survival [ ] . the use of autophagy inducer rapamycin, decreased the survival of b. pseudomallei in vitro [ ] . however, several bacteria exploit autophagic machinery as part of their intracellular life cycles (i.e., brucella abortus, c. burnetii, and f. tularensis). therefore infection may be exacerbated by the induction of autophagy (fig. . c ) [ ] . research to further understand the balance between infective and protective cellular targets in the autophagy pathway may enhance its utilization as a therapeutic target. hts of fda-approved drugs is another approach to identifying compounds that were previously approved for other disease indications but may have the potential to be repurposed as antiinfectives. trifluoperazine (an antipsychotic), amoxapine (an antidepressant), and doxapram (a breathing stimulant) mitigated fatal y. pestis infection in a pneumonic plague murine model [ ] . at h postinfection, these drugs provided animals with up to % protection against challenge with bubonic or pneumonic plague agents when administered in combination with levofloxacin [ ] . multiple fda-approved drugs targeting g-protein coupled receptors and calcium fluxes inhibited c. burnetii and b. abortus, whereas drugs targeting cholesterol traffic attenuated c. burnetii [ ] . similarly, increasing evidence suggested statin, a -hydroxy- -methylglutaryl-coenzyme-a reductase inhibitor, possesses antibacterial activity by the inhibition of sterols, prenylation, and isoprenoids (c. burnetii), the inhibition of antiinflammatory cytokines (y. pestis), and the modulation of phagosome maturation (c. burnetii) [ ] . it was demonstrated that a low dose of gleevec, an anticancer drug inhibiting abl , c-kit, and related protein tyrosine kinases, can increase the number of myeloid cells in the bone marrow, blood, and spleen and enhance antimicrobial responses in a mouse model of f. tularensis infection [ ] . in the case of viruses, small molecule targeting of innate immune receptors has also shown efficacy in several relevant viral models of infection. for example, treatment with poly(ic:lc) has also been protective against ebov infection in nhps [ ] . prophylactic pulmonary administration of tlr ligand (tmx ) significantly protected mice from lethal infection with veev [ ] . tlr and tlr agonists have also been shown to improve the efficacy of postexposure therapeutics against smallpox [ ] . sometimes modulation of host pathophysiological responses can be evaluated as a target. hemorrhagic fever virus pathophysiology includes the stimulation of procoagulant pathways and increased permeability of the vascular endothelium; therefore these processes are being evaluated as possible targets for therapeutic intervention. this could be accomplished by utilization of an anticoagulant, such as recombinant nematode anticoagulant protein c (rnapc ) that blocks initiation of the extrinsic coagulation pathway by inhibiting the tissue factor-factor viia complex [ , ] . rnapc has been shown to be highly protective in macaques infected with a lethal dose of ebola zaire virus, when treatment was initiated day post viral challenge [ , ] . whereas hdt targets the host directly, antibody therapy is the passive process of activating the immune system to respond to microbial threats. sources of antibodies can include individuals that survive infection or have received a prophylactic vaccine against a microbe. alternatively, antibodies can also be generated ex vivo using cell culture. historically, antibody-based serum or plasma therapy has been widely used to treat a variety of infectious diseases. limitations for clinical use arise however from the polyclonal nature of serum antibodies, resulting in lot-to-lot variation, approaches for determination of correct dose levels and regimens, and a risk for allergic reactions and transmission of transfusion-borne diseases. in general, limited clinical applications for antibody therapy existed until the development of technology that allowed the production of mabs through the use of hybridomas [ ] . hybridomas allow for the production of homogenous antibodies with the same specificity of a single immunoglobulin class and isotype. further advancements made it possible to humanize or generate fully human mabs. research advancements in the past - years have resulted in numerous mab-based therapies that have been approved for inflammatory and neoplastic diseases. infectious diseases have not been included in approved treatments. although many mab products targeting infectious diseases are in different stages of development, to date, one mab-based product, synagis (palivizumab), is currently approved for use in infectious diseases (rsv) [ ] , while two mabs abthrax (raxibacumab) and anthim (obiltoxaximab) have been approved under the fda's animal efficacy rule for treatment of inhalation anthrax [ ] . for treatment of ebola infection, the single mab mab and zmapp, a cocktail of three "humanized" mabs, have advanced in product development and are being tested for efficacy in the ongoing ebola outbreak in the democratic republic of the congo (nct , www.clinicaltrials.gov). it is clear that mabs offer a highly specific, potent, and generally safe platform for antimicrobials and may be a useful alternative to immune plasma. it is imperative to find appropriate niches in infectious diseases, specifically those caused by biothreat agents, where new antibody-based treatments could prove to be efficacious [ ] . table . summarizes key research in antibody therapy across different bacterial and viral families of some current biothreat agents. the utilization of mab therapy for the prophylactic or therapeutic treatment of biothreat agents varies depending on the agent. in all cases however, the challenge for the development of effective therapeutic antibodies against viruses is the viruses' heterogeneity and mutability. a related problem is the low binding affinity of cross-reactive antibodies that are capable of neutralizing a variety of primary isolates. finally, the cost of large-scale production of mabs is a limiting factor for continued use. a solution to the challenges with viral mutagenicity may be found in the identification of potent new mabs that target highly conserved viral structures, which are critical for virus entry into cells. alternatively, utilization of combination therapy, whereby, a cocktail of several mabs may be used or mabs may be combined with other drugs, such as antiviral compounds, may overcome mutagenicity issues. these areas of research will continue to be a major focus of biothreat agent therapeutic research [ ] . for countermeasures against lethal viral infections (i.e., category a), table . lists reported studies in either mice or nhps that have shown significant benefits to survival in challenge models. the table also includes in vitro potencies, viral strains, specific animal species, dosing regimens, routes of administration, pks, and benefits to survivaldata necessary for the reader to relate in vitro potency to in vivo efficacy, assess/interpret results, and make comparisons. the corresponding chemical structures are provided in fig. . . table . displays the status/results of clinical trials for therapeutics used for the treatment of infections caused by ebov and lasv. noteworthy, most of these clinical trials were underpowered without appropriate controls and hence results may be speculative. combination therapies are an excellent approach to improve treatment outcomes, shorten treatment duration, and overcome microbial resistance mechanisms caused by biothreat pathogens. combination therapy may incorporate antibiotics or antivirals with hdt or antibody therapy at rationally designed treatment schedule. in this way the usage of multiple treatment modalities can synergize to optimize the mechanism of action of biothreat-targeted therapies. table . includes combination therapies that have been used to treat each biothreat bacteria. in the case of viral infections, while combination therapy has been used for treatment of patients with human immunodeficiency virus (e.g., combination of nucleoside, nonnucleoside, protease, and/or host-targeted inhibitors) or chronic hepatitis c virus infection (e.g., combination of polymerase and rna-binding protein ns a inhibitors), to date there are no reported studies for biothreat viral agents. several β lactam antibiotic drugs have been able to overcome deactivation when delivered in combination with inhibitors that target extended-spectrum β lactamases (enzymes that are overexpressed in the mdr pathogens, inactivate the β lactam antibiotic by cleaving the β lactam ring and thus one of the major contributors of antibiotic resistance). the β lactam/β lactamase inhibitor combination drugs that have been fda-approved include augmentin xr (amoxicillin/clavulanate combination), unasyn (ampicillin/sublbactam combination), and zosyn (piperacillin/tazobactam combination). in the case of biothreat bacteria, the combination of ceftolozone and tazobactam exhibited increased in vitro susceptibility to a variety of clinical, environmental, and animal strains of b. pseudomallei, but to date it has not been evaluated in in vivo efficacy studies [ ] . challenges to developing countermeasures against biothreat agents are many, but some of the unique and key challenges are pk differences in healthy versus infected subjects, mapping the biodistribution of the countermeasure to the biodistribution of the pathogen, and limited opportunities to run randomized, controlled clinical trials. preclinical studies typically require a pk study in healthy animals to guide dose selection prior to testing a countermeasure in an animal model of infection. in that regard, it is critical to understand what cells and tissues the pathogen is infecting over time so that countermeasures can be properly designed to reach infected tissue. for example, countermeasures against pathogens causing encephalitis require drug to reach the central nervous system (cns). in contrast, ebov was found to infect lymph nodes, spleen, and liver in nhps - days following viral challenge, and by days - the virus was detected throughout the body (fig. . ) [ ] . thus if one was designing a countermeasure against ebov infection, it would likely require a wide tissue distribution in order to be effective. to complicate things further, infected animals often have altered metabolizing enzymes (e.g., cytochrome p s) [ ] , tissues, and barriers (e.g., blood-brain barrier) making drug exposure difficult to predict. running pk experiments in the presence of infection would eliminate many of these variables, but this is seldom done for countermeasures to biothreat agents, since it requires running these experiments in biocontainment labs. because biothreat pathogens cause infrequent human cases and outbreaks in generally remote areas of the world, planning a traditional human clinical trial with large numbers of participants is not feasible. even when the west african ebola outbreak of more than , cases was unfolding in - , and now that there is a large outbreak unfolding in the democratic republic of the congo, the amount of clinical efficacy data that have been collected for ebov therapeutics is quite limited. the limitations are due to difficulty of performing clinical research in a remote outbreak setting where cultural, geographical, and political barriers may hinder or halt trial planning [ ] . the bulk of the efficacy data for ebola published in the literature has been garnered through animal studies. to enable product development for viral, bacterial pathogens as well as for chemical, toxin, and radiological threat agents for which outbreaks or cases are sparse, the fda issued an animal rule, codified cfr . in , that proposes to permit consideration of product development and efficacy data obtained from animal studies for drug licensing, in lieu of human clinical trials when such trials would be unfeasible or unethical [ ] . since introducing the animal rule in , the fda has approved more than a dozen products, including several therapeutics for anthrax, plague, botulinum toxin, and smallpox [ , ] . the animal rule does not provide an expedited pathway to fda approval for drugs and can certainly be more challenging than traditional drug development pathways. the developer must compile a significant body of data to prove efficacy of the drug against the target therapeutic indication. first in , and updated now into a formal document published in [ ] , the fda has released guidance for industry describing critical data elements required for animal efficacy studies for drug approval under the animal rule: ( ) the pathophysiology of disease and the mechanism of action by which the drug prevents or ameliorates disease must be reasonably well understood; ( ) it is desired that the efficacy must be demonstrated in two animal species, although multiple studies in one species can be acceptable if the animal model is sufficiently well characterized and accurately predicts the human response; ( ) the animal study end point must be clearly related to the desired human efficacy end point, such as enhancement of survival; and ( ) pk and pharmacodynamics data must be generated in the animal studies to allow selection of an effective dose in humans. under the animal rule, efficacy studies are expected to demonstrate that drug effectiveness in animals reliably indicates efficacy in humans. thus while traditional human clinical efficacy studies require demonstration that the therapy is effective, the animal rule imposes an additional burden on investigators to establish a drug candidate's mode of action in at least one animal model that reproduces accurate human disease pathology. further, the animal rule outlines considerations for the development of the model(s), to include the use of an isolate of the etiologic agent that was known to cause human disease (e.g., agent was isolated from a fatal human case if it is a lethal disease, such as ebola) [ ] . there is also a requirement that the infection model using the chosen pathogen strain must present the same or similar pathophysiology as the human disease. definitive animal model efficacy evaluations should be performed only after careful model development studies have been performed and accepted by the regulators. these studies are known as natural history studies and are carefully designed to investigate and describe the course of the disease in the animal species, through clinical, serological, and histopathological evaluations, to compare the features of the disease in the model to the features of disease in human cases. it is important to consider the route of pathogen exposure (nasal, oral, and aerosol routes) to the animal because this will model the natural or unnatural modes of exposure predicted for humans, where a biorelease would constitute an unnatural exposure. a dose of challenge agent that is thought to be predictive of the human exposure level in a biorelease scenario should be used to develop the model, and that dose should be well characterized and reproducible by a quantitative measure. the route of drug delivery, dose administration timing, and treatment regimen in response to a biorelease scenario must also be considered when designing the animal model studies for a drug under development for such an indication. it is possible that a biorelease scenario would not be immediately known, and a period of time might pass before people begin to develop symptoms. studies evaluating the cutoff time for drug to still be effective, and what are the triggers for treatment should be investigated in the animal model. animal rule pivotal efficacy studies are essentially performed in place of traditional phase clinical studies, so they must be done in the containment laboratory under a quality system [ ] . use of fda good laboratory practice or other comparable quality system with high levels of documentation and data integrity is paramount, so data packages can withstand regulatory review and audit [ ] . the studies must be designed so that the program will collect the same results and conclusions one would expect from a well-designed traditional phase trial, but in addition a pivotal animal efficacy study must describe a mechanism of action for the treatment modality to prevent or block disease or tissue infection and damage [ ] . the pathologic mechanism needs to be consistent and well understood across both the human and animal models, such as the mechanism of pathogen entry into the target host cell, toxicological mechanism of lethal factors, or germination of spores and dissemination of bacterial infection in target cells and tissues, all of which may be mechanisms the drug under study is known to block; this must be proven in the animal model. products developed under the animal rule are subject to postmarketing or field studies when the product is actually used in the scenarios for which it was developed, and this is required to verify a product's clinical benefit [ ] . part of the approval process is a requirement to have postmarketing study plans in place, for quick execution should an event occur in which the drug would be field tested. approval may also come with restrictions for off-label use, distribution, or access. actual use will also come with requirements to inform patients of the conditions under which the drug was approved by virtue of only animal efficacy data, making them informed consumers as to the risks of possible nonefficacy or unknown effects in cases of human disease. with the advancement of systems and synthetic biology and the ease of genetic modification, biothreats are becoming more complex and there is a growing need for novel treatments that can have broad-spectrum activity against new, remerging, and engineered pathogens. developing novel countermeasures that can effectively treat and prevent massive casualties is an ongoing challenge that remains a central priority for future research. the development of novel therapies relies on an improved understanding of the host-pathogen interactions. key virulence factors have been identified and targeted for potential treatment options, including biofilm and t ss inhibitors for bacterial infections, and viral entry or polymerase 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studies key: cord- - fx w u authors: russell, clark d.; baillie, j. kenneth title: treatable traits and therapeutic targets: goals for systems biology in infectious disease date: - - journal: curr opin syst biol doi: . /j.coisb. . . sha: doc_id: cord_uid: fx w u among the many medical applications of systems biology, we contend that infectious disease is one of the most important and tractable targets. we take the view that the complexity of the immune system is an inevitable consequence of its evolution, and this complexity has frustrated reductionist efforts to develop host-directed therapies for infection. however, since hosts vary widely in susceptibility and tolerance to infection, host-directed therapies are likely to be effective, by altering the biology of a susceptible host to induce a response more similar to a host who survives. such therapies should exert minimal selection pressure on organisms, thus greatly decreasing the probability of pathogen resistance developing. a systems medicine approach to infection has the potential to provide new solutions to old problems: to identify host traits that are potentially amenable to therapeutic intervention, and the host immune factors that could be targeted by host-directed therapies. furthermore, undiscovered sub-groups with different responses to treatment are almost certain to exist among patients presenting with life-threatening infection, since this population is markedly clinically heterogeneous. a major driving force behind high-throughput clinical phenotyping studies is the aspiration that these subgroups, hitherto opaque to observation, may be observed in the data generated by new technologies. subgroups of patients are unlikely to be static – serial clinical and biological phenotyping may reveal different trajectories through the pathophysiology of disease, in which different therapeutic approaches are required. we suggest there are two major goals for systems biology in infection medicine: ( ) to identify subgroups of patients that share treatable features; and, ( ) to integrate high-throughput data from clinical and in vitro sources in order to predict tractable therapeutic targets with the potential to alter disease trajectories for individual patients. infection is the largest single cause of death in humans worldwide and many infectious agents provide relevant in vitro model systems that are both amenable to study with high-throughput techniques, and recapitulate key events in disease pathogenesis. in this review, we consider how systems biology approaches may be leveraged to address the major unmet needs in infection medicine in the st century, with the aim of improving outcomes for patients with infection. in clinical practice we are unable to therapeutically modulate the host immune response to infection, largely due to its inevitable complexity. despite this, we contend that host-directed therapies have a high probability of success, since there is already considerable innate variation in host responses to infectious disease, ranging from extreme susceptibility, to complete resistance, and tolerance. infectious diseases are survivable if you have the right genetics. the challenge is to make the same diseases survivable for patients who would otherwise succumb. a systems medicine approach to infection has the potential to combine and integrate relevant signals from clinical, genomic, transcriptomic, proteomic and pathogen biology data to draw inferences about disease pathogenesis. below we discuss examples of aspects of this approach applied to various infectious diseases, and suggest future goals for the application of systems biology to infection medicine. more than years after the discovery of penicillin [ ] , this same drug is still a prominent weapon in our antibacterial armamentarium. more broadly, the concept underlying this therapeutic approach -attempting to eradicate the pathogen from a patient's body using antimicrobial drugs -remains the only effective treatment. although spectacularly successful, the focus on the pathogen has two limitations. firstly, death frequently occurs in infectious disease despite effective antimicrobial therapy. alongside the direct effects of microbial virulence factors, tissue damage is also caused by the host immune response. immune-mediated damage leading to respiratory, cardiovascular and renal failure (sepsis) continues even after eradication of the pathogen [ ] . at present, no treatments exist to modify these deleterious aspects of the host immune response. secondly, antimicrobial resistance threatens to liberate pathogens from the range of our solitary weapon against them. unless something changes, deaths from infection are predicted to soar, overtaking malignant disease even in developed countries by [ ] . therapies to modulate the host response to infection would have the theoretical advantage that, in addition to promoting survival in the presence of effective antimicrobials, a host-targeted therapy may exert a less powerful selection pressure on pathogens, and may be more difficult for a pathogen to evolve to overcome. in our view, the development of such therapies is wellsuited to the application of systems approaches. inevitable complexity of the immune system the human immune system is arguably the most complicated organ system in the body, encompassing numerous effectors, inter-related feedback loops and extensive redundancy. this complexity is unsurprising when considering that our immune system has evolved in the face of microbial virulence factors that directly interfere with regulatory and effector mechanisms. examples of microbial interference with host immune mechanisms are numerous and diverse. for example, one of the first innate immune mechanisms encountered by many pathogens is phagocytosis, which serves to both prime the adaptive response and eliminate invading pathogens by intracellular killing. pathogenic yersinia species, a group of facultative intracellular pathogens, encode a type three secretion system to directly inject effector proteins into the host cell cytoplasm, modulating the cytoskeleton to prevent phagocytosis, and inducing apoptosis of immune cells and blocking the mapk and nf-kb pathways to reduce cytokine production [ ] . another bacterium, pseudomonas aeruginosa, secretes a protease that cleaves a host protein (corticosteroid-binding globulin) to release the corticosteroid hormone cortisol at the site of initial infection, incapacitating the local innate immune response [ ] . even the relatively tiny genome of the influenza a virus encodes a protein (ns ) which is nonessential for replication and seems to be dedicated to interfering with both the induction and action of the host antiviral interferon response by sequestering viral dsrna, preventing activation of rig- signalling and inhibiting protein kinase r and oas/rnase l [ ] . the adaptive response, mediated by t and b lymphocytes, is also a target. the human immunodeficiency virus encodes three proteins that each down-regulate cell surface mhc- expression by distinct mechanisms, preventing mhc-i signalling to activate the cytotoxic t-cell response to virus-infected cells [ ] . to prevent b-cells mounting an antibody response to infection, the staphylococcus aureus surface protein a binds to the fc-g portion of antibodies [ ] . these examples cover a few of the mechanisms pathogens have evolved to extensively interfere with the host immune response. the animal innate immune system is thought to have evolved over million years, starting with amebae able to phagocytose external material for nutrition [ ] . the adaptive immune system in mammals is thought to have arisen million years ago in fish [ ] . since these initial events, the immune system in each host species has participated in a genetic arms race, evolving alongside relentless exposure to these microbial immune interference strategies from innumerable pathogens. furthermore, the immune system must successfully distinguish self from non-self antigens, with deleterious consequences arising (i.e. autoimmune disease) when this fails. the requirement to overcome these microbial immune interference strategies whilst preserving the recognition of self-antigens has provided the necessary pressure to drive the human immune system to evolve into a hugely complex organ system. in the context of this inevitable complexity, it is no surprise that reductionist approaches to development of hosttargeted therapies in infectious disease have largely a subgroup within a population of patients who are distinguished by a shared disease process. treatable trait the pathophysiological feature (or, in a looser sense, a biomarker or group of biomarkers for that feature) that determines whether a given therapy will improve a given patient's outcome. the same trait may be present in many different clinical syndromes or disease processes. it is reasonable to expect that as-yet undiscovered therapeutic interventions could alter the host immune response to promote survival. we infer this from the simple fact that some hosts do better than others when confronted with the same pathogen: the host response to infection is, in all cases that we know to have been studied, heterogeneous. furthermore, much of this variation is heritable [ ] . we conclude that host factors exist that promote survival from specific infections, and that these must vary between individuals, and hence that it should be possible to identify and utilise these factors therapeutically to alter the biology of a susceptible host to induce a response more similar to a host who survives. the scale of the challenge of finding these targets is such that it is hard to imagine a solution being found without the power of systems approaches [ ] . conceptually, this could involve promoting resistance to (suppressing pathogen replication), or tolerance of (preventing damage associated with immune response to pathogen), an infecting pathogen [ ] . although theoretically attractive as a tool to limit damage to the host [ ] , inducing tolerance is not without potential dangers: a sustained high pathogen load could facilitate transmission (iatrogenic super-shedders) and, perhaps more worryingly in emerging zoonotic infections, provide the time required for the selection of mutants with better host adaptation. due to their reliable and broad acting anti-inflammatory effects, corticosteroids represent an intuitively attractive strategy to reduce inflammation during infection. indeed, a survival benefit has been demonstrated in a small number of uncommon infections (bacterial meningitis, tuberculous meningitis and pericarditis, hypoxaemic pneumocystis jiroveci pneumonia) [ ] . in contrast, there is uncertainty over safety and benefit in other infections (e.g. rsv bronchiolitis) and clear evidence of harm in others (viral hepatitis, cerebral malaria, influenza virus, sars coronavirus, hiv-associated cryptococcal meningitis) [ e ]. a more specific host-directed therapy, recombinant human activated protein c (rhapc), was licensed for treatment of severe sepsis based on the results of a single clinical trial [ ] . rhapc has anti-inflammatory and anti-thrombotic properties, and circulating levels are low in patients with sepsis [ ] . sadly, a subsequent trial in an overlapping patient group showed a trend towards increased mortality [ ] . a third trial, mandated by the regulatory authorities, did not detect any mortality benefit, and the drug was quickly withdrawn from the market [ ] . these negative trial results in sepsis are not necessarily conclusive. the heterogeneity of the host response, and the diverse range of microbes involved, means that amongst all patients with sepsis (itself an unrealistically broad syndrome) there are likely to be numerous biologically-different sub-groups, with distinct immune responses and more importantly, different risk: benefit balance for a given therapy. corticosteroids, for example, may save some patients but harm others. our current understanding of infection does not allow clinical differentiation of these sub-groups, so potentially beneficial therapies may be falsely rejected in clinical trials. we believe that a major output of systems approaches to infection will be the elucidation of previously hidden therapeutically-important subgroups of patients who share a 'treatable trait' (i.e. response to therapy). we have discussed two central problems in infection medicine. firstly, the development of therapies to modulate the host immune response is impeded by the inevitable complexity of the human immune system. secondly, the range and degree of characterisation of clinical syndromes in medicine is constrained by the range of observations that are available; we likely fail to identify sub-groups of patients with treatable traits due to a paucity of relevant observations. the promise of systems approaches to infection medicine is that new technologies may provide solutions to these old problems. large-scale biological data-sets from an increasing range of high-throughput technologies are becoming available, including (but not limited to) high-resolution transcriptional profiling [ ] , mass spectrometry-based proteomics [ ] and metabolomics, genome-scale crispr-cas knockout screening [ ] , and whole genome sequencing/genome-wide association studies. these modalities could provide new, biologically important observations that have not previously been observed in patients. it is very likely that some of these observations will be directly relevant to clinical management e the challenge will be to identify the ones that matter. there are three broad categories of clinical utility for these new data sources in infection medicine: identifying treatable traits and therapeuticallyrelevant subgroups; identifying new therapeutic targets in the immune response; and, improving prognostication. the value of prognostication in current clinical practice is, in our view, limited by the range of therapeutic options. put simply, it is only useful to predict the future if you have some capacity to change it. particularly in the case of acute and immediately lifethreatening infection, our range of therapeutic options is very limited, regardless of the degree of certainty attached to the prognosis, therefore we suggest the first two applications should be prioritised. in contrast, the identification of syndromes (collections of clinical observations that tend to occur in patients suffering the same disease) has been the primary mechanism for progress in the understanding of human disease since long before the time of hippocrates [ ] . finding a syndrome is the first step towards identifying the common biological processes that define a disease, and ultimately to identifying effective treatments. we hope that the application of systems technologies will help us define treatable traits in infection, thus providing a starting point for new therapeutic interventions ( figure ). importantly, a systems model of the disease process need not predict every transcript and metabolite in the massively multi-dimensional datasets generated by new technologies. the primary challenge is instead to identify those components of inter-host variation that are amenable to intervention; the evidence of disease processes that we can change. this is agusti's concept of "treatable traits" [ ] , a term coined in the field of chronic obstructive lung disease but no less relevant here. a computational model of infection could therefore include not only traditional measures of pathogen burden and evidence of systemic injury [ ] , but also independent components of the immune response or the metabolic consequences, detected using highthroughput technologies. the behaviour of the whole system may be impenetrably complex, but the components required to predict the effect of an intervention may be far simpler. the trajectory followed by each patient along an informative set of vectors may reveal different groups of patients that appear clinically similar, and may even have similar outcomes, but different disease processes ( figure ). by mapping the "flight path" of each patient through disease, we anticipate that summary of a systems medicine approach to infection. a wide range of data sources can be combined using various methods (see text) to achieve two fundamental goalsclinically-informative phenotyping of patients, and identification of therapeutic targets. important similarities and differences in immune response will become apparent [ ] . our ability to identify groups of patients sharing therapeutically-relevant similarities is dependent on measuring the relevant biological signal that determines classification. this is the fundamental attraction of high-throughput technologies e the probability of measuring important signals is greater if more signals are measured. that such groupings of patients, or disease endotypes, exist is already clear: therapeuticallyimportant sub-classifications have recently been discovered that redefine the clinical syndromes of asthma [ , ] , ards [ ] , and acute mountain sickness [ ] . in two related autoimmune conditions, anca-associated vasculitis (aav) and systemic lupus erythematosus (sle), a t-cell gene expression signature clearly delineates two distinct endotypes [ ] . subsequent work elucidated the immunological process underlying this sub-classification, cd t-cell exhaustion [ ] . this process is associated with better outcomes in autoimmune disease, but poor clearance of viral infection. in the future it may be possible to manipulate this pathway therapeutically in patients with aav or sle, to prevent relapse, or in the opposite direction in patients with chronic viral infection, to promote clearance. even in the best-case scenario, the distance between the identification of a tractable therapeutic target and successful exploitation in clinical practice is substantial, so it is no surprise that potential host-directed therapies discovered through high-dimensionality analytics have not yet been proven to be effective in clinical trials. nonetheless there are some promising leads, a few of which are described here. these exemplify different approaches: integrating cell culture, animal and human data sequentially to identify a host anti-viral factor (influenza virus); and using computational predictions from transcriptional data with proteomic and genetically modified animal studies to identify host pathways involved in pathogenesis (sars coronavirus). viruses are obligate parasites and undergo exclusively intracellular replication; properties that make them ideal for study in cell culture where host factors that affect viral replication can be expected to do the same in vivo. sirna screening has been used as a genomewide approach to identify such host factors for influenza virus infection. ifitm was identified as a novel host anti-viral factor by sirna screening with confirmatory in vitro work (including exogenous interferon administration and stable ifitm expression) demonstrating it is required for an effective interferon response to inhibit influenza virus replication [ ] . work in ifitm e/e mice was then undertaken, confirming that in vivo, influenza virus-infected mice suffer fatal viral pneumonia, even when infected with a low-pathogenicity virus [ ] . the genisis/mosaic groups identified a single nucleotide polymorphism hypothetical trajectories of two groups of patients through multidimensional space. each line indicates the path taken by a single patient, with periods of organ failure highlighted in red. a superficially similar group of patients may appear clinically indistinguishable (a), but different trajectories through illness are revealed by informative vectors derived from high-throughput data (b). it is reasonable to expect that such biological differences in disease process will underlie different responses to host-directed therapies. (rs -c) within the ifitm coding region in humans that was strongly over-represented in patients hospitalised with influenza virus infection [ ] , the majority of whom required invasive ventilation. metaanalysis of genomic studies of ifitm and influenza virus infection has confirmed this association between snp rs -c and increased susceptibility to infection in humans [ ] . work is now underway in many groups to investigate the impact of ifitm proteins in antiviral defence, with a view to generating hosttargeted antiviral therapies. this example highlights the potential of a systems-wide approach, integrating and cross-validating results from various experimental modalities (high-throughput screening in cell culture, followed by targeted studies in mice and humans) to converge from large-scale data onto a single critical host immune factor. acute lung injury (ali) and progression to acute respiratory distress syndrome are major features of the pathophysiology of sars coronavirus (and indeed other respiratory virus) infection. pathological changes in mice are very similar to humans and there is a dose-response relationship between viral inoculum and the severity of ali. host responses to the virus that result in ali are poorly understood. to investigate host responses associated with more severe acute lung injury in a murine model of sars coronavirus (sars-cov) infection, transcriptomic profiles of mice infected with lethal and sub-lethal doses of virus were compared and correlated with pathological data at multiple time points, then subject to bioinformatic network analysis [ ] . a module of genes involved in cell adhesion, extracellular matrix remodelling and wound healing was significantly up-regulated in the lethal infection model, and components of the urokinase pathway were found to be the most enriched and differentially regulated. massspectrometry proteomic analysis was then used to explore this transcriptional data further, demonstrating that sars-cov infection resulted in increased expression of fibrin b and g chains, factor viii and cytokeratins (all components of hyaline membranes), and reduced expression of surfactant proteins in the lung. these data are consistent with histological post-mortem findings in sars-cov infected humans, where extensive fibrin exudate, extensive hyaline membrane formation and alveolar collapse have been observed [ ] . serpine is part of the urokinase pathway and contributes to ecm remodelling. to confirm this finding from transcriptional and proteomic studies, serpine e/e knockout mice were infected with sars-cov and found to have a worse outcome compared to wild type mice. this systemsbased approach to sars-cov, sequentially applying transcriptional, proteomic then genetic modification techniques, demonstrates that the urokinase pathway contributes to ali, thus identifying a target for future experimental medicine work to determine if it can be therapeutically altered to benefit the host. these examples give us confidence that we can expect more progress along similar lines as the potential of systems medicine approaches becomes more widely appreciated, as expertise in computational methodologies grows, and as the cost of generating relevant data falls. physicians have long made progress by recognising patterns of similar observations in groups of patients, and by determining which biological features of disease are amenable to therapy. what has changed is the unprecedented rate of advance in new resources and tools with which to tackle the ancient problems of diagnosis and therapy in infectious disease. our responsibility is to ensure a similar acceleration in clinical progress. . sorensen ti, nielsen gg, andersen pk, teasdale tw: genetic and environmental influences on premature death in adult adoptees. n engl j med , : - . this prospective cohort study of adoptees demonstrated that genetic background contributes more to the relative risk of death due to infection (i.e. biologic parent also died of infection) than for death due to cancer, vascular disease or natural causes, thus affirming that susceptibility to infection is a strongly heritable trait. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest first clinical use of penicillin severe sepsis and septic shock review on antimicrobial resistance. tackling drug-resistant infections globally: final report and recommendations the yersinia ysc-yop 'type iii' weaponry pseudomonas aeruginosa elastase disrupts the cortisol-binding activity of corticosteroidbinding globulin the multifunctional ns protein of influenza a viruses mechanisms of hiv- to escape from the host immune surveillance role of protein a in the evasion of host adaptive immune responses by staphylococcus aureus evolution of innate immunity: clues from invertebrates via fish to mammals origin and evolution of the adaptive immune system: genetic events and selective pressures disentangling genetic variation for resistance and tolerance to infectious diseases in animals this study utilises a murine malaria model to demonstrate that resistance to and tolerance of infection represent genetically distinct host defence mechanisms disease tolerance as a defense strategy use of corticosteroids in treating infectious diseases adjunct prednisone therapy for patients with communityacquired pneumonia: a multicentre, double-blind, randomised, placebo-controlled trial adjunctive dexamethasone in hiv-associated cryptococcal meningitis corticosteroids as adjunctive therapy in the treatment of influenza sars: systematic review of treatment effects efficacy and safety of recombinant human activated protein c for severe sepsis role of activated protein c in the pathophysiology of severe sepsis drotrecogin alfa (activated) in severe sepsis drotrecogin alfa (activated) in adults with septic shock gateways to the fantom promoter level mammalian expression atlas mass spectrometry-based proteomics genome-scale crispr-cas knockout screening in human cells genome editing using the crispr-cas system can be applied to genome-wide knockout screening to identify genes with an important phenotype in an in vitro model, as exemplified in this study of genes required for cell viability in cancer and genes implicated in susceptibility to a therapeutic raf inhibitor used in melanoma hippocrates on ancient medicine phenotypes and disease characterization in chronic obstructive pulmonary disease. toward the extinction of phenotypes? health trajectories reveal the dynamic contributions of host genetic resistance and tolerance to infection outcome multidimensional endotypes of asthma: topological data analysis of crosssectional clinical, pathological, and immunological data asthma endotypes: a new approach to classification of disease entities within the asthma syndrome subphenotypes in acute respiratory distress syndrome: latent class analysis of data from two randomised controlled trials network analysis reveals distinct clinical syndromes underlying acute mountain sickness a systematic network analysis (using software originally designed for analysing microarray expression data) of symptoms associated with acute mountain sickness revealed three distinct sub-groups within the syndrome, resulting in a revision of international diagnostic criteria for a cd + t cell transcription signature predicts prognosis in autoimmune disease transcriptional profiling identified two distinct sub-groups (related to t-cell survival and memory) which could then be differentiated on the basis of only three genes, providing a useful biomarker for clinical trials of immunosuppressant therapies and focussing further studies on these pathways t-cell exhaustion, co-stimulation and clinical outcome in autoimmunity and infection the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus ifitm restricts the morbidity and mortality associated with influenza data was integrated from cell culture work, a murine infection model, human genotyping and patient clinical variables to validate ifitm as a critical host defence against influenza virus infection and thus a tractable therapeutic target interferon-inducible transmembrane protein genetic variant rs and influenza susceptibility and severity: a meta-analysis mechanisms of severe acute respiratory syndrome coronavirus-induced acute lung injury multiple organ infection and the pathogenesis of sars key: cord- -c dv zmi authors: rodriguez, william; macveigh-fierro, daniel; miles, jacob; muller, mandy title: fated for decay: rna elements targeted by viral endonucleases date: - - journal: semin cell dev biol doi: . /j.semcdb. . . sha: doc_id: cord_uid: c dv zmi for over a decade, studies of messenger rna regulation have revealed an unprecedented level of connectivity between the rna pool and global gene expression. these connections are underpinned by a vast array of rna elements that coordinate rna-protein and rna-rna interactions, each directing mrna fate from transcription to translation. consequently, viruses have evolved an arsenal of strategies to target these rna features and ultimately take control of the pathways they influence, and these strategies contribute to the global shutdown of the host gene expression machinery known as “host shutoff”. this takeover of the host cell is mechanistically orchestrated by a number of non-homologous virally encoded endoribonucleases. recent large-scale screens estimate that over % of the host transcriptome is decimated by the expression of these viral nucleases. while this takeover strategy seems extraordinarily well conserved, each viral endonuclease has evolved to target distinct mrna elements. herein, we will explore each of these rna structures/sequence features that render messenger rna susceptible or resistant to viral endonuclease cleavage. by further understanding these targeting and escape mechanisms we will continue to unravel untold depths of cellular rna regulation that further underscores the integral relationship between rna fate and the fate of the cell. from synthesis to decay, the regulation of rna steady state and turnover emerges as an important contributor of the gene expression cascade and is critical to the survival of the cell. a key component of this pathway is the role of rna structural elements in directing the many facets of rna life. these rna elements serve as signals utilized by the cell to monitor the stability of the rna pool and buffer gene expression accordingly. in particular, the steady-state levels of messenger rna (mrna) are tightly regulated through a number of features including transcriptional splice patterns, the ′ cap, the ′ poly-a tail, and internal rna secondary structures. collectively, these features define the identity of these rna as mrnas and direct their fate. any perturbation in this carefully regulated expression cascade can have widespread consequences on the overall cell fate. thus, it is not surprising that viruses extensively remodel these pathways for their own benefit. during infection, virus and host clash for control over gene expression pathways. for the host, holding control of gene expression equates to mounting a robust anti-viral response, while for the virus wresting control away from the host allows it to fulfill its life cycle and dampen host defenses. viruses have evolved to take over existing pathways governing mrna fate in order to get an edge over the host, and rapidly hijack the host gene expression environment [ ] [ ] [ ] [ ] [ ] . in fact, viruses are exceptionally powerful manipulators of rna decay and expertly exploit host rna surveillance pathways. emerging evidence has revealed that widespread rna decay triggers a massive down regulation of transcription and translation rates throughout the cell [ ] [ ] [ ] [ ] [ ] [ ] . interestingly, to reach this global level of gene expression remodeling, multiple viruses seem to have converged onto a strategy of accelerating rna decay using their own encoded endoribonucleases. viruses that express these mrna-specific endonucleases include alphaand gammaherpesviruses, influenza a virus, sars and mers coronaviruses, vaccinia virus, and african swine fever virus [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the effect of these viral rnases is extensive, as it is estimated that over twothirds of the host transcriptome is impacted by viral-mediated decay [ , [ ] [ ] [ ] . however, how the host reacts to this massive takeover, or how these viral endonucleases can target mrna in such a widespread manner remains elusive and is still an active area of research. in the past five years, studies of viral endonucleases and accelerated rna decay have greatly enhanced our understanding of rna regulation. in particular, recent studies have unveiled several rna elements that confer susceptibility or resistance to endonuclease cleavage [ ] [ ] [ ] [ ] [ ] [ ] [ ] . many of these elements consist of rna structural motifs and/ or internal sequences that recruit rna binding proteins. to date, it is still unclear how prevalent these rna elements are or their precise nature and characteristics. further study into their evolutionary implications in the virus-host battle for control of gene expression is also needed. below, we review our current understanding of rna elements that facilitate targeting or escape of cellular mrnas from viral endonuclease cleavage. rna fate is tightly coupled to the presence or absence of regulatory elements that direct rna-protein interactions, localization and turnover rates. it is well established that from the moment an rna emerges from the transcription machinery, it will undergo a number of modifications in both sequence and structure that will direct splicing, translocation, translation, and ultimately decay of mature transcripts [ ] [ ] [ ] . as the cell has evolved to utilize rna elements to regulate gene expression, so have viruses evolved to take advantage of them. viral endoribonucleases, an exemplary product of this co-evolution, utilize these defining elements as a means of selectively targeting mrna substrates. throughout this section we will discuss how each of these rna features render mrna susceptible toand in many cases directviral endonuclease cleavage or similar strategies aimed at degradation of the host transcriptome during viral infection. ′ caps are a common and conserved feature of mrna, so it is perhaps not surprising that viruses have converged onto a mechanism of accelerated rna decay relying on ′ cap targeting. several viruses encode their own "decappers" which are viral enzymes that are surprisingly similar to the prototypical human, plant, and yeast decapping enzyme dcp [ ] . ′ caps are rna modifications commonly found in the form of m gpppnm. once an mrna is decapped, what remains is the cap end with an m gdp modification and the uncapped monophosphoryltated rna products. this leftover rna product thus becomes the perfect substrate for ′- ′ exonucleoytic degradation, in particular by the cellular housekeeping exonuclease xrn [ ] . vaccinia virus (vacv) encodes two proteins (fig. a ) that exhibit decapping activity known as d and d [ ] [ ] [ ] . both of these proteins belong to a superfamily of hydrolases that contain a consensus nudix sequence, an essential motif for the decapping activity of cellular decapping enzymes such as dcp [ , ] . although both d and d are decapping enzymes, there are some differences in their expression kinetics and their target preferences. first, d is synthesized early while d is synthesized late during infections thus maintaining a prolonged stage of viral-controlled decapping throughout infection. intriguingly, d and d also widely differ in their affinity for capped mrna with d showing greater specificity for the biologically relevant mrna cap structure [ , ] . furthermore, mrna length seems to be an important criteria for d and d targeting, with d more efficiently cleaving small rna substrates whereas d was shown to cleave rna substrates of nucleotides or more with similar efficiency [ , ] . this suggests that both of these decapping proteins can target not only methylated caps but rna itself. both d and d were shown to be inhibited by uncapped mrna, however d needed a -fold molar excess of competitive uncapped mrna to reduce its decapping activity by %, suggesting that d has a greater affinity for rna [ , ] . since d and d have different expression kinetics, their affinity and activity for specific types of mrna may reflect a global attempt by the virus to control its cellular visibility. how viral mrna escape d and d activity remains unknown. another virus that employs a similar method of mrna degradation is the african swine fever virus (asfv), a large, double-stranded dna virus in the asfarviridae family. asfv carries a gene (d r in strain ba v and g r in strain malawi) that encodes a decapping protein (asfv-dp - fig. a ) that, similarly to d , d and the host dcp , has a nudix hydrolase motif [ ] that can hydrolyze a wide range of substrates [ ] . in particular, asfv-dp activitymuch like d was shown to depend on an rna body and that adding even -fold molar excess of uncapped mrna reduced decapping by more than %. asfv-dp n-terminus was hypothesized to be sufficient to bind rna whereas its c-terminal region encompassing the nudix motif is not required. the n-terminal region fold into a basic channel that may serve as an rna binding platform and it was proposed that the n-terminal domain interacts with the highly acidic c-terminus to increase the disassociation (off) rate of the rna-asfv-dp complex [ ] . furthermore, asfv-dp activity seems to be independent of methylated nucleotides suggesting that this endonuclease does not actually need a cap structure to locate its substrate [ ] . however, the cap structure seems to have a stabilizing influence on the binding of asfv-dp to its target mrna. more recently, asfv-dp was found to preferentially target the elongation-initiation factor elf e mrna compared to beta-actin mrna [ ] , raising the intriguing question of potential transcript specificity in asfv-dp targeting mechanism. given the high level of similarities between asfv-dp, and vacv d , it would be interesting to investigate whether d similarly has sequence-specific capabilities. influenza a virus (iav) has also evolved to target mrna ′ caps in a mechanism referred to as "cap snatching". the influenza rna genome is replicated in the host nucleus using a viral-encoded rna-dependent rna polymerase (rdrp). while host ′ caps are added to pre-messenger rna co-transcriptionally, the rdrp does not allow for the recruitment of this machinery. instead, it possesses a nuclease activity that will cleave fully formed and assembled ′ caps from host genes and use it as a primer for viral transcription [ ] . once a host transcript is used as a cap "donor", it is removed from the pool of viable mrnas and will be quickly decayed by the host machinery. there are limited studies on how this affects the host cell homeostasis but it would be interesting to investigate whether this could play a role in the viral-host interplay. influenza a virus (iav) also uses the power of rna decay to suppress host gene expression. pa-x is a recently identified endonuclease produced by a ribosomal frameshift during translation of the polymerase subunit pa and pa-x was shown to be responsible for host shutoff in influenza [ , , , ] . pa-x consists of the pa rnase domain fused to a unique carboxy-terminal domain known as the x-open reading frame (x-orf) [ ] . pa-x has been shown to selectively degrade rnas that are transcribed by the host rna polymerase ii (pol ii), but not by any other polymerase [ ] . this specificity allows the virus to protect its own viral rnas that are expressed from the viral rna dependent rna polymerase. pa-x accumulates in the nucleus, and previous research has shown that not all mrnas are degraded equally by pa-x [ , , , ] . this suggests that there is an additional layer or alternate molecular mechanism to the targeting of pa-x. interestingly, the x-orf is required for pa-x activity, specifically for nuclear localization and binding to other proteins [ ] . in particular, pa-x was shown to interact with host proteins involved in ′ rna processing as well as splicing regulators, and thus has been proposed to link splicing to polyadenylation during rna processing. furthermore, it was recently showed that even single splicing event can promote degradation by pa-x (fig. d ) and that prototypical splice sites ag/gt had higher pa-x susceptibly compared to imperfect matches like ta/gt [ ] . however, intronless reporters are still degraded by pa-x to some extent. this was attributed to their robust expression, which may promote association with cellular factors involved in pa-x targeting. it therefore emerges that the important signals that render mrna susceptible to pa-x are core features of host mrna, such as pol ii transcription and splicing leftover marks. this is a remarkable example of how viruses rapidly evolve to efficiently hinder host cell immunity while promoting expression of their own viral rnas. while the methods described above involved viral enzymes targeting the rna processing machinery, some viruses trigger rna decay later in the mrna life cycle. in particular, coronaviruses have a unique method of inducing host gene suppression by hijacking the host rna quality control pathways in a way reminiscent of non-sense and no-go decay pathways. the coronavirus nsp protein has been shown to be associated with widespread rna decay during infection (fig. c) . however, it has been documented that nsp itself is not directly responsible for endonucleolytic cleavage of host mrna and does not possess nuclease activity [ , [ ] [ ] [ ] . instead, nsp was shown to bind and stall the s ribosome leading to inhibition of mrna translation, rendering the transcript inactive and translationally incompetent [ , ] . the host cell machinery is prepared to deal with stalled ribosomes and once nsp triggers the inhibition of ribosome scanning a cascade of events is initiated: recruitment of an unknown cellular endonuclease and ′ cap removal followed by ′- ′ decay mediated by xrn [ ] . recently, it was discovered that the cellular endonuclease cue is responsible for rna decay during no-go decay [ ] , making cue a potential candidate for nsp -mediated decay during coronavirus infection. while the binding of nsp to the s ribosomal subunit is a major factor in the targeting mechanism, targeting can also be driven in a template dependent manner [ ] . earlier research has suggested that nsp -mediated rna cleavage can target internal ribosome entry sites as well as the ′ cap of non-viral mrna [ ] . it was shown that in transcripts containing internal ribosome entry sites, cleavage occurred upstream of the initiation codon, approximately - nucleotides away. nsp thus emerges as a thorough rna decay trigger that uses diverse and non-overlapping strategies to widely target host mrnas. how the viral transcripts escape nsp- mediated is still under investigation. clues explaining this resistance to decay came from structural studies of the sars-cov genome. located within the ′ utr of sars genome is a structure that has been dubbed stem-loop (sl ) that provides protection from nsp -mediated gene suppression [ , ] . while the mechanism driving the stabilization of sl containing-rna and escape from nsp remains unknown, it has been shown that direct binding by nsp to this structured region is important [ , ] . this highlights the dual role of nsp when recruited to host vs. viral mrna. overall, sars coronavirus nsp is an interesting regulator of rna stability: currently, nsp does not appear to have any endonucleolytic activity of its own, and instead binds to the s subunit exploiting the host's rna quality control pathways to trigger mrna degradation. at the same time, nsp is also directly responsible for the protection of viral transcripts. this intrinsic duality of nsp makes it a potent regulator of rna fate during infection. of note, the newly emerged sars-cov coronavirus encodes an nsp protein with high sequence similarity to the other known coronaviruses [ ] reinforcing how pivotal nsp is in the regulation of coronavirus infection. another master regulator of rna fate that uses its host translation machinery as a platform to trigger decay is the herpesvirus protein vhs. early exploration of the herpes simplex virus virion host shutoff protein (vhs) identified that this protein was responsible for destruction of almost all host and viral mrna [ ] . it became quickly evident that vhs preferentially targets mrna while rrna and trnas levels remain unaffected by vhs activity [ , ] . for these reasons, it was suggested that a particular mrna features, such as the ′ cap or the ′ polyadenylated tail may play an important role in vhs targeting. vhs was reported to interact with eif h, eif aii and eif f [ ] . in particular, it was shown that mutations in vhs blocking its interaction with eif h render vhs incapable of degrading mrna and that knock out of eif h expression is sufficient to abrogate vhs function [ ] . taken together, these results strongly suggest that eif h binding is necessary for vhs targeting (fig. b) . furthermore, vhs interaction with the other eif factors that form the cap-binding complex was demonstrated to contribute to the recruitment of vhs to the ′ end of its target transcripts [ , , , ] . vhs preferentially selects cleavage sites near translation initiation sites within the ′ utr and at the start codon which was suggested to be driven by secondary structures of mrna, blocking the movement of the s ribosomal subunit, or factors that may cause stalling [ ] . once initial cuts have been made by vhs, host factors such as xrn quickly detect vhs-mediated internal cuts and finish the decay process of the mrna [ ] . furthermore, it has been shown that in vitro-translated vhs is able to cleave viral mrnas containing ires slightly downstream of ribosome entry sites [ , , ] . there seems to be a clear distinction between the mechanisms of recruitment of vhs to cap complexes versus selective structural-dependent recognition of ires and this difference could play a role in the particular pool of transcript targeted by vhs [ ] . similar to vhs and nsp , the gammaherpesviral sox family of w. rodriguez, et al. proteins also targets the vast majority of host mrna for degradation. these gammaherpesviral nucleases include the kaposi's sarcoma-associated herpesvirus (kshv) sox, the murine herpesvirus (mhv ) musox and epstein-barr virus (ebv) bglf [ ] . while sox and its homologs selectively target translationally competent mrnas, the presence of ribosomes or other translation-associated structures is not a strict requirement for target recognition [ ] . rather, targeting by these nucleases has been demonstrated to be directed by recognition of a degenerate rna motif that facilitates substrate binding and cleavage. this targeting motif was first mapped within three sox target transcripts to a ugaag motif [ , ] . since this discovery, significant progress has been made in understanding how this element directs sox mediated cleavage. in this section of the review, we discuss the most recent advances made in further defining the sox-targeting element, its prevalence in the host transcriptome, and the contributions of rna structure to sox-target recognition and cleavage. in , gaglia et al. further defined the sox-targeting motif and proceeded to search the host transcriptome for the prevalence of this element amongst in vivo sox targets [ ] . they uncovered that the sox targeting mechanism can be simultaneously sequence-specific and promiscuous. they also further defined the targeting element itself as a degenerate pyrimidine-rich sequence pattern adjacent to an unpaired stretch of adenine residues. consistent with previous observations, these sox cut sites are present on endogenous mature mrnas and are not restricted to a particular region of these transcripts. astoundingly, despite the relatively large size of the targeting element, most wellannotated human and viral transcripts contained at least one sequence fitting the sox-targeting motif. by defining sox targeting element and its prevalence in the transcriptome this work set the foundation allowing in vitro analyses to further explore the contribution of secondary structures in sox targeting. given the existence of multiple cut-sites on target transcripts beyond this consensus sequence previously described, lee et al. argued that the presence/absence of this sequence alone was insufficient to capture the breadth of sox targeting [ ] . through structural exploration of known cleavage targets they identified several general stem loops and bulges flanking in silico predicted targeting sites and showed that sox substrate processing is entirely dependent on the recognition of loop or bulge rna folds within the target rna duplex (fig. e) . crystal structures of sox bound with rna further demonstrated that sox cleavage is restricted to regions within or flanked by an unpaired nucleotide tract, often within a loop fold. collectively, these results illustrated the critical involvement of rna secondary structures in sox targeting and further reinforced a targeting mechanism consistent with dynamic kshv-induced mrna decay. despite the further mapping of the sox targeting element and assessment of secondary structure participation one outstanding question remained: what is the direct impact of these rna features in target recognition? more specifically, how do these features influence sox affinity and/or the efficiency by which targets are degraded? to answer these questions, mendez et al. developed the first in vitro system that accurately mimicked sox in vivo cleavage activity [ ] and demonstrated the requirement of an exposed loop structure and specific upstream sequences for sox cleavage, both serving as a "binding platform" for sox. thus, it is the combination of features within the cut site that determines the binding affinity of sox for its substrate and modulates sox cleavage efficiency and target recognition. sox targeting therefore seems to depend on the cooperation of these various rna features, which can explain why although sox-mediated turnover is widespread, sox targeting is still selective. by further elucidating the prevalence and requirements for sox cleavage, each of these studies has expanded our understanding of the contributions of rna elements for endonuclease targeting. however, a broader challenge remains in determining how sox and related endonucleases preferentially target pol ii transcribed mrnas, a feature that is not preserved by purified sox in vitro [ , ] . one hypothesis is the involvement of cellular co-factors capable of directing sox to its targets. the aforementioned iav pa-x endonuclease actively interacts factors involved in mrna maturation and processing, thus allowing for efficient pa-x cleavage [ , ] . it is therefore possible that sox and its homologs are also recruited to pol ii-transcribed mrna in the cytoplasm via an unknown rna-binding proteins or related factors. characterizing the interactions of sox with these potential protein co-factors will also be instrumental in deciphering sox target specificity and may reveal the hidden mechanism behind transcripts escaping sox decay. it is commonly accepted that cis-acting elements play vital roles in determining mrna half-life either by extending the life of a transcript, or destabilizing the transcript and labeling it for decay [ , ] . oftentimes this stabilization is mediated by interactions with trans-acting factors that interact with the rna. these elements can be either sequence-based, such as au-rich elements (are), or they can be structurally based such as the iron-responsive element (ire) or like the subgeomic rna produced by flaviviridae (sfrna). as discussed above, rna elements play an integral part in viral nuclease targeting, but recently, studies have shown that these cisacting elements may also participate in regulating escape from viralinduced degradation [ , [ ] [ ] [ ] . the most studied example of this is in the context of kshv infection (fig. f) , where it was shown early on that while the vast majority of host transcripts are targeted for degradation, a portion of transcripts are only mildly affected by soxmediated degradation [ , , , ] . while most of these "escapees" likely simply lack the targeting features required for sox cleavage, some select transcripts were found to resist sox decay even in the presence of a robust sox targeting element [ , , , ] . this type of dominant negative effect was shown to stem from a sequence that is confided to the escaping transcript ′ utr and was dubbed the soxresistant element (sre) [ , , ] . strikingly, despite the limited sequence similarity between the known escaping mrna, there seems to be a conserved structurally important stem-loop located within the sox resistant elements [ , ] . this would suggest that from an evolutionary standpoint the structure of the sre is conserved over sequence and could suggest that it serves as a platform to recruitment of transacting factors. while au-rich elements have been shown to stabilize rnas, it was shown that there is a correlation between au-rich elements in a given mrna and its susceptibility to endonucleolytic cleavage. a study by escalatine et al. showed that the herpes simplex virus's endonuclease vhs discrimination between target and non-target mrna could be based on the presence of au-rich elements [ ] . the authors suggested that many au-rich elements containing mrna encode immune sensors that could disrupt viral replication, and vhs-mediated decay could thus alleviate some of this stress. furthermore, one of the vhs-resistant transcripts is tristetraprolin (ttp), a protein that binds au-rich elements and promotes the decay of the corresponding mrna. therefore, by sparing ttp, the effect of vhs on au-rich element-containing transcript is further enhanced. furthermore, while this review focuses on host rna elements that escape viral endonucleases, there also exist several examples of rna structures that escape from host nucleases to benefit viral infection. this type of escape relies on complex structural elements and have been identified in a diverse range of viruses including members of flaviviridae, phleboviruses, and arenaviruses [ ] . these structural elements hinder the host most prevalent exonuclease xrn [ ] , and have been dubbed xrn -resistant rna or xrrna. this highly structured rna is necessary for the production of sub-genomic flavivirus rna (sfrna), and is located within the ′ utr of the viral genome [ ] . these xrrna have been shown to adopt a pseudoknot conformation at which point xrn is blocked from proceeding [ ] . one structure of note is that of an xrrna element found in the phlebovirus rift valley fever virus, w. rodriguez, et al. where the element that stalls xrn is found within a g-rich region. this region has similar characteristics to a g-quadruplex, a secondary structure that forms between four stacked guanine that interact in hoogsteen hydrogen bonding to form stabilizing structures [ ] and suggest that the presence of g-quadruplexes may play an additional role in transcript stability and xrn resistance. overall, escape from viral nuclease cleavage may have widespread effects during infection and regulation of this escape mechanism may be viral or host driven. by understanding how rna elements can trigger targeting versus escape, it could be turned into powerful anti-viral drugs. with the advent of deep sequencing technology, it has emerged that regulation of rna decay relies heavily on "rna elements"both sequence and structural elements. as we discussed in this review, the characterization of these elements have helped us better understand of how viruses seize control of gene expression during infection. the never-ending battle to control the gene expression environment of the host cell has given rise to viral nucleases able to bypass several ratelimiting steps of the cellular rna decay pathways to accelerate mrna decay. insights gleamed from these mechanistic parallels between cellular and viral-mediated decay allow us to draw connections between seemingly distal stages of gene expression. these insights also draw attention to the critical role of rna structural/sequence elements in governing the fate of mrna and the widespread consequences of disturbing the equilibrium these rna elements govern. many challenges remain in the study of rna features that direct cleavage or protection from viral and host endonucleases. to date, very few large-scale screens of the targets/escapees across the eukaryotic transcriptome have been reported. this is due to a number of technical challenges, in particular the fact that most current rna-seq techniques require significant transcript expression levels to accurately assess shifts in gene expression [ ] . this is therefore inherently complicated by the expression of a viral nuclease with widespread rna decay activity. looking at rna stability in pools of infected cells has also historically been a problem during interpretation of sequencing results as infection may progress at different rates in cells. however, with the development single-cell sequencing technology, the next few years will be very essential to provide us with a more accurate overview of rna decay upon infection. identification of rna elements regulating rna stability is important to better understand gene expression and the past couple decades have seen a lot of effort in trying to identify and annotate novel regulatory sequences. using viral nucleases to uncover such elements has been very prolific and we anticipate it will continue to bring to light a vast network of rna regulatory elements that may be contributing to the regulation of rna decay. furthermore, while this review does not address this aspect of rna regulation, one of the main "rna elements" that has taken center stage the past couple of years is that of rna modifications. there are over of these chemical additions, and they are emerging as potent regulators of mrnas, mirnas, and lncrnas in all aspects of structure, function, and decay. rna post-transcriptional modifications can control when an mrna is degraded in a complex and nuanced manner [ , ] . even the localization along the mrna and stoichiometry of these modifications can influence the fate of the transcript. in the case of n methyladenosine (m a), where a methyl group is added to an adenine, these modifications serve as platform to recruit m a reader proteins. the composition of these m a reader complexes will impact multiple aspect of rna fate, from accelerated degradation by targeting to pbodies to regulation of splicing and export of mrnas [ ] . presence of an m a modification can also affect the structure of the mrna, acting like a switch, and creating new platforms for binding. with how deeply ingrained rna modifications are in the fate of mrna it only makes sense that an obligate parasite like a virus seeking control over host processes would attempt to subvert or utilize these as well. future work will reveal how viruses interface with the host post-transcriptional machinery and should yield important insights into the regulation of rna fate. finally, while viral endonucleases can trigger widespread mrna turnover, they only represent one of many strategies deployed by viruses to control rna turnover and reshape the host gene expression landscape. viruses can also encode their own non-coding rna [ , ] that can have widespread effect on dampening the host anti-viral response. furthermore, host non-coding rnas are known to be critical for numerous anti-viral and apoptotic processes, and therefore subverting this arm of the host gene expression machinery is also essential [ ] . for example, vacvin addition to encoding the deccaping enzymes d and d -encodes a poly(a)-polymerase (vp ) that polymerizes a polyadenosine tract tail onto the vast majority of host microrna (mirna), triggering their degradation by the ′-end uridylation-dependent and the ′- ′ exonuclease-mediated decay pathways [ ] . some viruses have a more specific strategy to selectively target anti-viral mirnas. hcmv, for example, encodes a long bicistronic non-coding rna, ul -ul known as mirde, that triggers the decay of the cellular mir- /mir- a family mirnas through a non-canonical mirna--mirna interaction [ ] . these studies emphasize the importance of accounting for the degradation of non-coding rnas during viral infection and the broader implications of this decay in regard to global rna interference pathways. the features that comprise a messenger rna, from cap-to-tail, define nearly all aspects of rna life. as such, it is unsurprising that viruses have evolved to take advantage of these rna elements and the regulatory pathways that are deeply intertwined with these elements. thus, by studying the mechanisms governing this takeover during infection, we can continue to reveal the integral relationship between the fate of rna and the fate of the cell. interplay between viruses and host mrna degradation a tale of two rnas during viral infection: how viruses antagonize mrnas and small non-coding rnas in the host cell rna regulatory processes in rna virus biology advances in analyzing virus-induced alterations of host cell splicing rna decay during gammaherpesvirus infection reduces rna polymerase ii occupancy of host promoters but spares viral promoters viral nucleases induce an mrna degradation-transcription feedback loop in mammalian cells herpesvirus infection reduces pol ii occupancy of host promoters but spares viral promoters rna polymerase ii subunits link transcription and mrna decay to translation a two-pronged strategy to suppress host protein synthesis by sars coronavirus nsp protein connections underlying translation the intimate relationships of mrna decay and translation a common strategy for host rna degradation by divergent viruses emerging roles for rna degradation in viral replication and antiviral defense targeting poxvirus decapping enzymes and mrna decay to generate an effective oncolytic virus characterization of the african swine fever virus decapping enzyme during infection mrna degradation by the virion host shutoff (vhs) protein of herpes simplex virus: genetic and biochemical evidence that vhs is a nuclease lytic kshv infection inhibits host gene expression by accelerating global mrna turnover host shutoff during productive epstein-barr virus infection is mediated by bglf and may contribute to immune evasion an overlapping protein-coding region in influenza a virus segment modulates the host response c orf broadly escapes viral-induced endonuclease cleavage and restricts kaposi sarcoma associated herpesvirus (kshv) transcriptome-wide cleavage site mapping on cellular mrnas reveals features underlying sequence-specific cleavage by the viral ribonuclease sox the influenza a virus endoribonuclease pa-x usurps host mrna processing machinery to limit host gene expression selective degradation of host rna polymerase ii transcripts by influenza a virus pa-x host shutoff protein kshv sox mediated host shutoff: the molecular mechanism underlying mrna transcript processing nuclease escape elements protect messenger rna against cleavage by multiple viral endonucleases site specific target binding controls rna cleavage efficiency by the kaposi's sarcoma-associated herpesvirus endonuclease sox rna splicing by the spliceosome rna localization: making its way to the center stage communication is key: ″- ″ interactions that regulate mrna translation and turnover the hdcp protein is a mammalian mrna decapping enzyme xrn ″→ ″ exoribonucleases: structure, mechanisms and functions decapitation: poxvirus makes rna lose its head characterization of a second vaccinia virus mrna-decapping enzyme conserved in poxviruses vaccinia virus d protein has mrna decapping activity, providing a mechanism for control of host and viral gene expression the nudix hydrolase superfamily decapping the message: a beginning or an end the african swine fever virus g r protein possesses mrna decapping activity the g r (d ) gene of african swine fever virus encodes a nudix hydrolase that preferentially degrades diphosphoinositol polyphosphates insight into influenza: a virus cap-snatching influenza a virus protein pa-x contributes to viral growth and suppression of the host antiviral and immune responses critical role of the pa-x cterminal domain of influenza a virus in its subcellular localization and shutoff activity mapping of a region of the pa-x protein of influenza a virus that is important for its shutoff activity specificity and functional interplay between influenza virus pa-x and ns shutoff activity coronavirus nonstructural protein : common and distinct functions in the regulation of host and viral gene expression severe acute respiratory syndrome coronavirus nsp protein suppresses host gene expression by promoting host mrna degradation middle east respiratory syndrome coronavirus nsp inhibits host gene expression by selectively targeting mrnas transcribed in the nucleus while sparing mrnas of cytoplasmic origin sars coronavirus nsp protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp -induced rna cleavage the endonuclease cue cleaves mrnas at stalled ribosomes during no go decay a two pronged strategy to suppress host protein synthesis by sars coronavirus nsp protein severe acute respiratory syndrome coronavirus nsp facilitates efficient propagation in cells through a specific translational shutoff of host mrna sars-cov- is sensitive to type i interferon pretreatment the herpes simplex virus vhs protein induces endoribonucleolytic cleavage of target rnas in cell extracts in vitro mrna degradation system to study the virion host shutoff function of herpes simplex virus control of mrna stability by the virion host shutoff function of herpes simplex virus mrna decay during herpes simplex virus (hsv) infections: protein-protein interactions involving the hsv virion host shutoff protein and translation factors eif h and eif a mrna decay during herpes simplex virus (hsv) infections: mutations that affect translation of an mrna influence the sites at which it is cleaved by the hsv virion host shutoff (vhs) protein herpes simplex virus vhs protein the herpes simplex virus host shutoff (vhs) rnase limits accumulation of double stranded rna in infected cells: evidence for accelerated decay of duplex rna rna degradation induced by the herpes simplex virus vhs protein proceeds ″ to ″ in vitro the vhs mutant form of herpes simplex virus virion host shutoff protein retains significant internal ribosome entry site-directed rna cleavage activity coordinated destruction of cellular messages in translation complexes by the gammaherpesvirus host shutoff factor and the mammalian exonuclease xrn mrna stability and the control of gene expression: implications for human disease cis-regulatory elements explain most of the mrna stability variation across genes in yeast a ribonucleoprotein complex protects the interleukin- mrna from degradation by distinct herpesviral endonucleases an rna element in human interleukin confers escape from degradation by the gammaherpesvirus sox protein deep sequencing reveals direct targets of gammaherpesvirus-induced mrna decay and suggests that multiple mechanisms govern cellular transcript escape host transcript accumulation during lytic kshv infection reveals several classes of host responses highly selective escape from kshv-mediated host mrna shutoff and its implications for viral pathogenesis the herpes simplex virus ul gene-dependent destabilization of cellular rnas is selective and may be sequencespecific identification of phlebovirus and arenavirus rna sequences that stall and repress the exoribonuclease xrn deadenylation of the unstable mrna encoded by the yeast mfa gene leads to decapping followed by ″- ″ digestion of the transcript zika virus noncoding sfrnas sequester multiple host-derived rna-binding proteins and modulate mrna decay and splicing during infection exoribonuclease-resistant rnas exist within both coding and noncoding subgenomic rnas the structure and function of dna gquadruplexes rna sequencing: the teenage years a molecular-level perspective on the frequency, distribution, and consequences of messenger rna modifications the emerging biology of rna post-transcriptional modifications reading, writing and erasing mrna methylation viral noncoding rnas: more surprises a combined computational and microarraybased approach identifies novel micrornas encoded by human gamma-herpesviruses viruses and mirnas: more friends than foes vaccinia virus infection suppresses the cell microrna machinery selective degradation of host micrornas by an intergenic hcmv noncoding rna accelerates virus production we thank all members of the muller lab for their insights. our work is supported by the umass microbiology startup funds to mm, a university fellowship in microbiology to wr and a spaulding smith fellowship to dmf. key: cord- -ydkigome authors: villarreal, luis p. title: the widespread evolutionary significance of viruses date: - - journal: origin and evolution of viruses doi: . /b - - - - . - sha: doc_id: cord_uid: ydkigome in the last years, the study of virus evolution has undergone a transformation. originally concerned with disease and its emergence, virus evolution had not been well integrated into the general study of evolution. this chapter reviews the developments that have brought us to this new appreciation for the general significance of virus evolution to all life. we now know that viruses numerically dominate all habitats of life, especially the oceans. theoretical developments in the s regarding quasispecies, error rates, and error thresholds have yielded many practical insights into virus–host dynamics. the human diseases of hiv- and hepatitis c virus cannot be understood without this evolutionary framework. yet recent developments with poliovirus demonstrate that viral fitness can be the result of a consortia, not one fittest type, a basic darwinian concept in evolutionary biology. darwinian principles do apply to viruses, such as with fisher population genetics, but other features, such as reticulated and quasispecies-based evolution distinguish virus evolution from classical studies. the available phylogenetic tools have greatly aided our analysis of virus evolution, but these methods struggle to characterize the role of virus populations. missing from many of these considerations has been the major role played by persisting viruses in stable virus evolution and disease emergence. in many cases, extreme stability is seen with persisting rna viruses. indeed, examples are known in which it is the persistently infected host that has better survival. we have also recently come to appreciate the vast diversity of phage (dna viruses) of prokaryotes as a system that evolves by genetic exchanges across vast populations (chapter ). this has been proposed to be the “big bang” of biological evolution. in the large dna viruses of aquatic microbes we see surprisingly large, complex and diverse viruses. with both prokaryotic and eukaryotic dna viruses, recombination is the main engine of virus evolution, and virus host co-evolution is common, although not uniform. viral emergence appears to be an unending phenomenon and we can currently witness a selective sweep by retroviruses that infect and become endogenized in koala bears. phylogenetic tools have greatly aided our analysis of virus evolution, but these methods struggle to characterize the role of virus populations. missing from many of these considerations has been the major role played by persisting viruses in stable virus evolution and disease emergence. in many cases, extreme stability is seen with persisting rna viruses. indeed, examples are known in which it is the persistently infected host that has better survival. we have also recently come to appreciate the vast diversity of phage (dna viruses) of prokaryotes as a system that evolves by genetic exchanges across vast populations (chapter ). this has been proposed to be the " big bang " of biological evolution. in the large dna viruses of aquatic microbes we see surprisingly large, complex and diverse viruses. with both prokaryotic and eukaryotic dna viruses, recombination is the main engine of virus evolution, and virus host co-evolution is common, although not uniform. viral emergence appears to be an unending phenomenon and we can currently witness a selective sweep by retroviruses that infect and become endogenized in koala bears. our understanding of virus evolution has reached a threshold in that it now appears to provide a much broader vista regarding its general signifi cance and infl uence on host evolution. several developments have brought us to this point. one has been the realization that viruses often evolve by processes involving the collective action of a consortia, or quasispecies. and the resulting adaptability and power of such evolution is unmatched by any other genetic entity. much of this volume is dedicated to this issue. in such consortia, the concept of a " wild-type " virus is no longer considered to be the fi ttest type, as the quasispecies itself provides fi tness (see chapters and ). the quasispecies model resembles population genetics in some ways, but it has led to some signifi cant departures from population genetics, and these departures are very well supported by experiments. another development that has recalibrated our view of the overall signifi cance of viruses is information on the scale and diversity of viruses. viruses are present at a previously unappreciated global level and appear to have affected the evolution of all life on earth. much of this realization has been brought about by the development of metagenomic methods as applied to various habitats. measurements of major habitats (the oceans, soil, extreme environments) have established that our biological world is predominantly viral, in terms of both numbers and diversity ( paul et al. , ; breitbart et al. , ; rohwer, a, b ; edwards and rohwer, ; comeau et al. , ) . these two developments would seem reason enough to consider virus evolution in a new light. however, there have also been numerous theoretical proposals suggesting viral involvement in some of the very earliest events and major transitions in the evolution of life. we no longer think of viruses as recent agents that escaped from the host chromosomes as run away replicons. viruses now appear very old to us and they relate to and trace all branches of life. the last years have been very active regarding virus evolution. major developments in theory, technology, medicine, and the study of human disease with respect to virus evolution have all occurred. and as we seek to grow and manage various life forms for human use, virus evolution has also had major impact on such efforts. as a science, virus evolution has benefi tted greatly from traditional evolutionary biology. however, since viruses are molecular genetic parasites that are inscrutable by casual observation, our understanding of virus evolution has been dependent upon measuring sequence variation and sequence diversity in a large number of virus genomes. because of this, these small genetic parasites have been the last domain of life to yield their secrets of evolution. and viruses harbor some clearly distinct evolutionary abilities. for one, they are polyphyletic. all major viral lineages have their own distinct origins. they are also difficult if not impossible to defi ne as species and are able to exchange genetic information across normal boundaries. even " dead " and defective viruses can participate in such exchanges, which confuses the defi nitions of fi tness. we now know that viruses can evolve by a consortia process and also exchange information by recombination across vast genetic pools to assemble new mosaic combinations of genes. we thus no longer think of a specifi c genetic lineage in understanding virus evolution, but instead think of a cloud, matrix, or a population as the basis of virus evolution. viruses are inherently fuzzy entities that can differ from their relatives in any specifi c feature. yet even with such fuzziness, it is clear that common themes also link them. patterns of evolution have become clear. diversity and variation are often (but not always) observed. stability and host congruence can also be observed. nevertheless, the evolutionary power of viruses has been learned at a human cost. the application of numerous analytical and phylogenetic tools have provided crucial insights into virus origin and evolution. yet these methods struggle to incorporate the fuzzy nature of viruses and have clear limits, especially regarding quasispecies and high recombination rates. structural biology now also adds tools that extend our vision of virus evolution beyond what can be seen in the genetic sequence. for example, common structural motifs from phage to eukaryotic dna viruses (t and herpesvirus) suggest very ancient links in virus evolution that span all domains of life (see below). nevertheless, our analytical methods are currently lacking as we struggle to understand complex genetic mixtures that provide fi tness, reticulated relationships, polyphyletic origins, and virus-host congruence. virus evolution has, for the most part, been considered to be a specifi c, esoteric part of broader evolutionary biology and has been given limited attention in reference works on evolutionary biology (see pagel, ) . historically, the focus has been on various rna viruses and some dna viruses that cause disease in humans and domesticated animals and plants . however, i have asserted that all life forms must be examined from the perspective of virus evolution ( villarreal, ) , not just those pathogens that impact on us. how viruses evolve in a more general sense informs us of evolutionary paradigms that have not been previously well understood (especially the evolution of consortia or the dynamics of vast reticulated gene pools). this volume now extends these traditional topics of virus evolution to include the vast virology of the prokaryotic world. in so doing, it illuminates the global consequences viruses have had on all life forms. prior to the s we saw some stunning successes in vaccine control for major viral human diseases, such as polio, measles, mumps, infl uenza, and especially smallpox virus. due to this historic success, american health agencies and educators considered virus disease as a thing of the past, no longer a serious threat. the era of infectious disease that they represented was now one for the historical record, an unfortunate part of human history, or so we thought. in what now seems to have been a clear case of hubris and naivety, we have been humbled by the evolutionary power of viruses, which was woefully underappreciated, even by most virologists. by the end of that decade, the evolution and emergence of hiv- permanently changed our views (see chapters and ) . this has also been followed by a seemingly never-ending series of viral threats as newly emerging viral diseases have come to our attention. hiv- provides the only example of a public health situation that has reversed centuries of progress for extending human health and lifespan. it now limits human life expectancy in many parts of the world, especially sub-saharian africa. this development could not have been imagined in the s. we are much less confi dent now about predicting the future of virus evolution and its potential impact on human health. diseases of domesticated microbiological, plant, and animal species have also experienced the trauma of the consequences of emerging viral diseases along with huge losses. however, the human hiv- story may not be a fl uke of virology but may be telling us something basic about human and primate evolution. as we sought to understand the origin of this new virus, we have come to appreciate a much broader virus-host story which involves simian immunodefi ciency virus (siv), foamy viruses, and the speciation of old world primates. we have also come to learn that the genomes of these primates show much evidence of past viral interaction and ongoing endogenous retrovirus colonization. the evolution of retroviral endogenization has taken on a much greater signifi cance in basic evolutionary biology. thus it is with great interest that we now study the ongoing endogenization of retroviruses in the koala bear genome (see below). historically, we are compelled to study viruses because they can cause serious disease. new viruses come to our attention also mainly because of disease. it is therefore understandable that most evolutionary biologists mainly think of viruses strictly as agents of disease. these are the products of run away replicons that provide negative selection to host survival. in this light, the application of predator-prey based mathematical models has seemed most appropriate. with such viral disease, variation has long been observed and was initially used for the generation of most vaccines. however, this disease-centric view has also occluded another more prevalent virus-host relationship. for example the emergence of hiv- has led us conclude that it likely evolved from various versions of siv. but siv is not pathogenic in its native african primate host. nor does it show the genetic diversifi cation of hiv- in these native primate hosts. it is a silent, asymptomatic infection. genomic and metagenomic analysis now allows us to identify many more silent, asymptomatic viruses that would not have previously been observed. we now know of many such viruses that are prevalent in a specifi c host. evolutionary biology must escape the confi nes of disease-centric thinking and seek to understand these relationships as well. in the last ten years i have attempted to provide another view concerning virus-host evolution. i have argued that viruses often attain evolutionary stability by species-specifi c persistence and that such states apply to all domains of life, including prokaryotes. on an evolutionary time-scale, the majority of viral lineages tend to exist as species-specifi c persistent (aka temperate, latent, and chronic) infections in which individual hosts will be colonized by mostly silent (asymptomatic) viruses for the duration of their life . such persistence can have major consequences to the evolution of both virus and host, which also leads us to more directly link virus evolution to broader issues of host evolution. it is from this perspective that we start to clearly see that viruses indeed belong on the tree of life as major participants ( villarreal, ( villarreal, , . persisting viruses are not simply agents responsible for destruction of life, but are also agents that create genetic novelty on a vast scale that infl uences all life and promotes symbiosis ( marquez et al. , ; ryan, ; villarreal, ) . the persistent lifestyle of such symbiotic virus-host relationships is not simply a less effi cient, acute infection; nor is it simply a " reservoir " for acute virus (as epidemiologists are prone to assert). neither can it be attributable to concepts of selfi sh dna. persistence represents a major virus life strategy that is both fundamental and highly adapted. it has distinct genetic, fi tness, and evolutionary characteristics that require intimate, host (tissue)-specifi c viral strategies and precise gene functions to attain stable maintenance in the presence of immunity and to allow biologically controlled reactivation. persistence also must resist displacement by similar viruses and competitors. it is virus-host persistence that provides the thread that allows us to link these polyphyletic viral lineages (and their clouds) with the entire tree of life. in turn, this link identifi es a much more fundamental role for viruses in the evolution of host, visible from the very earliest to the most recent events in host evolution. it is from such species-specifi c persistent states that the large majority of acute diseases evolve and emerge by various mechanisms. we know much about virus replication and disease. however, our understanding of the specifi c mechanisms of persistence is generally poor. persistence is a generally silent and inscrutable state, it does not lend itself to in vitro or cell culture experimental models. we are left with but a few examples from which to attempt to extrapolate the possible existence of general relationships. the study of virus evolution thus struggles to incorporate concepts of persistence. another recent and major development in virus evolution is the arrival of various proposals suggesting that viruses have been involved in some major innovation and transition in the evolution of life. in all these proposals, however, it is necessary that the virus in question has attained a stable genomic persistence with its host. these evolutionary events thus seem to be the products of viral-mediated symbiogenesis of host ( ryan, ; villarreal, ) . proposals include the possibility that viruses may have originated the dna replication system of all three cellular domains (archaea, bacteria, eukarya) of life ( forterre, ( forterre, , ( forterre, , a ( forterre, , b filee et al. , ; forterre et al. , ) . the discovery and analysis of the largest dna virus ( orf mimivirus), a lytic cytoplasmic virus of amebae (a distant relative of phycodnavirus and poxviruses), has also led to proposals that this virus lineage may represent an ancient fourth domain of life ( raoult et al. , ; desjardins et al. , ; claverie et al. , ) . it is interesting that in an initial structural analysis, the large complex replication centers for mimivirus were confused with the host nucleus ( suzan-monti et al. , ) . thus, it seems relevant that others have proposed that a distant relative of phycodnaviruses and poxviruses may have originated the eukaryotic nucleus ( villarreal, ; villarreal and defilippis, ; bell, bell, , takemura, ) . such proposals, although consistent with various observations, however, remain outside of the consensus of most evolutionary biologists. nevertheless, numerous other observations continue to suggest viral involvement in other major host innovations, such as a viral origin of rag / of the adaptive immune system ( dreyfus et al. , kapitonov and jurka, ; fugmann et al. , ) or the role of endogenous retroviruses (ervs) in the evolution of the placenta ( villarreal and villareal, ; harris, ; blond et al. , ; mi et al. , ; dupressoir et al. , ; caceres and thomas, ) . such possible roles for viruses in host evolution are at odds with accepted views of virus-host relationships, but might be the products of viral symbiogenesis. the metagenomic viral measurements mentioned above for prokaryotic dna viruses, along with the increasing realization that viruses and host can co-evolve, has led to various calls that a viral tree of life needs to be considered and developed ( forterre, ; villarreal, ; filee et al. , b ) . a virosphere clearly exists but its nature and boundaries are not so clear. multiple viral origins, their diversity and numerical dominance in distinct and sometimes harsh environments as well as their presence in host genomes suggest that any viral tree of life will be huge, multidimensional, and connected to the host tree of life. as discussed below regarding double-stranded (ds)dna viruses of prokaryotes, they have all the above characteristics. such viruses may represent the big bang of biological novelty. with their unmatched capacity to generate diversity they can function as the mass creators of biological novelty as well as destroyers of most species. surely, such capacity must have had big infl uences on the evolution of life. symbiosis, simply defi ned, is the stable coexistence of two previously separate lineages of organisms. there can be little doubt that many temperate phage can stably colonize a bacterial cell resulting in a stable descendent from two lineages. this is clearly symbiotic. endogenous retroviruses can similarly be found to persist in vertebrate genomes and also appear symbiotic. yet studies of symbiosis seldom consider a role for virus ( villarreal, ) . how important are viruses in general to evolutionary biology? the core concepts of evolutionary biology were developed well before we had a modern understanding and defi nition of viruses ( luria, ) . after all, the basic lysogenic model of phage integration was only clarifi ed in when developed by campbell (see campbell, ) . that cryptic and defective phage are ubiquitous in the genomes of all prokaryotes is generally considered uninteresting by many in the fi eld of evolutionary biology. i suggest that the seemingly applicable concepts of selfi sh dna effectively derailed any thinking that persisting genetic parasites might have a more germinal role in the evolution of life ( doolittle and sapienza, ; orgel and crick, ). yet as outlined above, virus footprints in major evolutionary transitions are clear and a direct role in such events now seems much more plausible. we therefore must seek to defi ning the nature of the virosphere and how its evolution relates to the tree of life. this book represents the fi rst integration of the entire fi eld of virus evolution, including both prokaryotic and eukaryotic life forms. however, because our understanding of virushost relationships remains uneven, the chapters necessarily focus on well-studied models (exemplars). these exemplars also tend to refl ect a historic disease focus (i.e., e. coli , fl owering plants, mouse, humans). it is unfortunate that the silent species-specifi c viruses that tend to exist in stable states with long evolutionary histories seldom provide our examplars. we understand these infections poorly and lack basic defi nitions concerning fi tness or selective advantages. only metagenomics tools now seem able to inform us of their presence, but not their biology. self-organization and the evolution of rna molecules as an origin of biological information are discussed in chapter . autocatalytic chemical reactions, such as replication of rna, presents issues such as how to optimize a rugged fi tness landscape yet allow the study of evolution in vitro with rna. rna in vitro reduces genotype-phenotype issues to rna secondary structure and minimal free energy states. this allows both continuity and discontinuity to be measured. these same issues are crucial for the study of rna viruses, whose sites of secondary structure often defi ne replicator identity. these models currently offer the best system to evaluate an early and simple biological world for evolutionary principles (see chapter ). viruses and viroids with their rna genomes may be the only extant survivors of this pre-dna world. since it was the consideration of error-prone replication that led to the development of the concept of quasispecies (see chapter ), such models have provided a conceptual foundation which led to several basic concepts. chapter discussed the foundations and various aspects of quasispecies theory. viruses appear to operate close to the error threshold, thus allowing maximum evolutionary exploration ( biebricher and eigen, ) . however, as presented below, the loss of the " fi ttest " type concept has also led to clear experimental evaluations of consortia-based evolutionary behaviors. such behaviors were not predicted by classical darwinian models. although virologists were initially attracted to quasispecies models, many evolutionary biologists were initially hostile to the application of the quasispecies concept to evolution. it was thought that the classical mathematical models of population biology, as originally developed by wright and fisher, and later applied by kimura and maruyama to asexual haploid populations at the mutationselection balance, had already fully developed the needed models and precluded additional need for the quasispecies concept. the classical models were thus argued to provide adequate mathematical coverage for viruses, including quasispecies and error threshold ( wilke, ) . however, these two approaches differ fundamentally with regard to the signifi cance of error-prone replication and it was the quasispecies approach that led to the clear experimental establishment that quasispecies selection, per se , is important for viral pathology and fi tness (see below). the development of quasispecies theory to virology does indeed demonstrate distinct differences with population genetics. various phenomena, such as complementation, cooperation, competition, and even defective mediated extinction ( domingo et al. , , domingo grande-perez et al. , ) have been observed, all of which fall outside of the parameters of classical population genetics. viral fi tness has indeed been shown to be due to interaction within a diverse population, and not to the fi ttest or master type. and with rna viruses, error threshold has become a central issue ( biebricher and eigen, ) . the collective experience has thus made clear the value of theory to biology. many working biologists understand that life seems overly complex and defi es most generalizations. thus they do not always appreciate attempts at general theory. although in reality, biology may indeed often be too complex for accurate theoretical predictions, these theories have nevertheless clearly stimulated crucial concepts and experimental evaluation, and biologists should be encouraged by them. by providing new ways of thinking, entirely new experimental approaches can be developed. the existence of error-prone replication and quasispecies also raises the issue of the conservation of information. how is information stability and higher fi tness attained with such errors? how can genetic complexity be created in such a circumstance? can cooperation (or consortia behavior) result from any of these models? these issues have yet to be resolved. some interesting suggestions, however, have been proposed. one involved cooperative evolution that results from ligated genomes. a model was proposed in by stadler and schuster in which they considered the dynamics of replicator networks resulting from higher order reactions involving the templated ligation of smaller genomes ( stadler et al. , ) . although this was based on concepts such as triple-stranded nucleic acids, this clearly has some elements that also resemble the ligation of recombinational processes for dna phage (presented below). a most interesting outcome of these models is that, depending on initial replicator concentrations, permanent coexistence of replicators could result in a cooperative network. such cooperation is a rare outcome for most models and given the conclusion that early dna based life was a " horizontal " consortium, such models are of special interest. the issue of consortia will come up often. consortia selection directly implies cooperation, but cooperation of selfi sh replicators presents dilemmas. replicator networks with interaction functions that give highly non-linear dynamics can result in complex mixtures, with behaviors ranging from survival of the fi ttest to also including attainment of globally stable equilibrium tantamount to permanent coexistence. the fi tness of populations, however, is inherent to current quasispecies concepts in virology. there may also be other ways to explain the genetic origin of cooperation (such as stable persistence involving addiction strategies). the stable persistence of a genetic parasite can compel cooperation and promote the conservation of information (see below). the defi nitions of fi tness with respect to a virus in a natural habitat are far from clear. although the concept of relative replicative fi tness is often applied to lab experiments of virus growth, we know many situations in which virus replication is not maximized in natural settings and many viruses can exist in relatively non-replicative states for long periods. even in the context of an acutely replicating virus in a host organism, the concept of fi tness is clearly conditional, as the virus must replicate through various in vivo habitats that can have opposing selection. as presented below, in vivo models that study fi tness and viral diversity have clearly indicated that diversity per se is important and fi tness is the result of consortia. how do we defi ne such fi tness since the mixture clearly matters? also, how do we defi ne information content or integrity of a consortia? currently, we cannot. in the lab, the viability of a virus is usually measured by the ability to produce plaques. this has been a crucial and main assay for many experimental systems that study virus population. here, the defi nition of fi tness seems direct: plaque formation equals fi tness. various highly useful models have thus been defi ned and developed that depend on these plaque assays (see chapter ). with this, populations and population growth are defi ned as relative growth of plaque-forming units. however, the concept has always been problematic when considered from a natural virology perspective. plaque formation is clearly not equal to fi tness in natural habitats. there are many examples of highly successful viruses that either plaque poorly or not at all. consider the roughly types of human papillomavirus (hpv), a simple small circular dna virus of epithelia; this does not form plaques in any known system (chapter ). hpv is clearly fi t, well adapted to, and stable in its human host. in addition, hpv evolution is phylogenetically congruent with their primate host, as are most persistent viral infections. we have yet to understand the definition of fi tness in this situation. in some cases, it seems selection for plaque propagation has clearly resulted in loss of highly conserved genes; such as with the plaque-adapted laboratory strains of cytomegalovirus (cmv). the problem posed by viruses with ineffi cient plaque formation is not limited to dna viruses. many persisting rna viruses also do not plaque well or at all, such as most rna viruses of plants or many insect picorna-like viruses, such as those found in drosophila and bees (which also conserve an extra orf). nor do most persistent infections make lots of virus. low-level persistence, such as hantavirus in rodents, for example, is common ( hart and bennett, ) . clearly, our simplifying assumptions of viral fi tness and population dynamics cannot apply to these stable evolutionary states. however, if we limit our defi nition of viral fi tness to relative replication or plaque formation we can perform some clear and quantitative evaluations. experimental evaluation forces us to study fi tness by only those defi nitions that we can currently measure. as fi tness appears to be a relativistic and transient concept, depending very much on the tissue, time, place, extant adaptive and innate immunity, and competition, it is likely that we can only measure with any accuracy one aspect of fi tness at any one time. hiv infection of humans shows evidence of this in that the r virus is more fi t for transmission and early disease whereas the x virus is fi t later during the aids disease phase. clearly conditional, time-dependent issues relate to fi tness defi nitions. however, much more problematic is that we have no theory for viral persistence or its fi tness. we lack specifi c or measurable parameters other than the simple maintenance of genetic material. yet it seems clear that some distinguishing features of persistence can already be recognized. for example, the possible participation of viral defectives (normally considered unfi t), which in numerous circumstances can modify or mediate persistence, would need to be included. clearly, a defective role in persistence would also preclude them from being considered as genetic " junk, " or selfish elements, since they would then matter in measurable ways to the biological outcome of virus persistent infection. persistence also requires an extended duration of infection, not simply maximized replication. in fact, persistence generally requires mechanisms to limit the replication of at least the same virus for at least some time. thus, limited replication must be an essential element for this life strategy. in my judgment, and much like the quasispecies concept, the concept of persistence will eventually be recognized for the fundamental (symbiotic) force it represents in virus evolution. the experimental work of domingo and holland spans the modern assessment of quasispecies theory that occurred in the s and s. these investigators were the chief proponents of this theory, bringing it to the attention of the broader virology community (chapter ). this work has transformed our thinking and laid the experimental foundations that we now build upon. this current volume is an extension from an earlier book on quasispecies and now encompasses both prokaryotic and eukaryotic viruses . since early experimental phage studies provided the foundations for quasispecies theory ( eigen et al. , ) , using mathematical descriptions (differential equations) of mutation rates in t-even phage ( luria and delbruck, ) , this inclusion is appropriate. interestingly, a second early paper measuring replication rates by these same authors also noted the problems of viral interference and defectives ( delbruck, ) . other early experiments of phage rna polymerase ( batschelet et al. , ) , especially with rna phage qβ ( domingo et al. , ) , helped set the stage for the subsequent experiments of the s and s. from the test tube to mouse models to the study of human disease, the work of domingo and colleagues has spanned the entire history of viral quasispecies ( domingo and gomez, ; chapter ) . quasispecies deals with the products of error-prone replication. however, it is worth repeating that products of error-prone replication are not behaving in a simple " selfish dna " capacity and are not devoid of biological relevance and phenotype. in their complex populations, they create clear and varied affects on viral adaptability, competition, and fi tness. since quasispecies necessarily involve defective and mutant virus, it is easy (and common) to think of these entities simply as genetic junk ( villarreal, ( villarreal, , . defective and even lethal or interfering variation in viral genomes can contribute to adaptability. thus, viruses can clearly adapt as a cloud with a mutant spectra. in addition, unending competition and exclusion, consistent with the red queen hypothesis, has also been observed ( clarke et al. , ) . the poliovirus-mouse model (see below) in particular has provided a solid experimental system for evaluating the adaptive consequence of quasispecies. it is thus ironic that these same experiments have also made clear that the original simplifying assumptions of the quasispecies ordinary differential as presented by eigen are violated by the resulting quasispecies. these products of error-prone replication do indeed strongly interact with each other in both positive and negative ways and such interactions contribute signifi cantly to the observed fi tness of the population. errors and interaction are important for fi tness. for example, defectives have been reported to mediate extermination of a competing wild-type virus ( grande-perez et al. , ). complementation has also been observed ( garcia-arriaza et al. , ) , as has trans -dominant inhibition ( crowder and kirkegaard, ) . genetic memory of past selection has been shown to be maintained in a minority of the population ( ruiz-jarabo et al. , ; briones et al. , ) . such cooperative (consortia) behavior, which can also depend on unfi t or defective members, is at odds with classical darwinian notions regarding survival of the fi ttest. consider, for example, the fi tness of a defective or mutant outside of its role in a quasispecies. such a consideration ignores the very nature of a quasispecies yet it is an issue that has often been posed and experimentally evaluated. we should refrain from thinking of viruses simply as fi t or non-fi t individual types since they clearly exist in populations that provide population-based adaptability. the selection of viral consortia or population raises some fundamental issues for evolutionary biology. this is essentially group selection in which a population, not the fi ttest individual, is selected. this view makes cooperation or interaction of individual genomes a signifi cant component of selection, which is not commonly thought to be a general or accepted process in evolution. yet population selection is no longer a contestable issue in rna virus evolution (see below). i expect that many classical evolutionary biologists might interpret this as evidence that viruses really are an oddity in this feature and are not representative of broader processes of evolutionary biology. furthermore, viral-based group selection may not be limited to quasispecies-based evolution. as presented below, persistent viral infections may also provide population-based selective advantage (see below for the p and mouse hepatitis virus (mhv) persistence exemplars; villarreal, villarreal, , . since viruses are ancient, numerically dominant, and the most diverse biological entities on earth, no life form can escape exposure to them. all extant life forms have evolved in a viral habitat. thus we should expect that the viral footprints (including defectives) that we now fi nd in all genomes have likely played an active role in their evolution; a role, i would argue, that is fundamental, dynamic, and unending. if we can accept this assertion, we may start to see and appreciate the vast evolutionary power that viruses can bring to bear onto host evolution. we can start to attain a global perspective and appreciation for their ability to assemble genetic function from enormous, complex mixtures of genomes, and select gene sets needed to solve multivariant, temporally dynamic evolutionary problems. we can then seek evidence for the role of viral elements in fundamental host innovation and be open to evaluating the occurrence of viral entities from a constructive perspective and not instinctively dismiss such observations as due to coincidence, " junk, " or selfi sh dna. the advantage of such a perspective is that it will promote the specifi c experimental evaluations that can better assess any constructive role genetic parasites might have played in host evolution. for example, there is much reason to think ervs have played an active role in human evolution (for references see ryan, ) . the quasispecies concept has provided the foundation for us to understand virus evolution and informed us of the evolutionary power viruses possess. if that power also links to host evolution, then the tree of life becomes enriched by virus, much larger and more dynamic. the recent experimental studies from andino and colleagues using poliovirus in the mouse model should, in my judgment, provide the keystone exemplar regarding the in vivo fi tness of quasispecies (see chapters and ; vignuzzi et al. , ) . these studies make clear the importance of quasispecies and error-prone replication. such detailed in vivo experiments were made possible by a long and detailed history of poliovirus studies that has identifi ed the nature of rna polymerase fi delity as well as developed mouse models for the study of pathogenesis. few other virus-host systems could have provided such potential for high resolution. these results also provide the experimental observations that distinguish quasispecies-based evolution from the classical fisher-based population genetics. the general importance of this story for understanding virus evolution thus deserves special emphasis. the very origins of modern animal virology stem from poliovirus studies with the need to develop in vitro cell culture technology in order to grow and evaluate poliovirus and generate variants. the live poliovirus vaccine is of special interest with regards to virus evolution and adaptability. the " live " oral sabin vaccine can be considered to have been a miracle of the practical approach to virology developed in the s ( horaud, ) in that it was used well before our understanding of the relevant evolutionary theory. the sabin vaccine strain was the result of rodentadapted virus and differs from the neurovirulent mahoney strain by point mutations (in the consensus sequence), although only a small number of these mutations were needed for neurovirulence ( christodoulou et al. , ) . one of the important neurovirulent mutations was within the rna polymerase gene ( tardy-panit et al. , ) . however, the signifi cance of this observation took many years to unravel and exploit. in time it became apparent that dpol mutants could affect replication fi delity. one poliovirus point mutant, dg s, was shown to have enhanced highfi delity replication and that selective pressure could be designed to increase fi delity in rna polymerase ( pfeiffer and kirkegaard, ) . another major development was the molecular identifi cation of the poliovirus receptor and the subsequent creation of transgenic mice expressing this receptor, making them susceptible to poliovirus infection. one of these transgenic lines allowed mouse brain infections with neurovirulent versions of poliovirus , and has provided a very useful animal model that allowed the evaluation of viral fi tness in the context of in vivo pathogenesis. although dg s replicates well in culture (with lowered error rate), it was less pathogenic in this mouse model and competed poorly with d wild-type virus. it seemed that the decreased viral diversity was less able to generate the variation needed to get past bottlenecks due to multiple selective differences presented in vivo in tissues in the host, such as brain infection ( pfeiffer and kirkegaard, ). this experimental system also makes clear the greater complexity of fi tness in vivo relative to that typically measured in culture. thus it seems that in vivo there may not be one fi tness but several that cannot be distinguished or individually measured. it is likely that various in vivo barriers require distinct fi tness solutions that tend to create bottlenecks and that the diversity per se is essential to get past such bottlenecks. a population, not a clone or a consensus, appeared more fi t as higher titer infections of dg s also failed to be pathogenic. thus, higher levels of a consensus virus are not equivalent to higher diversity. the relationship between rna polymerase structure, error rates, and ribavirin action is discussed by cameron in chapter and has been the subject of numerous studies ( crotty et al. , ; vignuzzi et al. , ) . knowledge of the structure and catalytic mechanism of rna polymerase function has allowed a greatly enhanced level of detail to be considered into what affects error rate (see castro et al. , ; korneeva and cameron, ; marcotte et al. , ) . this has provided insight into the likely action of ribavarin on product fi delity ( harki et al. , ) . thus, it appeared that even a mutant of rna polymerase with increased fi delity could still generate elevated diversity by various methods. such control of fi delity allowed for the design of control experiments in which the same consensus virus genome could be forced to generate either less or more diverse progeny populations. in no other virus-host system have we attained such detailed insight into issues of error rate as those that were put to such excellent use in the poliovirus-mouse system. how generally important is this poliovirus in vivo quasispecies result? although the poliovirus-mouse system provides us with a fi rm experimental result, it seems likely that the generality of this relationship will be questioned by evolutionary biologists for several reasons. for one, this was observed in a lab constructed model system, which, it could be argued, is not an accurate representation of in vivo virus-host fi tness. also, as mentioned above, group selection is a process that will not readily be accepted as representative by the broader community. is there any evidence that this result with poliovirus indeed represents a general virus-host evolutionary relationship in natural settings? as presented in chapters - , retroviruses and also human hepatitis virus c clearly exist as quasispecies populations that affect disease outcome. in the case of the retroviruses, viral populations show diversity that far exceeds that seen for other rna viruses. in both hiv- and hcv there is clear circumstantial evidence for the importance of quasispecies for in vivo disease outcome, drug resistance, and fi tness. in addition, with hcv, cns infection may sometimes result, and such brain infections appear to be mediated by distinct quasispecies ( forton et al. , ; forton et al. , ) , reminiscent of the polio virus mouse model. quasispecies memory, as mentioned above, also seems to be an important issue with regard to failure of antiretroviral therapy ( kijak et al. , ) and it appears that pol gene mutations could also be involved in this ( carobene et al. , ) . measurements of hiv quasispecies in individual patients indicates that multiple evolutionary patterns can be found in typical individual patients ( casado et al. , ) , thus mixtures of hiv exist in patients ( bello et al. , ( bello et al. , , . and hiv- recombination is clearly contributing to diversity ( kijak and mccutchan, ) . thus, with both hiv- and hcv, their capacity to cause human disease is clearly associated with quasispecies compositions that affect fi tness in complex ways. the poliovirus mouse system therefore appears to refl ect quasispecies issues as observed in natural virus-host situations. consideration of retrovirus-host evolution introduces another large issue in evolution: genomic viruses. unlike poliovirus and most rna viruses, retroviruses (e.g. non-lentivirus) have colonized the genomes of animal species in large numbers and represent a large fraction of these genomes. genomic retroviruses are present in vast numbers, most of which are defective and mutant copies. in this genomic colonization they resemble the dsdna viruses of prokaryotes (discussed below) that also colonize all prokaryotes although at a much lower numbers. the human genome has fewer than genes, but appears to have retroviral-related ltr elements. some of these elements are intact and conserved (human ervs (hervs)) and this genomic population has some clear characteristics of a viral quasispecies. such large amounts of genetic material have previously been dismissed simply as selfi sh or junk dna of no fi tness consequences to the host. however, given the importance of quasispecies mutant genomes for viral fi tness and persistence, we might need to re-evaluate this dismissal. retroviruses are clearly part of the human ancestry thus we should seek to understand, not dismiss their role in human evolution. in contrast to the story above in which polio infection of mouse brain was dependent on the quasispecies resulting from lowered fi delity replication, a different relationship has been proposed for the nidoviruses. these are also positive single-stranded polycistronic rna viruses ( gorbalenya et al. , ) . this group of virus includes the coronaviruses (e.g. mouse hepatitis virus and sars-associated coronavirus), which are the largest rna viruses known ( - kb). it has been proposed that such large genomes have required the adaptation of a high-fi delity rna polymerase in order to increase the error threshold and accommodate large rna genomes. based on the phylogenetics of this polymerase and other rna-processing enzymes, this group of viruses appears to be monophyletic and it is thought that the acquisition of a high-fi delity rna replicase was central to the origin of this lineage. this type of replicase is unique to rna viruses. the monophyletic view stems from an analysis of a small set of conserved genes. overall, however, these larger genomes have many other genes that show no similarities to related viruses. the origins and evolution of these more diverse and numerous genes cannot be currently traced. this is an inherent problem in the analysis of virus evolution: a small selected set of hallmark genes with some similarity are assumed to trace an apparently linear (tree-based) viral lineage whereas the larger number of genes are not included and cannot be traced. if most of rna virus evolution is indeed mediated by a mixed cloud of genomes, any role for mutant mixtures thus becomes obscure. but perhaps there is little else we can currently do given the lack of information. how might we explain the increased fi delity and genome size of the nidoviruses? was there some change in viral adaptation in which quasispecies and generation of mixtures was no longer as important for adaptation? did the need and selection for a larger genome override the use of error to generate adaptability as seen in poliovirus and hiv- ? if so, what selective pressures might have changed this seemingly basic feature? what do we know about the natural biology of these viruses, which might provide some insight into this? unfortunately, the natural distribution and gene functions of the nidoviruses are generally poorly understood. in terms of coronaviruses, numerous mammal and avian species can be infected and the virus will cause acute disease. in several of these acute infections, the virus involved seems to have recently been adapted to the new host from other, often unknown sources. with the recent emergence of the sars virus and human infections, however, much greater attention has been focussed on trying to understand the origin and evolution of this virus. it has recently become clear that there indeed appears to exist an evolutionary stable source of this virus from which adaptation to humans was possible. various bat species have been found to support persistent asymptomatic infections by specifi c versions of sars viruses ( tang et al. , ; vijaykrishna et al. , ) . these studies also indicate that there appear to be three different and independent groups of sars viruses in bats. in fact six novel coronaviruses were isolated from six different bat species showing an astonishing diversity in bats. furthermore, phylogenetic analysis indicates that all bat coronaviruses appear to have descended from a common ancestor. only one of these bat groups includes sars and sars-like coronaviruses that adapted to acute human infections. thus, a prevalent and speciesspecifi c persistence of sars viruses is found in particular geographical populations. why is this relationship stable? could the adaptation to a host-specifi c persistence-based basal life strategy provide some explanations for the evolution of the higher fi delity rna replicase of these coronaviruses? as i have argued, persistent viral infection represents the majority of evolutionary stable viral lineages ( villarreal, ) . however, we have almost no knowledge regarding how these bat sars viruses persist and escape elimination by innate and adaptive immunity and what, if any, role the high-fi delity replicase (or other genes) have in this life strategy. although we cannot yet evaluate natural sars virus persistence in native bat hosts, another coronavirus may be more informative regarding the effects of persistence on host populations. mouse hepatitis virus (mhv) may provide our best exemplar of virus-host relationships and show how the concept of virus addiction relates to population persistence. mhv is the best-studied coronavirus. as a natural and prevalent virus of rodents, mhv is our best natural model of persistent rna virus-host relationships for any mammal. in general, rodents are the most studied non-domestic mammals with regard to natural virus distribution. overall, we know that wild-caught rodents seldom show signs of acute virus infection ( kashuba et al. , ) . however, asymptomatic virus persistence is ubiquitous in wild rodents ( descoteaux et al. , ; gannon and carthew, ; schoondermark-van de ven et al. , ) , including voles ( descoteaux and mihok, ) . some fi eld studies have evaluated broader patterns of virus persistence in mice ( singleton et al. , ; becker et al. , ) which indicated that wild house mice are highly colonized with mhv ( - % prevalence). in addition to mhv, mouse cytomegalovirus, mouse parvovirus, mouse thymic virus, and mouse adenovirus are also prevalent. other well-studied mouse viruses, such as lymphocytic choriomeningitis virus (lcmv) and polyomavirus (pyv), were at low natural prevalence. interestingly, some non-native house mice that have colonized isolated islands may lack mhv ( moro et al. , ) , although most other isolated island populations retain mhv ( moro et al. , ) . other small mammals have yet to show any viral disease whatsoever (hedgehogs, chinchillas, prairie dogs, gerbils, sugar gliders) ( kashuba et al. , ) . thus, asymptomatic persistent viral infection is clearly the norm in rodents. yet, in spite of this usual asymptomatic viral persistence, historically, some zoonotic viral disease outbreaks have occasionally been documented in natural populations. one such early outbreak was an epizootic diarrhea that occurred in infant mice ( adams and kraft, ) . later, it was established that one such infection was due to mouse hepatitis virus ( carthew, ; ishida et al. , ) . in spite of this disease outbreak, with mhv, it has since become clear that asymptomatic persistent infections are the norm and are highly stable. yet mhv disease outbreaks, especially in virus-free mouse facilities, are also common and severe. how does mhv attain such stable and prevalent persistence in natural population yet retain the ability to cause disease in naive populations? what maintains the mhv fi tness of natural persistence? it is well known that once mhv is established in a mouse or rat colony it can be very diffi cult to eliminate ( gannon and carthew, ; lussier and descoteaux, ) , clearly indicating that stability is rapidly attained and likely genetically programmed by the virus. i propose that these stable evolutionary states of viral persistence are due to a strategy we can call virus addiction ( villarreal, ) and that mhv can provide the exemplar of such a state. with mhv, only persistently infected mice colonies are protected from the disease that is otherwise caused by the virus. in wild asymptomatic mice, mhv is found mostly as an enteric infection. the cns demyelinated disease that mhv can induce is most observed in newborn pups ( homberger, ; nash et al. , ) and once in the brain, mhv can persist in cns with recurring disease ( marten et al. , ) . this recurring cns disease is also associated with quasispecies (in the s gene) and recombination ( rowe et al. , ) . the most serious cns disease is in s-gene variant of mhv- (jhm), thus as with the poliomouse model, pathogenic fi tness with mhv is also associated with quasispecies. such mhv disease is the bane of all mouse colonies ( knobler et al. , ) . however, once mhv persistence is attained, the problem to a mouse facility is not due to acute disease, but because immunological measurements are signifi cantly affected by mhv persistence. thus mhv alters mouse molecular identity regarding immunological (t-cell) reactions ( wilberz et al. , ) . to establish stable asymptomatic persistence, however, mhv needs to infect newborns ( weir et al. , ) , in which acute disease is prevented due to maternal passive immune antibody transfer ( gustafsson et al. , ) . being born to immune mothers thus protects against cns disease and promotes enteric (not brain) virus colonization. in addition, it appears that persistence also promotes cross-species transfer ( baric et al. , ) . mhv persistence may involve genome stability and result in a distinct evolutionary dynamic. asymptomatic persisting infections in a lewis rat, for example, showed no variation in mhv s gene sequence, and no quasispecies as seen in brain infections ( stuhler et al. , ) . the need to establish stable persistence could then be providing a strong selection for increased genome complexity and stability and might better explain the selection for the enhanced rna polymerase fi delity in nidoviruses. how might such selection operate in natural populations? evolutionary biologists often consider what might differentiate one group from another very similar group in a way that leads to two isolated and distinct populations. consider two hypothetical adjacent hay stacks harboring two mus musculus colonies, one of which is persistently infected with mhv the other which is not. what is the fi tness consequence to the colony harboring mhv relative to its uninfected neighbor? our experience with mhv in mouse breeding colony provides a clear answer. the colony that is persistently infected with mhv will have a distinct advantage over its neighbor as mhv introduced into this uninfected colony will have severe effects on the offspring. eventually, we can expect only the mhv-harboring colony will prevail in both hay stacks. this is a state i have called virus addiction. only mice harboring persistent mhv are protected against the potential pathogenic consequence of acute mhv (or related virus) infection. the population is addicted to the virus. such a state, however, is clearly affecting colonies (or groups) of host, not individuals. an individual either quickly succumbs to the virus infection or, if infected, transmits it to others in the colony. a colony is thus under selection by mhv. to generalize this state, we expect that the persistence of sars in specifi c bat populations would be expected to also affect the fi tness of the corresponding specifi c bat populations. persistence is a more demanding phenotype than acute replication. it requires greater gene complexity to counter host immunity and also to promote self-regulation. thus the enhanced fi delity of rna replication is selected in order to conserve this greater genetic complexity and stability. we know that the high-fi delity rna replication system (including rna pol, helicase, endoribonuclease, and other activities) is also present in an ancient nidovirus relative of coronaviruses, such as fi sh-isolated white bream virus ( kb rna). i suggest there will also likely be species-specifi c persistent infections with this virus that require this enhanced replication fi delity and maintain this virus in its natural habitat. thus, i suggest, an ancient persistent life strategy could more easily explain the monophyletic character of the nidovirus virus lineage. it is particularly interesting that one of these unique and conserved replication proteins (adp ribose- -monophosphate) is dispensable for culture growth ( putics et al. , ) . i suggest it will not be dispensable for persistence. the hiv- pandemic is an unfi nished story. hiv- represents a real-time biological event in human evolution that confi rms for us the importance of quasispecies and retroviruses to human biology. however, even though its human toll is huge, modern medicine and culture has responded rapidly enough to limit the impact of hiv- to the point at which it will not likely be the cause of a selective evolutionary sweep that could have altered human genetic makeup (in contrast to the koala bear endogenization presented below). as described earlier, its amazing adaptability via quasispecies along with extensive recombination contribute directly to hiv- ' s diversity and makes it the most dynamic genetic entity ever studied. many studies track the dominant hiv population and fail to examine minority populations. yet it is precisely these minority populations, which evolve independently of the majority population, that can determine drug resistance phenotype and biological outcome ( charpentier et al. , ; briones et al. , ; morand-joubert et al. , ) . clearly, the specifi c makeup of a complex hiv population matters. furthermore, hiv defectives and variants can also have major consequences. in some cases, long-term non-progressors of hiv- have shown mixed populations and unusual polymorphism in the early phase of hiv infection, sometimes contributing to long-term non-progression (ltnp) ( alexander et al. , ) . one population of ltnps was reported to have been colonized by an hiv variant that showed low virus replication and slow or arrested evolution ( bello et al. , ) . in another case, a stable non-progressor was colonized by a replication incompetent version of hiv- ( wang et al. , ) . some of these non-progressors also appear to resist super-infection . it seems clear that at least in these exceptional situations, non-majority hivs are crucial to the outcome. there is also reason to think that other retroviruses have had a major infl uence on recent primate and human evolution, such as apathogenic persisting foamy virus in primates ( switzer et al. , ; murray and linial, ) . human antiretroviral genes seem to have undergone recent adaptations, such as apobec , which can interfere with exogenous retroviruses (such as mlv and siv) and underwent an expansion in the hominid lineage ( esnault et al. , ) . it thus seems clear that human and primate evolution has been signifi cantly affected by earlier, prevalent primate retroviruses. another important human-virus quasispecies story that has long been recognized is with hepatitis c virus (hcv), (see chapter ; domingo and gomez, ) . hcv seems to have adapted to humans in the recent past, possibly from asymptomatic enteric primate viruses currently found in africa ( smith et al. , ) . as hcv remains an infection predominantly transmitted by blood, it does not appear to have fully adapted to the tissues of and transmission within its human host. however, like hiv- , hcv has long been recognized to generate quasispecies in chronically infected people ( martell et al. , ) and it soon became apparent that the viral quasispecies are affected by and affects the outcome of antiviral therapy hohne et al. , ; kurosaki et al. , ; okamoto and mishiro, ) . thus, successful antiviral therapy is directly correlated with an initial dramatic reduction in genetic diversity. unfortunately, it has become clear that only a minority of hcv-infected individuals will respond favorably to a combination of interferon and ribivarin. thus it seems to be diversity per se and the resulting structure of an hcv quasispecies that has a direct consequence to human health. however, since hcv is less well-adapted to humans compared with hiv- , it does not pose the same threat to potentially provoke an evolutionary event in human evolution. vsv is a negative-stranded rna virus that has been a very important experimental model and has provided many laboratory measurements regarding quasispecies theory (see chapter ). using vsv, evidence supporting the red queen hypothesis, involving unending adaptation to greater competition and mueller ' s ratchet has been presented ( clarke et al. , ; novella et al. , ; elena et al. , ) . when vsv was evaluated as an arbovirus, requiring adaptation to alternating and opposing fi tness of insect and mammalian host, it was also apparent that minority quasispecies populations were responsible for maintaining the apparently antagonistic phenotypes ( novella et al. , ) . thus here too, the consortia character of a quasispecies is clear. yet in natural settings several very different virus-host relationships can be seen with rhabdoviruses. a distant relative of vsv (vhsv) is also known to be responsible for mass die-off of commercially important fi sh ( marty et al. , ) . this virus infects many teleost species and has shown % mortality in many experiments (i.e. with i.p. inoculation). in natural outbreaks, however, it has also shown surprising genetic stability ( einer-jensen et al. , ) . clearly error-prone rhabdovirus replication must be kept in check by purifying selection in this situation. in contrast, another rhabdovirus, sigma virus of drosophila , is associated with no mortality but is a vertically transmitted persisting virus in specifi c drosophila populations ( fleuriet, ) . yet in some recent population measurements, sigma virus infected drosophila are expanding for unknown reasons ( fleuriet, ) . clearly this particular virus-host persistent relationship has some undefi ned selective advantage that operates beyond the lab-based concepts as measured above. other rhabdoviruses also have peculiar host-specifi c relationships, such as bats that tend to support many persistent infections ( badrane and tordo, ; li et al. , ) , or birds that seem to be free of almost all rhabdoviruses. clearly, although vsv lab results have been highly informative, we still have much to learn regarding natural settings that affect rhabdovirus adaptation and evolution. another major paradigm for the high rates of negative-strand virus evolution is found with infl uenza virus. due to its history and potential for initiating great human epidemics, it has long held the special interest of evolutionary virologists (see chapter ; nelson and holmes, ) . however, this research has not much emphasized the quasispecies character of infl uenza virus evolution. instead, it concentrates on the evolution of the master template or clades of template for the purposes of vaccine development ( webster and govorkova, ) . the views stemming from this type of evolution have lent themselves well to master template-based phylogenetic analysis and have dominated how many researchers think of virus-host evolution. thus it is curious, given the above emphasis, that the quasispecies character of infl uenza populations often seems of low relevance to issues of acute disease and vaccination, other then to provide a source of diversity. in some situations, viral competitive interference may contribute to drift variation and displacement in antigenic epitopes ( levin et al. , ). yet outcomes of individual human and bird infections do not seem much affected by specifi c quasispecies structures, as we saw with hiv- and hcv. with infl uenza, we are mainly concerned with epidemic human disease. however, by shear numbers of infections and deaths worldwide, it must be admitted that infl uenza virus is really a virus that affects mostly birds. for example, during the outbreak in china, only humans died whereas million domestic birds died ( smith et al. , ) . although our concern on the large potential for human disease is understandable, these numbers should inform us of a more basic virushost biology. in this case, infl uenza shows a high affi nity for various birds; migratory water birds in particular can have high prevalence ( wallensten et al. , ) . some waterfowl, such as wild mallard ducks, have been called the stealth (asymptomatic) carriers of infl uenza h n and free grazing ducks seem to introduce virus into domestic bird populations ( gilbert et al. , ) . thus waterfowl represent the well-accepted epidemiological concept of a reservoir species ( louz et al. , ) . but these wild waterfowl, shorebirds, and gulls that are a natural host for avian infl uenza also seem to show a much slower rate of evolution ( spackman et al. , ) . in contrast, the much higher rate of evolution as seen in chickens and turkeys indicates that these hosts should not be considered as natural reservoirs ( suarez, ) . in waterfowl, infl uenza infections show several distinctions, such as virus co-infection or virus interference ( sharp et al. , ) as well as phylogenetically distinguishable waterfowl dendograms, including specifi c m lineages ( makarova et al. , ; widjaja et al. , ) . the diverse and stable avian pool of infl uenza virus appears to be ancestral to the infl uenza viruses that infected human populations. the phylogenetic methods that have been adapted from evolutionary biology have been tremendously helpful and have allowed us to trace the seemingly untraceable, virus evolution (see chapter ). thus, we have often been able to make informed judgments concerning broader patterns of virus evolution and this has become the major tool for the current study of virus evolution, such as infl uenza virus ( nelson and holmes, ) . infl uenza a, for example can be seen to show extended periods of stasis followed by periods of rapid adaptation that necessitates adaptations in vaccine strategy ( wolf et al. , ) . however, the evolutionary variation between seemingly similar viruses can be surprisingly large (see above vsv section). for example, the very different phylogenetic behaviors between infl uenza a and measles virus, both acute human respiratory infections due to membrane-bound negative-stranded rna viruses, are striking. the reasons for the maintained genetic stability of measles virus remain poorly understood, but may well involve more complex fi tness associated with systemic infections. phylogenetic methods can also be highly informative regarding the likely origins of viral lineages and possible sources of emergence. for example, the studies of dengue virus by holmes and colleagues suggest that this virus fi rst entered its human host about years ago, and that sylvatic (african jungle) asymptomatic infection of primates may have provided the origin of this virus that later became a human pathogen ( holmes and twiddy, ; holmes, ) . such insight provides valuable clues concerning the likely selective pressures that may lead to the emergence of dengue virus. phylogenetic methods are also highly informative regarding classifi cation and taxonomy relationships and have allowed us to understand viral relationships across broad species defi nitions ( zanotto et al. , ) . however, phylogenetic approaches necessarily assume the master template is the fi ttest type and that mutations or variants in the rna populations are a source of genetic load that are deleterious and limiting to virus adaptation ( pybus et al. , ) . such variation is mostly due to " unfi t " mutations, which indicates that a viral cloud is mostly and unfi t consortia. it would seem that such conclusions go against the concept of quasispecies as being fi t per se as described above. in this consideration we see a major weakness of extant phylogenetic methods. they were not developed to access the evolutionary relationship and fi tness of interacting mixtures. nor were they designed to follow the evolution of systems with high rates of recombination between numerous parental templates. we currently lack the analytical tools for such a population analysis. without such tools, however, it seems we can only evaluate those parameters we can defi ne and will remain confused by those we cannot. evolution of a consortia thus provides a new directions for theoretical and laboratory research. we should seek to investigate the mixture, not just its average. another major virus-host system that has been highly studied is the viruses of agricultural plants. our understanding of plant viruses has also been highly infl uenced by disease associated with agricultural domestic species, thus natural virus-plant relationships are much less understood, although some recent fi eld studies are starting to change this situation (see chapter ). we currently have a rather uneven understanding of broader virus-host relationships and evolution in plants. for example, viruses of the more ancient ferns, if they exist, are essentially unknown. the prevalence and diversity of positivestranded rna viruses in plants is striking. in addition, we are starting to appreciate that virus-virus interactions are also frequently involved, although this issue remains poorly studied. one well-studied family of plant virus are the tobamoviruses of angiosperms (see chapter ; gibbs, ) . progenitors of this virus family appear to also be found in algae and fungi consistent with a very long evolutionary history. both high transmission between host and virus-host congruence are observed with these viruses. virus-virus interactions also seem to be important. for example, tobacco mosaic virus (tmv) and tomato golden mosaic virus (tmgv) appear to have shown interactions in australia which have apparently led to the extinction of tmv, but the retention of tmgv with no increase in genetic diversity ( fraile et al. , ) . plant viruses have also been seen as quasispecies in some but not all settings (see chapter ; roossinck, ; roossinck and schneider, ) . besides the interactions expected for typical viral quasispecies, plants often show evidence of more extensive mixed virus infections. there are, for instance, many examples of satellite viruses that must necessarily interact with other rna viruses of plants. it is also clear that the subviral elements of even a single viral lineage can greatly affect the virus-host relationship. such subviral elements (dis) have been observed to both reduce and intensify disease, and also interact with satellite viruses ( qiu and scholthof, ) , thus virus-virus interactions are clearly crucial in many situations ( simon et al. , ) and viral interactions and synergism appear to have led to signifi cant events in plant virus emergence ( fargette et al. , ) . virus-virus interactions are not limited to plant rna viruses. the ssdna plant geminiviruses also display complex interactions with satellites as well as high diversity in fi eld isolates of east africa ( ndunguru et al. , ) . thus, plant viruses seem particularly prone to interactions. more recently, virus-mediated symbiosis with respect to host survival has been reported ( roossinck, ) (discussed below). phylogenetic methods also struggle to address the occurrence of high rates of recombination in viral lineages. such a situation complicates the analysis, creating hardto-defi ne, reticulated trees, although these limitations can be partially overcome by using sliding windows for the analysis. such approaches have allowed surveys of recombination in some viral lineages, such as with the plant potyviruses ( chare and holmes, ) . however, the rampant recombination and quasispecies generation of hiv- makes a quantitative assessment of the virus population problematic. one proposed solution is to use a composition vector method ( gao and qi, ) . the issue of measuring recombination and tracing evolution in large populations is especially a problem that applies to the dna viruses (phage) of prokaryotes (see below). cells. our perception regarding the overall importance of dna viruses of prokaryotes to the evolution of life on earth has undergone a major shift in recent years. the main realization is that dna phage are the numerically dominant genetic entity in most habitats on earth (mentioned above). in addition, as discussed in chapter , it is now clear that some of these viruses are surprisingly complex and that essentially the entire pool of dsdna viruses of prokaryotes may be exchanging dna via recombination at high rates. this would constitute by far the largest common gene pool on earth. historically, the evolution of the dna viruses of prokaryotes has seldom been considered in the broader context of virus evolution or evolutionary biology. although it has long been realized that there are many basic similarities between viruses of bacteria and eukaryotes ( luria et al. , ) , not until structural studies solved the capsid genes of prokaryotic and eukaryotic viruses did the evolutionary relationships between these viruses become clear. in addition, there have been a number of striking proposals that suggest that dna viruses of prokaryotes may be involved in the origin of several major systems used by cells and that viruses appear to be involved in several major transitions during host evolution. thus we now consider the possibility that these dna phage were fundamental to the origin and evolution of life on earth. it now seems likely that some large dna viruses infecting eubacteria, archaea, and eukaryotes share some common evolutionary histories. it also seems clear that such viruses can link all three domains of life. this realization was not apparent based on phylogenetic sequence conservation, which is absent. it stems from the structure and assembly of virion capsids in which t phage, halophage, and the herpesviruses all show clear similarity as well as similarity in replication strategies. in addition, phage prd and adenoviruses show similar broad structural and strategic conservation. some biochemical (dna pol family) and genetic similarities (gene order, gene programming) are also apparent, which taken together supports the common origins of these viruses ( hendrix, ( hendrix, , hendrix et al. , hendrix et al. , , . t -like viruses in particular seem to represent a major source of global genetic diversity. this giant genetic pool represents a huge potential to affect life ( filee et al. , ) and the viral genetic creativity represented by this pool would also be vast ( nolan et al. , ) . since t -like phage that infect cyanobacteria also encode virus-specifi c type ii photosynthetic core genes, viruses appear able to create the most complex of genes as well ( clokie et al. , ; sullivan et al. , ) . as presented in chapter , phage are now thought to evolve by distinct and highly mosaic " horizontal " processes of rampant recombination ( hendrix, ( hendrix, , . large dna phage appear to be ancient, present before the split of the three main branches of cellular life: bacteria, archaea, and eukarya ( benson et al. , ) . luca, the last universal common ancestor, would represent the putative cell ancestor prior to this split. however, phyogenetic analysis of common or conserved genes of luca identifi es only about or fewer genes in extant cellular genomes ( mushegian, ; koonin et al. , ; mirkin et al. , ) . ironically, the genes needed for dna replication are not part of this conserved set, calling into question the nature of the fi rst dna-based cell. large-scale " horizontal " transfer seems to have clearly prevailed early in the evolution of dna-based cellular life and it has recently been asserted that luca existed in a highly horizontal " consortia " of cooperative genes that developed the common genetic code ( vetsigian et al. , ) . since the dna replication proteins in the extant three domains of life have distinct compositions, it has been proposed by forterre that dna viruses and retroviruses were directly involved in the invention of the three extant cellular dna replication systems . according to this view, early cellular life was completely entangled with viral (phage) lineages; hence cells must have evolved from an ancestral " virus " -mediated population not a single genetic lineage. thus the evolution of early life would have clear similarity to the quasispecies (consortia) state of genetic information as seen in rna viruses above. thus the huge creative and adaptive potential of virus would have been directly involved in the very earliest evolution of life. clearly, such conjectures regarding the most ancient events in the evolution of life are hard to substantiate. but, these theories are as viable as any other and deserve serious consideration. in spite of this seemingly unending mosaic exchange in dsdna phage, some phage isolates show surprisingly stable genetic makeup. we now accept that t -related phage are an important source of the larger global phage genetic diversity and that most such viral genes are novel ( filee et al. , b ; nolan et al. , ). yet even with t -like viruses, there can be clear barriers to horizontal gene transfer which promote the evolution of stable viral lineages ( filee et al. , a ) . in t -type phage, similar core genes could be seen in all genomes, which seem to be inherited in gene blocks that preclude recombination. however, these blocks were not seen in the broader t-even and pseudo t-even genomes. other phage also show surprising genetic stability when repeatedly isolated from similar habitats, such as soil phages of burkholderia ( summer et al. , ) and bam ( saren et al. , ) as well as some hot spring isolates ( khayat et al. , ) . this bam capsid also identifi es another structural motif mentioned above that is broadly conserved in evolution and shows clear similarity to that capsids found in prd and pbcv- (discussed below). sh also has a clear prd related capsid, membrane, and genome; thus this halophilic euryarchaeon virus, although showing no sequence similarity to prd or any other bacterial phage, is clearly structurally related ( bamford et al. , a ) . it is interesting that overall the viruses of hyperthermophilic crenarchaeota generally show no sequence relationship to phage of bacteria. in addition, the use of the term phage for these viruses can also be questioned as most establish non-lytic chronic infections. many of these crenarchaeota viruses have unique morphologies not found in any other domain of life ( prangishvili et al. , a ( prangishvili et al. , , b ortmann et al. , ) . some, however, have clear structural and genetic similarity to specifi c phage (i.e. t ). considerations of phage evolution and rampant recombination (especially with t and t-even phage) often emphasize the viral lytic lifestyle and host death. in fact this lytic relationship was argued by many early phage researchers to be the fundamental and only character of phage-host relationships in general. we now know, however, that persisting (temperate) phage are also common, some of which have no independent lytic phase. the fundamental model of phage persistence by unique integration into host chromosomes (temperate lysogeny) marks a major development in our understanding of molecular virology and virus-host relationships which was fi rst clarifi ed by campbell in (see campbell, . all free-living prokaryotes show the presence of colonized phage in their genomes. both complete and defective genomes of dsdna viruses have been observed in the sequenced dna of all free living prokaryotic genomes ( gelfand and koonin, ) (exceptions are some intracellular parasites and plastids). thus, the massive genetic diversity and novelty of phage evolution as presented above has a direct conduit into the genetic composition of all prokaryotes via lysogeny. the fi tness and evolutionary consequences of such colonization to the evolution of the host and its virus should be considerable but is in need of theoretical development. fitness of temperate phage, however, is more complicated then that of a lytic virus and, like fi tness of persistence discussed above, cannot be simply described by relative replication or effi cient virion production. here too, successful phage colonization must inherently limit the replication of the same virus. thus, a temperate lifestyle also requires an autoinhibitory capacity. this generally involves an immunity gene set that not only limits self-replication but can also affect replication of other temperate and lytic viruses, i.e. lambda (even as a defective) precludes t and other t-even phage. uncolonized hosts are thus susceptible to lysis by highly prevalent acute tailed phage. host fi tness is thus strongly affected by a temperate phage due to its ability to preclude and survive other competing phage. i suggest this situation is similar to the mhv-mouse exemplar above, in that virus-colonized hosts are in a state of " virus addiction " in which persistence is needed to provide protection from the same or similar virus ( villarreal, ( villarreal, , . it is well established that most natural populations of bacteria have specifi c patterns of phage colonization, hence the utility of phage typing for strain identifi cation. from this, we can infer that virus-virus competition is a prevalent and major issue regarding the prokaryotic fi tness resulting from a symbiotic temperate phage-host combination. in addition, such virus-host symbiosis can also affect competition with other bacteria. this would be very much like the virus addiction concept outlined above for the mhv examplar. the original observation of a lysogenic process and coining of this term occurred in the s when two pure cultures of bacteria were grown together. it was observed that in some combinations, one strain would lyse the other strain (was lysogenic). later, it became clear that such lysis was mediated by reactivation of temperate phage present in the lysogenic strain, but absent from the non-lysogenic susceptible strain. in this relationship, we see another example of group selection operating on bacterial populations harboring a persistent virus. thus, what host is fi t depends very much on the prevalent viruses it will encounter as well as the viruses that colonize it. bacterial populations that are colonized by the same or similar phage express the appropriate immunity functions and are protected from lysis by the same or similar phage. such a situation has signifi cant implication for the evolution of immunity and group identity for cells. host stability becomes a major fi tness issue for a persistent virus life strategy. it is generally thought that a temperate virus attains a stable colonization of its host by simply integrating into and become one with the host genome. however, there are also clear examples of stable phage persistence that does not integrate and uses other strategies to attain host stability (similar to eukaryotic dna viruses; see below for the p phage exemplar of this). like a temperate phage, a host that is colonized by episomal persisting viruses has also been much affected in its evolutionary potential. it is clear that phage can have complex effects on host populations, but these phage themselves often exist in complex and mixed states that can be diffi cult to unravel ( harcombe and bull, ) . it has been known for some time that the presence of otherwise silent phage can greatly affect the growth of other virus and susceptibility of host. one such silent and common phage that has long been studied is p . p was initially discovered due to its effect on t and lambda. however, p has been a very interesting model, not because it causes disease or offers potential therapy against bacterial pathogens, but simply because it persists effi ciently as an episome and competes effectively with many other phage ( yarmolinsky, ) . since it does so without integrating, p provides us with one of the only well-studied models that can inform us regarding the molecular strategies and details of how stability in non-genomic persistence is attained. curiously, a main strategy by which p attains this stability was inapparent and not suspected after several decades of study. it became apparent only after replication mutations were made that induced self-destruction and uncovered the existence of what came to be called " addiction modules " ( lehnherr et al. , ) . p encodes several gene pairs (toxins/ antitoxins, such as the phd/doc pair) that protect bacteria harboring p , but kill daughter bacteria that have lost the p genome ( gazit and sauer, ) . this strategy compels colonized e. coli to maintain p or die (doc, death on curing). however, these very same addiction systems are also involved in protecting a p -colonized colony from t and lambda infection and will also induce self-destruction when cells are infected by those viruses, protecting the colony (population). p also provides an exquisite level of molecular self-identifi cation in that it will recognize a single second copy of its own genome ( yarmolinsky, ) . what then is the fi tness and evolutionary consequence to e. coli harboring p ? clearly it is major, but mostly host fi tness is affected relative to other viruses. accordingly, when contemplating the amazing complexity of the p immunity and how it evolved, yarmolinsky posed the question; " could the byzantine complexity of the controls at immi be the outcome, not of successive host-parasite accommodations, but of competition among related phages? " ( yarmolinsky, ) . if we answer yes to this question, then we would also conclude that virus-virus interactions and competition in general are major forces in the adaptability and evolution of persisting phage and surviving colonized host. in this light, viral persistence takes on a major role in virus and host evolution. the p exemplar has thus provided us the concept of viral addiction that also promotes host group selection. historically, we are biased to think of viruses (and phage) as agents that simply kill their host. some have proposed that the prokaryotic global biomass is phage partitioned into those populations that live and those that die due to viral lysis. from such a perspective, viral novelity would seen of little relevance to host evolution. metagenomic projects as noted above, have sequenced nearly million phage genomes and report that most of these phage genes are unique, not in the database, and likely not derived from host ( edwards and rohwer, ) . the protein repertoire of sequenced phage indicates that % of conserved phage genes are specifi c to phage and show an evolutionary independence from genes of host . this identifi es a massive genetic novelty from virus, which is especially apparent in large dna phage. as just discussed above, however, those hosts that live are also products of phage selection, and persisting temperate phage play a major role in this. such phage colonization allows this massive phage novelty to fi nd its way into host genomes, which allows viral complex gene sets to be applied to novel problems of host adaptation. host novelty can thus be introduced by phage ( comeau and krisch, ) . that persistence is a major life strategy of phage is confi rmed by the large numbers of genes associated with persistence (i.e. integrases, immunity) observed in metagenomic screens. there is also much practical experience that supports the crucial role of prophage in host evolution. one particularly well-studied system that has been studied for over years is the ongoing evaluation of phage evolution as observed in the dairy industry ( canchaya et al. , brussow et al. , ) . the temperate phage analysis of these bacteria follows a long tradition of lambda and e. coli studies ( campbell et al. , ; canchaya et al. , canchaya et al. , , . since lytic phage can severely disrupt dairy fermentation, it was of particular interest to understand and trace their evolution. these studies have led brussow to conclude that much of the more recent dairy bacteria evolution can be considered to have resulted from the action of temperate phage. a similar view applies to e. coli and cyanobacteria. in addition, the ecor collection of sequenced e. coli genomes of medical interest shows that they differ from each other mainly due to patterns of genetic colonization, mostly by prophage, but they also show the presence trna-adjacent defective prophage and plasmid elements that differentiate these strains ( hurtado and rodriguez-valera, ; mazel et al. , ; nilsson et al. , ) . cyanobacteria ( prochlorococcus ) is major model for the study of the origin of the type ii (plant-like) photosynthetic system. since such genes show much evidence of recent and massive horizontal movement, it seem quite likely that prophage are mediators of such transfers, especially as these phage encode their own version of these photosynthetic genes ( lindell et al. , ; sullivan et al. , ) . very similar prochlorococcus strains exist in distinct oceanic populations in various habitats known as ecotypes. some think that such ecotypes represent the initial type of genetic variation that leads to speciation. the sequencing of six ecotypes has shown that they are % similar to one another, but the genetic variation that distinguishes them is mostly due to patterns of prophage colonization (called phage islands) ( bouman et al. , ; coleman et al. , ) . thus in all these prokaryotic models, persisting viruses play a fundamental role in host evolution and host genetic novelty is mostly phage derived. such observations have led some to propose that " war is peace " regarding virus-host evolution ( comeau and krisch, ) . massive and complex innovation by phage appears to be a major force in the prokaryotic world. prokaryotes are the most adaptable of all cells. if we can accept the above conclusion concerning the role for viruses in the evolution of prokaryotes, we must then ask why such a successful evolutionary strategy was not apparently maintained in eukaryotes? in eukaryotes we see little evidence that largescale integration by dna viruses is an important evolutionary process (although the story with retroviruses is different). why should prokaryotes and eukaryotes differ is such a fundamental way? nevertheless, as noted at the start of this section, we do see good evidence that links the evolution of large dna viruses of prokaryotes to the large dna viruses of eukaryotes. in case we were becoming comfortable with the apparently clear distinctions between rna and dna virus evolution as outlined above (quasispecies vs. domain recombination respectively), the evolution of the parvoviruses informs us that dna viruses can also evolve by a quasispecies process. parvovirus evolution (see chapter ) can show a sharp contrast to the evolutionary pattern displayed by other small dsdna viruses above (hpv, py). with the emergence of an acute pandemic in domestic dogs and cats (as well as other wild carnivore species), we see what is essentially evolution driven by single point mutations, mostly affecting the capsid genes and host cell receptor binding. this system provides us with one of the better studied examples of the evolutionary dynamics of an emergent viral disease. in addition, in vivo mouse studies with minute virus of mouse (mvm) now make it clear that parvoviruses can behave much like rna viruses, generating quasispecies of diverse progeny that allow a high adaptability for the generation of fi tness and disease in vivo ( lopez-bueno et al. , ) . this story is very reminiscent of the study of poliovirus in mice mentioned above. human studies with b parvovirus are also consistent with high mutation rates ( parsyan et al. , ; shackelton and holmes, ) . although not specifi cally addressed in this volume, the viruses of eukaryotic unicellular green algae are of special interest from the perspective of dna virus evolution. these large, complex dsdna membrane-containing icosahedral viruses are abundant in some water habitats ( van etten, ; ghedin and claverie, ) . the reason they deserve special attention is that they clearly have many features that are characteristic of both prokaryotic and eukaryotic viruses. they resemble prokaryotic viruses in that their life cycle is clearly phagelike, such as external virion attachment, injection of dna and no pinocytosis. in addition, they also encode many phage-like genes, such as restriction-modifi cation enzymes and homing endonucleases ( filee et al. , c ) . they also resemble eukaryotic viruses in that they have eukaryotic dna replication proteins (dna polymerase beta and pcna; chen and suttle, ; nagasaki et al. , ; villarreal and defilippis, ) as well as many genes associated with eukaryotic signal transduction ( van etten et al. , ) . thus they represent a clear link between prokaryotic and eukaryotic dna viruses. for example, the dna polymerase of paramecium bursaria chlorella virus (pbcv- ) is the most conserved gene and most closely resembles that found in human herpesvirus and is distantly related to the similar family dna pol encoded by t . this polymerase is distinct from that of the poxviruses or prd /adenoviruses (associated with protein-primed dna replication). however, numerous other genes of the phycodnaviruses are similar to some genes found in the mimiviruses (giant dna virus of ameba), including the presence of conserved intenes in the dna pol gene ( ogata et al. , ) . in view of this it is most curious that in structural similarity, polydnavirus capsids clearly resemble prd capsid ( khayat et al. , ; nandhagopal et al. , ) . prd contains the double-barrel trimer capsid structure that was fi rst observed in adenovirus (for references see saren et al. , ) . adenovirus also closely resembles prd in dna replication strategy (i.e. linear dna with covalently closed ends ( benson et al. , ; khayat et al. , ) . the lineage of adenovirus-like dna viruses, however, is thought to be distinct from that herpes and poxviruses and its dna polymerase is clearly distinct from polyndavirus. it is clear that related elements of all these viruses can be found in phycodnaviruses. overall, the phycodnaviruses, like phage, also appear to be creating genes in large numbers and they encode many genes unrelated to their host. what then is the evolutionary relationship that links all of these seemingly distinct viruses? as outlined above, the pattern of evolution of dsdna phage involves lots of exchange by recombination from a vast gene pool. this pool resembles a cloud from which various mosaic subelements and substrategies are assembled to allow viral gene acquisition and novelty ( blum et al. , ; benson et al. , ) . does such a distributed pattern of evolution and gene novelty also apply to the phycodnaviruses? recently, another distinct phycodnavirus has been sequenced: coccolithovirus (ehv- ) ( allen et al. , a ( allen et al. , , b conserves only core genes in common with pbcv- and is unique to the phycodnaviruses in that it has acquired six dnadep rna polymerase subunit genes, which are absent in all other phycodnaviruses. as rna polymerase is considered a core viral gene function, it is clear that phycodnaviruses can alter some very basic molecular functions during their evolution. oceanic phycodnaviruses are thought to have large infl uence on the free-living populations of eukaryotic algae, such as the termination of algal blooms reported for emilian huxley virus ( martinez et al. , ; schroeder et al. , ) . however, not all phycodnaviruses are lytic. another lineage of phycodnaviruses is represented by two viruses of fi lamentous brown algae, esv- and firrv- ( delaroque et al. , ) . unlike the lytic phycodnaviruses noted above, these two viruses are " temperate phage " like. that is they exist as silent viruses whose dna is integrated into the germlines of their host. in this, they are unique to all known eukaryotic dna viruses; host chromosome integration is a normal part of their persistent life strategy. esv- has a -bp genome and encodes likely genes ( delaroque et al. , ) . these genes are mostly unique and only are clearly related to pbcv- genes. the gene differences include many replication genes and their gene order is completely different. like the temperate phage-host evolutionary relationship outlined above, it would be most interesting to understand how the integration of these large dna viruses has affected host evolution. thus, the phycodnaviruses appear to represent a basal but diverse viral lineage that has both acute and persistent lifestyle and have some clear relationships to most large eukaryotic dna viruses and many phage. the phycodnavirus exemplar above should leave us with several impressions regarding the nature and evolution of these large and ubiquitous dna viruses of algae, an early eukaryotic host. they show clear linkages by structure and function to both phage and various eukaryotic dna viruses. they also show major variation and novelty in their own genetic composition, including their core genes. in addition, they show clear relationships to distinct and seemingly separate viral lineages (adenoviruses, herpesviruses, poxviruses, iridoviruses). the picture we are left with is that they seem to resemble phage evolution in that they appear to have evolved from a diverse pool that has exchanged many basic viral features and created many new genes. this view, however, contrasts sharply with the work of iyer et al. ( iyer et al. ( , . by considering the small number of conserved genes in four families of eukaryotic dna viruses (poxviruses, asfarviruses, iridoviruses, phycodnaviruses), they suggest that these viruses are monophyletic, evolving from a common nucleo-cytoplasmic large dna virus (ncldv) with an icosahedral capsid. given the above information, i fi nd this view unhelpful and possibly confusing. it has numerous problems. the main problem is that it fails to acknowledge the clear link between prokaryotic and eukaryotic viruses. furthermore, by focussing on a small set of related genes, it represents a traditional perspective as found in evolutionary biology that assumes a common (fi ttest) linear lineage, not a cloud, cooperative, or mosaic pool as the main source of novelty resulting in the matrix pattern of virus evolution. the virosphere is clearly not disconnected from itself, but it is also clearly not a linear or tree-like evolutionary system as suggested above. we must learn to think of virus evolution in its own terms; fuzzy, mixed, reticulated, and cloud-like. as mentioned in the phage section, there have been various publications that suggest a deep evolutionary relationship between the herpesviruses and dsdna viruses of prokaryotes ( rice et al. , ; khayat et al. , ; duda et al. , ; akita et al. , ) . such enormously distant relationships, however, cannot now be measured by any reliable metric. although herpes-like viruses are found in invertebrates (such as ostreid herpesvirus (oshv- )) in both lytic and asymptomatic states ( barbosa-solomieu et al. , ) , our interest in their evolution has been mainly focussed on the vertebrate herpesviruses. vertebrate herpesvirus do tend to show clear sequence conservation that suggests broad patterns of evolution. one interesting feature of this evolution is the apparent link between the biology of the virus and its evolution. a common, but not universal pattern is that of virus and host co-evolution ( mcgeoch et al. , ( mcgeoch et al. , , mcgeoch and gatherer, ) . this trend has maintained several biological characteristics, such as highly species host-and tissue-specifi c persistence (i.e. neuronal and lymphoid persistence). the discovery of hhv- has further stimulated studies of herpesvirus evolution in that hhv- appears to have undergone much recombination with herpesviruses of related primate lineages ( mcgeoch and davison, ) . thus recombination seems prevalent in herpesviruses. the apparent link between herpesvirus evolution and recent human evolution, as well as an apparent link to primate retroviral evolution, is fascinating, but of unknown signifi cance ( kung and wood, ; lacoste et al. , ) . the herpesviruses lineages will often show the presence of lineage-specifi c genes. many of these genes affect innate and adaptive host functions, whereas others affect host metabolism. when the source of such genes has been contemplated, in contrast to phage, phycodnaviruses, or baculoviruses ( herniou et al. , ) , it is often proposed that most such herpes genes originate from the host. it is well accepted that the three major lineages of herpesviruses descended from a common ancestor in vertebrates ( mcgeoch et al. , ) . there have been numerous proposals that most new lineage-specifi c herpesvirus genes have originated from host (see becker and darai, ) . this includes herpesvirus dutpase ( davison and stow, ) , and viral chemokines and viral bcl- ( nicholas et al. , ) . in my evaluation of such claims, however, it seems clear that the possibility that there was an ancient viral source of such genes was not considered and cannot now be dismissed. we currently believe that ancient herpesvirus ancestors can be traced to tailed phage ( hendrix, ; bamford, ; baker et al. , ; duda et al. , ; mcgeoch et al. , ) . other phage lineages also appear to trace to eukaryotic viruses ( bamford et al. , b ) . within the herpesviruses, the same t- icosahedral structure, as well as invertable dna regions are also present in the very distant but much more recognizable oceanic ostreid herpesvirus . given the highly diverse and mosaic nature of large dna virus evolution in prokaryotes and lower eukaryotes described above, it seem quite possible that many other viral genes might also trace far back in virus evolution. consider the example of dutpase in avian and mammalian herpesvirus ( davison and stow, ; mcgeehan et al. , ) . the current view requires very complicated gene rearrangements to account for the viral source of this gene from its host. yet we know that diverse dutpases are found in many ancient viral lineages. for example, the ervs present in all vertebrate genomes also conserve dut-pase ( jern et al. , ) , as do exogenous retroviruses (i.e. lentiviruses) ( mcintosh and haynes, ) . in fact, since the herpesviruses genes are especially poor in introns, it would seem likely that any herpesviral gene acquisition would necessarily involve a retrovirus via a cdna. the oceans are especially fi lled with large complex dna viruses (such as mimivirus and phycodnavirus, plus numerous relatives of oshv- ) thought to be ancient ancestors of herpesvirus. the phycodnavirus (chlorella virus, pbcv- ) provides a clear bridge between phage and eukaryotic dna viruses. pbcv- also encodes a dutpase that has the highly conserved motif iii ( zhang et al. , ) . many phage are also known to encode dutpases of diverse types, such as b. subtilis (spbeta) ( persson et al. , ) , and a phage of thermus thermophilus ( naryshkina et al. , ) . this thermus phage (phiys ) is of special note since its dutpase gene is clearly related to the dutpases of eukaryotic viruses and has a version that has undergone multiple events of recombination from apparently distinct phage, exactly as expected for mosaic phage genes. thus, the origin of new herpesvirus genes might not be so different than that seen in other large dna viruses and a potential ancient source of new genes from these ancestral viruses remains plausible. similar considerations apply to other possible examples of herpesvirus gene capture. for example, the herpes thymidylate synthase (ts) has also been considered to have originated by host gene capture ). yet distinct versions of these genes are also found in different herpesviral lineages, which would necessitate multiple independent " capture " events of different version of host ts genes. ts genes are present in ancient virus sources. for example, bacillus phage beta encodes ts, which also has a self-splicing intron ( bechhofer et al. , ) . also, phage phikz has a highly conserved ts ( mesyanzhinov et al. , ), yet this virus lacks a dna polymerase or other replication proteins, clearly indicating that the viral ts genes has a basic viral role. similarly, the cytokines-like genes (such as il- ) as found in poxviruses and herpesviruses appear to have originated in at least three independent events prior to the divergence of mammalian eutherian orders. yet it is still presupposed that they are necessarily the products of host gene capture ( hughes, ) . comparative genomics supports the idea that the herpesviruse lineages are originating viral genes. a broader phylogenetic analysis of all herpesvirus genomes identifi ed only genes in common to all taxa of herpesvirus . thus only genes appear to be in common to all the herpesviruses. in this analysis, only a few genes of recent origin could be identifi ed as possibly having been transferred between virus and host (e.g. new genes found at tips of phylogenetic dendograms). thus, gene gain in the herpesviruses (as in dna phage and phycodnavirus) is prevalent but the origination of such genes from the host is not prevalent. i suggest that our tendency to assume that new viral genes are usually " stolen " from the host should be revised ( moreira and lopez-garcia, ). in contrast to the herpesviruses, the poxviruses evolution tend to have little congruence to host evolution (see chapter ). yet, they too show evidence of ancient linkages to other viruses. the replication of poxvirus dna is distinct in that it involves a linear genome with inverted ends that have covalently closed " snapback " dna. the resulting replication structures involve head-to-tail and tail-to-tail intermediates. this replication strategy is very different from that used by the host (and most other dna viruses), but is clearly related to that found in other eukaryotic and prokaryotic viruses. similar replication mechanisms are seen in all poxviruses, as well as african swine fever virus and phycodnaviruses (pbcv- ). this exact replication strategy is also present in archaeal lipothrixviruses (sirv and sirv ) which has been proposed to be ancestral to phycodnaviruses and poxviruses ( persson et al. , ) . a similar replication strategy is also seen with n ( lobocka et al. , ) , an unusual phage of e. coli that persists as a linear dna ( casjens et al. , ) . conservation of such replication similarities clearly suggests ancestral relationships, but no sequence similarity can be seen between these viruses. the similarity between poxvirus and pbcv- dna replication deserves some additional comment. pbvc- and herpesvirus have very similar dna polymerase genes, yet differ fundamentally in replication strategy. furthermore, the poxviral dna polymerase gene is very different from that found in the herpesviruses. yet, the pbcv- capsid was clearly similar to that of adenoviruses and prd phage (and iridovirus capsids). how then do we link poxvirus evolution to other more ancient dna viruses, such as pbcv- which has the same dna replication mechanism, but distinct replication proteins? such observations might seem confusing, but they are clearly consistent with mosaic, reticulated evolution of dna viruses. various distinct phage lineages can link in multiple ways to various distinct eukaryotic dna viruses. the concept of a net or matrix rather than a tree is thus a better way to describe the broad topology of dna virus evolution. the issue of gene gain and gene loss is also of central interest to orthopoxvirus evolution. typically, we seek to understand poxviruses evolution from the perspective of pathogenesis, such as the origin of human-specifi c smallpox virus. with the comparative genomics of several orthopoxviruses now possible, we see curious overall patterns of gene loss in their evolution ( randall et al. , ) . for example, comparing human smallpox to cowpox dna (a rodent virus that is phylogenetically basal to smallpox), we observe an overall diminution of gene content in smallpox virus. several poxviruses seem to have also lost genes relative to cowpoxvirus, especially genes that appear to affect immunity ( hughes and friedman, ) . i suggest that this evolutionary tendency for gene reduction is associated with a switch from a more demanding species-specifi c persistent life strategy to a less demanding, acute life strategy in a new host. cowpox is a naturally persistent infection in rodents (bank voles) ( feore et al. , ; chantrey et al. , ) , which has been called a natural virus reservoir ( hazel et al. , ) . smallpox is a strictly acute and human-specifi c disease. such gene loss in association with lost persistence could be a general situation and might also explain why clinical isolates of human cytomegalovirus isolates show a strong tendency to delete genes with passage in culture ( davison et al. , ) . most orthopoxviruses are not phylogenetically congruent with their vertebrate host. host switching and acute replication seem to be relatively common but recent occurrences in their evolution ( babkin and shchelkunov, ) . the avian poxviruses are not as well studied in this context, but curiously have signifi cantly more complex genomes than the orthopoxviruses ( jarmin et al. , ) . the entomopoxviruses are even less well understood from both a biological and molecular perspective, although they do conserve genes found in all poxvirus family members ( gubser et al. , ) . clearly these poxviruses share some degree of evolutionary history. it is most curious that entomopoxviruses have even larger, more diverse and complex genomes than the other poxviruses. why? as insects lack an adaptive immune system (the target of many orthopoxvirus genes), they would seem to present a simpler host for virus adaptation. this group appears to be the most basal phylogenetically, but evolutionary relationships between entomopoxvirus and insect evolution have not been studied. the enotomopoxviruses are particularly prevalent in grasshopper and locust species, often in unapparent states. interestingly, within these viruses we can fi nd examples of major shifts in core replication genes, such as the family of dna pol gene that is used (a shift from dna pol x to dna pol b in two entomopoxvirus lineages). we can recall that the dna pol b gene closely resembles that found in phycodnaviruses (and herpesvirus), but is distinct from that in orthopoxvirus ( zhu, ) . we also see in the entomopoxviruses some clear links to phage genes, such as t -like rna ligase found in all entomopoxviruses ( ho and shuman, ) as well as a lambdalike integrase seen in d epv ( hashimoto and lawrence, ) . this integrase in d epv implies possible integration and persistence, thus it is most signifi cant that d epv also shows a clear persistent host infection as well as symbiosis and apparent phylogenetic congruence between virus and host. this virus is symbiotic in its parasitoid wasp host in that virus is injected into larval host along with the wasp egg (and also along with a second d rhv rhabdovirus) and virus is needed for successful host parasitization. this symbiosis is clearly very reminiscent of the genomic polydnaviruses of other parasitoid wasp species. diepv is also expressed in the male poison gland. however, it is unknown if diepv is integrating into the host dna. clearly, d epv it is part of a complex virus-virus-host symbiotic interaction. the overall evolution of orthopoxviruses contrasts sharply with that of the papillomaviruses as presented by bernard in chapter . here, highly species-specifi c and tissuespecifi c host infection are the norm and the viral evolution is typically highly congruent with the host (with some exceptions). the resolution between virus and host can be high, in that human racial and geographical populations, for example, can often be differentiated based on the type of hpv they harbor. yet here too there is evidence of signifi cant shifts in core gene usage early during papillomavirus evolution. in the human and rodent viruses, a highly conserved gene function associated with replication and cell control are the e and e early genes. in particular, the prb-binding domain of the e gene is thought to be central to the biological strategy of the virus. thus, it is most curious that the papillomaviruses of lagomorphs, such as bovine and reindeer papillomavirus, lack an e rb-binding domain and instead appear to use e or e genes for this regulatory function ( narechania et al. , ) . it seems an early but signifi cant and bifurcating shift occurred in the molecular strategy during the virus-host evolution of this group of viruses for unknown reasons. other small dna viruses (jcv, bkv, py) can also show similar high-resolution host congruence ( shadan and villarreal, ) . as well as similar curious shifts in basic molecular strategies. for example, the presence of a middle tantigen in mouse virus (a third early gene), but its absence from primate viruses ( gottlieb and villarreal, ) , clearly differentiates these viral lineages. although the origins of these entire small dna viruses are obscure, and any links to prokaryotic viruses are unknown, it does appear they have tended to retain their overall biological strategy and show a strong tendency for tissue-specifi c (especially kidney) persistence and virus-host congruence. since persistence requires the stable coexistence of a virus and its host, it also fi ts the simple defi nition of symbiosis (the stable living together of two distinct lineages of organisms). viral involvement in symbiosis is a foreign idea to many and possibly presents a fundamentally different view of the role viruses may have in host evolution. a major role for persisting (temperate, cryptic) viruses in the evolution of prokaryotes is no longer a controversial idea. thus, at least in the prokaryotic world, virus persistence can be accepted as adaptive. in eukaryotes, however, viral persistence is seldom considered adaptive. the mhv-mouse exemplar as presented above has suggested how persistence can directly affect host survival. can this be considered an example of symbiosis in the accepted sense? a crowning achievement in the fi eld of symbiosis has been to explain the origin of plastids (chloroplasts, mitochondria) from symbiotic prokaryotes in eukaryotic cytoplasm ( margulis and bermudes, ) . this idea involves the high adaptability of prokaryotes to provide innovation but would seem not to involve virus in any way. yet here too we can fi nd viral footprints that suggest some involvement. for example, various plastid-specifi c rna and dna polymerases clearly resemble polymerases from t /t -like phage ( cermakian et al. , ; shutt and gray, ) . other models of symbiosis also show evidence of a viral role, such as the sexual isolation of buchnera ( moran et al. , ) . another very popular topic in the fi eld of symbiosis is the symbiotic origin of the photosynthetic sea slug, elysa chlorotica . what could be more fascinating than a green sea slug-an animal that can use light for photosynthesis? e. chlorotica eats photosynthetic eukaryotic algae ( vaucheria litorea ) and retains the functional chloroplast from algae for months. here too, however, there lies a viral footprint. this slug harbors an unusual endogenous retrovirus which is expressed in large numbers during sexual reproduction, following which all slugs die via synchronized apoptosis and in which the chloroplasts have accumulated numerous viral particles ( pierce et al. , ; mondy and pierce, ) . since there is reason to think gene movement from the algae to the slug genome is involved in this symbiosis, the presence of this retrovirus is a strong candidate to also be involved in symbiogenesis. clearly we should thus investigate retroviral elements as possible symbiotic participants and not dismiss them beforehand as irrelevant or " junk dna " (as is automatically done in many database screens). if viral persistence is a kind of symbiosis, viruses may also mediate the establishment of other symbiotic relationships ( villarreal, ) . the recent studies by roossinck and colleagues (see chapter ), in which a persisting virus, a plant, and a fungus were all symbiotically involved in altering the thermal tolerance of the plant could be an example of this ( marquez et al. , ) . many other virus-host relationships should also be examined for possible symbiosis. for example, placental vertebrate evolution has involved various endogenous retroviruses (i.e. herv-w, herv-frd). intact herv genomes, including env orfs, are important for placental trophoblast fusion (for references see ryan, ) . some will dismiss this situation as the quirky usurping of a viral gene for host function which is of little general signifi cance. the specifi c erv involved is simply selfi sh and mostly defective genetic material of no general consequence. if so, why is it that in sheep a distinctly different lineage of retrovirus (enjsrv) was also selected to provide a related placental function to a another mammal with signifi cantly different placental reproductive biology? it has been experimentally well established the enjsrv env is essential for sheep embryo implantation ( dunlap et al. , a ( dunlap et al. , , b . enjsrv is the endogenous version of jsrv, a problematic sheep-specifi c retrovirus that induces lung tumors (responsible for the death of dolly, the famous fi rst cloned sheep). the endogenous virus (enjsrv) is present in copies in the sheep genome and all sheep have this virus. sheep genomes also encoded a trans -dominant enjrsv gag that is inhibitory to exogenous jsrv ( mura et al. , ; oliveira et al. , ; murcia et al. , ) . it seems clear that this situation can also be considered from the perspective of viral symbiosis and/or virus addiction in host evolution. we should thus seek to understand why colonization by an erv population might generally provide a good solution to the evolutionary demands of placental biology. there are many other opportunities to examine the potential role of persistent and symbiotic viruses in the evolution of viruses, animals, primates, and humans. for example, as we seek to understand the origins of the adaptive immune system we should pay attention to viral footprints. we can ask, for example, why the major histocompatibility complex (mhc) locus, the most polymorphic, diverse, and rapidly evolving gene set in our chromosome, is so densely colonized with retroviral elements ( andersson et al. , ) . why is a retrovirus also the basic element of the duplication unit that was thought to have been the progenitor for the expansion of the mhc class i (and ii) genes ( gaudieri et al. , ; kulski et al. , kulski et al. , , ? why do similar herv element (l and ) also differentiate between human and chimpanzee mhc i ( watkins, ; kulski et al. , ) ? what was the role for siv in the evolution of primate mhc ( vogel et al. , ) ? humans and primates appear to have undergone some signifi cant and relatively recent evolution with regard to their endogenous and exogeneous retroviruses. along these lines, apobec-like genes are basic component of the adaptive immune response but they are also antiretroviral genes that act on retroviral cdna and gag ( ohainle et al. , ) . the apobec antiviral system has expanded recently in humans, but not chimpanzees ( sawyer et al. , ; ohainle et al. , ) . why? all african primates support unapparent foamy viruses (and also siv co-infection), but not humans ( murray and linial, ) . apobec c is active against foamy viruses ( delebecque et al. , ) . old world primates also underwent an expansion of ervl colonization (a clear relative of foamy virus) ( sawyer et al. , ) . was this ervl colonization of relevance to the ancient co-speciation of simian foamy virus and their primate host ( switzer et al. , ) ? what exactly was the relevance of herv endogenization to human survival and adaptations? curiously, human brain (neocortex) specifically expresses many of these more recent ervs as transcripts ( nakamura et al. , ; yi et al. , ) . if we consider these situations as possible examples of virus-mediated symbiosis in human evolution, perhaps they may make more sense of the otherwise confusing role or hervs. as noted, all primates, but especially humans show much evidence of recent endogenization by retroviruses. but these events mostly occurred in our extinct ancestors and we do not see ongoing evidence that any hervs remain active. however, we are currently witnessing a related virus-host evolutionary event of considerable interest. koala bears, native marsupials of australia, are currently experiencing a major epidemic caused by a leukemiainducing retrovirus. as a consequence, they are undergoing massive endogenization by a gammaretrovirus (mlv-related). this virus is similar to gibbon ape leukemia virus, but most likely originated from rodent ancestry ( tarlinton et al. , ; fiebig et al. , ) . the expectation is that extinction awaits those koalas that do not adapt or endogenize the retrovirus successfully ( stoye, ) . this event has the appearances of a retroviral-driven addiction that will result in a genetic variant of koala bear that has acquired a new antiretroviral state. this seems equivalent to the expansion of human apobec ; or perhaps a closer analogy is the endogenization of a suppressive gag as occurred with enjsrv. the surviving koala bears will likely tolerate or be persistently infected with this retrovirus pool. the genome of the species will have undergone considerable (but unpredictable) genetic perturbations and likely contain a large pool of variant and defective retrovirus. however, in so doing, the descendent koalas will likely present a biological hazard to any koala species that remain virus-free (as in virus addiction). currently, one island colony of koalas is suffi ciently isolated to have remained virus-free. this population will henceforth be under persistent threat from populations of endogenized koalas, now favored by group selection. from the very earliest events in evolution of prebiotic replicators to very recent events in human evolution, including the emergence of human-specifi c hiv, we expect viral evolution to show profound effects on the evolution of all life. unlike accepted host evolution, viruses also 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sue; craft, meggan e.; crooks, kevin r.; ernest, holly b.; fountain‐jones, nicholas m.; carver, scott title: pathogens in space: advancing understanding of pathogen dynamics and disease ecology through landscape genetics date: - - journal: evol appl doi: . /eva. sha: doc_id: cord_uid: qxkmngyk landscape genetics has provided many insights into how heterogeneous landscape features drive processes influencing spatial genetic variation in free‐living organisms. this rapidly developing field has focused heavily on vertebrates, and expansion of this scope to the study of infectious diseases holds great potential for landscape geneticists and disease ecologists alike. the potential application of landscape genetics to infectious agents has garnered attention at formative stages in the development of landscape genetics, but systematic examination is lacking. we comprehensively review how landscape genetics is being used to better understand pathogen dynamics. we characterize the field and evaluate the types of questions addressed, approaches used and systems studied. we also review the now established landscape genetic methods and their realized and potential applications to disease ecology. lastly, we identify emerging frontiers in the landscape genetic study of infectious agents, including recent phylogeographic approaches and frameworks for studying complex multihost and host‐vector systems. our review emphasizes the expanding utility of landscape genetic methods available for elucidating key pathogen dynamics (particularly transmission and spread) and also how landscape genetic studies of pathogens can provide insight into host population dynamics. through this review, we convey how increasing awareness of the complementarity of landscape genetics and disease ecology among practitioners of each field promises to drive important cross‐disciplinary advances. its formal inception in , facilitated by technological advances that have increased the availability of molecular and landscape data in conjunction with more powerful computational and analytical approaches. landscape genetics is fuelled by a steady stream of new ideas and methodologies, which, while exciting, can contribute to a lack of consensus or consistency in some key aspects. these aspects include the formulation of research questions, sampling strategies, analytical methods (balkenhol, waits, & dezzani, ; richardson, brady, wang, & spear, ; wagner & fortin, ) and even the identity of the field itself (dyer, ; storfer et al., ) . in fact, landscape genetics has yet to develop its own comprehensive, unifying theory for linking spatial and temporal landscape heterogeneity to genetic variation . while these issues are expected to be remedied as the field matures, many suggestions have been made to facilitate this progress. these have included calls for an increase in cross-disciplinary collaboration (balkenhol, gugerli et al., ) and an expansion of the scope of landscape genetic research beyond its current emphasis on vertebrates dyer, ) and, particularly, mammals (kozakiewicz, carver, & burridge, ) . one logical avenue for cross-disciplinary expansion of landscape genetics is in disease ecology (biek & real, ) . elucidating the specific influences of landscape features on pathogen transmission can provide key insights into the processes that affect disease risk and incidence. however, accomplishing this has been a challenge for disease ecologists (ostfeld, glass, & keesing, ) . indeed, the field of spatial epidemiology has only recently begun to emphasize the use of explicit landscape approaches in studies of spatial heterogeneity in infectious disease (i.e., "landscape epidemiology"; ostfeld et al., ; meentemeyer, haas, & václavík, ) . a major challenge for the study of landscape epidemiology, a field which does not traditionally implement genetic approaches, is that it is typically dependent on the ability to identify the location and timing of transmission events such that they can be compared to landscape features of interest. transmission events are essentially impossible to observe, so disease ecologists often assume that contacts between infected and susceptible individuals are a reasonable proxy for transmission. such contacts generally must be inferred indirectly using methods such as proximity collars, mark-recapture or telemetry, often using spatial overlap as a proxy for contact (craft & caillaud, ) . these methods are logistically challenging to employ, and whether an inferred contact resulted in transmission is uncertain (craft, ) . further, much landscape epidemiological research uses infection or exposure data to indicate past transmission, but these methods provide static snapshots of pathogen prevalence and may be inappropriate for inferring how transmission or spread has occurred (or is occurring) over time (meentemeyer et al., ) . the spatial distribution and movement of hosts are major factors affecting the likelihood, timing and spatial patterns of pathogen transmission and spread (dougherty, seidel, carlson, spiegel, & getz, ) . landscape genetics can identify landscape factors that are important drivers of host population structure. these landscape factors can determine the spatial configuration of a population, its density, its connectivity with other populations, its demographic structure and its genetic health-all of which have implications for the dynamics of microorganisms infecting the host species (ellis, václavík, & meentemeyer, ; prentice, marion, white, davidson, & hutchings, ; spielman, brook, briscoe, & frankham, ) . further, pathogen dynamics can be inferred directly using pathogen genetic data (archie, luikart, & ezenwa, ; decandia, dobson, & vonholdt, ) and incorporated into landscape genetic analyses. understanding specifically how infectious agents respond to the influence of landscape factors on hosts enables us to predict how such agents might spread based on present landscape configurations, as well as under potential future landscape scenarios (real & biek, ) . this knowledge can subsequently inform management efforts at the population level (such as vaccination targeted at key regions, culling), as well as broader decisions relating to the management of the landscape itself, which is a key aim of landscape genetics generally (manel & holderegger, ; segelbacher et al., ) . landscape genetics is being applied by managers at relatively low rates compared to related ecological fields such as landscape ecology, conservation biology and telemetry research (bowman et al., ) . therefore, studies that contribute to the management of disease agents within populations could increase the practical impacts of landscape genetics significantly. however, the conceptual underpinnings of pathogen landscape genetics are not fully developed, and the methodologies employed are diverse and potentially confusing for new practitioners. here, we investigate how landscape genetic techniques are being used to better understand dynamics of microorganisms infecting host species. in conducting this review, we aim to both advocate and facilitate landscape genetic research involving disease-causing organisms. we first evaluate the use of landscape genetics in disease ecology, including the types of questions addressed, the approaches used and the infectious agents studied. we then review established landscape genetic methods and their realized and potential applications to disease ecology. at last, we identify emerging frontiers in the landscape genetic study of pathogens that hold significant potential for advancing research in this field. landscape genetics was first implemented in the study of rabies virus by real et al. ( ) , offering an approach to overcome many feasibility issues associated with understanding landscape influences on pathogen transmission. the landscape genetic approach to studying disease was later reviewed by biek and real ( ) , who were optimistic about its growth and future use. in particular, they noted that microparasites, such as viruses, are well-suited to landscape genetic study due to their rapid mutation rate and potential spatial genetic structure that can be compared to heterogeneous landscape features at fine temporal and spatial scales. analyses could be conducted using both pathogenic organisms and agents that do not cause significant diseases in their hosts (biek, drummond, & poss, ) . they also identified that methodologies such as gis, which are commonly employed both in the wider landscape genetics literature and in spatial studies of infectious disease, had not been widely implemented in molecular epidemiology (archie et al., ) . further, other popular landscape genetic tools, such as those focused on differential landscape permeability (e.g., least-cost paths), were greatly underused despite compatibility with pathogen spatial genetic data. similar to landscape genetics, landscape epidemiology is an interdisciplinary field undergoing rapid development driven by technological advancements, and arguably still working to develop clear directions for future research (meentemeyer et al., ) . it is therefore likely that the interface of these two fields (i.e., where landscape genetics is used in epidemiology) is similarly challenged, perhaps to the extent that its potential is remaining unrealized. we thus believe it is timely to revisit the body of research that combines landscape genetics and landscape epidemiology, leveraging the work done both prior and subsequent to biek and real's ( ) earlier review into clear directions for future research. we conducted a literature search in february using the isi web of science database with the following terms: ts=(("landscape genetic*" or "landscape genom*") and (disease* or pathogen* or parasit* or virus* or virol* or epidem* or infect* or transmi*)) the search returned results. we read each article and retained the empirical papers that used landscape genetic methods to address questions related to pathogens (see supporting information appendix s ). we excluded reviews (n = ), meeting abstracts (n = ), purely methods-based papers (n = ) and articles that identified as or mentioned landscape genetics but did not sufficiently incorporate landscape factors or genetic data into the study (n = ), studies that referred to any of our pathogen-related search terms without it being a primary motivation for the study (n = ), and studies that used words like "transmit" or "parasite" outside of the context of infectious agents (such as the transmission of behaviours) (n = ). one paper was excluded due to a lack of access at our institutions. studies that qualitatively discussed landscape with respect to genetic variation were kept, although one might argue that landscape genetics requires quantitative testing of landscape effects. we classified each paper according to the type of host system studied (plant, wild animal, domestic animal and human), the type of pathogen studied (bacterium, protozoan, virus, prion, fungus, macroparasite and transmissible cancer) and the source of genetic data (host, pathogen and vector), and we estimated the severity of disease that each studied pathogen causes in its sampled host or vector. we also categorized each article according to its general conceptual approach. most examples described in this study were found in our literature search, while several other examples were cited by papers from our search and subsequently also discussed here. following publication of the first study using landscape genetics to investigate disease in , there was little further research in this area until , which saw a rapid increase in the number of publications (figure a ). this increase coincided with two prominent review articles (archie et al., ; biek & real, ) that were strong proponents of a landscape genetics approach to disease ecology and expressed optimism about its future use. the rate of publication has remained relatively steady (and arguably low) since then, with none of the subsequent years recording more publications than in , when six papers were published. however, articles using landscape genetics to investigate disease were published in , potentially indicating increasing interest in this area of research. a majority of studies ( of ) used genetic data from the host for comparison with landscape features (figure b ). this is likely because dna is easier to obtain from larger, free-living hosts than for pathogens, and methods for genotyping and characterizing host spatial genetic variation are more familiar to landscape geneticists, who predominantly study free-living organisms (storfer, murphy, spear, holderegger, & waits, ) . among pathogens that are associated with a particular animal vector, the vector is often genotyped ( of studies of vector-borne diseases), as vectors such as ticks or mosquitos are also easily sampled, and vector gene flow can be used as a proxy for pathogen spread. vectors can be targeted for population control as a means of limiting pathogen spread, which makes their study of immediate relevance to wildlife and livestock managers (townson et al., ) . pathogen genetic data are used in only of pathogen landscape genetic studies, which was somewhat surprising considering that the pathogen is the primary motivation behind many of the reviewed studies. one study included both host and pathogen genetic data (talbot, vonhof, broders, fenton, & keyghobadi, ) . viruses were the most frequently studied type of infectious agent ( of studies; figure c ). in general, viruses evolve more rapidly than other microparasites, which makes them well-suited to study of genetic variation for inference of transmission history (archie et al., ; grenfell et al., ) . however, a majority of landscape genetic studies involving viruses used host genetic data, potentially reflecting the relative difficulty of obtaining viral data, which we discuss later in this section. instead, the high representation of viruses is largely due to the considerable effort devoted to studying rabies, which comprised half of all landscape genetic studies on viral systems. rabies is one of the most well-known wildlife pathogens globally, due to its negative impacts on wildlife, domestic animal and human health (gordon et al., ) . large outbreaks have occurred in north american and european wildlife in recent years, where considerable resources have been devoted to its management (holmala & kauhala, ; slate et al., ) . animals infected with rabies also often exhibit behavioural changes that may make them easier to identify (lefèvre et al., ) , potentially aiding sampling of infected individuals. f i g u r e papers using landscape genetic approaches for the study of infectious agents. (a) number of publications per year that met our search criteria. (b) number of publications using genetic data from each of the host, agent or vector species. (c) number of publications studying pathogens by type, with genetic data source indicated for each type ("unspecified" typically involves studies of a hypothetical agent or estimates of overall pathogen exposure, such as inferred by immune-linked loci). (d) number of publications adopting each of our broadly identified conceptual approaches for applying landscape genetics to the study of pathogens/ infectious agents-using host/vector genetics to predict agent spread, using host/vector genetics to explain agent spread/distribution and using pathogen genetics to directly study agent spread we broadly define three distinct conceptual approaches by which landscape genetics has been used to study infectious agents ( figure d ). these are the prediction of agent spread using genetic information from the host or vector; the use of host or vector genetic information to explain existing spatial variation in infection risk or prevalence; and the use of genetic information from the infectious agent to directly study transmission and spread. the remainder of this section will address each of these approaches in turn. because the spread of many microparasites (particularly directly was unrelated to landscape features tested, determining that current rabies oral vaccination plans should be expanded given the high potential for long-distance host movement. in another rabies study, landscape genetics was used to characterize striped skunk dispersal across riverine and highway barriers to assess their utility as barriers to pathogen spread (talbot, garant, paquette, mainguy, & pelletier, ) . using host or vector genetic data to predict pathogen spread is attractive as it avoids sampling of the agent itself, which may be substantially more difficult, especially in wildlife populations. identification of infected hosts often requires laboratory testing and may require specific, potentially invasive sampling approaches (e.g., necropsy) for accurate diagnosis. in addition, extensive sampling may be required to obtain adequate sample sizes when prevalence is low and must be conducted strategically to capture spatial heterogene- therefore, studies using host or vector data alone have limitations for inferring or predicting pathogen spread, or lack thereof, directly. however, host landscape genetic studies can provide indications of the potential risk of spread of infectious agents, and the understanding gained about host movements can inform subsequent studies of pathogen dynamics. spatial variation in pathogen prevalence or infection risk can be represented in much the same way as any landscape variable , making spatial data relating to presence of an infectious agent well-suited for incorporation into host landscape genetic models. while spatial heterogeneity in pathogen prevalence could also be considered a component of the landscape that may influence spatial genetic variation in the host, typically only adaptive loci are investigated in this context. more commonly, host neutral genetic variation is used to explain spatial patterns of infection risk or prevalence. a prominent example is a study of chronic wasting disease (cwd) in white-tailed deer. blanchong et al. ( ) found that populations with lower cwd prevalence showed higher genetic differentiation from those that had high cwd prevalence. this genetic differentiation was found to be associated with roads and rivers, which were likely barriers to both host gene flow and cwd spread. these inferences have subsequently informed and been verified by additional landscape epidemiological research (robinson, samuel, rolley, & shelton, ) . spatial heterogeneity in pathogen infection risk can also drive microevolutionary responses in the host (epstein et al., ; monello et al., ) . host species are constantly being challenged by parasitic organisms, which, if not overcome, cause disease and can have fitness consequences. this can create strong selection that acts on various genes, and geographic variation in selection at loci that are known to be associated with adaptive immune genes may reflect variation in pathogen pressure, and individual infection or disease risk (fumagalli et al., ) . this variation may be tested for association with environmental features such as temperature, humidity or urbanization (tonteri, vasemägi, lumme, & primmer, ) , enabling insights into how future changes in climate or land use might influence overall pathogen prevalence. using the sampled disease agent as the source of genetic data is the most direct way to infer pathogen spread across landscapes, but can be challenging to accomplish. genetic material may be absent from, or uninformative in some infectious agents, such as prions or clonally transmissible cancers, necessitating genetic analysis of the host (kelly et al., ; storfer et al., ) . in addition to the aforementioned difficulties with pathogen diagnosis, pathogen nucleic acid can be difficult to isolate from samples taken from the host or vector and would ideally be present in the blood, saliva or other easily collected sample. samples may also require enrichment to obtain sufficient quantities of genetic material for analysis, which can be difficult to accomplish for many pathogens, particularly viruses. however, genetic information from viruses may be particularly useful for molecular epidemiologic analyses due to their rapid mutation rate that can closely infer transmission history (archie et al., ; brunker, hampson, horton, & biek, ) . further, viruses are prominent emerging pathogens and have relatively small genomes, aiding whole genome-analysis. there are a variety of methods available for implementing landscape genetics, some designed specifically for landscape genetics, while others have been adapted from other fields. the rapid development of landscape genetics means that new methods are regularly emerging, and it is difficult to comprehensively review all of them. however, there are some well-established methodological approaches that have either seen wide use for some time or are becoming increasingly popular at the cutting edge of the field . we describe the approaches (table ) and discuss their implementation in the study of pathogen transmission and spread. in landscape genetics, simulation models are usually agent-based and spatially explicit (landguth, cushman, & balkenhol, ) . genetic data are modelled for individuals which have discrete spatial locations with respect to one another and with respect to environmental heterogeneity. individuals move, behave and reproduce according to their own attributes in response to other individuals and in response to the simulated environment, and the model simulates changes in allele frequencies in response to these parameters. landscape genetic simulation modelling has been used to test and validate methodological approaches (cushman, wasserman, landguth, & shirk, ; zeller et al., ) , address theoretical questions about how and why landscape heterogeneity influences genetics (landguth et al., ) , and evaluate and explain empirical observations (shirk, cushman, & landguth, ) . further, simulation modelling can predict how a system might respond to certain changes, such as habitat fragmentation or future management activities. simulation modelling has been widely implemented in the study of pathogenic and nonpathogenic disease, beginning with medical research in the s (elveback & varma, (rees et al., ) . the spread of particular host genes relevant to disease can also be simulated to inform management efforts. for instance, landguth, holden, mahalovich, and cushman ( ) used landscape genetic simulations to determine optimal planting regimes to maximize the spread of blister rust resistant genes among whitebark pine populations. such simulations could undoubtedly be applied to vector species in particular, such as predicting the spread of pesticide resistance genes in mosquitos (chang et al., ) and selecting appropriate sites for introduction of genetically modified vectors (lavery, harrington, & scott, ) . in addition, with the need to develop further landscape genetic frameworks for the study of pathogens, simulation modelling can prove useful in testing and validating these techniques, as it has done in the broader landscape genetics field (cushman et al., ; zeller et al., ) . for example, leo, gonzalez, millien, and cristescu ( ) used landscape genetic simulations to validate their multitaxa integrated landscape genetic framework, which appears to be a promising solution to the challenge of studying pathogens with multiple hosts and/or vectors. landscape genetic simulations may also include epidemiological parameters such as mortality or activity responses to infection, or limited infectious periods, which may otherwise confound conventional (i.e., nonsimulation) landscape genetic approaches. edge detection methods, such as monmonier's maximum difference algorithm, (monmonier, ) have also been used to detect landscape barriers to transmission in pathogen studies (carrel et al., ; joannon et al., ) . ancestry estimates from model-based clustering algorithms can assign individuals to their populations of origin, enabling inference of landscape barrier permeability through the identification of migrants and thus estimation of the risk of pathogen spread across the barrier. most of the studies implementing clustering and assignment methods did not use approaches that incorporate environmental data. instead, spatially or nonspatially explicit methods were typically used to identify genetic discontinuities and relationships with landscape barriers were inferred ad hoc, or analyses proceeded to entirely different methods that explicitly include environmental data. associations between genetic discontinuities and landscape be applied to pathogens directly without these potential constraints. resistance surfaces are commonly used in landscape genetics for modelling hypotheses concerning the influence of landscape features (from gis landscape variables) on functional connectivity using techniques such as least-cost paths (adriaensen et al., ) or circuit theory (mcrae, dickson, keitt, & shah, ) . these techniques produce measures of landscape or "effective" distance among populations or individuals for each hypothesis, which can be tested against observed genetic variation. the primary applications of resistance surface modelling in landscape genetics have been the identification of dispersal corridors and predicting the impacts of landscape and environmental change, such as habitat fragmentation or climate change, on connectivity. similar to that, landscape genetic resistance surfaces can identify transmission corridors or future patterns of spread (e.g., streicker et al., ) , and such tools have been identified previously as having great utility for pathogen landscape genetic studies (biek & real, ) . however, resistance surface modelling remains infrequently applied among pathogen studies. careful consideration is required for identifying the most relevant landscape variables to be tested and correctly parameterizing (assigning costs to) the resistance surface(s) so that these variables are represented in a biologically meaningful way. developing landscape resistance hypotheses for transmitted agents may be more difficult as their interactions with the landscape are often indirect, mediated by the ecology of hosts and vectors. pathogen ecological niche models offer an empirical approach for constructing resistance surfaces based on ecological factors influencing pathogen prevalence fountain-jones, pearse et al., ) , but these also may not adequately represent host/vector movements. our literature search returned only one study that explicitly modelled landscape resistance based on pathogen-specific biology, testing elevation (as a proxy for temperature) as a predictor of plasmodium spread, in addition to resistance surfaces that modelled human movements and mosquito vector ecology (lo et al., graph theoretical approaches, which describe connections (edges) between discrete objects (nodes) (newman, ) , are a flexible yet powerful tool for use in landscape genetics (dyer, nason, & garrick, ; garroway, bowman, carr, & wilson, ) . in landscape genetics, nodes can represent individuals, populations or habitat patches, possessing genetic parameters such as diversity measures (dyer et al., ) , or landscape parameters such as percentage habitat or habitat quality (murphy, dezzani, pilliod, & storfer, ) . similar to that, edges can represent genetic relationships between nodes such as genetic distances, gene flow or dispersal (decout, manel, miaud, & luque, ) , or spatial/landscape relationships such as geographic distance or landscape resistance (dyer et al., ) . distinct from other landscape genetic analytical approaches, graphs allow inferences based on the overall shape, or topology, of the network, which can provide unique insights into systemwide processes, such as hierarchical population structure (dyer & nason, ) . network topology may be used to identify populations or habitat patches that form important "stepping stones" for maintaining genetic connectivity across an entire system. such an approach enables experimental simulation whereby nodes may be selectively removed and the overall effect on the system's topology (e.g., overall connectivity, population structure) assessed. metrics pertaining to the importance of individual nodes to network topology can be correlated with variables such as landscape to identify important drivers of network processes. despite their unique applications, graph theory and network approaches are relatively underutilized in landscape genetics compared to methods specifically derived from population genetics and landscape ecology. however, among studies of infectious agents, network approaches in wildlife are becoming increasingly popular (craft, ; craft & caillaud, ) . epidemiological network models are typically based on host contact networks, which are usually constructed using direct observations or indirect techniques such as mark-recapture, telemetry or proximity loggers, and pathogens are simulated on these contact networks. such approaches have already incorporated landscape and other environmental features. in addition, the potential for inferring host contacts in network models using pathogen genetic markers (see below) has been acknowledged in recent reviews (craft, ; gilbertson, fountain-jones, & craft, ; white, forester, & craft, ) , and some studies have directly compared host contact network parameters to parasite genotypes (bull, godfrey, & gordon, ) . despite this, to our knowledge, no published studies have used network models to investigate pathogen movement within a landscape genetic framework. while landscape genetics initially was used to investigate spatial genetic patterns using relatively few neutral markers, the more modern advent of landscape genomics allows the study of variation across the entire genome and effectively expands the scope of landscape genetics to include the study of functional, adaptive genetic variation. next-generation sequencing (ngs) techniques such as restriction-site-associated dna sequencing (radseq) require minimal prior knowledge of the genome under study and can genotype thousands of snps randomly distributed across the genome. some of these snps will by chance be located within or near (and thus linked to) genes or regulatory regions that are under selection. genomewide association studies (gwas) can make use of this information to identify loci linked to phenotypic variation such as disease susceptibility. genotyping of candidate loci identified using quantitative trait locus mapping and gwas can be expanded across a large number of individuals using methods such as targeted sequence capture (grover, salmon, & wendel, ) , and these data can be tested in a landscape genomic framework for associations with environmental variables. loci exhibiting a signature of selection can be identified using outlier tests (excoffier, hofer, & foll, ; luu, bazin, & blum, ) , which search for loci with allelic frequencies that are outliers relative to the majority. such loci are considered potentially under selection and may then be tested a posteriori for correlations with environmental variables. newer methods have focused on explicitly incorporating environmental variables into landscape genomic analyses, known as genetic-environment association (gea) tests (lotterhos & whitlock, ; rellstab, gugerli, eckert, hancock, & holderegger, ) . gea analyses test for correlations between environmental variables and individual genotypes, which eliminates problems due to underlying population structure that must be controlled when using outlier tests. ngs approaches also generate thousands of neutral loci, which provide greater power to detect fine-scale neutral genetic structure than conventional studies based on relatively few loci (allendorf, hohenlohe, & luikart, ) . however, for studies with a particular focus on functional genetic variation, ngs approaches can also be adapted specifically for this purpose through targeted sequencing of the exome (roffler et al., ) (roffler et al., ) . the spread of functional alleles has also been incorporated into landscape genetic simulations , enhancing predictions of future pathogen spread and its effects on host populations. this small body of research is promising for expansion of landscape genomic studies designed to couple pathogen-related functional genetic variation with landscape variables. while we believe that there remains much unexplored utility in established landscape genetic methods for the study of pathogen dynamics as we have described above, we also note new frontiers with significant potential for expanding research in this area. we complete this review by discussing three particularly promising frontiers. studies relating pathogen genetic data directly to the landscape using resistance surfaces are challenged by the mediating influence of distinct host and vector traits, as well as relative differences in the contributions of multiple host and/or vector species to microparasite gene flow. this necessitates frameworks that more holistically incorporate multiple host and vector factors into studies of pathogen gene flow, which can expand the potential insights provided by landscape genetic studies of infectious agents (figure ). single or multiple host or vector species can be added as "landscape variables" (e.g., as resistance surfaces) in addition to physical landscape and environmental variables to test as factors shaping spatial pathogen genetic structure. resistance surfaces for tests of microparasite gene flow can represent host/vector distributions or abundance, ideally inferred from empirically derived ecological niche or species distribution models. optimally, host/vector movement would be represented (dougherty et al., ) , using outputs from agent-based movement models informed by telemetry or mark-recapture data, or host/vector landscape genetic data representing spatial patterns of gene flow. we note that the common issue in conventional landscape genetics of spatio-temporal mismatches between landscape processes and genetic change (anderson et al., ; landguth et al., ) would apply even more strongly here. researchers must simultaneously consider the potentially different spatial and temporal scales over which host and pathogen genetic changes (and poten- explain observed spatial patterns of prevalence least-cost path models of water bird movement estimated from ecological niche models, and road networks representing human movement, as potential predictors of avian influenza spread (young et al., the rapid mutation of microparasites relative to their hosts has potential to provide greater power to detect subtle variation in host movement patterns in response to the landscape, as well as earlier detectability of changes in host movements (such as in response to a new barrier) that are yet to be reflected in host genetic structure (landguth et al., ) . in addition, movements of nonreproducing hosts are difficult to detect using host genetic markers, but instead might be inferred using markers from directly transmitted microorganisms. such an approach has demonstrated the utility of a chronic, relatively apathogenic infection of felids (feline immunodeficiency virus) for identifying demographic structure of mountain lions and recent population history (biek et al., ) , and has identified movement of bobcats across a highway barrier that was not detectable using host markers (lee et al., ) . however, these approaches have not been broadly applied, particularly in the study of landscape effects. phylogenetic approaches can reconstruct very recent epidemic histories, providing insights into particular transmission events and pathways that may be contextualized temporally and spatially (corman et al., ; faria et al., ; carroll et al., ; magee, beard, suchard, lemey, & scotch, ; fountain-jones, packer, et al., ; fountain-jones, pearse et al., ) . the majority of such work has been conducted on rna viruses owing to their small, rapidly mutating genomes, requiring relatively little sequencing effort to detect contemporary phylogenetic signals. other pathogens that evolve more slowly, such as bacteria or fungal pathogens, require the sequencing of larger portions of their genomes to capture equivalent phylogenetic signals (biek, pybus, lloyd-smith, & didelot, ) . while this is becoming increasingly feasible (kao, haydon, lycett, & murcia, ) , more complex computational analysis is required to make meaningful conclusions. several approaches may be used for relating phylogenetic information with landscape variables. neighbour joining trees can identify clusters for quantifying population-level landscape genetic relationships (joannon et al., ) . the calculation of genetic distances based on maximum likelihood trees (carrel, emch, tung, jobe, & wan, ; real et al., ; young et al., ) results in distance matrices that can be correlated with landscape resistance matrices using conventional landscape genetic approaches. relaxed random walk phylogeographic approaches (lemey, rambaut, welch, & suchard, ) that can reconstruct pathogen dispersal have been linked to landscape predictors using a "phylogeographic glm" method (faria, suchard, rambaut, streicker, & lemey, ; jacquot, nomikou, palmarini, mertens, & biek, ) . the phylogeographic glm approach has enabled a better understanding of how landscape and hosts can constrain pathogen spread. for example, using the phylogeographic glm approach on viral genomic data, roads and rivers, coupled with dog distribution, were found to impact rabies spread in tanzania (brunker et al., ) . however, this approach is limited to discrete sampling locations and is computationally intensive (dellicour, rose, & pybus, ) . a recent framework by dellicour et al. ( ) modifies the phylogeographic glm approach to use resistance surfaces to efficiently quantify landscape resistance along transmission pathways inferred by continuous phylogeographic analyses. these landscape resistances are then correlated with temporal estimates of transmission along these routes to estimate how the landscape has shaped rates and directions of pathogen spread. such approaches are yet to be broadly applied, but appear to be important developments that should see increasing application in the future. overall, landscape genetics has been relatively underutilized in disease ecology research. we believe this is partly due to a lack of cross-disciplinary awareness between the two fields, but also a lack of a clear landscape genetic framework specifically designed for tackling pathogen systems, which are often complex and do not facilitate easy translation of existing landscape genetic tools. however, we note there has been a recent effort to develop new frameworks for such research, expanding the utility of the landscape genetic toolset. these tools will increase our capacity to study complex multihost and host-vector systems, improving the integration of multiple genetic datasets and accounting for interspecific interactions. improved understanding of host-parasite associations will facilitate the use of microparasite genetic markers to provide insights into host processes that may be difficult to detect using conventional host landscape genetics. identification of idealized systems that are designed to target specific ecological questions will also facilitate progress in this field. recent methods that enable the incorporation of quantitative landscape data into spatio-temporal phylogenetic reconstructions of recent transmission events, coupled with advances in high-throughput sequencing, hold great promise for studying how the landscape shapes transmission processes. we believe that these recent developments represent a renewed interest in 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population structures: implications for disease transmission in a sympatric cervid community a conceptual framework for the spatial analysis of landscape genetic data the role of parasite-driven selection in shaping landscape genomic structure in red grouse (lagopus lagopus scotica) using contact networks to explore mechanisms of parasite transmission in wildlife how's the flu getting through? landscape genetics suggests both humans and birds spread h n in egypt using simulations to evaluate mantel-based methods for assessing landscape resistance to gene flow additional supporting information may be found online in the supporting information section at the end of the article. key: cord- -fc dn v authors: kato, kentaro; ishiwa, akiko title: the role of carbohydrates in infection strategies of enteric pathogens date: - - journal: trop med health doi: . /tmh. - sha: doc_id: cord_uid: fc dn v enteric pathogens cause considerable public health concerns worldwide including tropical regions. here, we review the roles of carbohydrates in the infection strategies of various enteric pathogens including viruses, bacteria and protozoa, which infect the epithelial lining of the human and animal intestine. at host cell entry, enteric viruses, including norovirus, recognize mainly histo-blood group antigens. at the initial step of bacterial infections, carbohydrates also function as receptors for attachment. here, we describe the function of carbohydrates in infection by salmonella enterica and several bacterial species that produce a variety of fimbrial adhesions. during invasion by enteropathogenic protozoa, apicomplexan parasites utilize sialic acids or sulfated glycans. carbohydrates serve as receptors for infection by these microbes; however, their usage of carbohydrates varies depending on the microbe. on the surface of the mucosal tissues of the gastrointestinal tract, various carbohydrate moieties are present and play a crucial role in infection, representing the site of infection or route of access for most microbes. during the infection and/or invasion process of the microbes, carbohydrates function as receptors for various microbes, but they can also function as a barrier to infection. one approach to develop effective prophylactic and therapeutic antimicrobial agents is to modify the drug structure. another approach is to modify the mode of inhibition of infection depending on the individual pathogen by using and mimicking the interactions with carbohydrates. in addition, similarities in mode of infection may also be utilized. our findings will be useful in the development of new drugs for the treatment of enteric pathogens. enteric pathogens, many of which are zoonotic, exert a major impact on public health worldwide including tropical regions. in humans and animals, the enteric pathogens, which include viruses, bacteria and protozoa, infect the intestine epithelial lining, resulting in food poisoning or diarrheal disease. when enteric pathogens enter humans or animals via the oral route, they must withstand the proteolytic conditions in the stomach before penetrating the mucus layer and accessing the underlying gut epithelium for attachment or cell invasion. adhesion of the enteric pathogens to the intestine epithelial tissue is a prerequisite for the initiation of infection. in many systems it is mediated by lectins present on the surface of the pathogen that bind to complementary carbohydrates on the surface of the host cells. carbohydrates such as heparan sulfate have been reported to play a crucial role in the entry or budding of viruses [ ] , and bacterial lectins typically act in the form of elongated submicroscopic multisubunit protein appendages, known as pili [ ] . recently, the surface proteins of apicomplexan parasites have also been reported to bind to carbohydrates on host cells [ ] . thus the initial steps of host cell recognition by enteric pathogens may incorporate common strategies. once pathogens invade the host cells, they initiate their survival mechanisms to avoid extermination by host immunity. ultimately, if infection of host cells could be inhibited, proliferation of the pathogens could be prevented and pathogenesis could be controlled. insights obtained from studies designed to address this concept will be invaluable to develop novel therapies using innovative drug design and engineered vaccine candidates to limit the infectivity of widespread enteric pathogens. here, we review the recent major advances in research on the role of carbohydrates in the infection strategies of enteric pathogenic viruses, bacteria and protozoa. we further discuss how our knowledge regarding these carbohydrates may influence prophylactic and therapeutic drug development for the treatment of diseases caused by enteric pathogens. carbohydrates function as receptors for virus entry. negatively charged carbohydrates, which are expressed on many types of cells and tissues such as sialic acid and heparan sulfate, are common viral receptors. orthomyxovirus, polyomavirus, reovirus, coronavirus, paramyxovirus and parvovirus recognize sialic acid as a receptor. adenoassociated virus, herpesvirus and flavivirus recognize heparan sulfate. on the other hand, the enteric virus norovirus recognizes histo-blood group antigens (hbgas), which are not charged. here, we focus on the association of carbohydrates with norovirus as the virus enters the host cell. norovirus, a member of the family caliciviridae, is a major cause of acute water-and food-borne gastroenteritis [ ] . norovirus infection is associated with up to % of epidemic nonbacterial acute gastroenteritis cases worldwide [ ] . noroviruses are divided into at least five genotypes, three of which (genogroups i, ii, and iv) infect humans. except for a few genotypes, all noroviruses bind to hbgas including abh antigens and lewis antigens [ , ] . in hbgas, carbohydrate core structures constitute antigenically distinct phenotypes, namely type (galβ - glcnacβ) and type (galβ - glcnacβ). h antigen (fuc-α - gal), i.e., o-type antigen, is generated by fucose transfer to a galactose residue with an α - linkage of type or type . the a antigen (galnac α - (fuc-α - )gal) and b antigen (gal α - (fuc-α - )gal) of hbgas are generated by transfer of galnac and gal, respectively, to an h structure irrespective of the carbohydrate core structure. fut and fut are α , futs that catalyze the transfer of fuc to the gal residue of type and chains, thereby resulting in the synthesis of h type and h type , respectively. hbgas are found in saliva and mucosal secretions from the intestinal epithelial cells of secretors (i.e., individuals who have the fut gene that encodes a fucosyltransferase). non-secretors, who do not express fut fucosyltransferase and consequently do not express h type or le b in the gut, are not infected after challenge with the prototype strain of norovirus, nv/ [ , ] . moreover, the association data between blood type and nv/ infection showed that, among secretor volunteers, blood groups o and a were associated with an increased risk of infection, while blood group b was associated with a decreased risk. on the other hand, epidemiological studies have shown that some norovirus strains with abh phenotypes that differ from that of nv/ can infect individuals. gii/ , which is known as a global epidemic strain, binds more hbgas than other strains, suggesting that the strength of transmission of gii/ strains is related to the broad recognition of hbgas [ ] . the recognition sites on hbgas by norovirus have been classified according to the interaction of the virus with the h, a, b, and le epitopes ( fig. ) [ , ] . hbgas are important factors for determining host specificity, although it is still unclear whether hbgas act as the primary receptor or enhance norovirus infectivity. researchers including ourselves have demonstrated that feline calicivirus (fcv), a member of the genus vesivirus, infects the upper respiratory tract by attaching to α - linked sialic acids and using junctional adhesion molecule- for internalization [ , ] . it is comparatively easy to study the life cycle of fcv because the virus replicates efficiently in cell culture without specific supplementation, whereas noroviruses are not cultivable in cell culture. carbohydrates also function as receptors for bacterial attachment at the initial step of infection. here, we describe the role of carbohydrates in bacterial infections, focusing on salmonella enterica and several assortative bacterial species that produce a variety of fimbrial adhesions (fig. ) . salmonella strains cause disease in diverse mammalian hosts. some salmonella strains have a narrow host range, such as salmonella enterica serovar typhi (s. typhi) and serovar paratyphi (s. paratyphi), which cause disease only in humans, whereas strains such as salmonella enterica serovar typhimurium (s. typhimurium) and serovar enteritidis (s. entertitidis) cause infection in numerous species including mice, poultry, pigs, sheep, cattle, horses and humans [ ] . infected orally, salmonella reach the intestinal tract and then mainly attach to the m cells of the intestinal epithelium to initiate invasion [ ] . after colonization of the intestinal epithelium, typhoid salmonella, s. typhi and s. paratyphi, invade m cells. however, s. typhi and s. paratyphi can also survive being engulfed by macrophages, which then spread throughout the body via the lymphatic and blood systems. non-typhoidal salmonella, s. typhimurium and s. enteritidis cannot survive within macrophages (fig. ) . they cause gastroenteritis in humans and animals by colonizing the intestinal epithelium and then invading and destroying the m cells and enterocytes [ ] . bacterial adherence requires both specific and nonspecific interactions. in the case of salmonella, the negative charge produced by sialic acid on the surface of the host cell is required as a non-specific adherence factor [ ] . for their specific interactions, salmonella and assor-tative bacteria possess various adhesion molecules such as a variety of bacterial fimbriae. at the initial infection step, bacterial attachment is mainly controlled by these bacterial fimbriae. individual fimbria recognize and bind to specific receptors to promote adhesion to the host cell surface [ , ] . long polar fimbriae (lpf) and plasmid-code fimbriae (pef) are categorized as type fimbriae (fig. ) . std fimbriae are categorized as π-fimbriae [ , [ ] [ ] [ ] . a previous report showed that when one of the fimbriae carried by salmonella typhimurium was deleted, only virulence for mouse was moderately altered, and that multiple fimbrial adhesins were required for full virulence [ ] . for salmonella and assortative bacteria, type fimbria is the std fimbriae are categorized as π-fimbriae. bacterial flagella are the moving apparatus of bacteria, but their components can also contribute to the binding to sugar-containing molecules. k. kato et al. best characterized [ ] . type fimbriae consist of a major component (fima) and a minor component (fimh) (fig. ) . fimh lies at the tip of type fimbriae, where it mediates binding to d-mannose-containing structures and enables the bacteria to colonize various host tissues [ ] . type fimbriae are also produced by other gram-negative bacteria, such as escherichia coli and klebsiella pneumoniae [ ] . the fimh protein of enterobacterial species including salmonella recognizes mannose-containing oligosaccharides [ ] . in the case of e. coli, previous reports have shown that fimh protein has a considerably high affinity for oligosaccharides containing manα - , such as manα - manβ - glcnac and manα - (manα - )manα - (manα - )man, which are constituents of cell surface glycoproteins [ ] . the process of bacterial adhesion to the epithelial cell surface mediated by type fimbriae (fimh) is conservative among enterobacteria. type fimbriae are highly expressed on the bacterial surface, allowing large quantities of bacteria to adhere via the fimh-mannose interaction (fig. ). type fimbriae are thin and flexible, and generally expressed at a lower level than type fimbriae. some type fimbriae are only present at low levels on the surface of bacteria, and are localized at the bacterial pole. although their expression level is low, type fimbriae frequently play an important role in bacterial infection. like type fimbriae, type fimbriae are thought to recognize carbohydrates as specific receptors, but the receptor molecules and precise functions of some type fimbriae have yet to be determined. for example, the function of the type fimbria bundle-forming pili (bfp), an important virulence factor for pathogenic e. coli strains, is not yet known (fig. ) . bfp may not be involved in initial adhesion; rather, it may participate in the formation of the bacterial colony by forming bundles that link one bacterium to another [ ] . although the function of and receptors for type fimbriae remain unclear, bacterial virulence has been shown to decrease when type fimbriae are deleted [ ] . lpf medi- salmonella and assortative bacteria express a variety of fimbriae. the minor component of type fimbriae, fimh, is present at the tip of type fimbriae, mediates binding to d-mannose-containing structures and enables bacteria to colonize various host tissues [ ] . type fimbria is highly expressed on the bacterial surface, allowing large amounts of bacteria to adhere via the fimh-mannose interaction. the various kinds of type fimbriae play an important role in bacterial infection. plasmid-encoded fimbria (pef) is required for bacterial attachment to intestinal epithelial cells. pef specifically binds to trisaccharide galβ - (fucα - )glcnac, also known as the lewis x (le x ) blood group antigen [ ] . long polar fimbria (lpf) mediates the adhesion of s. typhimurium to murine peyer's patches [ ] . extracellular matrix proteins (ecms) may act as receptors for lpf. ecms are modified with various carbohydrate moieties, and the presence of mannose inhibits the lpf-ecm interaction. mannose-containing carbohydrates may participate in bacterial adhesion via lpf [ ] . std fimbriae are categorized as π-fimbriae and are well conserved among s. enterica serotypes but absent from other related bacterial species. std fimbriae recognize and bind the h type histo-blood group oligosaccharide, the terminal fucα - galβ moiety. s. typhi and s. paratifi can survive within the macrophages after they are engulfed by phagocytosis. non-typhoidal salmonella, s. typhimurium and s. enteritidis, however, are unable to survive within macrophages. ates the adhesion of s. typhimurium to murine peyer's patches [ ] . lpf was first described in s. typhimurium, and is found in numerous pathogenic e. coli strains [ ] . although its specific receptor remains unclear, extracellular matrix proteins (ecms), which comprise an interlocking mesh of fibrous proteins and glycosaminoglycans, may act as a receptor for lpf of enterohemorrhagic e. coli o :h (fig. ). ecms are modified by various carbohydrate moieties, and the addition of mannose inhibits lpf-ecm interaction. then mannose-containing carbohydrates may participate in bacterial adhesion by lpf [ ] . in some cases, type fimbriae are encoded on plasmids. such plasmids frequently encode virulence factors for host bacteria, and are therefore called "virulence plasmids" [ ] . pef is required for bacterial attachment to intestinal epithelial cells. it specifically binds to trisaccharide galβ - (fucα - )glcnac, also known as the lewis x (le x ) blood group antigen (fig. ) [ ] . the le x antigen is defined by the presence of a terminal galβ - (fucα- )glcnac moiety on saccharide chains of glycoproteins or glycosphingolipids; in the human intestine, it is expressed mainly in crypt epithelial cells [ ] . s. typhimurium possesses pef as an adhesin that binds to a crypt-specific histo-blood group antigen that may be relevant to the pathogenesis of human infections. abundant crypt abscesses are commonly found in s. typhimurium patients, raising the possibility that the pathogen may bind to human crypt epithelium at a later stage of infection. in a situation where peyer's patches are unavailable because of an inflammatory reaction, salmonella can colonize at the crypt epithelium remaining intact and persist on the surface of the host intestinal tract [ , ] . on the other hand, some type fimbriae participate in "fimbria-mediated (pilus-mediated) conjugal transfer" of so-called "conjugative plasmids". conjugative plasmids can also be virulence plasmids if they encode not only the structural genes of the fimbriae but also other virulence factors, such as a drug resistance gene. these conjugative plasmids spread to other bacteria by horizontal transfer, and type fimbriae encoded on the plasmids play an important role in this event. for example, the r plasmid, which encodes the pilv gene and engages in the adhesion of type fimbriae, recognizes the di-saccharide moiety of bacterial surface polysaccharides (the core oligosaccharide or o-antigen unit of lipopolysaccharides, a unique structure of the bacterial cell surface) and determines the recipient bacteria of the conjugal transfer [ , ] . categorized as π-fimbriae, the std fimbriae are well conserved among s. enterica serotypes but absent from related bacterial species (fig. ) . std fimbriae recognize and bind the h type histo-blood group oligosaccharide, the terminal fucα - galβ - glcnac moiety. this structure represents the h type oligosaccharide of the o blood group antigen [ ] . the h type oligosaccharide of the o blood group antigen moiety is expressed as part of the mucin-type sugar chains of glycoproteins in the host cell. the terminal fucα - moiety of h type oligosaccharide of the o blood group antigen is essential for the recognition of std fimbriae (fig. ) . carbohydrate molecules act not only as "anchors" for pathogens but also as the determinants of host and tissue specificity. the variety of adhesion factors carried by a bacterium reflects its pathogenic profile, magnitude of virulence, host specificity, and tissue specificity. in the case of salmonella and assortative bacteria, the fimh adhesins show amino acid sequence diversity. this diversity in fimh structure results in the variation in affinity profiles. e. coli fimh shows a high affinity for aromatic αmannosides as well as manα - structures. on the other hand, the fimh of salmonella species shows a high affinity for α-mannosides and a low affinity for aromatic αmannosides [ ] . in the case of salmonella, allelic variation of fimh adhesion directs not only host cellspecific recognition but also distinctive binding to mammalian and avian receptors. this allele-specific binding profile parallels the host specificity of the respective fimh-expressing pathogen [ ] . similarly, the lewis b (le b ) blood group phenotype in combination with secretor status may hinder colonization of helicobacter pylori in certain populations [ ] . h. pylori express blood group antigen b-binding adhesion (baba), and baba binds to le b antigens. salmonella and assortative bacteria contain various adhesion factors, including several kinds of fimbriae, which contribute to bacterial virulence; however, analyses of their specific receptor moieties and functions are not yet complete [ , ] . carbohydrate moieties on the surface of pathogens are also recognized by hosts and trigger host defense mechanisms. the bacterial surface is covered with various kinds of carbohydrates. for gram-negative bacteria, including salmonella, the major carbohydrate component of the bacterial surface is lipopolysaccharide (lps). lps is categorized as a glycolipid, and is a major component of the bacterial outer membrane. because the saccharide moieties of lps differ structurally from mammalian carbohydrates, they function as targets of the host immune response. to avoid this host immune response, the lps of some bacteria, for example c. jejuni, is structurally similar to the glycosphingolipids of gangliosides [ , ] . similarly, the lps of most h. pylori strains expresses the le a and le b antigens [ ] . interestingly, the carbohydrate on the surface of the k. kato et al. host cell itself can be involved in the host defense mechanism. the salmonella flagella component flic contributes to bacterial attachment to the host cell by interacting with ganglioside molecules on the surface of the host cell, but gangliosides also act as co-receptors for salmonella enterica flic and promote flic induction of the human innate immune response [ ] . gangliosides, i.e. sialic acidcontaining glycosphingolipids, are ubiquitous components of eukaryotic cell membranes that have been identified as receptors for bacterial toxins and viruses. an in vitro assay showed that a nonflagellated mutant of s. enteritidis, constructed by disrupting the flic gene, was about -fold less invasive than the wild-type strain, but bacterial adherence was unaffected [ ] . at the attachment of salmonella enteritidis flic to the host cell surface, gangliosides thus function as receptors. on the other hand, the flagella component protein flic induces the host innate immune response by binding to toll-like receptor of the host cell, and gangliosides react as co-receptors with tlr on the flic-induced response. an in vitro assay showed that the incorporation of exogenous ganglioside gd a into the caco- cell membrane increased the effect of flic. incubation of caco- cells with a glucosylceramide synthase inhibitor reduced the innate immune response stimulated by flic [ ] . human enteropathogenic protozoas include the apicomplexans toxoplasma gondii and cryptosporidium as well as giardia and entamoeba histolytica. they are all zoonotic pathogens that invade and colonize their target tissues in the alimentary tract of the human host. they form hard cysts that resist degradation in the stomach. host-derived proteases and low ph trigger their excystation [ ] . in this section, we describe the role of carbohydrates in toxoplasma gondii invasion of intestinal epithelial cells. the ability of t. gondii to infect chinese hamster ovary (cho) cells deficient in sialic acids was reduced by . % compared to wild-type cells, indicating that sialic acid is critical for attachment and invasion of t. gondii (fig. ) [ ] . t. gondii microneme protein (tgmic ) forms a macromolecular complex with tgmic and tgmic . single deletion of the tgmic gene significantly decreases the invasion of host cells, suggesting an essential role for tgmic in host cell attachment and invasion of t. gondii [ ] . structural analysis of tgmic revealed a novel cellbinding motif called microneme adhesive repeat region (marr), which provides a specialized structure for glycan discrimination [ ] . carbohydrate microarray analyses showed that tgmic , tgmic and its homologue neospora caninum mic share a preference for α - -over α - -linked sialyl-n-acetyllactosamine sequences [ ] . p , a pan/apple domain-containing protein expressed at the apical end of the extracellular parasite, functions as a ligand in the attachment of t. gondii to chondroitin sulfate and other receptors on the host cell, facilitating invasion by the parasite (fig. ) [ ] . t. gondii display gpi-anchored surface proteins identified as surface antigen glycoprotein (sag) related sequences (srs) [ ] . sag , sag a and sag have some capacity for host cell attachment through glycan recognition (fig. ) [ , ] . sag binds to sulfated proteoglycans such as heparin, fucoidan, and dextran sulfate with high affinity [ ] . targeted disruption of sag significantly reduces host cell binding of t. gondii [ ] . e. histolytica fibronectin receptor (ehfnr) shows % homology to the intermediate subunit- of the gal/ galnac-specific lectin [ ] . electron microscopy revealed the close association of a purified ehfnr complex to adhesion plates and phagocytic invaginations. lipid rafts participate in interactions between e. histolytica and the host extracellular matrix, and it appears that raft-associated gal/ galnac lectin serves as a collagen receptor [ ] . cryptosporidium parvum surface receptors, gp and proteolytic fragments of the -kda precursor protein, gp and gp , are characterized as mucin-like and heavily o-glycosylated proteins [ ] [ ] [ ] . the gp and gp of sporozoites and merozoites have carbohydrate residues that are bound by αgalnac-specific lectins, suggesting that αgalnac residues are involved in the attachment of parasites to host cells via adherence to internal mucus. apicomplexan protozoan parasites also induce host innate immune responses via the carbohydrate molecules present on their cell surface [ ] . glycosylphosphatidylinositol (gpi) protein anchors are abundant in the membranes of tachyzoites and other apicomplexan protozoan parasites including trypanosoma, leishmania and plasmodium spp., where they can serve as ligands for innate recognition [ ] . the gpi moieties of t. cruzi and p. falciparum were found to be tlr ligands [ , ] , and t. gondii both stimulate cytokine production in macrophages and serve as tlr as well as tlr agonists. in the case of t. gondii, gpi induces tnf-α production in macrophages through the activation of the transcription factor nf-κb [ ] . carbohydrates serve as receptors for infections by viruses, bacteria and protozoa, but the usage of carbohydrates by these microbes varies depending on the microbe. at the initial infection step, these organisms do not simply utilize the electrical forces created by the positive and negative charges of the carbohydrates; rather, they make use of other systems in certain instances. one similarity shared by all three microbes regarding their interactions with carbohydrates, however, is that heparan sulfate plays an important role at entry or invasion of the host cell. blood group antigen oligosaccharides are highly expressed in the gastrointestinal epithelium [ ] . however, there are individual differences in terms of the presence of these antigens. in addition, there are individual differences in sensitivity to pathogens that recognize and bind to blood group antigens, such as norovirus and h. pylori. these individual differences in antigen expression profiles benefit the survival of the host species because the risk of an attack by a fatal virulent pathogen may be decreased to avoid extinction. a large array of glycoproteins, glycolipids and proteoglycans decorate the surface of animal cells. these glycoconjugates mediate many fundamental cellular processes, including cell-cell and cell-matrix adhesion, motility, growth and signaling [ ] [ ] [ ] . mucosal tissues represent the site of infection or route of access for most parasites, including viruses, bacteria and protozoa [ ] . on the surface of the mucosal tissues of the gastrointestinal tracts, various carbohydrate moieties are present and play a crucial role in infection. mucosal surfaces are coated with a layer of viscous mucus that ranges in thickness from μm in the stomach to μm in the intestine [ ] [ ] [ ] . mucin glycoproteins from mucus-producing cells in the epithelium or submucosal glands are the major macromolecular constituent of mucus and are responsible for the viscous properties of the mucus gel. in addition to forming a relatively impervious gel, which acts as a lubricant, a physical barrier and a trap for microbes, mucus provides a matrix for a rich array of antimicrobial molecules. underneath the mucus layer, the cells present a dense forest of highly diverse glycoproteins and glycolipids, which form the glycocalyx. membrane-anchored cell-surface mucin glycoproteins are a major constituent of the glycocalyx in all mucosal tissues. the oligosaccharide moieties of the molecules that form the glycocalyx and the mucus layer are highly diverse, and the average turnover time of the human jejunal glycocalyx is - h [ ] . consequently, both the secreted and adherent mucosal barriers are constantly renewed and can rapidly adjust to changes in the environment, for example, in response to microbial infection. epithelial mucins are a heterogenous family of large complex glycoproteins containing a dense array of olinked carbohydrates typically comprising over % of their mass. the carbohydrate structures present on mucosal surfaces vary according to cell lineage, tissue location and developmental stage [ ] . mucin glycosylation can alter in response to mucosal infection and inflammation, and this may be an important mechanism for unfavorable changes in the niche occupied by mucosal pathogens. the o-linked glycans of muchin proteins contain - residues, which occur both as linear and branched structures [ ] . in addition to the o-linked glycans, mucins contain a smaller number of n-linked oligosaccharides, which have been implicated in folding, oligomerization (muc ) and surface localization (muc ) [ ] [ ] [ ] . the terminal structures of mucin oligosaccharides are highly heterogeneous k. kato et al. and vary between and within species as well as between and even within tissues. the array of oligosaccharide structures on individual mucin molecules is also somewhat determined by stochastic events as the mucin protein moves through the golgi apparatus [ ] . the secreted mucins themselves likely function as decoys for adhesins that have been evolved by pathogens to engage the cell surface, as the mucins express many of the oligosaccharide structures found on the cell surface and are constitutively produced in large amounts, constantly washing the mucosal surfaces [ ] . proteoglycans are present on the cell surface [ ] and are also components of glycocalyx. glycosaminoglycan chains are composed of highly sulfated saccharides that give the cell surface a potent negative charge. one of the prototypical membrane proteoglycans is syndecan- , which carries conserved attachment sites for glycosaminoglycan chains [ ] . the syndecans exemplify hybrid proteoglycans because they contain mixtures of the two major types of glycosaminoglycan chains, heparan sulfate and chondroitin sulfate. the other major family of membrane proteoglycans is the glypicans, which contain gpi anchors in a tissue-specific and temporally regulated manner. their presence in the basolateral membranes of polarized cells varies [ ] . glycolipids are also a component of the cell membrane. a large variety of glycolipids is present on the surface of animal cells. the carbohydrate moieties vary, and each glycolipid may exhibit a special function, as an annular lipid, surface receptor marker or matrix lipid. for brain and neuronal cells, gangliosides (sialic acid-bearing glycolipids) are the major cell surface determinants [ ] . glycolipids function as the receptor for various biologic factors and also as the receptor for various pathogens. they are present at the undermost part of the glycocalyx. pathogens can recognize the glycolipids, directly bind to the cell membrane, and invade the host cell. glycolipids also function as receptors for certain effector molecules, such as bacterial toxins, produced by pathogens and directly react with the host cell. for example, cholera toxin binds to ganglioside gm [ ] . thus, for pathogens living in the outer mucus layer, it is difficult to make contact with the surface of normal epithelial cells because of the huge amount of mucin that functions as a "decoy" or "physical barrier". mucosal pathogens have, therefore, developed mechanisms to subvert these defense mechanisms of the mucosal layer. on the other hand, intestinal m cells, specifically designed to capture and present microbes to the underlying lymphoid tissue, can be regarded as a "hole" in the mucin barrier. the dome epithelium lacks goblet cells and therefore does not produce gel-forming mucins. their apical cell surface has only sparse microvilli and an apparently thin glycocalyx [ , ] . m cells are specialized epithelial antigen-transporting cells that constitute a minor proportion ( %~ % in humans and mice) of the follicle-associated epithelium that covers the lymphoid follicles of organized gut-associated lymphoid tissue such as peyer's patches [ ] [ ] [ ] [ ] . glycoprotein (gp ) was identified as an m cell-specific molecule [ ] . the gp expressed on m cells functions as a bacterial uptake receptor [ ] . gp recognizes fimh, a major component of the type fimbriae, which binds to certain glycoproteins on mammalian cells in a mannosedependent manner [ ] . consequently, even though m cells constitute only a very small percentage of mucosal epithelial cells, they are the major point of attachment and/or entry used by numerous mucosal pathogens including bacteria (e.g., s. typhimurium, shigella flexneri, yersinia enterocolitica and vibrio cholerae), viruses (e.g., reovirus, hiv- and polio virus) and parasites (e.g., cryptosporidia) [ , , ] . during cell-pathogen interactions (i.e., infection and/or invasion), carbohydrates function as receptors for various pathogens. on the other hand, carbohydrates (glycoconjugates) can also function as a barrier to infection. on the surface of mucosal tissue, the glycocalyx physically prevents microbes from accessing the cell membrane. some glycoconjugates, a component of the glycocalyx, contain carbohydrate structures that are recognized by pathogens. mucins often contain oligosaccharide moieties that correspond to the receptor for various pathogens. on the surface of the mucosal layer, microbes binds to these receptor moieties and are captured at the mucus layer, which consequently blocks the infection. moreover, when secretory mucins containing receptor carbohydrate structures "trap" pathogens, the pathogens are also carried away. m cells are specialized epithelial antigentransporting cells scattered in the follicle-associated epithelium that covers the gut lymphoid follicles such as peyer's patches. m cells can efficiently engulf particles as large as bacteria; however, the mucus layer of m cells and the surrounding area is relatively thin. glycoconjugates such as gp are expressed on the surface of m cells and function as receptors for bacterial attachment [ ] . in the case of the host-parasite interaction, the various kinds of glycoconjugates sometimes function as receptors for the invading pathogens, but they can also function as barriers tropical medicine and health vol. no. , and traps for the host defense system. in recent years, the damage caused by enteric pathogens, especially norovirus and salmonella, has expanded through the food chain [ , , ] . these pathogens cause food poisoning in humans and gastrointestinal diseases in animals all over the world. even today, they are often responsible for large-scale outbreaks of food poisoning. therefore, the prevention and treatment of infections caused by these pathogens is essential. in this review, we discussed the interaction between host cells and microbes such as viruses, bacteria and protozoa that involve carbohydrates such as sialic acids, heparan sulfate, and the carbohydrate moieties of abh and lewis antigens, mannose components, ecms and le x . the development and use of drugs that target these carbohydrates is anticipated, even though the microbes vary widely and have different modes of infection. accordingly, when an anti-microbial drug is developed on the basis of the interaction between a microbe and a carbohydrate, host cell modification of the drug's structure and/or inhibition of the mode of infection will need to be individualized while still taking advantage of the similarities between interactions. moreover, the host gastrointestinal tract cell surface, which is the object of microbial infection, is composed of glycoproteins, glycolipids, and proteoglycans. these molecules are potential targets for carbohydrate drugs used in the treatment of infectious diseases. oseltamivir and zanamivir are neuraminidase inhibitors that competitively inhibit the activity of the viral neuraminidase on the sialic acid that is found on glycoproteins on the surface of host cells [ ] . by blocking the activity of this enzyme, they prevent new viral particles being released from infected cells. there are various kinds of polysaccharides on the surface of bacteria. lipoteichoic acid (lta), a type of glycolipid, is a component of the bacterial cell wall of grampositive bacteria. studies have shown that lta stimulates the immune system [ , ] . recently, lta has been studied for use as a novel kind of biologically active substance. recently, sulfated polysaccharides have been analyzed as drug candidates for protozoan infectious diseases [ , , ] . according to our data, the sulfated positions in the carbohydrates can be critical for the inhibitory quality [ ] . collectively, these studies highlight the possibility that carbohydrate drugs may be developed for the prophylaxis and treatment of parasitic infectious diseases. the results of our studies highlight the possibilities for countermeasures against malaria and toxoplasmosis [ , ] . heparan sulfate glycosaminoglycans as primary cell surface receptors for herpes simplex virus salmonella enterica serovar typhimurium requires the lpf, pef, and tafi fimbriae for biofilm formation on hep- tissue culture cells and chicken intestinal epithelium analyses of interactions between heparin and the apical surface proteins of plasmodium falciparum norovirus disease: changing epidemiology and host susceptibility factors human susceptibility and resistance to norwalk virus infection noroviruses distinguish between type and type histo-blood group antigens for binding norovirus and histoblood group antigens: demonstration of a wide spectrum of strain specificities and classification of two major binding groups among multiple binding patterns junctional adhesion molecule is a functional receptor for feline calicivirus alpha , -linked sialic acid acts as a receptor for feline calicivirus the role of m cells in salmonella infection sialic acid is required for nonspecific adherence of salmonella enterica ssp. enterica serovar typhi on caco- cells contribution of fimbrial operons to attachment to and invasion of epithelial cell lines by salmonella typhimurium binding specificity of salmonella plasmid-encoded fimbriae assessed by glycomics salmonella enterica serotype typhimurium std fimbriae bind terminal alpha( , )fucose residues in the cecal mucosa salmonella enterica serovar typhi uses type ivb pili to enter human intestinal epithelial cells multiple fimbrial adhesins are required for full virulence of salmonella typhimurium in mice salmonella enterica serovar typhimurium binds to hela cells via fim-mediated reversible adhesion and irreversible type three secretion system -mediated docking direct evidence that the fimh protein is the mannose-specific adhesin of escherichia coli type fimbriae carbohydrates as future anti-adhesion drugs for infectious diseases role of intimin and bundle-forming pili in enteropathogenic escherichia coli adhesion to pediatric intestinal tissue in vitro the lpf fimbrial operon mediates adhesion of salmonella typhimurium to murine peyer's patches initial adherence of epec, ehec and vtec to host cells long polar fimbriae of enterohemorrhagic escherichia coli o :h bind to extracellular matrix proteins pathogenomics of the virulence plasmids of escherichia coli thin pilus pilv adhesins of plasmid r recognize specific structures of the lipopolysaccharide molecules of recipient cells pilv adhesins of plasmid r thin pili specifically bind to the lipopolysaccharides of recipient cells fimh alleles direct preferential binding of salmonella to distinct mammalian cells or to avian cells role of lewis a and lewis b blood group antigens in helicobacter pylori infection chemical structures of the core regions of campylobacter jejuni serotypes o: , o: , o: , and o: lipopolysaccharides expression of the human cell surface glycoconjugates lewis x and lewis y by helicobacter pylori isolates is related to caga status gangliosides act as co-receptors for salmonella enteritidis flic and promote flic induction of human beta-defensin- expression in caco- cells inactivation of the flagellin gene of salmonella enterica serotype enteritidis strongly reduces invasion into differentiated caco- cells active invasion and/or encapsulation? a reappraisal of host-cell parasitism by cryptosporidium host cell surface sialic acid residues are involved on the process of penetration of toxoplasma gondii into mammalian cells synergistic role of micronemal proteins in toxoplasma gondii virulence atomic resolution insight into host cell recognition by toxoplasma gondii members of a novel protein family containing microneme adhesive repeat domains act as sialic acid-binding lectins during host cell invasion by apicomplexan parasites a novel pan/apple domain-containing protein from toxoplasma gondii: characterization and receptor identification the surface of toxoplasma: more and less targeted disruption of the glycosylphosphatidylinositolanchored surface antigen sag gene in toxoplasma gondii decreases host cell adhesion and drastically reduces virulence in mice toxoplasma gondii: the role of parasite surface and secreted proteins in host cell invasion the surface anti- gen sag mediates the attachment of toxoplasma gondii to cell-surface proteoglycans physical, structural, and functional properties of the beta integrin-like fibronectin receptor (beta ehfnr) in entamoeba histolytica entamoeba histolytica: lipid rafts are involved in adhesion of trophozoites to host extracellular matrix components a novel multidomain mucin-like glycoprotein of cryptosporidium parvum mediates invasion mediation of cryptosporidium parvum infection in vitro by mucin-like glycoproteins defined by a neutralizing monoclonal antibody cloning and sequence analysis of a highly polymorphic cryptosporidium parvum gene encoding a -kilodalton glycoprotein and characterization of its -and -kilodalton zoite surface antigen products toll-like receptor recognition of toxoplasma gondii signaling of immune system cells by glycosylphosphatidylinositol (gpi) anchor and related structures derived from parasitic protozoa impaired production of proinflammatory cytokines and host resistance to acute infection with trypanosoma cruzi in mice lacking functional myeloid differentiation factor induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of plasmodium falciparum: cell signaling receptors, glycosylphosphatidylinositol (gpi) structural requirement, and regulation of gpi activity activation of tlr and tlr by glycosylphosphatidylinositols derived from toxoplasma gondii tissue distribution of histo-blood group antigens genetic analysis of proteoglycan structure, function and metabolism microbial adherence to and invasion through proteoglycans biological roles of oligosaccharides: all of the theories are correct mucins in the mucosal barrier to infection the adherent gastrointestinal mucus gel layer: thickness and physical state in vivo a novel method for the visualization of the in situ mucus layer in rat and man colonic mucin: methods of measuring mucus thickness functional morphology of the mucosa of the small intestine n-glycosylation is required for the surface localization of muc mucin characterization of the oligosaccharides associated with the human ovarian tumor marker ca roles of calreticulin and calnexin during mucin synthesis in ls and ht /a human colonic adenocarcinoma cells kinetic modeling confirms the biosynthesis of mucin core (beta-gal( - ) alpha-galnac-o-ser/thr) o-glycan structures are modulated by neighboring glycosylation effects biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans integral membrane heparan sulfate proteoglycans glycolipids at the cell surface and their biological functions studies on the molecular mechanism of anion transport across the red blood cell membrane the composition and function of m cell apical membranes: implications for microbial pathogenesis m cells and the pathogenesis of mucosal and systemic infections epithelial m cells: differentiation and function glycoprotein (gp ): grabbing the fimh bacteria into m cells for mucosal immunity uptake and transport of intestinal macromolecules and microorganisms by m cells in peyer's patches -a personal and historical perspective structure and functon of intestinal mucosal eptihelium uptake through glycoprotein of fimh(+) bacteria by m cells initiates mucosal immune response bacterial adhesion and entry into host cells entry of microbes into the host: using m cells to break the mucosal barrier cellular routes of invasion by enteropathogens salmonella and produce: survival in the plant environment and implications in food safety neuraminidase inhibitors for influenza b virus infection: efficacy and resistance lipotechoic acid in lactobacilli: d-alanine makes the difference structure-function relationship of cytokine induction by lipoteichoic acid from staphylococcus aureus effects of dextran sulfates on the acute infection and growth stages of toxoplasma gondii gellan sulfate inhibits plasmodium falciparum growth and invasion of red blood cells in vitro this study was supported by grants-in-aid for young scientists, and scientific research on innovative areas ( ) from the ministry of education, culture, science, sports, and technology (mext) and for research on global health issues from the ministry of health, labour and welfare of japan, the program for promotion of basic and applied researches for innovations in bio-oriented industry (brain), the science and technology research promotion program for agriculture, forestry, fisheries and food industry, the naito foundation, and the program to disseminate tenure tracking system from the japan science and technology agency (jst). we thank mr. tatsuya iwanaga for his help with the illustrations.