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Latest reviews on EID show that nearly 75% of zoonotic EID have a wildlife origin.3, 5, 13–15 In fact, the number of EID events caused by pathogens coming from wildlife has increased during the past six decades.3 The majority of pathogens recorded were of viral origin.16 Therefore viral zoonoses of wildlife origin represent the most significant and growing threat to global health among all EIDs.3, 13
As anthropogenic activities have been identified as the cause of a significant majority of outbreaks,16, 17 it is essential to fully understand the mechanisms driving contacts between wildlife and the human population as well as species-jumping infections to set up public health information campaigns. On the contrary, efforts to conserve areas rich in wildlife diversity (13 National Parks were created in 2002 in Gabon) by reducing anthropogenic activity may have an added value in reducing the likelihood of future zoonotic disease emergence in these areas.3 EIDs in free-living wild animals can be classified into three major groups on the basis of key epizootiological criteria:18 (i) EIDs associated with ‘spill-over’ from domestic animals to wildlife populations living in proximity; (ii) EIDs related directly to human intervention, via host or parasite translocations; and (iii) EIDs with no overt human or domestic animal involvement. These phenomena have two major biological implications: first, many wildlife species are reservoirs of pathogens that threaten domestic animal and human health; second, wildlife EIDs pose a substantial threat to the conservation of global biodiversity, with for example the disappearance of the most great ape populations in protected areas in Central Africa after the 2002–2003 ebola virus outbreaks.19–22
The 2009 A(H1N1)pdm09 influenza pandemic, the SARS epidemic in 2003, and the recent emergence of a novel coronavirus are recent reminders of the global health threat posed by zoonotic viruses. Prior to widespread emergence in human populations, such pathogens can cause occasional infections in sub-populations that have been exposed to reservoir species (common reservoir species include for example bats, birds, swine, non-human primates). Whilst viruses causing such “spill-over” infections are usually poorly adapted for sustained human-to-human transmission, they are under strong selection pressure to increase transmissibility once in humans. If the reproduction number R (i.e., the average number of persons infected by a case) evolves to exceed 1, a large scale epidemic in humans may result. Over the last decade, particular concerns were raised regarding highly pathogenic H5N1 avian influenza, due to the high mortality rate seen in humans and the virus's rapid spread in avian populations. However, as the A(H1N1)pdm09 influenza pandemic demonstrated, H5N1 is not the only influenza virus that may pose a pandemic risk. Recently, a swine-origin triple reassortant influenza A(H3N2) variant virus has emerged in the United States, carrying the matrix gene (M) from the H1N1pdm09 virus (H3N2v-M)–. Studies in animal models have suggested that the presence of the H1N1pdm09 M gene may increase transmissibility of the virus,. From January 2012 to September 2012, 307 laboratory-confirmed H3N2v-M human infections were reported to Centers for Disease Control and Prevention (CDC) as opposed to 12 throughout 2011. The majority of cases have been associated with agricultural fairs but there are documented events of human-to-human transmission. The surge in cases observed in summer 2012 raised public health concerns. Threats from zoonoses are not limited to influenza: more than half of all recent emerging infectious disease events were zoonotic.
For efficient prevention and control, quantitative and rigorous assessment of the risks associated with emerging zoonoses is desirable—in particular the risk that an emerging pathogen evolves to cause sustained human-to-human transmission. One approach to such risk assessment is by monitoring the reproduction number R of zoonoses in humans, with an alarm being raised if R increases or approaches 1–. However, until now, estimating R required detailed outbreak investigations of human clusters, and suffered from three important limitations: (1) the resources, access, and expertise needed to conduct investigations is not always available; (2) the proportion of cases that are missed during outbreak investigations may vary by setting and be difficult to assess; (3) even if the study is complete, the data collection process can be affected by a selection bias whereby larger outbreaks are more likely to be detected so that estimates of transmissibility may be biased upward. Consider for example a scenario where R = 0.7, where each case has the same detection probability ρ = 1%, and assume that once a cluster is detected, detailed outbreak investigation ensures that all cases in the cluster are detected. With an average size of 18.3 and a 21% probability of 1-case cluster, clusters that are detected are substantially larger than normal ones (average size: 3.3; 65% probability of 1-case cluster) (Figure 1A). As expected, this selection bias leads to R being overestimated as illustrated for methods that use the distribution of detected cluster sizes (Figure 1B).
Here, we present a new approach to estimate R during spillover events, aiming to address many of the limitations of existing methods. We apply our approach to assess the human-to-human transmissibility of swine-origin influenza A variant (H1N1v, H1N2v, and H3N2v) virus, in particular that of the H3N2v-M virus, from US surveillance data for the period December 2005–December 2011. We also present applications to another zoonotic virus (Nipah virus in Malaysia and Bangladesh) as well as to a non-zoonotic pathogen (Vibrio Cholerae in the Dominican Republic).
The orthopoxviruses (family Poxviridae, genus Orthopoxvirus) are a diverse group of large, enveloped viruses that contain a covalently closed, double-stranded DNA genome of approximately 200 kbp. The genus is comprised of at least 10 recognized species. Several viruses within this group are significant human pathogens, including Variola virus (the causative agent of smallpox), monkeypox virus, cowpox virus, and Vaccinia virus. Other members, including raccoonpox, camelpox, ectromelia (mousepox), taterapox, and volepox viruses have only been isolated from their respective mammalian hosts. Although many orthopoxviruses specifically infect certain animal hosts, others (e.g., monkeypox and cowpox viruses) can also infect humans and are considered zoonotic pathogens. In humans, symptoms of orthopoxvirus infections range from mild skin lesions to fatal systemic disease. For example, smallpox produced a generalized rash that progressed from the papular to vesicular to pustular stages and resulted in a greater than 30% mortality rate in unvaccinated persons. Although naturally-occurring smallpox was eradicated nearly three decades ago, official stocks of the virus still remain in two locations, one at the U.S. Centers for Disease Control and Prevention in Atlanta, GA and the other at the State Research Center of Virology and Biotechnology, Novosibirsk, Russia. This, in addition to waning immunity against smallpox within the human population, has led to concerns that Variola virus might be used as a bioweapon.
Monkeypox virus causes a disease similar to smallpox in humans, but results in a lower fatality rate. Monkeypox virus is primarily transmitted to humans through direct contact with infected animals, generally various species of rodents or squirrels in the rain forests of central Africa. However, additional attention was brought to bear on this virus when, in the spring of 2003, it emerged for the first time in the Western Hemisphere and caused a cluster of cases in the U.S. Midwest.
Vaccinia virus is famous for being the vaccine that was used to eradicate smallpox. It was also the first animal virus to be purified and chemically analyzed and was the first to be genetically engineered. Despite its notoriety, however, its origin and natural history remain obscure. Recent evidence suggests that Vaccinia virus and horsepox virus are very similar phylogenetically and share a relatively recent common ancestor. Vaccinia virus is often confused with cowpox virus; although it is now well established that they are distinct virus species. In fact, cowpox virus is considered a zoonotic pathogen and is seen in a broad range of host species, most notable wild rodents, but rarely in cattle, as its name would imply. Interestingly, wild and domestic cats and elephants appear to be highly susceptible to infection with cowpox virus. Numerous recent studies have uncovered several novel vaccinia-like viruses that have caused zoonotic outbreaks in Brazil,,,,,,. It is likely that as various ecological niches are examined further, even more species of Orthopoxvirus will be identified.
Several genus-specific assays have been described for the detecting and discriminating various Orthopoxvirus members that require conventional PCR followed by restriction endonuclease digestion and subsequent gel electrophoresis,. More recently, several real-time LightCycler-based PCR assays have been developed for pan-Orthopoxvirus or specific orthopoxvirus species detection,,. For these assays, differentiation of the various orthopoxvirus species requires the use of different TaqMan probes in separate reactions or melt-curve analysis of hybridization probes. Here, we describe a rapid, high-throughput, multi-locus method for identifying orthopoxviruses based on PCR amplification followed by electrospray ionization mass spectrometry (PCR/ESI-MS) performed on the Ibis T5000 instrument,,,. This technology has been applied to detection and identification of other viral pathogens, including alphaviruses, influenza viruses, adenovirus, and coronaviruses. The assay described here is extremely sensitive and able to detect and identify each species from a diverse collection of orthopoxviruses.
The recognition that the AIDS pandemic originated as a simian retrovirus transmitted to humans has increased public health concerns about the risk that humans become infected by other pathogens prevalent in NHP. The human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2), etiological agents of AIDS that cause about 1 to 2 million annual deaths, have been linked to cross-species transmission of simian immunodeficiency virus (SIV) from chimpanzees (Pan troglodytes) and sooty mangabeys (Cercocebus atys) (1). Humans might have been infected with SIV either by NHP hunting and wild meat consumption or by keeping infected NHP as pets (2). In the past decades, viruses as deadly as rabies, Herpes B virus, Marburg and Ebola viruses were transferred from NHP to humans. It is likely that during centuries and until recently, the main route of simian pathogen transmission to human was NHP hunting and wild meat consumption (3).
An ideal animal model for the study of a human disease is one which utilizes a route of infection that mimics the natural transmission of the pathogen; the ability to obtain disease with an infectious dose equivalent to that causing disease in humans; as well having a disease course, morbidity and mortality similar to that seen with human disease. Additionally, the animal model should have a mode(s) of transmission that mimics human cases. Factors which subsequently allow more detailed inferences about disease pathogenesis include the availability of reagents to evaluate host innate and adaptive immune responses to the pathogen, and histopathological changes in the host which result from infection or the host response to infection. These findings can then be compared to what is known of human disease. The utility of a small animal model of human disease for study of therapeutic efficacy is augmented when large numbers of animals are available for use in appropriately, well-powered studies. Even if all aspects of an animal model of disease are not completely faithful to what is known of human disease, important information regarding therapeutic efficacy can be gleaned from their use in “pre-clinical” studies.
The published literature on clinical manifestations of systemic human orthopoxvirus disease is derived from historic literature descriptions of human smallpox and more recent descriptions of human monkeypox disease. The clinical-descriptive literature on human monkeypox is expected to grow in the next five years, as data acquisition and analysis from an ongoing study in the Democratic Republic of Congo is finalized. Currently available literature is largely derived from WHO-sponsored surveillance efforts in West Africa and the Congo Basin in the 1980s, after the first recognition of human disease in these areas, and subsequent analyses of public health response data and human research studies following the introduction of West African clade virus into the U.S. in 2003. Human monkeypox, as described through the active surveillance and case ascertainment studies sponsored by WHO in the 1980s, was depicted as resembling discrete ordinary smallpox. In natural human infection, exposure leading to infection is believed to occur via a respiratory route, with subsequent progressive viremias/lymphemia, ultimately leading to seeding of the skin to generate a generalized rash. Percutaneous exposure, also leading to generalized rash formation, has also been described for both viral infections. The disease pathogenesis has been conjectured and modeled largely from animal studies; initial models were using ectromelia infection of mice; some kinetic observations of virus shedding and viremia have been made in human studies of smallpox and monkeypox. The time course of disease is generally thought to include an asymptomatic phase of 10–12 days, during which time the virus initially enters the host, replicates, seeds reticuloendothelial organs, replicates, then spreads via the bloodstream (inducing a febrile response) which is the first symptomatic hallmark of disease. The fever is usually described as occurring 10–12 days post initial exposure/infection. The range has been 7–17 days. Fever is accompanied by other symptoms, including headache, backache, myalgias, and or abdominal pain. Two to three days following the fever, rash develops—initially presenting as a macular, then papular, then vesicular and pustular eruption. Scabbing then begins. Each stage of rash lasts 1–2 days. Approximately 2–3 weeks post initial symptoms, scabs begin to separate from the skin. Death and disease severity have had some correlation with rash burden in epidemiologic studies of hospitalized smallpox patients. Severe outcomes are more frequent in unvaccinated, younger age groups; death occurs within the first week of illness in cases with hemorrhagic manifestations, and during the second or third week of illness in “ordinary” cases. In the human monkeypox cases studied in Zaire/DRC between 1981–1986, of the 33 deaths among 338 patients, all occurred in unvaccinated children less than eight years of age. Death occurred during the first week of illness in 21%, the second week in 52%, and the third week of illness in the remaining 27%.
The development of small animal models for the study of monkeypox virus (MPXV) has been quite extensive for the relatively short period of time this pathogen has been known. Initial animal models were designed to address natural history in potential or surrogate reservoir host species, as well as studies of disease in primates. Routes of exposure were designed to evaluate disease if respiratory or percutaneous exposures occurred, or in some cases to simply address whether virus would replicate in the animal model system. Factors that influence the outcome of a challenge study include the age of the animal at time of infection, inoculation route used, and the viral dosage given. Additionally, the strain of MPXV (currently delineated as belonging to Congo Basin clade or West African clade) used in the study may influence the disease severity.
Vaccinia virus (VV) is a member of the Poxviridae, which constitute a large family of enveloped DNA viruses and replicate entirely in the cytoplasm of the infected cells with a linear double-stranded DNA genome of 130–300 kilo base pairs. Poxviruses have a broad range of eukaryotic hosts including mammals, birds, reptiles and insects and can grow in many cell lines in vitro. Some poxviruses are causative agents of human diseases. Variola virus caused a deadly human disease smallpox until its global eradication in 1977, in which VV was used as a vaccine. Other poxviruses causing human diseases are molluscum contagiosum virus and the zoonotic monkeypox virus. Notably, variola and monkeypox viruses are transmitted to humans by respiratory route, whereas molluscum contagiosum virus is mainly transmitted through the skin. Variola and monkeypox viruses cause systemic infections with high levels of lethality, but the details of their pathogenesis are not well-understood.
Intranasal inoculation of different VV strains in mice shows different levels of virulence and only neurovirulent strains cause lethality. Western Reserve (WR) strain was generated by intracerebral mouse passages, and an intranasal inoculation results in an acute infection of the lung followed by dissemination of the virus to various organs. Intranasal infection with a low dose of WR strain induces an inflammatory infiltrate in the lung, and the virus was cleared 10 to 15 days after infection; however, infection with a high dose of WR strain caused lethality, which has been used as a challenge model to study the effect of antiviral drugs, immune IgG, soluble viral proteins and other vaccine strains. In one report intranasal infection with the WR strain caused pneumonia showing severe alveolar edema and acute necrotizing bronchiolitis and peribronchiolitis as well as neutrophilic infiltrates in the interstitium of the lung. The mechanisms of lethality in mice infected with the lethal dose of WR strain are, however, not well-understood.
In this study, we focused on the differences in virus replication and host immune responses between lethal and non-lethal respiratory infections with VV. We used two VV strains; neurotropic virulent WR strain and the less virulent Wyeth strain. Although BALB/c mice are frequently used for intranasal challenge of vaccinia virus, we used the C57BL6/J strain of mouse in these experiments for two reasons. One is that most knockout mice lacking genes involved in immune responses have been made with C57BL6/J genetic background. The other is that we and one other group have characterized cellular immune responses, especially CD8+ T cell responses, to vaccinia virus in C57BL6/J mice, when this study was planned. Infection of C57BL/6J mouse with a high dose (106 p.f.u. (plaque-forming units)) of the WR strain was lethal, whereas a high dose (106 p.f.u.) of Wyeth strain and a lower dose (104 p.f.u.) of WR strain were not lethal. The WR strain replicated and produced higher titers of virus in the lung and the brain compared to the Wyeth strain. There was, however, no difference between the virus titers in brains of mice infected with the high or low dose of WR strain. Lethal infection with WR strain resulted in fewer lymphocytes and an altered phenotype of T cells in the lung compared to non-lethal infection and uninfected controls, and induced severe thymus atrophy with a marked reduction of CD4 and CD8 double positive (DP) T cells.
Smallpox was eradicated worldwide in the 1970s. However, serious public health concerns due to the threat of bioterrorism and natural outbreaks of monkeypox at the start of the 21st century have highlighted the necessity for a vaccinia virus (VV)-based smallpox vaccine. Existing vaccine stockpiles have not been updated since the 1970s; because these early vaccines are lymph-derived vaccines produced by propagating vaccine viruses in the skin of animals (i.e., first-generation vaccines (Table 1)), they do not meet good manufacturing practice (GMP) standards. Therefore, they are at risk for adventitious microbial contamination. Moreover, these vaccines occasionally caused serious adverse effects (e.g., progressive vaccinia, eczema vaccinatum and post-vaccinial encephalitis) due to the pathogenicity of the vaccine viruses used.
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-1 and HIV-2 (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 host–pathogen equilibrium is a major challenge that differs substantially for each successful interspecies transmission and subsequent spread of the virus.
Through systematic evaluation of data reported in the scientific literature on zoonotic viruses, we identify several key virus characteristics and transmission mechanisms that are synergistic to zoonotic virus spillover, amplification by human-to-human transmission, and global spread. The majority (94%) of zoonotic viruses described to date (n = 162) are RNA viruses, which is 28 times higher (95% CI 13.9–62.5, exact P < 0.001) than the proportion of RNA viruses among all vertebrate viruses recognized, indicating that RNA viruses are far more likely to be zoonotic than DNA viruses, as has been reported among human pathogens6. Epidemiological circumstances involved in recent zoonotic transmission from animals to people are summarized here for 95 viruses with data on human activities enabling direct and indirect contact disease transmission and animal host taxa implicated in transmission. In general, wild animals were suggested as the source of zoonotic transmission for 91% (86/95) of zoonotic viruses compared to 34% (32/95) of viruses transmitted from domestic animals, and 25% (24/95) with transmission described from both wild and domestic animals (see Supplementary Table). Wild animals, which include a taxonomically diverse range of thousands of species, were significantly more likely to be a source for animal-to-human spillover of viruses than domesticated species (exact P = 0.001). Wild rodents were implicated as a source of spillover for 58% (55/95) of zoonotic viruses, particularly for zoonotic arenaviruses (n = 8/8, exact P = 0.019) and zoonotic bunyaviruses (n = 20/24, exact P = 0.004). Primates were implicated as a source of zoonotic retroviruses (exact P = 0.017), while bats were more implicated for zoonotic paramyxoviruses (exact P = 0.011) and most zoonotic rhabdoviruses (6/8, exact P = 0.002).
Emerging pathogens have been noted for their ability to infect a range of animal hosts578910. We find that most (63%) zoonotic viruses infecting humans were reported in animal hosts from at least two different taxonomic orders, and 45% were reported in four or more orders, in addition to humans. The virus-host unipartite network illustrates high connectivity among host groups sharing zoonotic viruses and the central role domestic animals play in cross-species transmission (Fig. 2). In a Poisson model predicting host range and evaluating common hosts and high-risk transmission interfaces, viruses with domestic animal hosts occurred in twice as many host orders than other viruses (Table 1). Most domestic animal groups clustered in the middle of the host network with high centrality measures and a high number of shared viruses (Fig. 2), indicating that domestic animals play a key role in cross-species transmission of zoonotic viruses. Among viruses from wildlife, we found higher host plasticity (ie, hosts from a higher number of taxonomic orders) in viruses transmitted at high-risk interfaces involving wild animals kept as pets, maintained in sanctuaries or zoos, and sold at markets, which were collapsed into one category due to similar effect and significance in the final Poisson model. We also found that vector-borne viruses were reported in three times the number of host taxonomic groups than non-vector-borne viruses, indicating that vector-borne pathogens have significantly broader host range than non-vector-borne viruses.
Based on data published to date, transmission of zoonotic viruses to humans occurs by direct or indirect contact with wildlife in a diverse array of interconnected animal-to-human interfaces, with little overlap with viruses transmitted primarily by vectors (Fig. 3). Zoonotic virus spillover from wildlife was most frequent in and around human dwellings and in agricultural fields, as well as at interfaces with occupational exposure to animals (hunters, laboratory workers, veterinarians, researchers, wildlife management, zoo and sanctuary staff). Primate hosts were most frequently cited as the source of viruses transmitted by direct contact during hunting (exact P = 0.051) and in laboratories (exact P = 0.009), while rodent hosts were more likely to be implicated in transmission by indirect contact in and around human dwellings (exact P < 0.001) and in agricultural fields (exact P = 0.001). Approximately 40% of zoonotic viruses involving wild animals required arthropod vectors for transmission to humans, with vectors providing an effective bridge for transmission of diseases from wild animals that do not normally contact humans. Zoonotic viruses with wild avian hosts were most likely to involve vectors (exact P < 0.001). Network analysis of disease transmission from wild animals illustrates that vector-borne viruses were the least connected to other transmission interfaces (Fig. 3), consistent with effective control of vector-borne diseases by elimination of vectors or contact with vectors. In contrast, 22% of viruses transmitted from domestic animals to humans were by vector only, with close proximity interactions with domestic animals enabling direct pathogen transmission to humans.
Once animal viruses have spilled over into humans, human-to-human transmission of zoonoses facilitates sustained spread of disease with a rapidity and reach infeasible for zoonotic viruses requiring contact with animal hosts for each transmission opportunity. Human-to-human transmissibility was described for 20% of zoonotic viruses investigated here (Supplementary Table). We find virus host plasticity to be positively correlated with capability for human-to-human transmission (Table 1). In a logistic regression model predicting virus capability for human-to-human transmission, we find viruses were significantly more likely to be human-to-human transmissible with each increase in virus host plasticity (count of host orders and ecological groups). Furthermore, we find viruses in the arenaviridae and filoviridae families to be more likely to possess human-to-human transmissibility, along with viruses transmitted by direct contact with hunted and consumed wildlife (Table 1). Hunting poses special risk for cross-species disease transmission of blood-borne zoonotic viruses1112 as evidenced by re-emerging threats, including ebolaviruses13 and primate retroviruses141516. Our findings therefore support speculation that hunting of high-risk host species carries an increased probability of spillover of zoonotic viruses that can be further spread by human-to-human transmission13.
We further characterized zoonotic virus capacity for spread by categorizing viruses according to geographic range in a single country (16%), >1 country in 1–3 World Health Organization-defined (WHO) regions (55%), or ≥4 WHO regions (29%), and used ordinal logistic regression to evaluate characteristics of viruses in broader range categories. We find viruses were more likely to be in broader geographic range categories with increasing host plasticity (Table 1). Among all high risk interfaces and hosts, only viruses transmitted to humans by contact with wild animals in the wildlife trade and in laboratories, such as lymphocytic choriomeningitis virus17, monkeypox virus18, herpes B virus19, and Marburg20, were more likely to have broader geographic reach.
Poxviruses are double-stranded DNA viruses with large genomes (up to 300 kb) belonging to the family Poxviridae. The family is divided into the invertebrate-infecting entomopoxvirinae and chordate-infecting chordopoxvirinae. The latter subfamily is further divided into ten genera and contains many important infectious agents of both animals and humans. The now-eradicated Variola virus (VARV, the causative agent of smallpox) illustrates the potential consequences of poxvirus infections having arguably caused more deaths in human history than any other infectious agent. Aside from humans, chordopoxviruses are also found in a multitude of terrestrial, aquatic and arboreal animal species from diverse taxa e.g., crocodiles, sea lions, birds, camels, etc. and many poxviruses are capable of infecting multiple host species and cause cross-species (including zoonotic) infections. For example, monkeypox virus has been recognized as a zoonotic agent since the 1970s and is classed a bioterrorism agent. Further to human disease burdens, cross species infections of poxviruses between non-human species can also have devastating consequences e.g., the near-extinction of red squirrels in the UK after the introduction of squirrelpox with grey squirrels from the USA. Owing to the significance of these zoonotic and cross-species poxvirus infections, poxvirus host range is a key area of research.
Poxviruses exhibit a heterogeneous host range with some poxviruses having a very broad host range (e.g., cowpox infects rodents, dogs, cats, horses, cows, primates including humans), and others being very specific (e.g., VARV is a human only pathogen). Although some poxvirus genera are known to exhibit broad host tropisms (e.g., orthopoxviruses) and are consequently thought to manifest greater zoonotic risks, phylogenetic relatedness among viruses is not indicative of poxvirus host range. In fact, determinants of poxvirus host range are poorly understood and viral tropism is not typically restricted at the level of cellular entry. Due to highly conserved virion proteins, most poxviruses can enter a wide variety of host cell types, with restriction of infection occurring downstream of entry (either through a lack of host factors or through the innate immune system). Consequently, changes in poxvirus host range are typically determined by changes in virus genome complement (e.g., gene duplication/gain/loss) that allow for subversion of host restriction rather than point mutations, as is the case for some viruses e.g. parvovirus and influenza. Genes that are known to cause shifts in poxvirus host range generally have functions relating to the interplay of the host innate immune mechanisms with the virus. These genes are termed poxvirus host range genes and although approximately 15 have already been identified, more work is needed to fully understand their restriction mechanisms and to identify novel determinants of poxvirus host range.
Bats are an ancient, highly diverse order of mammals that are known to be reservoirs for a large number of viruses. “Bats” is the collective term for some approximately 1200 species of mammals thought to have diverged some 50 million years ago (mya; comparatively humans and great apes are thought to have diverged ~5 mya). Second only in diversity to rodents, bats are subdivided into two suborders, commonly called megabats and microbats, on the basis of behavioral and physiological traits as well as molecular evidence. There has been a recent increase in interest regarding the relationship of bats with viruses (Figure 1) as some species of bats are reservoir hosts for lethal viral zoonoses such as SARS coronaviruses, paramyxoviruses (e.g., Nipah and Hendra viruses), and filoviruses (e.g., Ebola and Marburg virus) and numerous lyssaviruses. Outbreaks of disease attributable to bat-related zoonoses have high economic and human costs and their discovery has resulted in concerted research effort to isolate and characterize viruses from bat populations. Consequently, large numbers of previously unknown viruses have since 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 ≥58 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.
Transmission via the food- and waterborne route is a common mode of spread of a wide range of viruses. Many commonly recognized food- and waterborne infections are caused by viruses that are transmitted by the fecal-oral route. Particularly caliciviruses (norovirus, sapovirus) can cause diarrhea and vomiting and less commonly astroviruses, rotaviruses, and adenoviruses (Newell et al., 2010). Other viruses cause symptoms resulting from extra-intestinal spread, like hepatitis A (HAV), and hepatitis E (HEV). High levels of viral shedding through stool and vomit lead to dispersal in the environment. Moreover, the stability of many food- and waterborne viruses allows for prolonged persistence in the environment. Food- and water associated transmission is also suspected to enhance the spread and emergence of zoonotic viruses (e.g., Middle East Respiratory Syndrome-coronavirus and Nipah virus) and facilitates the occurrence of zoonotic events though the handling of bushmeat (Ebola virus) (Wolfe et al., 2005; European Food Safety Authority, 2014; Mann et al., 2015).
Challenges of detecting viruses transmitted by the food- and waterborne route are their diversity and the frequent secondary person-to-person transmissions, which may mask an initial food- or waterborne introduction. In addition, there is a lack of awareness among clinicians (Beersma et al., 2012), as the symptoms caused by foodborne viruses are not specific to the viruses causing the illness. Furthermore, there is limited coverage in surveillance of food- and waterborne viral disease, hampering detecting and tracing (Ahmed et al., 2014; Verhoef et al., 2015).
In the past years, high-throughput sequencing technologies have increased the ability to measure genomic material from diverse samples tremendously. These methods will most likely continue to improve in the future (Aarestrup et al., 2012). Specifically, metagenomic analysis using untargeted sequencing has received a lot of attention, because the high throughput of current sequencing technologies has made it possible to obtain multiple high coverage genomes from highly complex samples (Cotten et al., 2014; Smits et al., 2015). Even though it is still a developing field, metagenomics is starting to become mature enough for applications outside of the research environment.
With the development of multiplex real-time polymerase chain reaction (RT-PCR) protocols came the realization that unraveling etiologies of main disease syndromes is more complex than previously recognized. This led to questions about the detection of viruses for which the role as causes of illness remains to be evaluated, the importance of co-infections and recognition of less common disease etiologies (Binnicker, 2015). Similarly, high throughput metagenomic sequencing broadens the scope of detectable viruses, which, apart from making it more complex, make us further understand the role of viruses in health and disease. The biggest promise, however, is that of routine application of metagenomic sequencing in diagnostic context, facilitating viral detection and offering huge potential for tracing of viruses in (foodborne) outbreaks.
The Middle East respiratory syndrome coronavirus (MERS) is a zoonotic pathogen that has caused recurrent spillovers in the human population since March 2012. A total of 1,047 laboratory-confirmed cases of infection with MERS including 460 deaths have been reported in Saudi Arabia alone as of 15 July 2015; the concentration of human infections in this region is thought to be linked to the local population of dromedary camels, which may serve as an intermediate host for MERS [3, 4]. The human-to-human transmission potential of MERS is thought to be subcritical [1, 5, 6], although there is occasional amplification in the healthcare setting [5, 7–11]. While sporadic importations of MERS to Europe, Africa, Asia, and North America via returning travelers from the Middle East had not sparked local outbreaks until recently, a single importation into South Korea on 4 May 2015 triggered the largest cluster of cases outside the Middle East to date. The index patient was a 68-year-old businessman who visited several countries in the Middle East before returning to South Korea via Qatar [13, 14], where he developed respiratory symptoms on 11 May 2015. An accurate diagnosis of MERS was not established until 20 May 2015, after the index patient had sought treatment in several different healthcare facilities. A total of 186 MERS infections in South Korea have been linked to the healthcare facilities visited by the index patient and subsequent infections. As a result of this large cluster, more than 6,000 contacts have been monitored in South Korea [13–15].
Although large-scale community transmission has not been reported for MERS, large hospital clusters are not infrequent and can amplify transmission, which aligns with the transmission characteristics of severe acute respiratory syndrome (SARS), a related coronavirus that sparked global concern in 2002–2003 [10, 11, 16, 17]. Coronaviruses associated with both syndromes have high affinity to the lower respiratory tract and cause severe pneumonia [18–20], particularly among older adults with underlying medical conditions [21, 22]. Both viruses are thought to be associated with some degree of transmission heterogeneity, indicating that super-spreading events are expected [23, 24].
While the individual heterogeneity of MERS (i.e. variation in the transmissibility by individuals) has been explored recently [25, 26], here we focus exclusively on hospital outbreaks, where transmission is amplified. Further, we provide the first head-to-head comparison with SARS and carry out a comparative analysis of the transmission characteristics and exposure patterns of previously reported hospital clusters of MERS and SARS to assess the unexpected nature of the recent South Korean nosocomial outbreak and estimate the probability of future large hospital clusters.
Adult female C57BL6/J mice were inoculated with various doses of the WR and Wyeth strains intranasally. Weight change and survival of infected mice were recorded daily. Inoculation with higher doses (106 p.f.u. and 105 p.f.u.) of the WR strain induced rapid and severe weight loss, which became obvious at 3 days post-infection (Fig. 1A), and most of mice died at 7–10 days post-infection (with 106 p.f.u. all mice died by 8 days post-infection) (Fig. 1B). Lower doses (104 p.f.u. and 103 p.f.u.) of the WR strain caused mild weight loss, and all mice survived. These mice recovered their weight after 6–8 days post-infection (Fig. 1A). The 50% lethal dose (LD50) of WR strain was calculated as 4.2 × 104 p.f.u., which is similar to the LD50 reported for BALB/c mouse. Wyeth strain did not kill mice (Fig. 1B) or cause weight loss (Fig. 1A) even when 106 p.f.u. of the virus was inoculated.
For scenario 2, we denote the number of clusters that are investigated by M and the number of first detected cases of each cluster that were infected by the reservoir by m.
The likelihood is:(3)Estimation is performed conditional on overdispersion parameter, k, and the case detection rate, ρ. We also derive bounds on R when k and ρ are unknown (see Text S1).
To account for small sample sizes, we compute the bootstrap mean estimate and the bootstrap 95% confidence intervals. We compare these estimates with those obtained under asymptotic conditions (i.e., maximum likelihood and likelihood-ratio confidence intervals) and find that they are similar (Figures S2, S3, S4; Tables S1, S2, S3; Text S1).
For scenario 1, 1−G is an unbiased point estimate of R. A simple binomial likelihood function with probability 1−R can be used to derive confidence intervals for R. These intervals capture uncertainty arising from sampling; but may underestimate other sources of uncertainty if the total number of chains of transmission (both detected and undetected) in the study population remains small (see Text S1).
There used to be a criticism of diagnostic virology that by the time the result was known the patient was either dead or better. That it could be of practical use is illustrated by the following episode. In the 1960s, before smallpox had been eradicated from the world, an adult male was found in the busy out-patient department (OPD) of a major London hospital late on a Friday where he had been all afternoon. He said he had just returned from East Africa where smallpox was still endemic and claimed that he had had chickenpox as a child and that he had not been vaccinated. The staff of the OPD were faced with a man with a widespread vesicular skin rash and a slight fever and a dilemma—was this a case of smallpox? Should they alert the media to warn everyone who had been in the OPD to contact their own doctor over the weekend, or was there a way to defuse the situation quickly? One of us (DM), as an electron microscopist at a different London hospital, was telephoned to ask if a quick diagnosis could be made. After advice about collecting specimens, a taxi arrived soon afterwards and a doctor from the other hospital got out, carrying a small syringe containing fluid aspirated from the patient’s vesicles as if it was an unexploded bomb.
In the electron microscopy (EM) laboratory, ten minutes later numerous herpesvirus-like particles were seen in the specimen and everyone could relax. Despite the patient’s claim to have had chickenpox in childhood, it was clear that chickenpox was what he now had. No national alert was necessary, with all the widespread anxiety that would have followed. The speed and certainty of the diagnosis by EM offered a lesson that still has relevance.
Four valuable lessons can be drawn from this episode: (1) That a useful diagnosis can be made in minutes from the arrival of the specimen; (2) Diagnostic EM (DEM) required no specific reagents (such as primers or antisera) or special equipment. All that is required was an electron microscope, microscope support grids, stain and a competent virologist familiar with the appearance of relevant viruses; (3) That seeing is believing—knowing what the causative virus looks like is a useful confirmation for any other tests added later; (4) That other, more serious, causes could be discounted.
With concerns over the possible re-emergence of poxvirus infections, either through the spread of existing animal viruses to susceptible humans, survival and escape of old material, molecular manipulation and synthetic biology or even bioterrorism, there will be a need to provide accurate and quick diagnosis. This paper presents the possibilities and advantages of DEM and how DEM can help with organised future preparedness.
As shown in Figure 2, all of the four primer pairs produce amplicon that can distinguish Variola virus from any other orthopoxvirus. Primer pair VIR979 could resolve Variola major virus from Variola minor virus and camelpox strain CMS from strain M-96. Primer pairs VIR982, VIR985 and VIR988 could resolve the two strains of monkeypox tested: VR-267 and Zaire-96-1-16. All of the vaccinia isolates tested including rabbitpox and horsepox, which are sub-species of vaccinia, have a common base count signature for primer pair VIR9888 ( 34A, 16G, 19C, 30T). Vaccinia, Copenhagen strain and horsepox (gi|111184167) can be distinguished from each other and other vaccinia isolates based upon their unique base-count signatures for primers pairs VIR985 and VIR979.
In addition to providing sub-species resolution, the use of four PCR primers in the assay enabled the detection and identification of an orthopoxvirus at the stochastic limit of PCR: 4–8 copies/PCR reaction. For example if only 4–8 genomes/PCR are used in the assay there is a very high probability that at least one of the four primers would detect the virus and provide information sufficient for its identification.
The assay is specific to Orthopoxvirus. Nucleic acid extracts from the blood of non-infected rabbits (N = 4) and humans (20 ng and 500 ng/PCR reaction) failed to produce an amplicon other than the internal positive controls. As expected, swinepox, a suipoxvirus (ATCC VR-363), in the family Poxviridae failed produce an amplicon other than the internal positive controls using the assay. We further tested a panel of DNA viruses in the assay to further define specificity including HSV1, adenovirus types 1, 5, 8, 4, 7A, varicella zoster virus (VZV), HPV16 & 18, human parvo virus B19, BK virus and JC virus. None of these viruses cross-reacted using the assay.
In Central Africa, Asia and Latin America, wildlife is the primary source of meat for low-income people living in rural areas (4–6). The practice of NHP hunting is part of the culture; it has been happening for centuries and the sale of wild meat is considered legal in many countries despite being illegal in some. Even in France, in the French Guiana two species of NHP, the howler monkey (Alouatta maconnelli) and the squirrel monkey (Saimiri sciureus), are allowed on the hunt but prohibited for sale (7). This results in regular close contacts between animal carcasses and hunters as well as between raw meat and women who prepare food. The meat is usually cooked before being shared with children (8). Most recently, illegal hunting for wild meat consumption or traditional medicine, also known as the bushmeat trade, as well as extermination of wild animals by troops foraging for food during wars have accelerated the NHP populations decline. The impact on NHP populations varied from lightly to heavily hunted. Human predation went hand in hand with an increase in contacts between NHP carcasses and humans. Fa and collaborators (9) calculated that more 150,000 carcasses of NHP per year are traded in Nigeria and Cameroun. NHP meat in Congo basin's local market is a cheap source of food (10) (Figure 1). Although wild meat consumption is associated with an increased risk of acquiring zoonotic diseases, people eating NHP ignore or express indifference to the risk of contracting simian pathogens, mainly because their own experience suggests that they can do it without incident. Even when governments imposed a ban on the hunting and consumption of wild meat after the 2013 to 2016 outbreak of Ebola virus in West Africa, the trade and consumption of NHP meat were not deeply affected (14). Over the past decade, the washing-based Bush Meat Crisis Task Force has regularly reported alarming information about wild animals being harvested for food in the Congo basin every year (15). A study in Liberia reported that 9,500 NHP are trade annually on the Liberia-Ivory Coast wild meat markets. According to journalists from the Guardian, there has been a massive chimpanzee decline in DRC due to hunting, with more than 400 chimpanzees being slaughtered each year. The hunting of gorillas and chimpanzees by poachers in Cameroun was also reported by the Ape Action Africa in Mefou. However, in some tribes, women refuse to eat or cook ape as it goes against their beliefs. The consumption of NHP meat is not limited to people living in poverty in Central Africa, Asia, or Latin America; wealthier households consumed only slightly less wild meat than others. Spider monkey dishes are popular in Southern Mexico. Although currently banned from dishes, NHP brain has long been viewed as a prized delicacy in Asia. The CITES/GRASP (16), reported that in Indonesia orangutans could be purchased for $100 and that some restaurants prepare dishes containing orangutan meat if specifically requested by customers.
Emerging infectious disease (EID) epidemics and pandemics arise without warning, even with global efforts aimed at tracking pathogens early and at the source, a fact most recently evidenced by the swift global spread of influenza H1N1, and a current outbreak of ebolavirus affecting multiple West African countries simultaneously. Most major human EIDs are of zoonotic origin and include viral infections of both global (HIV-1, HIV-2, H1N1) and localized significance (ebolavirus, monkeypox, Marburgvirus, Nipah virus, severe acute respiratory syndrome [SARS]-associated coronavirus). Systematic monitoring of people and wildlife at hotspots of EID is one strategy for preventing human pathogens of animal origin from reaching a pandemic state. By detecting animal pathogens before or just as they emerge in humans, it may be possible to mitigate against their worldwide spread. Furthermore, in the case of some diseases such as Ebola virus disease (EVD), the monitoring of wildlife disease serves as a critical component of early warning systems aimed at preventing the transmission of zoonotic diseases to humans,. EVD has repeatedly passed from infected apes to hunters, leading to multiple epidemics and 360 human deaths (463 cases) in Gabon and the Republic of Congo (RoC) alone since 1994,–. More significantly, human epidemics are often preceded by observed animal outbreaks, underlining the human health implications of surveillance and control of epizootics,.
The International Union for Conservation of Nature (IUCN) currently lists the western lowland gorilla (G. gorilla gorilla) as critically endangered and cites infectious disease as one of the top two threats to this species. Ebolavirus is lethal in humans and nonhuman primates and has been described as a significant threat to the survival of western lowland gorillas and chimpanzees (Pan troglodytes) in Central Africa,,. Data from ecological surveys in Central African ape habitats illustrate declines in ape signs (nests, feces, prints) temporally and spatially linked with confirmed human EVD outbreaks–. Mathematical modeling suggests that, between 1983 and 2000, gorilla numbers in Gabon dropped by more than 56%, and it is hypothesized that infectious pathogens, including ebolavirus and Bacillus anthracis, may contribute to gorilla mortality in Africa,,.
Despite the significance to both human and wildlife health, direct evidence of great ape exposure to ebolavirus or other pathogens (either by pathogen or immune response detection) is scant, complicating our ability to monitor epizootics. Therefore, to fill this gap, there is a need for prospective epidemiologic studies combining ecological data with laboratory screening. Most currently available data regarding primate pathology and immune response comes from experimentally infected laboratory macaques,.
In direct response to the challenges associated with collecting blood or tissue from wildlife, non-invasively collected biological samples such as feces have been used for wildlife disease screening,. Primate feces have been screened for the presence of viral nucleic acids due to shedding of simian immunodeficiency virus (SIV), circoviruses, enteroviruses and hepatitis viruses–. For SIV, feces have also shown the presence of virus-specific antibodies. We developed a non-invasive immunological assay to detect ebolavirus antibodies in great ape feces, allowing us more insight into wild ape ebolavirus infections and their surveillance, and leading the way to identifying the best approaches for their protection. In addition, this new assay may prove valuable in the development and employment of prospective epidemiological ebolavirus studies in wild great ape populations.
The major source of anxiety over unexpected skin infections of unknown aetiology will be smallpox because of its possible fatal outcome and potential to cause a serious epidemic and panic in the community. Smallpox, caused by the variola virus, has been eradicated as an endemic pathogen from the world’s population since 1980 but stocks of the virus remain under WHO supervision under high-security at the CDC (Centers for Disease Control & Prevention), Atlanta, USA, and at VECTOR (State Research Center of Virology and Biotechnology), Koltsovo-Novosibirsk, Russia, for further research. Nevertheless, some variola virus may yet remain elsewhere, forgotten in a deep-freeze or as dried crusts, and escape, or a virulent variant may be generated through laboratory molecular manipulation. Other viruses also cause vesicular lesions which may mimic smallpox. In addition, other micro-organisms (including several virus families) or some non-infective conditions may cause similar skin lesions as listed in Table 1 and Table 2.
The poxvirus family comprises two subfamilies: the Chordopoxvirinae, specific for the vertebrates, and the Entomopoxvirinae, specific for insects. Consequent to the progress in molecular techniques, chordopoxviruses are currently classified into 10 genera, with the genus orthopoxviruses (OPV) being most relevant for man and many higher animals (for reviews see:).
The OPV include—besides the variola virus (VARV) itself, which is now extinct in the field—vaccinia virus (VACV), the virus originally used by Jenner late in the 18th century to vaccinate against smallpox and which may have evolved from cowpox and horsepox viruses. The widely used VACV “escaped into the wild” and variants of VACV are now globally endemic and are known as buffalopox virus (BPXV). As well as these, there are poxviruses native to animal species, some of which may infect man but without causing an epidemic. These less pathogenic OPV, as well as VACV, include cowpox virus (CPXV), monkeypox virus (MPXV) and camelpox virus (CMLV). All of them exist in distinct clades and can cause small, regional zoonotic outbreaks even with secondary and tertiary transmissions. Other members of OPV such as ectromelia virus (mousepox), squirrel- and volepox virus have not been shown to cause infections in humans. In immunocompromised patients, however, all OPV (and other febrile rash agents) can cause severe generalized rashes and systemic disease. The clinical appearances in man and the associated viruses as seen in DEM are shown in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6. OPV virions are large by virus standards and are brick-shaped with short surface protrusions.
The Parapoxviruses (PPV) are animal viruses that may be transmitted to man, causing a mild febrile zoonosis and single, nodular lesions containing some vesicular fluid. In immunocompromised patients they, too, may generalise. PPV include the species Orf virus (in sheep and goats), Pseudocowpox virus (in cattle, Milker´s nodule virus) and Bovine papular stomatitis virus, also in cattle. The clinical appearance in man and the associated viruses are shown in Figure 7 and Figure 8. The virions are slightly smaller than OPV, have a more oval outline and long, spiral surface “threads”.
Two other poxvirus genera may be involved in human disease: (1) the widespread human-specific Molluscipoxvirus (MCV) causing single or multiple small wart-like lesions in man, called molluscum contagiosum (Figure 9); and (2) Yatapox virus, which is an occasional cause of a single skin lesion and is found in the tropics. MCV infects man alone and is spread through direct contact or through contaminated clothing, towels, etc. By routine DEM, both viruses are indistinguishable from OPV. New animal poxviruses are still being discovered, with or without the potential for causing a zoonosis.
The Herpesviruses. Two virus species of the order Herpesvirales—varicella zoster virus (VZV) and herpes simplex virus (HSV) of the subfamily Alphaherpesvirinae—frequently cause infections in man. VZV is the cause of the common, and usually mild, chickenpox in children (Figure 10). During the primary infection, VZV enters the sensory root ganglia of the central nervous system where it remains dormant and may emerge again later as shingles (herpes zoster), as shown in Figure 11. This is usually limited to the distribution of one or two sensory nerves, as a painful vesicular eruption with, occasionally, a viral encephalitis. In contrast, there are two types of HSV—Type 1 causes small numbers of vesicles, often on the lips, as Cold Sores (Figure 12), while type 2 is a genital infection. Generally, type 1 occurs above the waist and type 2 below (genital infection, sexually transmitted) but either may be found anywhere on the body. Both infections are normally benign, though irritating to the patient, and often recur. The virions of the two types are indistinguishable by EM. In the immunocompromised, however, both VZV and HSV may cause serious life-threatening infections (Figure 13). Herpesvirus particles contain an icosahedral capsid core, 110 nm in diameter, which contains the DNA genome. This capsid is readily identifiable by its size and shape (Figure 6b, Figure 10, Figure 11, Figure 12, Figure 13b,c and Figure 14e,f) and is surrounded by an amorphous protein coat called the “tegument” and a loosely fitting envelope, 150–180 nm in diameter. The electron-dense stain used in DEM preparation often penetrates the capsid, giving a dark, “empty” appearance. The clinical appearances of chickenpox, shingles and cold sores, with their associated viruses, are shown in Figure 10, Figure 11, Figure 12 and Figure 13.
The enteroviruses are small RNA-containing viruses that may occasionally cause small epidemics of “hand, foot and mouth” disease—small aphthous ulcers in the mouth and vesicular lesions on hands and feet. However, the amount of virus in the lesions has not been shown to reach EM-detectable levels.
Other organisms, and none (see also Table 1): Anthrax can cause cutaneous skin lesions, usually a single “ulcer”, which evolves into a black scab later. It is not likely to be confused with smallpox, except in an immunocompromised patient. The lesions contain numerous large gram-positive rods, often containing a central spore. The treponemes of syphilis may also cause a single red papule, which later ulcerates. Similarly, allergic and drug-induced reactions may present as vesicular eruptions, as also may scabies and generalised dermatitis. All these may be substantially worse if the patient is immunocompromised.
At least 51 endemic or potential endemic viral infectious diseases have been reported in Gabon28 (Table 1). Among them, 22 are of zoonotic origin and involve 12 families of viruses. The most represented are Flaviviridae (dengue virus, yellow fever virus (YFV), zika fever virus), Poxviridae (monkeypox virus (MPXV)), Filoviridae (ebola and Marburg Viruses), Arenaviridae (lassa fever virus), Bunyaviridae (RVFV) and Togaviridae (chikungunya virus).
During the past two decades, several outbreaks of these zoonotic viral diseases have been reported in Gabon. All of them had a major impact on the public health:Zaïre ebola virus (ZEBOV): in Gabon, ZEBOV outbreaks occurred in 1994, 1996, 1997 and 2001;29 primary human cases were generally contaminated by direct contact with dead wild animals, such as great apes (chimpanzee and gorilla), which are highly susceptible to the disease, and therefore human outbreaks were often preceded by an animal epizootic (great apes). Since the first recorded outbreak in 1976, 20 human epidemics have occurred in Central Africa29, 30 with three recent outbreaks in RDC and Uganda in 2007 and 2008.Chikungunya virus (CHIKV): CHIKV has recently dispersed to new regions of the world including Gabon where two outbreaks in 2006 and 2007 mainly hit the capital, Libreville.31, 32 A total of 17,618 human cases were reported.33 The outbreaks appeared concomitantly with the spread in peridomestic urban areas of Aedes albopictus, the mosquito known as the main vector of the most recent epidemics of CHIKV.34 CHIKV disease had reemerged in 2001–200335 in the Indian Ocean after a 20-year gap with a new epidemiological pattern including A. albopictus as the main vector of epidemics and an adapted virus strain presenting an original mutation suspected to be responsible for an increase of pathogenicity.36
Dengue virus (DENV): a DENV outbreak occurred in Gabon simultaneously with the CHIKV outbreak in 2007,33 and concurrent infections of DENV and CHIKV have been reported in towns affected by the two outbreaks.34 Dengue fever and the severe form of the disease, dengue hemorrhagic fever (DHF), are caused by the world's most prevalent mosquito-borne virus.37 DENV is carried by Aedes aegypti mosquito, which is strongly affected by ecological and human drivers, but also influenced by climate (temperature, humidity and solar radiation).37 Although DENV was known to circulate among mosquitoes within limited areas in West Africa and East Africa, dengue fever first emerged among the African population during the epidemic of Nigeria in 1964–1968,38 then in Senegal in 198039 and Burkina Faso and Kenya in 1982.40, 41 Since then epidemic manifestations were recorded in East Africa (Mozambique, Sudan, Djibouti, Somalia, Eritrea), in Senegal and more recently in Gabon.34,42 It seems that dengue fever is on the edge of emergence in Africa with the potential appearance of the devastating DHF that is yet to be observed on the continent.Yellow Fever Virus (YFV): Gabon is officially designated as an infected country. A YFV outbreak occurred in 1994 in Ogooue-Ivindo Province, North East of Gabon with 44 cases reported.43 More recently, in 2009, Cameroon reported a laboratory-confirmed case of yellow fever (YF).44 YF has become an important public health issue because of its case-fatality rate of 50% and the estimated 200,000 cases and 30,000 deaths that occur each year worldwide. Also, despite the efficiency of the YF vaccine and its inclusion in the national vaccination program, human populations situated in remote areas have a limited access to the public health system.
Given the number of different viral pathogens potentially associated with food- and waterborne transmission their detection has not been straightforward. Partly because many of these pathogens lack cell culture systems that are sensitive and robust enough for application in routine settings (Amar et al., 2007). The entry point for disease-based surveillance of viruses spreading by food and water is the reporting of patients presenting to a clinician. However, patients only present themselves in case of a severe symptomatic infection, or in case self-help is not sufficient. Mild symptoms are therefore generally not registered creating a bias in surveillance. This phenomenon is captured in the surveillance pyramid (Figure 1), and the full extent of disease can only be captured through epidemiological studies addressing incidence and etiology at community level coupled with severity of a range of enteric pathogens (Sethi et al., 1999; de Wit et al., 2001; Tam et al., 2012). Additionally, it is challenging to distinguish between foodborne outbreaks and outbreaks caused by direct contact between humans. Classic clinical symptoms of foodborne disease vary, ranging from diarrhea and vomiting to abdominal cramps and general malaise, which makes it hard for clinicians to pinpoint the exact causative agent. This leads to misdiagnosis if the diagnostic workup is selective, and if there are no obvious signs of food-related exposure (Beersma et al., 2012). Moreover, heterogeneity in clinical interpretation can be caused by host factors, such as differences in the expression of histo-blood-group antigens that are receptors for rota- and noroviruses (Payne et al., 2015; de Graaf et al., 2016). Susceptibility to fecal-orally transmitted viruses may also be influenced by the established microbiome and virome in the host population, of which the prior is shown to differ between different locations and age groups (Yatsunenko et al., 2012). It is reasonable to think that the differences in the gut environment are more pronounced between countries with larger social and economic differences such as first and third world countries, which often differ in their resident pathogens (Ott et al., 2012; Yatsunenko et al., 2012; Hay et al., 2013). The role of the gut virome, in addition to the gut microbiome, is a relatively new concept and has been described as potentially having influence on gut health and therefore expression of disease (Cadwell, 2015). Because of under and miss-diagnoses, clinical surveillance likely only captures the tip of the iceberg of food- and waterborne viral disease cases.
Vaccines and vaccine vectors are termed genetically modified (GM) if recombinant gene technology is used to create the vaccine or vector. Genetically modified viruses (GMVs) or virus vectors of homologous or heterologous disease antigens are considered highly desirable for vaccinations against diseases that are difficult to treat or for which there exists no effective conventional vaccine, e.g., acquired immune deficiency syndrome (AIDS), malaria, tuberculosis and neoplastic disorders. Some of the advantages of GMVs in vaccination are the enhancement of immunogenicity without an adjuvant and induction of robust cytotoxic T lymphocyte response to eliminate virus-infected cells. Many viruses have been used as GMVs for vaccination purposes, the major ones being Poxviridae (Vaccinia virus; VACV), Retroviridae (including lentivirus), Adenoviridae (human Adenovirus; hAdV), Parvoviridae (Adeno-associated virus; AAV), Herpesviridae (cytomegalovirus; CMV) and Paramyxoviridae (sendai virus; SeV). Each has unique attributes and associated risks when used as a GM virus vaccine or vector.
VACV-based vectors have good historical precedence on safety in that VACV and modified vaccinia virus Ankara (MVA) were used in smallpox vaccination. VACV-based vectors were also well tolerated in clinical trials, although adverse effects were recorded in some studies at high dose (over 108 pfu) of MVA. Disadvantages of vectors based on VACV include limited immunogenicity and pre-existing immunity. Recombinant AdV vectors have the advantage of high transduction efficiency, a high level of transgene expression, broad cell tropism and the ability to infect both dividing and non-dividing cells. The AdV vectors are also well tolerated but the presence of pre-existing anti-AdV immunity is a disadvantage associated with them. Similar to AdV, AAV can infect dividing and non-dividing cells and has broad cell tropism. In addition, AAV vectors also provide long-term transgene expression, however, they may require host genome integration for viral gene expression. Retrovirus and AAV vectors provide long-term gene expression and they are not plagued with pre-existing immunity. However, their package size is limited to 4.5 and 7.5 kilobases respectively and retroviruses are associated with various diseases, e.g., leukemia, lymphoma and AIDS. Vectors derived from SeV show high efficiency in gene transfer and transduce both dividing and non-dividing cells. SeV infects human epithelial cells efficiently and can therefore be administered intranasally. This reduces the influence of pre-existing immunity compared to intramuscular administration. These advantages/benefits of the various GMVs are being exploited in improving virus-based GM vaccines, while efforts are underway to reduce their limitations.
Zoonotic pathogens, including Ebola virus, H1N1, MERS and SARS [1–5], impose significant threats to human health and are projected to increase in their distribution and impact in coming years. Currently, 61% of all pathogens that infect humans are zoonotic and 75% of emerging disease pathogens are zoonotic in origin. This pattern is driven in part by novel interactions between humans and previously undisturbed environments, and can be attributed to human modifications, land-cover change, climate change, unplanned urbanization and human migration.
Vaccinia virus (VACV) is one such example of an emerging, zoonotic pathogen. VACV is an Orthopoxvirus and is closely related to the virus that causes smallpox (Variola virus). VACV was used as the vaccine against smallpox during eradication efforts, but more recently, human infections of zoonotic origin have been reported [7–9] in Brazil, India and Mongolia. The natural history of VACV and its transmission cycle is not known, but several wild and peri-domestic species of mammals have shown evidence of orthopoxvirus infection, including horses, coatis, opossums, monkeys and rodents, which could be involved in the maintenance of the virus in nature [11–16]. In South America, the first VACV outbreak of zoonotic origin was identified in Brazil in 1999 and all documented VACV outbreaks on the continent since that year have been associated with dairy farms in Brazil [18–21] or Colombia. During an outbreak, the virus is presumably spread throughout a farm by direct cow-to-cow contact or via milkers who develop lesions on their hands and spread the virus to others during milking. The virus could be transmitted to neighboring farms by sharing infected cattle for breeding practices and/or infected milkers. Secondary human cases of VACV without direct physical contact with infected cattle, have also been reported [17, 23]. VACV is not a mandatory reportable disease and the current surveillance system is not designed to capture these infections. Further, only a limited number of epidemiologic studies have been conducted, which restricts the ability to estimate the burden of the disease and the use of other analytical approaches to research transmission patterns and risk factors that would aid in its control.
VACV infection causes moderate to severe illness in humans and reduces milk production in cows; disease manifestation in humans includes pruritus at the site of infection, papules, vesicles, and pustules surrounded by erythema and induration as well as fever, headache, exhaustion, enlarged lymph nodes, and malaise; symptoms last for up to 30 days. Experimentally infected cows show symptoms that last 1–32 days post inoculation (dpi), whereby vesicles, papules and ulcers form on teats, and in some cases the muzzle as well, and eventually scar. Milk production is affected by infection as mastitis begins early in infection and remains through the entirety of the disease. Milk volume drops by more than 70% by 3 dpi and milk quality, measured by somatic cell count (SSC), significantly decreases. Studies of milk experimentally contaminated with VACV showed a major reduction of infective viral particles (>94%), after the pasteurization process but a few were still infective.
The dairy industry in Brazil is currently the world’s 5th largest milk producer and is rapidly increasing. There are over 1 million dairy cattle farms in Brazil, which are heavily concentrated in the states which have experienced VACV outbreaks (Minas Gerais, São Paulo, Goiás, and Rio Grande do Sul). Studies of milk experimentally contaminated with VACV showed a major, but not complete, reduction of infective viral particles (>94%) after the pasteurization process, this opens the possibility for viral spread through consumption of milk.
Public health control of emerging pathogens is challenging when the origin and basic risk factors for pathogen acquisition are not well understood. The mechanism by which VACV is maintained in nature, cows become infected, transmission patterns, attack rate and basic risk factors are still unknown. In lieu of opportunities to collect more data from larger outbreaks or formal epidemiological studies, this work attempts to utilize the existing and publicly available information to gain insight into this emerging threat. Based on the premise that pathogen circulation depends, in part, on certain environmental conditions, identifying and mapping those conditions can be used to hypothesize the distribution of a pathogen across the landscape. Here, we aim to identify at-risk regions for VACV transmission in Brazil and Colombia by determining the environmental factors common among locations in which outbreaks have been recorded, and to identify the most relevant bioclimatic factors affecting its transmission.
Thousands of different microorganisms affect the health and safety of the world's populations of humans, animals, and plants. Infectious microorganisms include species of bacteria, viruses, fungi, and protozoa. Many different medical and governmental organizations have created lists of the pathogenic microorganisms most relevant to their missions. For example, the Centers for Disease Control and Prevention (CDC) maintains an ever-changing list of notifiable diseases, the National Institute of Allergy and Infectious Disease (NIAID) lists agents with potential for use in bioterrorist attacks, and the Department of Health and Human Services (HHS) maintains a list of critical human pathogens. Unfortunately, the nomenclature for biological agents on these lists and pathogens described in the literature is imprecise. Organisms are often referred to using common names, alternative spellings, or out-dated or alternative names. Sometimes a disease rather than a particular organism is mentioned, and often there may be multiple organisms or co-infections capable of causing a particular disease. Not surprisingly, this ambiguity poses a significant hurdle to communication among the diverse communities that must deal with epidemics or bioterrorist attacks.
To facilitate comprehensive access to information on disease-causing organisms and toxins, we have developed a database known as "The Microbial Rosetta Stone" that uses a new data model and novel computational tools to manage microbiological data. This article focuses on the information in the database for pathogens that impact global public health, emerging infectious organisms, and bioterrorist threat agents. It provides a compilation of lists, taken from the database, of important and/or regulated biological agents from a number of agencies including HHS, the United States Department of Agriculture (USDA), the CDC, the World Health Organization (WHO), the NIAID, and other sources. We curated these lists to include organism names that are consistent with the National Center for Biotechnology Information (NCBI) nomenclature and to provide sequence accession numbers for genomic sequencing projects (if available). Important synonyms or previously used names that identify the organisms are also shown. We have organized the lists according to phylogenetic structure. This paper provides graphic representations of the phylogenetic relatedness of important pathogenic organisms.
The goal of the database is to provide an informative, readily accessible, single location for basic information on a broad range of important disease causing agents. The database will help users to avoid the pitfalls of confusing nomenclature and taxonomic relationships and allow access to literature on in-depth studies. The database can be accessed at .
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 2), and although only partially genetically characterized, a small (100 amino acids) region of overlap in their RAP94 proteins has only 62% amino acid identity (please see Table S1 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.