To test the analytical sensitivity of the assay for Variola virus we performed a dilution to extinction using the Variola virus derived calibrant. The calibrant was quantified by OD260 and diluted down to determine the limit of detection by the assay. The calibrant is present in every PCR reaction at 100 copies.

Blood extract from rabbitpox-infected rabbits was taken six days after exposure to aerosolized rabbitpox virus. The first blood sample evaluated (sample ID: 3E-day 6) was found to have 4×103 plaque forming units (PFU) per mL of blood as determined by a plaque assay on Vero cells. DNA extract from this sample was analyzed by real-time PCR and found to contain 4×106 genomes/mL of blood, assuming 100% extraction efficiency. Blood taken on the 6th day post infection from a second infected rabbit (sample ID: 3D-day6) had 3.3×102 PFU/mL blood and 2.5×105 genomes/mL blood. The most sensitive primer pair, VIR985, detected eight genomic copies of rabbitpox genome isolated from infected rabbit blood with 100% efficiency in 10 out 10 PCR replicates, which is near or at the stochastic limits of PCR detection. The viral PFU in this blood samples was determined prior to DNA extraction and the diluted DNA extract would equate to less than 2 PFU/mL of blood. The three additional primer pairs are sensitive to 30 genomes per PCR reaction, which corresponds to a detection of 6.3 PFU/mL of blood, with 100% detection in 10 out 10 PCR replicates and provide added sub-species information and redundancy to the assay (Figure 3).

Today, orthopoxviruses are principally rare zoonotic pathogens, but historically Variola was a devastating human pathogen. Though Variola virus has been eradicated there is still the potential for its reintroduction into the human population through an act of bioterrorism. It is also possible that a zoonotic orthopoxvirus could emerge as a variola-like virus of humans or be used as a bioterrorist agent. A single assay that can detect and identify all orthopoxviruses is critical for effective surveillance. Though several methods exist for the detection and surveillance of orthopoxvirus, these assays are limited in their coverage or require large numbers of reactions to identify all Orthopoxvirus species. In this study, we describe an assay using four PCR primer pairs that can identify and distinguish all orthopox species and can provide subspecies level of identification of several important orthopoxviruses. Our primers target the highly conserved core viral DNA and RNA polymerase and helicase genes and thus it is very likely that novel orthopox viruses will also be detected using this strategy.

In this study we tested the assay on a diverse panel of orthopoxviruses and also tested detection of rabbitpox from infected rabbits. Rabbitpox is a vaccinia virus but does not infect humans and therefore is an ideal model for studying human smallpox. There was a 1,000-fold difference between PFU and genome levels from the blood samples studied; this also was true for several samples from different animals and probably represents a high level of non-viable or replicating virus in the blood. Similarly high genome to PFU ratios was observed for other viruses.

Overall we demonstrated that the pan-Orthopoxvirus assay can detect a wide and diverse range of orthopoxvirus and can be used to accurately speciate orthopoxvirus. The assay is simple and can be performed using premade PCR plates that contain all reaction components except genome. After extraction, the samples can be processed from PCR through analysis and reporting in five hours. Using the described assay, a single 96 well PCR plate can be used to analyze 24 samples and the system can process 16 plates in a 24 hour period for a total throughput of 384 samples per day on a single instrument. Such throughput is essential for surveillance and in times of an epidemic outbreak or acts of bioterrorism.

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).

We compared antibody prevalence between sampling locations using a log-likelihood ratio test (G-Test). A 95% confidence interval (CI) was constructed for the prevalence.

We analyzed a publicly available line list of MERS cases reported between March 2013 and May 2015 to the Kingdom of Saudi Arabia (KSA) Ministry of Health. For each case, we obtained the date of reporting, healthcare worker status, and whether the infection had been linked to healthcare facilities. Healthcare facilities in KSA report MERS cases to the Ministry of Health through an electronic case-reporting system once all appropriate testing is complete. Case confirmation is based on laboratory diagnosis through detection of viral nucleic acid or serology, regardless of the presence of clinical signs and symptoms.

We now consider surveillance scenario 2 (i.e., detection of a case may trigger an outbreak investigation). We only use data from the first detected case of each cluster to control for the change in surveillance intensity due to the outbreak investigation. We first make the stricter assumption that each cluster is made of one chain of transmission, but this assumption is relaxed in Figure S1 and Text S1. Conditional on detection of the chain, the probability F that the first detected case was the first case of the chain is:

(2)When the probability of detection is small (ρ≪1), we are left again with a simple linear relationship (see Text S1).

In order to examine ebolavirus exposure in wild great apes we sought to develop a strategy of detection in samples collected by non-invasive methods that would be sensitive and specific enough to detect multiple ebolavirus species with minimal false positive results. It has been shown previously that an enhanced chemiluminescent western immunoblot assay is able to successfully detect specific antibodies in RNAlater-preserved feces from simian immunodeficiency virus-infected chimpanzees (SIVcpz). The sensitivity and the specificity of SIVcpz antibody detection in fecal samples were estimated to be 92% and 100%, respectively. Viral SIVcpz nucleic acid could be amplified in an immunoblot-positive fecal sample, confirming SIVcpz infection. Furthermore, a similar approach was used to diagnose simian foamy virus infection in wild chimpanzees (SFVcpz). The sensitivities of SFVcpz antibody and viral nucleic acid detection in fecal samples from captive chimpanzees were 73% and 75% respectively, and assay specificities were 100%. These studies show the potential of assessing RNAlater-preserved fecal samples to document wild apes' exposure to viruses.

Given the success of this approach, we developed a fecal western blot assay to detect ebolavirus antibodies. We chose purified EBOV NP as the antigen for antibody detection since it is one of the most abundant structural proteins produced during infection and a major target of the host immune response. This is supported by previous studies showing that humans who have survived natural EBOV infection developed strong antibody responses mostly against NP–[37]. In addition, the NP sequence is well conserved among ebolavirus species (Figure S1), making it useful for detection of antibodies against multiple ebolavirus species, and potentially increasing the breadth of this detection method.

We first assessed the ability to detect NP antibodies in RNAlater-preserved fecal samples from captive cynomolgus macaques. Fecal specimens were experimentally spiked with different dilutions of positive serum containing polyclonal immunoglobulin from a monkey that was vaccinated with a genetic vaccine encoding NP. Serum from this vaccinated monkey displayed antibody reactivity with NP by both ELISA and western blot analysis (not shown).

Extracts from these positive serum-spiked feces were then used to incubate immunoblot strips containing immobilized NP. Anti-NP antibodies were detected by enhanced chemiluminescent western blot immunoassay in fecal samples at seropositive nonhuman primate (NHP) plasma dilutions of up to 105-fold (Figure 1), indicating a high sensitivity of the assay for fecal antibody detection. A similar level of sensitivity was observed for detection of anti-SIV and anti-HIV antibodies by western immunoblots using plasma samples from SIVsm-infected NHP diluted up to 10−4 and plasma samples from HIV-1 infected individual diluted up to 10−6

[40]. In contrast, fecal extracts from captive and uninfected nonhuman primates (cynomolgus macaque and western lowland gorilla species) treated in the same way showed no reactivity in the NP immunoblot, demonstrating low background for the assay and lack of cross-reactivity with serum antibodies directed against irrelevant proteins. These results demonstrated that NP antibodies present in primate fecal samples can be extracted and detected by immunoblotting.

Total SARS case counts for Canada, China, Hong Kong, Singapore, and Vietnam including cases among healthcare workers were obtained from the World Health Organization (WHO) website for the outbreaks in 2003. A probable case of SARS was defined as radiographic evidence of pneumonia or respiratory distress syndrome on a chest X-ray, positivity for SARS virus infection by one or more laboratory assays, or autopsy findings consistent with the pathology of respiratory distress syndrome. A confirmed case was defined based on a positive laboratory test combined with clinical evidence compatible with SARS.

Initially, survivor numbers were compared by the Mantel-Cox log-rank test. When statistical significance was found, pairwise analyses were performed using the Gehan-Breslow-Wilcoxon test. Differences in tissue virus titers and lung weights were statistically analyzed by the Tukey-Kramer multiple comparisons test. Lung hemorrhage scores were analyzed by the two-tailed Mann-Whitney U-test. Analyses were performed using the InStat® and Prism 5.0® computer software programs (GraphPad Software, San Diego, CA), comparing treated and placebo groups.

To date, four MPXV small animal models have been used for the testing of antiviral drugs Cidofovir, CMX001 and ST246 (tecovirimat). Herein we will summarize those studies, efficacy data, and discuss the advantages, and limitations, of the animal models used.

Sbrana et al. utilized ground squirrels to test the efficacy of ST-246 against a MPXV challenge. The authors used 100 pfu of MPX-ZAI-1970 (200 × LD50) via a subcutaneous route of inoculation. Squirrels (8–9 per group) were divided into five treatment groups; drug was given either at 0 hours of infection, 24 hours, 48 hours, 72 hours or 96 hours p.i. 100 mg/kg of drug was given once a day for 14 days. Two animals in each group were sacrificed at day 7 to measure objective morbidity; the remainder of the animals were used to calculate survival rates. Animals in the placebo group, that were not given ST-246, showed signs of illness beginning on day 4 and all died between days 6–9. Signs of disease included lethargy, anorexia, nosebleeds, and terminal respiratory distress. At day 7, a sampling of placebo-treated animals exhibited significant leukocytosis, transaminitis, and coagulopathy; almost 105 pfu/mL of infectious monkeypox was found in blood; at this time, between 107 and 108 pfu /mL of infectious MPXV was observed in 10% organ homogenates of liver, spleen and lung. Animals treated on days 0, 24, 48 or 72 hours, before symptomatic disease onset, all survived infection and showed no signs of disease. At day 7, in a sampling of animals treated at hour 0, 24, 48 or 72 p.i., no virus was found in the liver, spleen, lung, or blood; although some abnormal values were apparently recorded, no clear trends in leukocytosis, transaminitis or coagulopathy were noted with delay in treatment onset. In animals initiating treatment at 96 hours p.i., concurrent with symptomatic disease onset, 67% of animals survived infection. 2/4 survivors showed signs of disease. In those animals that succumbed to infection, ST-246 prolonged the time to death; the mean time to death was day 7 for animals receiving placebo and day 13 for those receiving ST-246 in the 96 hour p.i. treatment group. The sampling of animals at day 7, initiating ST-246 at 96 hour p.i., demonstrated lower levels of viremia (∼3 log decrease) and ∼5 logs less virus in liver, spleen and lungs than that seen on the placebo treated animals at day 7. Although some evidence of transaminitis was present, leukocytosis and coagulopathy were not observed in this treatment group. Pathologic examination of tissues in general showed greater tissue necrosis in animals treated at later times p.i. This study was able to demonstrate a survival benefit in animals treated prior to, or at the onset of disease symptoms, in a disease model that has a time course attenuated with respect to what is seen in human disease.

Schultz et al. infected African dormice with a lethal challenge of Congo Basin clade virus MPXV-ZAI-79 via an IN route of infection to evaluate the efficacy of Cidofovir as post exposure prophylaxis. Four hours post intranasal infection with 75, 4 × 103, or 5 × 103 pfu of MPXV, animals were intraperitoneally administered 100 mg/kg cidofovir (the calculated LD50 for the dormouse MPXV model was 12 pfu). Aggregate data from all challenges showed animals treated with cidofovir had a mortality rate of 19% (7/36), whereas vehicle treated animals all (41/41) succumbed to disease. Treatment initiation at later times p.i. was not evaluated; effects on viral load or histopathologic changes were not reported.

As inbred mice have historically shown little disease symptomatology or pathogenesis post monkeypox infection, Stabenow et al. utilized a laboratory mouse strain lacking STAT1 (C57BL/6stat-/-), which has been found to be sensitive to a range of viruses including SARS, murine norovirus 1, respiratory viruses, dengue virus and MPXV [19,22–25]. These animals are deficient in their ability to transcribe many of the Type I and Type II receptor interferon response genes. The authors used the Congo Basin clade virus MPX-ZAI-79, evaluated disease and the protective efficacy of CMX001 and ST246. In untreated mice, 0% mortality was observed with 4.7 pfu challenge, 90% mortality with 470 pfu of virus and 100% mortality with 4,700 pfu. Over 25% total body weight loss, and mortality was observed on or prior to day 10 p.i. in untreated animals. Animals in the treatment studies were subsequently challenged with 5,000 pfu via an IN infection. Animals were then treated with 10 mg/kg of CMX001 by gastric gavage on the day of challenge followed by every other day with 2.5 mg/kg until day 14 p.i. All C57BL/6 stat-/- mice that were treated with drug survived infection, demonstrated <10% body weight loss between days 10 and 20, and developed a serologic response to monkeypox. Similarly, mice treated daily, starting at the day of virus challenge, with 100mg/kg of ST246 for 10 days also survived infection and manifest <10% body weight loss between days 10 and 20. In this system, antiviral treated animals rechallenged with monkeypox at day 38 post initial infection (at least 10 days post reinitiation of steady weight gain), manifest 20% mortality. The model—again one with a short disease course—is useful for demonstrating immediate post exposure efficacy of antiviral treatment in the absence of a functioning interferon response system. Additionally, in this animal model system, perhaps due to the immune defect, a monkeypox protective immune response was not elicited in all animals receiving antiviral treatment. This observation merits further observation in other animal model systems.

Smith et al. tested the efficacy of ST246 in a prairie dog MPXV model. MPXV challenged prairie dogs have previously been shown to have an asymptomatic period followed by symptoms of disease including lethargy, nasal discharge, inappetence, weight loss and systemic lesion development most commonly between days 9–12. In the current study, animals were inoculated via an IN challenge with the Congo Basin clade virus ROC-2003-358. This is a different strain of MPXV than that used in the previous described studies, but is also a strain belonging to the Congo Basin clade. The challenge dose was 3.8 × 105, equal to 65 × LD50 for the prairie dog model. Animals were divided into three treatment groups; prophylactic (day 0), post exposure (day 3) and therapeutic (varying day based on rash onset), and a control vehicle treated group. ST246 was formulated at 30 mg/mL and administered daily, by oral gavage, for 14 days. Animals initiating treatment at day 0 or 3 were protected from death and apparent signs of illness. Animals treated at rash onset had symptoms similar to the placebo control group; however symptoms were less severe in the treated animals. Although all animals treated at rash onset survived infection, animals lost 10–24% of body weight and did develop generalized rash (however, lesions resolved more quickly when compared to untreated prairie dogs in previous studies. Although asymptomatic, viable virus was shed sporadically from animals in the prophylaxis and post exposure groups (from two oropharyngeal samples in the day 0 prophylaxis group, and five samples from the day 3 post exposure group). More, sustained virus was detected in the oropharyngeal samplings of the animals in the therapeutic treatment group, but levels were less than the virus levels in the untreated group. 1/4 sham-treated animals survived infection. Signs of disease and viral titers were all increased in this group of animals compared to the animals treated with ST-246. This is the first small animal study where a treatment and survival benefit has been demonstrated when animals are treated at later stages of illness. Initiation of treatment at rash onset is similar to expectations of a human treatment regimen. The observation of virus shedding after treatment cessation in the prophylactically or post exposure treated animals merits further study to assess whether this reflects viral resistance or a blunted and delayed immune recognition and ultimate clearance of virus.

Since the dawn of time, humans knew that the changing ecosystem exposes them to becoming sick. Today, traveling to regions of the world where hygienic conditions remain inadequate (lack of drinkable water, lack of sewerage), and touching wildlife with a special attraction for NHP, may still have undesirable consequences, especially that of being contaminated by a foreign pathogen infecting the NHP (the five main routes of pathogen' transmission being aerosol, direct contact, fomite, oral and vector). Conversely, there is also a risk of introduction of new infectious pathogens in the visited ecosystem. The “One Health” concept recognizes that human and animal health are intimately connected (39). Implementing this concept requires tracking the spread of pathogens from wildlife to humans. Insofar as part of the threat is unknown, it remains important to identify which behaviors increase exposure, how to quickly identify the type of pathogen which has passed the species barrier, how important is the risk to the health of the infected individual and that of the people he/she frequents, and what measures need to be taken. To this end, a public health approach to the problem is required. The risks will be very different depending on whether it involves bushmeat, contacts with NHP in laboratory, or NHP living in their natural ecosystem. The risk will also vary depending on the frequency of contact, the time spent in close proximity with NHP, the prevalence of the microorganism in the NHP population, the route of transmission (direct or indirect), the ability of hosts to transmit the pathogen, the time of incubation, the number of secondary infections produced in a completely susceptible population by an infected individual—known as R0 (basic reproduction ratio: for a pathogen to invade and spread, R0 must be >1)—(200, 201). Unfortunately, emergence of a new pathogen in the human ecosystem is impossible to predict (202, 203), and there is no guarantee of quick identification (e.g., HIV was discovered decades after its introduction and spread in the human population). Over the past decade, EID have increased, prompting the need for faster outbreak detection, monitoring, early warning, reports and intervention (74).

As for hunting, butchering, and consumption of NHP, serious health crises are very rare even if there are examples of major EID such as HIV or Ebola virus (2, 196). There is still no vaccine against HIV while the results for Ebola vaccine trials are encouraging (204, 205). An Ebola vaccine should help to prevent the spread of disease in countries where the epidemic is rife (206). Due to inadequate hygiene conditions (lack of drinkable water, lack of sewerage), bacterial, viral and parasitic intestinal infections are common, but they are rarely serious and most of them can be treated fairly easily. However, this can become a serious medical problem if the infected individual is sick in a rural area far away from any hospital. Regarding NHP caged in zoos, primate centers, and laboratories, the pathogens can be transmitted by scratches, bites, percutaneous inoculation, or contact with body fluids. In these working environments, (i) professionals have a good knowledge of the risks; (ii) the risk is limited because animals are subject to pre-import surveillance and post-import quarantine (e.g., in Europe Council Directive 92/65/EEC of 13 July 1992 laying down animal health requirements governing trade in and imports) (207); (iii) the workers adopt preventives measures (e.g., vaccine), and laboratory biosafety equipment with protective masks, glasses, gloves (208); and, (iv) prophylaxis actions are rapidly set up after an incident. In these workplaces, the pathogen is easy to identify because: (i) NHP are caged; (ii) the natural history of the animal involved in the incident is known; (iii) all NHP have regular veterinary and serological monitoring; (iv) the animal can be placed in quarantine and be subject to enhanced biological and veterinary surveillance. However, cases of accidental transmission of Marburg virus and Cercopithecine herpesvirus to laboratory staff should not be forgotten (179, 192). These accidents should serve as examples to strictly apply the precautionary principle in laboratories. Another source of worry comes from in situ NHP recovery centers, such as the Pan African sanctuary Alliance (209) in Africa or Wildlife Alliance in Asia (210), where there exist primate nurseries attended daily by workers and volunteers who come into very close contact with the animals to save them but also share microorganisms. It could become a potential public health problem and a conservation problem when trying to reintroduce these animals in a wild ecosystem. What remains the most difficult biohazard threat to assess is associated with the illegal detention of NHP as pets and tourists contact with NHP during trips (211). When an incident involves a wild NHP, it is frequently difficult to know the species and natural history of the NHP and the pathogens borne by this wild animal.

Whatever their destination, travelers are frequently victims of health problems because they are foreigners to the visited ecosystems. The ill rate of travelers varies from 15 to 70% according to the destinations, the conditions of stay and the epidemiological survey carried out. Diarrhea—mainly associated with bacteria or virus infections with a preponderance of bacterial infections—is still the most common undesirable incident encountered when traveling abroad (212, 213). It is followed by upper respiratory diseases, dermatitis and fever. Beside these common disorders, the threat might change in nature as more travelers end up moving into area where wildlife is present. Coming into contact with wildlife increases the risks of meeting pathogens whose presence was limited to weakly anthropized ecosystems. On some tourist sites in Thailand, Indonesia, India or Bali, it is not rare (incidence about 1/1,000) to be bitten by an NHP during feeding of the animals or when tourists refuse to give them food (214). As described in this review, when humans got into contact with NHP they could also come into contact with known pathogens such as C. tetani, rabies, Herpes B, monkeypox, Marburg, or Ebola viruses, and other pathogens—known or so far unknown—which could pass the species barriers. Rabies is a small part of the problem since high-risk travelers are usually vaccinated (215). On the other hand, there is no vaccine for most pathogens present in the NHP to which these tourists could be exposed. If we take the example of the Cercopithecine herpesvirus which can cause a potentially fatal meningoencephalitis in humans (case fatality rate above 50%), the review of the scientific literature indicates that the virus is widespread in wild NHP groups and those living in freedom on tourist sites (prevalence of 60 to 90% in adult macaques depending the NHP group studied). Although hundreds of thousands of tourists come annually into contact with these infected NHP, there is so far a lack of evidence of Cercopithecine herpesvirus infections among travelers. Yet, several serious cases have been reported in primate center research workers. A single case of human-to-human transmission of Cercopithecine herpesvirus was reported in a woman who became infected after applying hydrocortisone cream to her husband's Cercopithecine herpesvirus skin lesions (216). Recently, genomic sequence variations between Cercopithecine herpesvirus isolated from different macaque species have been reported confirming the existence of different genotypes of Cercopithecine herpesvirus (217). This might suggest that some genotypes of this Herpesvirus might be more suitable than others to cross the species barrier. There has also been no report of serious case in the population of people living in close proximity with NHP and it was claimed that monkey temple Thai workers had developed a protective immune response (not scientifically demonstrated) against the Cercopithecine herpesvirus. What this example tells us is that, despite knowing the threat, no current model can predict the probability of transferring Cercopithecine herpesvirus infection to tourists after an incident involving a NHP. The situation is totally different in Central Africa with the monkeypox virus threat. The seroprevalence of MPXV ranges between 5 and 10% in several NHP groups. Humans can be infected by MPXV and develop a Flu syndrome with a case fatality rate up to 10%. Once transmitted to humans, the virus is very contagious and person-to-person transmission of MPXV occurs through respiratory droplets or body fluids leading to larger outbreaks in human populations. However, there is evidence suggesting that without repeated zoonotic introductions of the virus, human infections would eventually cease to occur (218). In both cases discussed above, the threat is known and it is possible to take preventive measures or to promptly set up therapy after infection of an individual. Of course, it's even worse if we do not know the nature of the threat (unknown pathogen) and if a human undergoes a long incubation period during which the infectious agent is present, but it is not yet causing clinical signs. Travelers should endorse responsibility for taking protective measures aimed at reducing exposure to pathogens. They should follow strict hygiene protocols, including the appropriate vaccination, maintenance of distance with NHP, and not feeding wild NHP (219). It can't be ascertained that travelers are always aware of the biohazard risks. There is therefore a need for more information to travelers via public health professionals, national authorities, and media. In addition, proactive approaches to surveillance, health assessment and monitoring of NHP populations, should be encouraged.

Professionals in charge of travel medicine know perfectly that they should recommend standard vaccination (including tetanus, rabies) according to National Advisory Committees, and the greatest caution to those who wish to meet NHP in their natural environment (220). Pre- and post-travel clinical surveillance is strongly recommended. Even in the absence of animal scratches or bites, travelers/ecotourists should be encouraged to self-screen clinical signs following any meeting with NHP. Tetanus is a preventable disease that is declining worldwide due to vaccination, but surveillance is still required. Before a stay in an area known as high-risk for rabies, preventive vaccine (pre-exposure) may be recommended. In all cases of scratches and bites by NHP, medical consultation is needed. If it is assumed that it is not possible to predict which pathogen could be transmitted to humans during an incident involving a NHP, emergency physicians and medical professionals not familiar with the field of primatology must adopt an attitude based on the precautionary principle (221). In cases of suspected or proven exposures, post-exposure prophylaxis (PEP) with anti-rabies immunoglobulins (not always available on site) should be started. Pre-exposure rabies vaccine exempts of PEP. In case of superficial NHP scratches, patients often underestimate the seriousness of injuries. Wounds should be cleaned immediately by a 15 min deep irrigation with soapy water, and when possible by saline or antiseptic solution (e.g., chlorhexidine gluconate or povidone-iodine/betadine) to remove foreign bodies and pathogens. The injury may affect different layers of skin. Ischemic lesions promote microbial proliferation. Patients can be divided into low- and high-risk groups depending on the location and importance (superficial or severe) of the injury and the medical state (if known) of the animal that caused the injury. After adequate cleansing, evaluation of the risk of pathogen transmission (the patient's vaccine statute against tetanus and rabies should be questioned), examination, assessment of health status and investigation of any unusual symptom of the offending animal is required (when possible). Blood samples from the NHP and the victim should be collected and immediately sent for serological testing (a rapid transport time of the samples is critical; adequate information should be given to the laboratory for the research of unusual pathogens). In addition, buccal and conjunctival swabs from NHP should be used for culture and rapid PCR-identification of pathogens. The culture of pathogens classified BSL-3 or BSL-4 (for biosafety level), requires specialized facilities (e.g., herpes B virus that is of major concern with NHP bite, is classified BSL-4) (222). The victim should be directed to an emergency medical service where he/she should be considered for immunoprophylaxis and broad coverage antibiotic treatment against NHP's bacteria (such as Amoxicillin clavulanate and moxifloxicin or fluoroquinolone and metronidazole) (172). To prevent viral infections, initiating PEP with an antiviral drug such as valacyclovir (1g by mouth every 8 h for 14 days), or acyclovir (800 mg by mouth five times daily for 14 days or 5 mg/kg/8 h intraveinously for 3 days) and anti-rabies prophylaxis (20 IU/kg infiltrate around the wound and any remaining amount intramuscularly), may be needed (180, 181). Parenteral ganciclovir (5 mg/kg intravenously every 12 h for 2 days) is reserved for treatment of infection with central nervous system symptoms.

Post-exposure clinical survey of the patient is necessary to identify possible signs of illness (such as fever, pain, or shock). If there is evidence for a new pathogen, warning signal are needed for early detection and control of new infectious disease, and biosurveillance of humans and NHP in the area of emergence should be established to determine its evolutionary potential, its impact on health and the ability of leaders and stakeholders to control the phenomenon. The most serious risk for public health is a deadly pathogen able to spread through human-to-human transmission with high R0, or a deadly pathogen transmitted from NHP to humans via a flying blood-sucking vector insect.

The induction of immune responses by the delivery of inactivated pathogens has been a standard and successful vaccination approach for many years, and licenced, inactivated vaccines for diseases such as poliomyelitis 63 and rabies 64 are commercially available. The long history of this approach is underpinned by a well‐defined regulatory framework that can be readily applied to new disease targets 65. The major challenge for the inactivated virus approach is that infection is not established, and therefore a full adaptive immune response is generally not achieved. However, because of the absence of living pathogens, these types of vaccines are safe and a basic capability to prepare such vaccines, especially for emergency use, might be worthwhile as a stop‐gap while alternative longer‐term approaches are developed. In this regard, studies of virus inactivation with X‐ray radiation (as a simple and cheap alternative to gamma irradiation by the use of radioactive isotopes), which maintain the tertiary antigenic structures of virus particles while destroying infectivity, have shown useful promise for a range of applications including vaccination (B. Afrough, unpublished).

Animal models permit an advance beyond what can be gleaned from tissue culture evaluation of an antiviral effect. The evaluation of an antiviral, in the context of a host with a functioning immune system, enables better understanding of therapeutics’ potential efficacy. The evaluation of an antiviral in the context of an impaired immune system enables better understanding of therapeutic use in a particular immunosuppressed population. Pathogen host range, especially if not a simple issue of receptor utilization, can confound the ability to interpret, and extrapolate to the human, some of the nuances of the host pathogen interaction and prediction of potential human therapeutic benefit. Of the small animal models used to evaluate antiviral efficacy, all have used stringent virus challenges (all greater than 10 × LD50) and shown survival benefit. Routes of infection have used methods that attempt to simulate potential human routes of infection and resultant human illness courses. Given the uncertainties of what a human infectious or lethal monkeypox dose is, it is difficult to extrapolate the potential “best fit” of any of these models for human disease. The clinical time course of disease in the prairie dog model, however, has a temporal relationship that is close to what has been described with human systemic orthopoxvirus (variola or monkeypox) disease. However, a limitation of the prairie dog and some of the other described animal systems, with the exception of the mouse model, is a paucity of immune reagents. There are a handful of antiviral compounds which show promise in these small animal models using monkeypox virus as the challenge. Additional studies evaluating treatment benefit when used in later stages of disease, their effect on elicitation of a protective immune response, evaluation of antiviral resistance, and their effect on viral shedding will improve our understanding of how they may be used in treatment of human disease, or in response to epidemic disease.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Emerging pathogens represent one of the greatest risks to global health. There is already good evidence 1, 5 that zoonotic pathogens will most probably be transmitted from a few key animal species in resource‐poor areas of the world. Based on recent history, it is probable that such pathogens have never been seen before. The global impact of the West African outbreak of Ebola virus in 2014 underlines how stark differences in health‐care infrastructure can impact upon human‐to‐human transmission of emerging pathogens. Until basic health‐care infrastructure in all countries can be raised to a level that enables early identification and control of high‐risk pathogens at source, we will continue to respond to outbreaks of emerging disease long after epizootics have spilled over into human populations. Innovative strategies are therefore urgently required to control such pathogens, vaccination is a proven approach.

Many novel vaccination strategies that have been developed during recent years have the potential to specifically address the growing threat of new and emerging disease. The use of well‐defined vaccine vector platforms, with an extensive record of safety and efficacy against similar pathogens, can expedite the process of development, validation and production (Table 1). Accordingly, the design and licensure for particular platform vaccine technologies will help to accelerate the development of new vaccines, as only the simple substitution of a new antigen gene into the vector platform is required. This allows manufacturers to move to a new target disease with minimal changes in chemistry, manufacturing and controls. Thus, new vaccine development can focus on the safety and efficacy of the inserted gene. In addition, the ability of platforms to target multiple pathogens helps to justify the investment required to build and maintain manufacturing infrastructure that specializes in one platform, because a single manufacturing facility can be ready to produce multiple vaccines at any time.

In addition, further research into, and the development of, self‐disseminating vaccines to control potential pathogens in their wild‐life reservoirs should be encouraged. However, the progress of new vaccines through the necessary regulatory pathways to bring them to the clinic requires long‐term investment by governments and international organizations.

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.

Mice were anesthetized with ketamine/xylazine (50/5 mg/kg) by intraperitoneal (i.p.) injection for i.n. treatments and i.n. infection. I.p. infections required no anesthesia. Amounts of infecting virus (PFU/mouse) to achieve complete lethality varied with virus type, infection route, and infection volume, as follows: vaccinia (100-µl i.p.) - 5×106, vaccinia (50-µl i.n.) –1×105, cowpox (10-µl i.n.) –1×106, and cowpox (50-µl i.n.)- 5×105. A long-term (8-week) prophylaxis study that was performed used aged mice, which required an i.n. vaccinia virus challenge dose of 2.5×105 PFU/mouse to cause complete mortality. mDEF201 was administered i.n. to the nasal sinus or lung 24 h prior to vaccinia virus exposure for short-term prophylaxis, or 56 days prior to infection for long term prophylaxis. Placebo-treated mice were given saline by i.n. route. All animals were weighed every other day starting the day before and during the 21-day infection period. There were 10 mice in each mDEF201 or cidofovir treated group and 20 placebos per experiment.

In the long-term prophylaxis study, separate animals (5 mice per group) were maintained for determination of tissue virus titers, lung hemorrhage scores, and lung weights. These mice were sacrificed for removal of tissues after 5 days of infection. Lungs were given a hemorrhage score (color change from pink to plum which occurs regionally in the lung rather than gradually over the entire lung) ranging from 0 (normal) to 4 (entire lung affected). Lungs, spleens, livers, and snouts were weighed prior to homogenization that releases infectious virus for titration. Homogenization of soft tissues was performed in 1 ml of cell culture medium using a stomacher. Snouts were ground in 1 ml of medium using sterilized mortars and pestles. Samples were serially diluted in 10-fold increments and plaque titrated in 12-well microplates of Vero cells. Plaques were stained at three days with 0.2% crystal violet in 10% buffered formalin, followed by counting the plaques with the aid of a light box. Plaque numbers were converted to PFU per gram of infected tissue.

The strengths of DEM are that: (1) no specific reagents, no antibodies, no nucleic acid primers, nor any a priori decisions on what microorganisms to look for or which test to use, are required; (2) only the neutral (i.e., unbiased) sense of vision is used to recognise specific structures by their appearance alone; (3) DEM covers all the known (and even the unknown) agents; (4) can detect when there is more than one virus in the specimen; and (5) and most importantly, it is quick. The practical limits of resolution of a transmission electron microscope (TEM) on biological material is 2 nm, allowing the fine structures of any virus particles to be clearly visible. With this level of resolution, DEM is a catch-all method, able to detect unexpected viruses and other agents including bacteria and some parasites. DEM offers both speed and diagnostic certainty, while also allowing some of the other possible causes to be confidently discarded.

Speed in diagnostic virology matters in many instances—in severe, life-threatening clinical infections, in possible epidemic situations, as well as in possible bioterrorism. Currently, point-of-care or bed-side detection assays are being developed for many infections to enable diagnosis in less than an hour, always provided they contain the specific reagents necessary for detecting the actual causative virus. However, in contrast to these point-of-care systems, dedicated high tech methods like DEM will only be found in a small number of sites, mostly in universities and in centralized national facilities for cost and organisational reasons, but the “open view” of DEM is a valuable defence against the unexpected and, occasionally, double infections. Moreover, certainty over the diagnosis follows when the DEM result and the clinical history are compatible.

With skin diseases, several different specimens can be investigated by NS-DEM: vesicle fluids directly from the patient´s skin lesions, supernatants or cells from diagnostic culture, or solid tissues such as scabs or biopsies. The latter two can be evaluated by NS-DEM after grinding in distilled water and clarification with low speed centrifugation. TS-TEM can also be used in DEM, but preparing thin sections is more complex and more time-consuming (for details see), making the simple NS the preferable method for DEM. More sophisticated preparation techniques, such as the structure-preserving cryo-TEM, are not required to make the diagnosis and will diminish the essential speed advantage of DEM.

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

Initially, survivor numbers were compared by multiple group chi square analysis. When statistical significance was found, survivor numbers were evaluated using the two-tailed Fisher exact test. Differences in the mean day of death, tissue virus titers, lung weights, and lung hemorrhage scores were statistically analyzed using the two-tailed Mann-Whitney U-test. Analyses were performed using the InStat® computer software program (GraphPad Software, San Diego, CA), comparing treated and placebo groups.

In cases where a cluster of patients with similar symptoms presents itself, there can be an investigation to look for epidemiological clues of the link between the cases. Additional information is garnered from the use of viral genome sequencing, making it possible to track origins of outbreaks, and to estimate how much of the observed human disease is attributable to foodborne infection by computerized linking of epidemiologic data to aligned viral genomic sequences (Verhoef et al., 2011). However, often the original source or evidence of it being food- or waterborne cannot be found, which means that outbreaks often are merely registered. Of the 941 viral disease outbreaks reported as foodborne in the joint ECDC-EFSA surveillance report of 2015, only 9.1% had robust evidence of food- or waterborne transmission (Eurosurveillance editorial team, 2015). Routine application of genotyping of HAV in newly diagnosed cases quadrupled the number of cases in which food was the most likely source of infection a 3 year enhanced surveillance study in The Netherlands, but this is not commonly done (Petrignani et al., 2014). In an investigation of 1794 food- and waterborne outbreaks in Korea, roughly 75% of the outbreaks reported in schools and public restaurants were attributed to an unknown origin (Moon et al., 2014). Availability and costs of molecular testing combined with sequencing, additional to the limited success of virus detection in food products, are likely further limiting their use in food and water surveillance. This is demonstrated by the fact that formal confirmation of a viral outbreak associated with food- and waterborne transmission still requires extensive epidemiological analysis or confirmation of a virus in the infected individual, or both (ESFA, 2016). However, due to the increase of genomic information of viruses, sequence data is increasingly used to support and strengthen outbreak investigations. Nevertheless, the surveillance programs for these viruses in the human food chain is limited, in contrast with the American CDC1 and the European ECDC2 surveillance programs for bacteria and parasitic pathogens causing food- and waterborne diseases (Deng et al., 2016) and does not have widespread coverage. As an example, to comply with European food safety regulations, shellfish, a well-known source of foodborne pathogens, need to be tested for enteric bacteria. However, it has been well documented that shellfish that pass quality control based on bacterial counts may still contain human pathogenic viruses (Rodriguez-Manzano et al., 2014). To be able to recognize food- waterborne viral disease outbreaks and stop underestimation of its disease burden there should be innovations in the current foodborne surveillance system.

Multiple viruses were detected within some samples. Both LCV and SFV were detected in the bone marrow of AGM CII-051 and muscle of mangabey BM008 (Table 1). CMV, LCV, and SFV were detected in baboon CII-163 (Table 1).

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 microarray-assisted transcriptional profiling can provide us with a wealth of information regarding host genes and gene-interacting networks in 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.

The final challenge of metagenomic sequencing based surveillance is the interpretation of the annotated sequences. There is still little knowledge of the presence and dynamics of viruses in the environment and the food chain, which is of influence on the interpretation of food- and waterborne viral surveillance samples. Various factors are expected to influence the virome, and without knowledge of the typical viral content of a sample, the relevance of the detection of a virus is hard to determine. An example of this is a study showing a large discrepancy between the levels of HAV genotypes detected in sewage samples compared to the genotype infecting patients in the clinic in the same time (La Rosa et al., 2014). A potential sampling bias and asymptomatic shedding of one of the variants was proposed as an explanation of the discrepancy. However, this shows that a lack of knowledge of viral diversity in a population under surveillance could potentially lead to wrong conclusions in environmental surveillance studies.

Detection of a virus by molecular methods relies on intact genomic material of a virus being present in the sample. However, the relationship between the infectivity of a detected virus and the detection of a fragment of its genome is not unambiguous. Apart from intrinsic virus characteristics, infectivity and detection of a virus depends on its stability in the sample matrix (Cook and Rze, 2004) and during sample preprocessing steps (Conceição-Neto et al., 2015). Similarly, the detection of a virus using untargeted metagenomic sequencing does not confirm its infectivity. Cell culture based infectivity essays are the golden standard to determine virus infectivity, these methods are, however, not scalable and many viruses cannot be cultured in vitro (Hamza et al., 2011). High genome coverage combined with close sequence identity to a viral reference genome with a known pathogenic phenotype are currently the strongest links between metagenomic sequencing data and disease etiology. Nevertheless, currently employed PCR based methods, which are based on genome fragment detection, suffer from the same limitations (D’Agostino et al., 2011).

It is becoming increasingly clear that integration of different data sources and experimental results is crucial for the interpretation of metagenomic sequencing experiments. Therefore, browsing of these data and visualization of relationships between genome datasets and metadata should be facilitated. In the recent years, interactive web-based data browsing and visualization tools have increased in popularity to facilitate the interaction with and the browsing through highly complicated data in a user-friendly manner. Further development of tools that facilitate interaction with and visualization of metagenomic sequencing results, such as Kronatools (Ondov et al., 2011) and Taxonomer (Flygare et al., 2016), and frameworks for so-called data analysis “dashboards”3,4,5, should make the interpretation of metagenomics experiments easier in the future.

How to cite this article: Johnson, C.K. et al. Spillover and pandemic properties of zoonotic viruses with high host plasticity. Sci. Rep.

5, 14830; doi: 10.1038/srep14830 (2015).