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while destroying infectivity, have shown useful promise for a range of applications including vaccination (B. Afrough, unpublished).

In these studies, a single intranasal dose of mDEF201 was found to exhibit a potent prophylactic activity that endured for at least 56 days. An endpoint in the duration of the protection could not be projected because treatment at -56 days resulted in only one death and caused minimal weight loss during the infection. Indeed, weight loss during infection for the -56 day treatment group was equivalent to the -1 day treatment group. Investigation of other parameters of infection (tissue virus titers, lung weights, and lung hemorrhage scores) revealed that the inhibition of these parameters with a -28 day mDEF201 treatment was quite comparable to that of a -1 day treatment. Prophylaxis starting a day before infection could effectively be achieved with doses of 105 through 107 PFU/mouse. Extended prophylaxis up to 56 days pre-infection was only investigated at the 107 dose.

Other investigators have shown that DEF201 in both mouse and human constructs are active prophylactically,,,. Kumaki et al. showed 100% protection against SARS infection in mice by a 106 dose given only at -14 days (other time points were not assessed). Thus, it is possible that prophylaxis earlier than 14 days would be protective against SARS infection. Against yellow fever virus infections in hamsters, DEF201 was effective when given 7 days prior to infection but not at -21 days. This breadth of viral efficacy indicates the potential of DEF201 to function as a truly broad-spectrum antiviral.

Remarkably, mDEF201 was effective in mice against vaccinia virus infection when given nearly two months prior to virus exposure. This indicates that Ad5-vectored IFN induced a long-term antiviral state that was completely protective with a single dose. This extended prophylaxis appears to exist in the absence of measurable serum IFN levels, since Wu et al. were only able to detect interferon alpha protein in mice treated intramuscularly with mDEF201 at 24 and 48 h, but not at 7 days (times in between were not investigated).

This study raises a number of important questions relative to the long acting protective effect of mDEF201 against vaccinia virus infection in mice. Questions such as how much interferon is produced, how long is it produced, and what cells are responsible for producing it are being asked and are currently under investigation. It has already been shown that interferon is successfully produced by the vector when administered intramuscularly to mice and the protein is detectable in the serum. From that work we also know that the levels of interferon rise very quickly (within 3-5 h), are transiently high, and come down within several days. We can infer that the interferon protein will be delivered successfully to the lung and nostrils (and have since confirmed this in a separate study, unpublished). Given that the protection lasts longer than the previously measured levels of interferon protein persist, we assume that the interferon is activating an immunological cascade that then protects the animal from infection by inducing an antiviral state. Thorough investigation of induced genes, length of induction, and localization of induction are planned, but are beyond the scope of this study.

The choice of intranasal route of administration is important, as this route has been demonstrated to bypass pre-existing immunity to the adenovirus vector. Intranasal delivery is also less invasive and easier to administer to large numbers of patients in the event of a mass infection due to a natural outbreak or intentional release. Adenovirus was selected to be the vector for interferon due to its rapid infectivity, breadth of human safety data, and facility of intranasal administration. mDEF201, administered by intranasal route, has already been shown to be effective in the treatment of SARS coronavirus respiratory infections in mice. This is the first description of adenoviral vectored interferon alpha being used, by any route, to prevent or treat vaccinia virus infections.

A limited therapeutic window of time exists for treating vaccinia virus infections with interferon-based drugs like mDEF201, and to achieve treatment higher doses of mDEF201 are needed than for prophylaxis. Indeed, low dose mDEF201 (105–106 PFU/mouse) that were effective prophylactically were not effective when administered even 6 h after infection. Higher doses (108 PFU/mouse) were required for full protection from lethality when given at +24 h, however considerable body weight loss resulted due to infection (Figure 3). The single 108 dose administered at 24 h was not as effective as daily doses of 100 mg/kg of cidofovir that acts directly on vaccinia to inhibit replication. This may be due in part to the lag time between administration of mDEF201 and the production of therapeutic levels of IFN within 6 h. Secondly, in situ produced IFN must overcome a high level of IFN system down regulation caused by the established vaccinia infection. It has been demonstrated by other investigators that both mDEF201 and DEF201 are effective as a treatment only when given within 1–2 days after virus exposure,,,. How well Ad-vectored IFN protects infected animals will depend upon each virus' virulence including the number and expression of different IFN system antagonists. However, the treatment efficacy of DEF201 in these unrelated viruses indicates real potential as a broad spectrum antiviral, and the extrapolation of these treatment windows into the clinical scenario may be significant.

In these studies antiviral activity depended on the amount of mDEF201 administered, which was the case in other published studies with unrelated viruses,,,. The production of a steady-state level of interferon may bypass the need for bolus dosing associated with traditional interferon treatment, and thus reduce toxic side effects. Nevertheless, safety of DEF201 is a consideration for eventual human use, and safety and toxicology studies are ongoing. It will need to be determined how long the IFN-induced antiviral state persists and its effects on treated individuals.

This is the first study demonstrating that surviving mice treated with mDEF201 are protected against re-infection with the same virus. This is not surprising, as mDEF201 treatment did not completely prevent vaccinia virus production in the lungs and snouts of the animals (Figures 2A and 2F), thus driving an acquired immune response. The veracity of that immune response in mDEF201 treated animals most likely was weaker than it would be in more severely infected mice, based upon weight comparisons to surviving placebos (Figure 1B).

Assuming an adequate safety profile, one can envision the use of DEF201 as a means of quickly providing significant protection to first responders and medical chain personnel confronted with a deliberate release of variola or monkeypox virus into the environment. Even if infected, DEF201 treated individuals could still acquire an immunity to the virus, as demonstrated by the present studies, thus rendering them ‘vaccinated’ against future exposure. The intranasal method of delivery of DEF201 facilitates rapid prophylaxis in people entering a suspected poxvirus environment. Moreover, DEF201 has the potential to treat a large number of unrelated viruses simultaneously, for which there are no current treatments.

Vaccinia virus systemic infections that were initiated by i.p. virus challenges can be effectively managed prophylactically with mDEF201. Liver, lungs, and spleen are major targets of this infection. mDEF201 deposited primarily either in the sinuses (5-µl volume) or in the lungs (50-µl volume) would have to generate interferon that ultimately needs to reach the liver and other organs for protection. Complete protection was afforded by the 107 dose of mDEF201 administered to the nasal sinus (Figure 2) while lower doses provided partial protection. Each dose of mDEF201 delivered to the lungs was less protective (11/30 survived) than the same dose delivered to the nasal sinus (21/30 survived) (a difference of P<0.02 by two-tailed Fisher’s exact test). Thus, the nasal sinus delivered mDEF201 was more effective in treating this systemic infection, suggesting that more interferon was produced by the mDEF201 expression or that the gene product, interferon was better distributed when produced in the nose.

The viral strains used in this study were provided by the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID), the American type culture collection (ATCC), or the Biodefense and Emerging Infections Research Resources Repository (BEI Resources) and are listed in Table 2. Samples from BEI resources were supplied as DNA extracts. DNA from viral isolates from USAMRIID and ATCC were extracted using the QiaAmp MinElute Virus spin kit (Third Edition, February 2007 version) according to the manufacturers recommended procedure (Qiagen Inc, Valencia, CA).

The experiments were conducted in accordance with Protocol 552 approved by the Institutional Animal Care and Use Committee of Utah State University. The work was performed in the Biosafety Level 2 area of the AAALAC-accredited Laboratory Animal Research Center of Utah State University in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All individuals involved in working with infected animals and cells received prior vaccination with the standard smallpox vaccine.

Synthetic peptide‐based epitope‐vaccines (EVs) make use of short antigen‐derived peptide fragments that can be presented either to T cells or B cells 58. EVs offer several advantages over other forms of vaccines, particularly with regard to safety, ease of production, storage and distribution, without cold chain issues. They also offer the opportunity to vaccinate against several pathogens or multiple epitopes from the same pathogen. However, drawbacks include poor immunogenicity and the restriction of the approach to patients of a given tissue type [human leucocyte antigen (HLA) haplotype] 59 and, as such, they need to be tailored to accommodate the natural variation in HLA genes. Although initially this was thought to be a major impediment, new technologies have made this personalized‐medicine approach feasible 60, 61. Recently, bioinformatics tools have been developed to identify putative CD4+ T cell epitopes, mapped to the surface glycoproteins of the emerging viruses LASV, NipV and Hendra 62. While these vaccine candidates still need to be experimentally tested, the approach represents an interesting and novel strategy that shows promise for vaccination and which could also address immunity in particular target populations.

mDEF201 at two different doses was administered after infection to combat a vaccinia virus infection. Preliminary studies with low dose mDEF201 (105 or 106 PFU/mouse), given at 6, 12, or 24 h after infection provided no protection from the lethal infection (data not shown). However, higher doses of mDEF201 (108 PFU/mouse) were 100% protective when administered either at 6, 12, or 24 h after infection (Table 3). A 107 PFU/mouse dose was 80–90% protective when given at 6 and 12 hours, but only 30% protective when administered at 24 hours. Survival time in the lower dosage group receiving treatment at 24 hours was significantly increased relative to the placebo control. Cidofovir was 100% protective when administered at 100 mg/kg/day for two days starting at 24 h.

Body weight changes for treatments with 100% survival during the course of the infection are shown in Figure 3 (108 mDEF201 and cidofovir). Weight loss in mDEF201 treated mice increased with longer delay to commencement of treatment, with all animals regaining their pre-challenge weight by day 18 of the experiment. Given the weight loss in the +24h group, further delays in treatment would likely result in some mortality. Mice treated daily with cidofovir starting 24h post-vaccinia challenge lost less weight.

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.

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.

Viral extracts from rabbitpox virus-infected rabbits were obtained from USAMRIID. Rabbits were bled every other day after challenge with aerosolized rabbitpox virus. Blood was collected into EDTA tubes and 100 µL of blood was used to isolate viral DNA with the BioRobot M48 (Qiagen) using the QIAamp blood kit (Qiagen version February 2003) in accordance with manufacturer's instructions. Real-time PCR was carried out with the LightCycler (Roche, Indianapolis, IN) using a pan-Orthopoxvirus assay as previously described. Briefly, the oligonucleotide primers and a minor groove binder (MGB) protein-containing TaqMan probe were designed to hybridize to conserved regions of the orthopoxvirus hemagglutinin (HA) gene; sequences have been published elsewhere. Reactions were performed on a Roche LightCycler. Virus was quantified using standards based on the cloned Orthopoxvirus HA (J7R) gene were calculated using the LightCycler software version 4.0 as described by Kulesh et al[33]. Viral titers were determined by plaque assay on Vero cells grown in Earle's modified Eagle's medium supplemented with 10% fetal calf serum.

Despite its replication-competent phenotype, LC16m8∆ is highly attenuated and shows no pathogenic effects in SCID mice (similar to replication-defective VVs, such as MVA). However, it is a comparably effective smallpox vaccine with respect to Dryvax. Moreover, LC16m8∆-based vectors induce both antibody- and cell-mediated immune responses against foreign antigens more efficiently than non-replicating VV vectors. Therefore, LC16m8∆ is superior to non-replicating VV vectors and is suitable for use in humans. We also point out that LC16m8∆ recombinants may be useful as a dual vaccine against both smallpox and pathogens targeted with the inserted genes.

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 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.

The protective immune response elicited by LC16m8∆ was compared with that elicited by Dryvax, MVA, LC16m8 and LC16m8 derivatives (m8B5R and m8dTM, both of which express the B5 ectodomain at high levels) in a mouse model. This model, in which the immunized mice are challenged with a highly pathogenic VV (the Western Reserve (WR) strain), is one of the most popular methods of evaluating the efficacy of smallpox vaccines (Figure 3). We immunized each group of mice with a single dose (104, 105 or 106 PFU) of each VV via the intramuscular (i.m.) route. We found that the level of protective immunity elicited by LCm8∆ was comparable with that elicited by Dryvax and superior to that elicited by MVA. For example, the minimal dose (104 PFU) of LC16m8∆ or Dryvax fully protected mice from lethal infection with WR, whereas mice immunized with MVA, LC16m8, m8B5R or m8dTM, lost weight and, in some cases, died. The maximum dose (106 PFU) of MVA resulted in prominent weight loss after WR challenge. It is noteworthy that immunization with LCm8∆ was more efficient than that with m8B5R or m8dTM when compared at their minimal dose. In particular, m8B5R was significantly inferior to LC16m8∆ (t-test, p = 0.005). These results suggest that B5R does not play a major role in eliciting protective immune responses in these mice. In addition, LC16m8∆ elicited protective immune responses in cynomolgus monkeys and fully protected them against lethal infection with monkeypox virus. Taken together, these data suggest that LC16m8∆ is as effective as the first-generation smallpox vaccine, Dryvax. Although several studies report that the B5 protein is the major target of EEV-neutralizing antibodies, which are significant for protection against smallpox infection, immunization with B5-deficient vaccine viruses protects animals against lethal challenge by pathogenic orthopoxviruses. In addition, some reports show that smallpox vaccines do not always induce anti-B5 antibodies, and antibody response profiles against each viral protein are highly heterologous in humans. They also concluded that the key to inducing a strong neutralizing antibody response is to elicit antibodies that recognize multiple viral proteins; these antibodies then act synergistically to provide better protection.

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)–[4]. Studies in animal models have suggested that the presence of the H1N1pdm09 M gene may increase transmissibility of the virus,[6]. 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–[11]. However, until now, estimating R required detailed outbreak investigations of human clusters,[11] 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).

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

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.

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.

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).

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.

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.

In many situations, both the case detection rate ρ and overdispersion parameter k are unknown. Interestingly, 1−F always acts as a lower bound estimate of R. An upper bound for R can be obtained if it is possible to specify an upper bound for the case detection rate and a lower bound for the overdispersion parameter k (see Text S1). We specify that corresponds to the SARS scenario with superspreading events. Figure 5 shows how precision decreases as the upper bound increases. However, even in the scenario , our approach can provide useful insights on transmissibility. For example, R is expected to be in intervals 0.20–0.25, 0.40–0.59, 0.50–0.87 for F = 80%, 60%, and 50%, respectively. For F≤46%, we can only derive a lower bound on R. For example, if F = 20%, we find that R is ≥0.8.

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.

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,–[9]. 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–[14]. 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–[23]. 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.

Infectious diseases, including zoonoses, remain the major and increasing health threat in most developing countries.1, 3, 6, 67 Even if in industrialized countries, cardiovascular diseases and cancers are considered to be the main causes of illness and death, special attention still needs to be paid to zoonotic EID.67 This statement is now well described by the ‘one health—one medicine-one world’ concept which is a worldwide strategy for expanding interdisciplinary collaboration and communications in all aspects of health care for humans and animals and the interaction with environmental factors. Also, viral hemorrhagic fevers, because of their high infectiousity and the dramatic outcome, have attracted the attention of the medical world and the public in Africa and around the world to this particular category of EID.68

However, global effort in EID surveillance and investigation is inadequately allocated. Indeed, the majority of scientific resources focus on places from where the next important emerging pathogen is least likely to originate.3 Jones et al. advocated for the re-allocation of resources to EID hotspots in lower latitudes, such as tropical Africa because of the critical need for health monitoring and identification of new potentially zoonotic pathogens in African wildlife populations, and this to be used as a forecast measure for EIDs.3, 48, 67

Like other African countries, Gabonese resources for public health and health monitoring are unequally allocated; 60% are spent at a central level. Public health services and clinical practitioners need more resources to be able to actively educate the public about the risks of repeated contacts with wildlife or other sources potentially harmful for health.48 However, Gabon could be considered as a good model to investigate the emergence or re-emergence of zoonotic EID. On one hand, Gabonese forests are a hot spot for biodiversity (wild animals and unknown pathogens) and on the other hand there is a relatively small population (1.5 million of habitants), which is often in contact with surrounding wildlife. Also, the CIRMF, a research center advantageously located, offers high quality researchers and facilities that study pathogens and wildlife ecology. Altogether the combination of these factors should help to better understand the mechanisms of contact and transmission of new pathogens from wildlife to human, the emergence of zoonotic EID and the breaking of species barriers by the pathogens. Indeed the emergence of infectious diseases in wildlife is a continuous and ongoing process. The factors that give rise to zoonotic EID, such as ecosystem perturbations and modifications, climate changes, migrations of reservoirs species, pathogens or vectors, and intrinsic changes of pathogens may be of natural origin or due to human influences.17 To understand the underlying mechanisms that govern relationships between reservoir species, ecological factors and environmental perturbations with the emergence, transmission and dissemination of viral diseases in tropical forests, the CIRMF wishes to set up permanent surveillance of the health of the population by the establishment of (1) a network reference laboratories (WHO based reference laboratories including CIRMF, Pasteur Institute Network and other National laboratories or universities based) and (2) a Health Ecology Observatory (that is,. The CIRMF's Scientific Station in la Lopé National Park). Such measures will compile data from the public health system with the monitoring of the emergence of new pathogens. The collected information would favor better outbreak risk appraisal in the Gabonese human population as well as for the entire Congo basin region.