Viral bioterrorism: Learning the lesson of Ebola virus in West Africa 2013–2015

Virus Research 210 (2015) 318–326

Contents lists available at ScienceDirect

Virus Research journal homepage: www.elsevier.com/locate/virusres

Review

Viral bioterrorism: Learning the lesson of Ebola virus in West Africa 2013–2015 Orlando Cenciarelli a,b,∗,1 , Valentina Gabbarini c,1 , Stefano Pietropaoli d , Andrea Malizia a,b , Annalaura Tamburrini c , Gian Marco Ludovici c , Mariachiara Carestia a,b , Daniele Di Giovanni a,b , Alessandro Sassolini b , Leonardo Palombi b,e , Carlo Bellecci a,b , Pasquale Gaudio a,b a

Department of Industrial Engineering, University of Rome Tor Vergata, Rome, Italy Didactical Board of the International Master Courses in Protection Against CBRNe events, Department of Industrial Engineering and School of Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy c International Master Courses in Protection Against CBRNe events, Department of Industrial Engineering and School of Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy d Department of Science, University of Roma 3, Rome, Italy e Department of Biomedicine & Prevention, School of Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy b

a r t i c l e

i n f o

Article history: Received 30 June 2015 Received in revised form 2 September 2015 Accepted 3 September 2015 Available online 8 September 2015 Keywords: Ebola virus Bioterrorism BWA Hemorrhagic fever West Africa

a b s t r a c t Among the potential biological agents suitable as a weapon, Ebola virus represents a major concern. Classified by the CDC as a category A biological agent, Ebola virus causes severe hemorrhagic fever, characterized by high case-fatality rate; to date, no vaccine or approved therapy is available. The EVD epidemic, which broke out in West Africa since the late 2013, has got the issue of the possible use of Ebola virus as biological warfare agent (BWA) to come to the fore once again. In fact, due to its high case-fatality rate, population currently associates this pathogen to a real and tangible threat. Therefore, its use as biological agent by terrorist groups with offensive purpose could have serious repercussions from a psychosocial point of view as well as on closely sanitary level. In this paper, after an initial study of the main characteristics of Ebola virus, its potential as a BWA was evaluated. Furthermore, given the spread of the epidemic in West Africa in 2014 and 2015, the potential dissemination of the virus from an urban setting was evaluated. Finally, it was considered the actual possibility to use this agent as BWA in different scenarios, and the potential effects on one or more nation’s stability. © 2015 Elsevier B.V. All rights reserved.

Contents 1.

2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 1.1. Bioterrorism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 1.2. Ebola virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 Ebola virus as potential bioterrorist threat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 2.1. Ebola virus as potential biological warfare agent: learning from Africa 2013–2015 outbreak . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 Possible worst case scenarios of intentional Ebola virus release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 3.1. Airport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 3.1.1. The Ebola virus hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 3.2. Underground station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324 3.2.1. The Ebola virus hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324 3.3. Cruise ship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324

∗ Corresponding author at: Department of Industrial Engineering, University of Rome “Tor Vergata”, Via del Politecnico 1, 00173 Rome, Italy. E-mail address: [email protected] (O. Cenciarelli). 1 These authors contributed equally to the manuscript. http://dx.doi.org/10.1016/j.virusres.2015.09.002 0168-1702/© 2015 Elsevier B.V. All rights reserved.

O. Cenciarelli et al. / Virus Research 210 (2015) 318–326

4.

319

3.3.1. The Ebola virus hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

1. Introduction 1.1. Bioterrorism Bioterrorism is a criminal act that provides the deliberate use of biological agents such as viruses, bacteria or toxins as harmful means, causing diseases or death in humans, animals or plants (Centers for Disease Control and Prevention (CDC), 2015a). Biological agents can be spread through air, water or food and they could be modified to improve their capability to cause disease and to make them resistant to drugs. Depending on the severity of disease that they cause and on their ability to spread, biowarfare agents (BWA) are classified by the Centre for Disease Control and Prevention (CDC) into three categories: A–C (Centers for Disease Control and Prevention (CDC), 2015a). Category A pathogens would produce the greatest risk during a bioterrorism attack (Bray, 2005a) because they can be easily spread and transmitted from person to person; their release might cause public panic and require special actions for public health (Centers for Disease Control and Prevention (CDC), 2015a). Bacillus anthracis, Brucella spp., Clostridium botulinum, Yersinia pestis, Francisella tularensis, Variola virus and Ebola virus are the most likely biological agents to be used with bioterrorism aims. The phenomenon of bioterrorism represents a growing major threat of modern civilization, although examples of the use of biological agents as weapons date back thousands of years ago (Cenciarelli et al., 2013). In fact, if on one hand scientific and technological progress in molecular biology and genetic engineering fields has brought important benefits for mankind, on the other hand new knowledge could be exploited with terrorist purposes, determining serious repercussions on international communities (Bray, 2005b; Van Aken and Hammond, 2003). In recent times, the use of biological agents by terrorist organizations has become a worrying reality. Therefore, an assessment of the risks associated with the use of pathogenic microorganisms is required in order to develop countermeasures to limit unexpected scenarios, characterized by mass destruction, and to assure a ready response to them (Cenciarelli et al., 2013; Bray, 2003). Biological warfare and bioterrorism are very complex issues because many agents can be used and widespread, affecting environment and people. Two are the main factors in a biological event: one or more pathogens implied during the attack and the vehicle for their dissemination. Immediate diagnosis is very difficult due to the high spread ability, lethality, invisibility and difficulty in short-term detection (Cenciarelli et al., 2013). This is why the most effective resources to avoid critical bioterrorist episodes are prevention and collaboration. Both health intelligence and specialized medical units should cooperate to ensure rapid and effective response (Morse, 2007). A bioterrorist attack requires a large amount of biological agent to cause diseases in a target population (Cenciarelli et al., 2013). To be an effective weapon, a microorganism must first be highly pathogenic to humans. In addition, it should have a number of features including the ability to cause serious and predictable diseases in a short time and to resist outside the host organism for a sufficient period to infect a victim (Utgoff, 1993). Moreover, it should be easy to disseminate and difficult to detect through currently available techniques (Carus, 1991). The potential use of bioweapons represents a great concern, due to the serious impact that may cause and the lack of effec-

tive tools for detection and identification of the biological agent used (Cenciarelli et al., 2013; Carestia et al., 2014; Cenciarelli et al., 2014a). Bioterrorism detection represents a major issue: a possible bioterrorist attack may be announced or unannounced. In the first case primary health care providers and law enforcement agencies should be on alert, preparing isolation facilities and improving rapid response to contain the infection and take care of the victims; but if a bioterrorist attack occurs without any announcement, depending on the incubation period, the infectivity and the lethality of the biowarfare agent utilized, unusual diseases and death could spread in the community before anyone can really understand the situation (Üstun and Özgurler, 2005). Part of government policy in biological warfare and in terrorist groups is the manipulation and release of pathogens (Jansen et al., 2014). The real threat of a largescale bioterrorist attack makes the defense against bioweapons a priority in terms of security. 1.2. Ebola virus Ebola virus is the etiological agent of a hemorrhagic fever (EHF) in humans and non-human primates (monkeys, gorillas and chimpanzees) and in other wild animals; EHF is endemic in central Africa regions. Ebola virus, together with Marburg virus, is a Filovirus belonging to the family of Filoviridae, order of Mononegavirales (Feldmann and Geisbert, 2011). Ebola virus was first recognized in 1976, when in the Northern Zaire (actual Democratic Republic of Congo) and in the Southern Sudan EHF appeared. Five species of Ebola virus have been identified: Ebola virus (EBOV) and Sudan virus (SUDV), discovered in 1976; Reston virus (RESTV), discovered in 1989; Taï Forest virus (TAFV), discovered in 1994 and Bundibugyo virus (BDBV), discovered in 2007 (Bukreyev et al., 2014). All species of Ebola viruses are harmful to humans with the exception of Reston species: it originated in the Philippines and it does not cause disease in humans; however it can be fatal to monkeys and some evidences suggest also a pig-to-human transmission through contact, with the opportunity to become more communicable, by mutating in susceptible people (i.e. immune-compromised) (Morris, 2009). Filovirus viral particles are pleomorphic, thus they can take different shapes: long, branched, circular, as well as filaments shaped like “6” or “U” (Centers for Disease Control and Prevention (CDC), 2015b). Viral particles show a common diameter of 80 nm, while their length is variable, reaching 14 ␮m. Viruses are enveloped by a lipid membrane that encloses the ribonucleoprotein complex, including four of the seven structural proteins (NP, VP30, VP35, L protein) and viral genome, which is composed by a nonsegmented, negative-stranded RNA, approximately 19 Kb long. It consists of seven genes linearly arranged (Feldmann and Klenk, 1996) encoding 11 proteins. VP40 in association with VP24 serves as the matrix protein and mediates particle formation. GP (glycoprotein) is the only transmembrane surface protein of the virus; it is important to mediate binding to cellular receptors, such as ␤1integrins, and subsequent fusion with cellular membranes and it is the major viral antigen (Feldmann and Geisbert, 2011). Moreover, four soluble glycoproteins are produced during the infection by Ebola virus: sGP, delta peptide (-peptide), GP1 , and GP1,2 . These proteins seem to be involved in viral pathogenesis, mainly during the target cell activation phases (Wahl-Jensen et al., 2005). Viruses survival is closely related to the presence of a specific host (the natural virus reservoir) that allows viral replication

320

O. Cenciarelli et al. / Virus Research 210 (2015) 318–326

not developing the disease. Humans are not the natural reservoir for Filoviruses, but they can be infected when a contact with infected hosts occurs. For this reason EHF is considered a zoonotic disease; however, in the case of Ebola virus, interpersonal transmission is possible (Centers for Disease Control and Prevention (CDC), 2015b). New evidence implicates bats as the natural reservoir hosts for Ebola virus (Centers for Disease Control and Prevention (CDC), 2015c). Transmission occurs by direct contact with body fluids (saliva, feces, urine and semen) of an infected person or animal (Feldmann and Geisbert, 2011); airborne transmission among humans has not been reported yet, but literature shows that Ebola virus can infect the respiratory tract of non-human primates through aerosolized virus particles administration (Johnson et al., 1995; Weingartl et al., 2012). Host infection can occur through mucosae, wounds and abrasions on the skin or by parenteral route. Ebola virus can infect a wide range of cell types: monocytes, macrophages, dendritic cells, endothelial cells, hepatocytes and many types of epithelial cells. After infection, the incubation period ranges from 2 to 21 days and clinical manifestations are represented by weakness, myalgia, fever and chills. Subsequently symptoms involve gastrointestinal, respiratory, vascular and neurological systems (Colebunders and Borchert, 2000). Different species of Ebola virus cause different clinical symptoms. The peak of the disease is characterized by hemorrhagic manifestations; visceral hemorrhagy were found during post-mortem analysis. In later stages, metabolic disorders, convulsion, shock and coagulopathy are shown. Patient with fatal disease die for hypovolemic shock and multiorgan failure (MOF); in non-fatal cases convalescence is long and often associated with conseguences, such as myelitis, hepatitis, psychosis (Medecins Sans Frontieres (MSF), 2015). Ebola virus case-fatality (CF) in human is variable and it depends on the species or strain. An important key of Ebola virus pathogenesis is the inhibition of the type I interferon response: this results in an immunological disorder that partially provides to disease progression. EVD diagnosis is initially based on clinical evaluations, however its identification often results difficult because some of the first symptoms are common with other diseases, such as malaria, that are endemic in areas where Ebola virus is present. There is only one WHO approved rapid test for the screening of people which show some of the EVD-related symptoms (ReEBOVTM antigen rapid test); however the most diffused methods for the diagnosis of EVD are laboratory measurement of host-specific immune responses to infection (by IgM capture assay), the detection of viral particles (by antigen-ELISA) and, of course, specific viral genomic amplification (by PCR) (Centers for Disease Control and Prevention (CDC), 2015d). A virus-specific therapy does not exist: current treatment strategies are symptomatic and supportive and include isolation, antipyretic and antibiotics administration, fluid substitution to maintain effective blood volume and electrolyte balance. No approved vaccines against Ebola virus are actually available. The absence of effective therapies and vaccines, along with high CF rate, imply that Ebola virus is nowadays considered an important public health pathogen and bio-threat of category A (Feldmann and Geisbert, 2011; Centers for Disease Control and Prevention (CDC), 2015e). In fact, with a high CF rate around 40–50% (that, in some outbreak, reached 90%), it is the most lethal of human pathogens (Kagan, 2005). EVD was and still is a critical plague for the population of equatorial Africa; on March 23, 2014, the World Health Organization (WHO) communicated a new outbreak of EVD, which started in December 2013 in the Republic of Guinea (Gatherer, 2014). While previous EVD outbreaks involved only rural areas that were easily isolated (with the exception of the only one urban area previously affected by EVD, Kikwit (Democratic Republic of Congo), in 1995; however in that case the outbreak remained confined) (Sanchez et al., 1995), the recent outbreak spread to neighboring states including large cities such as the capital of Guinea. The access to

air travel could lead to a further spread of Ebola virus infection all over the world. While an individual that can board an aircraft during the symptomatic phase of EVD does not represent a very high risk, thanks to different types of medical controls before boarding, a disease incubating passenger, who still is in the asymptomatic phase of the infection, may be a worrying contagion danger. The Ebola emergency in West Africa accelerated the efforts for development of both effective treatments and vaccine. The importance of a working vaccine is crucial to prevent a further spread of EVD and future possible outbreaks; this is the reason why one of the priorities of WHO is to carry out further investigation on the most promising candidates through human testing (World Health Organizzation (WHO), 2015a). Two vaccines are currently in clinical trials: cAd3-ZEBOV (NIAID/GSK) and rVSV-ZEBOV (NewLink Genetics/Merk). The first one is an experimental vaccine developed collaboratively by scientists at the NIAID Vaccine Research Center (VRC) and at Okairos, an international biotechnology company acquired by GSK. cAd3-ZEBOV vaccine candidate is based on a recombinant non-replicating vector (chimpanzee-derived adenovirus 3) engineered to deliver Ebola virus genetic material from two virus species, EBOV and SUDV. Preliminary studies on nonhuman primates and phase I clinical trials assured the safety of the vaccine. Analyses demonstrated the presence of anti-Ebola antibodies within four weeks of receiving the vaccine and CD8 T-cell response. This vaccine is currently in phase II trials in Cameroon, Ghana, Mali, Nigeria and Senegal and entered in phase III trials in march 2015 under the NIAID supervision (National Institute of Allergy and Infectious Diseases (NIAID), 2015; U.S. National Institutes of Health, 2015; World Health Organization (WHO), 2015x). rVSV-ZEBOV vaccine candidate, originally developed by Public Health Agency of Canada, uses weakened and genetically modified vesicular stomatitis virus (rVSV) to express the glycoprotein (GP) of EBOV to induce an immune response against Ebola virus. Preliminary studies on non-human primates show that this vaccine is effective in protection against EBOV infection thanks to the production of anti-Ebola antibodies (Marzi et al., 2011). A single dose of the vaccine, administered one month before infection, protected 100% animals from a lethal dose of EBOV. After a successful phase I trials, this vaccine is currently undergoing phase II and will soon enter in phase III trials in EVD affected countries (World Health Organizzation (WHO), 2015a). WHO classifies putative drugs according to their stage of evaluation in 5 groups (World Health Organizzation (WHO), 2015b); main are groups A and B. Treatments categorized in the group A include drugs currently under evaluation in formal clinical trials; in the group B drugs that have been prioritized for testing in human efficacy trials but for which such trials are not yet underway are included. At the time, only a drug is included in the group A, Favipiravir, while in the group B are categorized six experimental drugs. Favipiravir (Toyama Chemical, Japan) is a small antiviral molecule able to inhibit the RNA-dependent RNA polymerase, currently used to treat influenza infections (Furuta et al., 2013; Oestereich et al., 2014). Clinical efficacy trial against Ebola virus began in Guinea in December 2014 and it is now under investigation. Among the drugs included in the group B, Zmapp (Mapp Biopharmaceutical) seems to be the most promising. It is a cocktail of three monoclonal antibodies produced in tobacco plants currently used to treat 8 patients on compassionate grounds. Phase I safety study started in January 2015 and phase II is currently ongoing with a multi-country, multisite randomized controlled trial opened to enrollment in Liberia and the United States in February 2015 and in Sierra Leone in March 2015 that will be further extended to Guinea. No data on efficacy is available yet (U.S. National Institutes of Health, 2015; World Healt Organization, 2015x). WHO categories C–E includes respectively drugs that have already been given to patients for compassionate reasons or in ad

O. Cenciarelli et al. / Virus Research 210 (2015) 318–326

hoc trials; drugs that demonstrate promising anti-ebola activity in vitro or in mouse models, but for which additional data should be generated prior to proceeding to clinical trials and drugs that had been prioritized or considered for prioritization and have now been de-prioritized based on new data or more detailed analysis of old data (World Health Organizzation (WHO), 2015b).

2. Ebola virus as potential bioterrorist threat Ebola virus is classified as Biological Level 4 (BSL4) agent by Health and Safety Executive (HSE) and as category A biological warfare agents (BWA) by Centers for Disease Control and Prevention (CDC) (Cenciarelli et al., 2014b). It can produce the greatest impact if used in bioterrorist attacks due to their potential widespread dissemination, causing severe and often fatal diseases (Bray, 2005a). This implies severe controls for diagnostic and for research laboratories to prevent the access to the virus by terrorist organizations: Ebola virus causes not only a highly contagious infection, an event already very worrying, but it may also be used intentionally, representing a serious threat as potential biowarfare agent (Üstun and Özgurler, 2005). Obtain and disseminate Ebola virus is not simple however, and requires the recruitment of individuals attempting to collect virus samples from a dead infected animal or from a patient, by collaborating with medical staff and secretly preserve the samples to be used later with insulting intent. Similarly, transfer of the samples from the site of acquisition to a predetermined place, is also needed. Moreover, cutting-edge laboratories with sophisticated protection devices, as well as special procedures to prevent infection of all personnel who handle it or might inadvertently enter into contact with it, are necessary; such a laboratory is unlikely to pass unnoticed (Berger and Shapira, 2002; Leitenberg et al., 2012). Generally, bioterrorist risk is therefore low, but it is not to be excluded. In 1993, the Japanese “Aum Shinrikyo” cult, led by Shoko Asahara, sent in Zaire (now Democratic Republic of Congo) a group of 16 people, including doctors and nurses for a false medical mission, used as a front to study Ebola virus as much as possible and to obtain samples to be used in terrorist attacks. In the early months of 1994, cult’s doctors were also cited by the Russian radio in a debate on the potential use of Ebola virus as a biological weapon (Olson, 1999). Thus, it is clear that Ebola virus introduction in non-endemic areas for offensive purposes is a threat to international security already taken into account by terrorist groups, and present in biological warfare programs of some states, including the Former Soviet Union (Groseth et al., 2007), who has studied and developed natural strains of Ebola virus to be used in biowarfare (Leitenberg, 2001). However, it is important to consider that the virus itself is not a weapon, but a biological agent that can become offensive after weaponization process. This implies that only experts in virology or genetic engineering might be able to turn it into a dangerous instrument of aggression (Teckman, 2013). Information on how to weaponize the simplest biological agents into BWA is of easy access: manuals on reproduction and growth of BWA are easily available on the internet and thousands of copies are sold each year (Katz, 2004; Garrett, 2001). To date, studies on Ebola virus manipulation, which is on its growth, storage and dispersion, have already been published, and allow understanding the processes needed for virus weaponization, even if complicated (Teckman, 2013). The high lethality after infection in humans, together with the absence of prophylactic and therapeutic regimens (Feldmann et al., 2003) and the potential transmission through mouth and conjunctivas (Lazarus and Decker, 2004) or air route (Johnson et al., 1995), demonstrated by the virus transmission in non-human primates through aerosol, makes Ebola virus a putative and extremely dangerous BWA (Cenciarelli et al., 2014b). The increasing frequency, in which Ebola virus is naturally occurring,

321

is raising concerns about the accessibility to such virus by terrorists and the recruitment of experts to prepare and intentionally use the virus with harmful purposes (Teckman, 2013). Great concern is aroused by terrorist groups, which members may perform terrorist actions using Ebola virus as a low-tech mean of massive annihilation, by global infection simply using human carriers to intentionally disseminate the virus through air transportation system, without sophisticated strategies. Recent articles suggest the possibility for terrorist to send operative groups into an Ebola outbreak area to intentionally expose themselves to the virus for this purpose. Once infected, they could easily spread the virus. In this regard, intelligence reports revealed that terrorists, including ISIS members, are searching BWA, although not specifically Ebola virus. Such a deadly virus could however be taken into account by any terroristic group or organization, representing a global health and security threat (Dorminey, 2014; Dvorin, 2014). 2.1. Ebola virus as potential biological warfare agent: learning from Africa 2013–2015 outbreak The study of EVD outbreaks can provide essential information to design security protocols and possible infection scenarios in order to react properly to a possible intentional release of a weaponized virus form during a bioterrorist attack. The magnitude of the EVD outbreak in West Africa in 2013–2015 is unprecedented in the history of mankind. Among the EVD outbreaks that have occurred so far, in fact, the largest was certainly the one that took place in Uganda in 2000. On October 8, 2000, the Ministry of Health (MoH) in Kampala reported unusual clinical cases that suggested EVD. A few days later, the National Institute of Virology in Johannesburg (South Africa), identified infections by Ebola virus in biological samples of a group of patients that included also health-care workers, thus confirming the initial clinical suspicions (World Health Organization (WHO), 2001). Since then, a surveillance system was immediately arranged in order to determine the outbreak extent and gravity, as well as to identify the main sites of diffusion, and to detect presumptive cases as soon as possible. To achieve this purpose, it was necessary to address to the coordination and logistical supports of the WHO, and to alert and train the whole population. On January 23, 2001, 425 suspected cases of EHF, including 224 deaths (CF rate = 53%) (World Health Organization (WHO), 2015y), were recorded in the three main affected districts: Gulu (393 cases), Masindi (27) and Mbarara (5). The areas interested by the outbreak stretched on a surface of approximately 31,000 square kilometers; involving about 1.8 million people (World Health Organization (WHO), 2001). It is reasonably a different matter whether the intentional spread of Ebola virus occurs in urban areas with a density of population far higher than the one affected in Uganda. The devastating effects of such an event are shown us by the EVD outbreak affecting the West Africa since December 2013 (Baize et al., 2014) and considered the most serious ever documented (Fig. 1). The current outbreak of EVD in West Africa, in fact, represents the first one that has been reported also in large urban sites as Conakry, the capital of Guinea (Gatherer, 2014; Dixon and Schafer, 2014). Furthermore, the constant movement of people among different countries undoubtedly increases the extent of the outbreak. “Even the dead are moving” affirmed Michel Van Herp, epidemiologist from Médecins Sans Frontières (MSF), referring to the common funeral practices that require transport of bodies in the burial sites (The Lancet, 2014). The first victim of Ebola virus in Guinea was a businessman returning from a trip in Dabola, who died the day after symptoms manifestations. The man’s body was taken to his native village, Watagala, located in the north of Dabola. His four siblings, also of Conakry, and four mourners who travelled with the body, were all tested positive for Ebola virus (Guineenews, 2015). Since

322

O. Cenciarelli et al. / Virus Research 210 (2015) 318–326

Fig. 1. Confirmed cases and deaths reported in all outbreaks since Ebola virus discovery in 1976. Year, country and virus strain are reported. EBOV, Ebola virus; SUDV, Sudan virus; BDBV, Bundibugyo virus; DRC, Democratic Republic of Congo; CDI, Côte d’Ivoire. Updated 23 August 2015 (Dorminey, 2014; World Health Organization (WHO), 2015c; Centers for Disease Control and Prevention (CDC), 2015f; World Health Organization (WHO), 1996).

Table 1 Intense-transmission countries involved in EVD outbreak 2014 in West Africa. Country

Reporteda

Number

Guinea

Cases Deaths Cases Deaths Cases Deaths Cases Deaths

3792 2527 10672 4808 13541 3952 28005 11287

Liberia Sierra Leone Totals

a Reported includes confirmed, probable and suspected cases or deaths. Updated 23 August 2015 (World Health Organization (WHO), 2015c).

then, 3792 cases (between confirmed and presumptive) of EVD have occurred in Guinea, including 2527 deaths (CF rate = 66.6%) until August 23, 2015 (World Health Organization (WHO), 2015c). To date, in addition to Guinea, two other nations have been interested by an intense transmission of Ebola virus: Sierra Leone and Liberia, with more than 28,000 reported cases of EVD and almost 8000 reported deaths until August 23, 2015 (Table 1) (World Health Organization (WHO), 2015c). The typology of involved urban areas is causing complexity in controlling the virus spread, with peaks that touch almost 130 more cases from day to day as well. The arrival of EVD for the first time in a capital city as Conakry, with more than 2 million inhabitants and such a rapid spread of the epidemic have brought to the fore the issue of bioterrorism.

A potential spread of the virus via aerosols cannot be excluded; experimental data suggest that virus dissemination by aerosol in non-human primates may putatively occur (Johnson et al., 1995). Inhalation is the most dangerous route of transmission that terrorists could exploit (Bray, 2003). The demonstration that inhalation by Rhesus monkeys of low doses of virus (400 PFU) contained in droplets administered via aerosol system may cause a disorder that leads quickly to death, underlines the importance to take suitable countermeasures in order to prevent the potential spread of Ebola virus by air (Johnson et al., 1995). Johnson’s experiment reveals, in fact, that aerogenic Ebola virus infection is a possible threat (Üstun and Özgurler, 2005). The potential use of Ebola virus as a bioweapon by terrorist groups is undoubtedly a source of panic, due to its lethality and to the strong impact it could have on social and economic country life. If the virus reaches urban areas, to not underestimate the situation is essential to avoid catastrophic effects of what should be considered immediately as a medical emergency. However, the intentional release of Ebola virus in an urban area requires several days before the first case of EVD will occur. Moreover, due to the non-specificity of initial symptoms and the improbability of such a disease, it must be remembered that an infection by Ebola virus will be unlikely diagnosed in the first affected patients in non-endemic sites. Only when more similar cases will be identified in a short period of time thoughts are focused on the occurrence of a bioterrorist attack (Centers for Disease Control and Prevention (CDC), 2001). Obviously, the awareness of the attack will be more difficult if affected people

O. Cenciarelli et al. / Virus Research 210 (2015) 318–326

are dispersed in different places and manifest the disease far from the area of first dissemination (Bray, 2003). Thus, the virus spread in areas with high density population is not the only concern; nowadays, world is more interconnected than ever (Hill-Cawthorne and Gilbert, 2014). Air travel could spread the virus all over the world, with important effects on sanitary level, as well as from a psychological point of view (Cenciarelli et al., 2014b). For these reasons, no matter how far is the outbreak and how likely is its spread into other areas, but such a situation will always create a great concern and anxiety worldwide (Greub and Grobusch, 2014), justified by the virus lethality and the lack of approved treatments and vaccines. For these reasons, pandemic fear has interested all the world (Isaacs, 2015). As in Conakry, special response plans are needed to control the outbreak, by providing for the implementation of countermeasures to avoid virus transmission in the affected countries and to prevent its spread to neighboring countries through surveillance, preparedness and response measures based on medical doctors and virologists expertise. The improvement of measures to prevent and detect suspected cases, as well as psychosocial support of affected populations is mandatory (World Health Organization (WHO), 2015d). However, a question that must be asked is: in the case of a bioterrorist attack that uses Ebola virus, are the world structures of bio-containment BSL-4 (Bio-Safety Level 4) available in a sufficient number to avoid the unpredictable virus spread and to isolate all suspected cases? The fast spread of West Africa outbreak teaches us that much effort will be needed to put an end to such a severe scenario. 3. Possible worst case scenarios of intentional Ebola virus release Each scenario using BWA must be divided in two different hypotheses: a) Overt attack. In this first hypothesis people take awareness that they have been infected with a BWA, so in this case a prompt intervention of authorities could restrict the number of infected people and with an efficient isolation and sanitation protocol, avoid further spread of the infection. This attack will then result in massive panic in the community but only a modest level of infection will be obtained. b) Stealthy attack. A stealthy attack would imply BWA release without anyone else realizing it. This strategy would have a postponed impact but could lead to a probable bigger spread of the infection, implying repeated attacks exploiting the incubation period of the BWA, if it is long enough. The choice between two strategies depends on the purpose of the terrorist organization: if the aim is to trigger an immediate panic and fear reaction in the population, the overt attack would be the best choice, but if their aim is to start an epidemic they would choose the stealthy type of attack, which would give better chances to spread the disease. 3.1. Airport Airports are maybe the most sensitive sites for a bioterrorist attack: every day thousands of people walk through each important international airport; everyone of them could be a potential target for a pathogen release. Moreover, the release of an infectious pathogen in an airport can maximize the bio-agent impact on the community and start different simultaneous outbreaks around the world minimizing the efforts. The most interesting and dangerous scenario implies that one person among the employees introduces clandestinely a significant amount of biowarfare agent into the

323

airport. Due to the frequent and close contact with people and a large availability of possible vehicles of infection, the best situation for the terrorist would be to work as bartender, waiter or in catering industry. A large number of waiting passengers spend their time in airports bars and restaurants, therefore each one of them could become an infected carrier of the pathogen around the world. Despite the possible extent of such a catastrophic scenario, there are many difficult steps for a terrorist to overcome in this situation: (a) airports are common hot spots for terrorist attacks and other illegal trafficking: this implies a large number of security agents and tight security protocols; however most of the attention is focused on drugs and explosive devices identification; (b) carrying an highly infective bioweapon in significant amount into an airport it is not a simple issue: biological agents must be handled with attention and they generally must be stored at low temperature and often in specific supportive condition to avoid their degradation; (c) according to the nature of the biological agent its transmission and administration can be very different: airborne agents are the most dangerous within bioweapons because they can be easily spread in a closed environment, while biological agents that imply close contact or parenteral transmission are more difficult to use in a massive infection attack; (d) each biological agent shows a different incubation period, therefore, the longer the incubation period lasts, the more chances there are of infecting people and the possibility for the disease to spread.

3.1.1. The Ebola virus hypothesis According to this scenario Ebola virus could be a quite good candidate, showing pros and cons. Due to the recent spread and intensity of the 2013–2015 EVD outbreak in West Africa and the global concerns about its spreading, the use of Ebola virus to perform a terrorist attack will result in huge public panic maximizing the terroristic effect of the attack. Ebola virus presents some features useful for this kind of bioterrorist attack: (a) it has a high case fatality rate (40–50%, reaching peaks of 90% depending on viral strains); (b) there is no approved vaccine or therapy available; (c) it has a quite long incubation period that would allow a continuous virus release in the airport for several days, before the diffusion being overt. The virus can survive for several hours at room conditions (between 20 and 25 ◦ C and 30–40% relative humidity) on objects or surfaces, especially in the dark (Sagripanti et al., 2010). Filoviruses were reported to be able to survive for weeks in blood or on contaminated surfaces at low temperatures (4 ◦ C); when dried in tissue culture media and stored at 4 ◦ C, Ebola virus survived for over 50 days (Piercy et al., 2010). The most challenging steps for virus release in this scenario are virus introduction and storage inside the airport, and the transmission of the infection. While the first problem depends on the security level of the airport, the second one could represent a real obstacle for spreading. Ebola virus transmission usually implies direct contact with concentrated virus solutions; infection occurs from direct contact through broken skin or mucous membranes with the blood, or other bodily fluids or secretions (feces, urine, saliva, semen) of affected people. Infection can also occur if broken skin or mucous membranes of a healthy person come into contact with objects, such as soiled clothing, bed linen or used needles, that were contaminated with EVD patient’s infectious fluids (World Health Organization (WHO), 2015e). There are no evidences of person to person transmission through air or ingestion, nevertheless studies on rhesus monkeys and guinea pigs showed that it is possible that pulmonary, nasopharyngeal, oral or conjunctival exposure to concentrated airborne droplets could lead to host infection (Jaax et al., 1995). In order to increase the efficiency of infection, Ebola virus weaponization should be required; it is a difficult, dangerous

324

O. Cenciarelli et al. / Virus Research 210 (2015) 318–326

and very expensive process, that requires BSL-4 laboratory facility and equipment and highly specialized personnel. Although the use of Ebola virus as BWA in such scenario seems to be improbable, security levels in airports and continuous controls not only on passengers but also on staff must be improved to avoid a putative large scale infection due to a terrorist attack. Moreover, different technology of active and passive biological agents’ detection devices should be involved in continuous control of airport environment to detect a possible bioterrorist attack in a short time and tactical emergency response teams, with specific training, should be located in each major airport. 3.2. Underground station Underground stations and trains represent a sensitive target to terrorism: as they are used by many people every day and a potential attack can have a great impact on the community. On March 20, 1995, in Japan, a terrorist group performed a coordinated chemical attack on Tokyo subway; during this attack liquid Sarin was released on the trains and in the train stations affecting passengers that came in contact with it; fortunately only 12 people were killed while more than a thousand resulted to be intoxicated; if Sarin had been released in aerosol form it would have been very much worse. On March 11, 2004, in Madrid, 191 train passengers were killed and more than two thousand injured by explosive devices in a terroristic attack; the following year (July 7, 2005) a series of four coordinated suicide terrorist attacks using improvised explosive device (IED) in London underground killed 52 civilians and injured more than 700. Such events teach that the real power of a bioterrorist attack is not the number of casualties that can be minimal also, but the great psychological impact resulting all over the world (Raevskiy, 2014). Underground trains provide a good environment for bioterrorist attack: (a) there are no particular controls to enter into an underground station and security is very low, therefore transport of a relevant amount of ceiled infective agent would go unnoticed; (b) on rush hours trains are very crowded with people staying in close contact; (c) the limited and enclosed space would offer an ideal environment for the release on an aerosolized agent; (d) each underground station hosts a continuous flux of coming and going passengers every day, offering many opportunities of infection in a short amount of time; (e) the air flow generated by the trains may spread the pathogen to other underground stations. 3.2.1. The Ebola virus hypothesis The use of Ebola virus as a BWA in this scenario would not be the best choice. The best chances to obtain Ebola virus infection are by direct contact, or by using high concentrated aerosolized virus. Both these options have different complications. As aforementioned before, the virus survival outside the host is very variable depending on several conditions. However, a strategy that exploits virus crystallization on surface would be probably quite useless: the virus would be continuously exposed to light and to temperatures (often higher than 25 ◦ C). According to this, the best scenario for an underground station or train BWA release using Ebola virus would imply virus aerosolization process, also using the air conditioning system, which requires virus weaponization. Another way for the virus release could be directly spraying high concentrated virus solution towards a target face exploiting nasopharyngeal, oral or conjunctival exposure to airborne droplets as route of infection. However this method would probably compromise the undercover terrorist, and would be difficult for him to infect a significant number of people. Certainly, an overt attack using Ebola virus would be useless except for the panic generation, because it will be prompted followed by isolation protocol by the authorities, and it will probably

lead to the blocking of any spread of the infection and a drastic potential lethal cases reduction. The air conditioning system could be an important resource to detect a possible airborne attack in underground stations: the use of passive automated detection devices, designed and programmed to execute quick and frequent controls, would be the best strategy to detect the release of BWA in short time. These devices should require minimum maintenance by specialized technicians and results of each detection should be automatically sent through a secure network to a team expressly hired. While nowadays different rapid detection devices for several BWA are available, till now the identification of Ebola virus has been performed by specific RT-PCR protocols: this procedure is the most accurate but it’s difficult to automate and requires at least two hours. Recently WHO approved the use of ReEBOVTM Antigen Rapid Test Kit for airport screening of passengers which show some of the EVD-related symptoms. This test has a 92% of confidence and if positive should be followed by specific RT-PCR for Ebola virus genome detection. ReEBOVTM antigen rapid test kit screens blood samples for the presence of Ebola virus VP40 antigen and for this reason it requires only 15 min to run (World Healt Organization, 2015z). This detection strategy could be exploited to create an automated device for rapid detection of Ebola virus in concentrated air flows. The detection of Ebola virus should result in complete lockdown of the sector involved and in the stop of air flow system to prevent further spread. To respond to a possible threatening scenario, emergency units should be deployed near crucial main underground interchanges, prepared and equipped to ensure prompt and effective intervention.

3.3. Cruise ship A cruise ship offers high density of potential target and lowermiddle security measures that characterize a putative scenario for the spread of a BWA; it would offer different routes of infection, sufficient time for spreading and the possibility to obtain international outbreaks thanks to the presence of tourists from all over the world. Cruise ships threats was an international concern in the latest years due to the increasing number of pirate attacks and intelligence reports that imply terrorist group’s interest (CNN, 2015). Cruise ships represent an attractive target for terrorism for different reasons: (a) large number of people confined in a single place; (b) worldwide resonance in the event of an attack; (c) lowermiddle security levels on passengers; (d) low presence of security if compared with the airports; (e) several possibilities to introduce undercover terrorists and weapons; (f) double faced attack. Many cruise companies perform X-ray scans on passenger’s luggage, this is an important measure that could prevent terrorist infiltration of weapons and explosives; however the chances to introduce BWA on a cruise ships are far higher respect to an airport scenario: terrorists could infiltrate with passengers or low level ship staff; in this second case it would be much easier in successing in secretly bring bioweapons on board. As the airport one, on the other hand, this scenario offers the opportunity of a continuous virus release if the terrorist infiltrate himself in the catering staff. Furthermore, the enclosed environment of a cruise ship offers constant chance to infect new people. Most dangerous BWA would be toxins and weaponized bacteria like Bacillus anthracis or Salmonella spp.; their use will result in many casualties in a short period of time. The use of viruses, especially if not airborne, is more complex; however, exploiting the long incubation time of virus such as Ebola virus, it could result unnoticed during the cruise and infected

O. Cenciarelli et al. / Virus Research 210 (2015) 318–326

passengers would become symptomatic when they have already returned home, generating multiple international outbreaks.

3.3.1. The Ebola virus hypothesis Suitability in using Ebola virus during a cruise ship scenario can have advantages in some situations. Ebola virus cannot be useful if the aim of the terrorist group is to kill passengers in a short time, due to the long incubation period. Contrariwise, if the aim of the terrorist organization is to unleash new epidemic outbreaks around the world, the choice to use Ebola virus could be suitable for the purpose. The best chances to spread the epidemic are letting the passengers be unaware that they have been infected, to do so terrorists must operate a stealthy attack. Ebola virus should be carried on the ship without raising any alarm. If passenger’s luggage is X-rayed before the boarding, the weaponized virus should be hidden in liquid or crystallized form; the amount of virus that can be transported on the ship in this way would be however minimal. Best chances to have a sufficient availability of the virus imply the presence of an undercover terrorist between low level staff, which could have more possibility to success in the infiltration of a relevant amount of virus. The major problem linked to Ebola virus used as BWA is how to perform the correct and effective infection without arousing suspicion. The use of weaponized airborne virus would be ideal, but as assumed earlier weaponization for a BSL-4 virus it’s unlikely for a terrorist group. Another possibility for Ebola virus spread of infection is the direct nebulization of high concentrated virus solution to exploit conjunctival and oral route of infection. As in the underground station scenario, even in cruise ships, air flow system could be used to control the presence of airborne BWA by passive automated or active detection devices.

325

mission, as well as better possibility for terrorists to obtain the virus. Currently, great concern is aroused by terrorist actions using Ebola virus as a low-tech mean of massive annihilation, by global infection using human carriers to intentionally disseminate the virus through different scenarios. Recent articles suggest the possibility for the Islamic State of Iraq and Syria (ISIS) to send operative groups into an EVD outbreak area to intentionally expose themselves to the virus. In this regard, intelligence reports revealed ISIS members researching BWA, although not specifically Ebola. Such a deadly virus could however be taken into account by any terrorist group or organization, representing a global health and security threat that cannot be estimated (Dorminey, 2014; Dvorin, 2014; Cronin, 2004). The battle against viral bioterrorism requires a correct and upto-date knowledge of the topic in order to carry into effect plans of prevention, surveillance and ready-response. Global biosecurity needs to be enhanced; early recognition of a bioterror event and a timely intervention to counter the effects are essential although not easy. Tools for BWA detection (for example by air monitoring systems) must be improved, and intelligence methods are required to acquire useful information to prevent terrorist actions. Physicians have to pose great attention to rapid diagnose the first victims of an attack, maintaining a high degree of suspicion at the onset of an unusual and severe multisystem symptoms. Timely identification of EVD may reduce its spread in either natural or deliberate outbreak (Teckman, 2013) and many efforts in effective therapies development are needed (Bray, 2005a). Furthermore, it is essential to equip teams that will provide an effective prompt response to a bioterrorist event acting with the utmost respect of international protocols on security. Acknowledgement

4. Conclusions Pathogens spread is an important field of analysis in matter of security (Van Aken and Hammond, 2003). Since the 1980s, terrorist groups have increasingly considered BWA as a highly destabilizing tool for civil society and global economy. The use of BWA by terrorist groups is mainly a strategy to defend extremist religious ideas by striking civilian populations or sensible government targets. Progress in biology allowed an improvement in knowledge concerning biological agents that could be exploited for illicit aims also by terrorists (Henderson, 1999). Thanks to the biotechnology, the production of more dangerous, more easily spread and more difficult to detect BWA is currently a great concern (Cenciarelli et al., 2013). Although terrorists already used bacteria and toxins as BWA (Cenciarelli et al., 2013), pathogenic viruses were never used yet; the destructive impact that such an event could determine, reveals that effective countermeasures as well as improved ability to detect and control natural outbreaks are needed (Bray, 2005b). The increasing natural appearance of EVD outbreaks is arousing reflections about security, due to the risk of its intentional acquisition and deliberate use. EVD represents one of the most serious viral diseases known, characterized by a CF rate around 40–50% (that achieves 90% in some virus strains). Actually, neither prophylactic measures nor approved therapies are available. Although obtaining, handling and weaponizing Ebola virus represent obstacles for bioterrorists, the extent makes natural outbreaks a threat due to its accessibility. The highly CF rate of EVD was once a spontaneous limitation of virus spread with most outbreaks started and ended in rural areas. The involvement of urban areas, during the ongoing West Africa outbreak, has made the risk of uncontrolled spread a real trouble. Wide outbreaks mean larger number of people involved and greater potential for person-to-person trans-

Special acknowledgments for the realization of this work go the International Master Courses in “Protection against CBRNe events” (www.mastercbrn.com). References Baize, S., Pannetier, D., Oestereich, L., Rieger, T., Koivogui, L., Magassouba, N., Soropogui, B., Sow, M.S., Keïta, S., et al., 2014. Emergence of Zaire Ebola virus disease in Guinea. N. Engl. J. Med. 371 (15), 1418–1425. Berger, S.A., Shapira, I., 2002. Hemorrhagic fevers and bioterror. IMAJ 4, 513–519. Bray, M., 2003. Defense against filoviruses used as biological weapons. Antivir. Res. 57 (1), 53–60. Bray, M., 2005a. Viral bioterrorism and antiviral countermeasures. In: Torrence, P.F. (Ed.), Antiviral Drug Discovery for Emerging Diseases and Bioterrorism Threats. John Wiley & Sons, Inc., Hoboken, NJ, USA. Bray, M., 2005b. Viral bioterrorism and antiviral countermeasures. In: Torrence, P.F. (Ed.), Antiviral Drug Discovery for Emerging Diseases and Bioterrorism Threats. John Wiley & Sons, NJ, USA. Bukreyev, A.A., Chandran, K., Dolnik, O., Dye, J.M., Ebihara, H., Leroy, E.M., Mühlberger, E., Netesov, S.V., Patterson, J.L., et al., 2014. Discussions and decisions of the 2012–2014 international committee on taxonomy of viruses (ICTV) filoviridae study group, January 2012–June 2013. Arch. Virol. 159 (4), 821–830. Carestia, M., Pizzoferrato, R., Cenciarelli, O., D’Amico, F., Malizia, A., Gelfusa, M., Scarpellini, D., Gaudio, P., 2014. Fluorescence measurements for the identification of biological agents features for the construction of a spectra database. In Photonics Technologies, 2014 Fotonica AEIT Italian Conference on (pp. 1–4). IEEE. Carus, S.W., 1991. The poor man’s atomic bomb? In: Biological Weapons in the Middle East. The Washington Institute for Near East Policy. Policy Papers Number 23., Washington DC, USA. Cenciarelli, O., Rea, S., Carestia, M., D’Amico, F., Malizia, A., Bellecci, C., Gaudio, P., Gucciardino, A., Fiorito, R., 2013. Bioweapons and bioterrorism: a review of history and biological agents. Defence S&T Tech. Bull. 6, 111–129. Cenciarelli, O., Pietropaoli, S., Gabbarini, V., Carestia, M., D’Amico, F., 2014a. Use of non-pathogenic biological agents as biological warfare simulants for the development of a stand-off detection system. J. Microb. Biochem. Technol. 6, 375–380. Cenciarelli, O., Pietropaoli, S., Frusteri, L., Malizia, A., Carestia, M., D’Amico, F., Sassolini, A., Di Giovanni, D., Tamburrini, A., Palombi, L., Bellecci, C., Gaudio, P.,

326

O. Cenciarelli et al. / Virus Research 210 (2015) 318–326

2014b. Biological emergency management: the case of ebola 2014 and the air transportation involvement. J. Microb. Biochem. Technol. 6 (5), 274–253. Centers for Disease Control and Prevention (CDC), 2001. Recognition of illness associated with the intentional release of a biologic agent. MMWR Morb. Mortal. Wkly. Rep. 50 (41), 893. Centers for Disease Control and Prevention (CDC). Emergency Preparedness and Response. Bioterrorism Overview. http://emergency.cdc.gov/bioterrorism/ overview.asp. Centers for Disease Control and Prevention (CDC). Viral Haemorrhagic Fevers (VHFs). http://www.cdc.gov/vhf/virus-families/filoviridae.html. Centers for Disease Control and Prevention (CDC). Travelers’ Health –Chapter 3: Infectious disease related to travel. http://wwwnc.cdc.gov/travel/yellowbook/ 2014/chapter-3-infectious-diseases-related-to-travel/viral-hemorrhagicfevers. Centers for Disease Control and Prevention (CDC). EBOLA (Ebola virus disease). www.cdc.gov/vhf/ebola/. Centers for Disease Control and Prevention (CDC). Emergency Preparedness and Response: Bioterrorism Agents/Disease. http://www.bt.cdc.gov/agent/ agentlist-category.asp. Centers for Disease Control and Prevention (CDC). Outbreaks Chronology: Ebola Virus Disease. http://www.cdc.gov/vhf/ebola/outbreaks/history/chronology. htmlhttp://www.cdc.gov/vhf/ebola/outbreaks/history/chronology.html. CNN. Documents reveal al Qaeda’s plans for seizing cruise ships, carnage in Europe. http://edition.cnn.com/2012/04/30/world/al-qaeda-documents-future/index. html. Colebunders, R., Borchert, M., 2000. Ebola haemorrhagic fever–a review. J. Infect. 40 (1), 16–20. Cronin, A.K., 2004. Terrorist motivations for chemical and biological weapons use: placing the threat in context. Def. Secur. Anal. 20 (4), 313–320. Dixon, M.G., Schafer, I.J., 2014. Ebola viral disease outbreak–West Africa, 2014. MMWR Morb. Mortal. Wkly. Rep. 63, 548–551. Dorminey, B., 2014. Ebola as ISIS bio-weapon? Forbes. Dvorin, T. (2014). Could ISIS target the West with Ebola? Arutz Sheva. Israel National News. http://www.israelnationalnews.com/News/News.aspx/ 186020#.VDvayfldWSo. Feldmann, H., Geisbert, T.W., 2011. Ebola haemorrhagic fever. Lancet 377 (9768), 849–862. Feldmann, H., Klenk, H.D., 1996. Filoviruses. In: Baron, S. (Ed.), Medical Microbiology. , 4th ed. University of Texas Medical Branch at Galveston, Galveston, TX, USA. Feldmann, H., Jones, S., Klenk, H.D., Schnittler, H.J., 2003. Ebola virus: from discovery to vaccine. Nat. Rev. Immunol. 3, 677–685. Furuta, Y., Gowen, B.B., Takahashi, K., Shiraki, K., Smee, D.F., Barnard, D.L., 2013. Favipiravir (T-705), a novel viral RNA polymerase inhibitor. Antivir. Res. 100 (2), 446–454. Garrett, L., 2001. Betrayal of Trust: The Collapse of Global Public Health. Hyperion, New York, USA. Gatherer, D., 2014. The 2014 Ebola virus disease outbreak in West Africa. J. Gen. Virol. 95, 1619–1624. Greub, G., Grobusch, M.P., 2014. Bioterrorism: myth or reality? Clin. Microbiol. Infect. 20, 485–487. Groseth, A., Feldmann, H., Strong, J.E., 2007. The ecology of Ebola virus. Trends Microbiol. 15, 408–416. Guineenews. Medium des dernières nouvelles guinéennes par les guinéens. http:// guineenews.org/2014/03/ebola-a-konakry-la-contamination-limitee-auxmembres-dune-seule-famille-et-a-ceux-qui-les-ont-approches/. Henderson, D.A., 1999. The looming threat of bioterrorism. Science 283 (5406), 1279–1282. Hill-Cawthorne, G., Gilbert, G.L. (2014). Don’t panic about Ebola’s spread, here’s what we can do instead. The conversation. http://theconversation.com/dontpanic-about-ebolas-spread-heres-what-we-can-do-instead-31559. Isaacs, D., 2015. Pandemic paranoia: a heretical view of Ebola and other infectious threats. J. Paediatr. Child Health 51 (2), 129–130. Jaax, N., Jahrling, P., Geisbert, T., Geisbert, J., Steele, K., McKee, K., Nagley, D., Johnson, E., Jaax, G., Peters, C., 1995. Transmission of Ebola virus (Zaire strain) to uninfected control monkeys in a biocontainment laboratory. The Lancet 346 (8991), 1669–1671. Jansen, H.J., Breeveld, F.J., Stijnis, C., Grobusch, M.P., 2014. Biological warfare, bioterrorism and biocrime. Clin. Microbiol. Infect. 20 (6), 488–496. Johnson, E., Jaax, N., White, J., Jahrling, P., 1995. Lethal experimental infections of rhesus monkeys by aerosolized Ebola virus. Int. J. Exp. Pathol. 76 (4), 227–236. Kagan, E., 2005. Update on Ebola virus and its potential as a bioterrorism agent. Clin. Pulmon. Med. 12 (2), 76–83. Katz, R., 2004. Biological weapons: a national security problem that requires a public health response. In: OPR Working Paper Series. Princeton University, Princeton, NJ, USA. Lazarus, A.A., Decker, C.F., 2004. Plague. Respir. Care Clin. N. Am. 10, 83–98. Leitenberg, M., Zilinskas, R.A., Kuhn, J.H., 2012. The Soviet Biological Weapons Program: A History. Harvard University Press.

Leitenberg, M., 2001. Biological weapons in the twentieth century: a review and analysis. Crit. Rev. Microbiol. 27, 267–320. Marzi, A., Ebihara, H., Callison, J., Groseth, A., Williams, K.J., Geisbert, T.W., Feldmann, H., 2011. Vesicular stomatitis virus-based Ebola vaccines with improved cross-protective efficacy. J. Infect. Dis. 204 (3), S1066–S1074. Medecins Sans Frontieres (MSF). Ebola: Surviving Survival - Life after recovery. http://www.msf.org/article/ebola-surviving-survival-life-after-recovery. Morris, K., 2009. First pig-to-human transmission of Ebola–Reston virus. Lancet Infect. Dis. 9 (3), 148. Morse, S.S., 2007. Global infectious disease surveillance and health intelligence. Health Aff. 26 (4), 1069–1077. National Institute of Allergy and Infectious Diseases (NIAID). NIAID/GSK Experimental Ebola Vaccine Appears Safe, Prompts Immune Response. Results from NIH Phase 1Clinical Trial Support Accelerated Development of Candidate Vaccine. http://www.niaid.nih.gov/news/newsreleases/2014/Pages/ EbolaVaxResults.aspx. ˜ C., Günther, S., 2014. Oestereich, L., Lüdtke, A., Wurr, S., Rieger, T., Munoz-Fontela, Successful treatment of advanced Ebola virus infection with T-705 (favipiravir) in a small animal model. Antiviral research 105, 17–21. Olson, K.B., 1999. Aum Shinrikyo: once and future threat? Emerg. Infect. Dis. 5, 513–516. Piercy, T.J., Smither, S.J., Steward, J.A., Eastaugh, L., Lever, M.S., 2010. The survival of filoviruses in liquids, on solid substrates and in a dynamic aerosol. J. Appl. Microbiol. 109, 1531–1539. Raevskiy, A.E., 2014. Psychological aspects of the Aum Shinrikyo affair. Psychology in Russia. State Art 7 (1), 34–39. Sagripanti, J.L., Rom, A.M., Holland, L.E., 2010. Persistence in darkness of virulent alphaviruses, Ebola virus, and Lassa virus deposited on solid surfaces. Arch. Virol. 155 (12), 2035–2039. Sanchez, A., Ksiazek, T.G., Rollin, P.E., Peters, C.J., Nichol, S.T., Khan, A.S., Mahy, B.W., 1995. Reemergence of Ebola virus in Africa. Emerg. Infect. Dis. 1 (3), 96. Teckman, A.M., 2013. The bioterrorist threat of ebola in East Africa and implications for global health and security. Glob. Policy Essay, 15. The Lancet, 2014. Ebola in west Africa: gaining community trust and confidence. Lancet 383 (9933), 1946. U.S. National Institutes of Health. Partnership for Research on Ebola Vaccines in Liberia (PREVAIL) NCT02344407. Üstun, C¸., Özgurler, O., 2005. Ebola: a significant threat as an infectious disease, and as potential bioterrorism agent. Turk. J. Med. Sci. 35 (1), 1–4. Utgoff, V.A., 1993. The biotechnology revolution and its potential military implications. Biological weapons: Weapons of the future. In: Roberts, Browder (Eds.), Biological Weapons: Weapons of the Future? Center for Strategic and International Studies, Washington DC, USA. Van Aken, J., Hammond, E., 2003. Genetic engineering and biological weapons. EMBO Rep. 4 (S1), S57–S60. Wahl-Jensen, V., Kurz, S.K., Hazelton, P.R., Schnittler, H.J., Ströher, U., Burton, D.R., Feldmann, H., 2005. Role of Ebola virus secreted glycoproteins and virus-like particles in activation of human macrophages. J. Virol. 79, 2413–2419. Weingartl, H.M., Embury-Hyatt, C., Nfon, C., Leung, A., Smith, G., Kobinger, G., 2012. Transmission of Ebola virus from pigs to non-human primates. Sci. Rep. 2, 811. World Healt Organization (WHO), 2015z. In vitro diagnostics and laboratory technology: Emergency Use Assessment and Listing (EUAL) Procedure for Ebola Virus Disease (IVDs). http://www.who.int/diagnostics laboratory/ procurement/150219 reebov antigen rapid test public report.pdf. World Health Organization (WHO), 1996. WHO influenza surveillance. Wkly. Epidemiol. Rec. 71, 353–360. World Health Organization (WHO). (2001). Outbreak of Ebola heamorrhagic fever, Uganda, August 2000–January 2001. http://who.int/csr/don/2001 02 09a/en. World Healt Organization (WHO), 2015x. Essential medicines and health products. Ebola vaccines, therapies, and diagnostics. http://www.who.int/medicines/ emp ebola q as/en/. World Health Organization (WHO), 2015y. Media Centre: Ebola Virus disease. Fact sheet N◦ 103. http://www.who.int/mediacentre/factsheets/fs103/en. World Health Organization (WHO). Ebola Situation Report - 26 August 2015. http:// apps.who.int/ebola/current-situation/ebola-situation-report-26-august-2015. World Health Organization (WHO). Media Centre: WHO Director-General, west African presidents to launch intensified Ebola outbreak response plan. http:// www.who.int/mediacentre/news/releases/2014/ebola-outbreak-responseplan/en/. World Health Organization (WHO). Media Centre: Frequently asked questions on Ebola virus disease. http://who.int/csr/disease/ebola/faq-ebola/en/. World Health Organizzation (WHO). Essential medicines and health products. WHO Ebola R&D Effort–vaccines, therapies, diagnostics. 30 January update. http://www.who.int/medicines/ebola-treatment/ebola r d effort/en/. World Health Organizzation (WHO). Essential medicines and health products. Categorization and prioritization of drugs for consideration for testing or use in patients infected with Ebola. http://who.int/medicines/ebola-treatment/cat prioritization drugs testing/en/.