4 Introduction

In 1967, in Marburg an der Lahn and Frankfurt ‘subtypes’ of a novel agent named ‘Ebola virus’ am Main, Germany1, and in Belgrade, Yugoslavia after the small Ebola river in Zaire [412, 1000, (now Serbia), laboratory workers accepted shipments 2410]. Today these ‘subtypes’ are called Sudan of African green monkeys (Chlorocebus aethiops) ebolavirus (SEBOV) and Zaire ebolavirus (ZEBOV), from Uganda. As they had done many times before respectively [805]. Studies of periodic hemorrhagic with such animals, workers performed routine ex- fever outbreaks in African countries and in the aminations for apparent ailments and then prepared Philippines indicated that at least two more ebola- tissue cultures from the monkeys’ kidneys for the viruses exist, which are now known as the Coote^ development of poliomyelitis vaccines. A few days d’Ivoire ebolavirus (CIEBOV) and Reston ebola- later, several workers were reported ill and were virus (REBOV) [805]. Molecular and other studies admitted to local hospitals. A total of 32 people revealed the close relationship of MARV and the fell sick with an apparently new disease, of which ebolaviruses, which resulted in their classification seven died. A hitherto unknown virus was isolated in the same viral family, Filoviridae (the filo- from patients and human tissues [2396] and called viruses2) [805]. ‘Marburg virus’ (today Lake Victoria marburgvirus, A substantial interest in filoviruses has developed MARV) [805]. Over the subsequent three decades, among the general public, in part because of novels, only individual MARV infections were recorded. popular science stories, and Hollywood productions In 1998, the virus reappeared in the Democratic that portrayed the horrendous diseases they cause. Republic of the Congo and caused at least 128 deaths Richard Preston’s articles and his very successful over a period of three years [270]. From the end of book, The Hot Zone [2057–2059], which is based 2004 to November 2005, MARV caused an outbreak on actual disease outbreaks caused by filoviruses, killing 227 people in Angola. captured the imagination of the public for months. In 1976, novel viruses were isolated from many Scholars remarked that the book was a bestseller patients of two large hemorrhagic fever outbreaks in mainly because Preston managed to describe filo- Maridi, Sudan, and Yambuku, Zaire (now Demo- viruses as an ‘‘external threat to Americans in a cratic Republic of the Congo) that resulted in the post-Cold War world with porous borders’’ [1097] death of 431 of 602 infected people. These new pathogens were characterized as two different 2 In 2006, taxon-specific suffixes were suggested for verna- cular virus names. According to that system, the suffixes 1 In this review, countries are designated according to their ‘-virad’, ‘-virid’, ‘-virin’, and ‘-virus’ are used for members English conventional short name as listed in The World Fact of a viral order (‘-virales’), family (‘-viridae’), subfamily Book [Online.] https:==www.cia.gov=cia=publications= (‘-virinae’), or genus (‘-virus’), respectively [2636]. This factbook=index.html [last accessed Sep. 1, 2007]. Names system appears to be practical but it is not yet widely accepted. are used for simplicity of recognition and are not intended as Therefore, in this review the terms ‘mononegaviruses’ and political statements regarding the official recognition (or ‘filoviruses’ are used rather than the more discriminating lack thereof) of a given country. novel terms ‘mononegavirads’ and ‘filovirids’. 14 Introduction that tap ‘‘into fears about travel and immigration’’ children, has fueled all these concerns. A new sub- [2440]. Accordingly, ebolaviruses ‘‘...emerge as a genre of horror movies, termed plague films [2728], key metaphor for the ambivalence, if not alarm, amplified the public’s concern. These productions about a new world order, one characterized by [41, 73, 87, 115, 116, 2252, 2666] include a very multi-sites of geo-political power and the displace- successful Hollywood movie, Outbreak [65], and ment of the privileged ... western European civi- individual episodes of popular U.S. television series lization in the face of rapid movement of peoples (Medical Investigation – Season 1 Episode 17, and cultural practices along expanding circuits of Millennium – Season 2 Episode 22 and Season 3 global capitalism’’ [1097]. In Hamilton, Canada, Episode 11, ReGenesis – Season 1 Episode 11, 24 – widespread panic was noted and fueled by the media season 3, 7 Days – Season 1 Episode 3, and CSI: in 2001 after a Congolese woman, who had just Crime Scene Investigation – Season 5 Episode arrived by plane from Ethiopia, became sick with 4). Most of the plague films disregard scientific an illness that was at first thought to be ebolavirus facts and focus on the ‘‘rhetorically constructed, disease [1825]. An analysis of the media response predatory nature of the virus’’ to attract interest suggests that increasing anxiety among Canadians [2728]. The same seems to be true for popular sci- over the growing presence of racial minorities was entific articles, which often display the filoviruses at the root of the ‘‘Ebola panic.’’ According to the as killers with a will [2255]. Comparative analyses study, immigration was made an issue by the demonstrated that fictitious work and Hollywood involved Canadian media by linking it to possible productions portraying filoviruses are construed future health risks for Canadians – that is, linking similarly [2302]. It was also found that scientific immigration to possible importation of filoviruses journal articles, popular science writing, and science [162]. Other researchers indirectly concur with this fiction have become ‘‘mutually, minutely entangled’’ assessment by interpreting the public’s morbid because emerging viruses ‘‘have attained a certain interest in emerging infectious diseases as a reflec- chic among the medical set’’ [2255]. Fascination tion of people’s fear of social change – and the fear with hazardous viral infectious diseases and the of ‘‘disintegration of self or of nation; Armageddon; ‘‘virus hunters’’ who track them [587] has grown the triumph of multiculturism and the global com- to such an extent that an increasing number of so- munity; the ecosystem’s anger and vengeance for cial scientists and scholars outside the fields of our meddling; the loss of the unknown; or the escape medicine and life sciences have begun to study the of the unknown into our society, where everything public interest in this topic [302, 303, 431, 1097, familiar will be destroyed ...’’ [2255]. Filoviruses 1295, 1408, 1609, 1708, 1766, 1804, 2255, 2302, seem to cause ‘‘the mythical disease of our time’’ 2419, 2440, 2584, 2626, 2702, 2728]. Poets [2517] [2440]. Another expert argues that, via Preston’s and artists [1498] seem to be inspired by filoviruses; book and a Hollywood movie, ebolaviruses have be- literature analysts have suggested that an ebolavirus come an icon of the anti-globalization movement. disease outbreak might have inspired Edgar Allan Accordingly, the viruses indicate a change of the Poe to write The Masque of the Red Death [2667]; public image of rain forests, which once were de- and investigators used ‘‘Ebola’’ as a catch phrase to picted as fragile, quasi-romantic places that had to draw attention to their articles, several of which did be protected for their own sake and which now are actually not pertain to filoviruses [241, 593, 1383, seen as threats to western civilization because of 2642]. By now, incorrect notions about these agents diseases they harbor and ‘‘export’’ (‘‘revenge of (high transmissibility, ‘‘liquefying organs,’’ ‘‘crash- the rain forest’’) [2936]. An ever increasing volume ing patients’’ that ‘‘bleed out’’, etc.) are stereotypi- of written work [184, 444, 569, 570, 604, 677b, cal wisdom or urban legends [2727] that are difficult 708, 750, 861, 970, 1009, 1013, 1147, 1156, to eradicate. Exaggerations and pseudoscientific 1189, 1337, 1610, 1654, 1984, 2068, 2110, 2415, descriptions of ebolaviruses have fueled doomsday 2425, 2744], computer games, and audio plays ad- scenarios focusing on possible pandemics. For ex- dressing the filoviruses, aimed at both adults and ample, an analysis of British tabloids and broad- Introduction 15 sheets revealed that many described ‘‘liquefying, published books addressing public fears and have disintegrating, combusting bodies’’ when referring attempted to correct false notions about these to filovirus-infected people [1295]. The public press pathogens [584, 592, 619, 791, 907, 1135, 1326, covers even the most remote and non-extensive 1395, 1701, 1932, 2006, 2069, 2102, 2188, 2215, occurrence of filoviral disease, perhaps stirring 2941]. Their success in correcting the public image the public’s fear of worldwide spread. In addition, of filoviruses was limited, in part because the lan- there have been insinuations that filoviruses were guage used clearly showed that some authors also man-made [1172, 2254] or are spread deliberately were fascinated with or frightened by the agents by the military [201, 1755] or ecological groups [2419]. For example, one researcher called Zaire [2641]. Such rumors were spread during a larger ebolavirus a ‘‘mysterious’’ agent with ‘‘epidemic outbreak in Kikwit, Zaire, in 1995, when a biolog- genius’’ [1053]. ical weapon based on an ebolavirus was suspected Infectious agents are assigned to biosafety catego- by locals to be the underlying cause of the outbreak ries, based on documented accounts of laboratory [1395]. The scarce and limited outbreaks of filo- or field infections and fatalities, their degree of viral disease also had a limited impact on the pathogenicity and virulence, induced case-fatality worldwide economy through restrictions on trade ratios, and the existence or absence of therapeutic in nonhuman primates [1960, 1961] and decisions remedies and prophylactic measures [2582]. Highly of airlines to curtail transport of nonhuman pri- virulent, infectious, and=or contagious pathogens mates. As a result, business professionals became for which there are no specific antivirals or vac- interested in the topic of emerging viruses [96]. cines are usually designated as Class 4 or Risk Using the language of the media, and in par- Group 4 pathogens [2582]. Since the filoviruses ticular that of Richard Preston, a virologist sum- are so virulent, and there are no licensed vaccines marized the public knowledge of filoviruses: or specific antivirals available to prevent or treat ‘‘...[b]ricks of bad information and fear-mon- infections caused by them, all work involving gering set up a highly-efficient, deadly cycle of these viruses must be performed in the few existing hysteria replication in the populace. The public maximum-containment facilities [2582]. Safety hemorrhages, spilling hysteria to the next unwitting concerns, and the resulting hindrance of research victim. Fear gushes from every media orifice. No progress due to the paucity of maximum-con- one is safe from the hype’’ (as cited in [2936]). The tainment laboratories have insured that filoviruses ‘‘hype’’ stands in contrast to the actual importance remain among the lesser characterized human of filovirus infections in the context of true global pathogens, which likely fuels the public concern infectious disease threats. After all, in the past the associated with these viruses. For example, the filoviruses had been known to cause only a few origins and whereabouts of the viruses remain outbreaks. The overall number of human filovirus unknown, and the severe diseases they cause in infections – 2,317 cases and 1,671 confirmed deaths humans, and their rapid emergence and disappear- (72.1% case-fatality rate) over almost 40 years – ance, still are poorly understood. seems negligible related to other diseases of known The terrorist attacks on the World Trade Center global significance (acquired immunodeficiency in New York City and on the Pentagon in Washing- syndrome (AIDS), dengue, malaria, river blindness, ton, D.C. on September 11, 2001 demonstrated the or tuberculosis) that have caused hundreds of thou- extent to which individuals with hostile intentions sands of cases or more per year [361, 437, 2615, can harm society as a whole. The ensuing spread by 2616]. As a result, many medical and scientific mail of powder containing lethal Bacillus anthracis professionals consider filovirus infections to be spores proved the possibility of covert biological only moderately important emerging infectious dis- attacks. Thus, it is no surprise that politicians and eases [531, 532]. Investigators and others in- the public consider the consequences of a hypothe- volved in characterizing filoviruses, and people who tical scenario combining the logistics of the first played important roles during the outbreaks, have attack (planes as explosives) with the contagious 16 Introduction possibilities of the second attack (infectious agents). [1551]. One conclusion of this mock exercise was Funding for anti-terrorist measures in general and that even a natural filoviral disease outbreak during biological defense (biodefense) in particular has a war or a military crisis could lead to serious and been increased dramatically in recent years in many damaging allegations about biological warfare countries. In the U.S. in particular, research on de- activities of involved military parties [2418]. fense against potential biological weapons agents Furthermore, recent studies indicate the involve- has been enhanced considerably [491b, 881b, 1502, ment of filoviruses in the dramatic decline of great- 2289, 2290]. Lists of such agents, which signifi- ape populations in Africa [212, 318, 319, 469, cantly overlap with the list of Select Agents that 1199, 1369, 1567, 1603, 2128b, 2159, 2643, 2681], pose a threat to the U.S. [518], have been estab- although both ideas (involvement of filoviruses, lished by the Australia Group [2530] and the U.S. and ‘‘dramatic’’ decline) are controversial [1922, National Institute of Allergy and Infectious Dis- 2371]. The filoviruses are the cause of exotic in- eases (NIAID) [1873]. Biosurety, i.e., ‘‘the combi- fections in humans. The actual risk of an attack nation of security, biosafety, agent accountability, with a filoviral biological weapon is low. However, and personnel reliability needed to prevent it is possible filoviruses cause recurring epizootics unauthorized access’’ to Select Agents [482], has in wild animals and that this alone might have a become a major part of biosecurity efforts [2240, profound effect on the environment and hence – 2897], which in their entirety attempt to prevent the indirectly – on the human population as well. production, stockpiling, and spread of potential or To achieve a valid filovirus threat assessment, it is actual biological weapons. of primary importance to summarize the current The first step in determining the actual threat knowledge of the agents and to review all relevant posed by a potential biological weapons agent is to published information to gain insights into past events describe the research that has been performed, who and to focus on relevant research to address these is in possession of the agent, and which type of re- agents. Filovirus research has been the focal point search will be necessary to develop counter-mea- of several scientific conferences (see Table 4-1) sures against intentional or natural outbreaks. MARV [113, 227, 412, 865, 1424, 1688, 1874, 1966], and and ebolaviruses are Class 4 pathogens [2582]. The the subject is of sufficient importance and interest to Soviet Union entertained an extensive covert bio- continue them. Many scientific reviews have been weapons program, which assessed, manipulated, and written on the subject of filovirus research and inter- produced filoviruses for weaponization [178]. Other est in these pathogens among scientists is now con- countries may have had similar undeclared pro- siderable. Indicators of worldwide interest in these grams. Hence, filoviruses have been classified as po- agents are the numerous brief reviews on the subject tential agents for biological warfare purposes by the that have been published in many languages other Australia Group [2530], NIAID [1873, 2158], and than English (for example, see [177, 589, 1054, by many experts [117, 171, 223, 267, 367, 399, 432, 1343, 1457, 1471, 1584, 1936, 1990, 2036, 2086, 460, 484, 625, 777, 883, 885, 1027, 1031, 1270, 2577, 2628, 2933, 2953, 3071, 3150, 3233, 3236, 1350, 1354, 1548, 1606, 1709, 1999, 2001, 2124, 3258, 3264]). Entire issues of scientific periodicals, 2192, 2218, 2485, 2588, 2645, 2694, 3100, 3234]. as well as books, have been devoted to the filoviruses Among the potential weapon agents, filoviruses have [1030, 1417, 1425, 2009, 2183, 3207], and these are been ranked as highly dangerous NIAID Category highly recommended for further reading on the sub- A Priority Pathogens [1873, 2158]. Thus, the exotic ject. The scientific compendia describing the dis- filoviruses are now considered important and real covery of MARV and the ebolaviruses also are threats to the global community. During a scien- suggested for additional reading [1666, 1979]. Addi- tific conference, epidemiologists constructed a fic- tional information on filoviruses can be obtained titious outbreak of an ebolavirus-like entity in an from viewing video productions [41, 153, 330, 537, imagined sub-Saharan country devastated by civil 585, 667, 834, 1326, 1375, 2252, 2579, 2930]. Char- war, with subsequent global spread of the agent acterization of these viruses on the World Wide Web Introduction 17

Table 4-1. International conferences and symposia focusing on filovirus research (see [94, 113, 129, 227, 412, 865, 1424, 1688, 1874, 1966])

Name of Conference=Symposium Date Location International Colloquium on Ebola Virus Research September 4–7, 1996 Antwerp, Belgium Russian–German Colloquium on Filoviruses: January 28–February 2, Koltsovo, Novosibirsk The Modern State of Problem [sic] 1997 Region, Russia Symposium on Marburg and Ebola Viruses October 1–4, 2000 Marburg an der Lahn, Hesse, Germany VRC Symposium on Viral Hemorrhagic Fevers October 14–17, 2003 Bethesda, Maryland, U.S.A. Outbreaks of Ebola Haemorrhagic Fevers September 7–8, 2004 Paris, France in Central Africa (2001–2003). Which Strategies Should We Adopt for the Control of Future Outbreaks – Workshop on Viral Haemorrhagic Fevers Workshop on Controlling the Impact of Ebola March 10–11, 2005 Washington, D.C., U.S.A. on African Apes Filoviruses: Recent Advances and Future Challenges – September 17–19, 2006 Winnipeg, Manitoba, An ICID Global Symposium Canada

Fig. 4-1. Number of filovirus-related scientific publications in languages other than English, 1967–2007. Of the 4,500 references listed in this review (cut-off: August 27, 2007), 1,100 have been written in languages other than English 18 Introduction

[1493] is mostly wrong or imprecise, with only a few with other, well-known human pathogens to gain truly informative sites accessible [860, 1549, 2638] structural and functional knowledge of them. Filo- and often in need of updating [1514]. Reasonable and viral genes or gene fragments in eukaryotic expres- useful discussions of infectious disease outbreaks sion systems have been made available recently. once thought to be due to filoviruses, as well as inter- These allow characterization and manipulation of views with major filovirus investigators, can be found subunits of these viruses in nonspecialized labora- as well [515, 1232, 2585]. A few filovirus research tories, rather than in maximum-containment facil- bibliographies are available [433, 1299, 1300, 2581]; ities, and open the field for investigators other than however, most of them focus on specific research virologists and pathologists. Presently, there is a aspects and do not provide comprehensive overviews. new generation of scientists and biodefense profes- Similarly, most published reviews in various lan- sionals interested in the characterization of filo- guages are not comprehensive, nor do they provide viruses, and one would anticipate the number of comprehensive citation of the scientific literature. filovirus research publications to continue to Much of the non-English literature, e.g. many increase in the future (see Fig. 4-2). research publications in French, German, Japanese, This literature review is based on a dissertation or Russian (see Fig. 4-1), has not been cited in written as part of the requirement for a doctorate these reviews. in medical sciences [1476], intended to summarize The threat of a biological attack with a MARV- most of the published filovirus research reports. It is or ebolavirus-based weapon compels the establish- hoped that it will serve as a main reference for scien- ment of countermeasures. Many investigators have tists involved in filoviral research and students inter- reported the molecular comparison of filoviruses ested in this aspect of virology. Journalists may use

Fig. 4-2. Number of filovirus-related scientific reports by year of publication. Filovirus research began with the discovery of Lake Victoria marburgvirus in 1967. After a first peak in 1968, the number of filovirus-related scientific publications declined, in part reflecting the fact that no further infections were reported until 1975. The two large hemorrhagic fever outbreaks leading to the discovery of Sudan ebolavirus and Zaire ebolavirus in 1976 sparked a second peak of filovirus research publications in 1978. Thereafter, there has been an almost steady increase in reports after the discovery of Reston ebolavirus and Cooted’Ivoire^ ebolavirus in 1989 and 1994, respectively, and after large hemorrhagic fever outbreaks caused by Zaire ebolavirus (1995), Lake Victoria marburgvirus (1998–2000, 2004–2005), and Sudan ebolavirus (2000–2001) Introduction 19 the review to verify information obtained elsewhere. rodent-borne with probable spread by inhalation Overall, this review addresses the worldwide concern of aerosols of rodent excreta or secreta (robo- about filoviruses, but from a scientific point of view. viruses); 3) of unknown etiology [1176, 2294]. Cur- rent information about viruses known to cause hemorrhagic fevers in humans is summarized in 4.1 Viral hemorrhagic fevers Table 4-2. It was predicted that new VHF-causing In humans, filovirus infections result in severe viruses could be assigned to the existing viral fam- clinical syndromes known as ‘‘viral hemorrhagic ilies summarized in Table 4-2 [1329]. fevers.’’ The term ‘‘hemorrhagic fever’’ was used Numerous other infectious diseases may have in a variety of different and confusing manners for hemorrhagic manifestations and can mimic VHF. many years in the medical literature, before being Bacterial examples are rickettsial diseases (e.g., authoritatively defined in 1962 [895]. The initial the various spotted fevers), chlamydial infections definition of hemorrhagic fever was based on a (psittacosis), typhoid fever, shigellosis, and spiro- relatively widespread disease now known as hem- chete-induced syndromes such as Canicola fever, orrhagic fever with renal syndrome (HFRS), caused relapsing fever, or Weil’s disease. Malaria, toxo- by certain hantaviruses (family Bunyaviridae). plasmosis, and trypanosomiasis, caused by pro- Most hemorrhagic fevers such as Crimean-Congo tozoa, are diseases whose manifestations may hemorrhagic fever, dengue hemorrhagic fever, include hemorrhage. Candidiasis and histoplasmo- Kyasanur Forest disease, Rift Valley fever, Omsk sis are fungal diseases that may induce hemorrhages hemorrhagic fever, and yellow fever were known in immunosuppressed or immunocompromised pa- at the time, and it was clear that agents causing the tients. In rare cases, even helminths can induce clinical picture of hemorrhagic fever were primar- severe bleeding. Viruses causing exanthemas, such ily viral in nature. Consequently, the term viral as human herpesvirus 3, measles virus, monkey- hemorrhagic fever (VHF) was coined [3157]. Even pox virus, mumps virus, rubella virus, and variola with many new diseases later added to the list of virus (eradicated), are known to cause bleeding VHFs, the first description of the syndrome is still complications in severe cases, as do certain ade- largely accurate: having a virus etiology, often with noviruses, echoviruses, Venezuelan equine enceph- a distinct vasotropism, sometimes resulting in cap- alitis virus [919], and severe acute respiratory illaropathy, fever, and a tendency towards bleeding. syndrome coronavirus. However, hemorrhaging is The syndrome is geographically focal in nature, not a principle sign of infection with these agents with a ‘‘distinct endemicity, a distinct geographical and therefore these viruses are not included among localization of the foci in rural areas and in sparsely the agents causing hemorrhagic fevers. populated regions ...where conditions capable of Viruses of various taxa have been used as model maintaining the circulation of the causative agent systems for VHF if they can cause VHF-like syn- in certain species of carriers and warm-blooded dromes in humans or naı¨ve or genetically modified animals prevail’’ (cited from the English translation experimental animals. Examples are Colorado tick of [3157]). The latter part of this definition refers to fever virus (family Reoviridae,genusColtivirus), epi- the occurrence of natural outbreaks. Obviously, zootic hemorrhagic disease virus (family Reoviridae, intentional spread of VHF agents could result in genus Orbivirus), Chikungunya virus (family vastly different epidemiologies. Togaviridae, genus Alphavirus), rabbit hemorrhagic Human infections with VHF-causing agents occur disease virus and European brown hare syndrome presumably by direct or indirect contact with in- virus (both family Caliciviridae, genus Lagovirus) fected animals. Occurrences of some known VHFs [183, 1387], simian hemorrhagic fever virus (fam- seem to be strictly seasonal, whereas others emerge ily Arteriviridae, genus Arterivirus), African swine in a seemingly random fashion. Known VHF- fever virus (family Asfarviridae, genus Asfivirus) causing agents are 1) transmitted during feeding [990], lymphocytic choriomeningitis virus (family of a hematophagous arthropod (arboviruses); 2) Arenaviridae, genus Arenavirus), the ungrouped 20 Introduction ] Geographic distribution Bolivia Brazil Bolivia Venezuela Europe Argentine pampas Middle East Asia Asia Central and West Africa sp. West Africa sp. Sub-Saharan Africa = Aedes Anopheles Mastomys Calomys Zygodontomys Apodemus Calomys Apodemus Apodemus Niviventer (Cellia) gambiae Natural reservoir vector Rodents: Rodent: callosus Mosquitoes: Rodent? Rodent? Rodent: brevicauda Rodent: flavicollis Rodent: musculinus Rodent: agrarius Mosquito: Rodent: peninsulae Rodent: confucianus 1 3 774, 793, 969, 1176, 1426, 1620, 1786, 1954, 2357, 2525, 2782, 2874, 2924 n (Argentinian) ´ Machupo (Bolivian) haemorrhagic fever (A96.1) Unnamed (A92.8) Haemorrhagic fever with Human disease Unnamed (‘‘Brazilian haemorrhagic fever’’) (A96.8) Haemorrhagic fever with Unnamed (‘‘Venezuelan Junı Unnamed (A96.8) renal syndrome (A98.5) haemorrhagic fever’’) (A96.8) haemorrhagic fever (A96.0) Lassa fever (A96.2) renal syndrome (A98.5) Haemorrhagic fever with (according to ICD-10) Unnamed (A92.8) Haemorrhagic fever with renal syndrome (A98.5) renal syndrome (A98.5)? )ssRNA Bipartite ambisense ssRNA Genome Tripartite ( 4 ’ (suggested) virus (SABV) 5 2 Biological agents causing viral hemorrhagic fevers in humans [389, 426, 530, ´ n virus (JUNV) virus ´ ´ n virus ´ Machupo virus (MACV) Sabia Dobrava-Belgrade virus (DOBV) Guanarito virus (GTOV) ‘Chapare virus’ (suggested) Saaremaa virus (SAAV) Virus(es) Junı Lassa virus (LASV) Amur virus (AMRV) Da Bie Shan virus (DBSV) Ngari virus (NRIV) Ilesha virus (ILEV) Chapare virus Machupo virus Sabia ‘ Dobrava-Belgrade virus Guanarito virus Species Junı Lassa virus Hantaan virus Bunyamwera virus Hantavirus Arenavirus Genus Orthobunyavirus Arenaviridae Table 4-2. Family Bunyaviridae Introduction 21 ) continued ( Asia, Europe Asia Asia Europe Africa Middle East, Sub-Saharan Africa, southeastern Europe, southwestern Asia, Russia and NIS Africa Africa Africa sp. sp. Africa, Middle East sp. Worldwide (tropics) sp. Worldwide sp. Aedes Aedes sp., Rattus Apodemus Clethrionomys Eothenomys Clethrionomys Dermacentor Rodent: agrarius Rodent: rufocanus Rodent: regulus Rodent: glareolus Rodents: Ticks: Hyalomma Rhipicephalus Mosquitoes: Mosquitoes: Haemorrhagic fever with renal syndrome (A98.5) Haemorrhagic fever with Haemorrhagic fever with renal syndrome (A98.5)? renal syndrome (A98.5)? Haemorrhagic fever with renal syndrome (A98.5) Haemorrhagic fever with renal syndrome (A98.5) Crimean-Congo haemorrhagic fever (A98.0) Marburg virus disease (A98.3) ? Ebola virus disease (A98.4) ? Rift Valley fever (A92.4) Ebola virus disease (A98.4) ? Ebola virus disease (A98.4) ? Dengue haemorrhagic fever (A91) )ssRNA þ)ssRNA Monopartite ( Monopartite ( ^ ooted’Ivoire ebolavirus (CIEBOV) Hantaan virus (HTNV) Hokkaido virus (HOKV) Muju virus (MUJV) Puumala virus (PUUV) Seoul virus (SEOV) Crimean-Congo hemorrhagic fever virus (CCHFV) Rift Valley fever virus (RVFV) Lake Victoria marburgvirus (MARV) C Sudan ebolavirus (SEBOV) Zaire ebolavirus (ZEBOV) Dengue viruses 1–4 (DENV1–4) ^ ooted’Ivoire ebolavirus Puumala virus Seoul virus Crimean-Congo hemorrhagic fever virus Rift Valley fever virus C Lake Victoria marburgvirus Sudan ebolavirus Zaire ebolavirus Dengue fever virus Nairovirus Phlebovirus Ebolavirus Marburgvirus Flavivirus Filoviridae Flaviviridae 22 Introduction is why ns. Siberia Western Siberia Geographic distribution India, Saudi Arabia South Africa, U.S.A. sp. Southern Africa sp. Africa, South America sp. Central and = Culex Aedes Aedes Ixodid ticks Ixodid ticks Natural reservoir vector Ixodid ticks Mosquitoes: Mosquitoes: 1 fevers based on pathological comparisons [2677]. called ‘‘Alkhumra’’) [530, 1620, 2924]. few human cases of VHF it allegedly caused are now in dispute. Health Problems, 10th Revision [2874]. The ICD-10 uses British English, which not included in this list since hemorrhages are not a hallmark of these infectio – contrary to the remainder of this book. Unnamed (A98.8) Omsk haemorrhagic fever (A98.1) Kyasanur Forest disease (A98.2) Unnamed (A98.8) Unnamed (‘‘Wesselsbron disease’’) (A98.8) Mosquitoes: (according to ICD-10) Yellow fever (A95) Genome Human disease 6 8 7 ) continued ( Tick-borne encephalitis virus, Far Eastern subtype (TBEV-FE) Omsk hemorrhagic fever virus (OHFV) Kysanur Forest disease virus (KFDV) West Nile virus (WNV) Wesselsbron virus (WESSV) Virus(es) Yellow fever virus (YFV) Tick-borne encephalitis virus Omsk hemorrhagic fever virus Kyasanur Forest disease virus West Nile virus Wesselsbron virus Species Yellow fever virus Genus ICD-10: International Statistical Classification of Diseases and Related Whitewater Arroyo virus hasSome been clinicians excluded doubt from that theIncludes Lassa list the fever because newly fits discovered the intoHantaviruses Garissa the that virus group cause [969]. of hantavirusIncludes hemorrhagic (cardio-)pulmonary the syndrome newly are discoveredOne Alkhurma particulate isolate isolate (in only theParticular [2525]. literature isolates also only [1954]. ‘‘hemorrhagic fever’’ is spelled ‘‘haemorrhagic fever’’ in this table Table 4-2 Family 1 2 3 4 5 6 7 8 Introduction 23 orthobunyavirus Wanowrie virus [2570], and at the time (1970) [486], the need for a new taxon Sindbis virus (family Togaviridae, genus Alpha- was obvious [2606]. virus) [1721]. ‘Ebola virus’ was named after the small Ebola river, which is the headwater of the Mongala River, which in turn is a tributary of the former Zaire (now 4.2 Filovirus taxonomy, evolution, and phylogeny Congo) River in Zaire (now Democratic Republic Filovirus taxonomy changed continuously since the of the Congo) [1000]. ‘Ebola virus’ shared certain discovery of the MARV in 1967. Several recom- features, including the filamentous-particle mor- mendations from taxonomy committees regarding phology and induced disease, with ‘Marburg virus.’ terminology were followed by the research commu- Therefore, a phylogenetic and associated taxonom- nity over the years, whereas others were not – the ic relationship between the agents was suggested net result is a plethora of circulating virus, species, [26, 1983, 2332]. The second ICTV report did not and genus designations that differ among publica- list the new pathogens, but in the third and tions and database entries. This subchapter explains fourth reports they appeared under the names the history of filovirus taxonomy from 1967 to the ‘Ebola virus’ and ‘Marburg virus’ in the ‘‘unclas- present, listing all terms found in the literature in sified’’ category [1683, 1684]. A genus name, comprehensive tables. Readers who are currently ‘Tuburnavirus,’ was later proposed for both ‘Ebola not directly involved in filovirus research may skip virus’ and ‘Marburg virus’ because of the tubular parts of the text and learn about the most current shape of the viral particles, but the proposal was filovirus taxonomy from the tables. not formally submitted to the ICTV. Molecular- Filovirions have a distinctive filamentous mor- biological studies demonstrated the actual rela- phology. Many unclassified mammalian virus par- tion of ‘Ebola virus’ and ‘Marburg virus’, and the ticles have been examined by electron microscopy viral family names ‘Nemaviridae,’ ‘Fibraviridae,’ and none were comparable to those of filoviruses ‘Funiviridae,’ and ‘Virgaviridae’ were considered. [735]. However, filovirions resemble certain animal However, a decision was made in favor of a newly and plant virions, which may be oval, elongated, or established genus, ‘Filovirus,’ in a newly estab- bullet-shaped. Because of its general resemblance to lished family, Filoviridae [1399, 2367, 3004]. A rhabdoviruses, the name ‘Rhabdovirus simiae3’was general description of this family was published once suggested for ‘Marburg virus’ [2027, 2030, by the ICTV in its fifth to eighth reports [805, 2325, 2948]. Other names, including ‘Arbovirus 881, 1265, 1883]. (tubulo-)hamatum,’ ‘Rhabdovirus (tubulo-)hama- All current molecular reports suggest an evo- tum’ [1690, 2104], and ‘Torovirus’ [181], were pro- lutionary position of the filoviruses between the posed because of the presence of tubular-hook and= pneumoviruses (Paramyxoviridae) and the vesiculo- or torus-like particles in cell cultures. ‘Rhabdovirus viruses (Rhabdoviridae). The amino-acid sequence b-1’ was the first name approved in the first report similarities between filoviral, pneumoviral, and of the International Committee on Nomenclature of vesiculoviral proteins indicate that the filoviruses Viruses (now the International Committee on Tax- are more closely related to the pneumoviruses onomy of Viruses, ICTV) in 1971 [2739]. However, [798, 2220, 2222]. Sufficient similarities among because ‘Rhabdovirus b-1’ was antigenically dis- viruses in the families Filoviridae, Paramyxoviridae, tinct from rhabdoviruses and any other virus known and Rhabdoviridae were found, allowing them to be grouped into a higher taxon, the order Mononega- virales [2065]. Later, the family Bornaviridae was

3 added to the order [2061]. Table 4-3 summarizes Taxon names that are no longer in use or have not yet the current taxonomic organization of the monone- been accepted by the International Committee on Taxonomy of Viruses (ICTV) are placed in inverted commas (‘...’) gaviruses. All mononegaviruses, with the exception because quotation marks (‘‘...’’) are used by the ICTV for of the proposed rhabdovirus ‘orchid fleck virus,’ official, but temporary vernacular taxon names. possess only one genomic nucleic acid. All mono- 24 Introduction

Table 4-3. Current organization of the viral order Mononegavirales [378b, 687, 805, 1233, 1448, 1485b, 1489, 1492b, 1503, 1571, 1693, 1694, 2062, 2065, 2687]

Order Family Subfamily Genus Type species Mononegavirales Bornaviridae Bornavirus Borna disease virus Filoviridae Ebolavirus Zaire ebolavirus Marburgvirus Lake Victoria marburgvirus Paramyxoviridae Paramyxovirinae Avulavirus Newcastle disease virus ‘Ferlavirus’ Fer-de-Lance virus (suggested ) ‘Jeilong virus’ ‘J virus’ (suggested ) (suggested ) Henipavirus Hendra virus Morbillivirus Measles virus Respirovirus Sendai virus Rubulavirus Mumps virus ‘‘TPMV-like viruses’’ Tupaia paramyxovirus Unnamed ‘Mossman virus’ (suggested ) Unnamed ‘Salem virus’ (suggested ) Pneumovirinae Metapneumovirus Avian metapneumovirus Pneumovirus Human respiratory syncytial virus Rhabdoviridae ‘Bracorhabdovirus’ ‘Itacaiunas virus’ (suggested ) (suggested ) Cytorhabdovirus Lettuce necrotic yellows virus ‘Dichorhabdovirus’ ‘Orchid fleck virus’ (suggested ) (suggested ) Ephemerovirus Bovine ephemeral fever virus Lyssavirus Rabies virus Novirhabdovirus Infectious hematopoietic necrosis virus Nucleorhabdovirus Potato yellow dwarf virus Vesiculovirus Vesicular stomatitis Indiana virus Unnamed Almpiwar virus Unnamed Flanders virus Unnamed Kern Canyon virus Unnamed Kolongo virus Unnamed Le Dantec virus Unnamed Tibrogargan virus Unnamed Unnamed ‘Nyamanini virus’ (suggested ) These names were suggested by individual researchers in original publications. The names of these viruses have no formal standing in virus taxonomy; and it is unclear whether proposals with these names have been formally submitted to the International Committee on Taxonomy of Viruses (ICTV). negaviruses share a similar genome organization, lope and membrane-associated proteins. Filoviruses with conserved regions at each end of their geno- encode a second minor nucleoprotein (VP30), which mic, negative-sense, single-stranded RNA [1503, is unique among the mononegaviruses, from the vari- 2066]. These regions encode the core proteins and able region [1503, 2066]. the viral polymerases. Variable regions, located Division of the genus ‘Filovirus’ into two genera, between the conserved regions, encode the enve- and their assignment to two families, ‘Alpha-Filo- Introduction 25 viridae’ and ‘Beta-Filoviridae’, respectively, was Since in filoviral genomes, synonymous nucleo- suggested in the late 1990s [2396] but not accepted tide diversity exceeds non-synonymous nucleotide by the ICTV. Nevertheless, further characterization diversity, it was suggested that purifying selection revealed substantial differences between ‘Marburg at certain polymorphisms is a mechanism for re- virus’ and ‘Ebola virus’ [2221], and the separation duced gene diversity [1195]. of the family into two genera was clearly supported Phylogenetic GP and L gene analyses place the by molecular studies [818, 1702]. Radioimmunoas- first discovered Zaire ebolavirus (1976 Mayinga says demonstrated that viruses of the two genera isolate) very near to the root of the ZEBOV tree and differ antigenically [2118], and oligonucleotide suggest that all other ZEBOV isolates have evolved mapping of the filoviral genomes provided evi- from a ZEBOV Mayinga-like virus after 1976 [327, dence that they are only distantly related [612]. 2679]. These data suggest that individual Zaire Evolutionary studies suggested that ‘Marburg virus’ ebolavirus disease outbreaks are epidemiologically and ‘Ebola virus’ diverged 7,100–7,900 years ago linked. Measurements of the geographical distance [2488]. The average rate of non-synonymous sub- between individual Zaire ebolavirus disease out- stitutions in the GP gene4 of what the agent known breaks suggested that ZEBOV spreads at a constant today as Zaire ebolavirus (ZEBOV) was estimated rate of 50 km per year. The genetic similarity of to be 3.6105 [2488] or 8104 per site per year individual ZEBOV isolates decreases with in- [2679], while a comparison of partial ZEBOV L gene creasing geographic distance of the outbreaks they sequences suggested a value of 1.1103 [327]. A cause, and this decrease in similarity occurs at the direct comparison of the fully determined ZEBOV same rate at all spatial scales. Together, these re- 1976 Mayinga isolate and ZEBOV 1995 Kikwit iso- sults indicate that ZEBOV is a relatively recent in- late genomes, which are 98.8% identical, yielded an troduction into human populations and that it has ad hoc evolutionary rate estimate of 6.2104 not been endemic at each outbreak area for an ex- nucleotide substitutions per site per year [327]. tended period of time [327, 2679]. Sequence analyses of the VP35, VP30, and VP24 Twenty-seven complete genomic sequences of filo- genes of various MARV isolates indicated that they viruses have been determined so far (see Table 4-4), are evolving at the rate of 105 to 104 substitu- but many more partial sequences of additional iso- tions per site per year. Rates of synonymous sub- lates5 are available (see Table 4-5). A comparison stitutions for MARV and ebolaviruses have been of the GP and VP24 genes of the ZEBOV isolates estimated to be 1.35102 and 1.77102 per site Ecran (Zaire 1976), Gabon (Gabon 1994), Kikwit per year at a maximum, respectively. Ninety per- (Zaire 1995), and Mayinga (Zaire 1976) revealed cent of the mutations in the third codon positions of a close relationship among them but that they are the MARV genomes were transitions, and the fre- not identical. Several passages of the Mayinga iso- quency of transitions was the same for purines and late in cell cultures did not result in significant pyrimidines. Taken together, these findings implied nucleotide sequence changes in the GP gene – con- that filoviruses evolve 100 times more slowly served regions, which differed slightly between dif- than do orthomyxoviruses or retroviruses [2488]. ferent isolates, did not mutate with further passage. Sequence analysis indicated that the Ecran and Mayinga isolates (both from the same outbreak) 4 The characteristics of the individual filoviral genes are were not the direct ancestors of the Gabon and discussed in detail in chapter 11. To understand filovirus Kikwit isolates [1108, 2651]. This hypothesis was taxonomy it suffices to know that filoviral genomes contain seven genes flanked by leader (l) and trailer (t) sequences (30- l-NP-VP35-VP40-GP-VP30-VP24-L-t-50). The GP and L genes, which encode spike proteins and RNA-dependent 5 In the filovirus literature, the term ‘‘strain’’ is almost RNA polymerases, respectively, are the least conserved filo- always used indiscriminately. The term is avoided in this viral genes, whereas the VP40 genes, which encode matrix review since criteria to differentiate filovirus strains from proteins, have very limited sequence diversity. individual isolates have not yet been published. 26 Introduction States Army GenBank accession number(s) Sequenced at the United States Army AY430366 AY430365 DQ44752 (listed as isolate ‘09DRC’) DQ447651 (listed as isolate ‘05DRC’) DQ447650 (listed as isolate ‘07DRC’) Medical Research Institute of Infectious Diseases inMaryland, Frederick, U.S.A. Sequence isavailable not to yet the public Medical Research Institute of Infectious Diseases inMaryland, Frederick, U.S.A. Sequence isavailable not to yet the public DQ447653-DQ447660 AY358025 Z12132; cDNA clone: DQ217792 Mt. Elgon) DQ447649 = Mt. Elgon) = South Africa ge municipalities) ´ Year and place of isolation of the Congo (Durba) of the Congo (Durba) of the Congo (Durba) Laboratory isolate 2004–2005: Angola (Bungo, Damba,and Songo, Uı 1967: Germany (Marburg an der Lahn) Sequenced at the United 1975: Rhodesia= (Johannesburg) 1987: Kenya (Mombassa 1980: Kenya (Nzoia 1967: Germany (Frankfurt am Main) Z29337, NC_001608 99-Nga 1998–2000: Democratic Republic 99-Dra 1998–2000: Democratic Republic 99-Aru 1998–2000: Democratic Republic = = = MARV-DRC-5 MARV-DRC-5 Guinea pig-adapted MARV-Ravn MARV-DRC-5 MARV-Angola MARV-Ci67 Abbreviation MARV-Ozo MARV-Musoke-pp4 Laboratory isolate MARV-Ravn MARV-Mus MARV-Musoke-pp3 Laboratory isolate MARV-Pop 2 1 Completely sequenced filovirus genomes (as of September 2007) 99-Nga isolate 99-Dra isolate 99-Aru isolate = = = Lake Victoria marburgvirus, Musoke isolate Lake Victoria marburgvirus, DRC-5 Lake Victoria marburgvirus, DRC-5 Lake Victoria marburgvirus, Ravn isolate, guinea pig-adapted Lake Victoria marburgvirus, DRC-5 Lake Victoria marburgvirus, Ci67 isolate Lake Victoria marburgvirus, Angola isolates Table 4-4. Virus Lake Victoria marburgvirus, Poppinga isolate Lake Victoria marburgvirus, Ravn isolate Lake Victoria marburgvirus, Ozolin isolate Lake Victoria marburgvirus, Musoke isolate, guinea pig-adapted plaque pick 4 Lake Victoria marburgvirus, Musoke isolate, guinea pig-adapted plaque pick 3 Introduction 27 under , it is not [2656], the AF499101 AF272001 Medical Research Institute of Infectious Diseases inMaryland, Frederick, U.S.A. Sequence isavailable not to yet the public AY354458 (listed as isolate ‘Zaire 1995’) AF086833, AY142960, NC_002549 AB050936 (listed as isolate ‘Reston’) AF522874, AY769362, NC_004161 Sequenced at the United States Army AY729654, NC_006432 V-Mayinga-8mc-N1-N5). According to the corresponding publication of the Soviet Union and its successor states. in the literature. of which are listed as hallmarks of this clone in the same publication. Hence GenBank by using accession number AF272001. However, the sequence deposited experiments reported in the Canadian, European, and U.S. literature. 1995: Zaire (Kikwit) 1976: Zaire (Yambuku) 1976: Sudan (Maridi) Laboratory isolate Laboratory isolate 2000–2001: Uganda (Gulu District) 4 REBOV-Pennsylvania 1989: UnitedREBOV-Philippines1996 States (Philadelphia) 1996: Philippines (Luzon) SEBOV-Bon None ZEBOV-May ZEBOV-Mayinga-8mc 3 c mouse-adapted) = clear to which of the five clones the deposited sequence refers. MARV-Mus is the LakeThus Victoria marburgvirus far, isolate MARV-Pop has used onlyZEBOV-May in is been most the used ebolavirus experimentallyFive isolate by different used investigators clones in of most ZEBOV-Mayinga-8mc experiments have been reported obtained (ZEBO complete sequence of ZEBOV-Mayinga-8mc-N4 can be retrievedthis from number does not show a U insertion and a nucleotide substitution, both Reston ebolavirus, Pennsylvania isolate 1 2 3 4 Reston ebolavirus, Philippines1996 isolate Sudan ebolavirus, Boniface isolate Sudan ebolavirus, Gulu isolateZaire ebolavirus, Mayinga isolate SEBOV-Gul Zaire ebolavirus, Kikwit isolate ZEBOV-Kik Zaire ebolavirus, Mayinga isolate (BALB Zaire ebolavirus, Mayinga isolate (guinea pig-adapted) 28 Introduction Sequence = (AY058897) (AY058896), Gene (DQ211657) (U23152) L VP24 VP40 (DQ978379) GP L (U28134) GP (U23458) L (AY058898), (AY058895), (EF183506) (EF183507) (EF183508) L NP L L GP (U81161) (AF034645), genome: AF522874, (AY526102), partial (U28006), partial (U23416) (AY344234, AY316199), genome: (U23069), (AY526100) (U23417) (AF173836), , not yet available to the public (GenBank accession number) GP Partial genomic sequences. GP AY769362, NC_004161 GP GP GP partial GP AY729654, NC_006432 GP NP GP GP GP No Complete genomic sequence determined No None No Yes No Partial No Partial No None No Partial Yes No Partial No None No No Yes Genomes: AB050936, No None No No No Yes Genome: AF499101 No None No None No None No Yes th of isolates also see Table 5-1) Yes Listed in the 8 ICTV Report Yes No No No No No No No No No Yes No Yes Yes No No No Yes (IC1), (CI-94) Yes Eckron (Eck), Eckron-76, 057878 Outdated isolate designation(s) (abbreviation) Manila, 920084 No Gabon96 Oct No 119810 (KUM) Nzara, 015176 Yes (AZ-1435) 12552 Maridi, VCP2D11 Yes 1 2 eee-96) 3 c mouse-adapted = eee-96(ZEBOV-Bou ^ ooted’Ivoire (CIEBOV-CI) Philippines (REBOV-Phi) Entsiami (ZEBOV-Entsiami) (abbreviation) Philippines1992 (REBOV-Philippines1992) Pennsylvania (REBOV-Pennsylvania) Maleo-Yambio0401 (SEBOV-Maleo-Yambio0401) Ekata November (ZEBOV-Ekata-Nov) Kum (SEBOV-Kum) Maleo (SEBOV-Mal) Maleo-Yambio0402 (SEBOV-Maleo-Yambio0402) Maleo-Yambio0403 (SEBOV-Maleo-Yambio0403) Ekata December (ZEBOV-Ekata-Dec) Philippines1996 (REBOV-Philippines1996) Gulu (SEBOV-Gul) Etakangaye (ZEBOV-Etakangaye) Siena (REBOV-Sie) E718 (ZEBOV-E718) Ecran (ZEBOV-Ecran) ZEBOV-Mayinga Gab275 (ZEBOV-Gab275) Bou Texas (REBOV-Tex) Partial genomic filovirus sequences (as of September 2007; for the origin CIEBOV C Table 4-5. Virus Current isolate designation REBOV 28H (REBOV-28H) ZEBOV BALB SEBOV Boniface (SEBOV-Bon) Introduction 29 ) continued ( (L11365), (X61274), (DQ205417) (DQ205416) VP24 L L VP35 (U28077), genome: (U77385) (U23187), (AJ001707), (L11365, U31033, (L11365), GP GP GP (L11365), VP24 VP40 VP35 (M33062), (DQ205418) VP35 (L11365) NP L L (X61274), (L11365), (AY526098), partial (AY526105), partial (AY526104) (U77384), (AY526101) (AY526099) (AY526103) (Y09358), (AF054908), (L11365), , not yet available to the public , not yet available to the public , not yet available to the public , not yet available to the public , not yet available to the public , not yet available to the public , not yet available to the public , not yet available to the public (X67110), genome: AF086833, GP GP NP GP GP NP GP GP AY354458 GP GP GP VP40 GP GP L AY142960, NC_002549 GP NP VP40 GP J04337), GP partial GP No No Yes No Yes Genome: AF272001 No None No None No No None No None No No No No None No No Yes Partial No No No No No Partial No No No No No No No No No No No No No No No No Yes No No No No No No No Zaire76, 186538 Yes Gabon-94 (Gab280) Yes Zaire 1995, 9510621 Yes ME, 057935 Yes 088296 Bonduni (BND), 5 4 Gab276 (ZEBOV-Gab276) Guinea pig-adapted laboratory isolate (ZEBOV-Mayinga-Ch-15) Guinea pig-adapted laboratory isolate (ZEBOV-Mayinga-K-5) Mvoula A (ZEBOV-Mvoula) ICR mouse-adapted laboratory isolate (ZEBOV-Mayinga-D-5) Mekambo-01 (ZEBOV-Mekambo-01) Mendemba B (ZEBOV-Mendemba-B) Mendemba A (ZEBOV-Mendemba-A) Gab277 (ZEBOV-Gab277) Kikwit (ZEBOV-Kik) Makokou (ZEBOV-Makokou) Olloba (ZEBOV-Olloba) Gab278 (ZEBOV-Gab278) Mayibout1996 (ZEBOV-Mayibout1996) Mayinga (ZEBOV-May) SA253 (ZEBOV-SA253) Gab279 (ZEBOV-Gab279) Guinea pig-adapted laboratory isolate (ZEBOV-Mayinga-8mc) Yembelengoye (ZEBOV-Yembelengoye) Zaire (ZEBOV-Zai) Tandala (ZEBOV-Tan) Gab281 (ZEBOV-Gab281) Gab282 (ZEBOV-Gab282) Gab293 (ZEBOV-Gab293) Gabon (ZEBOV-Gab) 30 Introduction Sequence = VP35 VP35 VP35 VP35 VP35 VP35 VP35 VP35 VP35 VP35 Gene (DQ466112), (DQ466189), (DQ466187), (DQ466190), (DQ466188), (DQ466195), (DQ466194), (DQ466193), (DQ466149) (DQ466151) L L VP35 GP GP GP GP GP GP GP (DQ466120), (DQ466122) (DQ466125), (DQ466121), (DQ466123), (DQ466179), partial (DQ466177), partial (DQ466186), partial (DQ466184), partial (DQ466175), partial (DQ466185), partial (DQ466178), partial (DQ466183), partial (DQ466182), partial (DQ466181), partial (DQ466145) (DQ466147) (DQ466146) (DQ466150) (DQ466153) (DQ466154) (DQ466155) (DQ466144) (DQ466156) (DQ466160) (DQ466157) (DQ466158) NP NP NP NP NP NP NP NP NP NP VP35 VP35 VP35 VP35 VP35 L L L L L L L L L L L L partial partial partial partial partial partial partial (DQ466109), partial (DQ466115), partial partial (DQ466174), partial (DQ466116), partial (DQ466110), partial (DQ466111), partial (DQ466114), partial (DQ466118), partial (DQ466119), partial (DQ466117), partial (GenBank accession number) partial partial partial partial Partial genomic sequences. NoNo Partial No Partial No Partial Partial No Partial NoNo Partial No Partial No Partial NoNo Partial No Partial Partial No Partial Partial Complete genomic sequence determined No Partial No Partial th ICTV Report No No Listed in the 8 Outdated isolate designation(s) (abbreviation) (MARV-05DRC99-2) (MARV-06DRC99-2) 6 6 (MARV-01DRC99) 01DRC99apr12(MARV-02DRC99) 02DRC99apr26(MARV-03DRC99) No 03DRC99apr30(MARV-04DRC99) No 04DRC99may01 No (MARV-06DRC99) No 06DRC99may01(MARV-08DRC99) No 08DRC99may09(MARV-10DRC99) 10DRC99aug06 No (MARV-11DRC99) 11DRC99aug18 No (MARV-12DRC00)(MARV-13DRC00) 12DRC00jan08 No (MARV-14DRC00) 13DRC00jan15(MARV-15DRC00) 14DRC00jan22 No (MARV-16DRC00) 15DRC00feb13 No No 16DRC00feb11 No No ) 6 6 6 6 6 6 6 6 6 6 6 6 6 continued ( 06DRC99 08DRC99 03DRC99 04DRC99 05DRC99-2 06DRC99-2 10DRC99 11DRC99 12DRC00 02DRC99 13DRC00 15DRC00 16DRC00 14DRC00 (abbreviation) MARV 01DRC99 Table 4-5 Virus Current isolate designation Introduction 31 ) continued ( (DQ466159) (DQ466170) (DQ466162) (DQ466161) (DQ466171) (DQ466163) (DQ466172) (DQ466164) (DQ466173) (DQ466165) (DQ466166) (DQ466167) (DQ466168) (DQ466169) L L L L L L L L L L L L L L GP GP (DQ466180), (DQ466176), NP NP (Z12132, M72714), (Z12132), (DQ466148) (DQ466152) L L (Z12132), NP (Z12132, M92834), VP40 L VP30 (DQ466140) (DQ466124), partial (DQ466126) (DQ466128) (DQ466129), partial (DQ466130) (DQ466132), partial (DQ466131), partial (DQ466143), partial (DQ466133), partial (DQ466134), partial (DQ466135) (DQ466136), partial (DQ466137), partial (DQ466138), partial (DQ466139), partial (DQ466141), partial (DQ466142), partial (DQ466127), partial (DQ466108), partial (DQ466113), partial VP35 VP35 VP35 VP35 VP35 VP35 VP35 VP35 VP35 VP35 VP35 VP35 VP35 VP35 VP35 VP35 VP35 VP35 VP35 VP35 VP35 (Z12132), (Z12132), (Z12132), leader (M36065), 0 (DQ466191), partial partial (DQ466192), partial partial VP35 GP VP24 genome: Z12132; cDNA cloneDQ217792 genome: Yes Genome: DQ447652 Yes Genome: DQ447651, partial NoNoNo Partial No Partial NoNo Partial No Partial No Partial No Partial No Partial No Partial No Partial No Partial No Partial No Partial Partial No Partial No Partial Partial Partial No None No None Yes 3 Yes Genome: DQ447650, partial Yes No None Yes Genomes: DQ447653-DQ447660 No Partial No Partial Yes Genome: AY430365 No No Yes No No No No No No ‘‘F’’ 09DRC, 09DRCmay26 No 05DRC, 05DRCmay08 No 07DRC, 07DRC99, 07DRC99may08 Marburg’67 and Cro) 99-Nga) 99-Dra) 99-Aru) = = = (MARV-32DRC00-2) (MARV-19DRC00) 19DRC00feb23 No 6 (MARV-17DRC00)(MARV-18DRC00) 17DRC00feb02(MARV-20DRC00) 18DRC00feb14(MARV-21DRC00)(MARV-22DRC00) 20DRC00feb12(MARV-23DRC00) No 21DRC00feb24(MARV-24DRC00) No 22DRC00may05(MARV-25DRC00) 23DRC00mar23(MARV-26DRC00) No 24DRC00apr04(MARV-27DRC00) No 25DRC00aug23 No (MARV-28DRC00) 26DRC00may20 No (MARV-29DRC00) 27DRC00jul03(MARV-30DRC00) No 28DRC00jul10 No (MARV-31DRC00) 29DRC00jul14 No (MARV-32DRC00) 30DRC00aug05 No 31DRC00aug11(MARV-33DRC00) No 32DRC00aug13(MARV-34DRC00) No No 33DRC00aug17 No 34DRC00aug23 No No No 6 6 6\ 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 99-Nga 99-Dra 99-Aru = = = Flak (MARV-Flak) Musoke (MARV-Mus) Mouse-adapted MARV-Flak (MARV-DRC-5 DRC-5 (MARV-DRC-5 DRC-5 (MARV-DRC-5 Ci67 (MARV-Ci67) 18DRC00 22DRC00 24DRC00 25DRC00 26DRC00 27DRC00 28DRC00 30DRC00 31DRC00 32DRC00 32DRC00-2 34DRC00 Angola (MARV-Angola) Cruickshank (MARV-Cru) (also called CRO DRC-5 20DRC00 17DRC00 33DRC00 19DRC00 21DRC00 23DRC00 29DRC00 Guinea pig-adapted plaque pick 3 isolate (MARV-Musoke-pp3) 32 Introduction V nk 99- = these under , it is not in Gabon. [2656], the Sequence the ZEBOV- = (Z29337, (Z29337, Gene VP30 VP35 (AF005733), genome: (AF005735) (AF005734), GP GP GP (Z29337, X64406), (Z29337, X64405), VP40 VP24 (AF005730), (AF005732), (AF005731), (Z29337, X68493), (Z29337, X68495), (Z29337, X68494), genome: Z29337, None (GenBank accession number) Genome: AY430366 None VP35 AY358025 NP X64406), GP X64405), VP35 VP35 genome: DQ447649 None L NC_001608 None Partial genomic sequences. None None No Yes No Yes Yes No No Complete genomic sequence determined No Yes No No ’’). ^ th 99-Mae, and DRC-99-Ova. Unfortunately, it is unclear which of = wever, in these years, three independent disease outbreaks caused by ZEBO as ‘‘Eckron (ZEBOV-Eck).’’ circumflex ‘‘ fully sequenced, but not even partial sequences are available. The listed GenBa V-Mayinga-8mc-N1-N5). According to the corresponding publication sequences [961, 965, 1565]. In this review, the Gab280 isolate is called of which are listed as hallmarks of this clone in the same publication. Hence No No Yes Yes No No ICTV Report No Yes Yes Listed in the 8 a, DRC-00-Doi, DRC-00-Kul, DRC-00-Mam, DRC-00-Man, DRC-00-Mbo, DRC-4 GenBank by using accession number AF272001. However, the sequence deposited listed in the report. 99-Lad, DRC-8 the 1994 outbreak, which was the first ebolavirus disease outbreak ever recorded = (RAV) (HGN), Hog No ‘‘H’’ (VOG) (RYT), Ryc Yes Outdated isolate designation(s) (abbreviation) 99-Buk, DRC-5 = 99-Wer, DRC-5 = ) 99-Kul, DRC-4 = continued Guinea pig-adapted plaque pick 4 isolate (MARV-Musoke-pp4) Hartz (MARV-Hartz) ‘‘L’’ (MARV-‘‘L’’) Hogan (MARV-Hogan) Ozolin (MARV-Ozo) Poppinga (MARV-Pop) Ravn (MARV-Ravn) Variant ‘‘U’’ (MARV-‘‘U’’) Voege (MARV-Voe) Porton (MARV-Porton) Ratayczak (MARV-Rat) (abbreviation) ( ICTV Report lists this virus as Cote d’Ivoire ebolavirus (without the ICTV Report and several publications refer to this isolate mistakenly ICTV Report lists the years 1994–1997 for the ZEBOV-Gab isolate. Ho ICTV Report mistakenly lists the Philippines1989 isolate of REBOVas th th th th The 8 The 8 The 8 The 8 Five different clones of ZEBOV-Mayinga-8mc have been obtained (ZEBO Some of these isolates also were reported as DRC-00-Alt, DRC-00-Av designations correspond to which particular isolate designation in the table. accession number is that of the Philippines1996 isolate, which is not clear to which of the five clones the deposited sequence refers. Bon, DRC-4 complete sequence of ZEBOV-Mayinga-8mc-N4 can bethis retrieved number from does not show a U insertion and a nucleotide substitution, both occurred in Gabon and the isolatesGab from isolate, each outbreak since differ it in their was genomic isolated from a sample collected during Table 4-5 1 2 3 4 5 6 Virus Current isolate designation Introduction 33 supported by a comparison of the GP gene se- position of these isolates in relation to the other quences from ZEBOV isolates obtained during three ebolaviruses [934, 2014]. The third species (Sudan outbreaks in Gabon in 1994, 1996, and 1996–1997, ebolavirus) is represented by ebolaviruses iso- in addition to the Kikwit isolate. These results lated during human disease outbreaks in Sudan in confirmed that isolates from individuals who were 1976 (Boniface), 1979 (Kum, Maleo), and 2004 ill during the same outbreak usually have almost (Maleo-Yambio0401-0403), and in Uganda in 2000 identical sequences, whereas viruses from different (Gulu). Tryptic peptide-mapping comparisons of outbreaks vary by only a few nucleotides. Further- the Boniface, Kum, and Maleo isolates on the more, 10–14 amino acid differences were found in one hand, and the isolates Ecran, Mayinga, and the spike (GP1,2) proteins among the Reston ebola- Tandala of the fourth lineage, Zaire ebolavirus, virus isolates from three independent outbreaks that on the other, demonstrated differences in the spike occurred in 1989, 1992, and 1996, whereas no amino- and VP40 proteins of the respective viruses [443]. acid differences were found among different iso- Partial sequencing of the Sudan ebolavirus isolate lates from each individual outbreak [961, 965, Gulu demonstrated that it was not derived from 1224, 1565, 2015, 2136, 2237]. Nucleic acid se- either the Boniface or the Maleo isolates, but that quence determination of the complete NP, VP40, all share a common ancestor [2561]. Using se- GP,andVP24 genes of ZEBOV isolates obtained quence analyses and mathematical models, it was during the 1996–1997 outbreak in Gabon con- estimated that Coote^ d’Ivoire ebolavirus and firmed the genetic stability of the ebolaviruses. ZEBOV diverged from each other 700–1,300 Isolates from fatal cases and isolates from survi- years ago and Reston and Sudan ebolaviruses sep- vors were identical. Isolates from three asymptom- arated 1,400–1,600 years ago. The two clusters atic individuals revealed a synonymous (C ! T) diverged from each other 1,000–2,100 years ago substitution in the VP40 gene, and one human ebo- [2488]. lavirus isolate with a G ! AmutationintheVP24 MARV and ebolaviruses differ antigenically gene [1565]. Isolates of ZEBOV from an outbreak [1405], although recent experiments suggest at least in 1976 in Zaire differ from the 1995 Kikwit some antigenic relationships between the mar- isolates in 1.5% of their GP-gene nucleotide burgviral and ebolaviral nucleoproteins (NP) and sequences. Similarly, the amino-acid sequences viral proteins 35 (VP35) [3046]. MARV isolates of the spike proteins derived from these genes dif- from different outbreaks possess nucleotide se- fer by only 1.5%. The amino-acid sequences of the quences that are very similar to each other and secreted glycoprotein sGP, which is encoded by are closely related antigenically. However, com- the same gene, were identical for both isolates parisons of the physicochemical and antigenic [2237]. properties of the Hogan (Rhodesia=South Africa Separation of ‘Ebola virus’ into four virus spe- 1975), Musoke (Kenya 1980), Ozolin (Rhodesia= cies was justified after results of in silico analyses. South Africa 1975), Ratayczak (Germany 1967), Genomic signatures for each of the viruses as- and Voege (Germany 1967) isolates suggested signed to the four species were identified and used that more than one MARV variant exists. Peptide for unambiguous identification [685]. One species mapping of the marburgviral NP, spike, and VP40 (Coote^ d’Ivoire ebolavirus) is represented by a hu- proteins has revealed extensive sequence homo- man ebolavirus isolate from Coote^ d’Ivoire in 1994 logies among various isolates [1405]. A 94% over- (isolate CI) [2232, 2235]. A second species (Reston all identity was recognized after alignments of ebolavirus) includes viruses exclusively isolated the complete genomic nucleic acid sequences of from nonhuman primates affected during epizoot- the Musoke and Poppinga (Germany 1967) iso- ics in 1989–1990 (28H, Pennsylvania, Philippines, lates [449, 450, 2981, 2984]. The nucleotide se- Texas), 1992 (Philippines1992, Siena), and 1996 quences of the VP35 and GP genes of the Musoke, (Philippines1996). Oligonucleotide mapping and Ozolin, Poppinga, and Ratayczak isolates were cross-neutralization tests confirmed the unique >90% identical [2234]. However, the Ravn isolate, 34 Introduction obtained in Kenya in 1987, is more distantly species (Cooted’Ivoire,^ Reston, Sudan, and Zaire related [1316, 2234]. Comparisons of partial GP- ebolaviruses) differ genetically by 37–41% at the gene sequences revealed only a 72.3% and a 71% nucleotide level (and from MARV by >65%) [821] nucleotide identity of the Ravn isolate with the (for the most up-to-date filovirus phylogeny see Musoke and Poppinga isolates, respectively. Figs. 4-3 through 4-7). Alignments of the deduced peptide amino acid Obviously, this plethora of data has led to at sequences derived from these GP regions revealed least some confusion, such that it has been difficult that those of the Ravn and Musoke isolates are for the ICTV to provide a rational yet useful ba- 72% identical, whereas a comparison of those of sis for the taxonomy of filoviruses. To reflect all the Ravn and Poppinga isolates revealed 67% the available information, the genus ‘Filovirus’ identity [1316]. A more comprehensive indication was subdivided in 1999. The genus ‘‘Ebola-like of homologies evolved after the recent determina- viruses’’ contained the four species ‘Coote^ d’Ivoire tion of the complete genomic sequences of the Ebola virus’, ‘Reston Ebola virus’, ‘Sudan Ebola Angola (Angola 2004–2005), DRC-5=99-Aru, virus’, and ‘Zaire Ebola virus’; these replaced DRC-5=99-Dra, DRC-5=99-Nga (Democratic the hitherto used designations ‘Ebola virus Coote^ Republic of the Congo 1998–2000), Ozolin, and d’Ivoire subtype’, Ebola virus Reston subtype’, Ravn isolates [2562]. Accordingly, at least five ‘Ebola virus Sudan subtype’ and ‘Ebola virus different lineages of MARV isolates exist. The Zaire subtype’. The genus ‘‘Marburg-like viruses’’ Angola lineage consists of isolates divergent from contained only one species: ‘Marburg virus’. The the Musoke and Poppinga=Ci67 lineage by 6.8 individual virus names were the same as those of and 7.1% genomic nucleotide differences, respec- the species, and abbreviated CIEBOV, REBOV, tively, and divergent by >7.4% from the Ozolin= SEBOV, ZEBOV, and MARV. These abbreviations DRC-5=99-Aru=DRC-5=99-Dra lineage. The Ravn replaced the previously used abbreviations EBOV- and DRC-5=99-Nga isolates represent a fifth line- CI, EBOV-R, EBOV-S, EBOV-Z, and MBGV, age with nucleotide differences reaching 21% rela- respectively [792]. Vernacular names had been tive to viruses of the other four lineages [2562]. assigned to the two genera because it was clear In comparison, members of the four ebolaviral that the taxonomic definition of each virus group

Figs. 4-3 to 4-7. Filovirus phylogeny. Phylogenetic analysis was performed using a nucleotide-sequence alignment of either complete genomic sequences, the polymerase (L) gene sequences, or the spike-protein (GP) gene sequences as indicated. For the complete genomic or GP-gene sequences, separate ebolavirus and Lake Victoria marburgvirus alignments were con- structed since sequence similarity between isolates of the two genera was not sufficiently high to construct reliable multiple sequence alignments. Only the L gene showed sufficient similarity between isolates of the two genera allow for construction of a reasonable alignment. Multiple sequence alignments were constructed using the program MUSCLE (Edgar Robert C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research (Oxford) 32(5): 1792–1797). Phylogenetic inferences were generated using Bayesian inference methods (inferences generated using maximum-parsimony analysis, or neighbor-joining analysis produced trees that were essentially the same as those generated using Bayesian inference). MrBayes version 3.1 (Ronquist Fredrik, Huelsenbeck John P. (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics (Oxford) 19(12): 1572–1574) was used for Bayesian inference methods. Bayesian inference used Markov chain Monte Carlo methods using a general time reversible (GTR) model of nucleotide substitution and allowed for gamma-distributed variation across sites with a proportion of invariable sites. For each alignment, the consensus phylogenetic tree obtained after Bayesian analysis is presented. Trees are drawn with horizontal branch lengths proportional to the number of nucleotide substitutions. Each figure provides a scale indicating the length of a branch that corresponds to 0.1 base substitutions per site (nucleotide). Each node (branch point) in a tree is labeled according to the probability that the particular partition or clade represented by that node is representative of all sampled trees. A probability of 1.00 indicates 100% confidence in that partition, while a lower probability indicates that some of the sampled trees showed alternative clade arrangements at that node (all phylogenetic trees courtesy of Elliot J. Lefkowitz) Introduction 35

Fig. 4-3. Phylogenetic analysis using filovirus RNA-dependent RNA-polymerase (L) genes still was incomplete [1883, 2062–2064]. At a plen- Zaire ebolavirus. The species name ‘Marburg ary session in Paris, 2002, the ICTV changed the virus’ was replaced by Lake Victoria marburg- ebolavirus species names to ‘Ivory Coast ebola- virus, and the genus names were finally established virus’, Reston ebolavirus, Sudan ebolavirus,and as Ebolavirus and Marburgvirus, respectively (see 36 Introduction

Fig. 4-4. Phylogenetic analysis using full-length ebolavirus genomic sequences

Tables 4-6 and 4-7) [1693]. These changes brought [805]. In this review, the latest filovirus nomencla- the taxonomy of filoviruses into agreement with the ture (see Tables 4-4 and 4-5) is used whenever stylistic taxonomy of other viruses, addressing the possible6. requirements that species (taxon) names be itali- cized, whereas the names of viruses (the entities) 6 th th The reader should be aware that the 8 ICTV Report does are not [472]. The latest (8 ) ICTV Report lists not list all filovirus-isolate names recorded in the literature the virus names, which are identical to the species and in nucleotide and protein databases. In this review, names except for the lack of italics, and assigns the abbreviations for isolates not mentioned in the ICTV Report abbreviations CIEBOV, REBOV, SEBOV, ZEBOV, are used as well (see Tables 4-4 and 4-5). Unfortunately, in ^ many publications it is not explicitly stated which filovirus and MARV for the viruses Coote d’Ivoire ebola- isolate had been used for experiments. Only the virus abbre- virus (which replaced ‘Ivory Coast ebolavirus’), viation without further designation is stated in this review in Reston ebolavirus, Sudan ebolavirus, Zaire ebola- these cases. Furthermore, some filoviruses have not yet virus, and Lake Victoria marburgvirus, respectively received a name or designation (e.g. the BALB=c mouse- [805]. According to this latest ICTV Report, adapted ZEBOV-Mayinga variant or several guinea pig- the abbreviation for an isolate is the respective adapted filoviruses). In cases of unclear origin, viruses are referred to in general as ‘‘mouse-adapted’’ or ‘‘guinea pig- virus abbreviation plus an abbreviation for the adapted’’ filoviruses. It will be important to develop a more isolate, e.g. SEBOV-Gul. Fourteen ebolavirus rigorous taxonomic standard, to achieve uniformity between and six MARV isolates are officially recognized electronic databases and publications. Introduction 37

Fig. 4-5. Phylogenetic analysis using ebolavirus spike-protein (GP) genes

In the life sciences, biosafety is ‘‘...the contain- referred to a recently published book on biosafety ment principles, technologies and practices that are [850] and to other references listed here. implemented to prevent the unintentional exposure to pathogens and toxins, or their accidental 4.3 Biosafety concerns in filovirus research release’’ [2873]. In this chapter, biosafety terminol- ogy will be briefly reviewed in regard to filovirus MARV and ebolaviruses have fascinated and fright- research. For additional information, the reader is ened the general public in part because these 38 Introduction

Fig. 4-6. Phylogenetic analysis using full-length Lake Victoria marburgvirus genomic sequences viruses are members of an exclusive and very small series [2728]. The term ‘‘biosafety level 4’’ became group of human and animal pathogens classified widely known and unfortunately is often used as highest risk pathogens. Images of researchers incorrectly and associated with the misconception (‘‘virus hunters’’) equipped with plastic ‘‘space’’ that highly contagious agents intentionally sur- suits to investigate these viruses and the horrific round the ‘‘space’’ suits, threatening scientists and results of infection can be found in the general the population in a premeditate, orchestrated man- media, as well as Hollywood productions and TV ner [2255]. Introduction 39

Fig. 4-7. Phylogenetic analysis using Lake Victoria marburgvirus spike-protein (GP) genes

Table 4-6. Current filovirus taxonomy according to the 8th Report of the International Committee on Taxonomy of Viruses (ICTV) [805]

Order Family Genus Species Virus (abbreviation) Mononegavirales Filoviridae Ebolavirus Cooted’Ivoire^ ebolavirus Cooted’Ivoire^ ebolavirus (CIEBOV) Reston ebolavirus Reston ebolavirus (REBOV) Sudan ebolavirus Sudan ebolavirus (SEBOV) Zaire ebolavirus Zaire ebolavirus (ZEBOV) Marburgvirus Lake Victoria marburgvirus Lake Victoria marburgvirus (MARV) The 8th ICTV Report lists the species and virus as Cote d’Ivoire ebolavirus and Cote d’Ivoire ebolavirus, respectively (without the circumflex ‘‘^’’).

In most countries, infectious agents are classified laboratory worker or of the environment with sub- by risk group [2582, 2873, 3279]. Agent classi- sequent infection of the general population. It fication emphasizes the potential risk and conse- reflects the overall knowledge of a given pathogen, quences of exposure to infectious agents of the in particular its infectivity, mode and ease of trans- 40 Introduction

Table 4-7. Differentiation between Lake Victoria marburgvirus and ebolaviruses according to the 8th Report of the Inter- national Committee on Taxonomy of Viruses (ICTV) [805]

Lake Victoria Ebolaviruses marburgvirus Antigenic cross-reactivity Minimal Minimal with members of the other genus Average particle length 665 nm 805 nm Genome length 19.1 kb 18.9 kb Gene overlaps One Several Co-transcriptional GP mRNA editing No Yes Protein profile Homologous sequences among all Species-specific sequence isolates, clearly distinct from differences, clearly distinct ebolaviruses from Lake Victoria marburgvirus Case-fatality rate in humans 25–90% 50–90% (exceptions are Cooted’Ivoire^ in larger outbreaks and Reston ebolaviruses at 0%) mission, pathogenicity and virulence (including Filoviruses are particularly hazardous infectious induced morbidity and case-fatality rate), sensitiv- agents. Globally, the consensus is that these agents ity to physical or chemical agents, and availability should be classified in the highest risk category or absence of countermeasures, including vac- and accordingly worked with at the highest bio- cines, therapeutic remedies, and cures [2582, safety level (see Table 4-8) [1429, 1899, 3097, 2873, 3279]. Guidelines for research are generally 3098, 3138]. This consensus was reached because published for each risk group, requiring research- these viruses cause serious disease with an extra- ers to perform techniques and to conduct experi- ordinary high case-fatality rate, pose a high risk to ments in a manner that will minimize risk of laboratory personnel, are very infectious (albeit, exposure or release. and contrary to wide-spread belief, not very conta-

Table 4-8. Filovirus biosafety and containment designations in selected countries

Country= Biosafety designation (filoviruses) Designation for containment facilities Organization required for work with filoviruses Australia Physical Containment (PC)-4 pathogen [1715], Biosecurity Level 4 [238], Risk Group 4 agent [601, 1711] Physical Containment (P)4 [2071] Canada Risk Group 4 [1746] Containment Level (CL) 4 [238, 1746] France Pathogeenede Classe (P)4 Niveau de SeecuriteeBiologique (NSB) [Class (P)4 pathogen] [97] 4 [Biological safety level 4] Gabon Pathogeenede Classe (P)4 Niveau de SeecuriteeBiologique (NSB) [Class (P)4 pathogen]? 4 [Biological safety level 4]? Germany Risikogruppe [risk group] 4 [459] Sicherheitsstufe [Safety level] (S)4 Japan 1 [class 1 infectious Maximum Biosafety Level (MBL) disease] [3245] [2317, 3245] Russia ’pyBB I [group I] pathogen [2433, 3007, 3091] ? South Africa Class 4 pathogen [2071] Physical Containment (P)4 U.K. Category A pathogen in the 1980s Physical Containment (P)4 [2071] [10, 25, 163, 637]; now Protection Level (P)-4 pathogen [638, 840, 2618] U.S. Class 4 pathogen [1272, 2582] Biosafety Level (BSL-)4 [238] World Health Risk Group IV pathogen [2873], Maximum Containment – Biosafety Organization (WHO) WHO Risk Group 4 pathogen [2873] Level 4 [238, 2873] Introduction 41 ) continued ( distribution Middle East, Sub-Saharan Africa, southeastern Europe, southwestern Asia, Russia and NIS worldwide Essentially Venezuela Argentine pampas Bolivia Brazil sp. sp. West Africa Vector Geographic sp. sp., Mastomys Zygodontomys Calomys Calomys Dermacentor Rodents Rodent: brevicauda Rodent: musculinus Ticks: Hyalomma Rhipicephalus Rodent: callosus Natural= reservoir Rodent? Rodents: 1 n (Argentinian) ´ Haemorrhagic fever with renal syndrome (A98.5), ‘‘Hantavirus (cardio-)pulmonary syndrome’’ Unnamed (‘‘Venezuelan haemorrhagic fever’’) (A96.8) Junı haemorrhagic fever (A96.0) (according to ICD-10) Crimean-Congo haemorrhagic fever (A98.0) haemorrhagic fever (A96.1) Human disease Unnamed (‘‘Brazilian haemorrhagic fever’’) (A96.8) Machupo (Bolivian) Lassa fever (A96.2) )ssRNA Bipartite ambisense ssRNA Genome Tripartite ( 3 virus (SABV) 2 ´ U.S. Class 4 pathogens [793, 1426, 2582, 2874] n virus (JUNV) virus ´ ´ n virus ´ Junı Guanarito virus (GTOV) All hantaviruses Virus(es) Crimean-Congo hemorrhagic fever virus (CCHFV) Machupo virus (MACV) Lassa virus (LASV) Sabia Guanarito virus Species Crimean-Congo hemorrhagic fever virus Junı Machupo virus Lassa virus Sabia Arenavirus Nairovirus Hantavirus Genus Family Table 4-9. Arenaviridae Bunyaviridae 42 Introduction Arabia distribution Philippines? Africa Africa Africa Africa Natural reservoir Geographic Macaques Worldwide Ixodid ticks Asia, Eastern Europe ? 1 Human disease Herpesviral encephalitis (B00.4)? Far Eastern tick-borne encephalitis (A84.0) Omsk haemorrhagic fever (A98.1) Ixodid ticks Western Siberia Kyasanur Forest disease (A98.2) Ixodid ticks India, Saudi Marburg virus disease (A98.3) ? None? Ebola virus disease (A98.4)Ebola virus disease (A98.4) ? ? Ebola virus disease (A98.4) ? (according to ICD-10) )ssRNA )ssRNA þ Monopartite ( dsDNA Monopartite ( Genome ) 4 continued ( ^ ooted’Ivoire ebolavirus (CIEBOV) Reston ebolavirus (REBOV) Sudan ebolavirus (SEBOV) Zaire ebolavirus (ZEBOV) C Cercopithecine herpesvirus 1 (CeHV-1) Subtype (TBEV-FE) Tick-borne encephalitis virus, Far Eastern Virus(es) Omsk hemorrhagic fever virus (OHFV) Lake Victoria marburgvirus (MARV) Kyasanur Forest disease virus (KFDV) ^ ooted’Ivoire ebolavirus Reston ebolavirus Sudan ebolavirus Zaire ebolavirus Cercopithecine herpesvirus 1 Tick-borne encephalitis virus Species Omsk hemorrhagic fever virus Lake Victoria marburgvirus C Kyasanur Forest disease virus Simplexvirus Marburgvirus Ebolavirus Flavivirus Genus Filoviridae Herpesviridae Table 4-9 Flaviviridae Family Introduction 43

gious), and also because specific antiviral treatment

is why or vaccines for them have not yet been developed

discovered past the research stage [2120]. Few biological agents are classified in the highest risk categories (see Table 4-9 for the U.S. Class 4 pathogens). Biosafety regulations in place not only recom-

work (e.g. inoculation of mend specific techniques for performing work with Australia Bangaladesh, Cambodia, India, Malaysia, Singapore Eradicated agents of a given category, but also require the work be performed in specialized facilities with in-vivo appropriate levels of physical containment. Conse- quently, laboratories are classified according to the approved containment level (see Table 4-8) [2582, 3279]. Agent risk group classification and labora- tory-containment classification are not interchange- foxes foxes Humans Frugivorous flying Frugivorous flying able, because the risk of infection with a particular agent is often dependent on the experiment per- formed. For example, experiments conducted with a highly concentrated agent classified as a medium level risk agent might require a biocontainment facility of a higher level. In the U.S., diagnostic work and typical research and development using Bacillus anthracis can be performed safely at biosafety level 2, while it is level. preparing and handling viral concentrates, and all recommended that work involving large quantities

called ‘Alkhumra’) [530, 1620, 2924]. or procedures prone to generating aerosols be con- Smallpox (B03) Unnamed (B34.8?) Unnamed (B34.8?) Health Problems, 10th Revision [2874]. The ICD-10 uses British English, which few human cases of VHF it allegedly caused are now in dispute; the newly ducted at biosafety level 3. Similarly, certain animal

– contrary to the remainder of this book. studies carry increased risk and are performed in a higher containment level facility, even though the )ssRNA

agentisnotahighriskagent.Anagentmayalso be classified at a higher level than need be, given the perception of hazard by the general public and among politicians, despite scientific data and in- formation that suggest the work can be done safely Monopartite ( dsDNA at a lower level of containment. In general, re- searchers should err on the side of safety, and store materials or perform experiments at the biosafety level deemed appropriate by most experts. Due to the (perceived) extreme risk filoviruses pose to in- dividual and potentially community health, an international consensus has been reached that regardless of the experiment performed, any work with infectious (‘‘live’’) filoviruses must be done in maximum-containment (in the U.S. biosafety level 4) facilities. Table 4-10 lists the known max- Variola virus (VARV) Hendra virus (HeV) Nipah virus (NiV) imum-containment facilities that have the capacity Variola virus Hendra virus Nipah virus to conduct experiments with filoviruses (see also Orthopoxvirus Henipavirus ICD-10: International Statistical Classification of Diseases and Related Whitewater Arroyo virus has been excluded from the listBSL-4 containment because is the required for large-scale growth of the viruses, including Includes the newly discovered Alkhurma isolate (in the literature also ‘Chapare virus’ has not yet been classified regarding biosafety or containment ‘‘hemorrhagic fever’’ is spelled ‘‘haemorrhagic fever’’ in this table Paramyxoviridae 1 2 3 4 Poxviridae virus-containing samples into permissive rodent species). [1023b]). 44 Introduction o o o No research Yes Yes Yes Not anymore? Filovirus Status Active Suit laboratory Active ??N ??N Glove cabinet Active Suit laboratory Active Suit laboratory Active Type ??N Combined suit laboratory and glove cabinet? Suit laboratory Inactive No eerieux’’], P46 H k eedicale(INSERM) y e ) H4H c 3>L x >Z =33]; 46 3> ( k eedicalesde Franceville (CIRMF) c :\ uuliode Mesquita Filho’’ (UNESP) e H @$@ ?Bac c *@&a eeet de la Recherche M @$4@:@(44 e p : eerieux[P4 Laboratory ‘‘Jean M cc uuliode Mesquita Filho’’ university], 3 - R>@ >@:@(44 4 ? ay x e = <4@:@(44 4 ;4k eedes Maladies Virales Emergentes [Emerging Viral Diseases Unit], e : #4@H e o Paulo [1615] http://www.fmb.unesp.br/index.php eepartementde Virologie [Departement of Virology], ˜ ?H* Commonwealth Scientific and IndustrialOrganization Research (CSIRO), Geelong [156,http://www.csiro.au/csiro/content/standard/pps84.html 664, 665] Virology Laboratory of theCoopers Queensland Plains Department of Health, National High Security QuarantineInfectious Laboratory Disease (NHSQL), Reference Victorian Laboratoryhttp://www.vidrl.org.au/labsandunits/nhsql/nhsql_idx.htm (VIDRL), Melbourne [1715] [International Center for Medicalhttp://www.cirmf.org/ Research], Franceville [1563, 1715] D [French National Institute forLyon Health [97, and 224–226, Medical 345, Research], 847, 957] http://www.cervi-lyon.inserm.fr/ Centre International du Recherche M [Department for Biotechnology andInfections], Especially Dangerous Winnipeg, Manitoba (Health CanadaInspection and Agency) the [207, Canadian 1180, Food http://www.hc-sc.gc.ca/ahc-asc/media/nr-cp/2000/2000_94bk2_e.html 1510, 1585, 2322, 2431] Central Public Health Laboratory, Toronto, Ontario [1585] Institut National de la Sant [Scientific-Research Institute for Epidemiology and Microbiology (SRIEM)], Minskhttp://www.riem.bn.by/opasnie.html [3125] Universidade Estadual Paulista ‘‘J [Estadual Paulista ‘‘J Sa ]B4* Campus de Botucatu [Botucatu campus], Location of maximum-containment facilities (as of September 2007) Table 4-10. Country Facility Belarus Australia Australian Animal Health Laboratory (AAHL), Gabon L’Unit Canada Canadian Science Centre for Human and Animal Health, France Laboratoire P4 Jean M Brazil Introduction 45 ) continued ( No Yes? No No Yes Yes Yes Yes Active Active Active (Test stage) Destroyed No Dismantled Not anymore Active? Yes Active Active ? Glove cabinet Active Combined suit laboratory and glove cabinet? ? Suit laboratory Active Suit laboratory Active Glove cabinet? Inactive No ? Combined suit laboratory and glove cabinet Glove cabinet Inactive No Combined suit laboratory and glove cabinet? Combined suit laboratory and glove cabinet? Combined suit laboratory and glove cabinet? a H y ) urg.de/ @$@ ) c H 5] P44 H4H y )9 c C epa =?A# H4H apace&4R O * >4b ? ( c e G a(C @:b 2 p >46 .A. a @6 K <>Z6 3> 46 3> k &a y k c @>H e b# c 4 4 94Re K a >4 9 k 46 :\ $@: >> e cc H4 e H @ c 1a @H4&@R P p x P44 4 a (>@ *@&a H@& 4Z : 46 A H42 @>Z - - pe :\ ?$@ >*ap k >@6 )4 e a e A p H a H R>@ x C H& H@ 4 a [Irkutsk Scientific-Research Anti-Plague ay H k 3>L k pa *@&a € oozpont[National Center for Epidemiology], y epc e x ec H@ H : $@ € uurTropenmedizin [Bernhard Nocht Institute 4 4= c b ?BZH>@ c a H4H k a cc c ec 3 R> - [National Institutes of Infectious Diseases], oogiaiK (@ %@ ay e R>@ #4@:@(4R 2@H4R [Institute of Physical and Chemical Research (RIKEN)], :\>@6 9 b= ay a k :\> @:@(44 Bp x a [Musashimurayama], Tokyo [2317, 2920, 3247, 3249] >>Z6 3> a k 4 e P4 k e 4] pyc c H& B 46 = € uurVirologie [Institute of Virology], Marburg an der Lahn, Hesse x k %4 44) c apc @$4@:@(44 ;4>4 gos Epidemiol p H pC p >Z * p ´ y B>@(@pc k *4P4> k >H >H e cy e e e p 4$4 H [National Institute for InfectiousRome Diseases [1236] Lazzaro http://www.inmi.it/ Spallanzani], [L. A. Tarasevich InstituteBiological for Preparations], the Moscow, Standardization Moscow and Region Control [3087] of Medicinal 3 Daura Foot and Mouth Disease Vaccine Facility, al-Manal [1715]? High-Security Animal Disease Laboratory (HSADL), Bhopal Budapest http://www.oek.hu/ Institut f [1715] http://www.med.uni-marburg.de/stpg/ukm/lt/hygiene/viro.htm for Tropical Medicine] (BNI), Hamburg [2271] http://www.bni-hamb ?Bac O Tsukuba http://www.riken.go.jp/ [Stepnogorsk Scientific Experimental-Industrial BaseStepnogorsk (SNOPB)], [178, 1742] http://www.nih.go.jp/niid/index.html C Institute of Siberia and the Far East], Irkutsk, Irkutsk Region [312 [Center of Special LaboratoryDangerous Diagnostics and and Exotic Treatment Infectious ofMoscow Diseases Region Especially (TsSDL)], [3104, Sergiyev 3156] Posad-6, O Istituto Nazionale per le Malattie Infettive Lazzaro Spallanzani (INMI) ;4 [Virological Center of theof Scientific-Research the Institute Ministry of of Microbiology Moscow Defense Region of [3125] the Russian Federation], Sergiyev Posad-6, ’@ ; 1 Iraq Italy Japan India Hungary Orsza Germany Bernhard-Nocht-Institut f Kazakhstan C Russia 46 Introduction Not anymore? No Yes No Yes No Yes Filovirus Probably not research Probably not No Active Active? Inactive inactive due to reconstruction and expansion Status ? ? ? Suit laboratory Active ? Combined suit laboratory and glove cabinet ? Suit laboratory Temporarily Type ? Glove cabinet Active ? Glove cabinet Active ? (SMI) Public >@:@(44 x e oon [Centers of ganization Communicable Diseases (NICD), @:@(44 4 #4@H usern http://www.ivi.ch/ ¨ pyc %4 p >H e p») [State Research Center for Virology H@ k R>Z6 O %e ay oonen Sanidad Animal [Center for Investigations « € oorMikrobiologisk Beredskap [Institute of Preventive Medicine] (Ministry of [Kwen Yang Laboratory], >>Z6 = e a Agraria y Alimentaria (INIA) [National Institute ’=O%# ´ H& € uurViruskrankheiten und Immunprophylaxe (IVI) [Institute of p» ( apc ) H@ * k cy kerhetslaboratorium och Forskning [Containment and research laboratory], ¨ %e Kunskapscentrum f [Center for Biological Preparedness] (KCB), Smittskyddsinstitutet [Institute for Infection Control],http://www.smittskyddsinstitutet.se/ Solna [1715] Virology and Immunoprophylaxis], Mittelha and Biotechnology ‘‘Vector’’ (SRCVB ‘‘Vector’’)]Health (Ministry and of Social DevelopmentKoltsovo, of Novosibirsk the Region Russian [3125] Federation), http://www.vector.nsc.ru/DesktopDefault.aspx?lcid=25 for Agricultural and FoodMadrid Scientific [1715] Research http://www.inia.es/ and Technology], Sandringham [1869, 1986, 2286, 2493, 2621] http://www.nicd.ac.za/ of Animal Health] (CISA),y Instituto Tecnologı Nacional de Investigaci « National Defense), Taipei ’@ Wiltshire (Health Protection Agency) [1, 2, 13, 24, 155, 2401, 2487] Emergency Preparedness and Response Centre, Porton Down, Disease Control], Executive Yuan[2901] (Department http://www.cdc.gov.tw/ of Health), Taipei Central Public Health LaboratoryAgency) (CPHL), [1715] London http://www.hpa.org.uk/ (Health Protection continued ( Switzerland Institut f Taiwan Singapore National Laboratories of the Defence Science Or South Africa Special Pathogens Unit, National Institute for Sweden Sa Spain Centro de Investigaci Table 4-10 Country Facility U.K. Introduction 47 these or the e, several No No No No No Yes No Yes Yes ? No? training facility ? Glove cabinet Active Suit laboratory Inactive Suit laboratory Active Suit laboratory Active Suit laboratory Active Suit laboratory? Active Suit laboratory Active Glove cabinetGlove Active cabinetGlove Active cabinet Active? ? viral Diseases ¼ as are individual institutes in the Irkutsk and Moscow Regions. Most of of Health facilities is probably incomplete. For instance, depending on the sourc of the U.S.S.R. Therefore, almost nothing is known about these institutes Antonio, biotech§ion ¼ Viral Rickettsial Diseases Maximum-containment Laboratory (MCL), National(NIH), Institutes Bethesda, Maryland [567, 2531] http://www3.niaid.nih.gov/ Southwest Foundation for Biomedical Research (SFBR), San Texas [483, 760, 794, 1378, 1379] http://www.sfbr.org/ Center for Biodefense andUniversity Emerging of Infectious Texas Diseases, Medical[759, Branch 794, (UTMB), 1715, Galveston, 2531] Texas http://www.utmb.edu/cbeid/ United States Army Medical(USAMRIID), Research Fort Institute Detrick, of Frederick,[794, Infectious Maryland 884, Diseases (Department 1095, of 1469, Defense) 2138, 2531]United http://www.usamriid.army.mil/ States Naval Medical Research UnitVirginia No. BioTechnology 3, Research Cairo, Park,Laboratory Egypt? Division Services of (DCLS) Consolidated ‘‘BioTech(Department Six’’, Richmond, of Virginia General Services[794, of 2531] the http://dcls.dgs.virginia.gov/ Commonwealth of Virginia) (DVRD), National Center for(NCZVED), Zoonotic, Coordinating Vector-Borne, Center and for Enteric Centers Infectious for Diseases Disease (CCID), Control(Department and of Prevention Health (CDC), and Atlanta,[176, Human Georgia 497, Services) 566, 794,http://www.cdc.gov/ncidod/dvrd/spb/index.htm 1318, 2119, 2531] Viral Immunology Center, GeorgiaAtlanta, State Georgia University, [99, 794,http://www.cas.gsu.edu:81/units/default.aspx?unit 1113, 2531, 2563] Defence Science and TechnologyWiltshire Laboratory (Ministry (dstl), of Porton Defence) Down, [1715] http://www.nibsc.ac.uk/ National Institute for BiologicalPotters Standards Bar and [1715] Control http://www.nibsc.ac.uk/ (NIBSC), National Institute for Medicalhttp://www.nimr.mrc.ac.uk/about/ Research (NIMR), London [1715] The list of Russian and Newly Independent States (NIS) maximum-containment scientific-research anti-plague institute are rumored to harborinstitutes BSL-4-like were laboratories, involved in the abandoned top secret biological weapons program work performed. 1 U.S.A. Special Pathogens Branch (SPB), Division of 48 Introduction

Fig. 4-9. Glove-cabinet maximum-containment labora- tory. Karl M. Johnson, epidemiologist and virologist and co-discoverer of the first recognized ebolavirus, Zaire ebolavirus, shown here working with MARV specimens through the sealed portals of a ‘‘glove box’’ in CDC’s building 8 in 1975 (courtesy of CDC=Dr. Lyle Conrad, obtained from CDC’s Public Health Image Library at http://phil.cdc.gov/phil/home.asp [last accessed September Fig. 4-8. ‘‘Space suit’’ maximum-containment laboratory. 1, 2007]) Two laboratorians, Luanne Elliot and Dave Auperin, working under a flow hood inside a BSL-4 suit laboratory (courtesy of CDC=Jim Gathany, obtained from CDC’s Pub- where the notably high cost of constructing a suit lic Health Image Library at http://phil.cdc.gov/phil/home. laboratory is deemed to be prohibitive (for guide- asp [last accessed September 1, 2007]) lines regarding the use of Class III BSCs see [2582, 2801]). Most existing and planned maximum-containment Facilities for containment of hazardous viruses, facilities are designed for basic research. Neverthe- such as those listed in Table 4-10, are commonly less, any research in maximum-containment labora- divided into two classes. In the first, research is tories is expensive, strenuous, and tedious due to performed with the investigator wearing a hermet- the level of caution required of personnel at every ically sealed, one-piece, impermeable, positive- working step [1885]. This necessary caution, in pressure ‘‘space’’ suit (see Fig. 4-8). In the second, combination with the shortage of maximum-con- infected material and the viruses are manipulated tainment facilities, slows the rate of scientific pro- inside a fully contained and double high-efficien- gress. This, in part, could explain why scientific cy particulate-absorbing (HEPA) filter-exhausted knowledge about highest risk agents is so limited Class III biological safety cabinet (BSC; ‘‘glove in comparison to more common, and less danger- box,’’ ‘‘crocodile,’’ see Fig. 4-9). The researcher ous pathogens. Additionally, a myriad of security conducts experiments through gloves that are measures has to be implemented at maximum-con- sealed to the cabinet. In either case, the researcher tainment facilities [613] to prevent theft of agents is separated from the virus, and if the Class III BSC or distribution of ‘‘sensitive’’ knowledge (‘‘brain is used the virus is also separated from the labora- drain’’) to hostile or criminal individuals, groups, tory environment [1522, 1885, 2618]. Although suit or nations interested or potentially interested in laboratories are increasingly popular (for a general perpetrating bioweapon attacks. Research with review see [1885]), Class III BSCs are in use in concentrated high risk agents or with infected ani- several maximum-containment facilities, including mals, and particularly performing necropsies or those in the U.K., in smaller laboratories primarily any other technique that might generate aerosols, used for diagnostic procedures, and in instances including aerosol challenges, is technically de- Introduction 49 manding and potentially very dangerous. For such location has yet been made. In Germany, the two research, custom designs often are developed for existing maximum-containment facilities in Marburg maximum-containment facilities [156, 157, 690, an der Lahn and in Hamburg are currently being 1099b, 1992, 2120, 2683, 2741, 2950, 3260]. Few expanded and modernized. Other facilities are facilities can or will be able to support research planned on the Island of Riems, Mecklenburg- on larger animals, such as nonhuman primates Western Pomerania [1824], and at the Robert Koch [1975]. Likewise, infecting arthropods with agents Institute7 in Berlin. In Switzerland, a maximum- of the highest biosafety level requires special containment facility is being built at the Depart- equipment and facilities, few of which exist, and ment of Defense’s Spiez Laboratory. In India, a none at the containment level required for work maximum-containment facility is under construc- with VHF agents [2298]. tion at the Centre for Cellular and Molecular Bi- Obviously, appropriate biosafety and facility se- ology (CCMB) in Hyderabad. High-containment curity are required for scientific progress in studies facilities were planned for the Netherlands [332], involving filoviruses. Hence, some experts believe and were requested for the French army [2555]; that our scientific knowledge of highest risk agents however, these plans have been shelved. Finally, a could increase rapidly only if many more modern maximum-containment facility is currently under maximum-containment facilities existed. In the construction at the Chinese Academy of Sciences’ U.S., the Centers for Disease Control and Pre- Wuuh aanInstitute of Virology8. vention (CDC) in Atlanta, Georgia [2531], and the All of these facilities would support only spe- United States Army Medical Research Institute of cialized work with certain pathogens. It remains Infectious Diseases (USAMRIID), Fort Detrick, to be determined how many requests for filoviruses Frederick, Maryland are currently expanding their will be put forth from new facilities. existing biosafety level 4 facilities. Maximum- Guidelines for manipulating exotic agents were containment facilities are also in the design stage criticized as early as 1977 [1619]. Several promi- or under construction at Boston University, Boston, nent investigators doubt whether the increasing Massachusetts (National Emerging Infectious number of high containment facilities is really nec- Diseases Laboratory) [2531], at Fort Detrick, essary to fuel scientific progress in the field of exotic Frederick, Maryland (Department of Homeland and especially dangerous pathogens [581], whereas Security’s National Biodefense Analysis and other investigators regard additional laboratories as Countermeasures Center [2531], NIAID Integrated welcome adjuncts to existing institutes. Many les- Research Facility [2531]), at the NIAID-associated sons regarding increased biosafety, learned from Rocky Mountain Laboratories in Hamilton, Mon- the construction and use of maximum-containment tana (Rocky Mountain Laboratories Integrated laboratories in the past, will be applied during the Research Facility) [636, 1352, 1353, 2051, 2531], construction of new facilities, and it is expected and at the University of Texas Medical Branch in that these will facilitate research [420, 614, 615, Galveston (Galveston National Laboratory) [635, 730, 889, 1541, 1885]. Indeed, it has been sug- 760, 2531]. Other U.S. institutions have declared gested that safety measures used today in the older a desire to construct maximum-containment facil- facilities are reminiscent of those thought necessary ities [635, 760, 2531]. The Department of Agricul- to protect personnel in the 1970s and that biosafety ture and Department of Homeland Security’s Plum protocols have not necessarily been adapted to novel Island Animal Disease Center (PIADC) in Long research techniques or to our increased knowledge Island, New York originally planned to upgrade of highest risk agents [1330, 1331, 1401]. In the its facility to accomodate a maximum-containment U.S., this is a direct result of scarce federal funding laboratory [760], but these plans have been shelved. available to conduct much needed biosafety re- A novel facility, which would replace that in Long Island, is now being planned (National Bio- and 7 German: Robert Koch-Institut. Agro-Defense Facility), but no decision about its 8 Chinese: 50 Introduction search that would provide reliable performance- public [1222, 1466, 3224]. The exact circumstances based data to better facilitate the development of of the 1990 case remain to be detailed [242, 3101]. safe and cost-effective containment and protection However, all other laboratory infections were due measures. Regardless of the current state of sophis- to accidental injection with filovirus-containing pre- tication of available containment technology [499, parations during animal experiments (see chapter 5 615, 616, 1885, 2463, 2710], sufficient protection for more details), demonstrating a need for im- only can be achieved by adherence of the re- proved adherence to biosafety protocols and im- searcher to appropriate safety guidelines, protocols, proved training, and to better equipment to restrain and standards for proper research behavior. Person- laboratory animals securely for injection experi- nel worldwide must be properly trained for work ments. One also should not forget that filovirus- in high and maximum-containment environments contaminated serologic reagents, reference isolates [2799]. Training programs are now in place at of other viruses, cell cultures, vaccines, transfu- Emory University and the CDC in Atlanta, Georgia, sions, and organ transplants may exist. Precautions to educate novices who might work in maximum- to prevent or detect such contaminations must be containment facilities in the near future [1372, taken [346, 534]. 1373]. A conference abstract describing a survey of investigators who had worked with MARV for 4.4 Biosecurity concerns in filovirus research 1–10 years claimed there were detectable enzyme- linked-immunosorbent-assay (ELISA) and indirect- In contrast to biosafety, the term ‘‘biosecurity’’ is immunofluorescent-assay (IFA) antibodies against less well defined. Originally, it referred to measures the agents in 30% of these people, and that mar- aimed at protecting indigenous or farm animals and burgviral proteins (NP, VP40, and VP35) were crops from the accidental introduction of nonen- detected by western blot. Subclinical infections demic pathogens. More recently, and especially with MARV were suggested as an explanation for after the terrorist attacks of September 11, 2001, these findings [1222], implying that laboratory biosecurity has been used to describe the means infections occurred despite employment of rigorous by which a country or region attempts to prevent safety measures. Alternatively, another survey, per- the deliberate introduction of pathogens into sus- formed by the same group, among 16 Russian ceptible humans, animals, and plants. Usage of the laboratory investigators who worked with either term differs from agency to agency and from ex- infectious or inactivated MARV for as long as 7 pert to expert. Some argue that biosecurity only years, did not detect antibodies by ELISA or IFA, encompasses physical measures, such as the pre- and western blots could not detect viral proteins vention of agent theft from research institutes, [1222], suggesting that the results of the first survey whereas others include all measures to prevent may have been due to faulty survey technology. In the use of biological weapons, such as national any case, one should not forget that accidents do policies, international treaties, and law. The WHO occur in maximum-containment facilities, and that has recently published a catalogue of recommended these accidents frequently end fatally or result in measures aimed at increasing ‘‘laboratory biosecur- serious disease. During filovirus research, labora- ity’’ in member states [2897]. A novel term coined tory accidents were recorded in 1976 in Porton by the U.S. Army, ‘‘biosurety,’’ refers to the inte- Down, United Kingdom (1 survivor) [385, 753], in gration of personnel security measures, biosafety 1988 (at least 1 fatal case) [178, 295, 1742], 1990 and biosecurity. (1 survivor) [242, 3101], and 2004 (1 fatal case) Although a thorough discussion of matters asso- in Koltsovo, Novosibirsk Region, U.S.S.R.=Russia ciated with biosecurity is far beyond the scope of [2959]; and in 1996 in Sergiyev Posad-6, Moscow this review, it is important to note that several hem- Region, Russia (1 fatal case) [3115]. Additionally, orrhagic fever viruses are considered to be potential several publications refer obliquely to further acci- threats if weaponized by nations (biowarfare), ter- dents, the details of which have not yet been made rorists (bioterrorism), or individuals (biocrimes). To Introduction 51 be considered a potential threat, a virus should be ensure a significant case-fatality rate; and the agent relatively stable in aerosols and transmissible by itself should induce fear in the target population, this route. The agent should replicate well to a re- thereby perturbing both the economy and societal latively high titer in easily obtainable cell cultures integrity. Since filoviruses fulfill several of these or other common sources, such as chicken eggs, to criteria, they are considered suitable for weaponi- obtain highly concentrated stock agent. If mass in- zation [117, 171, 223, 267, 367, 399, 432, 460, 484, fection is a goal of an aggressor, efficient human- 625, 777, 883, 885, 1027, 1031, 1270, 1350, 1354, to-human spread should be possible under certain 1548, 1606, 1709, 1999, 2001, 2124, 2192, 2218, circumstances (for instance, aerosol dissemination 2485, 2588, 2645, 2694, 3100, 3234]. Conse- of high concentrations of agent). Furthermore, di- quently, filoviruses appear on official lists of agnostic capabilities should be limited; genetic data ‘‘high-consequence’’ agents that pose a threat to should be incomplete [432, 885, 1270, 1709, 1999, the U.S. (‘‘Select Agents’’, ‘‘National Institute of 2485]; considerable virulence of the agent should Allergy and Infectious Diseases Priority Pathogens,’’

Table 4-11. U.S. ‘‘Select Agents’’ [518] (as of September 2007)

Category Agents HHS Non-Overlap Select Abrin, cercopithecine herpesvirus 1, Coccidioides posadasii, conotoxins, Agents and Toxins Crimean-Congo hemorrhagic fever virus, diacetoxyscirpenol, ebolaviruses, Flexal virus, Guanarito virus, 1918 H1N1 influenza A virus and viruses containing parts thereof, Junı´n virus, Kyasanur Forest disease virus, Lassa virus, Lake Victoria marburgvirus, monkeypox virus, Machupo virus, Omsk hemorrhagic fever virus, ricin, Rickettsia prowazekii, Rickettsia rickettsii, Sabia´ virus, saxitoxin, shiga-like toxins, tetrodotoxin, tick-borne encephalitis virus, variola virus, Yersinia pestis High Consequence Livestock Bacillus anthracis, Brucella melitensis (serovars abortus, melitensis, and suis), Pathogens and Toxins=Select botulinum toxin, Burkholderia mallei, Burkholderia pseudomallei, Agents (Overlap Agents) Clostridium botulinum (neurotoxin-producing strains only), Coccidioides immitis, Coxiella burnetii, eastern equine encephalitis virus, henipaviruses, Francisella tularensis, Rift Valley fever virus, shiga toxin, staphylococcal enterotoxins, T-2 toxin, Venezuelan equine encephalitis virus USDA High Consequence African horse sickness virus, African swine fever virus, Akabane virus, Livestock Pathogens and Toxins alcelaphine herpesvirus 1 and 2 (exotic strains only), bluetongue viruses (Non-Overlap Agents and Toxins) (exotic strains only), bovine spongiform encephalopathy prion, camelpox virus, classical swine fever virus, Ehrlichia ruminantium, foot-and-mouth disease virus, goatpox virus, human coxsackievirus B5, influenza A virus (avian highly pathogenic strains only), Japanese encephalitis virus, lumpy skin disease virus, Menangle virus, Mycoplasma capricolum, Mycoplasma mycoides capri, Mycoplasma mycoides mycoides, Newcastle disease virus (velogenic strains only), peste-des-petits-ruminants virus, rinderpest virus, sheeppox virus, vesiculoviruses (exotic strains only) Listed Plant Pathogens ‘‘Candidatus Liberobacter africanus,’’ ‘‘Candidatus Liberobacter asiaticus,’’ Peronosclerospora philippinensis, Ralstonia solanacearum (race 3, biovar 2), Sclerophthora rayssiae (var. zeae), Synchytrium endobioticum, Xanthomonas oryzae (pathovar oryzicola), Xylella fastidiosa (citrus variegated chlorosis strain) Agent names are adapted to current taxonomy schemes. HHS: (U.S. Department of) Health and Human Services. USDA: U.S. Department of Agriculture. 52 Introduction

Table 4-12. National Institute of Allergy and Infectious Diseases (NIAID) Category A, B, and C Priority Pathogens [1873] (as of September 2007)

Category A (highest priority) Category B Category C (lowest priority) Bacillus anthracis Brucellae Chikungunya virus Clostridium botulinum Burkholderia mallei Crimean-Congo hemorrhagic fever virus epsilon toxin Burkholderia pseudomallei Henipaviruses Dengue viruses Caliciviruses Influenzaviruses Filoviruses California encephalitis virus Mycobacterium tuberculosis Francisella tularensis Campylobacter jejuni (multidrug-resistant strains) Guanarito virus Chlamydophila psittaci Prions Hantaviruses Clostridium perfringens epsilon toxin Rabies virus Junı´n virus Coxiella burnetii Rickettsiae not covered in categories A and B Lassa virus Cryptosporidium parvum Severe acute respiratory syndrome coronavirus Lymphocytic Cyclospora catayanensis Tick-borne encephalitis virus choriomeningitis virus Eastern equine encephalitis virus Yellow fever virus Machupo virus Entamoeba histolytica Rift Valley fever virus Escherichia coli Variola virus and related (diarrheagenic strains only) poxviruses Giardia lamblia Yersinia pestis Hepatitis A virus Japanese encephalitis virus Kyasanur Forest disease virus LaCrosse virus Listeria monocytogenes Microsporidia Ricin Rickettsia prowazekii Salmonellae Shigellae Staphylococcus enterotoxin B Toxoplasma gondii Venezuelan equine encephalitis virus Vibrio sp. (pathogenic strains) West Nile virus Western equine encephalitis virus Yersinia enterocolitica Agent names are adapted to current taxonomy schemes. see Tables 4-11 and 4-12, respectively) [518, 1873, infections of humans were the result of an accident 2158] and Russia (see Table 4-13). The threat posed in a secret German biological weapons develop- by filoviruses is also emphasized by the Australia ment project [2254]. These allegations were coun- Group (see Table 4-14), which ‘‘is an informal tered by German scientists and authorities by gathering committed to ridding the world of chemi- conducting investigations of the outbreaks and cal and biological weapons,’’ currently consisting reporting results as openly as possible. Research of 39 member countries and the European Commis- materials, including the novel virus, were made sion [2530]. available to laboratories in Austria, Senegal, South The perceived filovirus threat was exemplified im- Africa, the U.K., the U.S. [1230, 1231, 2396], and mediately after the discovery of MARV in 1967 in the Soviet Union. German researchers provided in- Marburg an der Lahn and Frankfurt am Main, fected tissues, derived from a female patient of the Germany, when baseless allegations arose that the Frankfurt am Main outbreak, to the Institute of Introduction 53 Table 4-13. Russian ranking of potential bioweapons agents as described in English in [2733]

Group 1 (high probability of use) Group 2 (possible use) Group 3 (low probability of use) Bacillus anthracis Brucella melitensis Human immunodeficiency virus 1 Burkholderia mallei Corynebacterium diphtheriae Rabies virus Clostridium botulinum= Japanese encephalitis virus Salmonella Typhi Botulinum toxins Vibrio cholerae Shigella dysenteriae=Shiga toxins=Verotoxin Coxiella burnetii Yellow fever virus Staphylococcus aureus= Francisella tularensis Staphylococcus aureus toxins Influenzaviruses Lake Victoria marburgvirus1 Rickettsiae Variola virus ‘‘Viral lymphocytic encephalitis’’2 Yersinia pestis Agent names are adapted to current taxonomy schemes. 1 Note that ebolaviruses are not considered potential bioweapons agents in this ranking, perhaps reflecting the fact that only Lake Victoria marburgvirus, but not ebolaviruses, were successfully weaponized during the Soviet Union’s offensive biological warfare program [178]. 2 Lymphocytic choriomeningitis virus?

Poliomyelitis and Viral Encephalitides in Moscow,9 heavily involved in classified and offensive biolog- where MARV (isolate Poppinga, MARV-Pop) was ical weapons research [178, 179] and the later recovered, [108, 1449, 1689, 3229] and passaged creation of a MARV-based weapon with the code six times in guinea pigs [2183]. Samples from the name ‘‘N3’’ [178]. The first institute, the Scientific- last passage were distributed to all other Soviet Production Association (SPA) ‘‘Vector’’10 (now the Union institutes capable of handling them [2183, peaceful State Research Center for Virology and 3006]. Therefore, the often repeated story about Biotechnology (SRCVB) ‘‘Vector’’11) in Koltsovo, the filoviruses being acquired by a Soviet secret Novosibirsk Region, received MARV-Pop at an un- service agency from Germany [178, 1458, 3115] known time in the 1980s (see method section of most likely is incorrect and misleading [1472]. [2183]). Since this filovirus research program was According to the Russian literature, MARV-Pop classified, no formal scientific reports were published. is the principal MARV isolate used in experiments The first available reports on innocuous ongoing performed to date in Russia. experiments at this facility are dated 1988 and It is ironic that the good intentions of Germany’s 1989 [3130, 3132]. Conference abstracts, also re- researchers might have contributed to the estab- porting peaceful research, became available in 1989 lishment of a filovirus biological weapons pro- (for example see [3065]). The second institute that gram in the Soviet Union [178, 179, 2669]. The received MARV-Pop was the Scientific-Research first research publication on the characterization of Institute of Sanitation of the U.S.S.R. Ministry of MARV-Pop at the Institute of Poliomyelitis and Defense12 (now Virological Center of the Scientific- Viral Encephalitides was published in 1968 [3229]. Research Institute of Microbiology of the Ministry Judging from the literature (see [3125] and meth- ods sections of [2183, 3006]), MARV-Pop was 10 Russian: =ayR>@-Ap@42&@*cH&e>>@e ?$Xe*4>e>4e sent from there to at least two institutes that were (=A?)«%ekH@p». 11 Russian: ’@cy*apcH&e>>Z6 =ayR>Z6 Oe>Hp %4pyc@:@(44 4 #4@Hex>@:@(44 (’=O%#)«%ekH@p». 9 Russian: 3>cH4HyH A@:4@<4e:4Ha 4%4pyc>Zx 12 Russian: =ayR>@-3cc:e*@&aHe:\ck46 3>cH4HyH ]>PeLa:4H@&. Ca>4Hap44 ;4>4cHepcH&a ?$@p@>Z CCCP. 54 Introduction Table 4-14. The Australia Group’s ‘‘List of Biological Agents For Export Control’’ [2530] (as of September 2007)

Viruses Bacteria and Fungi Toxins and subunits thereof V1. Chikungunya virus B1. Bacillus anthracis T1. Botulinum toxins V2. Crimean-Congo hemorrhagic B2. Brucella melitensis T2. Clostridium perfringens toxins fever virus (serovar abortus) T3. Conotoxin V3. Dengue virus B3. Brucella melitensis T4. Ricin V4. Eastern equine encephalitis virus (serovar melitensis) T5. Saxitoxin V5. Ebolaviruses B4. Brucella melitensis T6. Shiga toxin V6. Hantaan virus (serovar suis) T7. Staphylococcus aureus toxins V7. Junı´n virus B5. Chlamydophila psittaci T8. Tetrodotoxin V8. Lassa virus B6. Clostridium botulinum T9. Verotoxin V9. Lymphocytic choriomeningitis virus B7. Francisella tularensis T10. Microcystin (Cyanginosin) V10. Machupo virus B8. Burkholderia mallei T11. Aflatoxins V11. Lake Victoria marburgvirus B9. Burkholderia pseudomallei T12. Abrin V12. Monkeypox virus B10. Salmonella Typhi T13. Cholera toxin V13. Rift Valley fever virus B11. Shigella dysenteriae T14. Diacetoxyscirpenol toxin V14. Tick-borne encephalitis virus B12. Vibrio cholerae T15. T-2 toxin (Far Eastern subtype) B13. Yersinia pestis T16. HT-2 toxin V15. Variola virus B14. Clostridium perfringens T17. Modeccin toxin V16. Venezuelan equine encephalitis virus (Epsilon toxin-producing T18. Volkensin toxin V17. Western equine encephalitis virus strains only) T19. Viscumin V18. White pox agent1 B15. Enterohemorrhagic T20. Verotoxin and Shiga-like toxins V19. Yellow fever virus Escherichia coli V20. Japanese encephalitis virus (serovar O:157 and other V21. Kyasanur Forest disease virus verotoxin-producing V22. Louping ill virus serovars) V23. Murray Valley encephalitis virus R1. Coxiella burnetii V24. Omsk hemorrhagic fever virus R2. Bartonella quintana V25. Oropouche virus R3. Rickettsia prowazekii V26. Powassan virus R4. Rickettsia rickettsii V27. Rocio virus F1. Coccidioides immitis V28. St. Louis encephalitis virus F2. Coccidioides posadasii V29. Hendra virus V30. Flexal, Guanarito, and Sabia´ viruses V31. Hantaviruses other than Hantaan virus V32. Nipah virus Agent names are adapted to current taxonomy schemes. 1 The most recent data suggest a bacterium, Serratia marcescens, to be the causative agent of white pox. of Defense of the Russian Federation13) in Zagorsk not be confirmed independently, and thus remains (today Sergiyev Posad-6), Moscow Region (see very dubious. [3125] and the methods section of [3006]). Accord- Belgian researchers, being unaware of the Soviet ing to one source, up to 250 metric tons of lyo- biological weapons program, provided the Voege philized MARV were allegedly produced in the isolate (MARV-Voe), also isolated from patients U.S.S.R. for weapons production [1742]. The exis- during the outbreak in Marburg an der Lahn in tence of this extremely high quantity of virus could 1967, during a routine scientific collaboration with the Soviet Union in the mid-1980s [3032, 3077]. 13 Russian: Oe>Hp %4pyc@:@(44 Bp4=ayR>@- Judging from the literature, this isolate was received 3cc:e*@&aHe:$ck46 3>cH4HyH;4kp@$4@:@(44 by the Byelorussian Scientific-Research Institute for ;4>4cHepcH&a ?$@p@>Z P@cc46ck@6 Ke*epaP44. Epidemiology and Microbiology of the Byelorussian Introduction 55

S.S.R. Ministry of Health14 (today Scientific- of Health in Minsk by Belgian researchers during a Research Institute for Epidemiology and Microbiol- scientific collaboration in the early 1980s [3032, ogy, SRIEM15) in Minsk and by the Irkutsk Sci- 3077]. The virus most likely was then sent to the entific-Research Anti-Plague Institute of Siberia Scientific-Research Institute of Sanitation of the and the Far East16, R.S.F.S.R. [2962, 3077]. The U.S.S.R. Ministry of Defense, where it was pas- latter might already have been in the possession saged twice in African green monkeys. From there, of filoviruses since 1978 [2940]. It is unclear the virus was sent to other institutes, including SPA whether MARV-Voe was used as starting material ‘‘Vector’’ [2183]. Reports indicate that ZEBOV- to develop bioweapons. May was used as starting material in a biological Like MARV, ebolaviruses also were widely dis- weapon development program [178], probably at tributed for further characterization after their dis- both institutes. Additionally, several filovirus infec- covery in 1976 in Sudan and Zaire. Isolates from tions occurred in personnel at SPA ‘‘Vector’’ [178, these outbreaks were received in Belgium, Germany, 242, 295, 1222, 1466, 1742, 2959, 3101, 3115, South Africa, and the U.K., shortly after or during 3224], but with the exception of two cases [2959, the actual outbreaks [1979]. It is unclear how and 3101], thorough descriptions of the exact circum- when ebolaviruses were obtained by the Soviet stances surrounding these infections have not been Union, but it has been reported that the Soviet published. The isolate from one fatal accident that Union was in possession of ebolaviruses as early as occurred in 1988 (MARV-‘‘U’’) was allegedly used 1978 [2940]. There have been assertions that the to produce the questionable tons of virus mentioned viruses were obtained by Soviet intelligence services above [178]. Anecdotal reports claim that the filo- [795]. The Mayinga isolate of ZEBOV (ZEBOV- virus-based weapons developed were tested in May) is the ebolavirus isolate used in all experi- explosive chambers at the Stepnogorsk Scientific ments reported in the Russian literature. In the Experimental-Industrial Base17, Kazakh S.S.R., as Soviet Union and its successor states, this isolate is late as 1990 [178, 243, 1742, 2060]. Again, it is called ‘1a4p’ (‘Zair,’ author’s transliteration) [2656], important to remember that this claim has not yet suggesting it was either obtained during the initial been confirmed independently. outbreak of 1976 or was provided later by wester- Certain conclusions regarding research per- ners (the genomic sequence of ‘Zair’ is identical to formed at Soviet institutes can be drawn from sev- that of ZEBOV-May). One report claims that this eral unclassified research reports published after the virus had been in the possession of the Institute of biological weapons development program had been Poliomyelitis and Viral Encephalitides, from where terminated. Amplification of MARV in suspension it was sent to an unspecified anti-plague insti- cultures of mammalian cells was achieved and pro- tute in 1978 – most likely the Irkutsk Scientific- vided the means for the accumulation of large Research Anti-Plague Institute of Siberia and the volumes of virus at high titers [3025, 3026, 3085, Far East [2940]. No confirmatory reports of this 3184]. Optimal lyophilization procedures for the claim are available. In any case, ZEBOV-May was virus were elucidated [3195, 3198], the effects of provided to Soviet researchers and via them to the repeated freeze-thawing of ZEBOV-May on its bio- Byelorussian Scientific-Research Institute for Epi- logical activity was studied, and optimal long-term demiology and Microbiology of the BSSR Ministry storage conditions and methods for the rapid de- termination of virus activity were developed [3196, 14 Russian: #e:@pycck46 >ayR>@-3cc:e*@&aHe:\ck46 3198]. Aerosols of ZEBOV-May were tested for 3>cH4HyH ]B4*e<4@:@(44 4 ;4kp@$4@:@(44 filovirus infectious potential [3211] and several ;4>4cHepcH&a 1*pa&@@xpa>e>4b #CCP. studies of aerosol challenges of animals, character- 15 = R>@ 3 : *@& H :\ k46 3> H4H H Russian: ay - cc e a e c c y izing filovirus transmissibility, were published [2303, ]B4*e<4@:@(44 4 ;4kp@$4@:@(44 (=33];). 16 Russian: 3pkyHck46 =ayR>@-3cc:e*@&aHe:\ck46 Ap@H4&@Ry<>Z6 3>cH4HyH C4$4p44)a:\>e(@ 17 Russian: CHeB>@(@pckab=ayR>ab ?BZH>@- %@cH@ka. Ap@>ab#a2a(C=?A#). 56 Introduction

2996, 3075, 3076, 3121, 3127]. Other studies report duct laboratory rotations with their Russian coun- on the development of protective measures against terparts in some of these facilities [154]. aerosol infection with filoviruses [3063]. Reports Soviet=Russian laboratories have been denounced describe methods used to obtain preparative and reviled for experiments performed with filo- quantities of filoviruses and their purification and viruses, but they are not the sole recipients of such concentration: patents for both ZEBOV-May and criticism. Past experimental aerosol challenges of MARV-Pop purification and concentration have nonhuman primates with ebolaviruses at the U.S. been filed [2983, 3025–3027, 3184, 3185, 3194, Army Medical Research Institute of Infectious 3220]. Sophisticated methods were developed to Diseases (USAMRIID) at Ft. Detrick in Frederick, increase the yield of filoviruses in cell cultures Maryland [1309], have raised suspicions of offen- [3024–3026, 3064, 3107, 3184], and a patented sive biological-warfare activities. The investigators method for the preparative production of Venezue- who were criticized defended their work as being lan equine encephalitis virus [3201] was used for of a peaceful nature [1380]. According to unpub- the preparation of MARV-Pop. It is true that all lished data from USAMRIID, MARV isolate Ci67, these experiments had legitimate justifications and the isolate most closely related to MARV-Pop, that they greatly contributed to our present under- replicates more rapidly and forms larger plaques standing of the biology of filoviruses. However, it is in Vero E6 cells than the more commonly used equally true that many of the results obtained are Musoke and Ravn isolates. MARV-Ci67 titers were also applicable to the production of biological almost 10-fold higher in the late stage of disease in weapons. The intentions behind these published stu- experimentally infected rhesus macaques compared dies have not yet been discussed openly. Certain to MARV-Mus-infected animals [2099]. A recent documents on past Soviet or Russian filovirus re- conference abstract from USAMRIID reports on the search are still classified (for example, most likely disease course and pathogenesis of guinea pigs [2962, 2998, 3198]), adding to existing doubts challenged with aerosolized MARV, and on the regarding the actual termination of the U.S.S.R.’s clinical and pathological differences observed after former biological weapons development program challenge of Hartley or strain 13 guinea pigs with in Russia and the Newly Independent States. isolates Ci67 or Musoke [1495]. Another abstract Inspections of Russian military-associated research reports on the clinical and pathological differences laboratories by external organizations are not per- observed in African green monkeys, cynomolgus mitted [179], and key persons, networks, organiza- macaques, and rhesus macaques challenged with tions, buildings, and safeguards from the old Soviet aerosolized Zaire ebolavirus [2098]. All these ex- biological weapons program are still in place [795]. periments have been performed for legitimate re- Conversely, Russia has opened some of its formerly asons to uncover functional differences between secret ‘‘civilian’’ bioweapons production facilities filovirus isolates observed to cause different pathol- for external inspections. Former key structures of ogies in different animal models or during human the civilian Soviet=Russian biological warfare pro- disease outbreaks, or to assess whether aerosolized gram ‘‘Biopreparat’’ (for example SRCVB ‘‘Vector’’ filoviruses could pose a threat in regard to bio- in Koltsovo) are now avowedly dedicated to peace- terrorism. However, Camp Detrick, the predecessor ful research within international collaborations, laboratory of USAMRIID, was one of the principle such as with the International Science and Technol- facilities in the U.S.A. for conducting biological ogy Center (ISTC) in Moscow, the U.S. National weapons research and developing biological weap- Academy of Sciences, and other organizations ons before the country officially canceled its offen- [1714, 1884, 2170, 2583, 3149]. Structural and or- sive program under President Nixon’s decree in ganizational changes at these open Russian insti- 1969. Since its inception, USAMRIID’s avowed tutes are proposed or ongoing to convert them into mission has been to develop countermeasures against major international reference centers [2447], and hazardous biological agents for the U.S. military western scientists have received permission to con- [1918]. Nonetheless, due to its history, unsubstan- Introduction 57 tiated allegations that this institute is still producing tem and attempts to disseminate Bacillus anthracis, biological weapons have been made [2254], em- unsuccessfully tried to obtain ebolaviruses in Zaire phasizing that proper independent oversight and for purposes of bioterrorism [210]. Events such as transparency at this facility may be necessary to these underscore the existing interest in filoviruses debunk such claims. by terrorists. It is important to note that the envir- To assess the actual threat of an attack using onmental stability of filoviruses is very limited, and filoviruses, the distribution and availability of these that sunlight or even moderately high temperatures viruses must be rigorously studied and monitored are sufficient to inactivate most of an infectious [1470]. The marburgvirus disease outbreaks of 1967 culture. Studies indicate that filoviruses are among originated with imported infected primates from the most sensitive RNA viruses regarding solar ra- Uganda. The same Ugandan primate exporter that diation. A 1-log filovirus inactivation (10% survi- supplied the affected laboratories also shipped mon- val) within 20–100 min. was estimated to be the keys to Japan, Sweden, Switzerland, the Netherlands, result of midday solar exposure at various locations and the U.S. No hemorrhagic fever-stricken or other- in the U.S. [1611]. Preparation of concentrated and wise sick animals were reported from these coun- stable filovirus aerosols in a clandestine laboratory tries [1117]. The mere suspicion of outbreaks of likely would prove to be demanding, challenging, REBOV in Germany and Switzerland fueled ru- and certainly hazardous for the producer and for mors of hidden outbreaks in other countries [2254]. those in proximity. The dispersion of filovirus-con- Laboratories in several countries, including Congo taining aerosols required to infect many people in (Brazzaville), Cooted’Ivoire,^ Democratic Republic urban settings also would not be a trivial task. Filo- of the Congo (former Zaire), Gabon, Italy, Kenya, viruses are at or near the top of the list of hazardous Sudan, Uganda, and Serbia (former Yugoslavia), may infectious agents (Tables 4-11 through 4-14), but still possess isolates of various filoviruses stem- they most likely are not at the top of the list of ming from prior local outbreaks or from archived agents of interest for use in biocrimes or bioterror- samples, which, at the time, contained the virus. It ism. Nevertheless, the psychologic, economic, poli- is of international concern to determine how these tical, and health-care impact of one or more clusters isolates (or infected tissue and contaminated mate- of filovirus infections on a sensitized population rials) are maintained and secured from theft. The should not be underestimated [1470]. An ebolavirus chaos that follows the first recognized stages of hoax perpetrated at an international airport clearly outbreaks also provides ample opportunity for demonstrated that few people with antisocial per- potential biocriminals, bioterrorists, or intelligence sonalities are aware of the effect filovirus threats agents to obtain infectious materials. Isolation of have on the public [1460]. Thus stated, national filoviruses from such acquired materials could be governments could provide the necessary manpower, achieved, albeit not necessarily using optimal resources, and time to overcome technical obstacles safety precautions, by following instructions in connected with the production of an efficacious, published or online literature or microbiological filovirus-based biological weapon. or biomedical textbooks. The existence of infectious cDNA clones of The introduction of mutations, which would ZEBOV-May [1374, 1886, 2560, 2659] and MARV- allow for replication of filoviruses in the respiratory Mus [766] will undoubtedly improve our under- linings of the lungs making them transmissible as standing of viruses of the family Filoviridae (for aerosols, into filoviral genomes is considered pos- reviews of the current state of reverse genetics for sible pathologists [3066]. Filoviruses might become VHF viruses see [724, 2533]). However, such sys- even more interesting to would-be bioterrorists if tems conceivably could be manipulated with harm- the existence of or ability to create such mutations ful intent. Site-directed mutagenesis and other becomes possible and known. The Japanese cult common methods could select for filoviruses more Aum Shinrikyo, which was responsible for the 1995 virulent, contagious or even with expanded host- dissemination of sarin gas in the Tokyo subway sys- range spectra compared to those isolated in nature. 58 Introduction

Illegally obtaining filoviruses in DNA form would apprehended. It is of indisputable concern that at be much easier than obtaining infectious RNA some point, perhaps even now, people with dubi- viruses because an engineered filoviral DNA ge- ous, mischievous or hostile motives could reassem- nome is much more stable than the natural RNA ge- ble a filovirus. There is also fear in some quarters nome. Therefore, cooling equipment or stabilizing that commonly acquired or ubiquitous agents could solutions would not be required to ensure ‘‘survi- be given new and pernicious characteristics should vability’’ in transport. Biosurety, ‘‘the combination genes of Class 4 pathogens be inserted into their of security, biosafety, agent accountability, and per- genomes [876, 2715]. In the U.S., biodefense bud- sonnel reliability needed to prevent unauthorized gets have increased dramatically in recent years access to select agents’’ [482], has to be a major [1502, 2289, 2290], implying that the threat of point of concern for institutes possessing either filo- attacks with especially dangerous viruses is taken virus cDNA clones or the wild-type pathogens. It is very seriously at the highest levels of government therefore of extreme importance that the distribu- [2289]. Indeed, Bioterrorism Risk Assessments, tion of these infectious agents be tightly controlled, which include viral agents such as filoviruses, are which of course holds true for all pathogens of con- being conducted by the U.S. Department of cern [2162]. A Dangerous Pathogens Advisory Homeland Security and provided to the U.S. gov- Group was established in the U.K. as long ago as ernment, as tasked by Homeland Security Presi- 1975 to control the distribution of hazardous viruses dential Directive 10 ([Online.] http:==www.fas. [10, 638]. More restrictions on the distribution of org=irp=offdocs=nspd=hspd-10.html [last accessed plasmids containing genes of Class 4 pathogens Aug. 1, 2007.]), to guide biodefense investments. have been proposed [876, 2715]. However, filovirus Expanded national and international committees sequences could be procured by means other than and guidelines should be developed to provide some acquiring plasmids. For one, amplification of se- form of oversight assuring that laboratories studying quences should be relatively simple for researchers filoviruses, and those that plan to perform filovirus with access to filovirus isolates. Second, after the research, do so in accordance with codes of conduct de-novo construction of several pathogens such as a for bioscientists that require them to pursue research poliovirus, an encephalomyocarditis virus, and 1918 only for peaceful goals [875]. Limitations of fund- H1N1 influenza A virus, it is only a matter of time ing resources and the few specialists working with before modern gene synthesizers can allow the highly hazardous infectious agents have been criti- assembly of entire filovirus genomes. The answer cized as resulting in delayed progress in advancing to the question how to provide biosecurity for sen- our knowledge of these agents and their ability to sitive genomes may lie in a regulatory mechanism cause disease, as well as impeding the development for reporting to a central institutional biosafety and of rapid assays for the detection of exposure or biosecurity committee the possession of these agents infection and advanced treatments for prophylaxis and the work being done with them. Such an [466, 2017]. A compromise must be achieved, which approach, even with existing biosecurity codes of ensures the co-existence of scientific progress and federal regulation, would not guarantee the appre- biosecurity measures without compromising public hension of those who break rules, but rather may health, the advancement of research, or national only provide for a means of prosecuting them once security interests.