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Molecular Epidemiology as a New Approach in Detecting Terrorist Use of Infectious Agents Sergey V. Netesov Corresponding Member of the Russian Academy of Sciences, Vector State Research Center for Virology and Biotechnology The underlying basis for the casualty effect of bioterrorist weapons is formed by biological agents, namely, microorganisms and certain products of their live activities (toxins) as well as a number of pests and insects capable of transmit- ting infectious diseases. These biological agents are capable of causing disease in humans, animals, and plants, as well as producing widespread panic. Bioter- rorist acts differ from other types of terrorist actions in that they may be open, announced, and demonstrative acts or hidden actions disguised as natural out- breaks or divine scourges. In the latter case, resources and procedures are re- quired to investigate the episode, document its unconventional characteristics, and in the best possible outcome, prove that it is artificially created. It should be noted that according to available data, the majority of bioterrorist episodes up to now have been of a disguised and masked nature. In particular, this was reported in the presentation of Dr. H. McGeorgei at the World Congress on Chemical and Biological Terrorism in Dubrovnik, Croatia, in April 2001. Therefore, the prob- lem of discriminating between natural and artificially created disease outbreaks is still a pressing one. Let us begin by considering the list of possible infectious agents that may be used for bioterrorist purposes. Such lists usually include biological agents that might be used as biological weapons. Table 1, which presents only viral agents, consolidates five versions of such lists compiled at different times by different expert teams from different institutions.2 It is evident that expert opinions differ considerably. Only variola virus, alphavirus chikungunya, yellow fever virus, and Rift Valley fever virus are present in all the lists. However, note that the World Health Organization (WHO) list was compiled in 1970, when Ebola and some other viruses were unknown. Note also that according to opinions of other 89
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9o HIGH-IMPACT TERRORISM TABLE 1 Viruses Considered Potential Biological Warfare Agents by Various International or National Agencies Agency or Country Viral Agents USA, USA, WHO, AHSC, BDRP, 1970 1983 1989 Australia and Germany, USA, 1992 1990 Chikungunya virus Crimean-Congo hemorrhagic fever virus Dengue virus Eastern equine encephalitis virus + + + Ebola virus Hantaan (HERS) virus Influenza virus Japanese encephalitis virus Junin (Argentine hemorrhagic fever) virus Lassa fever virus Lymphocytic choriomeningitis virus Machupo (Bolivian hemorrhagic + + fever) virus Marburg virus Monkeypox virus Rift Valley fever virus Smallpox virus Tickborne encephalitis virus Venezuelan equine encephalitis virus Western equine encephalitis virus + + Yellow fever virus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + NOTE: AHSC U.S. Army Health Services Command; BDRP Biological Defense Research Program. experts from our center as well as my own, this list is far from complete. In particular, it lacks hepatitis A virus, which causes a rather severe disease, espe- cially in older people, and which is easily transmitted through the fecal-oral route. Furthermore, more than 60 percent of the population in the United States and Western Europe lacks immunity to it. On the other hand, it is not clear why this list contains tick-borne encephalitis virus yet lacks St. Louis encephalitis virus, since the diseases they cause are similar in their severity and transmission routes. It is illustrative in this connection that the German team omits tick-borne encephalitis, whose foci are common in Germany, and includes Venezuelan equine encephalomyelitis virus, which causes a considerably milder disease but is absent in that country.
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BIOLOGICAL TERRORISM 91 Table 2 shows the list of biological agents selected by experts from the U.S. Centers for Disease Control and Prevention (CDC) as having the potential for terrorist used Interestingly, it contains virtually the same viral agents as Table 1. Again, hepatitis A virus is absent from the list. It is even more strange, given that the classic 1984 bioterronst episode at the salad bar in Oregon, when more than 700 people were infected, involved the bacterium Salmonella, which displays TABLE 2 Critical Biological Agents as Identified by the U.S. Centers for Disease Control and Prevention, 1998 Category A Highest Priority Variola major (smallpox) Bacillus anthracis (anthrax) Yersinia pestis (plague) Clostridium botulinum toxin (botulism) Francisella tularensis (tularemia) Filoviruses Ebola hemorrhagic fever Marburg hemorrhagic fever Arenaviruses Lassa (Lassa fever) Junin (Argentine hemorrhagic fever) and related viruses Category B Second-Highest Priority Coxiella burnetti (Q fever) Brucella species (brucellosis) Burkholderia mallet ("landers) Alphaviruses Venezuelan encephalomyelitis Eastern and Western equine encephalomyelitis Ricin toxin from Ricinus communis (castor beans) Epsilon toxin of Clostridium perfringens Staphylococcus enterotoxin B Salmonella species Shigella dysenteriae Escherichia cold 0157:H7 Vibrio cholerae Cryptosporidium parvum Category C Emerging Pathogens of Potential Future Use Nipah virus Hantaviruses Tickborne hemorrhagic fever viruses Yellow fever Multidrug-resistant tuberculosis
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92 HIGH-IMPACT TERRORISM the same transmission route as hepatitis A. In the Oregon case, the disease was first classified as a natural outbreak, and only after a year was it proven that extremists had added Salmonella to salads in order to disrupt local elections. However, this list contains Salmonella, although in category B. Generally, if we consider the biological agents listed in this table not from the standpoint of biological warfare (that is, the use of such agents by a state against another state) but as potential terrorist weapons, I believe that chances are not high that, for example, variola, Marburg, or Ebola viruses would be used for the following reasons: · The high danger to the terrorists themselves; · The great and hardly surmountable difficulties involved in obtaining the initial material for propagation this is true at least for variola virus because its stocks in Atlanta and Novosibirsk are very well guarded, and the protection systems are constantly being improved; and · The need for a high biosafety-level laboratory for propagation of these biological agents. From this standpoint, it is more likely that terrorists would use more com- mon pathogens that could be stolen from an average microbiological laboratory, easily produced, or isolated in considerable amounts for instance, hepatitis A from sewage collection systems, since human feces would contain it in great amounts during an outbreak. In addition, such common pathogens are safe for vaccinated persons or only mildly hazardous for terrorists due to available means of emergency prophylaxis. With this in mind, the list should be supplemented with several viruses: the hepatitis A virus; rotavirus, which is transmitted via drinking water and food and causes severe diarrhea lasting several days; differ- ent variants of influenza virus, including old strains from the 1960s and 1970s; and rabies virus. The same is true for bacterial agents; among them, attention should be paid to Salmonella, enteropathogenic Escherichia cold 0157:H7, ordi- nary diphtheria, and others. Here, I would like to give you a hypothetical example of the possibility of using a biological agent of the hepatitis A virus type. A paper analyzing morbid- ity in the Russian armed forces during the first armed conflict in Chechnya was published in a Russian journal five years ago.4 It is evident from Figure 1 that the majority (more than 90 percent) of infectious diseases within this contingent were anthroponoses with fecal-oral transmission routes. Figure 2 presents the infectious agents that caused these intestinal diseases and their rates. It is evident that the hepatitis A virus was the disease cause in more than 50 percent of the cases, with the dysentery-causing bacteria Shigella causing over 30 percent. The remaining cases were due to enterocolitis of different etiologies, including again those caused by Salmonella. Note that the hepatitis A morbidity rate in this contingent was essentially higher than in the other regions of Russia (Table 3~.
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BIOLOGICAL TERRORISM 93 Anthroponoses with fecal-oral transmission Aerosol infections Other infections FIGURE 1 Prevalence of Various Infectious Diseases Among the Russian Military Con- tingent in Chechnya During the Conflict (February-December 1995) ~ Hepatitis Avirus ~ Shigellosis ~ Enterocolitis FIGURE 2 Prevalence of Various Etiological Agents Causing Enteric Infections in the Russian Military Contingents During the Chechnya Conflict (February-December 1995) Unfortunately, the paper lacks the rate per 100,000 people; however, if we con- sider that the morbidity rate of hepatitis B and C is approximately equal in all three regions listed in Table 3, it is evident that the specific morbidity (per 100,000 people) of hepatitis A among Russian soldiers in Chechnya is about two times higher than the rates in military units located in other regions. The paper cited underlines this fact and indicates also that 82.4 percent of infection cases were found in recruits, and at times up to 20 percent of the contingent was hospitalized due to hepatitis A. This is the reason military hospitals in Chechnya were so overcrowded. A similar situation was recorded during the war in Af-
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94 HIGH-IMPACT TERRORISM TABLE 3 Etiological Structure of Acute Viral Hepatitis Among Servicemen from Different Regions of Russia (Monoinfections, in Percent) Military Northwest Region Nosological Forms and Forces (St. Petersburg Region South Region Serological Markers in Chechnya and Baltic Fleet) (Black Sea Fleet) Hepatitis A (anti-HAV IgM) 86.1 64.9 63.9 Hepatitis B (anti-HBc IgM) 4.3 14.9 8.2 Hepatitis C (anti-HCV) 0.5 2.0 1.6 Hepatitis E O 0.9 0 Mixed hepatitis 1.0 13.7 16.5 Nondifferentiated hepatitis 8.1 3.6 9.8 ghanistan, when at times up to 40 percent of Russian soldiers were unfit for action due to hepatitis A. Figure 3 illustrates the monthly morbidity plot from the same paper. It shows two waves of the disease: the first, in October-November, which is typical of this infection, and the second, in May-June, which is not typical and fails to coincide with the arrival of recruits. No correlations between the morbidity and type of dwelling (tents, houses, or trenches) were detected; however, there was a signif- icant correlation with certain water sources, which were limited in number. Ex- am~nation of water sources during the epidemic detected the viral antigen that 300 / / 200 100 / ""/ / / / / 11 111 IV V Vl Vll Vlil IX X Xl · Hepatitis A · Acute enteric infections FIGURE 3 Dynamics of the Incidence of Hepatitis Disease caused by Hepatitis A Virus during the Chechnya Conflict (February-December 1995)
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BIOLOGICAL TERRORISM 95 is, the virus itself in one water source. I am by no means saying that the hepa- titis A virus was deliberately used as a weapon in this case. But if somebody infected several water sources with the virus during September-November, the resulting increase in the morbidity rate would hardly cause any suspicion regard- ing deliberate use of the virus as a weapon. It would, however, create consider- able disorganization, require substantial additional expenses for hospitalization and treatment, and weaken or even temporarily halt military actions. Meanwhile, if such an outbreak occurred in the summer or in January-February, it would be very reasonable to suspect deliberate contamination of water sources. It should be noted that the Russian publications also lack the list of biologi- cal agents that may be used in bioterrorist acts. However, there is one official document that indirectly indicates such a list: President Yeltsin's Decree No. 298-rp of June 14, 1994, which imposed controls over the export of pathogens.5 The decree coincides completely with the Australia Group list presented in Table 1. It also lacks Salmonella and hepatitis A, which is reasonable for a list of this type (export control). Summing up, I would like to underline that in my opinion, the above-men- tioned lists on the whole pay appropriate attention to the most dangerous biolog- ical agents, and the special attention paid to corresponding protection systems is definitely reasonable. However, probably because of this special attention, it is the most common everyday pathogens that might now be being used and most likely will be used in the future. Let us now turn to discussing already existing and potential capabilities for detecting the covert use of biological agents. I will dwell in detail on the detec- tion of primary infection sources using standard epidemiological methods, since it is obvious that contamination not of an open water reservoir but rather of a closed vessel points in itself to deliberate contamination. Actually, if a water source is contaminated by a common pathogen and there is no direct evidence (a bottle labeled virus X) or reliable information on deliberate infection, there is no way to categorize the incident as being of a deliberate nature using conventional methods. What other methods are capable of detecting a deliberate but disguised bioterrorist action? Before the sequencing era, different viral strains of a patho- gen were characterized and differentiated only according to their phenotypic features: shape and size of plaques in cell culture, shape and size of viral parti- cles, differences in pathogenesis in animal models, differences in their antigenic characteristics, and so forth. In several cases, these methods allowed distinctions to be made between strains within taxonomic genera and species. If such distinc- tions were absent, it was considered that there was no difference between the strains. Early efforts in genomic sequencing and comparative analysis of strains with weak or no phenotypic differences detected such distinctions and facilitated the development of reliable and sufficiently precise methods for differentiating between viral strains based on molecular characterization. This strategy was soon dubbed molecular epidemiology.
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96 HIGH-IMPACT TERRORISM Here is one of the first examples of such research. Figure 4 shows a simpli- fied phylogenetic tree of influenza virus subtype HlNl.6 I would like to provide a few words on the history of this subtype. It is considered related to subtype HON1, the famous Spanish flu. It spread during 1946-1947, causing mass epi- demics all over the world, and circulated widely until 1956. The scale of its outbreaks then decreased considerably, and subtype H2N2 took its place. In the autumn of 1977, a mass outbreak of HlN1 influenza virus occurred among Rus- sian sailors in the Far East, and it was widely discussed in the mass media and scientific publications. Some publications included speculations on the artificial nature of this virus and its accidental or deliberate release from laboratories. In the mid-1980s, the main genes of this strain were sequenced; moreover, the sequences of one of the genes were published independently by three laborato- ries during one year. In the figure, these data are shown as differences in amino acid sequences of hemagglutinin HA-1 subunits, together with sequencing data of several other strains. The relations between the strains were calculated using the method of maximal likelihood between closest neighbors. It is evident from the figure that the strains circulating in Russia and the strains circulating in Australia since 1978 should actually have a common ancestor. In addition, this ancestor is antigenically close to the strain of 1950 (not the 1956 or 1946 strains, since almost no substitutions occur in the antigenic determinants). However, very important substitutions are localized in the region of amino acids 253-258 of the first Russian branch and at positions 56 and 295 of the Australian branch. The overall data obtained indicate unambiguously that the new epidemic caused by this subtype did not start from a single test tube, but involved a heterogeneous material that differed considerably from the strain circulated in the world in 1950 and kept in cold storage thereafter. Thus, the viral genome sequencing data demonstrated that the hypothesis on the artificial origin of the new epidemic wave of the influenza virus HlN1 is rather unlikely. I have one more comment regarding the figure. It is evident that sequencing data on the strains A/USSR/90/ 77 obtained in the three different labs differ from one another. This is the result of both the imperfect sequencing technique of the time and actual distinctions between subclones of the same strain and does not affect the conclusions made. It is now becoming clear that this method is very sensitive and sometimes even allows detection of changes in the virus population that occurred during its three- to seven-day circulation in the body of a single patient. It should be stated that hundreds of subsequent publications on the natural diversity of viruses and bacteria demonstrated that the genomes of these patho- gens are far from stable and uniform. For example, Figure 5 is a dendrogram describing the main genotypes of the hepatitis A virus. As shown in this figure, the scale of differences in percentages shows that the range of distinctions is wide enough to provide a reliable differentiation. In fact, a study tracing trans- mission of this pathogen at a molecular level in a stepwise manner from one person to another during an outbreak was presented in 1999 at the International
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BIOLOGICAL TERRORISM 99 Congress of Virology in Sydney.7 Shown in Figure 6 is the dendrogram of M segment nucleotide sequences of HERS (Hantaan) virus strains isolated in the Khabarovsk and Primorsky regions of Russia.8 It shows a small group of strains with numbers 4290 and 4029 sitting apart. This group of strains occurs only in a small part of this territory, and it can be isolated only from local rodents Apode- mus peninsular, which makes it possible to predict the place of infection solely based on sequence data of the isolate taken from the patient. There are many examples of the use of the molecular epidemiology approach and not only for viruses. For instance, a study of the molecular diversity of meningococcus in Russia during 1969-1997 was published recently.9 It provided the basis for a number of important predictions concerning this disease, including the hypothe- sis of the next serotypes that are expected to come soon to this territory. It is such methods that have led to the discovery of the source of the annual worldwide bacterial meningitis epidemics that result from the massive migration of Mus- lims to Medina and Mecca during the hajj. Another application of this approach is clarification of the sources of poliomyelitis cases, which are now rare in devel- oped countries. As it turned out, these cases were the result of an extremely rare reversion of the live vaccine strain to the wild-type pathogenic virus.~° Conse- quently, the worldwide program of poliomyelitis eradication now includes a five-year period when only inactivated vaccine will be used, and this step was included because of this finding. Similar findings led to the recent proposal to organize the worldwide PulseNet network for molecular epidemiological surveillance of intestinal infec- tions based on this approach. Although these studies still entail substantial costs, they will form the basis of a new epidemiology, as the opportunities and advantages they provide expand global prospects for combating infectious dis- eases.~2 An outstanding recent example of the advantages of this approach is a molecular epidemiological study of a case of deliberate HIV infection. A medi- cal employee of a U.S. hospital injected serum from an HIV-infected patient into his unwanted girlfriend, pretending it was a medication. It was the sequencing data and further analysis that unambiguously proved the origin of the straini3 and helped to convict this person. CONCLUSIONS 1. Existing lists of infectious agents with bioterrorist potential need to be expanded and should never be considered final. 2. The wide natural diversity of strains of infectious agents provides the principal possibility of investigating and differentiating between natural and arti- ficial origins of an infectious disease outbreak. 3. A detailed genomic inventory of the diversity of the strains of infectious agents will allow natural and artificial outbreaks to be differentiated on a routine basis.
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BIOLOGICAL TERRORISM 10 NOTES 1. McGeorge, H. 2001. An analysis of 404 non-military incidents involving either chemical or biological agents. World Congress on Chemical and Biological Terrorism. Dubrovnik, Croatia, April 22-27, 2001. Abstract Book, p. 53. 2. Geissler, E. 1994. Arms control, health care and technology transfer under the vaccines for peace programme. Control of Dual Threat Agents: The Vaccine for Peace Programme. E. Geissler and J. P.Woodall, eds. SIPRI Chemical and Biological Warfare Studies 15:10-37 (see especially p. 29). Anderson, W.C., III, King, J.M. 1983. Vaccine and antitoxin availability for defense against biological threat agents. U.S. Army Health Care Studies Division, Report No. 83-002. Fort Sam Houston, Tex.: U.S. Army Health Services Command. 3. Biological and Chemical Terrorism: Strategic Plan for Preparedness and Response. 2001. Recommendations of the CDC Strategic Planning Group. MMWR 49-#RR-4. 4. Ogarkov, P.I., V.V. Malishev, S.A. Tzutsiev, N.V. Mikhailov. 1996. The epidemiological characterization and laboratory diagnostics of viral hepatitis in the Russian federal military forces on the territory of the Chechen Republic. Voenno-Meditsinskii Zhurnal 8:48-55. 5. Russian Federation President's Directive No. 298-rp. June 14, 1994. 6. Beklemishev, A.B., V.M. Blinov, S.K. Vassilenko, S.Ya. Golovin, V.V. Gutorov, V.A. Karg- inov, L.V. Mamaev, N.N. Mikryukov, S.V. Netesov, N.A. Petrov, V.A. Petrenko, L.S. Sandakh- chiev. 1984. Synthesis, cloning and primary structure determination of full-length DNA copy of hemagglutinin gene of HlNl-subtype influenza A virus. Bioorganicheskaya khimiya (in Russian) 10(11): 1535- 1543. 7. Robertson, B., H. Khopraset, O.V. Nainan, T. Cromeans, K. Krawczynski, H.S. Margolis, et al. 1996. Transmission, excretion and genetic variants of hepatitis A virus within a community-wide outbreak. X International Congress of Virology, Jerusalem, Israel. Abstracts, p. 42. 8. Yashina, L.N., N.A. Patrushev, L.I. Ivanov, R.A. Slonova, V.P. Mishin, G.G. Kompanez, N.I. Zdanovskaya, I.I. Kuzina, P.F. Safronov, V.E. Chizhikov, C. Schmaljohn, S.V. Netesov. 2000. Genetic diversity of hantaviruses associated with hemorrhagic fever with renal syndrome in the Far East of Russia. Virus Research 70:31-44. 9. Achtman, M., et al. 2001. Molecular epidemiology of serogroup A meningitis in Moscow, 1969 to 1997. KID 7(3). 10. Macadam, A.J., et al. 1989. Reversion of the attenuated and temperature-sensitive pheno- types of the Sabin type 3 strain of poliovirus in vaccines. Virology 172:408-414. 11. Swaminathan, B., et al. 2001. PulseNet: the molecular subtyping network for foodborne bacterial disease surveillance, United States. KID 7(3). 12. Pfaller, M.A. 2001. Molecular approaches to diagnosing and managing infectious diseases: practicality and costs. KID 7(2). Centers for Disease Control. 2000. Preventing Emerging Infectious Diseases: A Strategy for the 21st Century. 13. Vogel, G. 1998. HIV strain analysis debuts in murder. Science 282(5390): 851-852.
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