5
Variola Virus Stocks Following Eradication of Smallpox

The World Health Organization (WHO) Intensified Smallpox Eradication Program was established in 1967. It included a global surveillance program, isolation of smallpox patients, and vaccination of the possible contacts of patients. Eradication of smallpox constitutes one of the greatest successes of modern medicine. After the World Health Assembly sanctioned the WHO declaration of 1980 that smallpox had been eradicated, WHO established a Committee on Orthopoxvirus Infections (COI) to advise on steps to be taken in the posteradication era and to monitor the conduct of those steps [15]. COI has continued to meet about every 4 years since that time. The question of whether smallpox virus should be retained and the conditions for its retention are among the most important issues faced by COI. The issue has been brought to formal vote at several COI meetings, usually with the majority favoring destruction.

Throughout its deliberations, COI has faced two strongly held, conflicting points of view [15]. Some scientists and many countries, especially those that experienced endemic smallpox most recently, greatly fear the reintroduction of smallpox and have argued passionately for taking all available measures to avert this possibility, including early destruction of all known stocks of the virus. It is recognized that this measure would provide no absolute guarantee against the possibility that some stocks of virus unknown to WHO might continue to exist. COI has also acknowledged that there is great scientific value in the stocks, but has expressed the belief that the risk of keeping the virus outweighs the potential benefits. It is generally believed that an international agreement to destroy the virus stocks would diminish the likelihood of the virus being released. On the other side, some scientists have argued for retention of the known stocks, expressing concerns



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--> 5 Variola Virus Stocks Following Eradication of Smallpox The World Health Organization (WHO) Intensified Smallpox Eradication Program was established in 1967. It included a global surveillance program, isolation of smallpox patients, and vaccination of the possible contacts of patients. Eradication of smallpox constitutes one of the greatest successes of modern medicine. After the World Health Assembly sanctioned the WHO declaration of 1980 that smallpox had been eradicated, WHO established a Committee on Orthopoxvirus Infections (COI) to advise on steps to be taken in the posteradication era and to monitor the conduct of those steps [15]. COI has continued to meet about every 4 years since that time. The question of whether smallpox virus should be retained and the conditions for its retention are among the most important issues faced by COI. The issue has been brought to formal vote at several COI meetings, usually with the majority favoring destruction. Throughout its deliberations, COI has faced two strongly held, conflicting points of view [15]. Some scientists and many countries, especially those that experienced endemic smallpox most recently, greatly fear the reintroduction of smallpox and have argued passionately for taking all available measures to avert this possibility, including early destruction of all known stocks of the virus. It is recognized that this measure would provide no absolute guarantee against the possibility that some stocks of virus unknown to WHO might continue to exist. COI has also acknowledged that there is great scientific value in the stocks, but has expressed the belief that the risk of keeping the virus outweighs the potential benefits. It is generally believed that an international agreement to destroy the virus stocks would diminish the likelihood of the virus being released. On the other side, some scientists have argued for retention of the known stocks, expressing concerns

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--> about losing experimental material that could provide important information regarding viral pathogenesis in humans, as well as about establishing a precedent for the deliberate destruction of an archived species and the associated loss of genetic information. These concerns are implicitly seen as outweighing the risks of keeping the virus. Many participants in the COI discussions have found themselves in broad agreement in perceiving merit in both points of view. Efforts to reconcile the two incompatible policy stances have been a continuing challenge for COI and others. Establishment of International Repositories Starting in 1977, the number of laboratories known to be holding stocks of variola virus was reduced until by the end of 1983 all known stocks were held in only two WHO Collaborating Centres—the U.S. Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, and the Research Institute for Viral Preparations in Moscow, Russia [1]. The Russian stocks were transferred in 1994 to the State Center of Virology and Biotechnology (VECTOR) in Kotsovo, Russia, which subsequently became the WHO Collaborating Centre for Orthopoxvirus Diagnostics. Use of these remaining variola stocks at CDC and VECTOR is restricted to approved Biological Safety Level 4 (BSL-4) conditions (see Box 5-1). WHO periodically conducts safety and security inspections of the facilities. Inventories of the samples at each institution are on file at WHO. COI recommended that cloned fragment libraries of selected strains be prepared and that selected prototypical strains be sequenced [15]. In addition, handling of cloned DNA fragments of the variola virus genome follows the recommendations issued in a 1994 COI report [16]: The two international repositories for storage, maintenance, and distribution of cloned DNA fragments of variola virus are CDC and VECTOR. Each sample is identified by nucleotide sequence determination. A register of cloned material is maintained, containing full descriptive and sequence information. Clones are distributed to research laboratories requesting them if:  The clones will not be further distributed to third parties.  The clones are not to be used for insertion of variola DNA into vaccinia virus or related poxviruses. No laboratory other than the international repositories is permitted to hold clones representing more than 20 percent of the variola virus genome at any one time.

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--> BOX 5-1 Laboratory Safety Because of its extreme virulence, variola virus must be handled in maximum containment facilities (BSL-4). These facilities consist of a separate building or clearly isolated section of a building with a sealed internal shell. Outer and inner change rooms separated by a shower are provided for personnel entering and exiting the facility. A double-doored autoclave, fumigation chamber, or ventilated airlock is provided for passage of materials not brought into the facility through the change room. Sewer and ventilation lines contain high-efficiency particulate air (HEPA) filters. Special individual supply and exhaust air ventilation is provided for laboratory workers, and pressure differentials are maintained to ensure the inward flow of air toward areas where the potential for hazard is highest [17]. There have been only two maximum containment facilities in the United States: one at CDC and one at Fort Detrick, in Frederick, Maryland. Recently a BSL-4 laboratory dedicated to the study of tuberculosis was opened at the National Institutes of Health. Since these limited facilities have generally been devoted to the study of pathogens perceived to pose a more immediate threat than smallpox, there has been very little research on variola. The scarcity of laboratory facilities suitable and authorized for studying live variola virus is a serious constraint on the U.S. capability to undertake the research discussed in this report. It would be possible to alter the variola virus genome to remove its capability for surviving outside the laboratory. Fox example, the uracil DNA glycosylase (UDG) gene could be knocked out, so that the virus could grow only in cells that expressed the viral UDG [18]. Currently, this approach would be limited to cell tissue studies, but it could, with a change in the regulations to recognize the inability of the altered virus to survive, enable use of the much more common BSL-3 facilities that utilize safety cabinets and other personal protective or physical containment devices [17]. Present regulations, however, require that no material handled in BSL-4 facilities can be opened at lower containment unless it is killed by means such as autoclaving, irradiating, or chemical treatment. Analysis performed mainly at CDC and VECTOR has produced about 750,000 bases of variola virus genome DNA sequence. The complete genome of variola major virus Bangladesh-1975 (GenBank #L22579) has been sequenced. With the exception of small regions at the ends of the DNA, the variola major virus India-1967 (GenBank X69198) and the variola minor alastrim virus Brazil/Garcia-1966 (EMBL Y167080)have also been fully sequenced.* *   Joseph J. Esposito, Personal communication, December 1998.

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--> The collections of variola virus samples held at CDC (approximately 450 samples, including CDC isolates and isolates deposited by the U.S. Army, the American Type Culture Collection, the National Health Institute of Japan, the National Health Institute of the Netherlands, and the United Kingdom Microbiological Research Establishment) and at VECTOR (120 samples comprising Moscow Institute of Viral Preparations isolates from about 20 countries) comprise isolates from clinical samples and material used in variolation that was collected from 1950 through the 1970s. The samples at CDC and VECTOR are preserved as frozen material (generally early passage material) from cell cultures or avian chorioallantoic membranes infected with lesion material; a portion of the samples is lyophilized. Approximately 27 samples at CDC and 17 at VECTOR comprise original lesion material from smallpox patients. Generally, the source of each isolate is documented, but the extent of that documentation varies. The samples, some of which are duplicated, do not represent a complete archive of taxonomically characterized strains from the different outbreaks in recent history.* Decision by the World Health Assembly to Destroy Variola Virus Stocks In 1986 and again in 1990, COI affirmed the desirability of destroying variola virus stocks and intact DNA (but not cloned DNA fragments) after satisfactory progress had been made with sequencing, mapping, and cloning [15]. COI subsequently decided that before making a final decision with regard to destruction of the virus, it would consult more widely with the scientific community [15]. In 1993, a special roundtable discussion took place in Glasgow at the International Congress on Virology. Alternative views regarding destruction of the virus were published in companion articles in Science, and the views of a number of professional bodies were solicited, several of which formally endorsed destruction. After a 1994 review of the issues and progress made in mapping, cloning, and sequencing of strains, COI voted unanimously to recommend that the virus be destroyed. All but two members recommended June 1996 as a target date, the others calling for a delay until June 1999. COI recommended to the Executive Board of the World Health Assembly that a resolution calling for the destruction of all known stocks of variola virus in June 1996 be endorsed and forwarded for action to the World Health Assembly. At the ensuing WHO Executive Board meeting, the United States supported the decision to destroy the virus at the end of June 1996. However, as a result of further discussion and debate at that meeting and at the subsequent meeting of the World Health Assembly, the decision was made to defer destruction of the *   Joseph J. Esposito, Personal communication, December 1998.

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--> remaining stocks of variola virus, intact viral genomic DNA, and clinical specimens or other material containing live variola virus until June 1999, and then only after a reaffirmation of the decision at the May 1999 World Health Assembly meeting [19]. At the most recent COI meeting in January 1999, the committee voted in favor of the destruction of the remaining stocks still kept at the WHO Collaborating Centres in the Russian Federation and the United States. The vote, however, was not unanimous. Five of the nine committee members voted for immediate destruction, two voted for eventual destruction following a review in 5 years, and two supported indefinite retention of the stocks [20]. U.S. Research on Smallpox In 1995, continuing disagreements within the U.S. government as to the appropriate policy on destruction of variola virus stocks led to the appointment of civilian advisers to the U.S. Department of Health and Human Services (DHHS) and the U.S. Department of Defense (DOD) to reexamine the issues involved. These advisors represented the Board of Scientific Counselors of the National Center for Infectious Diseases at CDC and the Armed Forces Epidemiological Board. They submitted a report containing the following conclusions and recommendations [15]: There is strong support for the ultimate destruction of viable smallpox stocks in all repositories. However, in the face of evidence of a public health threat posed by the potential use of unregistered stocks of smallpox as an agent of terrorism or biological warfare, destruction should be deferred until specific information is obtained on the following three critical issues:  The efficacy of currently available antiviral drugs.  The efficacy of current and new cell-culture-derived vaccine preparations for the prevention of smallpox induced by high-dose aerosol challenge.  Development and validation of potential animal models for the purpose of evaluating antivirals and new or improved vaccines. A short-term, focused research program to obtain this information should be established using the resources of CDC and the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). If the proposed research program cannot be given sufficient priority for the work to be carried out within a reasonable period of time—3 to 5 years—this should be taken as an indication that neither the research nor the variola virus stocks are needed.

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--> Elimination of existing smallpox stocks would lessen but not eliminate the public health threat of orthopox disease. Consequently, the United States must establish and maintain a strong diagnosis and disease prevention effort. DHHS and DOD also created a Joint Coordinating Group (JCG) to address critical scientific issues associated with destruction of variola virus stocks and to elaborate a research agenda involving collaboration among CDC, USAMRIID, the National Institutes of Health (NIH), and the U.S. Food and Drug Administration (FDA). The JCG identified three principal areas for research that encompassed the issues raised by the advisory group [21]. The first, dealing with antiviral substances, involves evaluation of treatment of smallpox with antiviral agents that have been approved by FDA for other purposes or are well on the way toward approval. The enormous costs of drug development, as well as the time involved, dictate the need to restrict the screening to these drugs. An initial step would be to undertake a screening of these compounds in cell cultures infected with variola virus or other orthopoxviruses and then to proceed through animal models using appropriate surrogate orthopoxviruses, recognizing that there is no satisfactory animal model for variola infection (see Chapter 6). A second research area identified by the JCG involves ascertaining the possible applicability of a model using monkeypox virus infection in monkeys to measure the effect of antivirals and assess the efficacy of vaccines. It was suggested that if this model were to prove unsatisfactory, further consideration would have to be given to the possible development of an animal model for smallpox, such as genetically modified rodents or immunosuppressed animals. Finally, the JCG noted the need to develop simple, rapid laboratory methods for identifying and differentiating among the various orthopoxviruses. Research at CDC and USAMRIID In addition to sequencing of variola virus isolates at CDC, collaborative studies at CDC and USAMRIID have pursued the research program outlined by the JCG. Antiviral studies have included screening of drugs for inhibition of variola and other orthopoxviruses in cell culture and for protection against monkeypox in macaque monkeys, an animal model system considered to be more closely analogous to smallpox in humans than is vaccinia infection in monkeys. Several new drugs being developed for other medical problems have shown potential. Cidofovir, a DNA polymerase inhibitor, has been found to be active when used systemically in a murine model of vaccinia infection [22], to be topically and systemically effective against molluscum contagiosum [23], and to protect monkeys when treatment was initiated within 2 days following monkeypox virus exposure [24]. However, these results do not guarantee that the drug will provide protection against smallpox in humans. Imitosol, for example, in-

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--> hibits cytomegalovirus (CMV) in humans, but not in guinea pigs or mice, the surrogate models of choice. Furthermore, cidofovir has significant side effects and must be injected intravenously with probenecid following hydration [24]. For these reasons, cidofovir appears unlikely to be an effective therapeutic agent for routine use against smallpox. A DOD vaccine derived in cell culture using plaque-purified Connaught smallpox vaccine as seed virus was also tested under this program. Monkeys vaccinated with the DOD vaccine or commercially available vaccine were subsequently exposed to aerosolized monkeypox virus. Both the DOD vaccine and the commercial vaccine partially protected the monkeys against monkeypox infection [25]. Again, however, this finding cannot be considered conclusive. Although such experiments cannot be done for ethical reasons, there is no substitute for challenging the target host with live virus to prove protective efficacy [25]. For rapid diagnosis, CDC and USAMRIID have incorporated fluorescence based polymerase chain reaction (PCR) technology in a suitcase-sized device that can identify and distinguish among the poxviruses. The assay is accurate with DNA prepared in the laboratory, but has not been proven with clinical materials in the field. Additional long-distance PCR techniques and restriction endonuclease assays that amplify and identify the entire genome have been developed. There are no routine serological assays that can specifically identify and differentiate variola virus antibodies in sera [26].

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