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INTERNATIONAL NOTE
SMALLPOX ERADICATION: DESTRUCTION OF VARIOLA VIRUS STOCKS
In May 1999, the Fifty-second World Health Assembly by resolution
WHA52.10 authorized temporary retention up to not later than 2002 of the existing
stocks of variola virus at the two current locations* for the purpose of further
international research. The resolution requested the Director-General of the
World Health Organization (WHO) to appoint a new group of experts which would
establish what research, if any, must be carried out in order to reach global
consensus on the timing for the destruction of existing variola virus stocks.
The WHO Advisory Committee on Variola Virus Research - composed
of 16 members from all WHO regions and advised by some 10 scientific academic
experts from such areas as public health, fundamental applied research and
regulatory agencies - was subsequently established and has met three times.
Reports from the first two meetings have already been submitted to the World
Health Assembly†. This article provides a report of the third meeting
(Geneva, 3 and 4 December 2001).
Third meeting of the WHO Advisory Committee on Variola
Virus Research
The Committee agreed that, despite the considerable progress
that had been made in investigating variola virus, significant components
of this research, most notably the refinement and use of an animal model developed
in 2001 and the development of antiviral drugs, were unlikely to be completed
by the end of 2002. Further, during extensive discussion about the potential
availability of an animal model, additional research was identified that would
necessitate access to live variola virus stocks after the expected 2002 destruction
date.
The Committee's main recommendation, therefore, was that serious
consideration should be given to further extending the deadline for the destruction
of variola virus in order to allow essential research to be completed. Furthermore,
this additional research with live variola virus should continue to be carefully
monitored and reviewed under the auspices of WHO, and steps should be taken
to ensure that all approved research would remain outcome-focused and time-limited
and periodically reviewed.
Review of variola virus strains in the two repositories
The Centers for Disease Control and Prevention held 451 viral
isolates obtained from different continents and countries when smallpox was
endemic. The current review and the studies reported at the meeting concentrated
on some 50 isolates in the Russian collection that were not present in the
American collection.
From these isolates, 23 strains from scab material and previously
lyophilized samples were viable in tissue culture. Isolation of deoxyribonucleic
acid (DNA) from these strains is continuing; already, two genomes have been
completely cloned and at least five others will be cloned by the end of 2002.
The Committee agreed that before the end of 2002 further consideration should
be given to the necessity of holding the wide range of isolates currently
available in the two repositories.
Nucleic acid-based diagnostics
Several methods have been devised recently for very sensitive
detection of variola virus DNA and to distinguish this DNA from that of other
orthopoxviruses, the most promising being analysis by polymerase chain reaction
(PCR) of restriction fragment length polymorphisms, multiplex PCR and real-time
PCR with fluorigenic probes. Some of these tests have been used in the definitive
diagnosis of a recent laboratory-acquired infection with a non-variola orthopoxvirus.
The results obtained indicate that single-gene restriction
fragment length polymorphisms and multiplex PCR detection methods are useful
for detecting variola virus in clinical samples. The Committee noted that,
although the real-time PCR test has greater sensitivity and can therefore
detect infection at an earlier stage, it requires the use of expensive equipment
and, so far, cannot consistently distinguish between species of orthopoxviruses.
An extended PCR test for restriction fragment length polymorphisms has proved
useful in defining the origin of an isolate, but may require prior tissue-culture
passage of clinical samples.
The Committee recognized the significant progress made in
the area of molecular diagnosis but agreed that there was still scope for
improving the sensitivity of the tests available. For example, it would be
useful to know how early infection with variola virus might be detected at
the prodromal stage. An ultimate goal might be the development of relatively
cheap hand-held equipment for the detection of variola virus DNA and diagnosis
of infection.
To further this important area of work, the Committee encouraged
investigators to share diagnostic reagents, essential primer sequences for
PCR assays and protocols where appropriate. This cooperation would be particularly
useful for enhancing capabilities in different countries for the rapid and
reliable detection and diagnosis of variola virus infections.
Sequence analysis of variola virus DNA
The Committee was informed that the complete genomes of an
additional seven isolates of variola virus had been sequenced, bringing the
total number of full-length genome sequences to 10 (nine variola major and
one variola minor strain). The sequences were highly conserved. To counter
the criticism that this result was a consequence of tissue-culture passage,
the Committee suggested that further thought be given to sequencing DNA directly
from scab material. The known degree of virulence of isolates has not yet
been correlated with identified variations in sequences.
The Committee noted that a considerable amount of information
on the nucleic acid sequences of variola viruses was now available. After
discussion, it was agreed that further sequencing of the more variable genomic
termini had priority over the derivation of sequences of additional whole
genomes. This would be useful for forensic purposes if there were ever a deliberate
release of variola viruses, and reference DNA should be kept for this purpose.
Serologic assays
Polyclonal and monoclonal antibodies against vaccinia virus
have been used in various enzyme-linked immunosorbent assays to evaluate their
usefulness in the detection of variola virus antigens. Polyclonal antibodies
detected all viral strains more readily than the monoclonal antibodies currently
available, but, although the methods appear to be relatively sensitive, they
do not facilitate the detection of all viral isolates. The Committee concluded
that a variola virus-specific serologic assay could usefully complement molecular
diagnostic techniques, particularly as a second method to detect infection.
However, further validation of the available tests was needed.
Animal models
The Committee was informed of the successful infection of
cynomolgus macaques with two different variola virus strains by intravenous,
or intravenous plus aerosol, routes. The disease induced shared several pathological
features with human smallpox but the course of disease is much faster and
the dose of virus needed to cause intravenous infection is particularly high.
Additional studies are needed to improve and validate this
animal model, but this would need work extending beyond 2002. The monkey model
has the potential to be used as an assay in prophylactic or therapeutic studies
with live variola virus and could also provide access to good diagnostic reagents.
Other surrogate animal models are being investigated in parallel, in particular
the infection of monkeys with monkeypox virus and the infection of rodents
with cowpox virus, in order to obtain data that relate more to models using
variola virus.
Drug development
Most studies have focused so far on the efficacy of cidofovir
against poxviruses. This compound has demonstrable activity against cowpox
in mice and against monkeypox in monkeys. In the United States, cidofovir
may be used in emergencies as an investigational new drug to treat significant
adverse events following immunization with the current smallpox vaccine, and
in the unlikely event of smallpox re-emerging.
In vitro screening of other chemical entities has identified
> 140 additional compounds with antiviral activity against poxviruses. The
finding that some of these compounds have selective activity, inhibiting one
or more orthopoxviruses but not necessarily variola virus, supports the premise
that access to live variola virus is necessary for the effective screening
of additional lead compounds. Most active compounds identified so far target
the viral DNA polymerase and it was considered important to identify other
viral gene products susceptible to drug intervention.
Vaccine development
The Committee agreed that the best safeguard against smallpox
was vaccination. This strategy had been successfully deployed during the eradication
program, but the smallpox vaccine currently available was associated with
a significant number of adverse events. This suggested that, although the
current vaccine had proved its efficacy and utility, improvements were needed,
particularly to facilitate the safe and effective immunization of vulnerable
sectors of certain populations (the immunocompromised, the elderly, pregnant
women and children with eczema).
The Committee therefore encouraged the delineation of further
research into vaccine strategies that might use more attenuated vaccinia virus
strains, subunit vaccines or other promising approaches, including DNA vaccines.
Results reported at the meeting and in numerous publications on attenuated
vaccinia virus recombinants encoding antigens from other pathogens indicate
the potential value of these alternative strategies for vaccine development.
It was recognized that access to live variola virus would be necessary to
assess the efficacy of new, improved smallpox vaccines and, ultimately, to
obtain regulatory approval.
Conclusions and recommendations
The Committee acknowledged that important progress has been
made in health-oriented research involving variola virus. However, it concluded
that much essential research will not be completed by the end of 2002. The
Committee recommended that further goal-oriented research, extending beyond
the expected 2002 destruction deadline, could be justified so that the world
population could be adequately prepared for the unlikely, but potentially
catastrophic, event of a re-emergence of smallpox.
It was further recommended that the current Advisory Committee
should continue its role in monitoring and reviewing all research involving
live variola virus, and that steps should be taken to ensure that all approved
research would remain outcome-focused and time-limited.
Recommendations of the Director-General
Having noted the report of the Advisory Committee for Variola
Virus Research, including the recommendations for research priorities, and
its conclusion that the research program will not be completed by the end
of 2002, the Director-General recommended that:
- the WHO Advisory Committee on Variola Virus Research should continue
to oversee the variola virus research program, and that the research program
should be conducted in an open and transparent manner;
- the research program should be completed as quickly as possible, and
a proposed new date for destruction should be set when the research accomplishments
and outcomes allows consensus to be reached on the timing of destruction
of variola virus stocks;
- regular biosafety inspections of the storage and research facilities
should be continued in order to confirm the strict containment of existing
stocks and to ensure a safe research environment for work with variola virus;
- depending on progress, a report on the research should be submitted to
the Executive Board and World Health Assembly in 2 to 3 years time.
Source: WHO Weekly Epidemiological Record, Vol 77, No 5,
2002.
* Centers for Disease Control and Prevention, Atlanta, Georgia,
United States, and the Russian State Centre for Research on Virology and Biotechnology,
Koltsovo, Novosibirsk Region, Russian Federation.
† See the WHO Weekly Epidemiological Record, No. 6, 2000,
pp. 45-48 and No. 19, 2001, pp. 142-45.
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