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OCR for page 111
8
Methods for Detection and Diagnosis
I
n the pre-eradication era, smallpox was usually diagnosed by its dis-
tinct clinical characteristics, particularly the vesicular–pustular rash
(see Chapter 2), in the context of a cluster of probable cases with an
epidemiologic link. Material from lesions could be analyzed by electron
microscopy in reference laboratories, providing morphologic identification
of the characteristic brick-shaped virions (Biel and Gelderblom, 1999; see
also Chapter 3), but did not distinguish variola from other poxviruses.
Recovery of infectious virus from infected persons using tissue culture
methods was feasible but was seldom used.
Despite the eradication of smallpox, the need remains for robust and
safe methods of detection of variola virus and diagnosis of the disease.
Diseases caused by poxviruses that can infect the human host, such as
monkeypox, continue to circulate and may be confused with smallpox,
necessitating precise methods for rapid differential diagnosis. Disseminated
vaccinia might also be misdiagnosed as smallpox, although a history of
recent vaccination or contact with a recently vaccinated person would
usually be obtained. Finally, the classification of smallpox as a category A
agent with the potential for aerosolization and broad distribution within
the environment requires new approaches to sensitive and specific detection
of the virus in nonclinical specimens.
The application of contemporary viral diagnostic tools, such as poly-
merase chain reaction (PCR) methods, to smallpox diagnosis has received
attention because rapid and accurate identification of index cases would be
essential for optimal containment of initial spread in a largely unimmunized
population in the event of an unintended or intentional release of the virus.
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LIVE VARIOLA VIRUS
These methods may allow diagnosis in respiratory secretions during the
12–14 day incubation period, which would be quite valuable for controlling
transmission. Additionally, recent and forthcoming advances in genomic
science mean that new approaches for identification of variola virus in
clinical or environmental samples can be developed that involve detecting
the presence of genomic DNA or viral proteins. Maximizing the specificity
of such tests will require knowledge of the genetic variability of related
poxviruses, the background against which variola must be distinguished to
maximize the sensitivity of the test, and the variability of variola and viral
proteins and their subdomains that are unique to variola. It will also be
important to develop new diagnostics that can be used to detect the virus in
different types of patient specimens (e.g., lesion material, secretions, organ
tissues) and environmental samples (e.g., air, surfaces, fomites). Developing
environmental detection and diagnostic methods that do not require the
isolation of infectious virus in tissue culture is important because of the
risk of human exposure during preparation of specimens to be tested in
the laboratory. Such advances in detection and diagnosis would facilitate
forensic investigations to determine the source of variola virus in the event
of an intentional release.
This chapter reviews the current status of methods to detect variola
virus and diagnose smallpox, relevant regulatory requirements, and the
need for live variola virus to achieve advances in the development of detec-
tion and diagnostic capabilities.
CURRENT STATUS OF DETECTION AND DIAgNOSTIC METHODS
The 1999 committee offered the following conclusion related to detec-
tion and diagnosis:
If further development of procedures for the environmental detec-
tion of variola virus or for diagnostic purposes were to be pursued,
more extensive knowledge of the genome variability, predicted pro-
tein sequences, virion surface structure, and functionality of variola
virus from widely dispersed geographic sources would be needed.
Since 1999, substantial work has been done on the development of
new techniques for the detection of variola virus and diagnosis of smallpox
and for the differentiation of variola virus from other orthopoxviruses that
infect humans (e.g., vaccinia, monkeypox, cowpox). Most of these assays
have been based on nucleic acid detection by PCR, and some have been
validated using clinical samples. Some experience has been reported with
the use of multiplex PCR to detect variola and differentiate it from other
poxviruses or unrelated viruses in laboratory-created specimens containing
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METHODS FOR DETECTION AND DIAGNOSIS
mixed genomic fragments. Limited experience exists with direct detection
of variola virus in stored patient specimens or in specimens from nonhuman
primates. Relatively little has been done to create assays that detect variola
virus proteins or to refine serologic approaches to smallpox diagnosis.
The capacity to carry out seroepidemiologic surveillance with rapid high-
throughput serologic assays for variola virus-specific IgG antibodies would
be valuable to characterize the extent of the spread of the virus in an out-
break setting, and serologic assays for variola virus-specific IgM antibodies
would be useful to document recent infection in individuals who were
asymptomatic when tested (see Appendix).
Polymerase Chain Reaction
PCR enables highly sensitive detection of viral nucleic acids to very low
copy numbers. PCR products can be sequenced to provide detailed genetic
information about the pathogen, and PCR can be performed as a quantita-
tive or multiplex assay in which the specimen is tested for multiple patho-
gen sequences at the same time. Several different regions of the variola virus
genome have been used to design primers that either detect all orthopox-
viruses of interest or are specific for individual poxviruses. Real-time PCR
for the hemagglutinin gene (J7R) of variola virus was sensitive and specific
when tested on variola virus samples from cell culture and infected tissues
that contained both viral and cellular DNA (Ibrahim et al., 2003; Aitichou
et al., 2008). This assay was evaluated with genomic DNA from 48 differ-
ent isolates of variola virus and 25 other poxviruses. Specificity for variola
detection was greater than 96 percent; the majority of these samples were
derived from virus-infected cell cultures and variola virus-infected tissues.
This poxvirus assay was applied successfully to the diagnosis of smallpox
from fixed human tissue from one fatal case (Schoepp et al., 2004), even
though specimens were obtained and stored under conditions not designed
to protect DNA integrity. The assay has been expanded to include other
variola virus genes (B9R and B10R) using prepared samples, detecting
12–25 genome copies (Kulesh et al., 2004). It has been adapted for use with
dried reagents and for multiplexing with probes for other orthopoxviruses
(Aitichou et al., 2008). The hemagglutinin gene has also been used to design
primers for detecting all orthopoxviruses for use with a probe that can
distinguish variola from other poxviruses by melting curve analysis, and
tested on plasmid DNA (Espy et al., 2002) and on tissue and blood spiked
with poxvirus DNA (Putkuri et al., 2009).
The CrmB (cytokine response modifier B) gene has also served as the
target for amplifying orthopoxvirus DNA using consensus primers. Viral
(genomic) amplicons may differ in size, but variola and other orthopox-
viruses can also be differentiated from each other by analysis of restriction
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LIVE VARIOLA VIRUS
fragment length polymorphism (RFLP) (Loparev et al., 2001). This assay
was validated on eight strains of variola virus. In a similar assay, TaqMan
probes were designed to be specific for all orthopoxviruses or for variola
virus and validated with poxvirus panels and plasmid DNA from the
European Network for Imported Viral Diseases (Fedele et al., 2006). A
multiplex PCR that distinguished orthopoxviruses from herpesviruses used
primers from the CrmB gene for poxvirus identification and RFLP of the
PCR product to differentiate one orthopoxvirus from another. This test
was developed and validated using plasmid DNA from only a single strain
of variola virus (Sias et al., 2007). A real-time PCR assay that combines
variola virus-specific and panorthopoxvirus primers targeted to the gene for
a 14 Kd protein (A30L) has been developed and validated on genomic DNA
from 12 strains of variola virus; variola was differentiated from cowpox,
vaccinia, monkeypox, and camelpox viruses (Scaramozzino et al., 2007).
A multiplex real-time PCR assay has been developed that includes indi-
vidual primers specific for variola (B11R–B12R), vaccinia, monkeypox, and
cowpox viruses, plus primers common for all orthopoxviruses, and results
in amplicons of different sizes. This assay was validated on DNA from virus
grown in culture and on scabs from smallpox skin lesions (Shchelkunov
et al., 2005). Another multiplex method targets the 14kD fusion protein
(A27L) for amplification from all orthopoxviruses and differentiates variola
from other orthopoxviruses by melting curve analysis (Olson et al., 2004).
This assay was validated on 14 variola virus samples from tissue culture
and from skin lesions in the VECTOR repository and detected four variola
genome copies. Multiplex PCR has also been performed using consensus
and variola virus-specific primers based on known single nucleotide poly-
morphisms (SNPs) in A13L and A36R genes that are different in variola
and other poxviruses; these SNPs were identified in PCR products from
43 variola strains but none of 50 other orthopoxviruses (Pulford et al.,
2004). These variola virus isolates had been collected over 40 years from
diverse geographic locations.
A number of PCR assays have been developed and tested for detection
and differentiation of variola virus using only plasmid DNA. The genes ana-
lyzed include hemagglutinin, RNA polymerase (rpo18), early transcription
factor VETF, and small membrane protein p8 (A13L). For each, melting
curve analysis was used to distinguish variola from other orthopoxviruses
(Nitsche et al., 2004; Panning et al., 2004). PCR has also been combined
with immobilization of synthetic oligonucleotides corresponding to variola
and other poxvirus genes on nylon membranes to allow direct visualization
of products that hybridize to specific oligonucleotides as a simplification,
but a PCR apparatus is still required (Fitzgibbon and Sagripanti, 2006).
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METHODS FOR DETECTION AND DIAGNOSIS
In Situ Hybridization
In situ hybridization was used to examine sections of tissue speci-
mens for the presence of variola virus DNA in skin lesion biopsies from
two South American smallpox cases. Specific molecular probes differenti-
ated skin cells containing variola from those caused by herpesviruses in
formalin-fixed tissue sections that showed no distinguishable differences by
standard histopathology methods (Nuovo et al., 2003).
gene Chip Analysis
Oligonucleotides specific for orthopoxviruses can be immobilized and
used to detect interaction with DNA extracted from samples suspected of
containing a poxvirus. Specific hybridization can be detected by fluorescent
probes (Lapa et al., 2002; Laassri et al., 2003; Ryabinin et al., 2006) or
use of electrochemical sensors (Komarova et al., 2005). Chips have been
designed using one or two individual variola virus genes (CrmB, Lapa et
al., 2002, and Komarova et al., 2005; C23L/B29R, Laassri et al., 2003;
C23L/B29R + B19R, Ryabinin et al., 2006) and the complete genomes of
multiple strains as resequencing tiling arrays (Sulaiman et al., 2007). These
assays can distinguish variola virus from other poxviruses and from herpes-
viruses. The resequencing array was tested on amplified DNA from 14
strains of variola virus and can also identify other human orthopoxviruses
(Sulaiman et al., 2008). This technology can be used for rapid identifica-
tion of a particular variola genome by comparison with known genomes
in sequencing databases. A variation on this approach is the development
of primers that span the orthopoxvirus genome followed by RFLP, which
is then used to distinguish one orthopoxvirus from another. This assay
was validated on genomic DNA from two strains of variola virus and on
monkeypox, camelpox, cowpox, tanapox, ectromelia, and vaccinia viruses
(Li et al., 2007). These whole-genome approaches would be useful to iden-
tify variola genomes that had been altered intentionally.
Protein-Based Methods
Little work has been done to develop direct protein detection methods
for variola. At present, these methods depend on developing antibody
reagents that bind specifically to variola proteins that are distinct from those
made by other orthopoxviruses. Utilizing ELISAs, Ulaeto and colleagues
(2002) have begun to characterize the reactivity of 23 strains/isolates of
variola virus, both γ®-ray inactivated and viable (under BSL-4 conditions),
with a panel of monoclonal antibodies and polyclonal antisera, raised
against either vaccinia or variola virus preparations. Polyclonal antibody
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LIVE VARIOLA VIRUS
reagents displayed more uniform detection of variola virus strains than was
obtained with monoclonal antibodies (Ulaeto et al., 2002). One monoclonal
antibody has been described that is specific for variola virus and can be
used to distinguish variola from other poxviruses (Damon, 2006). However,
monoclonal antibodies detect a single epitope in a single viral protein, and
most are conformation dependent. Specificity for geographically unrelated
variola isolates would depend on defining a fully conserved and stable
epitope or using a mix of monoclonals that would recognize epitopes in
several unique variola virus proteins having no homologues or differing
substantially from the related proteins in the other poxviruses.
Even when well-characterized reagents are available, designing antigen
detection methods that demonstrate the presence of viral proteins in patient
materials has been challenging for many human pathogens. Most successes
are achieved when the clinical material is a cutaneous lesion specimen,
which would be the case for variola at the symptomatic stage of infection.
In one example of a poxvirus detection method applied to respiratory secre-
tions, a biosensor technique using cyan-5 dye labeled antivaccinia antibody
was used to detect vaccina proteins in human throat swab specimens that
had been spiked with vaccinia virus from tissue culture (Donaldson et
al., 2004). One would expect such approaches to be feasible for variola
detection, but their development currently depends on generating panels
of antibodies that are highly specific for variola proteins. Pilot experiments
were conducted in which ELISAs were used to detect monkeypox virus
during the recent outbreaks in Africa and variola virus in specimens from
nonhuman primates (Karem et al., 2007). Nevertheless, although inhibi-
tors may be encountered, nucleotide detection methods are generally pre-
ferred for viral detection because nucleotides can be extracted from patient
materials and concentrated for PCR testing, whereas similar processes to
enhance sensitivity are difficult for protein detection in respiratory secre-
tions or other clinical specimens that would be available from patients in
the pre-eruptive phase of smallpox. Proteomics methods may emerge that
can identify a specific sequence of amino acid residues by direct analysis
of a sample using mass spectroscopy or other methods that do not require
antibody reagents, but these tools are not yet applicable for clinical use.
With the exception of measuring antibody titers by plaque reduc-
tion neutralization assay, serologic assays for IgG and IgM antibodies to
variola and other poxviruses are also protein-based detection techniques.
ELISA methods detect antibodies in serum samples through their binding to
immobilized viral antigens. The development of such an assay for detecting
variola virus IgG and IgM antibodies is feasible, but specificity requires the
identification of unique proteins that do not elicit cross-reactive antibodies
as a result of exposure to other poxviruses, such as by vaccination with vac-
cinia. It is anticipated that most variola infections would be symptomatic;
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METHODS FOR DETECTION AND DIAGNOSIS
however, a panpoxvirus serologic assay could be useful for assessing the
extent of asymptomatic infection in a population not previously vaccinated
should the need arise.
Currently, VECTOR is developing next-generation test kits to detect
orthopoxviral protein markers. These immunodiagnostic tests will rely on
hybridoma technology and technology for producing recombinant anti-
bodies to major neutralizing and protective antigens of variola virus and
those of other orthopoxviruses pathogenic for humans (Russian federation
Patent #2281327; Razumov et al., 2004, 2005; personal communication,
Ilya Drozdov, WHOCC for Orthopoxvirus Diagnosis and Repository for
Variola Virus Strains and DNA, March 27, 2009). In parallel, VECTOR is
working to develop and improve species-specific diagnostics for viruses such
as variola, monkeypox, and cowpox based on multiplex PCR, real-time
PCR, and microchip technology (Lapa et al., 2002; Laassri et al., 2003;
Ryabinin et al., 2006; personal communication, Ilya Drozdov, WHOCC
for Orthopoxvirus Diagnosis and Repository for Variola Virus Strains and
DNA, March 27, 2009).
Detection in the Environment
The technical capacity for environmental detection of variola virus
would be important in the event of an intentional release. Widespread
distribution of the virus could be achieved because poxviruses are stable
in aerosol form and can be lyophilized. The molecular methods for variola
virus detection that have been developed since 1999 use PCR and in situ
hybridization assays that have proven valuable for the clinical detection
of many viral pathogens in patient specimens, and a few of these methods
have been validated using archived tissues from variola cases. PCR-based
methods are also useful for detecting viruses in environmental samples,
including air samples, water, and soil, as well as in swabs taken from
potentially contaminated surfaces. These methods could be applied to the
identification of variola virus in such specimens with certain modifications
in the way the materials are prepared for testing. For example, it would
be necessary to take into account the inhibitory effects of detergents and
other materials on PCR sensitivity, as shown in experiments with vaccinia
virus (Kurth et al., 2008). The specificity of PCR for variola virus detection
should be preserved, but sensitivity in such samples is difficult to predict.
Ideally, tools for detecting the presence of variola virus in the environ-
ment would need to be rapid, portable, and easily deployable. Because pox-
virus genome detection methods require relatively complex equipment and
reagents, it would be necessary at present to bring materials suspected of
containing variola virus to a laboratory facility. A more practical variation
of the method for field use would be the use of dried reagents in a dual-
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LIVE VARIOLA VIRUS
probe real-time PCR assay for detection of variola or other orthopoxviruses
(Aitichou et al., 2008). Even if PCR or ELISA methods were used that could
differentiate variola from other poxviruses in environmental samples, their
sensitivity in field testing would need to be established. Criteria for speci-
ficity might need to be lowered to ensure that a positive sample was not
missed under field conditions, with the assumption that all specimens would
need to be retested and results validated in a reference laboratory. This
gap may be addressed by the development of tools such as direct electro-
chemical DNA sensors that can identify nucleotide sequences without the
need for PCR amplification and secondary analysis of the products by RFLP
or sequencing (Komarova et al., 2005). Nanotechnology-based tools may
be developed that can discriminate viruses based on their particle size and
other properties; if so, it would be necessary to have at least inactivated
variola particles to assess their sensitivity for environmental detection.
REgULATORy REqUIREMENTS
Currently available in vitro diagnostic devices (IVDs) for the detection
and diagnosis of variola infection are limited to research assays developed
by DOD, CDC, and academic laboratories. In the United States, licensure
of IVDs for various infectious agents, including variola, is regulated pri-
marily by the FDA’s Center for Devices and Radiological Health (CDRH),
which assesses benefits and risks according to the IVD’s analytical and
clinical performance. Medical devices, including IVDs, are categorized
as Class I, II, or III according to risk criteria and requirements listed in
21 Code of Federal Regulations (CFR) 800. Whereas most Class I devices
are exempt from premarket notification, most Class II devices do require
such notification [510(k)], and most Class III devices require premarket
approval (PMA), including submission of clinical data to support marketing
claims. The potential classification of IVDs for variola virus detection has
not been established, although it appears likely, given the critical impor-
tance of accurate detection methods, that premarket notification including
both general and special controls (Class II designation) would be required.
A new section (513(f)(2)) of the Food, Drug and Cosmetics Act as amended
by the FDA Modernization Act of 1997 includes a provision whereby
a sponsor can request a so-called “de novo” classification that may not
require premarket approval, but the sponsor would have to demonstrate
that the device would pose very little or no risk of harm, especially for
diagnosing suspected human cases. Finally, the use of a new IVD for variola
virus detection may also be approved via Emergency Use Authorization (see
Chapter 1).
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METHODS FOR DETECTION AND DIAGNOSIS
NEED FOR LIVE VARIOLA VIRUS
The identification and characterization of a series of variola virus-
specific genetic markers has paved the way for sensitive and specific multi-
plex nucleic acid methods, and further progress on refining these approaches
should not require live virus. Methods that detect viral proteins have been
pursued to a lesser extent but could also be expanded without the need for
live virus. Although not essential, better characterization of the sensitivity
and specificity of both nucleic acid and protein methods for variola virus
detection in relevant samples could be achieved by additional testing of
tissues from nonhuman primates infected with the virus. Preservation of
tissues for this purpose should be included in antiviral, vaccine, or patho-
genesis studies done in animals infected with variola. Since methods devel-
oped using only variola proteins could prove inadequate for their detection
in clinical materials from infected individuals, archived clinical specimens
could be tested to confirm the sensitivity and specificity of such tests, if
possible. Further work on protein-based detection would benefit particu-
larly from access to proteins made in variola virus-infected cells instead of
proteins made using expression vectors to ensure the reliability of the test
and to standardize reagents.
Environmental detection methods have seen little progress, but further
research in this area would use “mocked-up” specimens, so use of the live
virus would not be necessary. High-throughput assays, including serologic
methods to identify recently infected individuals, would be needed to test
large numbers of samples in a possible outbreak situation. However, the
development of most new methods would not require live virus as this
research could build on work with other validated methods and be scaled
up. Some future approaches that might prove valuable, such as those that
detect viral particles, could require access to variola virions made in culture
cells for their validation.
One caveat related to variola detection and smallpox diagnosis is that
genomic sequencing of enough geographically diverse isolates is necessary
to ensure that PCR tests have adequate specificity. PCR and sequencing of
the amplicons would be the first step in a forensic analysis of the source of a
variola isolate should a reintroduction of the virus occur, and would also be
accomplished most effectively if background information were available on
the complete genome sequence of as many variola isolates as possible. It is
expected that use of the live virus would not be necessary for this purpose,
assuming that sufficient DNA is still available in stored specimens in the
U.S. and Russian stocks.
Finally, it is not yet clear whether the FDA will require the use of live
variola virus in the evaluation of new diagnostic methods.
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0 LIVE VARIOLA VIRUS
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