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Highlights
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The upper respiratory tract is the most frequent
site of smallpox infection. |
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It is likely that a low dose, perhaps as little
as one virion, is needed to cause infection. |
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More work is needed to clarify smallpox pathogenesis,
including how the virus is transmitted from person to person and spreads
from the upper respiratory tract to the rest of the body. |
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A number of vaccines and antivirals are in development
to combat smallpox. |
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The virus’ potential use as bioweapon has
revitalized interest in smallpox research. |
In a comprehensive and insightful presentation on smallpox
research, R. Mark Buller of the Saint Louis University School of Medicine
in St. Louis provided a succinct look at our current knowledge of how
smallpox is spread, the course of the disease, and vaccines and treatments
in development.
Smallpox is caused by variola virus, which is a poxvirus that is in
many ways similar to poxviruses that infect animals. Therefore, most of
what is known about smallpox comes from various animal models. These include
ectromelia virus (mousepox), cowpox virus, vaccinia virus, rabbitpox virus,
and monkeypox virus. Studies of these poxviruses focus both on poxvirus
biology and on testing of vaccines and antivirals to treat potential smallpox
outbreaks.
“Currently, there are only two official repositories for the smallpox
virus, at the Centers for Disease Control and Prevention (CDC) in Atlanta,
Georgia, USA, and the VECTOR Institute in Kotsovo of the former Soviet
Union,” Buller noted. Although work on variola virus at these sites
has been restricted, two recent, albeit, preliminary, decisions by the
World Health Organization (WHO) could open up new avenues of research,
he said. One would allow the introduction into variola virus of a foreign
gene—a reporter gene—to aid in the development of
antivirals; the other would allow the recombination of variola virus genes
into other orthopoxvirus species to evaluate their function. If approved,
such work would help research “tremendously,” he observed.
In the meantime, he focused in his talk on what is currently known about
the virus.
 How smallpox spreads
Smallpox is less contagious than such viral illnesses as influenza or measles,
but it is still considered to be highly contagious. Person-to-person transmission
most often occurs through exposure to large droplets of aerosol from an infected
individual. The smallpox virus can also be spread through contaminated bedding
and clothing. According to Buller, the upper respiratory tract is likely to
be the most frequent site of infection.
Five factors contribute to transmission, Buller observed. Most important is
the amount of virus produced in the upper respiratory tract of infected individuals.
Other factors include whether or not the infected person is coughing and sneezing;
the proximity of susceptible people to the infected person during the transmission
period (two to five days after a rash appears see Henderson); how long
the virion remains stable
in the environment (the smallpox virus tends to be very stable regardless of
temperature and humidity); and the form of the virus (virus particles in scabs
and skin debris are less infectious than those in the air).
Smallpox is less contagious than influenza or measles, but is still highly
contagious.
How much virus is needed to cause disease? Very little, Buller
said. Studies in animal models suggest that perhaps as little as a single
virion is sufficient. Two epidemiological studies seem to support this conclusion:
one concerning the 1970 outbreak in Meschede Hospital, which involved the
importation
of smallpox from an endemic country into smallpox-free Germany (see Poland and Henderson); and another a 1971 outbreak in the Aral’sk region of Kazakhstan.
In 1971, in Aral'sk, a Russian biologist working on a trawler called the Lev Berg in the Aral Sea contracted smallpox. At the time, she was in an area that was not friendly to the Soviets, and was not allowed to leave the ship. Thus, said Buller, she must have been infected on board the ship—probably during periods on deck, where she spent considerable amounts of time. No other shipmates were infected. The source of the virus was likely the island of Vozrozhdeniye, where the Soviets maintained a biological weapons field testing facility that was 15 kilometers north of the Lev Berg. Therefore, the biologist's infection was deemed to be an example of long-distance transmission of smallpox, from the island to the trawler.
"Both epidemiological studies suggest that the infectious dose of variola is probably very, very low," said Buller, and if it were given in the form of an aerosol that was intentionally released, the efficacy of the aerosol could be "very, very high in the presence of a non-vaccinated population."
Under the microscope
As noted earlier, smallpox is thought to be transmitted primarily by large
droplets of aerosol, and most frequently to the upper respiratory tract.
After reviewing the complex processes involved in viral replication, Buller
went
on to give his vision of how human smallpox infection occurs, noting that
there is still much work to be done in elucidating both the cellular pathogenesis
of smallpox and host cell-mediated immunity to smallpox infection.
Virus infection of the upper respiratory epithelium is mediated by extracellular
enveloped virus (EEV) and/or intracellular mature virus (IMV), he explained.
The EEV is an IMV virion wrapped in a second membrane [SLIDES 7-9 provide a
high-resolution electron micrograph of the binding of EEV to the cell], which
disrupts the outer membrane of the EEV; this allows the attachment of the IMV
particle and the fusion of its membrane with the cellular plasma membrane,
which releases the core into the cytoplasm. The transcription machinery in the
virion
is then activated, and the virus replication cycle commences.
The life cycle of the virus occurs in entirely in cytoplasm, although it requires
certain nuclear functions. During replication, the virus produces an array of
molecules that modulate the internal and external host responses to infection,
contributing to virus persistence at the site of infection and spread in the
animal host. The virus has a very complex morphogenesis pathway [SLIDE 11 partially
describes the morphogenesis pathway of smallpox virus].
Using the example of an ectromelia virus infection in the epidermis, Buller
explained that infection induces a host response that leads to the production
of a wide spectrum of cytokines, chemokines, and interferon. These proinflammatory
substances activate the proximal epithelial cells as well as macrophages and
NK cells in the dermis.
Next, the cytokines and chemokines form a gradient and upregulate
the adhesion molecules ICAM-1, E-selectin, and VCAM, which normally would facilitate
adhesion and movement of inflammatory cells into the tissue surrounding the
site of infection; however, virus-encoded binding proteins for key cytokines
and chemokines are thought to prevent this process.
In addition to encoding homologs of host cytokines and chemokines receptors,
the virus also encodes other host-response modifiers that contribute to the
inflammatory process: serpins (protease inhibitors), which can affect a wide
range of biological processes; an initiation factor 2α homolog, which is thought
to be important in allowing the virus to escape the antiviral effect of interferon;
the proinflammatory cytokine interleukin-1β-like antagonist; the inhibitor of
complement enzyme, which may be an important virulence factor in variola; inhibitors
of apoptosis to damp down apoptosis signals; CD47-like homolog, whose function
is not yet known; inhibitors of the antiviral enzymes PKR and RNase L; and superoxide
dismutase-like protein.
Host-response modifiers coded by the virus contribute to blocking the inflammatory
process.
But although the factors that allow viral replication have been identified,
how the virus spreads from the site of primary infection in the upper respiratory
tract to the rest of the body is not clear, Buller emphasized. “The conventional
wisdom is that the virus spreads through the body as cell-associated viremia,
but there’s never been a very thorough study to support this. The pathogenesis
scheme for variola is based on that of ectromelia [mouse poxvirus].
However, the two viruses are very different, and so this scheme really represents
only our best guess.”
Combating
smallpox
Although the correlates for immunity in smallpox are not yet known, it is
likely that protective approaches will need to focus on both cell-mediated and
humoral (antibody-mediated) responses, Buller noted. Among traditional vaccines,
Dryvax (dried, calf lymph type) is the “gold standard.” Also in
development are derivatives of Dryvax, which include Acambis 2000, a Dryvax
clone; MVA, which comes from a different strain of vaccinia virus and has not
yet been tested against smallpox; and LC16m8, a live virus derived from the
Lister vaccine strain that also has not been tested against smallpox.
Novel vaccine candidates include a conditionally lethal vaccinia virus with
an inactivated E3L gene, which has been shown to be effective in a mouse model;
a live recombinant Streptococcus gordonii vaccine that encodes certain protective
antigens; and DNA and subunit vaccines that use antigens from the EEV or IMV
forms.
Among the candidate antivirals, only cidofovir, which is licensed for treating
human cytomegalovirus infections, has been tested for safety in humans and shown
in vitro and in animal models to be active against variola virus. An orally
available derivative of cidofovir, hexadecyloxypropyl-cidofovir (HDP-cidofovir),
has not yet been safety-tested in humans, but is active against variola virus
in vitro.
A third compound, ST-246, works by a different mechanism. It seems to block
EEV formation, thereby blocking viral spread. It, too, has yet to be safety-tested
in humans, although it has been shown in vitro to be active against variola
virus. A fourth compound, TTP-6171, targets a protease, 17L, that is encoded
by the vaccinia virus and conserved in the variola virus, so it has a high degree
of specificity. “It hasn’t been tested for safety or for efficacy
against variola virus, but it’s a very promising idea,” Buller concluded. |
