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THE FUTURE
OF SARS
Part 1: The origins
By John Parker
The world was stunned recently by news out of Singapore that severe acute
respiratory syndrome (SARS), the disease that swept around the globe early this
year, had apparently resurfaced. On July 5, Gro Harlem Brundtland, the outgoing
director general of the World Health Organization (WHO), had announced to
reporters in Zurich that the SARS epidemic had been contained.
Brundtland's announcement was a milestone in the history of an epidemic that
had killed hundreds, sickened many times that number, caused countless
thousands to be quarantined, and brought normal life almost to a halt across a
vast region of the globe since it began to spread in late February. The new
Singapore case, although not confirmed as SARS at this writing, has brought
back bad memories.
 In
the Greater China region, which was most directly affected, SARS presented
every government with an unprecedented public-health challenge; the political
effects of the SARS crisis will reverberate for years. SARS turned Chinese
cities into eerily quiet, no-touch ghost towns and took a dreadful economic
toll in the process, bringing proud firms such as Cathay Pacific Airlines to
their knees, and striking another body blow to an Asian tourist industry
already reeling from post-September 11 syndrome and Middle Eastern turmoil.
Now that the "containment" of SARS has been brought into doubt, it is an
appropriate time to try to answer fundamental questions about the epidemic and
its future. What is the cause of SARS? Where did it come from? Will the disease
return in earnest, perhaps seasonally like influenza? If it does return, will
it be more or less severe in terms of symptoms and mortality rates? Which
public-health measures are most likely to prevent a recurrence? Which countries
are most vulnerable and why? And what are the prospects for drugs and/or
vaccines directed against SARS?
Fortunately, all these questions, and others, can be answered far more fully
today than in the frightening and uncertain days of March, when the disease
first appeared and almost nothing was known about it. Although the SARS
epidemic was a frightening harbinger of how easily a lethal disease can appear
and spread in the modern world, it was also a reassuring example of how quickly
biomedical researchers can organize to deal with an unknown pathogen, using
tools that are geometrically faster and more powerful than those that were
available even 10 years ago.
What is the cause of SARS?
This question can now be unequivocally answered. The etiologic (causative)
agent of SARS is a new type of coronavirus, now officially known as SARS-CoV.
Because of the lingering controversy over etiology that has dogged other
diseases, such as AIDS, it is worth going over the accumulated evidence that
SARS-CoV indeed causes SARS.
Scientists use Koch's postulates to judge whether a pathogen, or dangerous
micro-organism, can be considered the cause of a disease (Robert Koch was the
German bacteriologist who first isolated the tuberculosis bacterium in 1882).
In 1937, T M Rivers published a modified version of Koch's postulates that he
considered to be more appropriate for viral diseases, and it is the Rivers
criteria, listed below, that scientists used this year to assess whether
SARS-CoV is the cause of SARS.
1) It must be possible to isolate the virus from diseased hosts and not from
healthy individuals.
2) It must be possible to culture the virus (ie, grow it) in appropriate host
cells.
3) It must be shown that the virus can be removed from an infectious solution
with a sufficiently fine filter.
4) It must be possible to produce comparable disease in the original host
species or a closely related one.
5) Having met the fourth criterion, it must be possible to re-isolate the virus
from the new host.
6) A specific immune response to the virus must be detected, as shown by the
production of antibodies specific to that virus in infected individuals.
In the first days of the epidemic, germ-containing samples - eg, sputum, blood
serum, stool, and oropharyngeal washes - were collected from SARS patients in
Vietnam, Hong Kong and elsewhere. These samples were provided to various
laboratories around the world, which then used them to try to identify the
infectious agent.
These investigations produced a fair number of false leads. For example, one
major group of scientists, consisting of researchers in Germany, France and the
Netherlands, worked initially with samples from three patients. The "index", or
first, patient examined was a 32-year-old male physician who had become
infected with SARS while treating a Hong Kong man in Singapore. The physician's
symptoms began suddenly while he was visiting New York on March 9; he attempted
to return to Singapore, but the illness took hold so quickly that he was
hospitalized during his Frankfurt stopover. His sputum and throat-swab samples
were examined under the electron microscope and showed what seemed to be
paramyxovirus particles.
This result probably led to suspicions focusing on a paramyxovirus early on,
and also to premature media reports in March speculating that a paramyxovirus
was the cause of SARS. But when highly sensitive PCR (polymerase chain
reaction) tests for various known members of the paramyxovirus family came back
negative, and other investigators failed to find any paramyxoviruses, it
gradually became clear that a paramyxovirus was not the culprit; most likely,
the index patient was coincidentally infected with a paramyxovirus at the same
time he acquired SARS (coincident infections with different viruses are not
uncommon, especially during the cold and flu season). The European group also
found Chlamydia pneumoniae - the bacterium that causes pneumonia - in
their index patient, but only after he had been sick for 11 days. It eventually
became clear that this was a secondary infection, not the primary cause of his
illness.
Another group of investigators consisted of scientists at the Centers for
Disease Control (CDC) in Atlanta, collaborating with scientists from Hong Kong,
Singapore, Thailand and Taiwan, as well as Dr Carlo Urbani of the WHO, who was
himself tragically struck down by SARS. The Atlanta group attempted to grow the
infectious agent in cell culture (viruses cannot reproduce without host cells,
so bottles of cells are required to grow viruses in the laboratory). These
attempts initially produced another blind alley: a rhinovirus was isolated from
cells that had been inoculated with oropharyngeal wash taken from "Patient 16",
a 46-year-old male Vietnamese physician. But further investigations showed that
this rhinovirus, too, was not present in other patients, and thus could not be
the cause of SARS.
Both the European scientists and the Atlanta group would finally succeed with
the same approach: Vero cells, a cell line originally derived from monkeys,
proved to be a suitable host for SARS-CoV. Cultured cells are sickened by viral
infection: instead of lying flat on the floor of their dish, and growing in a
steady, ordered manner, they can "round up", detach from the dish, and
eventually exude virus particles; sometimes they even explode from the force of
the virus particles being built up within them. When the Atlanta group found
sick Vero cultures that had been infected with samples from five different SARS
patients - three from Hong Kong and two from Vietnam - and the European group,
using virtually identical procedures, observed a sick Vero culture, inoculated
from their index patient, at almost exactly the same time, attention quickly
turned to isolating and characterizing whatever virus was present in these
cultures.
Two main methods were used to do this: electron microscopy and RT-PCR. The
electron microscope, developed in the 1950s and '60s, uses an electron beam to
obtain images of objects much smaller than a light microscope can resolve
(viruses are generally much too small to be seen in a light microscope). When
the researchers turned their electron microscopes on the sick Vero cultures,
they saw the unmistakable crown-shaped forms of a coronavirus.
The next step was RT-PCR (reverse-transcriptase polymerase chain reaction), to
confirm directly the presence of coronavirus RNA (ribonucleic acid). RT-PCR is
a variation of the PCR technology for amplifying small amounts of DNA
(deoxyribonucleic acid), which gained such notoriety during the O J Simpson
trial in the United States. Basically, RT-PCR amplifies RNA, by converting it
to DNA and then amplifying the DNA. Because of the extreme, almost unbelievable
sensitivity of PCR - it can show the presence of as little as one copy of the
molecule it is amplifying - it is ideal for detecting the presence of small
amounts of an infectious agent in a patient sample; and the RT-PCR variant is
necessary for detecting RNA viruses such as coronaviruses and retroviruses.
Here, the European and Atlanta groups used slightly different techniques. But
both groups found overwhelming evidence that a coronavirus was, indeed,
present. And when the Europeans checked the sequence they had isolated against
that obtained by the CDC, they found that the two sequences were 100 percent
identical, showing that both groups had independently isolated the same virus.
Once the virus was found, grown, and known to be a coronavirus, it was a simple
matter, with present-day DNA-sequencing technology, to find its genetic
sequence. Doing so provided further proof that the new virus was, indeed, a
coronavirus, but also showed that it was a previously unknown type, with a
genetic sequence considerably different from previously studied coronaviruses.
Coronaviruses are not rare; about 30 percent of common colds are caused by
them, which means that most people reading this article have probably been
infected by coronaviruses several times. The family also causes several
well-known veterinary diseases, such as infectious peritonitis in cats. But
sequencing showed that the particular coronavirus making SARS patients sick had
never been detected in humans, or anywhere else, before.
The availability of a sequence for the virus made it possible to develop
patient test kits that used the PCR technology. This was done almost
immediately, and by April, these tests were being used in affected areas. The
results left little room for doubt. Scientists in Hong Kong found the new
coronavirus in 45 of 50 SARS patients tested, whereas all their healthy
controls tested negative for the virus. The European group, the CDC, another
group in Canada, and later a group in Shenzhen, China, confirmed the presence
of the new virus in numerous patient samples.
Once it was shown that filtration did, indeed, render virus-containing liquids
non-infectious, the first three of Koch's postulates had been met. In mid-May,
a Dutch group, assisted by two Hong Kong researchers, fulfilled the fourth
postulate by successfully infecting macaque monkeys with SARS-CoV. The Dutch
group easily re-isolated the virus from the sick monkeys, using both microscopy
and RT-PCR, which met the fifth postulate. All that remained was to meet the
sixth postulate, by showing a specific immune response to the virus. The Dutch
showed that antibodies from the macaques reacted with viruses from the cell
cultures, a finding which confirmed numerous similar results obtained by other
labs using serum samples from human SARS patients. By the time the Dutch
group's paper appeared in the journal Nature, on May 15, the WHO had already
been convinced: the public announcement that SARS-CoV was the cause of SARS
came on April 16.
Where did SARS come from?
From the very earliest days of the SARS epidemic, scientists believed that SARS
would prove to be a zoonotic infection (ie, a disease transmitted from animals
to humans). This was a safe hypothesis, because most new diseases that have
appeared in recent years - for example, avian flu, Nipah virus, West Nile
virus, Ebola virus, Lyme disease, and AIDS - have been zoonotic in origin. In
addition, the geographical origin of the epidemic - southern China - argued in
favor of a zoonotic infection; as noted ad nauseam in the global press, the
high population density and close proximity of humans and animals in southern
China has historically made the area an incubator for new, recombinant
pathogens that can cause worldwide pandemics.
If SARS had an animal origin, the obvious question then became, which animal,
or animals, did it come from? Because the known coronaviruses mostly infected
ordinary domestic animals such as the pig, rat, cow, chicken and dog,
researchers immediately tried to infect these animals with the new virus. This
proved to be difficult; in one such attempt, Canadian scientists at the
National Microbiological Laboratory in Winnipeg, Manitoba, tried and failed to
infect chickens and pigs. Researchers eventually noticed that domestic cats
could become infected with SARS-CoV; for example, numerous pet cats in Amoy
Gardens, the notorious Hong Kong apartment complex that generated more than 100
SARS cases, were found to harbor the virus. But since it seemed extremely
unlikely that a totally new virus, which did not resemble known feline
coronaviruses, could have suddenly emerged from domestic cats, scientists
realized that it was necessary to cast a wider net and screen wild animals as
well as domestic species.
A key breakthrough came in mid-May, when a team from the University of Hong
Kong's Queen Mary Hospital, assisted by the Shenzhen Center for Disease Control
and Prevention, discovered the presence of a virus nearly identical to SARS-CoV
in animals purchased at a wildlife market in Shenzhen, China. Although the team
tested eight different species and detected signs of infection in three of
these, the most likely candidate for the animal reservoir of SARS, based on
this group's data alone, was an obscure raccoon-like animal known as the masked
palm civet, or civet cat (Paguma larvata).
The civet cat, in spite of its name, is not a true feline; rather, it is a
member of the family Vivveridae, a close relative of the mongoose. Civets are
small, tree-dwelling fructivores, native to parts of southern China and
Southeast Asia. Ironically, before SARS came along, the civet family was
perhaps best known for the role of the closely related common palm civet (Paradoxurus
hermaphroditus) in producing the so called "cat dung" coffee made in
Indonesia and Vietnam. Apparently, these civets like to eat coffee berries, and
when they do, the coffee bean itself, which is indigestible, passes through and
is released in their dung. The dung is then collected, cleaned and dried. The
coffee made with this process is considered to be extremely high quality,
because the civets are more selective than any human picker, and choose only
the ripest berries to eat! (There is absolutely no evidence, to my knowledge,
that drinking this coffee presents any risk of acquiring SARS.)
In any event, the masked palm civet is considered tasty by Chinese diners;
civet meat is one of the main ingredients in the ye wei ("wild taste")
dish called dragon-tiger-phoenix soup. As a result, a market in the animals has
developed. Although most civets sold for food are now captive-bred, others have
been trapped in southern China and northern Vietnam, depleting the wild
population. Eating civet meat is almost certainly not a risky activity in
itself, since the virus is not normally present in muscle tissue, and even if
it was, the viral particles would be killed by the cooking process. Rather,
danger would arise from breathing the same air as infected civets, inhaling
infected fecal particles when cleaning their cages (since the virus is known to
exist in feces), and possibly from blood during the butchering process.
The UHK/SZCDC group was able to directly isolate a SARS-like coronavirus from
the civets. When scientists sequenced the genome of this virus, they discovered
that it was 99.8 percent identical to the SARS-CoV found in human patients.
Furthermore, researchers tested human workers in the market for antibodies to
SARS and found that not only did many of them have antibodies to SARS-like
coronaviruses, showing that they had been infected some time in the past, but
the likelihood of prior infection was correlated with the degree of contact
with wild animals: 40 percent of wild-animal traders and 20 percent of those
who slaughtered the animals had antibodies, but only one of 20 vegetable
sellers that were tested (5 percent) showed evidence of infection. These two
findings, coupled with the fact that the epidemic originated in the same part
of southern China where the testing was done, constituted very strong
circumstantial evidence that the SARS epidemic originated in an animal-to-human
transmission, probably from one of the three species tested.
Although it is theoretically possible that the Shenzhen animals could have been
infected by humans carrying SARS-CoV, which would imply that the true animal
reservoir has not yet been discovered, several observations made by the UHK
group showed this to be unlikely. First, the animal virus contained 29 extra
bases (letters) in its RNA genome compared with the SARS-CoV isolated from
human patients. This suggested that the virus had gone from one of these
species to humans, and not vice versa, since viruses often lose genetic
information when they jump to a new species, but rarely gain new information.
Second, the animal viruses, although they were obtained from only one location,
were even more genetically diverse than the SARS-CoV strains taken from
patients all over the globe. Because lineages of viruses accumulate genetic
differences over time, one expects to find more diverse viral strains in a
virus's natural reservoir than in a species that has only recently been
infected. Thus, the greater genetic diversity of SARS-like coronaviruses in
animals suggests that the virus has been in animals for longer than it has been
in humans, and this also argues for animal-to-human transmission rather than
the reverse.
In spite of the indisputable evidence that the civet can carry SARS-CoV, it is
still possible that the 2003 epidemic originated in some way other than a
civet-to-human transmission, for several reasons. First, the UHK group also
found the virus in two other animals, namely, the raccoon dog (Nyctereutes
procyonoides) and a Chinese ferret badger (Melogale moschata). SARS-CoV
was directly isolated from the raccoon dog, as it had been from the civets.
Although researchers failed to find the virus itself in the ferret badger, they
did find antibodies to the virus, suggesting that the badger had been infected
with a SARS-like coronavirus some time in the past. Thus, either one of these
species conceivably could have been the source that actually started the
epidemic, although the civet seemed more likely, since 100 percent (six of six)
of the civets that the group purchased in Shenzhen showed evidence of
infection. Second, it is possible (though perhaps unlikely) that all the
virus-bearing animals examined were in fact infected by another animal during
transport, and the ultimate animal reservoir of the virus has not yet been
found.
Also, some contrary evidence to the "civet hypothesis" has emerged since the
UHK group made its preliminary report in May. In mid-June, researchers from
China Agricultural University reported the results from their own study, which
was much larger in scope than the UHK study, examining a total of 732 animals
from 65 different species. Although this group used the same RT-PCR technique
to find SARS-CoV as other labs, remarkably, they failed to find SARS-CoV in a
single animal. They did, however, find three new coronaviruses in various
species, including one in the palm civets that they collected from the same
Shenzhen market that the UHK group had used. This virus was not SARS-CoV: it
was yet another strain, with 23 percent of the letters in its genetic sequence
proving to be different from SARS-CoV.
The UHK group's virus, by contrast, was almost identical to SARS-CoV, with the
29-base deletion in the human virus being the most important difference. These
results are not necessarily contradictory; it is perfectly possible that the
two groups' civets came from different vendors and/or different geographic
sources, meaning that they could quite reasonably have been infected with
different coronaviral diseases. In addition, the UHK group's finding that 100
percent of the civets it examined were carrying a SARS-like virus seemed very
compelling evidence that the civet was the natural source of SARS, but there
are alternative explanations that exonerate the civet. For example, the six
civets could have been infected by a different species on the way to market,
then quickly infected each other, since they could have been kept in the same
cage. (Coronaviral diseases are respiratory diseases, and confined animals
would be in an ideal circumstance to quickly infect each other, once one of
them had acquired a respiratory virus.)
One of the first known Chinese SARS patients was a 34-year-old Shenzhen cook
named Huang Xingchu. Tellingly, Huang's restaurant served wild animal dishes.
Was Huang the "Patient Zero" of the SARS epidemic? In fact, there are many
candidates for that dubious honor: another one of the earliest cases was a bird
and snake merchant in the city of Shunde, and it is actually possible that more
than one individual may have acted as a "Patient Zero". In late May,
Singaporean scientists published a report showing that two significantly
different subtypes of SARS-CoV were involved in the epidemic: one type caused
all the cases traceable to the Metropole Hotel, where Hong Kong's first cases
were infected in mid-February. The other type was responsible for all other
known cases. This finding raises the possibility that "the SARS epidemic" may
in fact have been two different epidemics, caused by genetically distinct
coronavirus strains, initiated at around the same time. And each "epidemic"
could have been touched off by a different infected person.
Once the presence of SARS-CoV in civets became publicly known, the Chinese
government reacted speedily. Guangdong authorities vowed to stop the civet
trade and plastered pictures of the animal all over Guangdong newspapers in
late May. It was not entirely clear, however, whether all trade in civets had
been banned, or only trade in wild civets. In Beijing, the Beijing Zoo
authorities removed their nine civets from public display and placed them in
isolation. Banning was not an issue in Hong Kong, where the sale or consumption
of civets has been prohibited for some time.
As of mid-September, the case against the masked palm civet remains
circumstantial; although the "smart money" is on the civet, other possibilities
exist, and much more research is needed. Still, if the hypothesis proves to be
correct, the implications are sobering: the SARS epidemic could have been
prevented quite easily if the measures to curtail the civet trade that were
carried out in May and June had been carried out in February instead. The civet
hypothesis also has great implications for the future control of SARS. If
civets are indeed the ultimate source, simply eliminating human contact with
the animals could avert future outbreaks of SARS at an incomparably lesser cost
than the burdensome measures of quarantining, disinfection, and masking that
have been used to date.
Next: Will SARS return?
John Parker is a freelance writer based in Vietnam. He has a Master
of Science degree in cell biology.
(Copyright 2003 Asia Times Online Co, Ltd. All rights reserved. Please contact
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