(China)The Future of SARS

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http://www.atimes.com/atimes/China/EI11Ad04.html

 

Sep 11, 2003

China

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 led by

Dr Yuen Kwok-yung, a University of Hong Kong

microbiologist, discovered the presence of a virus

nearly identical to SARS-CoV in six animals purchased

at a wildlife market in Shenzhen, China. The guilty

species could hardly have been more obscure: it was

the masked palm civet, or civet cat, known

taxonomically as 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. This species is a small, raccoon-like,

tree-dwelling fructivore, 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.)

 

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.

 

When the coronavirus found in the civets was compared

to SARS-CoV, scientists discovered that the wild virus

was almost identical, except that it had an extra 29

bases (letters) in its RNA genome. This was an

important clue to the origins of SARS: the wild virus

would have been adapted to its animal hosts, and

probably infected humans only with difficulty. But the

29 lost bases could have created a genetically

different virus with a greater infectivity and

lethality to humans: SARS-CoV. This hypothesis has not

been proved, however; to show that the natural civet

virus, with the extra 29 bases, is less dangerous,

researchers would need to infect humans with it and

show that this results in less severe illness than

SARS. Obviously, ethical considerations would prevent

this experiment (unless some extremely brave

volunteers can be found).

 

However, Yuen's scientists, and others, discovered

that many of the wild-animal sellers at markets in

Guangdong were already carrying antibodies against a

SARS-like coronavirus. It isn't possible to tell from

antibody tests whether the virus that infected these

people was SARS-CoV or its wild relative, but it was

probably the latter. Therefore, this unethical

" experiment " may already have been inadvertently

performed by the animal sellers themselves, when they

handled civets, or whatever animal was the ultimate

source. In any case, when a virus jumps between

species, it often loses bases, but it rarely gains

them. Thus, the deletion finding implies that the SARS

virus originated in animals and migrated to humans,

not the reverse.

 

Although there is now indisputable evidence that the

civet can carry SARS-CoV, that does not necessarily

mean that the 2003 epidemic originated in a

civet-to-human transmission, for several reasons.

First, Yuen's 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

SARS-CoV at 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 Yuen's group purchased in Shenzhen

were infected. Second, it is possible that all the

infected animals examined by Yuen's team were in fact

infected by another animal during transport, or even a

human SARS carrier, and the true animal source of the

virus has not yet been discovered.

 

Third, some contrary evidence to the " civet

hypothesis " has emerged since Yuen's group made its

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

Yuen's, 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 Yuen 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 Yuen group's virus, by

contrast, was almost identical to SARS-CoV, with the

29-base deletion being the only 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, when Yuen found that 100 percent of the

civets he examined were carrying a SARS-like virus, it

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.)

 

Still, one of the first known Chinese SARS patients

was a 34-year-old Shenzhen cook named Huang Xingchu.

Since Huang's restaurant served wild-animal dishes, it

is tempting to speculate that he may have been the

" Patient Zero " of the SARS epidemic, much as the

French-Canadian flight attendant Gaetan Dugas was for

the AIDS epidemic. Huang, however, is not the only

candidate for that dubious honor: another one of the

earliest cases was a bird and snake merchant in the

city of Shunde.

 

In fact, 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.

 

When discussing the civet connection, it is important

to note that 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.

 

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.

 

Although research published in recent days have

strengthened the case against the masked palm civet,

it remains circumstantial; ongoing research will

eventually shed more light on the question. 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.

 

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