Influenza: Five Questions on H5N1

by Ed Yong
Nature 486, 456-458 (28 June 2012) doi:10.1038/486456a
June 21, 2012
www.nature.com/news/influenza-five-questions-on-h5n1-1.10874

     Scientists now know that the deadly bird flu virus
     is capable of causing a human pandemic. That makes
     tackling the remaining unknowns all the more
     urgent.

The biology of the H5N1 avian influenza virus is rife
with paradoxes. The virus is widespread, but hard to
detect. It kills more than half of the people known to
be infected, but thousands of those exposed have no
apparent problems. It seems to be just a few mutations
away from gaining the ability to spread from person to
person, but despite more than 16 years of fast-paced
evolution, it has failed to do so.

This week saw the publication of the second of two
papers identifying mutations that give H5N1 the ability
to spread through the air between ferrets. The papers,
the latest [1] from a group led by Ron Fouchier at the
Erasmus Medical Center in Rotterdam, the Netherlands,
and the earlier one [2] by Yoshihiro Kawaoka at the
University of WisconsinMadison and his colleagues, have
been controversial because they offer what some see as a
recipe for disaster -- that they increase the risk of
accidental or intentional release of a deadly human
pathogen. But what is most unsettling about them, say
many in the flu community, is the evidence they provide
that the wild virus could spark a pandemic on its own.
That threat makes the outstanding scientific mysteries
about this tiny RNA virus -- its genome just 14,000
letters long -- even more pressing. Here are five of the
biggest puzzles, and what researchers are doing to
solve them.

QUESTION 1 Why Is It So Successful?

H5N1 influenza gets its name from the combination of two
proteins on its surface: haemagglutinin (HA) and
neuranimidase. But there are many different strains of
H5N1. The highly pathogenic strain that has grabbed
headlines for more than a decade was first identified in
1996. Called Gs/Gd because it was found in domestic
geese in China's Guangdong province, it is "totally
different to any avian influenza virus in the past",
says Robert Webster, a virologist at St Jude Children's
Research Hospital in Memphis, Tennessee. Most avian
influenza viruses ride harmlessly aboard wild fowl,
occasionally flaring into lethal but short-lived
outbreaks in domestic birds. The Gs/Gd lineage, however,
has jumped back from poultry into wild fowl. It also
infects mammals, including humans, tigers, pikas, civets
and more. It has spread to 63 countries, and is endemic
in bird populations in six of them.

"What is so special about this virus that allows it to
spread through the animal world so effectively?" asks
Jeremy Farrar, a tropical-medicine specialist at the
University of Oxford, UK. There are no firm answers.

China's crowded farms and markets, which offer a
smorgasbord of potential hosts, might have selected for
viruses that are adept at crossing the species barrier.
The virus evolved quickly in 1999 and 2000, its family
tree sprouting many new branches after efforts to stamp
it out among domestic birds failed. During this time,
one clade of the Gs/Gd lineage, known as 2.2, picked up
a mutation in PB2, one of three polymerase genes that
allow the virus to copy its genome.

The mutation is widely considered to be an adaptation to
mammalian hosts.

In 2002, for reasons that are still unclear, the viruses
started hopping back into wild birds and killing them.
At first, there were just a few isolated deaths, but in
May 2005, clade 2.2 viruses killed more than 6,000
geese, gulls and ducks at Qinghai Lake -- China's
largest lake, and a major breeding spot for migratory
birds. This outbreak heralded the start of a global tour
in which the virus spread through bird trade and wild
migrations to the rest of Asia, Europe and Africa.
Vaccination controlled the virus in Hong Kong and
Vietnam, but where applied haphazardly, it has helped to
speed up the virus's evolution. In Egypt, it led to the
birth of several new sub-clades [3] , and the country
has had more new human cases than any other nation every
year since 2009.

There is some good news: infections in wild birds have
fallen sharply since 2006. But even as old lineages
wane, new ones arise, such as clade 2.3.2.1, which has
swept through poultry in Asia since early 2011. "That's
the one that is of great concern to me," says Webster.
"It seems to be becoming dominant and it goes into wild
birds readily." H5N1 may be evolving faster than our
ability to understand it."

QUESTION 2 Where Is It Now?

H5N1 seems to be both everywhere and nowhere at the same
time, making it hard to predict when, where and whether
it will bloom into a human pandemic.

Wild birds carry H5N1, but the virus can be hard to
detect because very few become ill. No one knows how
widespread it is in humans, either. As of 7 June, the
World Health Organization had counted 606 H5N1
infections in humans, 357 of them fatal. Many think that
the real number is much larger, which would mean that
the death rate would be much lower than 60%. Peter
Palese, a virologist at Mount Sinai School of Medicine
in New York, looked at a number of studies that had
found evidence of H5N1 infection in the blood of healthy
people. He estimates that 1-2% of people in populations
exposed to the virus become infected, but most have only
mild or no symptoms [4].

Palese's arguments are controversial. Farrar, who has
treated patients in Asia, says that most of the cases he
has seen have been severe. "If many mild infections were
occurring, we'd expect to see some less severe patients
in hospital, given the heightened awareness in the
public and the medical profession," he says.

Underlying the debate about the infection rate is a poor
understanding of how the immune system responds to the
virus. People who become infected produce antibodies and
T cells that recognize the virus, but no one knows how
these responses rise and fall over time, or how they
manifest in people who show no symptoms. The signals
could also be false alarms. "If you go into henhouses
every day to clean up bird droppings that are loaded
with virus antigen, you may get an antibody response
without being infected," says Fouchier. More thorough
surveillance of suspected cases will be needed to
resolve the debate about how often people are infected,
says Fouchier. Farrar adds that to understand how immune
responses change over time, such studies will have to be
done over several years. Many researchers have called
for better surveillance of domesticated and wild
animals, too.

To Ilaria Capua, a veterinary virologist at the
Experimental Animal Health Care Institute of Venice in
Legnaro, Italy, the distribution of H5N1 is the most
important issue, and the hardest to work out. "Any
prediction about whether this virus will go pandemic is
a function of where it is, how much of it there is and
how possible the human-animal contacts are. But there
are big black holes of information."

QUESTION 3 How Does It Kill?

Bit by bit, scientists are teasing out the genetic
factors that make H5N1 so deadly. The virus has several
mutations in its three polymerase genes that allow it to
replicate aggressively, and patients who die carry the
highest levels of viral RNA. Certain changes to HA,
which codes for a protein that latches onto host cells,
also allow the virus to infect tissues beyond the lungs
and gut, including the brain. This all-access pass helps
the virus to kill ferrets, mice and birds, but it is
apparently less important in primates. "In humans, it
still looks predominantly like a respiratory disease
kills the patients," says Malik Peiris, a clinical
virologist from the University of Hong Kong. Autopsies
might paint a clearer picture, but Peiris says that
these are rarely allowed in Asia because of cultural
demands for wholebody burials.

H5N1 also drives the immune system berserk. Immune cells
flock to sites of infection and produce inflammatory
chemicals called cytokines, which attract more immune
cells. The result, a cytokine 'storm' that floods the
lungs with fluid and fatally damages surrounding
tissues, is often what kills people. H5N1 triggers a
more extensive storm than the human flu viruses H1N1 or
H3N2 [5].

These factors may explain the severity of recorded
cases, but not why infections are so rare. "Why is it
that there are tens of thousands of kids running around
playing with sick chickens, but we've only had 600
infections over nine years?" asks immunologist Anthony
Fauci, who heads the National Institute of Allergy and
Infectious Diseases in Bethesda, Maryland. Palese
suspects that the severe cases have simply inhaled high
doses of the virus. But Peiris says that this cannot be
the sole explanation. "Infection and disease are not
directly proportional to exposure. Ninety-nine point
nine per cent of the people who are massively exposed
don't have disease, and don't have antibodies in their
blood. But in people who get sick, the virus replicates
like crazy."

Notably, cases are often clustered within families,
specifically blood relatives. These people might be
genetically susceptible to H5N1 infection, or others may
have genetic variants that protect them. Identifying
such variants will be hard because fewer than 300 people
around the world have survived the infection, but
studies are starting to reveal clues.

A few months ago, a group from the Wellcome Trust Sanger
Institute in Hinxton, UK, found that the gene IFITM3
plays a pivotal part in responses to some flu infections
[6]. One variant of the gene, which encodes a stunted
protein, was overrepresented in people who had been
hospitalized with pandemic and seasonal flu strains, and
even a mild H3N2 virus ran amok in the lungs of mice
that lacked the gene [6]. Farrar has just completed a
similar study, of 67 Asian patients who had been
hospitalized with H5N1. The results, which have been
submitted for publication, identify variants in two
other genes that seem to confer susceptibility to the
virus.

QUESTION 4 Will It Become Transmissible in Humans?

So far, people seem to catch H5N1 only through close
contact with infected birds. To spread from person to
person, the virus would have to become transmissible
through airborne droplets. The two papers just published
[1, 2] have shown that that is possible.

Fouchier's strategy was to tweak HA so that its protein
recognized receptors in the upper airways of mammals
rather than those on the surface of bird cells [1]. He
then allowed the virus to pass between ferrets until it
evolved such that it started spreading through coughs
and sneezes. Kawaoka took a similar approach [2], but he
fused a mutated HA from H5N1 with other genes from a
2009 pandemic H1N1 strain. "In principle, H5N1 can
become airborne," says Fouchier. "The critical question
is whether it will." One of the biggest questions about
H5N1 is why it hasn't become transmissible after
circulating for so many years -- but noone has a good
answer. Many of the mutations that Fouchier and Kawaoka
identified are already found in the wild. By searching
surveillance databases, Derek Smith, a bioinformatician
from the University of Cambridge, UK, found that many
wild clades are already two to four mutations away from
the sets that Fouchier and Kawaoka identified [7].

Smith was unable to determine the actual risk because
surveillance data masks the genetic diversity of the
virus.

H5N1 reproduces with errors, so any one patient carries
a swarm of viruses with subtle genetic differences. The
databases contain just the 'consensus' sequence,
essentially a mash-up of the most common variant at
every position in the genome. Only deeper sequencing, in
which each position is read many times over, will reveal
all the variants.

Even if the same combination of HA mutations that
Fouchier and Kawaoka identified arises naturally, no one
knows whether the resultant viruses would spread between
humans as easily as they do between ferrets in the lab.
It is also not clear how H5N1's other genes contribute
to transmissibility, or whether different combinations
of mutations would achieve the same effect. "These guys
have only scratched the surface," says Webster.

Fouchier and Kawaoka say that the value of their work
lies in identifying the physical traits that make H5N1
transmissible. Some mutations allowed HA to recognize
mammalian receptors, whereas others stabilized the
protein. "If you take those traits, can you then make
any flu virus go airborne?" asks Fouchier. The virulence
of a transmissible strain is another unknown. One
hypothesis suggests that as transmissibility goes up,
virulence will become muted. An airborne H5N1 might
recognize receptors in the upper airways, for example,
but be less likely to descend into the lungs to cause
the extensive damage inflicted by wild strains.
"Theoretically, one could imagine such a scenario," says
Pereis. "But I wouldn't want to stake my life on it."

Fouchier and Kawaoka's mutant viruses caused milder
disease in ferrets than their wild counterparts do, but
both men note that such comparisons are misleading
because wild H5N1 has to be administered to the animals
directly, which can introduce high doses deep within the
lungs. And, Kawaoka notes, transmissible strains do not
have to have a fatality rate of 60% to kill millions of
people: the H1N1 pandemic of 1918 had a mortality rate
of 2.5%, yet claimed around 50 million lives.

QUESTION 5 What Else Could Cause a Pandemic?

The Gs/Gd strain is what is known as a reassortant. It
was born from a flu version of sex, in which different
viruses infecting the same cell swap genes, and it
probably includes genes from the H6N1 and H9N2 viruses
[8] . Since then, H5N1's descendants have swapped genes
mostly with each other. "H5N1 is not very sexually
promiscuous," says Capua. "It likes to reassort within
its own lineage." But the H1N1 strain responsible for
the 2009 pandemic could shake H5N1 from its insularity.
That strain is itself a cocktail of genes from swine
H1N1, avian H1N1 and human H3N2 and includes a set of
genes called the triple-reassortant internal gene (TRIG)
cassette, which seems to make flu viruses more prone to
reassortment. "That virus loves to mate," says Webster.

Kawaoka's team has shown that the two viruses are
compatible, and will reassort spontaneously when they
infect the same cells [9] . This is made more likely by
their shared ability to infect pigs. Furthermore, Stacey
Schultz-Cherry, a virologist at St Jude Children's
Research Hospital, has found that reassortant viruses
containing HA from H5N1 and other genes from pandemic
H1N1 are better at replicating in human lung cells than
either parent is, and that they become more virulent
after a few rounds of replication [10] .

Wendy Barclay, a virologist at Imperial College London,
cautions that although these experiments reveal that
reassortment is possible, they do not quantify the odds
that it will happen. "If you force the event, it'll
happen, but I haven't seen anyone do the experiment in a
more natural way," she says. That would involve housing
uninfected pigs with ones carrying pandemic H1N1, and
poultry carrying H5N1. "Do they catch both viruses and
do the viruses mix up?" asks Barclay. "It's an unknown
and a pretty important one."

The upside of an H5N1-H1N1 reassortant is that many
people have already been infected with H1N1 and so might
have some immunity. But few people have encountered any
of the flu viruses that circulate in birds. "I think the
great worry is that a purely avian virus somehow crosses
over to us," says Farrar. H5N1 tops the list of concerns
because of the severe nature of the known infections,
but other subtypes could escalate into pandemics first.
"H9N2 may be an equally plausible pandemic candidate,"
says Peiris. It generally goes unnoticed, but has
hunkered down among Asia's poultry, caused occasional
outbreaks in humans and can reassort with seasonal flu.
Some strains already have mutations that are associated
with greater transmissibility in mammals. H7N7 is
similarly widespread and under-reported. In 2003, it
flared up in the Netherlands, infecting 89 people and
killing a veterinarian.

Virologists hope that by understanding the secrets that
allow H5N1 to spread and kill, they are in a better
position to assess the risk posed by other subtypes.
"With flu, nothing is predictable," says Capua.

References

1. Herfst, S. et al. Science 336, 1534-1541 (2012).

2. Imai, M. et al. Nature
http://dx.doi.org/10.1038/nature10831 (2012).

3. Cattoli, G. et al. Vaccine 29, 9368-9375 (2011).

4. Wang, T. T., Parides, M. K. & Palese, P. Science 335,
1463 (2012).

5. de Jong, M. D. et al. Nature Med. 12, 1203-1207
(2006).

6. Everitt, A. R. et al. Nature 484, 519-523 (2012).

7. Russell, C. A. et al. Science 336, 1541-1547 (2012).

8. Hoffman, E. et al. J. Virol. 74, 6309-6315 (2000).

9. Octaviani, C. P., Ozawa, M., Yamada, S., Goto, H. &
Kawaoka, Y. J. Virol. 84, 10918-10922 (2010).

10. Cline, T. D. et al. J. Virol. 85, 12262-12270
(2011).

Author information

1. Ed Yong is a freelance writer based in London and
author of the blog 'Not Exactly Rocket Science'.

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