Commentary

7F22

H1N1-influenza as Lazarus: Genomic resurrection from
the tomb of an unknown

Joshua Lederberg*

The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399

he 1918-1919 pandemic of H1N1 vi-

rus influenza was the greatest acute
plague of the 20th century. Incurring over
20 million human fatalities, however, was
not a good strategy for sustaining the
evolutionary fitness of the virus, because it
is no longer extant; whereas, say, measles
and chickenpox remain with us with no
evidence of remarkable genetic change,
although this may become more evident if
they were to face total or near eradication
through vaccination programs. The folly
of flu virulence remains our chagrin, be-
cause the threat always looms over us that
this family of viruses, endemic in birds,
again may generate human-lethal gene
reassortments. We had valid scares about
that contingency with the appearance of
H5N1 variant flu in Hong Kong just 3
years ago. Influenza can be regarded as a
zoonosis prevalent in birds, many of them
world travelers, with occasional outbreaks
in humans and other animals mainly
rooted in nature’s own experiments in
genetic engineering. Special importance is
attached to reassortments between bird-
and human-adapted strains most likely to
occur in habitats with close contact be-
tween birds, e.g., ducks, humans, and
swine (as a mixing reservoir; ref. 1). For
these reasons, high urgency attaches to
efforts to resurrect genetic information
about the singularities of H1N1-1918. The
intact virus is nowhere to be found, but
genomic fragments can still be detected
sensitively and diagnosed. Exemplifying
the latest technical advances in the use of
DNA amplification, reverse-transcrip-
tase-PCR (RT-PCR), Jeffery Tauben-
berger and his associates at the Armed
Forces Institute of Pathology initiated the
tour de force of recovering sequences of
flu from paraffin-embedded pathological
specimens preserved since 1918 in the
AFIP collections (2). These sources then
were augmented by samples from frozen
remains of an Inuit woman who suc-
cumbed to the flu in 1918 and was buried
in permafrost at Brevig Mission on the
Seward Peninsula of Alaska’s western
coast, not far from the Bering Strait. This
nameless woman has left an indelible
mark on world medical history (3). Now,

www.pnas.org/cgi/doi/10.1073/pnas.05 1000798

as reported in this issue, the AFIP team
has joined forces with teams from the U.S.
Department of Agriculture and the Peter
Palese/Adolfo Garcia-Sastre groups at
Mt. Sinai Medical School in a further
quest for the RNA sequences of H1N1-
1918 that might account for its historic
human virulence (4).

The flu genome comprises about 13,500
bases of single-stranded RNA, disposed in
eight segments varying from approxi-
mately 900 to 2,341 each. This genome is
only a few millionths of the complexity of
the human genome, but it is organized
with great efficiency, lacks “junk
R/DNA,” and encodes for a short dozen
of identified gene products (Fig. 1). Many
strains of flu have been sequenced fully;
this feat will be achieved for HIN1-1918
with arduous labor, because the RNA,
although frozen, is fragmented into snip-
pets no larger than approximately 120
bases each. The practical way now avail-
able is to devise probes by using segments
from extant flu strains, guessing at possi-
ble homologous strings, or synthesizing
probes with calculated degeneracy. Until a
complete genomic sequence is achieved,
and it is hard to see how that will be
authenticated, it is possible even that
H1N1-1918 contains extraneous inserted
sequences quite foreign to the canonical
flu strains. Very reasonably, initial efforts
focus on flu genes already identified in
viruses recovered from recent outbreaks
in humans, birds, swine, and other animals.

Previous work has focused on two well
studied gene products: hemagglutinin
(HA) and neuraminidase (NA), which
dominate the surface specificities of the
virus and underlie most of its taxonomy
(e.g., HIN1 refers to type 1 hemaggluti-
nin, type 1 neuraminidase). These gene
products are also the chief determinants
of specificity in vaccine prophylaxis for flu
strains circulating at any given time. HA
variation can account for fluctuations of
virulence and host specificity of extant flu
viruses. However, nothing remarkable was
seen in the HA or the NA of H1N1-1918.
The next gene to be scrutinized now is
NS1 (nonstructural protein 1), which the
Palese/Garcia-Sastre groups have fin-

PNAS | February 27, 2001

INFLUENZA VIRUS

 

encodes
NS1 and NEP

Fig. 1. Diagram of an influenza-virus particle.
The surface of each influenza virion consists of a
lipid envelope in which two major viral surface
antigens, the hemagglutinin (HA) and the neur-
aminidase (NA), are found. Within the particle are
the eight negative-sense viral RNA segments en-
coding the viral proteins. The smallest viral seg-
ment, the NS segment, encodes two proteins: the
NS1, an antagonist of the cellular type | interferon
system, and the nuclear export protein (NEP),
which functions in viral assembly.

gered recently as an interferon antagonist
and as gene essential for flu virulence in a
mouse model. A reasonable conjecture
was that the hypervirulence of HINI-
1918 might be lodged in its NS1, and this
might be revealed in reinsertions of the
1918-NS1 segment into mouse-adapted
flu strains. This challenging construct was
generated in the laboratory—one hastens
to footnote, under BL-3+ conditions, and
under the USDA’s stern regulatory scru-
tiny—and tested in mice. The unexpected
and perhaps disappointing result was the
mitigation not enhancement of virulence
in this species. The incapacitation of the
NS1-virulence function in the mouse was

See companion article on page 2746.

*E-mail: lederberg@mail.rockefeller.edu.

| vol.98 | no.5 | 2115-2116

ee aad
ascribed to interaction with its host fac-
tors; the other variable would be other
elements of the genome of the mouse-
adapted flu strain. NS1 singularity for
the human virulence of HIN1-1918 is
neither falsified nor corroborated by these
findings.

There still remain a handful of gene
candidates, including the polymerases es-
sential for the replication of the virus. This
label does not preclude any of them from
also functioning in networks and pathways
that are expressed as virulence. It should
caution us about the nominalist fallacy to
recall that the 6 crystallin of the bird’s lens
does double duty as argininosuccinate
lyase, an enzyme in the urea cycle.

In principle, the NS1 hypothesis (and its
alternatives) might be tested by using sim-
ilar gene constructs based on flu viruses
adapted to other animal species, including
primates, and challenging the correspond-
ing hosts. Negative results would be as
inconclusive as those with the mouse. Pos-
itive results, namely the association of
hypervirulence with a gene sequence bor-
rowed from N1H1-1918, would be a great
advance in medical science and would
offer constructive models for the devel-
opment of prophylactic and therapeutic
measures. They would also induce great
alarm about the potential hazards to hu-
man health, if humans were also suscep-
tible, and the virus might escape. Any such
experiments should be done with strains
for which current vaccines are dissemi-
nated widely and have proven effectiveness.

To conduct such experiments with hu-
man-adapted strains and challenge to hu-
man subjects as the probative step, is well
nigh unthinkable. But nature is under no
such restraint! The current results are a

1. Webster, R. G., Bean, W. J., Gorman, O. T.,
Chambers, T. M. & Kawaoka, Y. (1992) Microbiol.
Rev. 56, 152-179.

2. Taubenberger, J. K., Reid, A. H., Krafft, A. E.,

caution to look closely at the involvement
of NS1 (as well as HA and NA) variation
in natural outbreaks in many species and
to look out for their reassortment into
human strains. In addition, it might be well
to undertake a special search for close
homologues to 1918-NS1 in viruses circu-
lating in avian and other species, in which
they may appear to be benign in their
current hosts (as in the present mouse
experiments). That would be nature’s in-
verse of the current report.

The publication by Basler et al. (4) will
attract great admiration for its technical
finesse and will serve as an example of the
fruits from convergence of natural his-
tory, field exploration, clinical insight, and
sophisticated molecular wizardry. It also
will awaken anxieties about the obvious
opportunities for abuse. The really fateful
step was taken with the very first cultiva-
tion of pathogenic bacteria and viruses a
century ago—perhaps most importantly
with the discovery of the concepts of
germs and communicable diseases. The
notion of using ever more sophisticated
technology for intentionally constructing
or reconstructing ever more pathogenic
variants lends further weight to that anx-
iety. The great debate of the mid-1970s led
to sensible measures for the regulation of
recombinant DNA research. There has
been increasing understanding that some
of nature’s pathogens deserve equal or
greater respect. We should be sure that we
continue to devote as much reasoned in-
genuity to the design of safeguards and to
informed and transparent third-party
scrutiny of potential hazards as we do
generally to the authentication of scien-
tific claims. We cannot afford to forego
the deepest research into the plagues that

Bijwaard, K. E. & Fanning, T. G. (1997) Science
275, 1793-1796.

3. Reid, A. H., Fanning, T. G., Hultin, J. V. &
Taubenberger, J. K. (1999) Proc. Natl. Acad. Sci.
USA 96, 1651-1656.

2116 | www.pnas.org/cgi/doi/10.1073/pnas.05 1000798

beset humankind. Nor can we afford to
blunder into mistakes that will do primary
injury to bystanders and incur incommen-
surate social sanctions.

My deepest anxieties pertain to the
smoldering technology and arms race that
attends the power struggles in the Middle
East and the economic instabilities of the
former Soviet Union. Although the 1975
Biological Weapons Convention (BWC)
has demilitarized the main drivers of bio-
weaponry technical advance, in the US.
and in the overt activities of other formi-
dable powers, the BWC has not been
enforced successfully against Iraq and is
more or less openly flouted in a handful of
other countries. The United Nations
(UN) Security Council is too splintered on
other issues to take a firm stand on the
defiance by Iraq of the UN-mandated
inspections. It would not be child’s play for
defiant small countries to adopt advanced
biotechnology into their weapons pro-
grams. But we have seen that the climactic
high-science successes in one decade be-
come fodder for high-school projects in
the next. Influenza is an unlikely candi-
date for rational weapons development,
because new strains promptly embrace the
world. But that logic is insufficient reason
to neglect the contingency. More likely
similar principles would be applied to
more governable bioagents, but any bio-
agents in warfare are an affront and a
threat to the entire human species. In-
formed professionals throughout the
world should be leading campaigns to
insist on universal compliance with the
BWC as a major bulwark of human health
and associating that with the most positive
measures to apply advanced biotechnol-
ogy in a constructive way for dealing with
nature’s continued scourges.

4. Basler, C. F., Reid, A. H., Dybing, J. K., Janczewski,
T. A., Fanning, T. G., Zheng, H., Salvatore, M.,
Perdue, M. L., Swayne, D. E., Garcfa-Sastre, A., et al.
(2001) Proc, Natl. Acad. Sci. USA 98, 2746-2751.

Lederberg