H5N1 Outbreaks

and Enzootic Influenza

Robert G. Webster,*† Malik Peiris,†‡ Honglin Chen,†‡ and Yi Guan†‡

Ongoing outbreaks of H5N1 avian influenza in migra- continuing human-to-human transmission by now, it is
tory waterfowl, domestic poultry, and humans in Asia during unlikely to do so in the future.” Such complacency is akin
the summer of 2005 present a continuing, protean pandem- to living on a geologic fault line and failing to take precau-
ic threat. We review the zoonotic source of highly patho- tions against earthquakes and tsunamis.
genic H5N1 viruses and their genesis from their natural
reservoirs. The acquisition of novel traits, including lethality
to waterfowl, ferrets, felids, and humans, indicates an The Source
expanding host range. The natural selection of nonpatho- Influenza A viruses are perpetuated in the wild birds of
genic viruses from heterogeneous subpopulations cocircu- the world, predominantly in waterfowl, in which the 16
lating in ducks contributes to the spread of H5N1 in Asia. subtypes (which differ by 30% in their hemagglutinin
Transmission of highly pathogenic H5N1 from domestic [HA] nucleotide homology) coexist in perfect harmony
poultry back to migratory waterfowl in western China has with their hosts (2,3) (Figure 1). In these natural hosts, the
increased the geographic spread. The spread of H5N1 and viruses remain in evolutionary stasis, showing minimal
its likely reintroduction to domestic poultry increase the evolution at the amino acid level over extended periods.
need for good agricultural vaccines. In fact, the root cause
This fact indicates that the influenza-bird association is
of the continuing H5N1 pandemic threat may be the way
the pathogenicity of H5N1 viruses is masked by cocirculat- ancient; this lack of change is surprising because influenza
ing influenza viruses or bad agricultural vaccines. viruses are segmented, negative-stranded RNA viruses that
have no quality-control mechanisms during replication and
are highly prone to variation. After transfer to a new type
nfluenza is an ancient disease that has infected humans
I at irregular intervals throughout recorded history (1).
While the 1918 “Spanish” influenza is the best recorded
of host, either avian or mammalian, influenza viruses
undergo rapid evolution. However, all 16 HA subtypes,
including H5 and H7, have until recently been considered
catastrophic influenza pandemic, similarly severe pan- to be benign in their natural hosts. This benign equilibrium
demics occurred earlier, when the human population of the between the influenza virus and its host may have
world was much smaller, and they will occur again. Our changed.
challenge is to understand all aspects of the influenza
virus, the hosts and their response, and the virus’ global Genesis of the H5N1 Virus
impact so that we may be better prepared to face the Before 1997, no evidence had indicated that H5
inevitable next influenza pandemic. influenza viruses could infect humans and cause fatal dis-
The influenza virus that appears most threatening is the ease. The H7 influenza viruses were known to cause con-
avian H5N1 strain that since 2003 has infected >130 per- junctivitis in humans, and serologic studies provided
sons in Vietnam, Thailand, and Cambodia and has killed evidence of subclinical human infection with the subtypes
more than half of them. Nonetheless, the H5N1 influenza prevalent in avian live poultry markets (4). The precursor
threat is viewed with disturbing complacency; a frequent- of the H5N1 influenza virus that spread to humans in 1997
ly heard statement is “since the virus has not adapted to was first detected in Guangdong, China, in 1996, when it
caused a moderate number of deaths in geese and attracted
*St. Jude Children’s Research Hospital, Memphis, Tennessee, very little attention (5). This goose virus acquired internal
USA; †University of Hong Kong, Hong Kong, SAR, China; and
‡Shantou University Medical College, Shantou, Guangdong,
gene segments from influenza viruses later found in quail
China (A/Quail/HK/G1/97 [H9N2]) and also acquired the

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 1, January 2006 3

INFLUENZA OVERVIEW

Japan and Korea were genetically different from those in

the other countries (the V genotype) (12,13). The phyloge-
ny of the recent Z genotype viruses showed that viruses
isolated in Vietnam and Thailand formed a cluster that
remained distinct from those isolated in Indonesia.
To date, >140 million domesticated birds have been
killed by the virus or culled to stem its spread; as of
December 2005, >130 persons have been infected in
Vietnam, Thailand, Indonesia, Cambodia, and China, and
70 have died (42 in Vietnam, 14 in Thailand, 8 in
Indonesia, 4 in Cambodia, and 2 in China). These recent
H5N1 influenza viruses have acquired the unprecedented
Figure 1. Emergence of H5N1 influenza virus and control options.
and disturbing capability to infect humans; to cause neu-
A nonpathogenic H5 influenza virus is believed to have spread to rotropic disease and a high proportion of deaths in water-
domestic ducks and geese, then to domestic chickens. In chick- fowl in nature; to cause death in and be transmitted among
ens, the H5 virus became highly pathogenic before it was trans- felid species, including domestic cats (14); and to cause
ferred back to domestic ducks and geese. The highly pathogenic neurotropic disease and death in ferrets and mice (15).
H5 virus reassorted its genome with those of other influenza virus-
es in aquatic birds, and the resulting viruses spread to domestic
These incremental changes intensify concern about this
poultry farms, humans, and occasionally to pigs. These viruses H5N1 virus’ pandemic potential. These traits are likely to
acquired mutations in their PB2, HA, NA, and NS genes that made have been acquired initially by reassortment in 2001 and
them lethal to domestic and wild waterfowl and humans. Solid 2002, when a plethora of different genotypes were detect-
lines, transmission demonstrated; dotted lines, transmission pos- ed in poultry markets and later in farms in Hong Kong
tulated but not demonstrated. Multiple opportunities exist for con-
trol of highly pathogenic avian influenza: 1) prevent contact
(13). These genes were presumably acquired from viruses
between wild and domestic poultry by use of screened poultry found in waterfowl in Southeast Asia, but the actual gene
houses and treated water; 2) prevent contact between domestic donors have not yet been identified. Since late 2002, the Z
waterfowl and gallinaceous poultry by use of screened houses and genotype has become dominant, but phylogenetically dis-
treated water and by exclusion of waterfowl from “wet markets”; tinguishable viruses have continued to cocirculate in
3) eradicate H5/H7 influenza viruses from gallinaceous poultry by
culling or the use of vaccines that prevent disease and transmis-
Indonesia and western China. These characteristics have
sion; 4) prevent contact between poultry, pigs, and humans and been acquired mainly through mutations in the RNA poly-
make vaccines and antiviral drugs available. merase (PB2) gene, insertions in the HA gene, and dele-
tions in the NA and nonstructural (NS) genes. Thus, the
H5N1 viruses continue to evolve, initially by reassortment
and more recently by mutation and deletion (16,17). While
neuraminidase gene segment from a duck virus most H5N1 influenza viruses isolated from avian species
(A/Teal/HK/W312/97 [H6N1]) before the goose virus in Asia since 1997 are highly pathogenic in gallinaceous
became widespread in live poultry markets in Hong Kong poultry, they show heterogeneous pathogenicity in other
and killed 6 of 18 infected persons (6,7). This H5N1 virus species.
was eradicated by culling all domestic poultry in Hong In domestic ducks, the pathogenicity of the H5N1
Kong, and the genotype has not been detected since that viruses varies from high to nonpathogenic. In ferrets, most
time. However, different reassortants continued to emerge avian isolates replicate and cause respiratory tract infec-
from goose and duck reservoirs (8) that contained the same tion, while a few strains are highly pathogenic and neu-
H5 HA glycoprotein but had various internal genes. The rotropic (causing hind leg paralysis), and the virus has
H5N1 viruses continued to evolve, and in late 2002, a sin- been isolated from the brain (15). In contrast, all isolates
gle genotype was responsible for killing most wild, domes- from humans are highly pathogenic to ferrets. A similar
tic, and exotic waterfowl in Hong Kong nature parks pattern is found in experimental infection of mice, in
(9,10). This genotype of H5N1 spread to humans in Hong which most avian isolates cause respiratory infection.
Kong in February 2002, killing 1 of 2 infected persons
(11), and was the precursor of the Z genotype that became Mechanisms of Spread
dominant. The Z genotype spread in an unprecedented Were the highly pathogenic H5N1 viruses transferred
fashion across Southeast Asia, affecting Vietnam, within and between countries by persons, poultry, or
Thailand, Indonesia, Cambodia, Laos, Korea, Japan, fomites? In previous outbreaks of highly pathogenic H5
China, and later Malaysia. Further analysis showed that the and H7 infection in multiple countries, the spread was
H5N1 influenza viruses that caused outbreaks in poultry in directly attributable to humans. The main way influenza

4 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 1, January 2006

 H5N1 Outbreaks and Enzootic Influenza

virus is spread in poultry is by movement of poultry and distinguishable in HI tests. Therefore, H5N1 viruses circu-
poultry products; establishing good biosecurity measures lating in avian populations in Southeast Asia are clearly
on poultry farms is therefore an important defense. The heterogeneous. Notably, this phenomenon has repeatedly
poultry industry is a huge, integrated complex in Asia, and been reported for other influenza viruses that are in the
a number of firms have branches in China, Vietnam, process of altering their interspecies transmission, includ-
Thailand, and Indonesia. Nonetheless, the involvement of ing European avian H1N1 viruses that were transmitted to
multiple lineages of H5N1 argues against human-mediated pigs (21), H9N2 viruses that were transmitted to pigs and
spread from a single source. Live poultry markets are an humans, and now H5N1 viruses that are transmitted from
amplifier and reservoir of infection (18) and probably play ducks to humans. How these mixtures of codominant
a role in the maintenance and spread of the virus in the viruses are generated in a quasispecies is unresolved.
region. However, a number of other factors unique to Suggested mechanisms include mutator mutations or par-
affected Asian countries make control difficult. Backyard tial heterozygotes, but a satisfactory explanation is not
flocks are common in the region, and these domesticated available (22).
birds are not subject to any biosecurity measures. Fighting A subdominant population of H5N1 viruses is presum-
cocks are prized possessions and are often transported long ably selected in ducks after the immune response clears the
distances. Fighting cocks may also play a role in the spread dominant virus. The subdominant population appears to be
of infection and in transmission to humans. Many of the uniformly nonpathogenic to ducks, as if this is the natural
affected countries have a weak veterinary infrastructure situation for influenza in the duck. Whether further selec-
and are facing highly pathogenic avian influenza outbreaks tion will occur against the polybasic cleavage site in the
for the first time. The migrant ducks that commonly wan- HA and the pathogenicity determining sites in PB2 and NS
der through rice fields scavenging fallen rice seeds are remains to be seen. These viruses’ loss of pathogenicity to
another potent mechanism for the spread of infection. ducks, but retention of pathogenicity to chickens and pre-
sumably to humans, has been a problem associated with
Role of Domestic Ducks their eradication. In Vietnam, for example, disease signs
After late 2002, when H5N1 viruses had killed water- were used as the criteria for identifying H5N1 infection in
fowl in Kowloon Park in Hong Kong, most avian H5N1 ducks. Thus, the duck has become the Trojan horse of
isolates isolated in Vietnam, Thailand, and Indonesia were highly pathogenic H5N1 influenza in Asia (20).
highly pathogenic to chickens and domestic ducks.
However, by late 2003 and early 2004, some avian isolates Role of Migratory Birds
were nonpathogenic to ducks but retained their patho- Migratory waterfowl are generally believed to be the
genicity to chickens (19). Genetic analysis of these isolates main reservoir of all 16 subtypes of influenza A viruses,
showed evidence of multiple variants within single speci- including H5 and H7 subtypes. However, less agreement is
mens (20). On Madin-Darby canine kidney (MDCK) cells, found regarding the role of migratory waterfowl in the ini-
these viruses formed a mixture of small and large plaques tial spread of highly pathogenic H5N1 viruses across east-
that had different biologic properties. Viruses that formed ern Asia in 2003. The isolation of highly pathogenic H5N1
large plaques were usually highly pathogenic to ducks and from herons, egrets, and peregrine falcons in Hong Kong
ferrets, whereas viruses that formed small plaques were in 2003 and 2004 leaves no doubt that wild migratory birds
usually nonpathogenic to both birds and ferrets. Some can be infected and may spread disease to local poultry
virus isolates formed small plaques that were pathogenic to flocks. The outbreak in Qinghai Lake (16,17) proves that
ducks. Thus, plaque size was not a marker of pathogenici- these highly pathogenic H5N1 influenza viruses are trans-
ty. When ducks were orally infected with the original missible among migratory waterfowl. The migration route
mixed population of H5N1 viruses, most birds died, but of shorebirds in the east Asian-Australasian flyway does
some excreted virus for an extended period (up to 17 overlap the areas that have had H5N1 outbreaks, although
days); during this time, viruses that were nonpathogenic to the virus has been notably absent in Taiwan, Malaysia
ducks were selected. Serologic testing of these ducks (except for occasional outbreaks near the Thai border), and
showed hemagglutination inhibition (HI) and neutralizing western Australia (Figure 2). The role of migratory birds in
antibodies against the original dominant virus in the mix- the transmission and spread of highly pathogenic H5N1
ture; thus, immune clearance had caused the selection of viruses is still unclear. However, the recent outbreak of
the minor variants. The viruses shed on day 17 had become H5N1 infection in bar-headed geese and other species in
nonpathogenic to ducks, although they remained highly Qinghai Lake is a cause for concern because these birds
pathogenic to chickens. Sequence analysis of the HA migrate southward to the Indian subcontinent, an area that
showed that these viruses differed from the original domi- has apparently not been affected by H5N1 avian influenza.
nant virus at multiple amino acids and were antigenically If the virus were to become entrenched in India, its

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INFLUENZA OVERVIEW

Although culling domestic poultry to contain the spread

of highly pathogenic H5N1 virus is considered an accept-
able agricultural practice, culling migratory birds is not
acceptable to any international authority (Food and
Agriculture Organization of the United Nations [FAO], the
World Organization for Animal Health [OIE], the World
Health Organization [WHO]). The idea of culling migrato-
ry birds must be strongly discouraged, for it could have
unknown ecologic consequences. Instead, since highly
pathogenic H5N1 has been demonstrated in migratory
birds, the poultry industries of the world must adapt meas-
ures such as increased biosecurity (Figure 1), the use of
vaccines, or both.
Early detection and aggressive control measures
allowed Japan, South Korea, and Malaysia to eradicate
H5N1 virus soon after its introduction into those countries’
poultry flocks, demonstrating that rapid and determined
responses can keep the virus from gaining a foothold. In
other countries in Asia, delayed detection and response
caused the virus to become entrenched across a wide
region, and eradication at this stage has become a formida-
ble undertaking.

Agricultural Vaccines
The need for H5N1 vaccines for domestic poultry is
increasing. Adopting a policy to use vaccines in poultry is
an important decision for agricultural authorities in coun-
Figure 2. Migration routes of Asian birds. A) Distribution and migra- tries such as Thailand (a major poultry exporter) and
tion routes of bar-headed geese (courtesy of P. Leader). B) The Vietnam. Both countries are investigating their specific
Asia-Pacific region contains >240 species of migratory birds. The
3 flyways run primarily in a north-south direction, overlapping and
needs. While considerable data exist on the efficacy of
extending from Australia/New Zealand to India, Central Asia, and influenza vaccines in domestic chickens, little comparable
Siberia. The outbreak of highly pathogenic (HP) H5N1 in migrato- information is available regarding ducks. The pros and
ry waterfowl at Qinghai Lake, China, affected primarily bar-head- cons of the use of vaccines in poultry have been reviewed
ed geese (Anser indicus); however, other species, including gulls (23). Current technologies permit discrimination between
and ducks, were affected (16,17). The outbreak started in early
May 2005, and by June >5,000 birds had died. The birds exhibit-
vaccinated and naturally infected birds; however, vaccines
ed neurologic signs, inability to stand, diarrhea, and death. are not standardized on the basis of antigen content.
Systemic infection was detected in all organs tested. C) Bar-head- “Good” and “bad” agricultural vaccines are in use.
ed goose infected with HP H5N1 influenza virus. D) Immunostain
of goose pancreas, using H5 monoclonal antibodies (magnifica- Good Agricultural Vaccines
tion ×400). (C and D, courtesy of H. Chen). Countries shown in red
have had outbreaks of HP H5N1 since 2004. The geographic
Good agricultural vaccines provide protection from dis-
range of H5N1 may be extended by bar-headed geese or by ducks ease despite lack of a close antigenic match between the
that are less susceptible to lethal infection. vaccine and circulating strain and reduce the virus load
below the level of transmissibility. They do not provide
sterilizing immunity: vaccinated birds may excrete low
levels of virus after challenge infection. Sentinel unvacci-
geographic range would be substantially extended, and the nated birds are kept in each house to monitor for virus
pandemic threat would increase accordingly (17). A shedding, antigenic drift, or both.
mutation in the PB2 gene (residue E627K) associated with
pathogenicity in mammals (16,17) has been found in virus- Bad Agricultural Vaccines
es isolated from birds in Qinghai Lake; this finding has Bad agricultural vaccines prevent disease signs but do
caused concern that this mutation will be transferred to not prevent shedding of transmissible levels of virus. They
other migratory birds (e.g., wild ducks) and will be spread also promote undetected spread of virus on farms and to
because not all infected birds die. live poultry markets and promote antigenic drift. China

6 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 1, January 2006

 H5N1 Outbreaks and Enzootic Influenza

and Indonesia have adopted poultry vaccination to control that a pandemic is inevitable and that the only question
H5N1, and Vietnam has begun vaccine trials in poultry. remaining is “When?” The H5N1 virus continues to evolve
However, the resurgence of H5N1 in Indonesian poultry and spread, with additional human infections occurring in
and pigs (24) and the detection of H5N1 in apparently Vietnam, Cambodia, Indonesia, China, and Thailand. If
healthy birds in live poultry markets in China (17) suggest this virus acquires human-to-human transmissibility with
that some vaccines are of suboptimal quality or that coin- its present fatality rate of 50%, the resulting pandemic
fection masks disease. The adoption of a vaccine strategy would be akin to a global tsunami. If it killed those infect-
for H5N2 virus in Mexico in the 1980s reduced disease ed at even a fraction of this rate, the results would be cat-
signs but has not eliminated the H5N2 virus from the astrophic. While the high pathogenicity of the Qinghai
region; instead, vaccination may have contributed to the bar-headed goose isolate is a continuing threat to poultry
virus’ widespread presence in Central America and to its and humans, perhaps the most insidious threat comes from
antigenic drift (25). unobserved transmission through wild and domestic
ducks. The isolation of H5N1 virus from bar-headed geese
H9N2 and Cross-protection in Qinghai Lake in southern China in 2005 originated from
The clinical signs of infection with highly pathogenic unobserved infection in poultry markets and suggests that
H5N1 virus may be masked by cross-protection by other highly pathogenic H5N1 viruses continue to circulate
influenza subtypes, but this fact is often overlooked. unseen among poultry in China (17). We cannot afford
During the initial outbreak of highly pathogenic H5N1 in simply to hope that human-to-human spread of H5N1 will
Hong Kong in 1997, chickens in the live poultry markets not happen and that, if it does, the pathogenicity of the
exhibited no disease signs, yet samples from apparently virus will attenuate. Notably, the precursor of the severe
healthy chickens, ducks, and quail showed highly patho- acute respiratory syndrome (SARS)–associated coron-
genic H5N1 in each of the poultry markets surveyed (26). avirus (31) repeatedly crossed species barriers, probably
Surveillance showed that multiple influenza subtypes were for many years, before it finally acquired the capacity for
cocirculating, including 2 lineages of H9N2, the first rep- human-to-human transmission, and its pathogenicity to
resented by the G1 lineage (A/Quail/Hong Kong/G1/97 humans was not attenuated. We cannot wait and allow
[H9N2]) and the other by G9 (A/Chicken/Hong nature to take its course. SARS was interrupted by early
Kong/G9/97 [H9N2]). The G1 lineage has the same 6 case detection and isolation, but influenza is transmissible
internal gene segments as the index H5N1 human isolate early in the course of the disease and cannot be controlled
(A/Hong Kong/156/97 [H5N1]) and is believed to have by similar means. Just 1 year before the catastrophic tsuna-
been the donor of these genes during reassortment that pro- mi of December 2004, Asian leaders rejected a proposed
duced the original H5N1 human strain in 1997 (27). In lab- tsunami warning system for the Indian Ocean because it
oratory studies, chickens previously infected with H9N2 was too expensive and the risk was too remote. This mis-
(A/Quail/Hong Kong/G1/97 [H9N2]) were protected from take must not be repeated in relation to an H5N1 avian
disease signs and death when challenged with highly path- influenza pandemic. We must use this window of opportu-
ogenic H5N1, but the chickens shed H5N1 virus in their nity to prepare and to begin prepandemic implementation
feces (28). Further studies in inbred chickens established of prevention and control measures.1
that the cross-protection was due to cell-mediated immuni-
ty and that it could be transferred by CD8+ T cells but not Acknowledgments
by antibodies (29). We thank Carol Walsh for manuscript preparation and
The possible effect of cocirculating influenza viruses Sharon Naron for editorial assistance.
on the pathogenicity of highly pathogenic H5N1 in
This work was supported by Public Health Service grants
Vietnam, Thailand, and elsewhere in Asia has not been
AI-95357 and CA-21765 and by the American Lebanese Syrian
resolved. To date, no other subtypes of influenza A viruses
Associated Charities.
have been reported in poultry in Vietnam or Thailand.
Surveillance of live poultry in Hong Kong and in Dr Webster is a professor in the Division of Virology,
Nanchang (30) suggests that other influenza A viruses are Department of Infectious Diseases at St. Jude Children’s
cocirculating in live poultry markets and on duck farms. Research Hospital. The major focus of his research concerns
Definitive information is required to understand the ecolo-
gy of influenza and the possible masking of disease signs 1Since this article was written, the H5N1 influenza virus has con-
caused by H5N1. tinued to spread in migratory birds to Turkey, Croatia, and
Romania. The global spread of this H5N1 in migratory birds and
Conclusion domestic poultry is inevitable. The question is, “When will it
acquire sustained human-to-human transmission?”
Conventional wisdom about pandemic influenza holds

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 1, January 2006 7

INFLUENZA OVERVIEW

influenza viruses in wild aquatic birds and their role in the evolu- 17. Chen H, Smith GJD, Zhang SY, Qin K, Wang J, Li KS, et al. Avian
tion of new pandemic strains for humans and animals. flu: H5N1 virus outbreak in migratory waterfowl. Nature.
2005;436:191–2.
18. Kung NY, Guan Y, Perkins NR, Bisset L, Ellis T, Sims L, et al. The
impact of a monthly rest day on avian influenza virus isolation rates
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