2006, Vol 1 No 2, Article 6
H5N1 Outbreaks and Enzootic Influenza
Robert G. Webster,*† Malik Peiris,†‡
Honglin Chen,†‡ and Yi Guan†‡
Ongoing outbreaks of H5N1 avian influenza in migratory waterfowl, domestic poultry, and humans in Asia during the summer of 2005 present a continuing, protean pandemic threat. We review the zoonotic source of highly pathogenic H5N1 viruses and their genesis from their natural reservoirs. The acquisition of novel traits, including lethality to waterfowl, ferrets, felids, and humans, indicates an expanding host range. The natural selection of non-pathogenic viruses from heterogeneous subpopulations co-circulating in ducks contributes to the spread of H5N1 in Asia. Transmission of highly pathogenic H5N1 from domestic poultry back to migratory waterfowl in western China has increased the geographic spread. The spread of H5N1 and its likely reintroduction to domestic poultry increase the need for good agricultural vaccines. In fact, the root cause of the continuing H5N1 pandemic threat may be the way the pathogenicity of H5N1 viruses is masked by co-circulating influenza viruses or bad agricultural vaccines.
Influenza is an
ancient disease that has infected humans at irregular intervals
throughout recorded history (1). While the 1918 "Spanish" influenza
is the best recorded catastrophic influenza pandemic, similarly
severe pandemics occurred earlier, when the human population of the
world was much smaller, and they will occur again. Our challenge is
to understand all aspects of the influenza virus, the hosts and
their response, and the virus global impact so that we may be
better prepared to face the inevitable next influenza pandemic.
Influenza A viruses are perpetuated in the wild birds of the world, predominantly in waterfowl, in which the 16 subtypes (which differ by 30% in their hemagglutinin [HA] nucleotide homology) coexist in perfect harmony with their hosts (2,3) (Figure 1). In these natural hosts, the viruses remain in evolutionary stasis, showing minimal evolution at the amino acid level over extended periods. This fact indicates that the influenza-bird association is ancient; this lack of change is surprising because influenza 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 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 to be benign in their natural hosts. This benign equilibrium between the influenza virus and its host may have changed.
GENESIS OF H5N1 VIRUS
Before 1997, no evidence had indicated
that H5 influenza viruses could infect humans and cause fatal disease.
The H7 influenza viruses were known to cause conjunctivitis in humans,
and serologic studies provided evidence of sub-clinical human infection
with the subtypes prevalent in avian live poultry markets (4). The
precursor of the H5N1 influenza virus that spread to humans in 1997 was
first detected in Guangdong, China, in 1996, when it caused a moderate
number of deaths in geese and attracted very little attention (5). This
goose virus acquired internal gene segments from influenza viruses later
found in quail (A/Quail/HK/G1/97 [H9N2]) and also acquired the
neuraminidase gene segment from a duck virus (A/Teal/HK/W312/97 [H6N1])
before the goose virus became widespread in live poultry markets in Hong
Kong and killed 6 of 18 infected persons (6,7). This H5N1 virus was
eradicated by culling all domestic poultry in Hong Kong, and the
genotype has not been detected since that time. However, different re-assortants
continued to emerge from goose and duck reservoirs (8) that contained
the same H5 HA glycoprotein but had various internal genes. The H5N1
viruses continued to evolve, and in late 2002, a single genotype was
responsible for killing most wild, domestic, and exotic waterfowl in
Hong Kong nature parks (9,10). This genotype of H5N1 spread to humans in
Hong Kong in February 2002, killing 1 of 2 infected persons (11), and
was the precursor of the Z genotype that became dominant. The Z genotype
spread in an unprecedented fashion across Southeast Asia, affecting
Vietnam, Thailand, Indonesia, Cambodia, Laos, Korea, Japan, China, and
later Malaysia. Further analysis showed that the H5N1 influenza viruses
that caused outbreaks in poultry in Japan and Korea were genetically
different from those in the other countries (the V genotype) (12,13).
The phylogeny 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.
Figure 1. Emergence of H5N1 influenza virus and control options.
A nonpathogenic H5 influenza virus is believed to have spread...
Figure 2. Migration routes of Asian birds. A) Distribution and migration routes of bar-headed geese
(courtesy of P. Leader)...
MECHANISM OF SPREAD
Were the highly pathogenic H5N1 viruses transferred within and between countries by persons, poultry, or fomites? In previous outbreaks of highly pathogenic H5 and H7 infection in multiple countries, the spread was directly attributable to humans. The main way influenza virus is spread in poultry is by movement of poultry and poultry products; establishing good bio-security measures on poultry farms is therefore an important defense. The poultry industry is a huge, integrated complex in Asia, and a number of firms have branches in China, Vietnam, Thailand, and Indonesia. Nonetheless, the involvement of multiple lineages of H5N1 argues against human-mediated spread from a single source. Live poultry markets are an amplifier and reservoir of infection (18) and probably play a role in the maintenance and spread of the virus in the region. However, a number of other factors unique to affected Asian countries make control difficult. Backyard flocks are common in the region, and these domesticated birds are not subject to any bio-security measures. Fighting cocks are prized possessions and are often transported long distances. Fighting cocks may also play a role in the spread of infection and in transmission to humans. Many of the affected countries have a weak veterinary infrastructure and are facing highly pathogenic avian influenza outbreaks for the first time. The migrant ducks that commonly wander through rice fields scavenging fallen rice seeds are another potent mechanism for the spread of infection.
Role of Domestic Ducks
After late 2002, when H5N1 viruses had
killed waterfowl in Kowloon Park in Hong Kong, most avian H5N1 isolates
isolated in Vietnam, Thailand, and Indonesia were highly pathogenic to
chickens and domestic ducks. However, by late 2003 and early 2004, some
avian isolates were non-pathogenic to ducks but retained their
pathogenicity to chickens (19). Genetic analysis of these isolates
showed evidence of multiple variants within single specimens (20). On
Madin-Darby canine kidney (MDCK) cells, these viruses formed a mixture
of small and large plaques that had different biologic properties.
Viruses that formed large plaques were usually highly pathogenic to
ducks and ferrets, whereas viruses that formed small plaques were
usually non-pathogenic to both birds and ferrets. Some virus isolates
formed small plaques that were pathogenic to ducks. Thus, plaque size
was not a marker of pathogenicity. When ducks were orally infected with
the original mixed population of H5N1 viruses, most birds died, but some
excreted virus for an extended period (up to 17 days); during this time,
viruses that were non-pathogenic to ducks were selected. Serologic
testing of these ducks showed hemagglutination inhibition (HI) and
neutralizing antibodies against the original dominant virus in the
mixture; thus, immune clearance had caused the selection of the minor
variants. The viruses shed on day 17 had become non-pathogenic to ducks,
although they remained highly pathogenic to chickens. Sequence analysis
of the HA showed that these viruses differed from the original dominant
virus at multiple amino acids and were antigenically distinguishable in
HI tests. Therefore, H5N1 viruses circulating in avian populations in
Southeast Asia are clearly heterogeneous. Notably, this phenomenon has
repeatedly been reported for other influenza viruses that are in the
process of altering their interspecies transmission, including European
avian H1N1 viruses that were transmitted to pigs (21), H9N2 viruses that
were transmitted to pigs and humans, and now H5N1 viruses that are
transmitted from ducks to humans. How these mixtures of co-dominant
viruses are generated in a quasi-species is unresolved. Suggested
mechanisms include mutator mutations or partial heterozygotes, but a
satisfactory explanation is not available (22).
Role of Migratory Birds
Migratory waterfowl are generally
believed to be the main reservoir of all 16 subtypes of influenza A
viruses, including H5 and H7 subtypes. However, less agreement is found
regarding the role of migratory waterfowl in the initial spread of
highly pathogenic H5N1 viruses across eastern Asia in 2003. The
isolation of highly pathogenic H5N1 from herons, egrets, and peregrine
falcons in Hong Kong in 2003 and 2004 leaves no doubt that wild
migratory birds can be infected and may spread disease to local poultry
flocks. The outbreak in Qinghai Lake (16,17) proves that these highly
pathogenic H5N1 influenza viruses are transmissible among migratory
waterfowl. The migration route of shorebirds in the east
Asian-Australasian flyway does overlap the areas that have had H5N1
outbreaks, although the virus has been notably absent in Taiwan,
Malaysia (except for occasional outbreaks near the Thai border), and
western Australia (Figure 2). The role of migratory birds in the
transmission and spread of highly pathogenic H5N1 viruses is still
unclear. However, the recent outbreak of H5N1 infection in bar-headed
geese and other species in Qinghai Lake is a cause for concern because
these birds migrate southward to the Indian subcontinent, an area that
has apparently not been affected by H5N1 avian influenza. If the virus
were to become entrenched in India, its geographic range would be
substantially extended, and the pandemic threat would increase
accordingly (17). A mutation in the PB2 gene (residue E627K) associated
with pathogenicity in mammals (16,17) has been found in viruses isolated
from birds in Qinghai Lake; this finding has caused concern that this
mutation will be transferred to other migratory birds (e.g., wild ducks)
and will be spread because not all infected birds die.
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 countries such as Thailand (a major poultry exporter) and Vietnam. Both countries are investigating their specific needs. While considerable data exist on the efficacy of influenza vaccines in domestic chickens, little comparable information is available regarding ducks. The pros and cons of the use of vaccines in poultry have been reviewed (23). Current technologies permit discrimination between vaccinated and naturally infected birds; however, vaccines are not standardized on the basis of antigen content. "Good" and "bad" agricultural vaccines are in use.
Good Agricultural Vaccines
Good agricultural vaccines provide protection from disease despite lack of a close antigenic match between the 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 unvaccinated birds are kept in each house to monitor for virus shedding, antigenic drift, or both.
Bad Agricultural Vaccines
Bad agricultural vaccines prevent disease signs but do not prevent shedding of transmissible levels of virus. They also promote undetected spread of virus on farms and to live poultry markets and promote antigenic drift. China and Indonesia have adopted poultry vaccination to control H5N1, and Vietnam has begun vaccine trials in poultry. However, the resurgence of H5N1 in Indonesian poultry and pigs (24) and the detection of H5N1 in apparently healthy birds in live poultry markets in China (17) suggest that some vaccines are of suboptimal quality or that co-infection masks disease. The adoption of a vaccine strategy for H5N2 virus in Mexico in the 1980s reduced disease signs but has not eliminated the H5N2 virus from the region; instead, vaccination may have contributed to the virus widespread presence in Central America and to its antigenic drift (25).
H9N2 AND CROSS-PROTECTION
The clinical signs of infection with
highly pathogenic H5N1 virus may be masked by cross-protection by other
influenza subtypes, but this fact is often overlooked. During the
initial outbreak of highly pathogenic H5N1 in Hong Kong in 1997,
chickens in the live poultry markets exhibited no disease signs, yet
samples from apparently healthy chickens, ducks, and quail showed highly
pathogenic H5N1 in each of the poultry markets surveyed (26).
Surveillance showed that multiple influenza subtypes were
co-circulating, including 2 lineages of H9N2, the first represented by
the G1 lineage (A/Quail/Hong Kong/G1/97 [H9N2]) and the other by G9
(A/Chicken/Hong Kong/G9/97 [H9N2]). The G1 lineage has the same 6
internal gene segments as the index H5N1 human isolate (A/Hong
Kong/156/97 [H5N1]) and is believed to have been the donor of these
genes during re-assortment that produced the original H5N1 human strain
in 1997 (27). In laboratory studies, chickens previously infected with
H9N2 (A/Quail/Hong Kong/G1/97 [H9N2]) were protected from disease signs
and death when challenged with highly pathogenic H5N1, but the chickens
shed H5N1 virus in their feces (28). Further studies in inbred chickens
established that the cross-protection was due to cell-mediated immunity
and that it could be transferred by CD8+ T cells but not by antibodies
Conventional wisdom about pandemic influenza holds that a pandemic is inevitable and that the only question remaining is "When?" The H5N1 virus continues to evolve and spread, with additional human infections occurring in Vietnam, Cambodia, Indonesia, China, and Thailand. If this virus acquires human-to-human transmissibility with its present fatality rate of 50%, the resulting pandemic would be akin to a global tsunami. If it killed those infected at even a fraction of this rate, the results would be catastrophic. While the high pathogenicity of the Qinghai bar-headed goose isolate is a continuing threat to poultry and humans, perhaps the most insidious threat comes from unobserved transmission through wild and domestic ducks. The isolation of H5N1 virus from bar-headed geese in Qinghai Lake in southern China in 2005 originated from unobserved infection in poultry markets and suggests that highly pathogenic H5N1 viruses continue to circulate unseen among poultry in China (17). We cannot afford simply to hope that human-to-human spread of H5N1 will not happen and that, if it does, the pathogenicity of the virus will attenuate. Notably, the precursor of the severe acute respiratory syndrome (SARS)–associated coronavirus (31) repeatedly crossed species barriers, probably for many years, before it finally acquired the capacity for human-to-human transmission, and its pathogenicity to humans was not attenuated. We cannot wait and allow nature to take its course. SARS was interrupted by early case detection and isolation, but influenza is transmissible early in the course of the disease and cannot be controlled by similar means. Just 1 year before the catastrophic tsunami of December 2004, Asian leaders rejected a proposed tsunami warning system for the Indian Ocean because it was too expensive and the risk was too remote. This mistake must not be repeated in relation to an H5N1 avian influenza pandemic. We must use this window of opportunity to prepare and to begin pre-pandemic implementation of prevention and control measures.
We thank Carol Walsh for manuscript
preparation and Sharon Naron for editorial assistance.
Bird flu threatens to be a pandemic of huge magnitude. The only way to face it and hopefully defeat it is preparedness and information. In this context this article from the journal Emerging Infectious Diseases" is reproduced.
Kashvet and VetScan are thankful to
National Center for Infectious Diseases, Centers for Disease Control
and Prevention, Atlanta, USA for allowing the reproduction of this
article from Webster RG, Peiris M, Chen H, Guan Y. H5N1 outbreaks and
enzootic influenza. Emerg Infect Dis [05-1024]. 2006 Jan. Available
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