D1014.PDF

Rev. sci. tech. Off. int. Epiz., 2004, 23 (2), 453-465
Microbial adaptation and change: avian influenza
R.G. Webster (1, 2) & D.J. Hulse (1)
(1) Saint Jude Children’s Research Hospital, Department of Infectious Diseases, Division of Virology,
332 North Lauderdale, Memphis, TN 38105, United States of America
(2) University of Hong Kong, Department of Microbiology, Pokfulam Road, Hong Kong, People’s Republic of
China
Summary
The evolution of influenza is a continuing process involving viral and host factors.
The increasing frequency of emergence of the highly pathogenic H5N1, H7N3
and H7N7 influenza viruses and the panzootic spread of H9N2 influenza virus, all
of which can be potentially transmitted to humans, are of great concern to both
veterinary and human public health officials. The question is how soon the next
pandemic will emerge. A convergence of factors, including the population
densities of poultry, pigs and humans, are likely factors affecting the evolution of
the virus. Highly concentrated poultry and pig farming, in conjunction with
traditional live animal or ‘wet’ markets, provide optimal conditions for increased
mutation, reassortment and recombination of influenza viruses. Strategies to
reduce the evolution of influenza and the emergence of pandemics include the
separation of species, increased biosecurity, the development of new vaccine
strategies and better basic knowledge of the virus. More effective co-operation
between scientists and veterinary and public health officials is required to
achieve these goals.
Keywords
Avian influenza – Emerging disease – Influenza H5 – Influenza H7 – Influenza H9 –
Microbial adaptation – Pandemic potential – Prevention – Vaccine – Variation –
Zoonosis.
Introduction
The emergence and re-emergence of influenza viruses with
pandemic potential for both human and veterinary public
health is of great concern to humans globally. The
convergence of factors affecting contemporary human and
animal health issues has led to changing roles for
veterinarians and public health officials worldwide.
Humans influence global ecology with their increasing
population density, growing demands on land use and
high intensity farming to feed ever-increasing human
populations. In addition, the development of rapid
transportation ensures the global spread of any virus. Each
of these factors has implications for the emergence of novel
disease agents. This is particularly the case for influenza,
since poultry, pigs and humans are all factors in its viral
evolution. These issues were addressed in a recent Institute
of Medicine report of the National Academy of Sciences of
the United States of America (USA) (35). In this paper, the
authors will consider the adaptation of influenza viruses to
the changing world and the urgency for co-operation
among disciplines in planning for a pandemic that many
believe is imminent.
There are 16 haemagglutinin (HA) subtypes of influenza
viruses, which are perpetuated in the aquatic birds of the
world. This gene pool of influenza viruses in aquatic birds
is largely benign, but evolves rapidly after transfer to
domestic avian and mammalian species. Of the
16 HA subtypes, two that can evolve into highly
pathogenic strains (H5 and H7) are of great concern to
agricultural authorities, including the World Organisation
for Animal Health. Other subtypes, including H9, H6 and
H3, have established, or are in the process of establishing,
permanent lineages in chickens and the severity of disease
depends on co-infecting agents. From a human
perspective, H1, H2 and H3 have caused pandemic and
epidemic influenza in humans, while H5, H7 and H9 have
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Rev. sci. tech. Off. int. Epiz., 23 (2)
been transmitted to humans with mortality caused by
Asian H5N1 strains. The remaining subtypes of influenza
A viruses may evolve into pathogens of concern in the
future but, at present, they are not considered to have
pandemic potential. Nevertheless, all influenza A subtypes
have the potential to contribute to the emergence of
a pandemic strain through genetic reassortment. This is a
continuous process in nature, which allows the exchange
of gene segments (22, 24, 35).
Influenza virus
This section will consider the mechanisms used by
influenza viruses to adapt to their host range, as well as
possible control strategies. Special attention will be given
to the H5, H7 and H9 subtypes, which are considered to
have high pandemic potential.
Avian influenza is classified as a member of the
Orthomyxoviridae family. The virus is enveloped and the
genome consists of eight segments of linear negative-sense,
single-stranded ribonucleic acid (RNA). The negativestranded RNA of influenza serves as a template for the
synthesis of messenger RNAs. It also serves as a template
for the positive-sense strand. The eight segments of viral
RNA encode for ten proteins. Three of these are
polymerase proteins: PA, PB1 and PB2. Two are surface
proteins: neuraminidase (NA) and HA. One is a
nucleocapsid (NP), two are matrix proteins (M1 and M2)
and two are non-structural proteins (NS1 and NS2).
A constantly varying virus:
mechanisms of variation
Frequent mutations: antigenic drift
Influenza viruses lack ‘proof-reading’ mechanisms and are
therefore unable to repair errors that occur during
replication (46). These mutations accumulate amino acid
changes within the viral genome, resulting in the existing
strain being replaced by a new antigenic variant (Fig. 1).
This mechanism of acquiring new mutations is known as
‘antigenic drift’ and the accumulation of point mutations is
seen in each of the gene products of the virus. However, it
is most pronounced in the surface proteins HA and NA,
where selection is antibody-mediated.
Antigenic drift is a continuous process in mammalian
influenza viruses and is the basis for the evolution of
human epidemic influenza. Antigenic drift is most
apparent in human influenza viruses, less so in swine and
equine influenza viruses and usually least apparent in
Fig. 1
The three mechanisms used by influenza viruses to adapt to
their host ranges
A. Antigenic drift occurs through errors during the replication of
influenza viruses, which are unable to be repaired. These mutations
accumulate amino acid changes within the viral genome, resulting in
the existing strains being replaced by a new antigenic variant
B. Reassortment or antigenic shift is the exchange of ribonucleic acid
(RNA) segments between two genotypically different influenza viruses
infecting a single cell, which can result in the generation of a novel
strain and/or subtype
C. Recombination occurs when two different sources of RNA
contribute to a single influenza RNA segment
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Rev. sci. tech. Off. int. Epiz., 23 (2)
avian influenza viruses. It can be mimicked in the
laboratory by the selection of escape mutants with
monoclonal antibodies (6).
Antigenic drift in avian influenza viruses in their original
aquatic bird reservoirs is limited but, after the virus has
spread into domestic poultry, it can become more
pronounced. Recent examples of antigenic drift, which are
relevant to recently emerging influenza viruses, are found
in the H5 and H9 subtypes in Asia (Tables I and II). Since
antigenic drift is mediated by antibody pressure,
researchers must accept that antigenic variation in
influenza viruses is caused by immune pressure in poultry.
Thus, vaccination can potentially contribute to increasing
the rate of antigenic drift (20).
infecting a single cell, can result in the generation of a
novel strain and/or subtype (Fig. 1). Theoretically, one pair
of influenza viruses with eight segments each can produce
256 different combinations. Reassortment has been
detected between influenza viruses in wild aquatic birds
(12, 49), in live poultry markets (24) and in the evolution
of the Z genotype of H5N1, which spread across Asia in
2004 (22) (Fig. 2).
Reassortment: antigenic drift
The available evidence is that the human pandemics of
1957 (the Asian pandemic) and 1968 (the Hong Kong
pandemic) each arose from reassortment between human
and avian influenza viruses (18). The acquisition of novel
HA and NA glycoproteins, together with a novel avian
PB1 gene, allowed the reassorted viruses to circumvent the
humoral immunity of the host while they gained the ability
to spread from human to human from their human
influenza precursor.
Influenza viruses exhibit a high frequency of reassortment.
Owing to the segmented nature of the influenza viral
genome, ‘reassortment’, or the exchange of RNA segments
between two genotypically different influenza viruses
Other recent examples of reassortment among the
influenza viruses of humans, pigs and birds are the H3N2
viruses that are circulating in pigs in the USA (16, 42, 50).
Table I
Cross-reactivity (titres determined by haemagglutinin inhibition) of H9N2 viruses (isolated between 1997 and 2003) with post-infection
serum to the H9N2 viruses
A/quail/HK/G1/97
A/ck/HK/G9/97
320
< 10
A/quail/HK/G1/97
Post-infection sera from H9N2 virus strains
A/ck/HK/FY313/00
A/ck/Nan/1-0016/00 A/duck/Shantou/4808/03
20
< 10
40
A/HK/2108/03
< 10
A/ck/HK/G9/97
20
≥ 1,280
≥ 1,280
640
160
80
A/ck/HK/FY313/00
20
≥ 1,280
≥ 1,280
640
80
80
10
≥ 1,280
≥ 1,280
640
160
160
A/duck/Shantou/4808/03
A/ck/Nan/1-0016/00
< 10
40
160
40
640
160
A/HK/2108/03
< 10
80
320
160
320
≥ 1,280
HK: Hong Kong
ck: chicken
Nan: Nanchang
Table II
Cross-reactivity (titres determined by haemagglutinin inhibition) of H5N1 viruses (isolated between 1997 and 2004) with post-infection
serum to the H5NI viruses
A/HK/156/97
A/HK/156/97
A/gs/HK/437-4/99
Post-infection sera from H5NI virus strains
A/HK/213/03
A/VN/1203/03
A/VN/3046/04
≥ 1,280
≥ 1,280
640
640
160
A/gs/HK/437-4/99
80
640
160
80
640
A/HK/213/03
40
320
640
320
640
80
A/VN/1203/03
10
< 10
160
160
≥ 1,280
640
A/ck/VN/c58/04
< 10
< 10
40
40
640
320
A/VN/3046/04
< 10
< 10
40
40
640
320
HK: Hong Kong
gs: goose
VN: Vietnam
ck: chicken
80
A/ck/VN/c58/04
≥ 1,280
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Rev. sci. tech. Off. int. Epiz., 23 (2)
the cells found in pig tracheas. Having both receptor
specificities could allow for human and avian viruses to
replicate in the same cell, allowing reassortment to occur.
Pigs not only provide ideal conditions for reassortment,
they have also been implicated in interspecies transmission
events between swine, avian and human hosts (1). Quail
have also been identified as intermediate hosts, since they
permit the replication of all the subtypes of influenza
found in wild aquatic birds and may be involved in
transmitting these viruses to chickens (28).
Recombination
Recombination results in a single influenza RNA segment
containing genetic material from two different sources
(Fig. 1). Recombination is a rare occurrence in influenza
viruses. However, it has recently been documented more
frequently (40). Recombination can occur through several
mechanisms, including the ‘crossing-over’ or ‘copy-choice’
mechanism.
Current status of H5, H7 and
H9 avian influenza strains
and their microbial adaptation
Gs/Gd: goose/Guandong
Fig. 2
The emergence of a single H5N1 genotype in 2004
The Gs/Gd/96-like viruses reassorted with viruses from wild aquatic
birds (genotype C), resulting in multiple genotypes appearing in 2001.
Some 2001 H5N1 viruses may have been transmitted from domestic
poultry back into wild aquatic birds, resulting in a new set of genotypes
for 2002. In 2002, the haemagglutinin gene of the H5N1 strains
showed significant antigenic drift. The antigenic drift was pronounced
in the genotype Z and Z+ viruses isolated in late 2002, as well as in the
human viruses isolated in early 2003. To date, in 2004, these
circulating viruses have evolved to include only one genotype, genotype
Z. The colour coding of the schematically shown virus particles
indicates the virus lineages. A gap in the neuraminidase or
non-structural protein gene segments indicates a deletion
Thus, reassortment occurs much more frequently than was
previously realised and live bird markets in both Asia and
the USA provide optimal conditions for the reassortment
of influenza viruses (32, 44).
It has been published that pigs can serve as the
intermediate host or reassortment vessel (31). Both
2-3 and 2-6 terminal sialic acid linkages are present in
Influenza A viruses of all 16 subtypes are perpetuated in
aquatic birds throughout the world. After transfer to an
alternative avian or mammalian host, influenza viruses
undergo rapid evolution (26, 51). For avian influenza
viruses, including H5, this transfer can occur in backyard
poultry flocks or live poultry markets where ducks, geese,
quail, pheasants, chickens, etc. are raised or housed
together (44). Since avian influenza viruses are primarily
transmitted through faeces, inadequate disease control
measures between the live markets and industrial poultry
farms, such as insufficient hygiene measures for people and
trucks, can result in the spread of influenza. Once an
influenza virus invades a commercial poultry farm, it has
an optimum number of susceptible poultry for rapid
viral evolution.
H9 subtype: a low-pathogenic avian influenza
problem
The H9 subtypes in Asia became established in domestic
poultry some time before 1990 and have evolved into three
phylogenetic sub-lineages, as follows:
– G1
– Y280 (a G9-like subtype)
– Y439 (7, 21).
Rev. sci. tech. Off. int. Epiz., 23 (2)
The G1 lineage is thought to have undergone reassortment
with H5N1/97 viruses to generate new H9N2 viruses,
which infected humans (23), as well as generating new
highly pathogenic H5N1 viruses (7). The Y280 lineage was
also involved in human infections, which arose from the
virus undergoing reassortment with A/chicken/HongKong/
G9/97 virus (10, 11). The H9N2 viruses have acquired the
ability to bind to both the 2-3 and 2-6 sialic acid
linkages, indicating specificity for both human and avian
cells (27). It has been reported that H9N2 viruses in the
People’s Republic of China were transmitted from chickens
or quail back to domestic ducks, which provided a perfect
environment for further reassortment. These multiple
genotypes of H9N2 were generated between 2000 and
2001 in the People’s Republic of China (21). A recent study
of H9 viruses circulating in Hong Kong bird markets in
2003 showed that the G1-like viruses were no longer
present (3). This was most probably due to the removal of
quail from the live bird markets. However, these viruses
were replaced by a new, highly diverse set of H9N2 viruses.
The haemagglutinin inhibition test shows the crossreactivity of some of these H9N2 viruses and indicates that
antigenic drift is occurring (Table I). Reassortment, linked
with the re-introduction of H9N2 viruses from domestic
poultry into ducks, contributes to the constant evolution of
the viruses.
H7 subtype: shifts in virulence cause health
concerns for poultry and people
The H7 subtype of avian influenza has always been of
concern, due to the evolution of highly pathogenic
variants, which have caused large economic losses in
poultry flocks. Recently, it has also become a human health
concern. In March 2003, the Netherlands experienced a
large outbreak of highly pathogenic H7N7.
Eighty-five people became infected, with the virus causing
conjunctivitis and one death (4, 19). This highly
pathogenic H7N7 variant originated from another virus
which was isolated from a duck in the Netherlands in
2000. All of the internal genes of the H7N7 isolate were of
avian origin (19).
The variant H7N2 has been a recurring problem for the live
bird markets of the northeastern USA since 1994, and can be
linked to three outbreaks in domestic poultry (39). These
viruses once again caused problems in 2004. Data indicate
that reassortment between HA, NA, M and NS genes played
a large role in the genetic diversity found in H7N2 viruses
before 1997 (38). However, after 1997, there does not seem
to be further evidence of reassortment (36).
In 2002, a highly pathogenic H7N3 outbreak occurred in
Chile (29). The highly pathogenic avian influenza (HPAI)
virus emerged from a low pathogenic virus that had been
isolated a few months earlier. Interestingly, the increase in
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virulence of the HPAI virus was linked with the insertion of
10 amino acids in the HA by a recombination event (40).
The sequence inserted into the HA was similar to a portion
of the NP gene of A/gull/Maryland/704/77 (H13N6) from
positions 1,268 to 1,297 (40). The exact mechanism of the
recombination event is unclear.
In February 2004, an HPAI H7N3 outbreak occurred in
British Columbia, Canada. This outbreak, like the
H7N3 outbreak in Chile, was also linked to a virulence shift
from low pathogenic avian influenza to HPAI. This shift in
virulence was also caused by a recombination event.
However, in this British Columbian outbreak, the
recombination occurred between the HA and M genes (14).
Two human cases of H7N3 infections have been reported.
Such recent occurrences indicate that recombination events
can play a significant role in creating genetic diversity,
resulting in virulence shifts for H7 viruses.
H5N1: the continuing evolution of avian strains
In 1997, H5N1 infected humans, killing six of the eighteen
people who were clinically infected. The H5N1 virus that
infected these people acquired all eight gene segments
from Eurasian avian sources. It is speculated that this
1997 H5N1 virus was generated from an H5N1 gooseGuangdong-like virus from its natural reservoir in geese,
which then crossed into chickens, hence acquiring internal
genes from H9N2 and/or H6N1 by reassortment (8). The
1997 H5N1 strain was eradicated in Hong Kong. However,
the HA gene of the 1997 strain continued to circulate in
geese in the southeastern People’s Republic of China (48).
In 2000, H5N1 viruses were isolated from aquatic poultry
and from 2001 onwards, these viruses were isolated from
aquatic birds as well as domestic chickens. In 2002, the HA
gene of the H5N1 strains showed significant antigenic drift
(9). The antigenic drift was pronounced in the genotype Z
and Z+ viruses isolated in late 2002, as well as the human
viruses isolated in early 2003 (9). In addition, these
Z genotypes of H5N1 were lethal for aquatic poultry (37).
Between 2003 and 2004, the H5N1 epidemic spread across
much of East Asia. Outbreaks in Indonesia, Thailand and
Vietnam in late 2003 and early 2004 were all of genotype Z
viruses (22) (Fig. 2). Moreover, H5N1 viruses were also
isolated from the People’s Republic of China, Japan,
South Korea, Cambodia, Laos and Malaysia. To date,
11 human deaths from 16 infections of H5N1 have been
reported from Thailand and 20 human deaths out of
27 infections have been reported from Vietnam. The
antigenic drift of these viruses can be illustrated by showing
their cross-reactivity (Table II). Post-infection sera collected
from earlier strains (between 1997 and 2003) no longer react
with the new 2004 viruses. Interestingly, the new 2004 virus
post-infection sera react more strongly with the older viruses
than with the homologous 2004 virus.
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The resurgence of HPAI H5N1 viruses on poultry farms in
Thailand, Vietnam, the People’s Republic of China and
Malaysia in the summer months (between July and
September 2004) and the subsequent infection and deaths in
humans in August 2004 raise the possibility that
HPAI H5N1 viruses are now endemic in poultry in Asia. The
available evidence is that H5N1 is widespread in domestic
ducks in the southern People’s Republic of China (2), and the
above information supports the likelihood that H5N1 is
endemic in domestic ducks throughout southern Asia.
Although the Z genotype of H5N1 influenza virus has been
dominant in Asia, there is heterogeneity in pathogenicity in
ducks. Examination of multiple H5N1 isolates of the
Z genotype for pathogenicity in ducks reveals that these
isolates can be divided into three broad groups, as follows:
– those that kill all inoculated ducks with neurological
signs of disease
– those that kill a percentage of ducks without
neurological signs of disease
– those that replicate but cause no signs of disease
(K.M. Sturm-Ramirez et al., unpublished findings).
All these H5N1 viruses found in ducks are highly
pathogenic in gallinaceous poultry.
Controlling the spread of avian
influenza
Whether the highly pathogenic H5N1 viruses that are
probably endemic in domestic ducks have also been
transmitted back into wild migrating birds is of great
concern. This question is currently unresolved. If the
hypothesis is correct, how will it affect control strategies?
Since wild birds are the source of all influenza viruses (47),
they must be excluded from poultry farms. Thus, the
biosecurity measures of screening commercial poultry
houses for the presence of virus, treating water and food
supplies and controlling access to farms are crucial, whether
it is migrating or local birds which are spreading highly
pathogenic or non-pathogenic influenza viruses. While
poultry raised indoors can be separated from wild birds,
poultry raised in open sheds cannot be separated. Thus, the
practice of raising ducks on rice farms in Asia for insect
control and retrieving residual grain at harvest is not in
accordance with influenza control. Another practice that is
not commensurate with influenza control is fish farming in
Asia, where the faecal wastes from poultry sheds are dropped
directly into fish ponds as fertiliser (30). The recently
evolving intensive farming practice in the USA, of raising
pigs and poultry in adjacent sheds with the same staff, is yet
another unsound agricultural practice.
Rev. sci. tech. Off. int. Epiz., 23 (2)
The first strategy for controlling influenza is preventing the
virus from gaining access to domestic poultry and pigs.
However, if biosecurity measures fail to prevent the
transmission of an influenza virus to domestic animals and
the virus becomes highly pathogenic, what are the control
options?
Culling is the time-honoured strategy recommended by
agricultural authorities. This involves:
– the establishment of quarantine zones
– banning the movement of poultry, poultry products and
people to and from the affected areas
– repopulating the affected areas with livestock only after
the ‘clean-up’ (successful application of disease-control
measures) is complete and the area is free of the virus.
There is concern that the second wave of H5N1 in Asia
between July and September 2004 was due, in part, to an
inadequate clean-up, insufficient testing and premature
restocking.
If the virus is not widespread, and it is not endemic in local
poultry and wild birds, then culling is likely to be a
successful strategy. Culling has been successfully used in
many countries, including the USA for H5N2 in
Pennsylvania in the 1980s, and in Japan for H5N1 in early
2004. If the virus is widespread in domestic animals or has
become endemic in domestic animals and wild birds, then
it is unlikely that culling alone will be successful.
The alternative strategy is culling plus vaccination. This
method was adopted by the People’s Republic of China and
Indonesia in 2004 for the control of highly pathogenic
H5N1/04 influenza virus. There are diverse opinions on
this strategy, which was used by Mexico to control highly
pathogenic H5N2 in the 1990s. Although highly
pathogenic H5N2 has not been reported in poultry in
Mexico since this measure, the precursor virus was not
eradicated, continues to circulate and has become highly
pathogenic in poultry in Central America and the USA in
2004 (20). The arguments against the use of vaccination
are as follows:
a) the use of vaccine does not encourage farmers to
improve isolation, disease control or biosecurity measures
b) vaccination can successfully prevent signs of the disease
but does not completely prevent virus shedding
c) vaccine use has been reported to promote the selection
of antigenic drift variants (20)
d) vaccine use may cause the virus to become endemic, as
appears to have happened in Mexico and Central America
and is probably occurring in Asia
e) the use of vaccine can affect trade and mask residual
infection.
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Rev. sci. tech. Off. int. Epiz., 23 (2)
Poultry-exporting countries do not use vaccine. Thus, at
the time of writing, Thailand has made the use of
vaccination illegal.
The arguments for the use of vaccine are as follows:
a) vaccines can prevent disease and save the poultry
industry of a country from collapse, especially if the
outbreak is widespread and culling is beyond the economic
capacity of the country concerned
b) vaccines reduce the virus load and the likelihood of
transmission, including transmission to humans and the
possibility of emergence of a pandemic strain
c) the use of sentinel unvaccinated birds can detect
transmission in a flock and warn of vaccine failure
d) vaccinated birds are eventually eliminated by slaughter
and processing for food, and virus eradication is declared
only when all the poultry in the area are serologically
negative
e) there is no disagreement on control strategies for viral
diseases in humans: vaccination is the first and most
efficacious approach.
was developed recently (15). This technique provides a
powerful new tool for the generation of vaccines for both
poultry and humans. A recent study showed the efficacy of
a reverse-genetic-derived H5N3 vaccine for agricultural
use (25). The H5N3 vaccine was developed as follows: the
HA gene came from A/goose/HK/437-4/99, which was
altered by the deletion of basic amino acids from the
connecting peptide region. The internal genes were from
A/PuertoRico/8/34, and the NA was derived from
A/duck/Germany/1215/73 (H2N3), which allowed
differentiation between vaccinated and non-vaccinated
birds (Fig. 3) (25). The vaccine induced antibodies in the
vaccinated birds, prevented death and clinical signs of
disease and reduced virus shedding after challenge (25).
There are several advantages in using reverse genetics to
generate vaccines for poultry, as follows:
– rapid and easy development of vaccines
– uniform vaccination
– low cost
– safety and effectiveness.
Rapid and easy development of vaccines
H5 vaccines for poultry
Two strategies are currently being used to prepare
H5 poultry vaccines. One strategy is the use of fowl pox
recombinants containing the HA gene; such a vaccine has
been approved for use in Mexico. The other strategy is the
use of conventional, egg-grown inactivated vaccine:
A/chicken/Mexico/232/94 (H5N2) vaccine, which has
been used in Hong Kong (25, 34). A/turkey/England/
N-28/73 (H5N2) has been used on the mainland of the
People’s Republic of China. Studies on the efficacy of the
A/chicken/Mexico/232/94 (H5N2) vaccine against the
H5N1 virus from Hong Kong in 2001, which has
94% homology in the HA gene between the vaccine and
challenge virus, showed that the vaccine gave complete
protection from signs of disease but some challenged birds
shed low levels of the virus (25). The use of
A/chicken/Mexico/232/94 (H5N2) vaccine on Hong Kong
poultry farms in 2004 probably contributed to the absence
of H5N1 in domestic poultry in Hong Kong during that
year, while surrounding countries, including the rest of the
People’s Republic of China, were affected.
Reverse genetic vaccines
for poultry and humans
A new technique for the rescue of infectious influenza A
virus from cloned complementary deoxyribonucleic acid
(DNA), using an eight-plasmid DNA transfection system,
With the constant evolution of pathogenic viruses in the
field, it is essential to find a method for developing
vaccines that are immediately protective, so that action can
be taken as soon as a problem is detected.
Uniform vaccination
At present, the commercial H5N2 vaccine is not
standardised for antigen content. As a result,
H5N2 vaccines have been shown to vary substantially
among the batches of prepared virus (5). Conventional
methods, which are used to standardise the human
influenza vaccine, can be applied to standardise the
antigen content of the reassortant vaccine in the
unconcentrated allantoic fluid. This allows the vaccine to
have standardised antigen content. Thus, during
vaccination, vaccinated poultry will consistently receive
the same amount of antigen, resulting in
uniform protection.
Inexpensive vaccine
Using reverse genetics, it is possible to generate high-yield
virus that does not require concentration or purification
(25). Since the vaccine virus is non-pathogenic, it does not
kill embryonated chicken eggs, allowing the virus to reach
high titres.
Safety and effectiveness
Vaccines generated by reverse genetics are safe, due to the
removal of the basic amino acid peptides which are
necessary for H5 viruses to retain their high pathogenicity.
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Rev. sci. tech. Off. int. Epiz., 23 (2)
vaccine virus to the 2004 Vietnam H5N1 strains, which
has been produced by the same methods, is about to enter
clinical trials.
Reverse genetics techniques may also be applied to the
production of human vaccines. In 2003, a pandemic alert
was issued by the World Health Organization due to
human infections caused by H5N1 influenza viruses. In
response, the rapid development of a human vaccine
generated by reverse genetics was initiated (43). The
vaccine virus that was generated contained the HA from
A/Hong Kong/213/03, which again had the basic amino
acids at the HA cleavage site removed, and the internal
genes came from A/Puerto Rico/8/34 (43). The new vaccine
was found to be:
– non-pathogenic for chickens and ferrets
– stable after several passages in chicken eggs
– rapid, in that it took less than four weeks to generate
– effective in conferring protection.
PA: polymerase protein
NP: nucleocapsid
M: matrix protein
NS: non-structural protein
PR: Puerto Rico
HA: haemagglutinin
NA: neuraminidase
HK: Hong Kong
Fig. 3
Construction of an H5N3 influenza A virus containing a modified
haemagglutinin gene (in which the polybasic amino acids were
deleted) derived from A/goose/Hong Kong/437-4/99, a
neuraminidase gene derived from A/duck/Germany/1215/73, and
the internal genes of the high-growth, egg-adapted influenza
A/Puerto Rico/8/34 by reverse genetics, using the eight-plasmid
system
Seventy-two hours after transfection, recombinant H5N3 viruses
contained in the supernatant of a mixed tissue culture of 293T and
Madin-Darby-canine-kidney cells (MDCK) were injected into ten-day-old
embryonated chicken eggs for further propagation
Stability testing of the reverse genetic H5N3 vaccine
showed that, after 14 passages in chicken embryos, the
virus had not reverted to an increased virulence and the
HA sequence remained the same (25). The vaccine used is
inactivated, which also increases safety. The reverse genetic
vaccine was also shown to be effective. While it did not
provide sterilising immunity in a single dose, it did protect
the chickens from clinical signs of disease and death, and
reduced shedding after challenge. At present, a chicken
Avian H5 vaccines, unlike human vaccines, do not need to
be so closely matched antigenically to provide protection
from signs of disease. The basic mechanism of crossprotection between widely divergent antigenic variants in
poultry is unresolved. It may, in part, be due to differences
between the systemic spread of a highly pathogenic virus
in gallinaceous poultry when compared to the respiratory
tract infection of mammals, including humans. An
important consideration with poultry vaccines is that they
do not necessarily prevent virus shedding and
unvaccinated sentinel birds are required in each poultry
house to monitor viral spread to contacts. Unfortunately,
agricultural vaccines are not required to be standardised
for antigen content, as human influenza vaccines are, and
unvaccinated sentinel birds are not obligatory in the field.
Thus, it is probable that antigenic drift is being driven by
the use of poultry H5 vaccines. There is clearly room for
improvement of agricultural vaccines; the methodology is
available but to date the political will is missing.
Conclusions
The transmission of H5N1, H7N3, H7N7 and H9N2 avian
influenza viruses to humans since 1997, and the
accompanying occurrence of disease outbreaks in domestic
poultry, are a continuing threat to veterinary and human
public health. What has changed since 1997? Before
1997, sporadic transmissions of H7N7 to humans caused
conjunctivitis (41, 45), but since then multiple human
deaths have occurred. Human infections have been more
frequent in Eurasia (H5N1, H7N7, H9N2) and have been
associated with changes in virus genes, including the
acquisition of multiple basic amino acids at the cleavage
site of the HA (17), mutations in the PB2 gene resulting in
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a lysine residue at position 627 (13), and mutations in the
NS gene resulting in a glutamic acid at position 92 that
modulates the host cytokine responses (33). These changes
are the result of mutational, reassortant and recombination
events and result from huge numbers of replicative cycles
in susceptible hosts. Highly concentrated poultry raising
provides optimal conditions for the evolution of influenza
viruses. Acquisition of the multiple basic amino acids
occurs mainly in gallinaceous poultry, while the PB2 and
NS mutations are detected after transmission to humans. It
is unknown if they are first selected in poultry or
in humans.
The much-dreaded event of continued human-to-human
transmission of novel H5, H7 and H9 influenza viruses
would most easily be realised through reassortment with a
circulating human strain, as occurred in the human
pandemics of 1957 (Asian) and 1968 (Hong Kong) (18).
Alternatively, the viruses could continue to acquire
multiple mutations that would support human-to-human
transmission, as may have happened with Spanish
influenza in 1918. Optimal conditions for reassortment
occur when influenza viruses from different species are
brought together in ‘wet’ or live bird markets (44). What
has changed is the direct and rapid transmission of
infection between wet markets and highly concentrated
poultry farming. Traditional wet markets were village
operations. Now, poultry can move from a mega-farm to a
wet market and the cages, trucks and humans can return
to the mega-farm (with viruses) in sufficient time to ensure
occasional transmission. The continuing problems with
H7N2 influenza in the wet-market system of the
northeastern USA is indicative of this process, with an
apparent inability to prevent non-pathogenic viruses
becoming pathogenic by the steady acquisition of basic
amino acids at the cleavage site of the HA.
What can be done? Hong Kong has demonstrated that
changes in poultry marketing practices probably prevented
an H5N1 outbreak in 2004, when the rest of the People’s
Republic of China and surrounding countries were
experiencing a widespread epidemic. The steps that were
taken included: banning all aquatic birds (the original
source of influenza viruses) and quail from the markets
and the introduction of two ‘clean days’ per month, during
which all markets were simultaneously closed, emptied
and cleaned. Subsequently, all birds sold in the markets
were vaccinated and screening was conducted to ensure
the desired level of antibody (34).
There is continuing debate in Hong Kong on whether to
close all wet markets and move to poultry that is killed and
chilled in the abattoir. While this debate may serve as an
illustration of what could be done to reduce the evolution
of influenza viruses, it is unlikely to reduce the risk of an
emergence of pandemic influenza in Hong Kong, unless
similar changes are introduced in the rest of the
People’s Republic of China. Severe acute respiratory
syndrome is the most recent example of transmission of a
newly emerging virus to neighbouring areas.
Thus, it is apparent that influenza will continue to be a reemerging, zoonotic infectious disease that requires
attention from researchers in veterinary and human
infectious diseases. Much remains to be clarified about the
molecular mechanisms of interspecies transmission of
influenza viruses and how to prevent it. The tools to
produce better vaccines are available and more rigorous
standards of quality control are greatly needed.
Acknowledgements
The authors thank Carol Walsh for preparing the
manuscript and Julia Cay Jones for editorial assistance.
This work was supported by Public Health Service grants
AI-95357 and CA-21765 and by the American Lebanese
Syrian Associated Charities.
462
Rev. sci. tech. Off. int. Epiz., 23 (2)
L’influenza aviaire : un exemple de modification et d’adaptation
microbienne
R.G. Webster & D.J. Hulse
Résumé
L’évolution de l’influenza s’inscrit dans un processus continu mettant en jeu des
facteurs liés au virus et à ses hôtes. L’émergence de plus en plus fréquente des
souches H5N1, H7N3 et H7N7 hautement pathogènes du virus de l’influenza et la
propagation panzootique de sa souche H9N2 suscitent une vive inquiétude
auprès des responsables de la santé publique et de la santé animale dans la
mesure où toutes ces souches sont susceptibles d’être transmises à l’homme. La
question est de savoir combien de temps nous sépare de la prochaine pandémie.
Un faisceau de facteurs convergents (la densité des populations avicoles,
porcines et humaines, par exemple) peuvent influer sur l’évolution du virus. Les
élevages à forte concentration de volailles et de porcins, ainsi que les marchés
traditionnels d’animaux vivants à ciel ouvert, créent des conditions optimales
pour les mutations, les réassortiments et la recombinaison des gènes des virus
de l’influenza. La séparation des espèces, le renforcement de la biosécurité, la
mise au point de nouvelles stratégies de vaccination et l’amélioration de notre
connaissance du virus sont autant de stratégies capables de ralentir l’évolution
de l’influenza et l’apparition de pandémies. La réalisation de ces objectifs passe
par une coopération plus efficace entre les chercheurs en recherche
fondamentale et les responsables de la santé publique et de la santé animale.
Mots-clés
Adaptation microbienne – Hémagglutinine 5 – Hémagglutinine 7 – Hémagglutinine 9 –
Influenza – Influenza aviaire – Maladie émergente – Potentiel pandémique – Prophylaxie
– Vaccin – Variation – Zoonose.
La influenza aviar como ejemplo de adaptación y cambio
microbiano
R.G. Webster & D.J. Hulse
Resumen
La evolución de la influenza es un proceso continuo en el que intervienen
factores ligados tanto al virus como a sus anfitriones. La creciente frecuencia
con la que aparecen los virus de la influenza altamente patógenos H5N1, H7N3
y H7N7, así como la diseminación panzoótica del virus H9N2, todos los cuales
pueden transmitirse al ser humano, preocupan sobremanera a los responsables
de la salud pública y veterinaria. La cuestión es cuánto va a tardar en surgir la
próxima pandemia. Hay una serie de factores convergentes, entre ellos la
densidad de población de las aves de corral, los porcinos y el ser humano, que
probablemente influirán en la evolución del virus. Las explotaciones avícolas y
porcinas con poblaciones animales muy densas, combinadas con los
tradicionales mercados de animales vivos, ofrecen un contexto idóneo para que
los virus experimenten más mutaciones, redistribuciones y recombinaciones
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Rev. sci. tech. Off. int. Epiz., 23 (2)
génicas. Para frenar la evolución de la influenza y reducir la aparición de
pandemias se requieren, entre otras cosas, la separación de las especies, el
aumento del nivel de seguridad biológica, la concepción de nuevas estrategias
en materia de vacunas y un mejor conocimiento básico del virus, objetivos todos
ellos que exigen una colaboración más eficaz entre los científicos dedicados a
la investigación fundamental, por un lado, y las instancias de salud pública y
veterinaria por el otro.
Palabras clave
Adaptación microbiana – Enfermedad emergente – Hemaglutinina 5 – Hemaglutinina 7
– Hemaglutinina 9 – Influenza – Influenza aviar – Potencial pandémico – Prevención –
Vacuna – Variación – Zoonosis.
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