9Ecology of Influenza Viruses in Nature, Birds, and Humans
Hiroshi KIDA1,2,3
1

Department of Disease Control, Graduate School of Veterinary Medicine
2
Research Center for Zoonosis Control
3
OIE World Reference Laboratory for Highly Pathogenic Avian Influenza
Hokkaido University
Kita 18, Nishi 9, Kita-ku, Sapporo 060-0818, Japane-mail: kida@vetmed.hokudai.ac.jp

Abstract
Recent outbreaks of highly pathogenic avian influenza (HPAI) have spread from Southeast Asia to 62
Asian, Middle-Eastern, African and European countries. H5N1 viruses isolated from dead waterbirds inMongolia on the way back to their nesting areas in Siberia in May 2005 and 2006 were genetically closely
related to those isolated from birds in China, Iraq, Croatia, Nigeria, Korea and Japan, but different fromthose from poultry in Vietnam, Thailand and Indonesia. It is a serious concern that these HPAI viruses may
perpetuate in lakes where migratory water birds nest in summer. In intensive surveillance studies on avian
influenza fulfilled in the autumn of 2005, 2006 and 2007 in Mongolia and Japan, no HPAI viruses were isolated from migratory water birds that flew from Siberia, indicating that the virus has not yet persisted intheir nesting lakes in Siberia. Careful surveillance of avian influenza in migratory water birds needs tocontinue.
These H5N1 HPAI virus strains have caused severe disease with high mortality in a few humans who
appear particularly susceptible to infection with the virus strain in Asia, the Middle East and Africa since2003. More than 240 fatalities have occurred out of 380 confirmed cases in fifteen countries in four years
worldwide (as of June 2008). The second concern is that the H5N1 avian influenza virus alone is expected
to cause the next pandemic influenza in humans. Since each of the known subtypes of influenza viruses
perpetuates among migratory ducks and their nesting lake water in nature and avian viruses of any
hemagglutinin (HA) subtype could contribute genes in the generation of reassortants in pigs, none of the 16
HA and 9 neuraminidase (NA) subtypes can be ruled out as potential candidates for future pandemic strains.
Surveillance of swine influenza is, thus, extremely important as well as that of avian influenza to ensure
preparedness for the emergence of pandemic strains in humans.
Avian influenza virus strains isolated from migratory ducks in our global surveillance studies havebeen stored for use in vaccines and diagnosis. Influenza virus isolates of 61 combinations of HA and NA
subtypes have been isolated from fecal samples of ducks and viruses of 78 other combinations have been
generated by genetic reassortment procedure in chicken embryos. Thus, 246 avian influenza virus strains of
139 combinations of HA and NA subtypes have been stocked in a library of a panel of influenza virus
strains and their genes. Their pathogenicity, antigenicity, genetic information and yield in chicken embryos
have been analyzed, registered in the database and opened for the member countries of the World HealthOrganization (WHO) and Office of International Epizootics (OIE) through the Internet.
Key words: antigenic and genetic variation of influenza viruses, host range, interspecies transmission,
natural reservoir, perpetuation, vaccine

1. Introduction
Although many infectious diseases have been eradicated or controlled in the last century, influenza remains
to be overcome, with influenza A viruses causing occasional catastrophic pandemics in poultry and mammals
including humans. In addition, recent outbreaks ofhighly pathogenic avian influenza (HPAI) around the
Global Environmental Research
12: 9-14 (2008)
printed in Japanworld cause us alarm to realize that there are no borders
for infections and give rise to concern for human healthas well as for the livestock industry. H5N1 avian influenza virus strains have jumped the species barrier andcaused severe disease with high mortality in humans,
although the number of cases remains very small, first in
Hong Kong in 1997, then in Viet Nam, Thailand andCambodia through 2004. It is obvious that live bird
©2008 AIRIES

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H. KIDA

markets have played important roles in the generationand supply of highly pathogenic avian influenza virus
strains. Subsequently, the new HPAI virus strains havereturned to feral water birds and spread to 62 countries.
More than 240 victims have died out of 380 confirmed
cases in fifteen countries worldwide in the four years
since 2005. It is also obvious that the reason why the
H5N1 HPAI virus strains have persisted for more thanten years in Asia is attributable to the failure of control
measures, using inactivated vaccines, forgetting aboutbasically essential stamping-out strategies in three countries. In addition, vaccination has accelerated the selection of multiple antigenic variants (Wu et al., 2008).
Moreover, these three countries are ironically ranked as
the top three for the number of human cases of H5N1
virus infection.
To elucidate the origin and evolution of pandemic
influenza viruses, we have conducted global surveillance of influenza in birds, animals and humans. On thebasis of antigenic and genetic analyses of influenza
virus isolates from migratory ducks, domestic ducks,
pigs and humans and experimental infection studies onbirds and mammals with those viruses, we proposed thatthe hemagglutinin (HA) gene of A/Hong Kong/68(H3N2) strain was introduced into the precedent human
H2N2 Asian influenza virus by genetic reassortment,
occurring in the epithelial cells lining the upper respiratory tract of pigs, through domestic ducks from an H3
influenza virus circulating in migratory ducks in southern China (Kida et al., 1987; Kida et al., 1988; Yasuda
et al., 1991; Kida et al., 1994).
Ecological studies have revealed that a vast influenza
virus gene pool for avian and mammalian influenza exists in migratory ducks. Each of the known subtypes ofinfluenza A viruses perpetuates among migratory ducks
and their nesting lake water in nature (Ito et al., 1995;
Okazaki et al., 2000). Experimental infection studies
have established that influenza viruses preferentiallyreplicate in the columnar epithelial cells of the colon,
causing no disease signs and are excreted in highconcentrations in ducks (Kida et al., 1980). Influenza
viruses have been isolated from freshly deposited fecal
materials and from lake water, indicating that migratory
ducks have an efficient way to transmit viruses, i.e., via
fecal material in the water supply.
In comparison with influenza viruses of humans,
influenza viruses of ducks are in evolutionary stasis(Kida et al., 1987). The remarkable lack of amino acidchanges among influenza viruses isolated from migratory ducks suggests that the virus genes in this host are
subject to stringent stabilizing selection. The genetic andantigenic conservation of influenza viruses in ducks
suggests that the virus and host have reached a longestablished adaptive optimum. Such a stasis in migratory ducks is further evidence that duck populations are
the natural reservoir of influenza viruses.
In experimental infection of pigs, most tested strainsof each of the known HA subtypes of avian influenza
viruses replicated in the upper respiratory tract of theanimals. Co-infection of pigs with a swine virus and an
avian virus unable to replicate in this animal generated
reassortants, whose polymerase and HA genes were entirely of avian origin, that could be passed in pigs (Kidaet al., 1994). The results indicate that avian viruses ofany subtype can contribute genes in the generationof reassortants so that none of the 16 HA and 9neuraminidase (NA) subtypes can be ruled out as potential candidates for future pandemics in humans andpoultry. The direct transmission of H5N1 influenza
viruses from domestic poultry to humans in fifteen
countries in Asia, the Middle East and Africa in recent
years have further emphasized the need to have information on all influenza A subtype viruses circulating inavian species worldwide.
To provide information on influenza viruses in theirnatural hosts, virological surveillance of avian influenza
was carried out in Alaska and Siberia during their breeding season, summer. A number of influenza viruses ofdifferent subtypes were isolated from fecal samples ofducks and surprisingly also from fresh water samples ofthe lakes where they nest. Even in autumn when the
ducks had left for migration to the south, viruses could
still be isolated from the lake water, indicating that
influenza viruses are maintained in duck populations bywater-borne transmission in nature and revealing themechanism of year-by-year perpetuation of the virusesin the frozen lake water while ducks are absent in winter.
Phylogenetic analysis of the isolates from Alaska indicates that ducks carrying these viruses migrate to thesouth through the continent of North America and not toAsia (Ito et al., 1995). Influenza virus isolates fromfecal samples of ducks in their nesting areas in Siberia
phylogenetically belong to the Eurasian lineage and areclosely related to those from birds, pigs and horses in
Asia. It was noted that these isolates are closely
correlated to the H5N1 influenza virus isolates fromchickens and humans (Okazaki et al., 2000). Phylogenetic analysis of the HA of H5 influenza virus isolates
from ducks in Japan reveals a close relationship withthose of H5N1 influenza viruses from Hong Kong,
southern China, Thailand, Viet Nam and Japan indicating that the H5HA of these viruses originated frominfluenza viruses maintained in migratory ducks nestingin Siberia. These results indicate that the precursor
genes of pandemic influenza viruses are perpetuated inwater in the lakes where ducks nest in the northern
regions. Vaccine strains should be antigenically closely
related to the pandemic strain, avirulent for humans andchicken embryos, of high yield in chicken embryos, andimmediately prepared when the causative virus ischaracterized. We tested the immunogenicity to mice ofinactivated avirulent H5N4 influenza viruses isolated
from reservoir host ducks. The results showed that they
were potent enough to protect animals from a challengewith a lethal dose of the highly pathogenic H5N1 virus(Takada et al., 1999).

Ecology of Influenza Viruses in Nature, Birds, and Humans

11

2. Influenza is a Zoonosis.

3. Perpetuation of Influenza Viruses in Nature

Influenza A viruses are widely distributed in birds
and mammals including humans. Among those, virusesof each of the known hemagglutinin (HA) and neuraminidase (NA) subtypes (H1-H15 and N1-N9, respectively) other than H16 (the H16N3 virus has only been
isolated once, from a seagull in 2005, Olsen et al., 2006)
have been isolated from migratory ducks. Ecologicalstudies have revealed that a vast influenza virus genepool for avian and mammalian influenza exists in migratory ducks and their nesting lake water and that influenza is a typical zoonosis (Figs. 1 and 2). Influenza
viruses in ducks are in evolutionary stasis, suggestingthat the virus and host have reached a long establishedadaptive optimum.

Ducks are orally infected with influenza viruses bywaterborne transmission at their nesting lakes in Siberia,
Alaska and Canada around the Arctic Circle during theirbreeding season, summer. These viruses then replicate in
the columnar epithelial cells forming crypts in the large
intestine, and are excreted together with feces. The
viruses in the lake water become frozen after the ducks
migrate to the south and are preserved there over the
winter. In other words, northern nesting lakes for migratory ducks serve as influenza virus gene pools in nature.

InfluenzavirionHemagglutinin（HA, H1-16
subtypes）
Neuraminidase（NA, N1-9
subtypes）
Envelope

Fig. 1 Electron micrograph of an influenza virus particle of A/Hong Kong/68(H3N2) and its schematic diagraph.
The influenza A virus contains eight segmented negative-strand RNA genomes within
its bi-layered lipid membrane and envelope. Two spike glycoproteins, the hemagglutinin(HA) and the neuraminidase (NA) are embedded in the envelope. On the basis of
antigenic specificity of the HA and NA, the influenza A virus is divided into 16 HA and
9 NA subtypes.

Fig. 2 Host animal species and HA and NA subtypes of the influenza A virus.
Influenza A viruses infect humans, pigs, horses, various species of birds, mink, seals,
and whales. Of these, from waterfowl such as ducks, each of the known subtypes of the
HA and NA, except H16 that has only been isolated from a seagull, has been isolated.

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H. KIDA

4. Mechanism of Emergence of New
Pandemic Influenza Viruses in Humans
The phrase “a new virus” describes an influenza A
virus of a subtype of the HA or NA other than H1N1,
H2N2 and H3N2, which have spread among humans in
the last century, that is transmitted to humans and causesa pandemic (Fig. 3). We have revealed that the A/HongKong/68 (H3N2) pandemic influenza virus emerged in1968 via genetic reassortment in pigs concurrently
infected with an H3Nx influenza virus circulating inmigratory ducks through domestic ducks and the H2N2virus, which was circulating among humans at the time,in southern China (Figs. 4 and 5). Furthermore, experimental infection studies have revealed that pigs arehighly susceptible to avian influenza viruses of all HA
subtypes, and that since epithelial cells lining the upper
respiratory tract of pigs have receptors for both human
Pig
Cell

A/duck/S China/x/67

A/S China/x/67(H3NX)(H2N2)

A/Hong Kong/1/68 (H3N2)

HumansFig. 4 Genetic reassortment.

Fig. 3 History of the occurrences of pandemic influenza in humans andhighly pathogenic avian influenza.
There are three instances of naturally occurring emergence of new pandemicstrains in humans in the last century. A new pandemic strain is defined as a virus
possessing new HA and/or NA subtypes that humans have not experienced for thelast several decades. On the other hand, highly pathogenic avian influenza viruses
with H5 or H7 HA must have emerged everywhere in the world since feral water
birds were domesticated.

If two different influenza A viruses concomitantly infect one host
cell, viruses of different constellations of the gene segments
derived from either parental viruses, are produced when theymaturate and are released from the cell. Thus, the mathematicalnumber of different combinations of genome segments is 256.
This is the mechanism of genetic reassortment, and some of thesereassortant viruses bearing the hemagglutinin gene derived from aparental virus of non-human origin, which can infect and betransmitted to humans, are potential candidates for new pandemicstrains. The A/Hong Kong/68 (H3N2) pandemic strain bears PB1and HA genes from an H3Nx virus of duck origin and the othersfrom a human H2N2 Asian influenza virus strain prevailing at thattime.

Fig. 5 Route of transmission of the genes of pandemic influenza virus strains.
Ecological studies of animal influenza viruses and antigenic and genetic analyses of H3 influenza viruses
isolated from migratory ducks, domestic ducks and pigs, together with experimental infection studies
indicate that the hemagglutinin gene of A/Hong Kong /68 (H3N2) influenza virus was introduced into theprecedent human H2N2 virus from an influenza virus circulating in migratory ducks through domestic
ducks and pigs in southern China. It is now believed that the Asian influenza virus (H2N2) strain musthave emerged similarly. It is also assumed that the 1918 Spanish influenza pandemic strain originatedfrom a swine influenza virus strain in early 1918.

Ecology of Influenza Viruses in Nature, Birds, and Humans

13

and avian viruses, genetic reassortment takes place andnew viruses are thus produced in pigs. These findings
indicate that genetic reassortants of any HA subtype will
be generated in pigs, and new viruses will emerge as aresult.

tion, no highly pathogenic avian influenza virus has
been isolated, indicating that the highly pathogenic
H5N1 avian influenza virus strain has not yet perpetuated in the lakes where the migratory birds nest insummer.

5. Highly Pathogenic Avian Influenza Viruses

7. Transmission of Highly Pathogenic Avian
Influenza Viruses to Humans

Since late 2003, the highly pathogenic avian influenza virus of the H5N1 subtype has seriously affected
poultry in Asia. Well over 400 million birds have diedfrom infection or have been killed for control purposes.
A highly pathogenic avian influenza virus is generated
when a nonpathogenic virus brought in by migratory
birds from nesting lakes in the north is transmitted tochickens via domestic ducks, geese, quails, turkeys, etc.
and acquires pathogenicity for chickens with repeatedmultiple infections in the chicken population. This
pathogenicity does not apply to other kinds of birds ormammals, nor does it to humans (Fig. 6).

6. Do the Highly Pathogenic Avian Influenza
Viruses that were Conversely Transmitted
from Poultry to Feral Water Birds
Perpetuate in the Lakes where Migratory
Birds Nest?
During over-wintering, some migratory birds wereconversely infected with the highly pathogenic H5N1
virus from poultry and have been found dead at lakes innorthern China, Mongolia, Russia, Europe and Africa in
May on their way back to their nesting lakes in northern
regions (Fig. 6). It was found that all viruses isolated
from these birds were the same viral strains isolated
from poultry in China. Since it is of concern that this
H5N1 virus may perpetuate in lakes in Siberia where
migratory ducks nest in summer, intensive surveillanceof avian influenza has been carried out in autumn when
migratory birds fly from Siberia to the south inMongolia, China and Japan. In the four-year investiga-

It has been confirmed that more than 380 people
have been infected with the H5N1 virus, 60% of whom
have died in Asia, the Middle East and Africa since2003. Against this backdrop, it is assumed that the
H5N1 virus will acquire the ability to be transmitted
among humans and wreak havoc as a new virus. The
WHO has warned for the past decade that this avian
influenza virus is in the final stages of acquiring theability to infect humans. In response, industrialized
nations have formulated emergency plans, including
measures such as stocking an inactivated H5N1 vaccine
for humans.
The world is now confused as to the extent that theavian influenza can pass as a human disease. Both avian
influenza and human influenza are infections with the
influenza A virus. Although the potential for a virus with
the H5HA gene to cause an influenza pandemic inhumans cannot be denied, we should not focus on the
H5N1 virus alone. Rather, we should keep in mind thepossibility of influenza viruses of other subtypes emerging as new pandemic influenza virus strains.
Research to date has revealed that all the genes ofinfluenza viruses for birds and mammals including
humans are derived from non-pathogenic viruses maintained in migratory ducks. The basics of practical avian
influenza control measures involve minimizing damage
and protecting human health and food safety by eradication policies to contain avian influenza within poultry(Fig. 7).
Efforts should also be made to conquer human influenza through prediction and prevention. It is necessary

gooseturkeyAPAIV

6-9Months
quail
Low Pathogenic
AIVchickens

HPAIV
HPAIV

duck

> 6-9
monthsHuman virus
pig

LPAIV
HPAIV
(H5 or H7)

Fig. 6 Return of the H5N1 highly pathogenic avian influenza virus
strain from domestic poultry to migrating water birds.
During over-wintering, some migratory birds were conversely infected withthe highly pathogenic H5N1 virus from poultry in southern China and were
found dead at lakes in northern China, Mongolia, Russia, Europe and Africa
in May on the way back to their nesting lakes in northern regions.

Apathogenic
AIVX
Genetic
Reassortantvirus

Pandemic
Pandemic
virus
virus

Fig. 7 The origin and routes of transmission of the genes ofhighly pathogenic avian influenza virus and humanpandemic influenza virus strains.

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H. KIDA

Isolates from ducks

Reassortants

Fig. 8 Present status of the library of vaccine strain candidates.
Influenza viruses of 61 combinations of HA and NA subtypes have been
isolated from fecal samples of ducks in Alaska, Siberia, Mongolia,
Taiwan, China and Japan. So far, 77 other combinations have been
generated by genetic reassortment procedures in the laboratory. Thus,
246 avian influenza virus strains of 138 combinations of HA and NA
subtypes have been stocked as vaccine strain candidates and for
diagnostic use. Their pathogenicity, antigenicity, genetic information andyield in chicken embryos have been analyzed, compiled in a database,and published at the web site (http://virusdb.czc.hokudai.ac.jp/vdbportal/
view/index.jsp).

to establish and implement a preemptive strategy to control highly pathogenic avian influenza and humanpandemic viruses by investigating mechanisms of theemergence of human pandemic influenza virus strains
and the acquisition of pathogenicity of avian influenza
viruses. To this end, we have completed a library of all
HA- and NA-subtype virus strains and genes that exist
in nature, created a database and provided it over theInternet (Fig. 8). It has already been used for vaccine
production and diagnosis by 26 research institutions inten countries.

8. Zoonosis Control Measures
The strategy fostered in influenza research has been
applied to combat other zoonotic infections. First of all,it is necessary to identify the natural host of the causal
agent and clarify the root of transmission. That is to say,the ecological elucidation of wildlife and agents innature leads to fundamental control of infections. The
Research Center for Zoonosis Control, inaugurated at
Hokkaido University in 2005, conducts global surveillance studies and engages in research and education
toward the establishment of methods of diagnosis,
prevention and control. Its aim is to predict and preventthe outbreaks of influenza, West Nile fever, EbolaMarburg hemorrhagic fever, Hantavirus infections,rabies and other unknown infections.
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Hiroshi KIDA
Hiroshi KIDA, DVM, PhD, is a Professor
at the School of Veterinary Medicine andDirector of the Research Center for
Zoonosis Control, Hokkaido University,
Sapporo, Japan. He has devoted himself
to clarifying the origin, evolution, perpetuation mechanisms and ecology of
influenza A viruses in nature and among
birds, and mammals including humans
through extensive global surveillance, molecular epidemiology andexperimental infection studies. He has demonstrated that aquatic birds andtheir nesting lake water are the natural reservoir for influenza A viruses
and that past pandemic influenza viruses arose via genetic reassortment
between human and avian viruses in pigs. In 2005, he was awarded theJapan Academy Prize for a series of accomplishments and selected as amember of the Japan Academy in 2007. He has been designated Directorof the OIE Reference Laboratory for Highly Pathogenic Avian Influenza
and is also a member of the OIE Scientific Committee, the WHO
Human/Animal Interface in Influenza Surveillance Network and theScientific Committee of the OIE and FAO Influenza (OFFLU) Network.
He has established an influenza virus and gene library consisting of all the
known HA and NA subtype combinations, opened it to other researchers
as a database and started to provide these strains and genes as vaccine
seed candidates and for diagnostic use. He has also demonstrated that test
vaccines prepared from H5N1 and H7N7 influenza virus strains selectedfrom the library induced protective immunity against a challenge withhighly pathogenic avian influenza viruses in a monkey model.

(Received 12 August 2008, Accepted 18 August 2008)