JOURNAL OF VIROLOGY, Mar. 2003, p. 3816–3823 Vol. 77, No.

6
0022-538X/03/$08.00⫹0 DOI: 10.1128/JVI.77.6.3816–3823.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Neurovirulence in Mice of H5N1 Influenza Virus Genotypes Isolated

from Hong Kong Poultry in 2001
Aleksandr S. Lipatov,1 Scott Krauss,1 Yi Guan,2 Malik Peiris,2 Jerold E. Rehg,3
Daniel R. Perez,1 and Robert G. Webster1,4*
Division of Virology, Department of Infectious Diseases,1 and Department of Pathology,3 St. Jude Children’s Research
Hospital, and Department of Pathology, University of Tennessee,4 Memphis, Tennessee 38105, and Department
of Microbiology, University of Hong Kong, Hong Kong Special Administrative Region,
Hong Kong, People’s Republic of China2
Received 30 August 2002/Accepted 3 December 2002

We studied the pathogenicity of five different genotypes (A to E) of highly pathogenic avian H5N1 viruses,
which contained HA genes similar to those of the H5N1 virus A/goose/Guangdong/1/96 and five different
combinations of “internal” genes, in a mouse model. Highly pathogenic, neurotropic variants of genotypes A,
C, D, and E were isolated from the brain after a single intranasal passage in mice. Genotype B virus was
isolated from lungs only. The mouse brain variants had amino acid changes in all gene products except PB1,
NP, and NS1 proteins but no common sets of mutations. We conclude that the original H5N1/01 isolates of
genotypes A, C, D, and E were heterogeneous and that highly pathogenic neurotropic variants can be rapidly

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selected in mice.

The outbreak of avian influenza A virus of subtype H5N1 in
Hong Kong in 1997 killed 6 of 18 infected persons (4, 5, 35)
and was the first documented case of direct transmission of
avian influenza virus from poultry to humans. The outbreak led
to the development of serious lethal diseases and was regarded
by some as an incipient pandemic situation (27). The slaughter
of all poultry in live poultry markets in Hong Kong in Decem-
ber 1997 removed the source of infection and prevented fur-
ther transmission to humans (28). The human H5N1 isolates
(H5N1/97) are thought to be naturally occurring reassortants
of the avian virus in which the hemagglutinin (HA) gene is
highly homologous to that of A/goose/Guangdong/1/96
(H5N1) (Go/Gd) (30, 34) and the replicative complex is highly
homologous to that of A/quail/Hong Kong/G1/97 (H9N2) and
A/teal/Hong Kong/W312/97 (H6N1) viruses (8, 15). The neur-
aminidase (NA) gene of H5N1/97 viruses is also closely related
to that of A/teal/Hong Kong/W312/97 (15).
During 1999 and 2000, the Go/Gd-like H5N1 viruses con-
tinued to circulate in geese in Southeastern China (3, 9, 33).
These viruses have reassorted with viruses of aquatic avian
origin, and multiple genotypes of highly pathogenic H5N1 vi-
ruses containing HA and NA genes from Go/Gd-like viruses
reappeared in land-based poultry in the markets in Hong Kong
between February and May 2001. This reappearance of H5N1
viruses in terrestrial poultry—the first to occur after the out-
break in December 1997—resulted in the second slaughter in
4 years of all live poultry in Hong Kong’s Special Administra-
tive Region (10, 33). All of the H5N1 viruses isolated from
poultry in the retail markets during winter and spring 2001
were reassortants that contained HA and NA genes of Go/Gd
* Corresponding author. Mailing address: Division of Virology, De-
partment of Infectious Diseases, St. Jude Children’s Research Hospi-
tal, 332 North Lauderdale St., Memphis, TN 38105-2794. Phone: (901)
495-3400. Fax: (901) 523-2622. E-mail: robert.webster@stjude.org.
lineage and “internal genes” of other aquatic avian viruses
(10). Five types of reassortants, with genotypes designated A,
B, C, D, and E, were observed and grouped according to the
phylogenetic origins and constellation of the “internal” genes
(Fig. 1). Viruses of all five genotypes are highly pathogenic for
chickens and quail; all five genotypes are also lethal to mice
inoculated with a high dose of infective virus. Moreover, vi-
ruses of four of the five genotypes have been isolated from the
brains of mice that had signs of central nervous system (CNS)
disorders (10).
Influenza A virus pathogenicity in mice usually requires ad-
aptation to the new host, which occurs during growth of several
consecutive generations (serial passages) of the virus in the
lungs or brain. Studies of the acquisition of virulence during
adaptation in the mouse have shown that pneumovirulence
and neurovirulence of mouse-adapted viruses are associated
with mutations in HA, NP, NA, M, NS, and one or more
polymerase genes (1, 2, 18, 24, 29, 31, 32).
Unlike other human and avian influenza A viruses, the hu-
man and avian Hong Kong H5N1/97 isolates are pathogenic in
mice without adaptation (6, 7, 12, 19, 20). The H5N1/97 viruses
are heterogeneous in their pathogenicity for mice: some iso-
lates are highly pathogenic and replicate systemically, whereas
others are less pathogenic and replicate only in the respiratory
tract (7, 19). The pathogenesis of H5N1/97 viruses in mice is
distinct from that of other highly pathogenic H5 viruses (6),
and the molecular features of H5N1/97 phenotypes that are
highly pathogenic in mice have been determined (14, 17). Fur-
thermore, Hatta et al. showed, by using plasmid-based reverse
genetic techniques, that a lysine at residue 627 in PB2 protein
and a polybasic cleavage site in HA are crucial for high viru-
lence and systemic replication of A/Hong Kong/483/97 (H5N1)
virus in mice (13). The pathogenicity in mice of H5N1/97
phenotypes of low and high pathogenicity has been studied in
an outbred ferret model: both phenotypes were highly virulent

3816
VOL. 77, 2003 NOTES 3817

FIG. 1. Genotypes of Hong Kong 2001 H5N1 viruses. Initial genotyping of H5N1/01 viruses on the basis of partial gene sequences (10) was
confirmed in the present study by determination of the full-length sequences. The solid bars indicate genes of A/goose/Guangdong/1/96-like
lineage, the hatched bars indicate genes of A/duck/HK/Y280/97-like lineage, the shaded bars indicate genes of wild aquatic bird lineage, and the
open bars represent genes of unknown lineage.

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in ferrets, unlike the differential pathogenicity observed in level 3⫹ (23, 36) laboratory approved by the U.S. Department
mice (37). Pathogenesis of human A/Hong Kong/156/97 of Agriculture.
(H5N1) virus has been studied in cynomolgus macaques. This Pathogenicity and organ tropism of original H5N1 virus
virus replicated efficiently in the respiratory tract, and viral isolates in mice. Female, 1.5-month-old, BALB/c mice (The
gene segments were detected by PCR in brain and some in- Jackson Laboratory, Bar Harbor, Maine) were used for these
ternal organs of infected macaques, but no systemic replication studies. To determine the 50% lethal dose in mice (MLD50),
of this virus was observed (22). groups of four mice were anesthetized by isoflurane inhalation
In the present study, we used a mouse model to characterize and infected intranasally with 0.1 ml of allantoic fluid in phos-
the pathogenicity of the five genotypes of Hong Kong 2001 phate-buffered saline (PBS) (10-fold serial dilutions from 10⫺1
H5N1 viruses in mammalian hosts. Viruses of four of the five to 10⫺9). Animals were weighed daily and observed for 20
genotypes were isolated from mouse brain after intranasal days.
inoculation. We compared the pathogenicity for mice and the MLD50 titration revealed that all five genotypes of the
molecular features of these neurotropic, mouse brain (MB) H5N1/01 viruses were of low pathogenicity in mice; that is, they
variants with those of the original H5N1/01 virus isolates. The caused significant signs of illness and mortality only at an
results allowed us to assess the influence of naturally occurring extremely high dose. (Table 1). Because the viruses had similar
constellations of internal genes on the pathogenicity of avian EID50 values, we chose the 10⫺1 dilution of allantoic fluid that
H5N1 influenza viruses in mammalian hosts and the possibility contained ⬃106.5 EID50 of virus as the infective dose for fur-
of the rapid selection of neurovirulent, highly pathogenic vari- ther studies of pathogenicity. Groups of 18 to 26 mice were
ants. inoculated intranasally with 0.1 ml of allantoic fluid diluted
Viruses. Five avian H5N1 viruses, one of each of the five with PBS. Three mice from each group were sacrificed on days
genotypes isolated in Hong Kong in spring 2001 (10), were
used in the present study (Fig. 1): genotype A, A/chicken/Hong
Kong/YU822.2/2001 (Ck/HK/YU822.2/01); genotype B, A/chick- TABLE 1. Pathogenicity of H5N1 viruses in experimentally
infected micea
en/Hong Kong/YU562/2001 (Ck/HK/YU562/01); genotype C,
A/pheasant/Hong Kong/FY155/2001 (Ph/HK/FY155/01); geno- Virus detected no. of virus-
positive mice/total no. of
type D, A/chicken/Hong Kong/FY150/2001 (Ck/HK/FY150/ MLD50b dead micec in:
Virus Genotype
01); and genotype E, A/chicken/Hong Kong/NT873.3/2001 (EID50)
Internal
(Ck/HK/NT873.3/01). Viruses were isolated by one passage in Lungs Brain
organs
embryonated hens’ eggs (10). To prepare stocks for studies in
mice, viruses were propagated for one more passage in the Ck/HK/YU822.2/01 A 106.0 11/11 1/11 1/11
Ck/HK/YU562/01 B 106.5 13/13 0/13 0/13
allantoic cavity of 10-day-old embryonated hens’ eggs for 40 to Ph/HK/FY155/01 C 105.75 17/17 5/17 0/17
48 h at 37°C. Allantoic fluids containing virus were harvested, Ck/HK/FY150/01 D 106.0 8/8 8/8 0/8
and their infectivity was titrated in eggs; virus titers were ex- Ck/HK/NT873.3/01 E 106.75 6/6 6/6 0/6
pressed as the log10 of the 50% egg infective dose per 0.1 ml of a
Mice were inoculated with 0.1 ml of PBS-diluted allantoic fluid containing
fluid (log10 50% EID50 per 0.1 ml), according to the method of 106.25 to 106.75 EID50s of virus.
b
Reed and Muench (21). Virus stocks were divided into aliquots MLD50 values are shown as the number of EID50s resulting in 50% mortality
in mice.
and stored at ⫺80°C until use. c
Brains, lungs, and internal organs (liver, spleen, kidney, and heart) were
All studies with H5N1 viruses were carried out in a biosafety removed from mice that died or were sacrificed at the terminal stage of illness.
3818 NOTES
FIG. 2. Rate of survival of mice infected with different genotypes of
H5N1/01 viruses. Mice were observed for 20 days after intranasal
inoculation with 0.1 ml of PBS-diluted allantoic fluid containing ca.
106.5 to 106.75 EID50 of infective virus. Symbols: ⽧, Ck/HK/
YU822.2/01 (A); ■, Ck/HK/YU562/01 (B); Œ, Ck/HK/FY155/01 (C);
E, Ck/HK/FY150/01 (D); 䊐, Ck/HK/NT873.3/01 (E).
3 and 7 after inoculation to determine the virus titer in lung
(day 3) and in brain and internal organs (days 3 and 7). The
remaining mice in each group were weighed daily and observed
for 20 days for signs of disease and mortality. The lungs, brain,
spleen, liver, kidney, and heart were removed from each dead
mouse or animals sacrificed at the terminal stage of disease.
Organs were washed in cold PBS, weighed, ground, and ho-
mogenized in cold PBS with antibiotics to obtain an approxi-
mately 10% homogenate; solid debris was pelleted by centrif-
ugation at 2,000 ⫻ g for 10 min. The tissue homogenates were
injected into the allantoic cavities of 10-day-old embryonated
hens’ eggs to detect and isolate the viruses and to determine
the EID50. The lower limit of virus detection was 0.1 log10
EID50 per 0.1 ml of tissue homogenate. No virus was detect-
able in the brain or internal organs of mice 3 or 7 days after
inoculation. Virus was, however, detected in the brain and
internal organs of mice showing neurologic signs (mainly hind
leg paralysis) 8 to 14 days after inoculation (Table 1).
The results of pathogenicity and tissue tropism analyses di-
vided the five H5N1/01 genotypes into three groups. The first
group included viruses of genotypes A and B, which replicated
primarily in mouse lungs. Infection with virus of genotype A
(Ck/HK/YU822.2/01) killed 73.3% of the mice infected (Fig.
2). In one mouse infected with Ck/HK/YU822.2/01, virus was
isolated from the lungs, brain, and internal organs (Table 1).
Virus replicated efficiently in the brain of this animal (Fig. 3).
Titers of Ck/HK/YU822.2/01 in the liver, spleen, and kidney
were very low (virus was detected in undiluted tissue homog-
enates only). Virus of genotype B (Ck/HK/YU562/01) was
detected only in the lungs of infected mice (Table 1). Infection
with this virus caused a 55% mortality rate (Fig. 2).
The second group comprised a single member, Ph/HK/
FY155/01 (genotype C), which replicated in the lungs and
brain of infected mice (Table 1). Infection with this virus re-
sulted in the highest mortality rate (Fig. 2). This virus was
isolated from the brains of mice that had developed hind-leg
J. VIROL.
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FIG. 3. Titers of original H5N1 isolates in mouse lungs (A) and
brains (B). Virus titers were determined in ca. 10% tissue homoge-
nates prepared in PBS with antibiotic-antimycotic solution. Mice were
inoculated intranasally with 0.1 ml of PBS-diluted allantoic fluid con-
taining ca. 106.5 to 107.5 EID50 of infective virus. Lungs were obtained
from mice sacrificed on day 3 after inoculation; brains were removed
from mice showing signs of CNS disorders (hind-leg paralysis, tremor)
that died or were sacrificed on days 8 to 14 after inoculation. ❋, Virus
of genotype A was isolated from the brain of one mouse only.
paralysis and were dead or were sacrificed on days 7 to 10 after
inoculation. Twenty-nine percent of the dead (27.7% of in-
fected) animals had virus in the brain (Table 1). Virus of
genotype C replicated in mouse lungs and brain at similar
titers, which were the highest in comparison with those of the
other genotypes (Fig. 3).
The third group included viruses of genotypes D and E,
which also replicated in the lungs and brain of infected mice
(Fig. 3) but were more neurotropic than other genotypes. All
mice that died after infection with these viruses had virus in the
brain. The mortality rates for mice infected with Ck/HK/
FY150/01 (genotype D) and Ck/HK/NT873.3/01 (genotype E)
were 61.5 and 33.3%, respectively (Fig. 2). Mice infected with
viruses of genotypes D and E exhibited hind-leg paralysis,
paresis, and tremors before death 10 to 14 days after inocula-
tion with the virus.
Pathogenicity and organ tropism of H5N1 variants isolated
from mouse brain. Neurotropic variants of the original viruses
of genotypes A, C, D, and E (MB variants) were isolated from
the brain by culturing virus-positive brain homogenate in 10-
day-old embryonated hens’ eggs for one passage. The EID50
and MLD50 values were determined as described above. For
VOL. 77, 2003 NOTES 3819

TABLE 2. Pathogenicity of MB variants of H5N1 viruses in

experimentally infected micea
Virus detectedc in:
MLD50b
Virus Genotype Internal
(EID50) Lungs Brain
organs

Ck/HK/YU822.2/01-MB A 101.0 ⫹ ⫹ ⫹
Ph/HK/FY155/01-MB C 100.25 ⫹ ⫹ ⫹
Ck/HK/FY150/01-MB D 101.25 ⫹ ⫹ ⫺
Ck/HK/NT873.3/01-MB E 101.5 ⫹ ⫹ ⫺
a
Neurotropic variants of the original viruses (MB variants) were isolated from
the brain by one passage of virus-positive brain homogenate in 10-day-old em-
bryonated hens’ eggs. Brains, lungs, and internal organs (liver, spleen, and
kidney) were removed from mice that died or were sacrificed at the terminal
stage of illness. Mice were inoculated with 0.1 ml of PBS-diluted allantoic fluid
containing 101.5 to 102.5 EID50s of MB variant virus.
b
MLD50 values are shown as the number of EID50s resulting in 50% mortality
in mice.
c
Virus was detected in all dead or sacrificed mice (⫹) or was not detected (⫺).

studies of pathogenicity and organ tropism, groups of four

animals were intranasally inoculated with 0.1 ml of PBS-di-

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luted allantoic fluid containing 101.5 to 102.5 EID50 of infective
virus. Brain, lungs, and internal organs were removed from
mice that died or that were sacrificed at the terminal stage of
illness.
The MB variants, designated Ck/HK/YU822.2/01-MB, Ph/HK/
FY155/01-MB, Ck/HK/FY150/01-MB, and Ck/HK/NT873.3/01-
MB, were markedly more virulent than the original viruses
(Table 2). These variants had MLD50 values similar to their
EID50 values, which indicates that these viruses are highly
pathogenic for mice.
The MB variants fell into one of two groups according to
their organ tropism. The first group included the highly patho-
genic variants of genotypes A and C, which replicated system-
ically and infected brain and internal organs (Table 2). High
infective doses of these MB variants resulted in death within 3
to 5 days; lower doses killed the mice 6 to 11 days after inoc-
ulation. Inoculation with doses of 102.5 to 100.5 EID50 of either
virus caused neurologic signs such as hind-leg paralysis,
tremor, and paresis. Viral titers of the MB variants of geno-
types A and C in liver, spleen, kidney, and heart were at least
3.5 to 2.0 log10 lower than those in lungs and brain (Fig. 4),
where these variants replicated efficiently; this result suggests
that these MB variants were pneumotropic and neurotropic.
The second group included MB variants of genotypes D and
E, which were detected only in lungs and brain of infected
mice. The genotype D variant killed mice 4 to 8 days after
inoculation and 101.25 EID50 of infective virus caused hind-leg
paralysis and paresis on day 8 after inoculation. MB variant of
genotype D was detected in lungs and brain of all dead or
sacrificed mice (Table 2). The pathogenicity of Ck/HK/
NT873.3/01-MB (genotype E) was similar to that of the geno-
type D variant: most animals were dead at day 9, and neuro-
logic signs were observed in mice infected with doses of 103.25,
102.25, and 101.25 EID50. Virus of genotype E was isolated from
the lungs and brain of each dead or sacrificed animal (Table 2).
The titers of the genotype D and E variants in lungs and brain
were similar (Fig. 4).
Histopathologic analysis. To localize the pathological
changes in the CNS that caused the neurologic signs, samples
FIG. 4. Titers of MB variants of H5N1 virus in the lungs (A) and
brains (B) of infected mice. Mice were intranasally inoculated with 0.1
ml of PBS-diluted allantoic fluid containing 101.5 to 102.5 EID50 of
infective virus. Virus titers were determined in approximately 10%
tissue homogenates prepared in PBS with antibiotic-antimycotic solu-
tion. The lungs and brains were removed from mice that were sacri-
ficed at the terminal stage of illness on day 8 after infection.
of brain and spinal cord from mice infected with Ck/HK/
YU822.2/01-MB (genotype A) and sacrificed at the terminal
stage of disease were fixed in 10% neutral buffered formalin,
embedded in paraffin wax, sectioned at 5 ␮m, stained with
hematoxylin and eosin, and evaluated for histopathologic al-
terations. Foci of neuronal degeneration and neuronophagia
associated with inflammatory infiltrates of granulocytes and
mononuclear cells were restricted to the brain stem and spinal
cord (Fig. 5A and B). Foci of mononuclear cell infiltrates were
also seen in the meninges and in the perivascular spaces of the
brain stem and spinal cord neuropil (Fig. 5C). In the spinal
cord, there was also an occasional degenerating glial cell, and
there were neurons with marginated chromatin and an eosin-
ophilic inclusion that partially or completely filled the nucleus
(Fig. 5A and D). Necrotic ependymal cells were scattered
among the ependyma lining the central canal of the spinal cord
(Fig. 5A). Neuronal degeneration and inflammatory infiltrates
were also evident in some dorsal root ganglia, and there were
many foci of necrosis and inflammatory cells associated with
the perivertebral adipose tissue (Fig. 5D).
Plaque morphology. One of the important characteristics of
influenza viruses is their ability to form plaques in cultured
cells. Like pathogenicity, this feature can be changed during
selection in mice. Plaque morphology can illustrate the heter-
3820 NOTES J. VIROL.

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FIG. 5. Hematoxylin-and-eosin-stained sections of spinal cords and brain stems from mice infected with Ck/HK/YU822.2/01-MB. Brains and
spinal cords were collected from mice sacrificed at the terminal stage of illness on days 10 and 11 after intranasal inoculation with 10 EID50 of virus.
(A) Karyorrhexis in two degenerating ependymal cells lining the spinal cord’s central canal (two split arrows). An intranuclear inclusion is present
in a neuron (broken arrow) and in degenerating glial cells (solid single arrows). The inset shows a section from a deeper part of the same tissue.
(B) Inflammatory cells and a focus of degenerating neurons undergoing neuronophagia (arrows) in the spinal cord of a mouse with hind-leg
paralysis. (C) Inflammatory cells in the meninges and white matter of the spinal cord and (inset) in the perivascular (Virchow-Robin) space of the
medulla oblongata. (D) Dorsal root ganglion (arrow) with degenerating neurons, some of which have an intranuclear inclusion. In the adjacent
adipose tissue there is a focus of necrosis with inflammatory cells (arrow head). Original magnifications: (A) ⫻260, (B) ⫻260, (C) ⫻130 (inset,
⫻260), (D) ⫻130.

ogeneity of the virus population. Plaque assay was performed
in MDCK cells essentially as described previously (11). The
original viruses of genotypes A, B, C, and D formed large,
well-defined plaques characteristic of highly pathogenic avian
influenza viruses. Virus of genotype E (Ck/HK/NT873.3/01)
formed very small, turbid plaques. The plaques formed by the
original viruses were homogeneous. The plaques formed by the
MB variants were larger than those formed by the original
viruses, and the genotype E MB variant (Ck/HK/NT873.3/01-
MB) formed plaques that were better defined than those of the
original virus. No plaques like those formed by the MB variants
were observed in MDCK cells infected with the original vi-
ruses. Similar increases in plaque size were observed in MB
variants of genotypes C and D (data not shown).
Sequence comparison of original viruses and MB variants.
Studies in mice revealed that inoculation of original isolates of
H5N1/01 viruses of genotypes A, C, D, and E in mice resulted
in the selection of highly pathogenic, neurotropic variants. To
compare the molecular features of the original isolates and the
MB variants and to determine the molecular basis of mouse
virulence and neurotropism, all genes of the MB variants gen-
erated were sequenced. Viral RNA was isolated from virus-
containing allantoic fluid by using the RNeasy Mini kit (Qia-
gen, Valencia, Calif.) as specified by the manufacturer. Uni-12
primer was used for reverse transcription. PCR was performed
with the set of universal primers specific for gene segments of
influenza A viruses (16). PCR products were purified with the
QIAquick PCR purification kit (Qiagen). The sequencing re-
VOL. 77, 2003 NOTES 3821

TABLE 3. Comparison of amino acid sequences of the original viruses and their MB variantsa
PB2 sequence at amino acid: PA sequence at amino acid:
Virus (genotype)
305 307 463 627 738 10 97 212 317 331 343 349 357 361 364 373 395 501 519 528 542

HK/483/97 E A I K K N T R W N A E T K S N S Y N T V
HK/486/97 E A I E K N T R W N A E T K S N S Y N T V
Ck/HK/YU822.2/01 (A) E A I E K N T R W N A Q T R S K S Y N T I
Ck/HK/YU822.2/01-MB (A) E A I E N I T P W N A E T K G N S Y N T V
Ck/HK/YU562/01 (B)b E A I E K N T R W N A E T K G N S Y N T V
Ph/HK/FY155/01 (C) E A V E K N T R W N T E T K G N G Y N T V
Ph/HK/FY155/01-MB (C) S G I K K N T R W N A E T K G N G F N T V
Ck/HK/FY150/01 (D) E A I E K N T R W N A E T K G N S Y S T V
Ck/HK/FY150/01-MB (D) E A I E K N I R W N A E T K G N G Y N T V
Ck/HK/NT873.3/01 (E) E A I E K N T R C T A E A K G N S Y N S V
Ck/HK/NT873.3/01-MB (E) E A I E K N T R W N A E T K G N S Y N T V
a
Positions of amino acid differences observed among the original viruses and their MB variants were compared to those in phenotypes of human H5N1/97 isolates
with high and low pathogenicity in mice (13) and with those in virus of genotype B, which did not infect mouse brain. HK/483/97 and HK/486/97 are phenotypes of
Hong Kong 1997 viruses (13) of high and low pathogenicity, respectively, in mice. Amino acids in boldface indicate amino acid changes between original isolates and
MB variants.
b
Virus of genotype B not isolated from mouse brain.

action and analysis of samples were performed at the Hartwell mutations may be involved in the acquisition of high virulence
Center for Bioinformatics and Biotechnology at St. Jude Chil- and neurovirulence by MB variants, but there were no com-

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dren’s Research Hospital. DNA sequences were completed, mon patterns of such mutations observed among the MB vari-
edited, translated, and analyzed by using the Lasergene se- ants of different genotypes. The absence of such unique
quence analysis software package (DNASTAR, Madison, changes in the MB variant of genotype E can be explained by
Wis.). the initially high neurotropism and low virulence of the origi-
Phylogenetic analysis of full-length genome sequences of all nal isolate in mice (Table 1). Subsequent changes in the MB
genes of the H5N1/01 MB variants, the original viruses, and variant may have affected pathogenicity but not neuroviru-
Ck/HK/YU562/01 (virus of genotype B, which was not isolated lence.
from mouse brain) showed that the original isolates distributed The human and avian Hong Kong H5N1 viruses isolated in
into the same five genotypes (Fig. 1) as were determined pre- 1997 had the same genotype (7, 12, 15); the heterogeneity of
viously on the basis of partial gene sequences (10). The neu- their pathogenicity in mouse was a result of minor differences
rotropic variants isolated from mouse brain belonged to the in sequence of the viral genomes (13, 14, 19). In contrast, the
same set of genotypes as were initially inoculated intranasally H5N1 viruses isolated in Hong Kong poultry markets in 2001
in mice. were of five different, naturally occurring genotypes. These
Amino acid sequence variations between the original viruses H5N1/97 and H5N1/01 viruses have only the HA gene (which
and their MB variants were compared to those found between is phylogenetically close to that of the Go/Gd-like viruses) in
mouse phenotypes of human H5N1/97 isolates of high and low common (10, 33).
pathogenicity (Tables 3 to 5). There were amino acid changes The Go/Gd-like viruses isolated in Hong Kong in 1999, the
in the highly pathogenic variants in all gene products except for progenitors of the HA, NA, and some of the internal genes of
PB1, NP, and NS1 proteins. Most of these changes distin- the H5N1/01 viruses, have been shown to be of low pathoge-
guished the original viruses from the MB variants, as well as
from other viruses of high and low pathogenicity, and were not
unique. The random nature of these differences suggested that TABLE 4. Comparison of amino acid sequences of the original
viruses and their MB variantsa
the viruses were heterogeneous and indicated that rapid selec-
tion of highly pathogenic variants was possible (Tables 3 to 5). HA
NA sequence at
sequence at
This suggestion was confirmed by the heterogeneity of the Virus (genotype) amino acid:
amino acid(s):
original sequences (shown by electrophoretograms) of the PB2
50 17 20 34 Dc 70 258
and PA genes, especially in genotypes A, C, and D (data not
shown). The MB variant of genotype A (Ck/HK/YU822.2/01- HK/483/97 E V I I 53
D 71
I
53
MB) was distinguishable from the original virus by unique HK/486/97 E V I V D71 I
changes at residue 738 in polymerase PB2, residues 10 and 212 Ck/HK/YU822.2/01 (A) E V I V ND S M
49 68
Ck/HK/YU822.2/01-MB (A) K I V V D G M
in polymerase PA, residue 50 in HA, and residue 86 in NS2 Ck/HK/YU562/01 (B)b E I I A ND S I
and by deletion of 20 amino acids and a S703G substitution in Ph/HK/FY155/01 (C) E I V I ND S I
NA. The MB variant of genotype C had four unique amino Ph/HK/FY155/01-MB (C) E I V V ND S M
acid replacements: at positions 305, 307, and 627 in PB2 and at Ck/HK/FY150/01 (D) E I V V ND S I
Ck/HK/FY150/01-MB (D) E I V V ND S I
position 501 in PA. In the MB variant of genotype D there was Ck/HK/NT873.3/01 (E) E I V V ND S I
one unique substitution at residue 97 in PA, whereas the MB Ck/HK/NT873.3/01-MB (E) E I V V ND S I
variant of genotype E had no such changes (i.e., all amino acid a
See Table 3, footnote a.
differences distinguished the original virus from its MB variant b
See Table 3, footnote b.
and other viruses of high and low pathogenicity). These unique c
D, deletion; ND, no deletion.
3822 NOTES J. VIROL.

TABLE 5. Comparison of amino acid sequences of the original viruses and their MB variantsa
M2 sequence NS2
M1 sequence at amino acid: at amino sequence at
Virus (genotype) acid: amino acid
15 17 78 98 104 142 206 224 21 30 86

HK/483/97 I S R K K V A S D A R
HK/486/97 V S R K K V A S D A R
Ck/HK/YU822.2/01 (A) V A L N R V A S D A R
Ck/HK/YU822.2/01-MB (A) V S R K K V A S D A I
Ck/HK/YU562/01 (B)b I S R K K V A N G A R
Ph/HK/FY155/01 (C) I S R K K V G N D A R
Ph/HK/FY155/01-MB (C) V S R K K G A S D A R
Ck/HK/FY150/01 (D) I S R K K G A N G S R
Ck/HK/FY150/01-MB (D) I S R K K V A N D A R
Ck/HK/NT873.3/01 (E) I S R K K G A N G S R
Ck/HK/NT873.3/01-MB (E) I S R K K G A N G S R
a
See Table 3, footnote a.
b
See Table 3, footnote b.

nicity in mice: these viruses replicated efficiently in mouse Virus of genotype A is different from that of genotype B in
lungs, and only one of the isolates studied was detected, at a having PA and M genes of Go/Gd-like phylogenetic lineage

Downloaded from jvi.asm.org by on December 12, 2008

very low titer, in the brain (10, 33). We have found that most
of the original H5N1/01 viruses, like the Go/Gd-like H5N1/99
viruses, were of low pathogenicity in mice and were pneumo-
tropic. However, unlike the Go/Gd-like viruses, H5N1/01 vi-
ruses of four of the five genotypes (A, C, D, and E) were
detected in mouse brain. Virus-specific lesions in the brain
stem and spinal cord and inflammatory lesions in dorsal root
ganglia (Fig. 5) indicated that these neurotropic MB variants
had spread from the lungs to the brain trans-synaptically, via
sensory nerves. A neuronal route of viral transmission has also
been shown in mice infected with avian H5N3 viruses that
adapted in chickens and became highly virulent (25, 26). The
virus of genotype B replicated efficiently in mouse lungs and
caused some mortality in infected mice (Table 1, Fig. 1), but in
contrast to viruses of genotypes A, C, D, and E, genotype B
virus was not isolated from the brain.
Inoculation of mice with the MB variants isolated from the
brain showed that these variants are highly pathogenic in mice
and have at least the same pathogenicity in mice as do the
highly pathogenic phenotypes of the H5N1/97 viruses. The
selection of the four neurotropic variants of the five H5N1/01
genotypes occurred rapidly during replication in mice—the
highly pathogenic MB variants were generated during only one
passage of the viruses. Such rapid selection was probably fa-
cilitated by the unusually long persistence of these viruses in
the lungs (we isolated virus from mouse lungs at days 8 to 12
after inoculation). Our findings indicate that the combination
of internal genes in the H5N1/97 viruses, which were removed
from circulation by slaughter of all poultry in 1997, is not the
only combination of genes that could result in high pathoge-
nicity of these viruses in poultry and mammals: multiple dif-
ferent internal gene segments from viruses currently circulat-
ing in Southeastern China can complement Go/Gd-like HA to
generate viruses that are highly pathogenic and neurotropic in
the mammalian host. Therefore, the HA of Go/Gd-like viruses,
which has multiple basic cleavage sites, may play a key role in
the pathogenicity of avian Hong Kong H5N1 viruses in mam-
malian species only when it is complemented by an appropriate
combination of internal genes.
(Fig. 1). Genotype C contains PB1 and NP genes from Go/Gd-
like viruses; these genes differentiate genotype C from geno-
types A and B (Fig. 1). Genotypes D and E differ from each
other and genotype C in their NP gene only. The virus of
genotype B contains HA and NA genes that are phylogeneti-
cally close to those of Go/Gd-like viruses; its remaining genes
are of unknown wild aquatic bird lineage (Fig. 1). We suggest
that the virus population of genotype B is homogeneous; there
were no highly pathogenic variants of this genotype selected
during one passage in mice. The HA of genotype B virus,
which has multiple basic cleavage sites and which originated
from Go/Gd-like viruses, was not alone sufficient to endow the
virus with high pathogenicity and neurotropism in mice, at
least during one passage. In contrast to the population of
genotype B virus, the populations of viruses of genotypes A, C,
D, and E are heterogeneous; one passage of these viruses in
mice resulted in the selection of highly pathogenic variants.
Sequence comparisons between the MB variants and the
phenotypes of Hong Kong H5N1/97 viruses that were highly
pathogenic for mice (14, 17) and mouse-adapted viruses (2, 31,
32) did not reveal any common sets of mutations. Only the
substitution E6273K in PB2, observed in the MB variant of
genotype C, was similar to that responsible for the high viru-
lence of HK/483/97 virus (13), but this mutation was found only
in genotype C virus. The absence of a common set of mutations
in the highly pathogenic, neurotropic variants suggests that
there are many different ways for Hong Kong H5N1/01 influ-
enza viruses to achieve high virulence and neurotropism in
mice. The essential condition for the neurovirulence of Hong
Kong H5N1/01 viruses in mice and the rapid selection of highly
pathogenic phenotypes in the new host may be a particular
constellation of replicative complex genes. Multiple basic
cleavage sites in the HA are necessary, but not enough, to
make these viruses highly virulent and neurovirulent in mice:
specific changes in polymerase proteins PB2 and PA are im-
plicated in this process. Therefore, we speculate that the se-
lection of highly pathogenic neurotropic variants during natu-
ral reassortment is facilitated by viral heterogeneity arising
from the insertion of PA (genotype A) and PB2 (genotypes C,
VOL. 77, 2003
D, and E) genes of Go/Gd-like origin into the replicative
complex background of another phylogenetic lineage.
It is of questionable validity to connect the pathogenicity of
Hong Kong H5N1 viruses in mice with their potential patho-
genicity in humans and other mammalian species. However,
the rapidity of selection of neurotropic variants in mice raises
concern about the possibility of a similarly rapid selection of
variants that are pathogenic for other mammals. The decision
to slaughter poultry in Hong Kong in 2001 was a useful mea-
sure to eradicate these viruses and prevent the selection of
such highly pathogenic variants.
Nucleotide sequence accession numbers. Nucleotide se-
quences obtained in the present study have been deposited in
the GenBank database under the accession numbers
AY221521 to AY221592.
These studies were supported in part by Public Health Service grants
AI29680 and AI95357, by Cancer Center Support (CORE) Grant
CA-21765 from the National Institutes of Health, and by the American
Lebanese Syrian Associated Charities.
We thank Richard J. Webby for critical reading and helpful discus-
sion, Patrick Seiler for excellent technical assistance, and Janet R.
Davies for editorial assistance.
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