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Journal of Virology, November 2000, p. 10807-10810, Vol. 74, No. 22
Influenza Branch, Division of Viral and
Rickettsial Diseases, National Center for Infectious Diseases,
Centers for Disease Control and Prevention, Atlanta, Georgia 30333
Received 12 June 2000/Accepted 23 August 2000
Highly pathogenic avian influenza A H5N1 viruses caused an outbreak
of human respiratory illness in Hong Kong. Of 15 human H5N1 isolates
characterized, nine displayed a high-, five a low-, and one an
intermediate-pathogenicity phenotype in the BALB/c mouse model.
Sequence analysis determined that five specific amino acids in four
proteins correlated with pathogenicity in mice. Alone or in
combination, these specific residues are the likely determinants of
virulence of human H5N1 influenza viruses in this model.
The molecular determinants and
related mechanisms that make certain influenza viruses highly
pathogenic for mammalian species, including humans, remain poorly
understood. Both viral factors and host factors may determine
virulence. Numerous studies have shown that influenza virus virulence
in mammalian species is a polygenic trait, which may require a critical
constellation of genes (4, 21, 32, 33). The phenotypes of
virulence and pathogenicity are distinct from that of host range.
Influenza viruses that infect one species, such as birds, are often
restricted in their ability to replicate in other host species, such as
humans, by host range determinants. However, in 1997, highly pathogenic avian H5N1 viruses infected poultry in the live-bird markets of Hong
Kong and caused an outbreak of 18 human cases of respiratory illness,
including six deaths (6, 8, 30, 34). The majority of
individuals who experienced severe illness or died from the H5N1
infection were 13 to 60 years old and had no known risk factors for
complications from influenza (5).
The H5N1 viruses are the only highly pathogenic avian viruses that have
been documented to cause an outbreak of respiratory disease in humans.
An earlier study of humans exposed to chickens infected with an H5N2
virus failed to find any evidence of human infection with this highly
pathogenic avian virus (2). The 16 H5N1 viruses isolated
from humans during the 1997 outbreak had avian virus genomes; the
hemagglutinin (HA) and neuraminidase (NA) genes showed no evidence of
adaptive change for humans (3). The outbreak created a new
awareness that avian influenza viruses could spread directly from
poultry to humans and cause severe respiratory disease in humans, but
the molecular basis of the H5N1 virus virulence in humans was not evident.
The BALB/c mouse was previously shown to be a useful mammalian model
for the evaluation of human H5N1 virus pathogenesis (11, 13,
17). H5N1 viruses replicate efficiently in the respiratory tract
of mice without prior adaptation. Viruses exhibiting high lethality
(pathogenicity) replicated in extrapulmonary sites, including the
brain, while growth of viruses of low lethality was restricted to the
respiratory tract of mice (11, 17). All 16 human H5N1
viruses possessed a multiple basic amino acid motif at the cleavage
site between HA1 and HA2 and were lethal for experimentally infected
chickens (3, 6, 11, 27, 30). The basic amino acid motif, a
key molecular feature of avian viruses of the H5 and H7 subtypes that
are highly pathogenic for chickens (23), allows the HA to be
cleaved by ubiquitous proteases of the subtilisin family
(26) and enables these viruses to replicate systemically in
birds. The fact that human H5N1 viruses of low pathogenicity for mice
possessed the motif but did not replicate systemically or cause lethal
disease in mice (11, 17) suggested that other molecular
features are associated with the high pathogenicity of H5N1 viruses in
mammalian species. We now investigate the molecular determinants
that distinguish 15 H5N1 viruses of high and low pathogenicity in mice.
The origin and outcome of human disease associated with 15 H5N1 viruses
isolated from confirmed cases in Hong Kong in 1997 and the antigenic
profile and relative pathogenicity of the viruses for mice are shown in
Table 1. Viruses were grown in
Madin-Darby canine kidney (MDCK) cells and/or the allantoic cavity of
10-day-old embryonated hens' eggs at 37°C for 24 h. Fifty
percent egg infectious dose (EID50) titers were determined
by serial titration of viruses in eggs and were calculated by the
method of Reed and Muench (20). The 50% lethal dose
(LD50) of the viruses for 6- to 8-week-old female
BALB/c mice (Charles River Laboratories, Wilmington, Mass.) was
determined as previously described (17) and used as a marker for pathogenicity. LD50 titers were expressed as the
EID50 value corresponding to 1 LD50. Viruses
with an LD50 of >106.5 were considered to be
of low pathogenicity, while viruses with an LD50 of
<103.0 were considered to be of high pathogenicity.
0022-538X/00/$04.00+0
Molecular Correlates of Influenza A H5N1 Virus Pathogenesis
in Mice
ABSTRACT
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TABLE 1.
Characteristics and mouse pathogenicity phenotypes of
influenza A H5N1 viruses isolated from humans
Previously, the H5N1 viruses were distinguished into two antigenically and genetically distinct subgroups based on the absence (group A) or presence (group B) of a potential N-linked glycosylation site at residues 154 to 156 (H3 numbering) in the HA1 region of the HA molecule (3, 27). While all five viruses of low pathogenicity for mice belonged to group A, the nine viruses that were highly pathogenic for mice included all seven group B viruses and two group A viruses. A/Hong Kong/156/97 (HK/156), a group A virus, gave an intermediate phenotype of pathogenicity in mice (Table 1). Therefore, the presence or absence of the glycosylation site in HA1 alone could not explain the differences in pathogenicity observed in mice.
The complete nucleotide sequences for all coding regions of all gene
segments of nine of the H5N1 viruses were determined by direct cycle
sequencing of PCR products generated by reverse transcription-PCR from
MDCK cell-passaged (XC1 or XC2) stocks by using gene-specific primer
sets as previously described (3; M. Shaw, L. Cooper,
X. Xu, et al., submitted for publication). This analysis identified
five residues that segregated with the mouse pathogenicity phenotype in
genes that encoded the NA, matrix (M1) protein, and viral polymerases
PB1 and PB2. To confirm this finding, partial sequence analysis was
conducted on these four gene segments from the same virus stocks that
were used to determine the pathogenicity phenotype in mice (Table
2). Amino acid residues I or T at residue
223 in the NA, K or R at 198 and I or M at 317 in PB1, and K or Q at
355 in PB2 correlated with high and low pathogenicity, respectively, in
all 15 viruses analyzed. The amino acid at position 15 (I or V) in the
M1 protein correlated with high (I) and low (V) pathogenicity in 14 of
the H5N1 viruses. HK/485/97, a virus of high pathogenicity, possessed a
unique codon (ACC) relative to all other H5N1 viruses analyzed,
encoding a threonine at position 15 in the M1 protein. This result
suggests either that threonine at this position is permissive for the
high-pathogenicity phenotype or that substitution of this residue alone
is insufficient to alter the mouse pathogenicity phenotype of the
virus.
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Amino acid 223 in the N1 NA corresponds to residue 222 of N2 NA, a conserved framework residue in the enzyme active site in the head of the NA molecule (7). H5N1 viruses of the low-pathogenicity phenotype possess a threonine at this position, creating a potential glycosylation site (N-X-T) in the enzyme active site. Previously, the neurovirulence of mouse-adapted A/WSN/33 virus was correlated with the loss of a glycosylation site at residue 146 in the head of the N1 NA (16). Goto and Kawaoka (12) reported that the absence of this glycosylation site together with the presence of a carboxyl-terminal lysine (residue 453) was associated with the binding of plasminogen by the NA, which facilitated and enhanced HA cleavage and conferred a broader tissue tropism on this virus. However, the molecular features important for this mechanism of virulence have been observed only in the laboratory-derived A/WSN/33 virus and not in any wild-type viruses, including the H5N1 viruses. Other studies have suggested a role for the NA in influenza virus-induced apoptosis, which has been implicated as a mechanism of pathogenicity among influenza viruses (18, 22). Interestingly, the highly pathogenic H5N1 virus HK/483/97 induces peripheral blood lymphocyte depletion and apoptosis in the spleens and lungs of infected mice, whereas HK/486/97, a virus of low pathogenicity, does not (31). Because the specific residues in the PB1, PB2, and M1 proteins that correlated with mouse pathogenicity were not located in any of the defined functional domains of these proteins, their contribution to the mechanism(s) of virulence remains unknown.
Because the human H5N1 viruses are a genetically closely related group of viruses (3, 15), it was possible to associate the five specific molecular markers in the NA, PB1, PB2, and M1 genes with the two distinct phenotypes of pathogenicity observed in mice. To investigate the prevalence of these specific residues in other influenza A viruses, nucleotide sequence alignments for the four gene products were performed using available sequence data (downloaded from the GenBank viral nucleotide database in March and April 2000), including sequences from human, avian, swine, and equine influenza A viruses. No significant distribution of the specific residues associated with high or low pathogenicity for mice in this study was observed, most likely because of the relatively low genetic relatedness of influenza A viruses from different species. Nevertheless, the analysis revealed that residues Thr-223 in the NA and Arg-98 in PB1 were unique to the H5N1 viruses of low pathogenicity and that Glu-355 in PB2 was found only in the human H5N1 viruses of low pathogenicity and in four avian H5N1 viruses isolated from birds in Hong Kong in 1997.
In earlier studies, investigators compared wild-type viruses with either mouse-adapted virus or reassortants derived from parental viruses of distinct pathogenicity phenotypes. In mice, pneumovirulence and neurovirulence were associated with the HA, NA, M, and one or more polymerase genes (4, 21, 32, 33). However, the specific amino acid residues identified in the NA, M1, PB1, and PB2 genes in the present study have not been previously associated with pathogenicity. It is possible that additional molecular markers for pathogenicity were not detected because the findings from complete sequence analysis of nine of the human H5N1 viruses were used to direct partial analysis of the remaining viruses. Mutations in the polymerase genes of influenza viruses have previously been reported to determine host range (1, 29), temperature sensitivity, and, in some instances, attenuation of influenza viruses for mice, ferrets, and humans (25, 28). Mutations in the M protein have been associated with host range, growth, and virulence phenotypes. Growth properties of influenza viruses can be determined by specific mutations in individual gene segments or by the constellation of genes present in the virus.
Although HK/156/97 was shown to be of intermediate pathogenicity in this study, the genotype was that of the high-pathogenicity viruses, a finding consistent with the results of others who have characterized HK/156/97 as being highly pathogenic for BALB/c mice (9, 11, 13, 24). The molecular basis for the apparent attenuation of virulence of the HK/156/97 virus used in this study is unknown, but biological and molecular heterogeneity of this virus isolate has been reported (11, 14). Passage of HK/156/97 in mice was shown to increase the virulence of the virus (13, 14, 24). One other virus, HK/482/97, consistently yielded an indeterminate mouse pathogenicity phenotype and a genotype that consisted of a mixture of the residues associated with high and low pathogenicity in M1, PB1, and PB2. Gao et al. (11) found that HK/482/97 virus passaged exclusively in MDCK cells exhibited low pathogenicity in mice. Therefore, it is possible that the original HK/482/97 isolate, like HK/156/97, was biologically heterogeneous.
While two distinct pathogenicity phenotypes were observed in this
inbred mouse model, a broader spectrum of pathogenicity was observed in
humans infected with the H5N1 viruses. In particular, the age of an
infected individual was an important factor associated with severity of
disease (34). Some of the deaths associated with H5N1
infections occurred late in the course of hospitalization, following
extended periods of mechanical ventilation and other complications. The mouse pathogenicity phenotype of four
viruses failed to correlate with the severity of disease observed in
humans. Three of the viruses that were highly pathogenic for mice were isolated from children 
4 years of age who had mild disease. One virus
of low pathogenicity for mice was isolated from a 34-year-old woman
with systemic lupus erythematosus who succumbed to a lethal H5N1
infection. Therefore, in addition to the general virulence of the H5N1
viruses, age, underlying medical conditions, and other unknown risk
factors may have contributed to the severity of disease in humans.
Nevertheless, the fact that H5N1 viruses of high
pathogenicity induced symptoms of disease similar to those observed in
severe and fatal human cases, including viral pneumonia,
multiorgan involvement, leukopenia, and death, suggests that
the mouse is an appropriate model with which to better understand the
molecular basis of influenza virus virulence in mammalian species.
However, at present it is not possible to distinguish between molecular
determinants responsible for general virulence in mammals and those
responsible for specific virulence in mice.
The use of plasmid-based reverse genetics techniques (10, 19) will enable an evaluation of the contribution of each of the specific amino acid residues identified here, either alone or in various combinations, to the pathogenicity phenotype in mice. While it is likely that the polygenic nature of pathogenicity differs among influenza viruses and among host species, the molecular determinants of pathogenicity in this mammalian model may provide a framework for the future identification of influenza A viruses with the potential to cause severe disease.
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ACKNOWLEDGMENTS |
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We thank the Epidemiology Section of the Influenza Branch, CDC; Paul Saw, K. H. Mak, Wilina Lim, and others from the Hong Kong Department of Health for the acquisition of specimens and isolation of the H5N1 viruses; Xiyan Xu, Sarah Cantrell, Mark Hemphill, and Alexander Klimov for contributing to the sequence analysis; and Nancy Cox for critical review of the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Influenza Branch, Mailstop G-16, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333. Phone: (404) 639-3591. Fax: (404) 639-2334. E-mail: JKatz{at}cdc.gov.
Present address: USDA/ARS/Southeast Poultry Research Laboratories,
Athens, Ga.
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Journal of Virology, November 2000, p. 10807-10810, Vol. 74, No. 22
0022-538X/00/$04.00+0
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Hatta, M., Gao, P., Halfmann, P., Kawaoka, Y.
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