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Journal of Virology, February 1999, p. 1146-1155, Vol. 73, No. 2
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Surface Glycoproteins of H5 Influenza Viruses Isolated from
Humans, Chickens, and Wild Aquatic Birds Have Distinguishable
Properties
Mikhail
Matrosovich,1,2,*
Nannan
Zhou,1,3
Yoshihiro
Kawaoka,4 and
Robert
Webster1,5
Department of Virology and Molecular Biology,
St. Jude Children's Research Hospital, Memphis, Tennessee
381051;
M. P. Chumakov Institute of
Poliomyelitis and Viral Encephalitides, 142 782 Moscow,
Russia2;
Department of Microbiology,
Jiangxi Medical College, Nanchang 330006, China3;
Department of Pathobiological
Sciences, School of Veterinary Medicine, University of
Wisconsin
Madison, Madison, Wisconsin 537064;
and
Department of Pathology, University of Tennessee at
Memphis, Memphis, Tennessee 381635
Received 7 August 1998/Accepted 20 October 1998
 |
ABSTRACT |
In 1997, 18 confirmed cases of human influenza arising from
multiple independent transmissions of H5N1 viruses from infected chickens were reported from Hong Kong. To identify possible phenotypic changes in the hemagglutinin (HA) and neuraminidase (NA) of the H5
viruses during interspecies transfer, we compared the receptor-binding properties and NA activities of the human and chicken H5N1 isolates from Hong Kong and of H5N3 and H5N1 viruses from wild aquatic birds.
All H5N1 viruses, including the human isolate bound to Sia2-3Gal-containing receptors but not to Sia2-6Gal-containing receptors. This finding formally demonstrates for the first time that
receptor specificity of avian influenza viruses may not restrict initial avian-to-human transmission. The H5N1 chicken viruses differed
from H5 viruses of wild aquatic birds by a 19-amino-acid deletion in
the stalk of the NA and the presence of a carbohydrate at the globular
head of the HA. We found that a deletion in the NA decreased its
ability to release the virus from cells, whereas carbohydrate at the HA
head decreased the affinity of the virus for cell receptors. Comparison
of amino acid sequences from GenBank of the HAs and NAs from different
avian species revealed that additional glycosylation of the HA and a
shortened NA stalk are characteristic features of the H5 and H7 chicken
viruses. This finding indicates that changes in both HA and NA may be
required for the adaptation of influenza viruses from wild aquatic
birds to domestic chickens and raises the possibility that chickens may
be a possible intermediate host in zoonotic transmission.
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INTRODUCTION |
Global outbreaks of human
influenza (pandemics) arise from influenza A viruses with novel
neuraminidase (NA) and/or hemagglutinin (HA) molecules
to which humans have no immunity. These pandemic strains derive from
viruses of wild aquatic birds by interspecies transmission of the
whole virus or by genetic reassortment between avian and human viruses
(reviewed in reference 44).
Avian influenza viruses do not replicate efficiently in humans. For
example, high doses of virus were found to be required for the
replication of avian influenza virus strains in volunteers even at a
limited level (3), and no cases of influenza virus infections were documented in workers exposed to highly pathogenic avian viruses during the 1985 outbreak in poultry in the United States
(2). It is believed that a growth restriction of avian influenza viruses in humans limits the emergence of new pandemic strains. HA and NA are regarded as possible restrictive factors because
of the differences between influenza virus receptors on the target
cells of birds and humans. For example, the HAs of avian viruses bind
to Sia2-3Gal-terminated sialylglycoconjugates and not to
Sia2-6Gal-containing receptors, whereas those of human viruses
display the opposite receptor-binding specificity (reviewed in
reference 32; see also references
9, 18, and
27). The viral NA removes sialic acid from the HA
and NA of progeny virus particles, intercellular
glycoproteins, and host cell receptors, and thus
facilitates virus release from infected cells and from intercellular
inhibitors. The specificity of the NA has to match the specificity of
the HA. Thus, the NA of the N2 avian virus cannot hydrolyze the
Sia2-6Gal linkage, but it acquired this ability during evolution in
humans (1). A poor match between the HA and NA can serve as
a potential barrier to the reassortment of influenza viruses
(35). Because of these host-range restrictions, it has not
been clear whether an avian virus can be directly transmitted to humans
and start a new pandemic. Scholtissek et al. (38) hypothesized that the pig may serve as a mixing vessel for the reassortment of avian and human viruses. Moreover, adaptation of the
avian virus to pigs leads to changes in the specificity of receptor
binding (22, 34), thus potentially facilitating transmission
of the virus to humans.
In 1997, 18 Hong Kong residents were infected with H5N1 influenza
viruses, which proved closely related to the H5N1 avian viruses that
had caused influenza outbreaks in chickens in Hong Kong at the
beginning of 1997 (7, 11, 41). Subsequent surveillance of
live bird markets in Hong Kong indicated wide dissemination of H5N1
viruses, contributing to the decision to depopulate all birds in these
markets. No additional human cases have been reported through July
1998. This incident demonstrated, for the first time, that avian
viruses can cause severe disease in humans without reassortment with
human viruses and without any apparent intermediate mammalian host.
The HA and NA of the human H5N1 isolates have been sequenced and
compared with those of H5 chicken and duck viruses (7, 40).
All avian and human isolates from live bird markets in Hong Kong 1997 contained multiple basic amino acids at the cleavage site of the HA, a
feature known to be associated with high levels of virulence among
avian influenza viruses. Two variants of the chicken and human viruses
were identified that differed by the presence or absence of a
glycosylation sequon at the top of the HA head (position 158 by H3
numbering), this sequon was shown to be glycosylated (40).
The NA of chicken viruses and of HK/156/97 human virus contained a
19-amino-acid deletion in a stalk region (7).
In this study, we sought to identify and characterize possible
phenotypic changes in the surface glycoproteins of H5N1
influenza viruses that accompanied their transmission from wild aquatic birds to chickens and from chickens to humans. In particular, we asked:
Did H5N1 viruses from chickens and humans acquire the ability to
recognize Sia2-6Gal determinants that are believed to be the major
sialyloligosaccharide determinants on ciliated cells of human
respiratory epithelia? What effect does the carbohydrate at position
158 have on the receptor-binding properties of the viruses? What is the
effect of the deletion in the NA on the enzyme activity? Do additional
glycosylation of the HA and deletion in the NA represent unique
features of 1997 Hong Kong H5N1 virus lineage, or do they also occur in
other influenza viruses? Thus, we compared the receptor-binding
properties and NA activity of the first human virus isolate, A/Hong
Kong/156/97, with those of chicken isolates identified in Hong Kong in
1997, and of the H5 isolates from aquatic birds. We also compared the
HA and NA amino acid sequences of H5 and H7 chicken viruses with those
of viruses from other hosts.
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MATERIALS AND METHODS |
Viruses.
The isolation and characterization of the 1997 H5N1
human and chicken influenza viruses (A/HK/156/97, ck/HK/258/97,
ck/HK/220/97, ck/HK/728/97, ck/HK/786/97, and ck/HK/915/97) were
described earlier (7, 40). Other influenza A virus strains
were from the repository at St. Jude Children's Research Hospital. All
viruses were grown in 9- to 10-day-old chicken eggs. Because of their
high pathogenicity, chicken and human H5N1 isolates were inactivated by
treatment with 
-propiolactone (BPL; 1/2,000) for 3 days at 4°C. To
assess the effect of inactivation on the properties of the viruses, we prepared three nonpathogenic duck H5 viruses (dk/HK/205/77,
dk/HK/698/79, and dk/Minnesota/1525/81) and divided them into two
portions; one was treated with BPL, and the other was used as a
control. None of other viruses used were inactivated. The allantoic
fluids were clarified by low-speed centrifugation, and the viruses were pelleted by high-speed centrifugation, resuspended in 0.1 M Tris buffer
(pH 7.2) containing 50% glycerol, and then stored at 
20°C.
Peroxidase-labeled glycoproteins.
Bovine fetuin
and type III-O chicken egg white ovomucoid were purchased from Sigma.
Pig 
2-macroglobulin (PM) was isolated from total pig
serum by gel chromatography on a Sephacryl S-300 column. To increase
the binding affinity of ovomucoid to the virus, the
glycoprotein was aggregated by heating of its 5% water
solution at 95°C for 2 h. Conjugates with horseradish peroxidase
(HRP) were prepared from PM, fetuin, and heat-aggregated ovomucoid by using the periodate method described by Boorsma and Streefkerk (5).
To selectively destroy minor amounts of Sia2-3Gal moieties present in
PM-HRP conjugate, we treated it with the avian influenza virus N2 NA,
which shows strict specificity for cleavage of this moiety and does not
substantially affect the Sia2-6Gal sequence (1).
Concentrated avian influenza virus A/mallard/NY/6750/78 (H2N2) was
added to the PM-HRP conjugate in 0.1 M Tris buffer containing 10 mM
CaCl2 to a final hemagglutination titer of 1:80. The
mixture was incubated at 37°C for 4 h and centrifuged at 14,000 rpm for 10 min, and the precipitate was then discarded. Stocks of all
conjugates were stored at 20°C in 50% glycerol-0.1 M Tris buffer
(pH 7.3).
Binding of HRP-labeled sialylglycoproteins.
The
binding of the HRP-labeled sialylglycoproteins to the
viruses was evaluated in a solid-phase assay as described earlier (17). In brief, 96-well polyvinyl chloride microplates
(Costar) were coated with fetuin (10 µg/ml in phosphate-buffered
saline [PBS], 50 µl/well) at 4°C overnight, washed with water,
and air dried. Purified viruses diluted with PBS to a hemagglutination titer of 1:20 were adsorbed to the wells of fetuin-coated plates at
4°C overnight (40 µl/well). After unbound virus was removed with
washing buffer (WB, 0.01% Tween 80 in 0.2× PBS), twofold dilutions of
the HRP-labeled sialylglycoproteins (30 µl/well) were
added to the plate. The dilutions were prepared in reaction buffer (RB;
PBS supplemented with 0.02% bovine serum albumin, 0.02% Tween 80, and
1 µM of the NA inhibitor zanamivir
[2,3-didehydro-2,4-dideoxy-4-guanidino-N-acetyl-D-neuraminic acid; GG167], kindly provided by R. Bethell, Glaxo Wellcome). After
incubation for 1 h at 4°C, the plates were washed with WB, and
the amount of labeled sialylglycoprotein bound was
quantified by evaluating the peroxidase activity present in the wells
by using the standard o-phenylenediamine chromogenic substrate.
The binding data were converted to
A490/C versus
A490 Scatchard plots, where
A490 represents the absorbancy of the colored
product of peroxidase reaction and C represents the concentration
of
labeled sialylglycoprotein added to the corresponding
wells.
Because we were interested only in the relative binding affinity
of different viruses for the same receptor analog, the concentration
of
the lowest dilution of each HRP-labeled sialylglycoprotein
was arbitrarily taken to be 1 U, without any regard to the actual
content of the protein, sialic acid, or peroxidase. The dissociation
constants of the virus-sialylglycoprotein complexes were
determined
from the slopes of the Scatchard
plots.
Binding of sialic acid and sialyloligosaccharides.
Free
N-acetylneuraminic acid (Neu5Ac) and 3'-sialyllactose
(3'-SL; Neu5Ac2-3Gal1-4Glc) were purchased from Sigma. The
virus-binding affinity for these receptor analogs was assessed with the
solid-phase fetuin binding inhibition assay (17, 26). This
assay is based on the competition for binding sites on the viral
particle between nonlabeled sialic acid-containing compound and
enzyme-labeled sialylglycoprotein fetuin. In brief,
influenza viruses were adsorbed in the wells of fetuin-coated wells as
described above. Twenty-five-microliter portions of solutions
containing a fixed amount of fetuin-HRP conjugate and a variable amount
of receptor analog in RB were added to the plate, which was then
incubated for 1 h at 4°C. After this competitive binding step,
the amount of fetuin-HRP bound was determined by using the
o-phenylenediamine substrate.
The association constants of the virus-receptor analog complexes were
calculated as described before (
17) for each concentration
of the compound used in the competitive reaction, and the results
were
averaged.
Virus elution from chicken erythrocytes.
The virus stocks
were diluted with PBS to a hemagglutination titer of 1:32.
Fifty-microliter portions of these solutions were mixed with equal
volumes of 1% chicken erythrocytes (CRBCs) in U-bottomed microtiter
plates. The plates were left on ice for 1 h and transferred to a
water bath (37°C), and the precipitation of the agglutinated
erythrocytes was monitored for 24 h to determine the time required
for complete deagglutination ("elution"). In parallel, the NA
activity and the agglutination of CRBC preparations treated with
variable concentrations of Vibrio cholerae NA (see below)
were determined for the same virus dilutions that were used in the
elution experiment.
NA activity of the viruses.
Viral NA activity was determined
with 4-methylumbelliferyl N-acetylneuraminic acid
(4-MU-NANA; Sigma) used as a substrate (43). To 1 or 5 µl
of virus dilutions in U-bottomed microtiter plates, we added 50 µl of
a 40 µM solution of 4-MU-NANA in calcium-TBS buffer (6.8 mM
CaCl2, 0.85% NaCl, 0.02 M Tris; pH 7.3). The mixtures were
incubated for 10 to 30 min at 37°C in a water bath, and the reaction
was stopped by the addition of 0.1 ml of 0.1 M glycine buffer (pH 10.7)
containing 25% ethanol. The fluorescence of released 4-methylumbellyferone (4-MU) was determined with a Labsystems Fluoroskan II spectrophotometer (
exc = 355 nm,
em = 460 nm). The specific NA activity was expressed in
moles of 4-MU liberated per hour per microliter of virus suspension.
Virus agglutination of CRBCs pretreated with NA.
To estimate
the ability of soluble exogenous NA to elute the virus from CRBCs and
to compare the affinity of different viruses for the receptors on
CRBCs, we assayed the effect of NA pretreatment on cell agglutination
by the viruses (45). In brief, 10% suspensions of CRBCs in
calcium-TBS buffer were incubated with twofold dilutions of V. cholerae NA (RDE; Center for Disease Control, Atlanta, Ga.) for
2 h at 37°C. The NA activity of the stock RDE preparation, determined as described above, was 3.2 nmol of 4-MU/h · µl;
final dilutions of the stock during treatment ranged from 2 to 160. Treated erythrocytes were washed with PBS. To 50-µl virus suspensions in PBS with an HA titer of 1:32, 50 µl of 1% NA-treated CRBCs suspensions were added in U-bottomed microplate, and hemagglutination was scored after 1 h of incubation of mixtures on ice. The results were expressed as the lowest activity of RDE (nanomoles of 4-MU/µl) used for the treatment that completely prevented agglutination. Both
assays that utilized CRBCs (adsorption/elution and agglutination of
NA-treated cells) were done in duplicate and on the same day. As a
rule, the HA titers of parallel probes were identical.
RNA extraction, PCR, and sequencing.
Viral RNA was extracted
from allantoic fluid with the RNeasy Mini-Kit (Qiagen, Santa Clarita,
Calif.). Amplification of the viral RNA was done by reverse
transcription PCR (RT-PCR) as described previously (39).
After being purified with the Quiquick PCR Purification Kit (Qiagen),
the PCR products were subjected to sequencing. The sequencing reactions
were performed by the Center for Biotechnology at St. Jude Children's
Research Hospital on template DNA with Prism BigDye terminator cycle
sequencing ready reaction kits with Ampli-Taq DNA polymerase FS
(Perkin-Elmer/Applied Biosystems, Inc. [PE/ABI], Foster City, Calif.)
and synthetic oligonucleotides. Samples were electrophoresed, detected,
and analyzed on PE/ABI model 373 and 377 DNA sequencers. The Wisconsin Sequence Analysis Package, version 9.0 (Genetic Computer Group, Inc.,
Madison, Wis.) was used for the analysis and translation of nucleotide
sequence data. The HA1 sequences of H5 viruses determined in this study
are available under GenBank accession numbers from AF082034 to
AF082043.
Analysis of the HA and NA amino acid sequences.
The
sequences of the influenza virus HAs and NAs were obtained from GenBank
(release 105.0) and were studied with GeneDoc 2.3 software
(28; K. B. Nicholas and H. B. Nicholas,
Jr., 1997, GeneDoc: a tool for editing and annotating multiple sequence
alignments. Distributed by the authors.
http://www.cris.com/~Ketchup/genedoc.shtml). The HA sequences
were inspected for the presence of glycosylation sequons on the
membrane distal end of the HA globular head (positions 90 to 260 of
HA1). The H3 numbering system, in accord with the alignment of Nobusawa
et al. (29), is used throughout this study. Two overlapping
sequons (for example, NNSS) were counted as one; the sequences NPT and
NPS were assumed to be nonglycosylated and were disregarded
(14).
The NA sequences were analyzed for the presence of deletions of 10 or
more amino acids in the stalk region of the enzyme,
as, starting from
residue 36 and ending with the conserved cysteine
in position 92 (
4). The N2 numbering system, in accord with
the alignment
of Colman et al. (
8), is used
here.
Phylogenetic relationships between the virus HAs and NAs were estimated
with the PHYLIP 3.572 software package (
15; J. Felsenstein,
1993, PHYLIP [Phylogeny Inference Package] version 3.5c.
Distributed
by the author. Department of Genetics, University of
Washington,
Seattle, Wash.;
http://evolution.genetics.washington.edu/phylip.html).
The trees
shown on the figures were obtained for the nucleotide
sequences of
coding regions of the whole HA1 and NA genes, respectively,
by using
the neighbor-joining algorithm and the Jukes-Cantor distance.
The
TREEVIEW 1.5.2 program (
31) was used to draw the
trees.
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RESULTS |
H5 chicken and human viruses from Hong Kong display an
avian-like receptor-binding phenotype and do not bind to
Sia2-6Gal receptor determinants.
To evaluate possible
changes in the receptor-binding specificity of chicken and human
viruses from Hong Kong, we utilized two
sialylglycoproteins, ovomucoid (Ovo) and PM, that contain Sia2-3Gal-determinants and Sia2-6Gal determinants, respectively (24, 36). Ovo and PM were labeled with peroxidase, and
their binding to the viruses was determined in a solid-phase assay.
Table
1 shows the dissociation constants
of complexes between different viruses and Ovo-HRP or PM-HRP
conjugates. Neither
virus isolate from chickens or virus isolate from
wild aquatic
birds bound PM-HRP (
Kass <0.2),
indicating the low affinity of
these viruses for the terminal Sia2-6Gal
moieties represented
in PM. In contrast, two of the earliest human
virus isolates from
the pandemics of 1957 (RI/5+/57) and 1968 (Aichi/2/68) bound PM-HRP
at least 20 times better than did
avian viruses, in accord with
the known preference of these human
strains for Sia2-6Gal determinants
(
9). The H5N1 virus
A/HK/156/97, isolated from a child in Hong
Kong, did not bind PM-HRP,
suggesting that this virus has not
yet acquired the ability to bind
Sia2-6Gal.
The binding affinity of the viruses for the Ovo-HRP conjugate varied.
Isolates from aquatic birds bound Ovo-HRP with the highest
affinity,
while human H2N2 and H3N2 strains clearly displayed
the lowest binding
(if any), in accord with the known low affinity
of human viruses for
Sia2-3Gal-terminated sequences. Among the
H5N1 viruses isolated in
1997, those with a carbohydrate at position
158 bound Ovo-HRP somewhat
more weakly than did strains without
this site
(T
160
A
160 mutation), suggesting that a loss
of the
carbohydrate from the tip of the HA increased the binding
affinity.
A known characteristic feature of the receptor-binding phenotype of
avian viruses, a feature which clearly separates them
from human virus
strains, is the ability of the avian HA to bind
to the galactose ring
of the Sia2-3Gal moiety (
27). To determine
whether this
feature is changed in H5 chicken viruses, we compared
their
binding to Neu5Ac and to 3'-SL (Neu5Ac2-3Gal1-4Glc) (Table
2). Like the duck viruses, two chicken
strains and A/HK/156/97
bind 3'SL more than an order of magnitude more
strongly than they
bind free Neu5Ac, a finding indicative of
favorable interactions
in the receptor-binding site of these viruses
with the penultimate
galactose residue of 3'SL. This is in a marked
contrast to the
Aichi/2/68 human strain, which shows no binding to the
3-linked
galactose (its affinity for 3'SL does not differ from its
affinity
for Neu5Ac). Thus, with respect to both their inability to
bind
Sia2-6Gal receptor determinants and their specific recognition
of
Sia2-3Gal determinants, H5N1 chicken viruses and the A/HK156/97
human
isolate display a typical avian receptor-binding phenotype.
Comparison of the binding affinity and NA activity of H5
viruses.
Partial sequencing of the NA gene of H5N1 chicken viruses
from Hong Kong used in this study revealed that all of them contained the same 19-amino-acid deletion in the stalk that was previously identified in the NA of A/HK/156/97 human isolate (data not shown). To
evaluate possible effects of this deletion on the NA activity, we
prepared suspensions of different H5 viruses with the same hemagglutination titers and tested each suspension for (i) its ability
to agglutinate CRBCs treated with V. cholerae NA, (ii) adsorption-elution from CRBCs, and (iii) NA activity against a low-molecular-weight substrate, 4-MU-NANA. The results of these experiments are summarized in Table 3.
In the assay, in which CRBCs were treated with gradually increasing
concentrations of bacterial NA, viruses from aquatic birds
required the
highest concentrations of NA to destroy receptors
on the CRBCs,
indicating that they possessed the highest affinity
for the receptors
(
45). In the case of H5N1 viruses from Hong
Kong, their
affinity correlated with the presence of CHO
158. Chicken
virus strains that carried carbohydrate at this position had a
lower
affinity than did variants without the site. The relative
affinity of
H5 viruses for CRBC receptors correlated well with
their affinity for a
soluble labeled sialylglycoprotein, Ovo-HRP
(Table
1).
To compare the abilities of the NA of different H5 viruses to induce
virus release from CRBCs, we adsorbed the viruses to
erythrocytes at
4°C and monitored their elution at 37°C. The results
indicated
three distinct groups. H5 viruses from aquatic birds
eluted most
readily, with elution being complete after 1.5 h at
37°C. All
Hong Kong 1997 viruses eluted at a slower rate. For
these viruses, the
elution rate correlated with the presence of
a glycosylation site at
position 158 of the HA. Isolates with
this site were able to elute
during 3 to 12 h of observation,
whereas strains without this
site failed to elute even after 24
h of incubation. Thus, the rate
of elution from CRBCs of chicken
and human H5N1 viruses corresponds to
their affinity for CRBC
receptors: the lower the affinity the faster
the elution. However,
there is an apparent discrepancy between the
highest binding affinity
of duck and gull viruses and their highest
elution rate. To explain
this finding, we attributed the slower elution
rate of chicken
viruses to the deletion in the NA stalk, which appears
to have
decreased the ability of the enzyme to destroy receptors on the
erythrocytes.
To test this hypothesis, we determined the NA activity
against a low-molecular-weight substrate, 4-MU-NANA, in the same virus
preparations that were used in the elution experiments (Table
3). The
NA activity of chicken and human virus preparations against
this
substrate was not lower than that of viruses from aquatic
birds,
indicating that a lower activity of the chicken virus NA
against virus
receptors on CRBCs most likely results from the
steric hindrance of the
enzyme active site. This effect is consistent
with the presence of the
deletion in the NA stalk of chicken
viruses.
Chicken influenza viruses of the H5 and H7 subtypes often carry
additional carbohydrates at the head of their HA and contain a deletion
in their NA.
To evaluate whether the carbohydrate at position 158 of the HA and the deletion in the NA stalk were unique to the H5N1
influenza viruses from Hong Kong, we analyzed the HA and NA amino acid
sequences of different avian viruses available from GenBank.
Table
4 summarizes data on the
glycosylation of the HA globular head of H1, H2, H3, and H4 viruses
from wild aquatic birds.
Generally, only one glycosylation sequon is
present on the upper
part of the HA1 of these viruses.
Figure
1 shows the HA glycosylation
patterns of the H5 and H7 subtype viruses from different avian species,
including chickens.
Some viruses of these two subtypes are known to be
highly pathogenic
in poultry, a property that has been attributed to
the presence
of multiple basic amino acids at the HA cleavage site
(reviewed
in reference
23). None of the 11 nonpathogenic avian H7 viruses
(three or fewer basic amino acids at the
HA cleavage site) isolated
mainly from aquatic birds contained more
than one glycosylation
sequon. Remarkably, among the seven other
strains that carry one
or two additional glycosylation sequons, six
were highly pathogenic,
four were isolated directly from chickens, and
one belongs to
the avian-like virus lineage that caused disease in
salts (
19).
Phylogenetic analysis of the HA sequences (Fig.
1, H7) shows that
these
H7 viruses with additional glycosylation of the HA do not
belogn to the
same phylogenetic lineage but emrged through independent
evolution from
nonpathogenic precursors.
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FIG. 1.
Analysis of glycosylation sites on the HA1 globular head
of H5 and H7 influenza viruses. Columns: A, Phylogenetic relationships
based on the HA1 nucleotide sequences of the virus strains; B, NA
subtype; C, GenBank accession number; D, number of basic amino acids at
the HA cleavage site, if there were more than three such amino acids at
this site; E, positions of glycosylation sequons by H3 numbering (see
Fig. 2 for the location of sequons on the three-dimensional model of
the HA). The asterisks next to the GenBank accession number mark the H5
HA sequences determined in this study.
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FIG. 2.
Positions of glycosylation sites on the upper part of
the HA1 subunit (amino acid residues 90 to 260) of avian influenza
viruses shown on a model of the complex between X31 virus HA and 3'-SL
(1HGG structure; Brookhaven Protein Databank [37]).
The HA monomers are depicted in shades of gray. Positions corresponding
to the asparagine residues in the N-linked glycosylation triplets are
shown in black and numbered. The sialyllactose molecules are shown as
stick models. The figure was generated with WebLab ViewerPro 3.1 (Molecular Simulations, Inc., San Diego, Calif.).
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Similarly, none of nonpathogenic H5 isolates from aquatic birds has
more than one glycosylation sequon on the HA head, and
the presence of
additional sequons clearly correlates with isolation
of such H5 viruses
from chickens. As with H7 strains, H5 strains
with additional
glycosylation sites belong to different lineages
that emerged from the
viruses of aquatic birds. However, unlike
the H7 strains analyzed, not
all H5 viruses with additional glycosylation
of the HA are highly
pathogenic. For example, all H5N2 Mexican
viruses isolated in 1994 and
1995 contain an additional site at
position 240, but only some late
isolates from 1995 have insertions
of basic amino acids at the cleavage
site. This feature may indicate
that additional glycosylation of the HA
precedes the development
of pathogenicity of the influenza viruses
during their evolution
in
chickens.
Table
5 presents the results of the
analysis of the influenza virus NA sequences for the presence of
deletions in the stalk
region that would be comparable in size to the
19-amino-acid deletion
in the NA of H5N1 strains from Hong Kong. We
noticed that the
length of the stalk does not vary significantly
between NA subtypes:
the N9 NA has the longest stalk, the stalks of
most other type
A and type B virus NAs were only 1 to 3 amino acids
shorter (not
shown). Nine of more than 100 NA sequences from GenBank
were found
to contain deletions in the stalk that ranged from 15 to 22 amino
acids in length (Fig.
3). One of
them (X-7-Stubby) was a laboratory
mutant (
13). Five NAs
with shortened stalks belong to H1N1 human
virus isolates from
1933 to 1935. Remarkably, three of the five
available NA sequences of
the chicken viruses (FPV/Rostock/34,
ck/PA/8125/83, and
ck/PA/1370/83) contain deletions in the NA
stalk that are similar in
size and position to that of H5N1 viruses
from Hong Kong. Thus,
although only a small number of NA sequences
of chicken viruses were
analyzed in this study, we think the above
finding is striking.
Moreover, like some of the Hong Kong H5N1
viruses, all three chicken
H5N2 and H7N1 strains with deletions
in their NAs have additional
glycosylation sites on the top of
their HAs (Fig.
1), suggesting an
interrelationship between these
features.
View larger version (60K):
[in this window]
[in a new window]
|
FIG. 3.
Amino acid sequences of the stalk region of N1 and N2
influenza virus NAs (top) and a phylogenetic tree for some of the N1
NAs (bottom). The positions of deletions are shown by dashes; potential
glycosylation sites are shaded. Only a partial sequence was available
for the NA stalk region of the three human H1N1 strains marked with an
asterisk (*). Numbers at the tree nodes show bootstrap values (100 replications); the horizontal lengths of the branches are proportional
to these values.
|
|
In the case of the N1 NA, deletions were observed in three distinct
groups of viruses (FPV/Rostock.34 [H7N1]; H5N1 viruses
from Hong
Kong, 1997; and the earliest human H1N1 strains). Phylogenetic
analysis (Fig.
3, tree) suggested that these groups belong to
separate evolutionary lineages. The region of the NA stalk that
is
deleted in these viruses is conserved among all N1 NAs that
lack
deletions (Fig.
3, N1). This feature indicates that deletions
in the
stalk of the of ancestral N1 NA (rather than stalk insertions)
occurred
independently in each of the three lineages after their
diversion.
| |
DISCUSSION |
Poor binding of avian viruses to Sia2-6Gal-terminated
sialyloligosaccharide receptor determinants on human respiratory cells is thought to be one of the factors that limit the replication of avian
influenza viruses in humans. We found in this study that the H5N1 virus
isolate from a human A/HK/156/97 displays the receptor-binding properties that are typical of avian but not of human viruses. This
finding indicates that an avian virus can replicate and cause a fatal
disease in humans without a significant change in its binding
specificity for sialyloligosaccharide receptor determinants. In
contrast, the earliest available H2 and H3 virus strains from the
previous human pandemics of 1957 and 1968 clearly differ from avian
viruses in their ability to bind Sia2-6Gal-specific receptors (9; the present study). The difference between these
pandemic viruses and the H5N1 viruses isolated in Hong Kong in 1997 is that the former were isolated when the pandemic was already under way,
that is, when the viruses had been present in humans for some time and
were effectively being transmitted from human to human. This scenario
contrasts with the situation in Hong Kong, where cases of H5N1
influenza in humans resulted from independent and direct introductions
of the virus from chickens into humans without evidence of
human-to-human transmission (7, 41). Two conclusions can be
drawn. First, the receptor-binding specificity of the avian HA does not
change immediately after the HA is introduced into humans but rather
evolves only after a certain number of replications in this host.
Second, alterations of receptor-binding specificity appear necessary
for effective virus transmission among humans. Therefore, changes of
the receptor-binding phenotype of the avian HA in humans could serve as
a marker of ongoing enhancement of the epidemiologic potential of the virus.
Wild aquatic birds are regarded as the source of all human pandemic
viruses (reviewed in reference 44). The H5N1 human
cases in Hong Kong demonstrate that chickens can serve as an
intermediate host for the introduction of these viruses into humans.
Indeed, our study indicates that influenza viruses from aquatic birds undergo significant selective pressure in chickens, leading to definite
changes in both the HA and the NA during the adaptation process. Thus,
although the number of isolates available for the study of HA and NA
sequences of chicken influenza viruses was limited, our analysis
indicates a marked correlation between the isolation of the virus from
chickens and (i) the presence of a deletion in the stalk of the NA and
(ii) increased glycosylation of the HA globular head. These features of
the HA and NA clearly separate chicken viruses from the viruses of wild
aquatic birds.
The stalk region of the influenza virus NA is often regarded as being
rather variable, both with respect to its amino acid sequence and with
respect to its length. However, as revealed by our analysis, natural
variation in the stalk length is limited to a few distinct virus
lineages that have large deletions in the stalk compared to the stalks
of most other NAs. Three of these lineages belong to chicken viruses
with N1 and N2 NAs (FPV/Rostock/34 [H7N1]; H5N1 isolates from
Hong Kong; and chicken H5N2 isolates from Pennsylvania, 1983). The
fourth known lineage includes several human H1N1 viruses isolated
from 1933 to 1935 (4). Partial sequencing of the NA of the
A/North Carolina/1/18 strain, which has been implicated in the
"Spanish flu" pandemic, indicates that the NA of this strain
belongs to the same human virus lineage (42). Given that a
deletion in the NA stalk is a characteristic feature of chicken
influenza viruses, one can speculate that this human virus lineage
originated from a chicken virus precursor.
The mechanisms for the selection in chickens with a shortened NA stalk
is unclear. One possibility could be that there is a defect in a
formation of enzymatically active NA in chicken cells, which is somehow
corrected by the deletion. For example, a deletion in the N1 and N2 NAs
of different viruses leads to a loss of several glycosylation sites in
the same position of the stalk (Fig. 3), and it can be speculated that
this feature may influence the efficiency of co- and posttranslational
protein folding (20). Alternatively, the variant with the
deletion could be selected because the NA enzymatic activity of the
progenitor virus from aquatic birds is too high for its effective
multiplication and/or transmission in chickens. Previous studies showed
that deletions in the stalk impaired the ability of the enzyme to
release influenza virus from erythrocytes (6, 13, 25). We
found that the same is true for a deletion in the NA of H5N1 Hong Kong viruses. This means that the chicken virus NA destroys
sialylglycoconjugates on the surface of cells less effectively than
does the NA of the virus from wild aquatic birds. The selective
advantage that could be conferred by this property to the chicken virus
remains unresolved.
Inkster et al. (21) were the first to notice that avian H1
influenza viruses differ from human strains by virtue of reduced glycosylation of the HA. Our analysis confirms the notion about low
glycosylation of the HA for the other influenza virus subtypes from
wild aquatic birds. In contrast, H5 and H7 chicken strains had
additional carbohydrates on the top of the HA globular head in close
proximity to the receptor-binding site (Fig. 2). Numerous studies
indicated that glycans attached at the tip of the HA decrease the
binding of the virus to solid-phase exposed receptors, erythrocytes, and cells (10, 12, 16, 27, 30), presumably because of steric
hindrance of the receptor-binding site. In accord with these
observations, we found that the carbohydrate attached at position 158 of the H5N1 virus HA decreased the virus affinity for soluble
sialylglycoproteins and for CRBCs. It can be suggestd, therefore, that additional glycosylation of the HA of chicken viruses
modifies the receptor-binding properties of the progenitor aquatic bird
virus in chickens. This adjustment could be connected to the deficiency
in the NA activity of the chicken virus. Thus, in the case of H5N1
strains, we found that carbohydrate at position 158 compensates for the
decreased enzymatic activity of the viral NA and significantly improves
elution of the virus from erythrocytes. Alternatively, it cannot be
excluded that a deletion in the NA does not occur as a result of an
adjustment of the NA activity to match a decreased affinity of
additionally glycosylated HA.
Our observation that the HAs of highly pathogenic H5 and H7
viruses (in poultry) often carry additional glycosylation sites agrees with the finding of Perdue et al. (33), who
showed that a single substitution in the HA resulting in the addition
of a new glycosylation site at the tip of the HA in position 197 (see Fig. 2) renders the virus highly pathogenic in chickens. It seems reasonable to suggest that this mutation increases the release of virus
from cells, facilitating its spread and replication in different
tissues of chickens.
Our data introduce the notiion that chickens represent a separate
natural reservoir of influenza viruses, with the functional properties
of their HAs and NAs being substantially different from those of
influenza viruses from wild aquatic birds. Further study is required to
understand the relation of these distinctions to the biologic
characteristics of chicken viruses, incuding the potential for their
transmission to humans.
| |
ACKNOWLEDGMENTS |
We thank Richard Bethell of Glaxo Wellcome Research & Development
for providing the NA inhibitor zanamivir, Scott Krauss and Melissa
Norwood for the technical assistance, and John Gilbert for editing the manuscript.
This work was supported by Public Health Service research grants
AI08831 and AI29680 from the National Institute of Allergy and
Infectious Diseases, Cancer Center Support (CORE) grant CA-21765, and
the American Lebanese Syrian Associated Charities (ALSAC). M. N. Matrosovich was supported by a Karnofsky fellowship from St. Jude
Children's Research Hospital.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Virology and Molecular Biology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. Phone: (901) 495-3412. Fax: (901)
523-2622. E-mail: Mikhail.Matrosovich{at}stjude.org.
| |
REFERENCES |
| 1.
|
Baum, L. G., and J. C. Paulson.
1991.
The N2 neuraminidase of human influenza virus has acquired a substrate specificity complementary to the hemagglutinin receptor specificity.
Virology
180:10-15[Medline].
|
| 2.
|
Bean, W. J.,
Y. Kawaoka,
J. M. Wood,
J. E. Pearson, and R. G. Webster.
1985.
Characterization of virulent and avirulent A/chicken/Pennsylvania/83 influenza A viruses: potential role of defective interfering RNAs in nature.
J. Virol.
54:151-160[Abstract/Free Full Text].
|
| 3.
|
Beare, A. S., and R. G. Webster.
1991.
Replication of avian influenza viruses in humans.
Arch. Virol.
119:37-42[Medline].
|
| 4.
|
Blok, J., and G. M. Air.
1982.
Sequence variation at the 3' end of the neuraminidase gene from 39 influenza type A viruses.
Virology
121:211-229[Medline].
|
| 5.
|
Boorsma, D. M., and J. G. Streefkerk.
1979.
Periodate or glutaraldehyde for preparing peroxidase conjugates?
J. Immunol. Methods
30:245-255[Medline].
|
| 6.
|
Castrucci, M. R., and Y. Kawaoka.
1993.
Biologic importance of neuraminidase stalk length in influenza A virus.
J. Virol.
67:759-764[Abstract/Free Full Text].
|
| 7.
|
Claas, E. C. J.,
A. D. M. E. Osterhaus,
R. Vanbeek,
J. C. de Jong,
G. F. Rimmelzwaan,
D. A. Senne,
S. Krauss,
K. F. Shortridge, and R. G. Webster.
1998.
Human influenza A H5N2 virus related to a highly pathogenic avian influenza virus.
Lancet
351:472-477[Medline].
|
| 8.
|
Colman, P. M.,
P. A. Hoyne, and M. C. Lawrence.
1993.
Sequence and structure alignment of paramyxovirus hemagglutinin-neuraminidase with influenza virus neuraminidase.
J. Virol.
67:2972-2980[Abstract/Free Full Text].
|
| 9.
|
Connor, R. J.,
Y. Kawaoka,
R. G. Webster, and J. C. Paulson.
1994.
Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates.
Virology
205:17-23[Medline].
|
| 10.
|
Crecelius, D. M.,
C. M. Deom, and I. T. Schulze.
1984.
Biological properties of a hemagglutinin mutant of influenza virus selected by host cells.
Virology
139:164-177[Medline].
|
| 11.
|
De Jong, J. C.,
E. C. J. Claas,
A. D. M. E. Osterhaus,
R. G. Webster, and W. L. Lim.
1997.
A pandemic warning.
Nature
389:554[Medline].
|
| 12.
|
Deom, C. M.,
A. J. Caton, and I. T. Schulze.
1986.
Host cell-mediated selection of a mutant influenza A virus that has lost a complex oligosaccharide from the tip of the hemagglutinin.
Proc. Natl. Acad. Sci. USA
83:3771-3775[Abstract/Free Full Text].
|
| 13.
|
Els, M. C.,
G. M. Air,
K. G. Murti,
R. G. Webster, and W. G. Laver.
1985.
An 18-amino acid deletion in an influenza neuraminidase.
Virology
142:241-247[Medline].
|
| 14.
|
Feldmann, H.,
E. Kretzschmar,
B. Klingeborn,
R. Rott,
H. D. Klenk, and W. Garten.
1988.
The structure of serotype H10 hemagluttinin of influenza A virus: comparison of an apathogenic avian and a mammalian strain pathogenic for mink.
Virology
165:428-437[Medline].
|
| 15.
|
Felsenstein, J.
1989.
PHYLIPphylogeny inference package (verison 3.2).
Cladistics
5:164-166.
|
| 16.
|
Gambaryan, A. S.,
V. P. Marinina,
A. B. Tuzikov,
N. V. Bovin,
L. A. Rudneva,
B. V. Sinitsyn,
A. A. Shilov, and M. N. Matrosovich.
1998.
Effects of host-dependent glycosylation of hemagglutinin on receptor-binding properties of H1N1 human influenza A virus grown in MDCK cells and in embryonated eggs.
Virology
247:170-177[Medline].
|
| 17.
|
Gambaryan, A. S., and M. N. Matrosovich.
1992.
A solid-phase enzyme-linked assay for influenza virus receptor-binding activity.
J. Virol. Methods
39:111-123[Medline].
|
| 18.
|
Gambaryan, A. S.,
A. B. Tuzikov,
V. E. Piskaev,
S. S. Yamnikova,
D. K. Lvov,
J. S. Robertson,
N. V. Bovin, and M. N. Matrosovich.
1997.
Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6'-sialyl(N-acetyllactosamine).
Virology
232:345-350[Medline].
|
| 19.
|
Geraci, J. R.,
D. J. St. Aubin,
I. K. Barker,
R. G. Webster,
V. S. Hinshaw,
W. J. Bean,
H. L. Ruhnke,
J. H. Prescott,
G. Early,
A. S. Baker,
S. Madoff, and R. T. Schooley.
1982.
Mass mortality of harbor seals: pneumonia associated with influenza A virus.
Science
215:1129-1131[Abstract/Free Full Text].
|
| 20.
|
Hebert, D. N.,
J. X. Zhang,
W. Chen,
B. Foellmer, and A. Helenius.
1997.
The number and location of glycans on influenza hemagglutinin determine folding and association with calnexin and calreticulin.
J. Cell Biol.
139:613-623[Abstract/Free Full Text].
|
| 21.
|
Inkster, M. D.,
V. S. Hinshaw, and I. T. Schulze.
1993.
The hemagglutinins of duck and human H1 influenza viruses differ in sequence conservation and in glycosylation.
J. Virol.
67:7436-7443[Abstract/Free Full Text].
|
| 22.
|
Ito, T.,
J. N. S. S. Couceiro,
S. Kelm,
L. G. Baum,
S. Krauss,
M. R. Castrucci,
I. Donatelli,
H. Kida,
J. C. Paulson,
R. G. Webster, and Y. Kawaoka.
1998.
Molecular basis for the generation in pigs of influenza A viruses with pandemic potential.
J. Virol.
72:7367-7443[Abstract/Free Full Text].
|
| 23.
|
Ito, T., and Y. Kawaoka.
1998.
Avian influenza, p. 126-136.
In
K. G. Nicholson, R. G. Webster, and A. J. Hay (ed.), Textbook of influenza. Blackwell Science, London, England.
|
| 24.
|
Kato, A.,
S. Hirata, and K. Kobayashi.
1978.
Nature of the carbohydrate side chains and their linkage to the protein in chicken egg white ovomucin. Part III. Structure of the sulfated oligosaccharide chain of ovomucin.
Agric. Biol. Chem.
42:1025-1029.
|
| 25.
|
Luo, G.,
J. Chung, and P. Palese.
1993.
Alterations of the stalk of the influenza virus neuraminidase: deletions and insertions.
Virus Res.
29:141-153[Medline].
|
| 26.
|
Matrosovich, M. N.,
A. S. Gambaryan,
A. B. Tuzikov,
N. E. Byramova,
L. V. Mochalova,
A. A. Golbraikh,
M. D. Shenderovich,
J. Finne, and N. V. Bovin.
1993.
Probing of the receptor-binding sites of the H1 and H3 influenza A and influenza B virus hemagglutinins by synthetic and natural sialosides.
Virology
196:111-121[Medline].
|
| 27.
|
Matrosovich, M. N.,
A. S. Gambaryan,
S. Teneberg,
V.E. Piskarev,
S. S. Yamnikova,
D. K. Lvov,
J. S. Robertson, and K. A. Karlsson.
1997.
Avian influenza A viruses differ from human viruses by recognition of sialyloligosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site.
Virology
233:224-234[Medline].
|
| 28.
|
Nicholas, K. B.,
H. B. Nicholas, Jr., and D. W. Deerfield.
1997.
GeneDoc: analysis and visualization of genetic variation.
EMBNEW.NEWS
4:14-14.
|
| 29.
|
Nobusawa, E.,
T. Aoyama,
H. Kato,
Y. Suzuki,
Y. Tateno, and K. Nakajima.
1991.
Comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza A viruses.
Virology
182:475-485[Medline].
|
| 30.
|
Ohuchi, M.,
R. Ohuchi,
A. Feldmann, and H. D. Klenk.
1997.
Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety.
J. Virol.
71:8377-8384[Abstract].
|
| 31.
|
Page, R. D. M.
1996.
TREEVIEW: an application to display phylogenetic trees on personal computers.
Comput. Appl. Biosci.
12:357-358[Free Full Text].
|
| 32.
|
Paulson, J. C.
1985.
Interactions of animal viruses with cell surface receptors, p. 131-219.
In
M. Conn (ed.), The receptors, vol. 2. Academic Press, Orlando, Fla.
|
| 33.
|
Perdue, M. L.,
J. W. Latimer, and J. M. Crawford.
1995.
A novel carbohydrate addition site on the hemagglutinin protein of a highly pathogenic H7 subtype avian influenza virus.
Virology
213:276-281[Medline].
|
| 34.
|
Rogers, G. N., and B. L. D'Souza.
1989.
Receptor binding properties of human and animal H1 influenza virus isolates.
Virology
173:317-322[Medline].
|
| 35.
|
Rudneva, I. A.,
E. I. Sklyanskaya,
O. S. Barulina,
S. S. Yamnikova,
V. P. Kovaleva,
I. V. Tsvetkova, and N. V. Kaverin.
1996.
Phenotypic expression of HA-NA combinations in human-avian influenza A virus reassortants.
Arch. Virol.
141:1091-1099[Medline].
|
| 36.
|
Ryan-Poirier, K. A, and Y. Kawaoka.
1993.
Alpha 2-macroglobulin is the major neutralizing inhibitor of influenza A virus in pig serum.
Virology
193:974-976[Medline].
|
| 37.
|
Sauter, N. K.,
J. E. Hanson,
G. D. Glick,
J. H. Brown,
R. L. Crowther,
S. J. Park,
J. J. Skehel, and D. C. Wiley.
1992.
Binding of influenza virus hemagglutinin to analogs of its cell-surface receptor, sialic acid: analysis by proton nuclear magnetic resonance spectroscopy and X-ray crystallography.
Biochemistry
31:9609-9621[Medline].
|
| 38.
|
Scholtissek, C.,
H. Burger,
O. Kistner, and K. F. Shortridge.
1985.
The nucleoprotein as a possible major factor in determining host specificity of influenza H3N2 viruses.
Virology
147:287-294[Medline].
|
| 39.
|
Shu, L. L.,
W. J. Bean, and R. G. Webster.
1993.
Analysis of the evolution and variation of the human influenza A virus nucleoprotein gene from 1933 to 1990.
J. Virol.
67:2723-2729[Abstract/Free Full Text].
|
| 40.
|
Suarez, D. L.,
M. L. Perdue,
N. Cox,
T. Rowe,
C. Bender,
J. Huang, and D. E. Swayne.
1998.
Comparison of highly virulent H5N1 influenza A viruses isolated from humans and chickens from Hong Kong.
J. Virol.
72:6678-6688[Abstract/Free Full Text].
|
| 41.
|
Subbarao, K.,
A. Klimov,
J. Katz,
H. Regnery,
W. Lim,
H. Hall,
M. Perdue,
D. Swayne,
C. Bender,
J. Huang,
M. Hemphill,
T. Rowe,
M. Shaw,
X. Y. Xu,
K. Fukuda, and N. Cox.
1998.
Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness.
Science
279:393-396[Abstract/Free Full Text].
|
| 42.
|
Taubenberger, J. K.,
A. H. Reid,
A. E. Krafft,
K. E. Bijwaard, and T. G. Fanning.
1997.
Initial genetic characterization of the 1918 "Spanish" influenza virus.
Science
275:1793-1796[Abstract/Free Full Text].
|
| 43.
|
Warner, T. G., and J. S. O'Brien.
1979.
Synthesis of 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and detection of skin fibroblast neuraminidase in normal humans and in sialidosis.
BIochemistry
18:2783-2787[Medline].
|
| 44.
|
Webster, R. G.,
W. J. Bean,
O. T. Gorman,
T. M. Chambers, and Y. Kawaoka.
1992.
Evolution and ecology of influenza A viruses.
Microbiol. Rev.
56:152-179[Abstract/Free Full Text].
|
| 45.
|
Yewdell, J. A.,
A. J. Caton, and W. Gerhard.
1986.
Selection of influenza A virus adsorptive mutants by growth in the presence of a mixture of monoclonal antihemagglutinin antibodies.
J. Virol.
57:623-628[Abstract/Free Full Text].
|
Journal of Virology, February 1999, p. 1146-1155, Vol. 73, No. 2
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[Full Text]
-
Tumpey, T. M., Maines, T. R., Van Hoeven, N., Glaser, L., Solorzano, A., Pappas, C., Cox, N. J., Swayne, D. E., Palese, P., Katz, J. M., Garcia-Sastre, A.
(2007). A Two-Amino Acid Change in the Hemagglutinin of the 1918 Influenza Virus Abolishes Transmission. Science
315: 655-659
[Abstract]
[Full Text]
-
Maines, T. R., Chen, L.-M., Matsuoka, Y., Chen, H., Rowe, T., Ortin, J., Falcon, A., Hien, N. T., Mai, L. Q., Sedyaningsih, E. R., Harun, S., Tumpey, T. M., Donis, R. O., Cox, N. J., Subbarao, K., Katz, J. M.
(2006). Lack of transmission of H5N1 avian-human reassortant influenza viruses in a ferret model. Proc. Natl. Acad. Sci. USA
103: 12121-12126
[Abstract]
[Full Text]
-
Zhou, J.-Y., Shen, H.-G., Chen, H.-X., Tong, G.-Z., Liao, M., Yang, H.-C., Liu, J.-X.
(2006). Characterization of a highly pathogenic H5N1 influenza virus derived from bar-headed geese in China. J. Gen. Virol.
87: 1823-1833
[Abstract]
[Full Text]
-
Wong, S. S. Y., Yuen, K.-y.
(2006). Avian Influenza Virus Infections in Humans. Chest
129: 156-168
[Abstract]
[Full Text]
-
Rudneva, I. A., Ilyushina, N. A., Timofeeva, T. A., Webster, R. G., Kaverin, N. V.
(2005). Restoration of virulence of escape mutants of H5 and H9 influenza viruses by their readaptation to mice. J. Gen. Virol.
86: 2831-2838
[Abstract]
[Full Text]
-
Lee, C.-W., Swayne, D. E., Linares, J. A., Senne, D. A., Suarez, D. L.
(2005). H5N2 Avian Influenza Outbreak in Texas in 2004: the First Highly Pathogenic Strain in the United States in 20 Years?. J. Virol.
79: 11412-11421
[Abstract]
[Full Text]
-
Shinya, K., Hatta, M., Yamada, S., Takada, A., Watanabe, S., Halfmann, P., Horimoto, T., Neumann, G., Kim, J. H., Lim, W., Guan, Y., Peiris, M., Kiso, M., Suzuki, T., Suzuki, Y., Kawaoka, Y.
(2005). Characterization of a Human H5N1 Influenza A Virus Isolated in 2003. J. Virol.
79: 9926-9932
[Abstract]
[Full Text]
-
Johansson, P., Nilsson, J., Angstrom, J., Miller-Podraza, H.
(2005). Interaction of Helicobacter pylori with sialylated carbohydrates: the dependence on different parts of the binding trisaccharide Neu5Ac{alpha}3Gal{beta}4GlcNAc. Glycobiology
15: 625-636
[Abstract]
[Full Text]
-
Govorkova, E. A., Rehg, J. E., Krauss, S., Yen, H.-L., Guan, Y., Peiris, M., Nguyen, T. D., Hanh, T. H., Puthavathana, P., Long, H. T., Buranathai, C., Lim, W., Webster, R. G., Hoffmann, E.
(2005). Lethality to Ferrets of H5N1 Influenza Viruses Isolated from Humans and Poultry in 2004. J. Virol.
79: 2191-2198
[Abstract]
[Full Text]
-
Puthavathana, P., Auewarakul, P., Charoenying, P. C., Sangsiriwut, K., Pooruk, P., Boonnak, K., Khanyok, R., Thawachsupa, P., Kijphati, R., Sawanpanyalert, P.
(2005). Molecular characterization of the complete genome of human influenza H5N1 virus isolates from Thailand. J. Gen. Virol.
86: 423-433
[Abstract]
[Full Text]
-
Zhang, M., Gaschen, B., Blay, W., Foley, B., Haigwood, N., Kuiken, C., Korber, B.
(2004). Tracking global patterns of N-linked glycosylation site variation in highly variable viral glycoproteins: HIV, SIV, and HCV envelopes and influenza hemagglutinin. Glycobiology
14: 1229-1246
[Abstract]
[Full Text]
-
Hulse, D. J., Webster, R. G., Russell, R. J., Perez, D. R.
(2004). Molecular Determinants within the Surface Proteins Involved in the Pathogenicity of H5N1 Influenza Viruses in Chickens. J. Virol.
78: 9954-9964
[Abstract]
[Full Text]
-
Lipatov, A. S., Govorkova, E. A., Webby, R. J., Ozaki, H., Peiris, M., Guan, Y., Poon, L., Webster, R. G.
(2004). Influenza: Emergence and Control. J. Virol.
78: 8951-8959
[Full Text]
-
Lee, C.-W., Senne, D. A., Suarez, D. L.
(2004). Effect of Vaccine Use in the Evolution of Mexican Lineage H5N2 Avian Influenza Virus. J. Virol.
78: 8372-8381
[Abstract]
[Full Text]
-
Chen, H., Deng, G., Li, Z., Tian, G., Li, Y., Jiao, P., Zhang, L., Liu, Z., Webster, R. G., Yu, K.
(2004). The evolution of H5N1 influenza viruses in ducks in southern China. Proc. Natl. Acad. Sci. USA
101: 10452-10457
[Abstract]
[Full Text]
-
Sturm-Ramirez, K. M., Ellis, T., Bousfield, B., Bissett, L., Dyrting, K., Rehg, J. E., Poon, L., Guan, Y., Peiris, M., Webster, R. G.
(2004). Reemerging H5N1 Influenza Viruses in Hong Kong in 2002 Are Highly Pathogenic to Ducks. J. Virol.
78: 4892-4901
[Abstract]
[Full Text]
-
Thompson, C. I., Barclay, W. S., Zambon, M. C.
(2004). Changes in in vitro susceptibility of influenza A H3N2 viruses to a neuraminidase inhibitor drug during evolution in the human host. J Antimicrob Chemother
53: 759-765
[Abstract]
[Full Text]
-
Iwatsuki-Horimoto, K., Kanazawa, R., Sugii, S., Kawaoka, Y., Horimoto, T.
(2004). The index influenza A virus subtype H5N1 isolated from a human in 1997 differs in its receptor-binding properties from a virulent avian influenza virus. J. Gen. Virol.
85: 1001-1005
[Abstract]
[Full Text]
-
Matrosovich, M. N., Matrosovich, T. Y., Gray, T., Roberts, N. A., Klenk, H.-D.
(2004). Human and avian influenza viruses target different cell types in cultures of human airway epithelium. Proc. Natl. Acad. Sci. USA
101: 4620-4624
[Abstract]
[Full Text]
-
Gamblin, S. J., Haire, L. F., Russell, R. J., Stevens, D. J., Xiao, B., Ha, Y., Vasisht, N., Steinhauer, D. A., Daniels, R. S., Elliot, A., Wiley, D. C., Skehel, J. J.
(2004). The Structure and Receptor Binding Properties of the 1918 Influenza Hemagglutinin. Science
303: 1838-1842
[Abstract]
[Full Text]
-
Spackman, E., Senne, D. A., Davison, S., Suarez, D. L.
(2003). Sequence Analysis of Recent H7 Avian Influenza Viruses Associated with Three Different Outbreaks in Commercial Poultry in the United States. J. Virol.
77: 13399-13402
[Abstract]
[Full Text]
-
Flandorfer, A., Garcia-Sastre, A., Basler, C. F., Palese, P.
(2003). Chimeric Influenza A Viruses with a Functional Influenza B Virus Neuraminidase or Hemagglutinin. J. Virol.
77: 9116-9123
[Abstract]
[Full Text]
-
Matrosovich, M., Matrosovich, T., Carr, J., Roberts, N. A., Klenk, H.-D.
(2003). Overexpression of the {alpha}-2,6-Sialyltransferase in MDCK Cells Increases Influenza Virus Sensitivity to Neuraminidase Inhibitors. J. Virol.
77: 8418-8425
[Abstract]
[Full Text]
-
Perez, D. R., Lim, W., Seiler, J. P., Yi, G., Peiris, M., Shortridge, K. F., Webster, R. G.
(2003). Role of Quail in the Interspecies Transmission of H9 Influenza A Viruses: Molecular Changes on HA That Correspond to Adaptation from Ducks to Chickens. J. Virol.
77: 3148-3156
[Abstract]
[Full Text]
-
Tsuchiya, E., Sugawara, K., Hongo, S., Matsuzaki, Y., Muraki, Y., Nakamura, K.
(2002). Role of overlapping glycosylation sequons in antigenic properties, intracellular transport and biological activities of influenza A/H2N2 virus haemagglutinin. J. Gen. Virol.
83: 3067-3074
[Abstract]
[Full Text]
-
Gubareva, L. V., Nedyalkova, M. S., Novikov, D. V., Murti, K. G., Hoffmann, E., Hayden, F. G.
(2002). A release-competent influenza A virus mutant lacking the coding capacity for the neuraminidase active site. J. Gen. Virol.
83: 2683-2692
[Abstract]
[Full Text]
-
Guan, Y., Peiris, J. S. M., Lipatov, A. S., Ellis, T. M., Dyrting, K. C., Krauss, S., Zhang, L. J., Webster, R. G., Shortridge, K. F.
(2002). Emergence of multiple genotypes of H5N1 avian influenza viruses in Hong Kong SAR. Proc. Natl. Acad. Sci. USA
99: 8950-8955
[Abstract]
[Full Text]
-
Tumpey, T. M., Suarez, D. L., Perkins, L. E. L., Senne, D. A., Lee, J.-g., Lee, Y.-J., Mo, I.-P., Sung, H.-W., Swayne, D. E.
(2002). Characterization of a Highly Pathogenic H5N1 Avian Influenza A Virus Isolated from Duck Meat. J. Virol.
76: 6344-6355
[Abstract]
[Full Text]
-
Zitzow, L. A., Rowe, T., Morken, T., Shieh, W.-J., Zaki, S., Katz, J. M.
(2002). Pathogenesis of Avian Influenza A (H5N1) Viruses in Ferrets. J. Virol.
76: 4420-4429
[Abstract]
[Full Text]
-
Wagner, R., Heuer, D., Wolff, T., Herwig, A., Klenk, H.-D.
(2002). N-Glycans attached to the stem domain of haemagglutinin efficiently regulate influenza A virus replication. J. Gen. Virol.
83: 601-609
[Abstract]
[Full Text]
-
Campitelli, L., Fabiani, C., Puzelli, S., Fioretti, A., Foni, E., De Marco, A., Krauss, S., Webster, R. G., Donatelli, I.
(2002). H3N2 influenza viruses from domestic chickens in Italy: an increasing role for chickens in the ecology of influenza?. J. Gen. Virol.
83: 413-420
[Abstract]
[Full Text]
-
Wentworth, D. E., Holmes, K. V.
(2001). Molecular Determinants of Species Specificity in the Coronavirus Receptor Aminopeptidase N (CD13): Influence of N-Linked Glycosylation. J. Virol.
75: 9741-9752
[Abstract]
[Full Text]
-
Seo, S. H., Goloubeva, O., Webby, R., Webster, R. G.
(2001). Characterization of a Porcine Lung Epithelial Cell Line Suitable for Influenza Virus Studies. J. Virol.
75: 9517-9525
[Abstract]
[Full Text]
-
Ha, Y., Stevens, D. J., Skehel, J. J., Wiley, D. C.
(2001). X-ray structures of H5 avian and H9 swine influenza virus hemagglutinins bound to avian and human receptor analogs. Proc. Natl. Acad. Sci. USA
10.1073/pnas.201401198v1
[Abstract]
[Full Text]
-
Rimmelzwaan, G. F., Kuiken, T., van Amerongen, G., Bestebroer, T. M., Fouchier, R. A. M., Osterhaus, A. D. M. E.
(2001). Pathogenesis of Influenza A (H5N1) Virus Infection in a Primate Model. J. Virol.
75: 6687-6691
[Abstract]
[Full Text]
-
Tumpey, T. M., Renshaw, M., Clements, J. D., Katz, J. M.
(2001). Mucosal Delivery of Inactivated Influenza Vaccine Induces B-Cell-Dependent Heterosubtypic Cross-Protection against Lethal Influenza A H5N1 Virus Infection. J. Virol.
75: 5141-5150
[Abstract]
[Full Text]
-
Yao, Y., Mingay, L. J., McCauley, J. W., Barclay, W. S.
(2001). Sequences in Influenza A Virus PB2 Protein That Determine Productive Infection for an Avian Influenza Virus in Mouse and Human Cell Lines. J. Virol.
75: 5410-5415
[Abstract]
[Full Text]
-
Suzuki, T., Portner, A., Scroggs, R. A., Uchikawa, M., Koyama, N., Matsuo, K., Suzuki, Y., Takimoto, T.
(2001). Receptor Specificities of Human Respiroviruses. J. Virol.
75: 4604-4613
[Abstract]
[Full Text]
-
Leneva, I. A., Goloubeva, O., Fenton, R. J., Tisdale, M., Webster, R. G.
(2001). Efficacy of Zanamivir against Avian Influenza A Viruses That Possess Genes Encoding H5N1 Internal Proteins and Are Pathogenic in Mammals. Antimicrob. Agents Chemother.
45: 1216-1224
[Abstract]
[Full Text]
-
Donatelli, I., Campitelli, L., Di Trani, L., Puzelli, S., Selli, L., Fioretti, A., Alexander, D. J., Tollis, M., Krauss, S., Webster, R. G.
(2001). Characterization of H5N2 influenza viruses from Italian poultry. J. Gen. Virol.
82: 623-630
[Abstract]
[Full Text]
-
Perkins, L. E. L., Swayne, D. E.
(2001). Pathobiology of A/Chicken/Hong Kong/220/97 (H5N1) Avian Influenza Virus in Seven Gallinaceous Species. Vet Pathol
38: 149-164
[Abstract]
[Full Text]
-
Horimoto, T., Kawaoka, Y.
(2001). Pandemic Threat Posed by Avian Influenza A Viruses. Clin. Microbiol. Rev.
14: 129-149
[Abstract]
[Full Text]
-
Fouchier, R. A. M., Bestebroer, T. M., Herfst, S., Van Der Kemp, L., Rimmelzwaan, G. F., Osterhaus, A. D. M. E.
(2000). Detection of Influenza A Viruses from Different Species by PCR Amplification of Conserved Sequences in the Matrix Gene. J. Clin. Microbiol.
38: 4096-4101
[Abstract]
[Full Text]
-
Miller-Podraza, H., Johansson, L., Johansson, P., Larsson, T., Matrosovich, M., Karlsson, K.-A.
(2000). A strain of human influenza A virus binds to extended but not short gangliosides as assayed by thin-layer chromatography overlay. Glycobiology
10: 975-982
[Abstract]
[Full Text]
-
Matrosovich, M., Tuzikov, A., Bovin, N., Gambaryan, A., Klimov, A., Castrucci, M. R., Donatelli, I., Kawaoka, Y.
(2000). Early Alterations of the Receptor-Binding Properties of H1, H2, and H3 Avian Influenza Virus Hemagglutinins after Their Introduction into Mammals. J. Virol.
74: 8502-8512
[Abstract]
[Full Text]
-
Lin, Y. P., Shaw, M., Gregory, V., Cameron, K., Lim, W., Klimov, A., Subbarao, K., Guan, Y., Krauss, S., Shortridge, K., Webster, R., Cox, N., Hay, A.
(2000). Avian-to-human transmission of H9N2 subtype influenza A viruses: Relationship between H9N2 and H5N1 human isolates. Proc. Natl. Acad. Sci. USA
10.1073/pnas.160270697v1
[Abstract]
[Full Text]
-
Hoffmann, E., Stech, J., Leneva, I., Krauss, S., Scholtissek, C., Chin, P. S., Peiris, M., Shortridge, K. F., Webster, R. G.
(2000). Characterization of the Influenza A Virus Gene Pool in Avian Species in Southern China: Was H6N1 a Derivative or a Precursor of H5N1?. J. Virol.
74: 6309-6315
[Abstract]
[Full Text]
-
Wagner, R., Wolff, T., Herwig, A., Pleschka, S., Klenk, H.-D.
(2000). Interdependence of Hemagglutinin Glycosylation and Neuraminidase as Regulators of Influenza Virus Growth: a Study by Reverse Genetics. J. Virol.
74: 6316-6323
[Abstract]
[Full Text]
-
Tumpey, T. M., Lu, X., Morken, T., Zaki, S. R., Katz, J. M.
(2000). Depletion of Lymphocytes and Diminished Cytokine Production in Mice Infected with a Highly Virulent Influenza A (H5N1) Virus Isolated from Humans. J. Virol.
74: 6105-6116
[Abstract]
[Full Text]
-
Dybing, J. K., Schultz-Cherry, S., Swayne, D. E., Suarez, D. L., Perdue, M. L.
(2000). Distinct Pathogenesis of Hong Kong-Origin H5N1 Viruses in Mice Compared to That of Other Highly Pathogenic H5 Avian Influenza Viruses. J. Virol.
74: 1443-1450
[Abstract]
[Full Text]
-
Basler, C. F., Garcia-Sastre, A., Palese, P.
(1999). Mutation of Neuraminidase Cysteine Residues Yields Temperature-Sensitive Influenza Viruses. J. Virol.
73: 8095-8103
[Abstract]
[Full Text]
-
Takada, A., Kuboki, N., Okazaki, K., Ninomiya, A., Tanaka, H., Ozaki, H., Itamura, S., Nishimura, H., Enami, M., Tashiro, M., Shortridge, K. F., Kida, H.
(1999). Avirulent Avian Influenza Virus as a Vaccine Strain against a Potential Human Pandemic. J. Virol.
73: 8303-8307
[Abstract]
[Full Text]
-
Lu, X., Tumpey, T. M., Morken, T., Zaki, S. R., Cox, N. J., Katz, J. M.
(1999). A Mouse Model for the Evaluation of Pathogenesis and Immunity to Influenza A (H5N1) Viruses Isolated from Humans. J. Virol.
73: 5903-5911
[Abstract]
[Full Text]
-
Zhou, N. N., Shortridge, K. F., Claas, E. C. J., Krauss, S. L., Webster, R. G.
(1999). Rapid Evolution of H5N1 Influenza Viruses in Chickens in Hong Kong. J. Virol.
73: 3366-3374
[Abstract]
[Full Text]
-
Reid, A. H., Fanning, T. G., Janczewski, T. A., Taubenberger, J. K.
(2000). Characterization of the 1918 "Spanish" influenza virus neuraminidase gene. Proc. Natl. Acad. Sci. USA
97: 6785-6790
[Abstract]
[Full Text]
-
Lin, Y. P., Shaw, M., Gregory, V., Cameron, K., Lim, W., Klimov, A., Subbarao, K., Guan, Y., Krauss, S., Shortridge, K., Webster, R., Cox, N., Hay, A.
(2000). Avian-to-human transmission of H9N2 subtype influenza A viruses: Relationship between H9N2 and H5N1 human isolates. Proc. Natl. Acad. Sci. USA
97: 9654-9658
[Abstract]
[Full Text]
-
Ha, Y., Stevens, D. J., Skehel, J. J., Wiley, D. C.
(2001). X-ray structures of H5 avian and H9 swine influenza virus hemagglutinins bound to avian and human receptor analogs. Proc. Natl. Acad. Sci. USA
98: 11181-11186
[Abstract]
[Full Text]
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Abstract
Introduction
