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Journal of Virology, November 2000, p. 10714-10728, Vol. 74, No. 22
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Evolution of Bovine Respiratory Syncytial
Virus
Jean-François
Valarcher,1,2
François
Schelcher,1 and
Hervé
Bourhy2,*
UMR INRA-ENVT de Physiopathologie Infectieuse
et Parasitaire des Ruminants, ENVT, 31076 Toulouse Cedex
3,1 and Unité de la Rage, Institut
Pasteur, 75724 Paris Cedex 15,2 France
Received 28 March 2000/Accepted 26 July 2000
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ABSTRACT |
Until now, the analysis of the genetic diversity of bovine
respiratory syncytial virus (BRSV) has been based on small numbers of
field isolates. In this report, we determined the nucleotide and
deduced amino acid sequences of regions of the nucleoprotein (N
protein), fusion protein (F protein), and glycoprotein (G protein) of
54 European and North American isolates and compared them with the
sequences of 33 isolates of BRSV obtained from the databases, together
with those of 2 human respiratory syncytial viruses and 1 ovine
respiratory syncytial virus. A clustering of BRSV sequences according
to geographical origin was observed. We also set out to show that a
continuous evolution of the sequences of the N, G, and F proteins of
BRSV has been occurring in isolates since 1967 in countries where
vaccination was widely used. The exertion of a strong positive
selective pressure on the mucin-like region of the G protein and on
particular sites of the N and F proteins is also demonstrated.
Furthermore, mutations which are located in the conserved central
hydrophobic part of the ectodomain of the G protein and which result in
the loss of four Cys residues and in the suppression of two disulfide
bridges and an 
helix critical to the three-dimensional structure of
the G protein have been detected in some recent French BRSV isolates.
This conserved central region, which is immunodominant in BRSV G
protein, thus has been modified in recent isolates. This work
demonstrates that the evolution of BRSV should be taken into account in
the rational development of future vaccines.
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INTRODUCTION |
Bovine respiratory syncytial
virus (BRSV) and human respiratory syncytial virus
(HRSV) are members of the genus Pneumovirus, subfamily
Pneumovirinae, and family Paramyxoviridae
(38, 39, 40, 49). These two related viruses share common
epidemiological, clinical, and pathological characteristics. The
respiratory syncytial viruses (RSV) are the most common and important
cause of lower respiratory tract illness in cattle and young infants
(71). More than 70% of calves exhibit a positive
serological response against BRSV by the age of 12 months.
Neutralization tests (9) and reaction patterns with specific
monoclonal antibodies (2, 48) have revealed that HRSV contains two major groups, A and B. The main differences between these
two groups are located in the glycoprotein (G protein), while
others are located in the fusion protein (F protein) and nucleoprotein
(N protein) (48, 51). Subgroups within the G and F proteins
of BRSV have been characterized more recently by serological analysis
of a limited number of isolates and confirmed by phylogenetic analysis
(20, 54, 60). Further studies of BRSV variability have
focused on the G protein, which was shown to be the most variable
protein and, with the F protein, one of the targets for neutralizing
antibodies. A low percentage of sequence divergence between and within
the G proteins of BRSV subgroups has been reported, suggesting that
BRSV has the same extent of diversity as HRSV (15, 21, 36, 44,
66).
The G protein is responsible for virus binding to the cell surface
receptor (41). It is a type II G protein with a
signal/anchor domain between residues 38 and 66. It is synthesized as a
32-kDa polypeptide precursor which is extensively modified by the
addition of both N- and O-linked oligosaccharides to achieve the mature form of 80 to 90 kDa (10, 26, 32, 77). This protein is structurally different from its counterparts
(hemagglutinin-neuraminidase and hemagglutinin) in other
paramyxoviruses. It has been proposed that the BRSV G protein
contains several independently folding regions, in which the ectodomain
consists of a conserved central hydrophobic region located between two
polymeric mucin-like regions (33, 34). This conserved
central hydrophobic region contains four conserved cysteines which form
two disulfide bridges. Peptides based on this region were found
to be immunodominant in the G proteins of both HRSV (1)
and BRSV (34). They also conferred protection against
HRSV challenge in mice (69). Recently, the three-dimensional
structure of a segment of this conserved central region was elucidated
by nuclear magnetic resonance spectroscopy (13). The major
epitope of this region was described as being located at the tip of a
loop, overlapping a relatively flat surface formed by the double
disulfide-bonded cysteine noose and lined by highly conserved residues
(33, 34).
Attempts to develop a vaccine against HRSV in humans have been
unsuccessful (28). However, inactivated and modified live vaccines against BRSV have been developed and used for more than 15 years in France and Belgium and for 2 or 3 years in other countries, such as Denmark or Sweden. Three types of vaccines are mostly used in Europe. Rispoval RS (strain RB-94 attenuated vaccine) is the
vaccine most commonly used in bovines. It has been commercialized in Belgium and The Netherlands since 1978 and in France since 1983. Bayovac BRSV (strain Lehmkuhl 375 attenuated vaccine) has been
commercialized in The Netherlands, France, and Belgium since 1994, 1996, and 1997, respectively. The third vaccine is Vacores (strain
220/69 inactivated with beta-propiolactone and associated with the
adjuvants aluminum hydroxide and saponin). It has been commercialized
in France and Belgium from 1996 to 1999. Such vaccination prevents the
disease and its economic consequences in herds. It does not suppress
the circulation of BRSV, however. In fact, some of the
characteristics of RSV infection are that the virus can replicate
in spite of detectable levels of specific antibodies (3, 72)
and that previous immunization of animals through infection or
vaccination does not completely eliminate the circulation of the virus
(53, 61). In addition, reinfection occurs in human and
bovine adults (14, 25, 27, 72).
Given the high rates of mutation of RNA viruses (12), it
would not be surprising if previously stimulated immunity (i.e., former
infection or vaccination) could select new variants, at least in the G
protein, which is known to favor the accommodation of sequence changes.
Studying the sequence of BRSV isolates in Europe therefore could
provide an indication of how the evolution of RSV might be modified in
nature by immune escape mechanisms. We therefore examined the
changes that have occurred in the N, F, and G protein genes of 87 BRSV
isolates over the past 32 years. Some of these isolates originated from
vaccinated or nonvaccinated calves in geographical areas where
vaccination had been widely used for many years, while others came from
nonvaccinated calves in countries where vaccination had been absent or
very limited. In this study, we demonstrate that a continuous evolution
of the sequences of the N, G, and F proteins of BRSV has been occurring in isolates since 1967. The exertion of a strong positive selective pressure on the G protein and on particular sites of the N and F
proteins is also demonstrated.
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MATERIALS AND METHODS |
Viruses and field samples. (i) BRSV with cell culture
passages.
The five BRSV strains isolated from Belgium (220/69,
RB-94, FV 160, MVR553, and 5761) between 1969 and 1990 were kindly
provided by Wellemans and Leunen (76) (Table
1). The
Lelystad strain isolated in 1974 from The Netherlands and strain 4642 isolated in 1976 from the United Kingdom were kindly provided by
T. J. Kimman. The Lelystad strain has been described by Kimman et
al. (29). Strain V347 isolated in 1990 from The Netherlands
was kindly provided by R. S. Schrijver. D80 was isolated by C. Delacourt in 1986 in the Somme Veterinary Laboratory (Dury, France).
B35 and 90504, which were kindly provided by E. Le Drean, were isolated in 1986 and 1990, respectively, in the Ille-et-Vilaine Veterinary Laboratory (Rennes, France). W6 was isolated in our laboratory in 1993 from a calf from a herd involved in a respiratory tract infection
outbreak. Isolates A1 and A2Gelfi were obtained from the same herd in
1995. Vaccine strains were obtained from vaccine bottles for RB-94
(Rispoval RS; batch 31863L) and Lehmkuhl 375 (Bayovac; batch 2071); the
strain 220/69 vaccine (Vacores) was kindly provided by Merial.
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TABLE 1.
Origins of virus strains and classification according to
nucleotide variability in segments of the N, G, and F
protein genesa
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(ii) BRSV without culture passage.
Twenty-three calves from
11 herds exhibiting an acute respiratory tract infection outbreak
compatible with BRSV were sampled between 1995 and 1997 by
bronchoalveolar lavage (A1, A2Gelfi, B1, B2, C1, C2, D1, D2, 394, 673, Bioch, F1, F2, I1, I2, I3, J1, J2, L1, L2, G2, K1, and K2). In 1998, nasal secretions (8346, 8352, and 8356) and a necropsy sample of lung
tissue (7313) from four calves in the same herd with BRSV-associated
respiratory tract infection signs were sampled. During the same year,
lungs from calves which had died of BRSV infection were collected from three French herds (58P, 75P, and 88P) and nine Belgian herds (P1, P2,
P3, P4, P5, P6, P7, P8, P9, and P10). All the samples were stored at
80°C until analysis. These isolates were never passaged in cell
cultures before RNA extraction.
Extraction of RNA and PCR. (i) RNA extraction.
Tissue
samples were homogenized directly in their storage tubes with a Diax
600 homogenizer (Heidolph, Bioblock, Illkirch, France). The homogenizer
arbor was replaced between each tissue sample and sterilized (NaOH for
10 min and then 30 min at 120°C) before further use. The RNA used as
a template for cDNA synthesis was extracted from cells and tissues as
previously described (70). Briefly, RNA was extracted with a
solution of guanidine isothiocyanate and phenol (TRIzol reagent; Gibco
BRL), purified with chloroform, and precipitated with isopropanol
overnight at 4°C. After being washed with 70% ethanol, the RNA
pellet was dried and then resuspended in 50 ml of diethyl pyrocarbonate
(DEPC)-treated water.
(ii) Nested RT-PCR.
Different nested reverse transcription
(RT)-PCRs were used to amplify the BRSV RNA of the N protein gene, the
G protein gene, and the F protein gene (Table
2). The nested RT-PCR for the N protein
gene was performed as previously described (70). Primers used for the G and F protein gene nested RT-PCRs were based on the
published sequences of the BRSV G protein (GenBank accession numbers
M58307, L10925, L08410, L08411, L08412, L08413, L08414, L08415, L08416,
and L08417) and the BRSV F protein (GenBank accession numbers D00953,
M58350, and M82816). Primer sequences were selected using the program PRIMER, version 0.5 (43). The different nested RT-PCRs were performed with a GeneAmp PCR system 480 (Perkin-Elmer, Courtaboeuf, France).
RT of viral RNA into specific cDNAs of the N, G, and F proteins was
done using primers N2.1, G2.5, and F2.1, respectively.
One microliter
of each primer (2 pmol) was mixed with 9 µl of
DEPC-treated water
containing RNA, incubated at 68°C for 10 min,
and finally chilled on
ice. Each tube received 4 µl of a solution
containing each nucleoside
triphosphate (10 mmol), 1 µl of RNasin
(40 U) (Promega,
Charbonnières, France), 2 µl of dithiothreitol
(Gibco BRL), 4 µl of Superscript TM II buffer (250 mM Tris HCl,
375 mM KCl, 15 mM
MgCl
2) (Gibco BRL), and 1 µl of SuperScript
TM II (200 U)
(Gibco BRL) and was incubated at 42°C for 50 min.
The reverse
transcriptase was inactivated at 70°C for 15 min.
The RNA-cDNA
hybrids were diluted 10 times in DEPC-treated
water.
The first set of primers, N2.1 and N2.2, was used for the first round
of PCR for the N protein gene, and the internal set
of primers, N2.3
and N2.4, was used for the second round of PCR.
The first set of
primers used to amplify a segment of the G protein
gene consisted of
G2.5 and F2.7, and the second set consisted
of VG1 and VG4
(
73). The primer sets F2.1-F2.2 and F2.3-F2.4
were
successively used to amplify a segment of the F protein gene.
The
dilutions at each different step and the compositions of the
PCR
mixtures were identical for all three nested RT-PCRs. The
first round
of PCR was performed with 10 µl of diluted cDNA which
was mixed in a
final volume of 100 µl with 5 µl of each primer
(50 pmol) from the
first set, 200 mM each nucleoside triphosphate,
10 mM Tris HCl (pH
8.3), 50 mM KCl, 1 mM MgCl
2, and 2.5 IU of
AmpliTaq Gold
polymerase (Perkin-Elmer). The products of the first
PCR were diluted
10 times. Ten milliliters was taken to perform
the second round of PCR
using the same mixture except for the
substitution of a set of internal
primers.
Sequence analysis.
Sequencing of amplified products was
performed using primers of the second set for each corresponding nested
RT-PCR according to the Applied Biosystems (Foster City, Calif.)
protocol. The samples were loaded on an Applied Biosystems 373A
sequencer. The nucleotide sequences of BRSV isolates were analyzed
using the program TRANSLATE of the University of Wisconsin GCG 9.1 package (11). They were compared with the sequences of other
BRSV isolates, when available, obtained from GenBank (the accession
numbers of the sequences are reported in Table 1). Multiple sequence
alignments were generated with the CLUSTAL W 1.60 program
(68). Representative isolates of HRSV subgroups A and B and
ovine RSV (ORSV), when available, were also included in this analysis.
Phylogenetic trees of nucleotide sequences were constructed using the
maximum-likelihood (ML) method available in version
1.2 of the
FastDNAml program (
52) derived from version 3.3 of
the DNA
Maximum Likelihood program (
17). Trees were computed
using
empirical base composition and transition/transversion ratios
obtained
from the data sets. Localized rearrangements were made
to improve tree
search efficiency. To reduce computing time, all
calculations were
performed with starting trees obtained by the
neighbor-joining (NJ)
method (
57). The robustness of the topologies
was assessed
with 100 bootstrap replicates (
18) using the same
parameters
and program options. The NJ method implemented in CLUSTAL
W
(
68) was also used for comparisons, in which case 1,000 bootstrap
replicates were
used.
Finally, the numbers of nucleotide substitutions per synonymous site
(
ds) and nonsynonymous site
(
dn) were estimated using
the method of Nei and
Gojobori (
50) as implemented in the MEGA
sequence analysis
package (version 1.01) (
30) for all possible
pairs of BRSV
sequences from each coding region. They were also
plotted for
individual codons using the SNAP program (available
at
http://hiv-web.lanl.gov/SNAP/WEBSNAP/SNAP.html). For both
types
of substitution, only non-zero values were retained for the
analysis
so that the nonsynonymous/synonymous substitution rate ratio
could
be calculated for each pair of fragments, together with the
average.
The rates of nonsynonymous and synonymous substitutions were
calculated
using the method of Li et al. (
42), in which the
rate is calculated
for pairs of sequences with a third sequence as an
outgroup (
46).
Modeling of secondary and tertiary structures.
Hydrophilicity was calculated using PEPTIDESTRUCTURE in the GCG package
according to the algorithm of Kyte and Doolittle (31) with a
window set to seven residues. The prediction of solvent accessibility
was performed using PHDacc (55) (available at http://dodo.cpmc.columbia.edu/predictprotein).
Secondary structures for the conserved central region of the G protein
of BRSV were obtained from the Brookhaven Protein Data
Bank
(
http://www.pdb.bnl.gov/index.html). The structures were
aligned with the computer program SWISSPDBVIEWER
(
http://www.expasy.ch/spdbv/mainpage.html).
Amino acid replacements
were performed on the alignments, and
the new structure was modelled
using version 4 of the MODELLER
program. This program models the
three-dimensional structure of
proteins by satisfaction of spatial
restraints (
58,
59). The
output of MODELLER was then
graphically visualized using
SWISSPDBVIEWER.
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RESULTS |
Nucleotide sequences of BRSV isolates are closely related.
The
nucleotide sequences of fragments of the N protein gene (690 nucleotides corresponding to codon positions 50 to 279), of the G
protein gene (489 nucleotides corresponding to codon positions 53 to 215), and of the F protein gene (741 nucleotides corresponding to
codon positions 167 to 413) from 54 BRSV isolates were determined
and compared with those of 33 isolates taken from GenBank. These
included the sequences of the three vaccine strains (RB-94 [Rispoval
RS], 220/69 [Vacores], and Lehmkuhl 375 [Bayovac]) used mostly in
Europe. Alignment of the nucleotide sequences determined in this study
did not reveal any gaps or insertions. After removal of the nucleotide
sequences which were identical, 25 partial N protein gene sequences
(690 bases), 63 partial G protein gene sequences (437 to 489 bases),
and 25 partial F protein gene sequences (741 bases) were available for
analysis. The vaccine strains RB-94 and 220/69 were identical in all
three coding regions. The average percentage of pairwise divergences
between fragments of BRSV was lowest for the N and F protein genes
(2%) and highest for the G protein gene (8%). The average percentage
of pairwise divergences between fragments of BRSV and HRSV was lowest
for the N protein gene (20%), intermediate for the F protein gene
(22%), and highest for the G protein gene (48%).
Several isolates were collected within the same herd. Twenty-nine
isolates were collected from different calves exhibiting
respiratory
tract illness in 11 different herds located in France
and 1 herd
located in Belgium. The isolates of a definite herd
were obtained on
the same day, except for those from herd A, which
was sampled over an
interval of 2 years (Table
1). The nucleotide
sequences of the N, F,
and G protein gene regions obtained from
five of these herds (C, I, J,
M, and K) were identical. Differences
were found in the F protein gene
sequence between samples from
four of the other herds. Only a very
limited number of differences
were apparent, within the N and G protein
gene sequences, in the
three remaining herds. The sequences of the N,
F, and G protein
genes of the viruses (W6, A1, and A2Gelfi) isolated at
a 2-year
interval from the same herd (A) were different. Thus, a
single
virus or a group of very closely related viruses would seem to
predominantly infect a given herd at a given
time.
Temporal and geographical clustering of BRSV sequences.
The ML
method was used to compute phylogenetic trees, based on the alignment
of the different gene fragments. For clarity, it was possible, from the
phylogenetic tree obtained with the G protein fragment, to arbitrarily
divide the data into six subgroups (I to VI) (Fig.
1 and Table 1). The topology and
significance of the different nodes giving rise to these six subgroups
were verified by bootstrap analysis using both the ML and the NJ
methods (data not shown). However, the node giving rise to the isolates of subgroup IV was not statistically corroborated by bootstrap analysis, but the subgroup was retained because of its intermediate location between the isolates of subgroups V and VI and the others. Isolate NMK7 from Japan was not related to any of these subgroups and
was arbitrarily included in group II. Trees were also constructed using
the alignments of the nucleotide sequences of the N and F protein genes
(Fig. 2 and
3). In this case, as for the G protein gene, the topology and significance of the important nodes of the trees
were verified by bootstrap analysis using both the ML and the NJ
methods (data not shown). No sequence from subgroup III isolates was
available to be included in these trees, except for the Lehmkuhl 375 and 391.2 strains. These trees showed the same general clustering of
isolates as that already described for the G protein gene. In both
cases, the subgroup IV isolates were still in an intermediate position
between those of subgroups V and VI and the others. A striking
difference between these trees and the tree obtained for the G protein
gene is that the isolates belonging to subgroup VI (K1, K2, and 75P)
could no longer be distinguished from those of subgroup V. The
vaccine strains RB-94 (and 220/69) and Lehmkuhl 375 were
localized in subgroups II and III, respectively.
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FIG. 1.
ML phylogenetic tree showing the relationships among the
G protein genes of 87 BRSV isolates, 1 ovine RSV (ORSV) isolate, and 2 human RSV isolates (18537 and hrsvA2). Horizontal branches are drawn to
scale, and the tree is rooted with isolate hrsvA2. Designations at the
ends of the branches refer to the identifying code of the isolate
(Table 1).
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FIG. 2.
ML phylogenetic tree showing the
relationships among the N protein genes of 58 BRSV isolates, 1 ovine
RSV (ORSV) isolate, and two human RSV isolates (18537 and hrsvA2).
Horizontal branches are drawn to scale, and the tree is rooted with
isolate hrsvA2. Designations at the ends of the branches refer to the
identifying code of the isolate (Table 1).
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FIG. 3.
ML phylogenetic tree showing the relationships among the
F protein genes of 56 BRSV isolates and 2 human RSV isolates (18537 and
hrsvA2). Horizontal branches are drawn to scale, and the tree is rooted
with isolate hrsvA2. Designations at the ends of the branches refer to
the identifying code of the isolate (Table 1).
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Subgroup I, which consisted of strains isolated before 1976, no longer
seems to exist in Europe, as has previously been suggested
(
15). The isolates of subgroup III came exclusively from the
United States. Those belonging to the remaining subgroups, II,
IV, V,
and VI, came exclusively from Europe, except for the VC464
isolate,
which originated from the United States. A correlation
therefore exists
between the continent of origin of the isolates
and their clustering in
the phylogenetic tree. In Europe, the
isolates from northern Europe,
Denmark, and Sweden clustered in
subgroup II, whatever their date of
isolation (from 1983 to 1995).
Those from The Netherlands, Belgium, and
France were found in
subgroups II, IV, V, and VI, depending on their
date of isolation.
In general, the close clustering of European
isolates is related
to their date of isolation, although several
isolates could be
distant from each other for a particular year. For
example, when
the strains originating from The Netherlands, Belgium,
the United
Kingdom, and France are considered, the dates of isolation
of
the subgroup II, IV, V, and VI isolates are 1969 to 1986, 1971
to
1990, 1993 to 1998, and 1997 to 1998, respectively (Fig.
1 and Table
1). All the recent isolates from these countries are
located at the top
of the tree in subgroups V and VI. This result
could indicate the
presence of a temporal clustering in addition
to the geographical
clustering already described. The recent isolates
of subgroups V and VI
are more distantly related to the vaccine
strains than to the other
isolates.
During 1998, field samples were obtained from 11 calves that had been
vaccinated approximately 2 to 3 months before the onset
of symptoms.
These calves were less than 6 months old and exhibited
respiratory
tract illness. Seven of them had previously been vaccinated
with strain
220/69 (Vacores), and four of them had been vaccinated
with strain
RB-94 (Rispoval RS). Analysis of the N, F, and G protein
gene sequences
indicated that 10 of these isolates belonged to
subgroup V and 1 belonged to subgroup VI, in accordance with their
dates of isolation
(Fig.
1,
2,
3, and
4). These
results suggest
also that vaccination sometimes does not prevent
infection of
calves with BRSV of subgroups V and VI, indicating that
vaccinated
calves may be poorly protected against infection by recent
BRSV
isolates of subgroups V and VI.
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FIG. 4.
Sequences of amino acids 53 to 215 of the G protein of
BRSV isolates. Designations to the left of the sequences indicate the
isolate code (Table 1). Subgroup designations are shown to the left of
the isolate designations.
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Four highly conserved Cys residues have been replaced by Arg in the
sequence of the G protein of recent French isolates.
The mutations
observed in the amino acid sequences of the different European
subgroups are shown in Fig. 4. The sequences can be divided into three
domains: two highly variable domains corresponding to the mucin-like
regions and one central conserved domain. The two mucin-like regions of
the BRSV G protein, the first of which was located between the
transmembrane domain and the central conserved domain and the second of
which was located from this central conserved domain to the COOH
terminus of the molecule, had very high Ser and Thr contents
31% for
the first region (amino acid positions 67 to 153) and 30.4% for the
second region (amino acid positions 193 to 215)thereby confirming the results of previous studies (34).
The central conserved domain also exhibited important amino acid
changes (Fig.
4 and
5). Based on the
availability of the
structural coordinates of and the experimental
restraints on the
central conserved region of the G protein deposited
in the Protein
Data Bank of Brookhaven National Laboratory, we were
able to examine
the modifications induced in the three-dimensional
structure by
the mutations observed in this domain in field isolates.
This
domain is particularly important, as it was shown to be
immunodominant
in the G protein (
34). The initial resolution
of the structure
was performed on strain 391.2 (
13), which
belongs to subgroup
III. As most of the important residues in subgroups
I to VI were
conserved, the characteristic structure of the central
conserved
region of the G protein, as indicated by MODELLER, was
probably
also conserved. This structure consists of two disulfide
bridges:
Cys173-Cys186 (outer bridge) and Cys176-Cys182 (inner bridge).
It begins with 1 turn of the
helix (Cys173-Cys176) which runs
antiparallel to the 1.5
-helical turn formed by Leu180-Leu185
(
13) (Fig.
6). The two helices
are linked by a type I turn (Cys176-Asn179).
Asparagine 179 is involved
in three hydrogen bonds.
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FIG. 5.
Consensus sequence of BRSV subgroups from residues 170 to 209. Horizontal arrows indicate linear epitopes from the study of
Langedijk et al. (33). Vertical arrows indicate mutations
determining antigen subgrouping (33). Boxes indicate helices.
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FIG. 6.
Representation of the superimposition of the cysteine
noose of BRSV strain 391.2 (left) and of the consensus sequence of
isolates from subgroup VI (right). Helices are indicated in blue
and red. Disulfide bridges are drawn in yellow. Residues 171 to 189 in
the representation of strain 391.2 correspond to residues 1 to 19 in
that of subgroup VI.
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The most striking mutations are located in the isolates of subgroup VI.
In these isolates, the two disulfide bridges no longer
exist due to
replacement of the four Cys residues by four Arg
residues. Resolution
of the structure indicates that the first
helix is disrupted (Fig.
6). As a consequence, Asp179 is involved
in only one hydrogen bond with
Arg176 (data not shown). Near the
central conserved region, the
Ala205
Thr mutation distinguishing
the G protein sequences of
subgroup II (which includes most of
the currently used vaccine strains)
from those of subgroups III,
IV, V, and VI is sufficient to cause
escape from antibody binding.
This mutation results in a
potential additional O-glycosylation
site which could modify the
antigenicity of the linear epitope
described for positions 199 to 209 (
33).
Nonsynonymous changes are localized in specific regions.
The
following analysis is restricted to the European BRSV isolates and to
the vaccine strains Lehmkuhl 375 (subgroup III), RB-94 (subgroup II),
and 220/69 (subgroup II). This analysis includes 38 different
nucleotide sequences of the G protein gene which were available for all
489-base sequences, 24 different N protein gene nucleotide sequences
(690 bases), and 24 different F protein gene nucleotide sequences (741 bases). The percentages of amino acid divergence between the European
BRSV sequences were 0.7, 0.8, and 16.8 for the N, F, and G protein gene
regions, respectively.
ds and
dn as well as the
dn/
ds ratios for these
three coding regions were assessed.
ds,
dn, and the
dn/
ds ratios were
calculated
as the means of the ratios determined in the pairwise
sequence
comparisons. Overall differences in the
dn/
ds ratios between the
N, F, and G protein gene regions were significant (Pearson chi-square
analysis). As expected,
ds,
dn, and the
dn/
ds ratios of the G
protein fragment were significantly higher than those of the N
and F
protein fragments (Table
3). However, the
dn/
ds ratio was
not
identical over the entire G protein gene sequence (Fig.
7)
and rose to values exceeding 0.8 in
the two different coding parts
of the genes corresponding to the
mucin-like regions described
above. The first region that exhibited a
major increase in the
dn/
ds ratio was located
between codons 82 and 153, in particular,
between codons 104 and 132, where two
dn/
ds
ratios reached 1.587.
Another, minor region was located between
codons 192 and 202.
These ratios are considered evidence of
positive selection (
16)
in the G protein. Our analysis shows
a slight increase in
ds between
these two
regions, starting from codon 149 and reaching a maximum
of
0.545 between codons 174 and 183.
ds
then decreases slowly
toward codons 184 to 203, where
ds is 0.206 (Fig.
7). The region
between
codons 154 and 192 seems to be particularly well conserved.
View larger version (25K):
[in this window]
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|
FIG. 7.
Genetic changes in the G protein gene according to
codon number. ds (diamonds) and the
dn/ds ratio (squares) are
represented along the G protein length from codons 53 to 215, with
a window of 20.
|
|
Similarly, to evaluate the effects of natural selection on individual
amino acids that large-scale pairwise comparisons are
unlikely to
reveal, we calculated the mean
dn for each
codon in
the N and F protein gene sequences and identified the
codons with
rates higher than the mean
ds across
all codons (
4). In the
N protein gene, two regions that
exhibited a major increase in
the average
dn/
ds ratio (the average
dn/
ds ratio rose to
values
exceeding 0.62) were located between codons 56 and 67 (region
N1) and codons 147 and 153 (region N2). The mean
dn for codon
98 in the N protein gene
sequence was also above the mean
ds.
Three such
regions in the F protein gene sequence (the average
dn/
ds ratio rose to
values exceeding 0.66) were identified between
codons 202 and 218 (region F1), codons 294 and 305 (region F2),
and codons 389 and
401 (region F3). These results indicate that
these regions concentrate
most of the positive selection in the
N and F proteins. Regions N2, F1,
and F3, which exhibit high values
for hydrophilicity and relative
solvent accessibility (the accessibility
of F3 being the lowest of the
three regions), are potentially
exposed at the surface of the proteins
(data not shown). In contrast,
region F2 is hydrophobic and has a very
low relative accessibility.
Region N1 has values for hydrophilicity and
relative solvent accessibility
that decrease from its amino terminus to
its carboxy terminus
(data not
shown).
Rates of evolution of the synonymous and nonsynonymous
substitutions in the G protein gene.
The relative selective
pressures exerted on the G protein gene of BRSV isolates in the
presence or in the absence of vaccination were investigated. First,
this analysis was carried out with the three vaccine strains and 29 subgroup I, II, IV, V, and VI isolates originating from different herds
in the United Kingdom, The Netherlands, Belgium, and France between
1969 and 1998 (29 years). This time frame includes the period when
vaccination was widely practiced, at least in Belgium, France, and The
Netherlands. Second, the same analysis was performed with 11 isolates
of subgroups I and II, including the three vaccine strains, beginning
with the oldest Belgian isolate (1969) and ending with a Danish isolate
with the most recent complete sequence (1995). After 1977, the date of first use of vaccination against BRSV in Europe, this analysis was
restricted to isolates from Denmark and Sweden. This time frame
represents 26 years of BRSV evolution in the absence of selective
pressure due to cattle vaccination. ds and
dn as well as the
dn/ds ratios were
calculated for these two groups of isolates. The
dn/ds ratio for the
32 sequences potentially subjected to vaccine-induced
selection (dn/ds
ratio, 0.62) was slightly higher than that obtained for the 11 sequences not subjected to any vaccine selective pressure
(dn/ds ratio, 0.47)
(Table 3).
To more precisely evaluate the effects of selection in these two
epidemiological situations, the rates of evolution of the
synonymous
and nonsynonymous substitutions were determined. The
vaccine strain
Lehmkuhl 375 (subgroup III) was chosen as the outgroup.
For the
isolates potentially subjected to vaccine selective pressure,
the correlations obtained between time of isolation and synonymous
(syn) or nonsynonymous (nsy) substitutions were highly
significant
(
n = 703,
Rsyn = 0.189,
Rnsy = 0.458,
P < 0.001). The rates were
5.2 × 10
4 and 6.4 × 10
4 nucleotide changes per year per synonymous
site and per nonsynonymous
site, respectively. These results are
indicative of a continuous
evolution of BRSV G protein gene sequences
from subgroups I and
II to subgroup VI. For the isolates collected in
the absence of
vaccination, no significant correlation was obtained
between the
intervals of isolation and synonymous or nonsynonymous
changes.
Rates of evolution of the synonymous and nonsynonymous
substitutions in the N and F protein genes.
The same analysis
concerning the rate of evolution was performed with 31 N and 40 F
protein gene sequences belonging to subgroups II, IV, V, and VI (all
the European sequences were included in this analysis, except for the
identical sequences from the same herds). This sample includes viruses
isolated when and where vaccination was widely practiced in Europe.
This analysis could not be performed in the absence of selective
pressure due to cattle vaccination, as the N and F protein gene
sequences of isolates from Denmark and Sweden were not available from
GenBank. The correlations obtained were highly significant for the N
protein gene (n = 465, Rsyn = 0.731, Rnsy = 0.396, P < 0.001)
and for the F protein gene (n = 780, Rsyn = 0.574, Rnsy = 0.751, P < 0.001). For the N protein gene, the rates
were 1.6 × 103 and 5.4 × 105
nucleotide changes per year per synonymous site and per nonsynonymous site, respectively. For the F protein gene, the rates were 6.3 × 104 and 1.9 × 104 nucleotide changes
per year per synonymous site and per nonsynonymous site, respectively.
These values are also indicative of a continuous evolution of BRSV N
and F protein gene sequences in countries where vaccination was widely used.
| |
DISCUSSION |
The genetic variability of the two antigenically distinct
HRSV subgroups has been well characterized (8).
However, this type of analysis has, in the case of BRSV, been limited
until now to a small number of isolates and mostly to the G protein (15, 20, 21, 36, 44, 54, 60, 66). In the work reported here,
we examined the divergence within nucleotide and amino acid sequences
of the G protein in 87 BRSV isolates. The coding sequences of the N and
F proteins in at least 58 of these isolates were also compared.
The rate of evolution of the BRSV sequences varies according to the
gene, with the G protein gene evolving more rapidly and being highly
tolerant to the fixation of mutations, whereas the N and F protein
genes appear to be under stronger selective structural constraints
which limit their evolution. However, a progressive accumulation of
nucleotide changes was noted for the three fragments of these genes, as
previously described for HRSV (8).
This work confirms the primary structure of the G protein determined
for HRSV (for a review, see reference 47). It can be divided into three different parts, with one region located between amino acids 67 and 153 and another, starting with amino acid 193, constituting two mucin-like regions, with their characteristic high Ser
and Thr contents. The end of this second region could not be determined
in our study due to partial sequencing of the G protein gene. As with
the G protein gene of HRSV (7, 22), nonsynonymous mutations
are not randomly distributed along the G protein gene of BRSV but are
localized predominantly in two areas of the gene (codons 82 to 153 and codons 192 to 202) coding for the mucin-like regions;
presumably, these areas represent antigenic regions in the BRSV G
protein. Both regions have already been shown to be of high antigenic
importance in both BRSV and HRSV (8, 23, 34, 56, 66).
In contrast, the central hydrophobic region of the G protein, located
between the two mucin-like regions and comprising amino acid positions
154 to 192, has remained highly stable during evolution. This region
has been shown to accept predominantly synonymous mutations. It
has been postulated to contain the region interacting with the putative
RSV cell receptor. However, a very limited number of changes
still occurred in the sequence; these changes modified the
three-dimensional structure of the internal loop in the central hydrophobic region and an O-glycosylation site at codon position 205. The cysteine noose motif connecting the strands of the loop was
disrupted due to mutation of Cys to Arg. Cysteine noose motifs are
known to induce a high surface accessibility for the residues contained
in the loop (35).
Some of these mutations presented by field isolates were similar to
some of those already described for in vitro HRSV antibody escape
mutants (45, 74). However, this work constitutes the first
evidence that natural in vivo infections can occur with mutants lacking
the four cysteines involved in the two disulfide bridges. Since at
least one calf died of its infection, there is nothing about the
clinical illness due to these viruses to suggest that they are
attenuated. These mutations probably lead to important modifications in
the antigenic sites located in this region. Some of these mutations had
been tested by Pepscan analysis and had been shown to severely modify
the recognition of antibodies (33). This finding is of
concern with regard to recent BRSV isolates from France, where
vaccination is widely used. Whether or not these mutations are linked
to the positive selection exerted on other parts of the molecule
remains to be proved. However, it has been shown that this central part
of the G protein molecule constitutes a major domain involved in
protection against HRSV infection (63, 67). Furthermore,
this region, particularly amino acids 193 to 203, is involved in the
enhancement of illness and lung eosinophilia in mice (65).
The consequences of the modification of the conformational structure
within the G protein in recent isolates from France should be
investigated to determine if this modification could have an effect on
illness enhancement and on protection. Indeed, strains from vaccination
failures (subgroups V and VI) described in this paper are the most
divergent from the vaccine strains and also the most recent.
The rate of evolution per nonsynonymous sites indicated 6.4 × 104 nucleotide changes per year for isolates originating
from countries where vaccination was widely used (France, The
Netherlands, and Belgium) during the period from 1969 to 1998. This
high value cannot be related to Taq polymerase-induced
errors (5, 64). Conversely, the same evaluation performed on
isolates originating from countries where calf vaccination was limited
and very recent (Denmark and Sweden) or from countries before the start
of extensive vaccination (France, The Netherlands, and Belgium before
1977) did not indicate any significant evolution of the sequence. This result also favors the existence of a stronger positive selection pressure on the BRSV G protein in countries where BRSV vaccination had
been more widely used. In some ways, this finding is equivalent to the
sequence evolution due to immunological pressure on the hemagglutinin
of influenza A viruses (for a review, see reference 24). The rates of evolution per synonymous and
nonsynonymous sites determined for the N, G, and F protein fragments
also indicate continuous evolution of BRSV sequences in the presence of
vaccination. The values obtained for the evolution of BRSV are not
surprising compared to those obtained for other RNA viruses (for a
review, see reference 12). They are higher than
those obtained for stable G protein genes, such as those of
lyssaviruses (4), but lower than that of the hemagglutinin
gene of influenza A virus, which is known to show rapid and temporal
rather than geographical variations (19). In this respect,
the molecular epidemiology of BRSV is intermediate between those of
influenza B virus (6, 78) and influenza A virus
(75). The clustering of BRSV isolates according to their
continent of origin has not been observed for HRSV (8). This
difference might reflect different circulation patterns for the
human populations and calf herds between the American and European continents.
Some localized regions in the N protein (regions N1 [positions 56 to
67] and N2 [positions 143 to 153]) and in the F protein (regions F1
[positions 202 to 218], F2 [positions 294 to 305], and F3
[positions 389 to 401]) contained most of the amino acid variability
of these proteins. No data are available concerning the localization of
antigenic sites in the BRSV N protein. However, region N2, which is
predicted to be exposed at the surface of the molecule, overlaps with a
peptide of the HRSV N protein (positions 131 to 150) which was shown to
be a linear antigenic domain (37). There is complete
identity in this region between the two N proteins. The regions defined
in the F protein do not correspond to any of the antigenic sites
described for BRSV and HRSV. Further work will be needed to study the
relationships between these relatively hypervariable regions in the N
and F proteins and the immunological response against BRSV. An
alternative explanation is that some of the variable sites are of high
flexibility and accumulate accompanying mutations that are not directly
implicated in positive immune selection.
In conclusion, the evolution of BRSV combines a high rate of sequence
evolution (providing local genetic differentiation, which explains some
of the geographical clustering observed in the phylogenetic trees) with
a greatly elevated rate of amino acid changes in some regions of the G
protein. Since these regions of the G protein are believed to be
antigenically important, these changes provide an opportunity for the
virus to potentially escape from previously established immunity, as
determined in vitro with HRSV (67). As a consequence, recent
isolates originating from countries where vaccination is widely used
exhibit greater changes in the amino acid sequences in these critical
regions. Further studies are now needed to reevaluate the level of
protection provided by vaccine strains, taking into account this rapid
evolution and, in particular, some of the mutations in the G protein
described here. In addition, the ability of RSV to evade immune
responses in humans should continue to be investigated. We are
suggesting that particular attention should be given to the development
of candidate HRSV subunit vaccines including only the central region of
the G protein (62). Considering the mutations in this region described here, this subunit-vaccine approach should be used with caution.
| |
ACKNOWLEDGMENTS |
We are greatly indebted to veterinarians, practitioners, and
farmers who allowed us to collect BRSV isolates; to Chantal Delacourt for strain D80 isolated in the Veterinary Laboratory of the Somme; to
Eric Le Dréan for viruses B35-1 and 90504 isolated in the Veterinary Laboratory of Ille et Vilaine; and to D. Desmecht for lung
samples from Belgium (P1 to P10). We also thank Suzanne Bonhoure, Martine Deplanche, Frédéric Lasserre, Martine Moulignier,
and Jean-Luc Pingret for expert technical assistance and Edward Holmes, Nicolas Le Novere, and Wayne Sullender for helpful comments and critical reading of the manuscript.
This study was supported in part by grants from ENVT and from Institut
Pasteur (Air, Environnement, et Santé grant).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité de
la Rage, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris
Cedex 15, France. Phone: 33.1.45.68.87.85. Fax: 33.1.40.61.30.20. E-mail: hbourhy{at}pasteur.fr.
| |
REFERENCES |
| 1.
|
Åkerlind-Stopner, B.,
G. Utter,
M. A. Mufson,
C. Örvell,
R. A. Lerner, and E. Norrby.
1990.
A subgroup-specific antigenic site in the G protein of respiratory syncytial virus forms a disulfide-bonded loop.
J. Virol.
64:5143-5148[Abstract/Free Full Text].
|
| 2.
|
Anderson, L. J.,
J. C. Heirholzer,
C. Tson,
R. M. Hendry,
B. N. Fernie,
Y. Stone, and K. McIntosh.
1985.
Antigenic characterization of respiratory syncytial virus strains with monoclonal antibodies.
J. Infect. Dis.
151:626-633[Medline].
|
| 3.
|
Beem, M.
1987.
Repeated infections with respiratory syncytial virus.
J. Immunol.
98:1115-1122[Abstract/Free Full Text].
|
| 4.
|
Bourhy, H.,
B. Kissi,
L. Audry,
M. Smreczak,
M. Sadkowska-Todys,
K. Kulonen,
N. Tordo,
J. F. Zmudzinski, and E. C. Holmes.
1999.
Ecology and evolution of rabies virus in Europe.
J. Gen. Virol.
99:2545-2557.
|
| 5.
|
Bracho, M. A.,
A. Moya, and E. Barrio.
1998.
Contribution of Taq polymerase-induced errors to the estimation of RNA virus diversity.
J. Gen. Virol.
79:2921-2928[Abstract].
|
| 6.
|
Buonagurio, D. A.,
S. Nakada,
U. Desselberger,
M. Krystal, and P. Palese.
1985.
Noncumulative sequence changes in the hemagglutinin genes of influenza C virus isolates.
Virology
146:221-232[CrossRef][Medline].
|
| 7.
|
Cane, P. A.,
D. A. Matthews, and C. R. Pringle.
1991.
Identification of variable domains of the attachment (G) protein of subgroup A respiratory syncytial viruses.
J. Gen. Virol.
72:2091-2096[Abstract/Free Full Text].
|
| 8.
|
Cane, P. A., and C. R. Pringle.
1995.
Evolution of subgroup A respiratory syncytial virus: evidence for progressive accumulation of amino acid changes in the attachment protein.
J. Virol.
69:2918-2925[Abstract].
|
| 9.
|
Coates, H. V.,
D. W. Alling, and R. M. Chanock.
1966.
An antigenic analysis of respiratory syncytial virus isolates by plaque reduction neutralization assay.
Am. J. Epidemiol.
83:299-313[Free Full Text].
|
| 10.
|
Collins, P. L., and G. Mottet.
1992.
Oligomerization and post-translational processing of glycoprotein G of human respiratory syncytial virus. Altered O-glycosylation in the presence of brefeldin A.
J. Gen. Virol.
73:849-863[Abstract/Free Full Text].
|
| 11.
|
Devereux, J.,
P. Haeberli, and O. Smithies.
1984.
A comprehensive set of sequence analysis programs for the Vax.
Nucleic Acids Res.
12:387-395.
|
| 12.
|
Domingo, E., and J. J. Holland.
1997.
RNA virus mutations and fitness for survival.
Annu. Rev. Microbiol.
51:151-178[CrossRef][Medline].
|
| 13.
|
Doreleijers, J. F.,
J. P. M. Langedijk,
K. Hard,
R. Boelens,
J. A. C. Rullmann,
W. M. Schaaper,
J. T. van Oirschot, and R. Kaptein.
1996.
Solution structure of immunodominant region of protein G of bovine respiratory syncytial virus.
Biochemistry
35:14684-14688[CrossRef][Medline].
|
| 14.
|
Dowell, S. F.,
L. J. Anderson,
H. E. Gary,
D. D. Erdman,
J. F. Plouffe,
T. M. File,
B. J. Martson, and R. F. Breiman.
1996.
Respiratory syncytial virus is an important cause of community-acquired lower respiratory infection among hospitalized adults.
J. Infect. Dis.
174:456-462[Medline].
|
| 15.
|
Elvander, M.,
S. Vilcek,
C. Baule,
A. Uttenthal,
A. Ballagi-Pordany, and S. Belak.
1998.
Genetic and antigenic analysis of the G attachment protein of bovine respiratory syncytial virus strains.
J. Gen. Virol.
79:2939-2946[Abstract].
|
| 16.
|
Endo, T.,
K. Ikeo, and T. Gojobori.
1996.
Large-scale search for genes on which positive selection may operate.
Mol. Biol. Evol.
13:685-690[Abstract].
|
| 17.
|
Felsenstein, J.
1981.
Evolutionary trees from DNA sequences: a maximum likelihood approach.
J. Mol. Evol.
17:368-376[CrossRef][Medline].
|
| 18.
|
Felsenstein, J.
1985.
Confidence limits on phylogenies: an approach using the bootstrap.
Evolution
39:783-791[CrossRef].
|
| 19.
|
Fitch, W. M.,
R. M. Bush,
C. A. Bender, and N. J. Cox.
1997.
Long term trends in the evolution of H(3) HA1 human influenza type A.
Proc. Natl. Acad. Sci. USA
94:7712-7718[Abstract/Free Full Text].
|
| 20.
|
Furze, J.,
G. Wertz,
R. A. Lerch, and G. Taylor.
1994.
Antigenic heterogeneity of the attachment protein of bovine respiratory syncytial virus.
J. Gen. Virol.
75:363-370[Abstract/Free Full Text].
|
| 21.
|
Furze, J. M.,
S. R. Roberts,
G. W. Wertz, and G. Taylor.
1997.
Antigenically distinct G glycoproteins of BRSV strains share a high degree of genetic homogeneity.
Virology
231:48-58[CrossRef][Medline].
|
| 22.
|
Garcia, O.,
M. Martin,
J. Dopazo,
J. Arbiza,
S. Frabasile,
J. Russi,
M. Hortal,
P. Perez-Brena,
I. Martinez,
B. Garcia-Barreno, and J. A. Melero.
1994.
Evolutionary pattern of human respiratory syncytial virus (subgroup A): cocirculating lineages and correlation of genetic and antigenic changes in the glycoprotein.
J. Virol.
68:5448-5459[Abstract/Free Full Text].
|
| 23.
|
Garcia-Barreno, B.,
A. Portela,
T. Delgado,
J. A. Lopez, and J. A. Melero.
1990.
Frame shift mutations as a novel mechanism for the generation of neutralization resistant mutants of human respiratory syncytial virus.
EMBO J.
9:4181-4187[Medline].
|
| 24.
|
Garnett, G. P., and R. Antia.
1994.
Population biology of virus-host interactions, p. 51-73.
In
S. S. Morse (ed.), The evolutionary biology of viruses. Raven Press, New York, N.Y.
|
| 25.
|
Glezen, W. P.,
A. Paredes,
J. E. Allison,
L. H. Taber, and A. L. Frank.
1981.
Risk of respiratory syncytial virus infection for infants from low-income families in relationship to age, sex, ethnic group, and maternal antibody level.
J. Pediatr.
98:708-715[Medline].
|
| 26.
|
Gruber, C., and S. Levine.
1985.
Respiratory syncytial virus polypeptides. IV. The oligosaccharides of the glycoproteins.
J. Gen. Virol.
66:417-432[Abstract/Free Full Text].
|
| 27.
|
Hall, C. B.,
R. G. Douglas, and J. M. Geiman.
1976.
Respiratory syncytial virus infections in infants: quantification and duration of shedding.
J. Pediatr.
89:11-15[CrossRef][Medline].
|
| 28.
|
Kim, H. W.,
J. G. Canchola,
C. D. Brandt,
G. Pyles,
R. M. Chanock,
K. Jensen, and R. H. Parrott.
1969.
Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine.
Am. J. Epidemiol.
89:422-434[Abstract/Free Full Text].
|
| 29.
|
Kimman, T. G.,
G. M. Zimmer,
F. Westenbrink,
J. Mars, and P. W. de Leeuw.
1986.
Diagnosis of bovine respiratory syncytial virus infections improved by virus detection in lung lavage samples.
Am. J. Vet. Res.
47:143-147[Medline].
|
| 30.
|
Kumar, S.,
K. Tamura, and M. Nei.
1993.
MEGA: Molecular Evolutionary Genetic Analysis, version 1.01.
The Pennsylvania State University, University Park.
|
| 31.
|
Kyte, J., and R. F. Doolittle.
1982.
A simple method for displaying the hydropathic character of a protein.
J. Mol. Biol.
157:105-132[CrossRef][Medline].
|
| 32.
|
Lambert, D. M.
1988.
Role of oligosaccharides in structure and in function of respiratory syncytial virus glycoproteins.
Virology
164:458-466[CrossRef][Medline].
|
| 33.
|
Langedijk, J. P. M.,
R. H. Meloen,
G. Taylor,
J. M. Furze, and J. T. van Oirschot.
1997.
Antigenic structure of the central conserved region of protein G of bovine respiratory syncytial virus.
J. Virol.
71:4055-4061[Abstract].
|
| 34.
|
Langedijk, J. P. M.,
V. M. M. Schaaper,
R. H. Meloen, and J. T. van Oirschot.
1996.
Proposed three-dimensional model for the attachment protein G of respiratory syncytial virus.
J. Gen. Virol.
77:1249-1257[Abstract/Free Full Text].
|
| 35.
|
Lapthorn, A. J.,
R. W. Janes,
N. W. Isaacs, and B. A. Wallace.
1995.
Cystine nooses and protein specificity.
Nat. Struct. Biol.
2:266-268[CrossRef][Medline].
|
| 36.
|
Larsen, L. E.,
A. Uttenthal,
P. Arctander,
K. Tjornehoj,
B. Viuff,
C. Rontved,
L. Ronsholtved,
S. Alexandersen, and M. Blixenkrone-Moller.
1998.
Serological and genetic characterisation of bovine respiratory syncytial virus (BRSV) indicates that Danish isolates belong to the intermediate subgroup: no evidence of a selective effect on the variability of G protein nucleotide sequence by prior cell culture adaptation and passages in cell culture or calves.
Vet. Microbiol.
62:265-279[CrossRef][Medline].
|
| 37.
|
Leonov, S. V.,
M. Waris, and E. Norrby.
1995.
Linear antigenic and immunogenic regions of human respiratory syncytial virus N protein.
J. Gen. Virol.
76:357-364[Abstract/Free Full Text].
|
| 38.
|
Lerch, R. A.,
K. Anderson, and G. W. Wertz.
1990.
Nucleotide sequence analysis and expression from recombinant vectors demonstrate that attachment protein G of bovine respiratory syncytial virus is distinct from that of human respiratory syncytial virus.
J. Virol.
64:5559-5569[Abstract/Free Full Text].
|
| 39.
|
Lerch, R. A.,
K. Anderson, and G. W. Wertz.
1991.
Nucleotide sequence analysis of the bovine respiratory syncytial virus fusion protein mRNA and expression from a recombinant vaccinia virus.
Virology
181:118-131[CrossRef][Medline].
|
| 40.
|
Lerch, R. A.,
E. J. Stott, and G. W. Wertz.
1989.
Characterization of bovine respiratory syncytial virus proteins and mRNAs and generation of cDNA clones to the viral mRNAs.
J. Virol.
63:833-840[Abstract/Free Full Text].
|
| 41.
|
Levine, S.,
R. Klaiber-Franco, and P. R. Paradiso.
1987.
Demonstration that glycoprotein G is the attachment protein of respiratory syncytial virus.
J. Gen. Virol.
68:2521-2524[Abstract/Free Full Text].
|
| 42.
|
Li, W.-H.,
M. Tanimura, and P. M. Sharp.
1988.
Rates and dates of divergence between AIDS virus nucleotide sequences.
Mol. Biol. Evol.
5:313-330[Abstract].
|
| 43.
|
Lincoln, S. E.,
M. J. Daly, and E. S. Lander.
1991.
PRIMER: a computer program for automatically selecting PCR primers, Version 0.5.
MIT Center for Genome Research and Whitehead Institute for Biomedical Research, Cambridge, Mass.
|
| 44.
|
Mallipeddi, S. K., and S. K. Samal.
1993.
Sequence variability of the glycoprotein gene of bovine respiratory syncytial virus.
J. Gen. Virol.
74:2001-2004[Abstract/Free Full Text].
|
| 45.
|
Martinez, I.,
J. Dopazo, and J. A. Melero.
1997.
Antigenic structure of the human respiratory syncytial virus G glycoprotein and relevance of hypermutation events for the generation of antigenic variants.
J. Gen. Virol.
78:2419-2429[Abstract].
|
| 46.
|
McGuire, K.,
E. C. Holmes,
G. F. Gao,
H. W. Reid, and E. A. Gould.
1998.
Tracing the origins of louping ill virus by molecular phylogenetic analysis.
J. Gen. Virol.
79:981-988[Abstract].
|
| 47.
|
Melero, J. A.,
B. Garcia-Barreno,
I. Martinez,
C. R. Pringle, and P. A. Cane.
1997.
Antigenic structure, evolution and immunobiology of human respiratory syncytial virus attachment (G) protein.
J. Gen. Virol.
78:2411-2418[Medline].
|
| 48.
|
Mufson, M. A.,
C. Örvell,
B. Rafner, and E. Norrby.
1985.
Two distinct subgroups of human respiratory syncytial virus.
J. Gen. Virol.
66:2111-2124[Abstract/Free Full Text].
|
| 49.
|
Murphy, F. A.,
C. M. Fauquet,
D. H. L. Bishop,
S. A. Ghabrial,
A. W. Jarvis,
G. P. Martelli,
M. A. Mayo, and M. D. Summers.
1995.
Virus taxonomy: sixth report of the International Committee on Taxonomy of Viruses.
Springer-Verlag, Vienna, Austria.
|
| 50.
|
Nei, M., and T. Gojobori.
1986.
Simple methods for estimating the number of synonymous and nonsynonymous nucleotide substitutions.
Mol. Biol. Evol.
3:418-426[Abstract].
|
| 51.
|
Norrby, E.,
M. A. Mufson, and H. Sheshberadaran.
1986.
Structural differences between subtype A and B strains of respiratory syncytial virus.
J. Gen. Virol.
67:2721-2729[Abstract/Free Full Text].
|
| 52.
|
Olsen, G. J.,
H. Matsuda,
R. Hagstrom, and R. Overbeek.
1994.
FastDNAml: a tool for construction of phylogenetic trees of DNA sequences using maximum likelihood.
Comp. Appl. Biosci.
10:41-48[Abstract/Free Full Text].
|
| 53.
|
Piazza, F. M.,
S. A. Johnson,
M. E. R. Darnell,
D. D. Porter,
V. G. Hemming, and G. A. Prince.
1993.
Bovine respiratory syncytial virus protects cotton rats against human respiratory syncytial virus infection.
J. Virol.
67:1503-1510[Abstract/Free Full Text].
|
| 54.
|
Prozzi, D.,
K. Walravens,
J. P. M. Langedijk,
F. Daus,
J. A. Kramps, and J. J. Letesson.
1997.
Antigenic and molecular analyses of the variability of bovine respiratory syncytial virus G glycoprotein.
J. Gen. Virol.
78:359-366[Abstract].
|
| 55.
|
Rost, B., and C. Sander.
1994.
Conservation and prediction of solvent accessibility in protein families.
Proteins
20:216-226[CrossRef][Medline].
|
| 56.
|
Rueda, P.,
T. Delgado,
A. Portela,
J. A. Melero, and B. García-Barreno.
1991.
Premature stop codons in the G glycoprotein of human respiratory syncytial viruses resistant to neutralization by monoclonal antibodies.
J. Virol.
65:3374-3378[Abstract/Free Full Text].
|
| 57.
|
Saitou, N., and N. Nei.
1987.
The neighbor-joining method: a new method for reconstructing phylogenetic trees.
Mol. Biol. Evol.
4:406-425[Abstract].
|
| 58.
|
Sali, A., and T. L. Blundell.
1993.
Comparative protein modelling by satisfaction of spatial restraints.
J. Mol. Biol.
234:779-815[CrossRef][Medline].
|
| 59.
|
Sanchez, R., and A. Sali.
1997.
Evaluation of comparative protein structure modeling by MODELER-3.
Proteins
1(Suppl.):50-58.
|
| 60.
|
Schrijver, R. S.,
F. Daus,
J. A. Kramps,
J. P. M. Langedijk,
R. Buijs,
W. G. J. Middel,
G. Taylor,
J. Furze,
M. W. C. Huyben, and J. T. van Oirschot.
1996.
Subgrouping of bovine respiratory syncytial virus strains detected in lung tissue.
Vet. Microbiol.
53:253-260[CrossRef][Medline].
|
| 61.
|
Schrijver, R. S.,
J. P. M. Langedijk,
W. G. J. Middel,
J. A. Kramps,
F. A. M. Rijsewijk, and J. T. van Oirschot.
1998.
A bovine respiratory syncytial virus strain with mutations in subgroup-specific antigenic domains of the G protein induces partial heterologous protection in cattle.
Vet. Microbiol.
63:159-175[CrossRef][Medline].
|
| 62.
|
Siegrist, C. A.,
H. Plotnicky-Gilquin,
M. Cordova,
M. Berney,
J. Y. Bonnefoy,
T. N. Nguyen,
P. H. Lambert, and U. F. Power.
1999.
Protective efficacy against respiratory syncytial virus following murine neonatal immunization with BBG2Na vaccine: influence of adjuvants and maternal antibodies.
J. Infect. Dis.
179:1326-1333[CrossRef][Medline].
|
| 63.
|
Simard, C.,
F. Nadon,
C. Seguin, and M. Trudel.
1995.
Evidence that amino acid region 124-203 of glycoprotein G from the respiratory syncytial virus (RSV) constitutes a major part of the polypeptide domain that is involved in the protection against RSV infection.
Antiviral Res.
28:303-315[CrossRef][Medline].
|
| 64.
|
Smith, D.,
J. McAllister,
C. Casino, and P. Simmonds.
1997.
Virus "quasispecies": making a mountain out of a molehill?
J. Gen. Virol.
78:1511-1519[Medline].
|
| 65.
|
Sparer, T. E.,
S. Matthews,
T. Hussel,
A. J. Rae,
B. Garcia-Barreno,
J. A. Melero, and J. M. Openshaw.
1998.
Eliminating a region of respiratory syncytial virus attachment protein allows induction of protective immunity without vaccine-enhanced lung eosinophilia.
J. Exp. Med.
187:1921-1926[Abstract/Free Full Text].
|
| 66.
|
Stine, L. C.,
D. K. Hoppe, and C. L. Kelling.
1997.
Sequence conservation in the attachment glycoprotein and antigenic diversity among bovine respiratory syncytial virus isolates.
Vet. Microbiol.
54:201-221[CrossRef][Medline].
|
| 67.
|
Sullender, W. M., and K. G. Edwards.
1999.
Mutations of respiratory syncytial virus attachment glycoprotein G associated with resistance to neutralization by primate polyclonal antibodies.
Virology
264:230-236[CrossRef][Medline].
|
| 68.
|
Thompson, J. D.,
D. G. Higgins, and T. J. Gibson.
1994.
CLUSTAL W: improving the sensitivity of progressive multiple alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res.
22:4673-4680[Abstract/Free Full Text].
|
| 69.
|
Trudel, M.,
E. J. Stott,
G. Taylor,
D. Oth,
G. Mercier,
F. Nadon,
C. Seguin,
C. Simard, and M. Lacroix.
1991.
Synthetic peptides corresponding to the F protein of RSV stimulate murine B and T cells but fail to confer protection.
Arch. Virol.
117:59-71[CrossRef][Medline].
|
| 70.
|
Valarcher, J.-F.,
H. Bourhy,
J. Gelfi, and F. Schelcher.
1999.
Evaluation of a nested reverse transcription-PCR assay based on the nucleoprotein gene for diagnosis of spontaneous and experimental bovine respiratory syncytial virus infections.
J. Clin. Microbiol.
37:1858-1862[Abstract/Free Full Text].
|
| 71.
|
Van der Poel, W. H. M.,
A. Brand,
J. A. Kramps, and J. T. van Oirschot.
1994.
Respiratory syncytial virus infections in human beings and in cattle.
J. Infect.
29:215-228[CrossRef][Medline].
|
| 72.
|
Van der Poel, W. H. M.,
J. A. Kramps,
W. G. J. Middel,
J. T. van Oirschot, and A. Brand.
1993.
Dynamics of bovine respiratory syncytial virus infections, a longitudinal epidemiological study in dairy herds.
Arch. Virol.
133:309-321[CrossRef][Medline].
|
| 73.
|
Vilcek, S.,
M. Elvander,
A. Ballagi-Pordany, and S. Belak.
1994.
Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples.
J. Clin. Microbiol.
32:2225-2231[Abstract/Free Full Text].
|
| 74.
|
Walsh, E. E.,
A. R. Falsey, and W. M. Sullender.
1998.
Monoclonal antibody neutralization escape mutants of respiratory syncytial virus with unique alterations in the attachment (G) protein.
J. Gen. Virol.
79:479-487[Abstract].
|
| 75.
|
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].
|
| 76.
|
Wellemans, G., and J. Leunen.
1975.
Le virus respiratoire syncytial et les troubles respiratoires des bovins.
Ann. Med. Vet.
119:359-369.
|
| 77.
|
Wertz, G. W.,
M. Krieger, and L. A. Ball.
1989.
Structure and cell face maturation of the attachment glycoprotein of human respiratory syncytial virus in a cell line deficient in O glycosylation.
J. Virol.
63:4767-4776[Abstract/Free Full Text].
|
| 78.
|
Yamashita, M.,
M. Krystal,
W. M. Fitch, and P. Palese.
1988.
Influenza B virus evolution: co-circulating lineages and comparison of evolutionary pattern with those of influenza A and C viruses.
Virology
163:112-122[CrossRef][Medline].
|
Journal of Virology, November 2000, p. 10714-10728, Vol. 74, No. 22
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
