Download Very virulent infectious bursal disease virus

Journal of General Virology (2006), 87, 209–216
DOI 10.1099/vir.0.81184-0
Very virulent infectious bursal disease virus:
reduced pathogenicity in a rare natural segment-Breassorted isolate
Cyril Le Nouën,1 Gaëlle Rivallan,1 Didier Toquin,1 Pierre Darlu,2
Yannick Morin,3 Véronique Beven,4 Claire de Boisseson,4
Christophe Cazaban,5 Sylvain Comte,5 Yannick Gardin5
and Nicolas Eterradossi1
1,3,4
Correspondence
French Agency for Food Safety (AFSSA), Avian and Rabbit Virology, Immunology and
Parasitology Unit, OIE Reference Laboratory for Infectious Bursal Disease1, Experimental
Services for Avian Pathology (SEEPA)3 and Virus Genetics and Biosecurity Unit4, BP 53,
22440 Ploufragan, France
Nicolas Eterradossi
[email protected]
2
INSERM U535 Genetic Epidemiology and Structure of Human Populations, 94817 Villejuif
Cedex, France
5
CEVA-santé animale, BP 126, 33501 Libourne Cedex, France
Received 16 May 2005
Accepted 5 October 2005
The purpose of this study was to compare the molecular epidemiology of infectious bursal disease
virus (IBDV) segments A and B of 50 natural or vaccine IBDV strains that were isolated or
produced between 1972 and 2002 in 17 countries from four continents, with phenotypes ranging
from attenuated to very virulent (vv). These strains were subjected to sequence and
phylogenetic analysis based on partial sequences of genome segments A and B. Although there
is co-evolution of the two genome segments (70 % of strains kept the same genetic relatives
in the segment A- and B-defined consensus trees), several strains (26 %) were identified with the
incongruence length difference test as exhibiting a significantly different phylogenetic
relationship depending on which segment was analysed. This suggested that natural reassortment
could have occurred. One of the possible naturally occurring reassortant strains, which
exhibited a segment A related to the vvIBDV cluster whereas its segment B was not, was thoroughly
sequenced (coding sequence of both segments) and submitted to a standardized
experimental characterization of its acute pathogenicity. This strain induced significantly less
mortality than typical vvIBDVs; however, the mechanisms for this reduced pathogenicity remain
unknown, as no significant difference in the bursal lesions, post-infectious antibody response or
virus production in the bursa was observed in challenged chickens.
INTRODUCTION
Infectious bursal disease virus (IBDV) is a pathogen of
worldwide importance to the poultry industry. IBDV
destroys B lymphocytes in the bursa of Fabricius in young
chickens, causing both mortality and immunosuppression
(Lasher & Shane, 1994). Very virulent (vv) IBDV strains
The GenBank/EMBL/DDBJ accession numbers for the full sequences
of segments A and B of IBDV strain 02015.1 are AJ879932 and
AJ880090, respectively. The GenBank/EMBL/DDBJ accession numbers for the partial sequences determined in this study are AJ878882–
AJ878913 (segment A) and AJ878638–AJ878687 (segment B) as
listed in Supplementary Table S1.
Details of the strains used in this study and the oligonucleotide primers
used are available as supplementary material in JGV Online.
0008-1184 G 2006 SGM
emerged in Europe in the late 1980s, causing up to 60 %
mortality (Chettle et al., 1989; van den Berg et al., 1991).
IBDV belongs to the family Birnaviridae, genus Avibirnavirus, and has a bisegmented dsRNA genome (Delmas
et al., 2004). Genome segment A (3?2 kbp) encodes in a
major open reading frame (ORF) a precursor polyprotein
which is cleaved by autoproteolysis to yield mature VP2
(outer capsid), VP4 (protease) and VP3 (inner capsid)
(Kibenge et al., 1988). Segment A also encodes, from a
small ORF which precedes and partially overlaps the
large ORF, a non-structural protein, VP5 (Spies et al.,
1989), possibly involved in virus release (Lombardo et al.,
2000; Yao & Vakharia, 2001). Genome segment B (2?8 kbp)
encodes the virus polymerase, VP1 (Macreadie & Azad,
1993), the polymerase activity of which has recently been
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209
C. Le Nouën and others
characterized unequivocally in vitro (von Einem et al.,
2004).
Molecular characterization of IBDV has been based mainly
on the study of the VP2 gene, the middle third of which contains a variable region (vVP2) (Bayliss et al., 1990) where
stretches of hydrophilic amino acids have been recognized
as the molecular basis for antigenic variation (Schnitzler
et al., 1993; Vakharia et al., 1994). Several amino acids in
vVP2 have also been proposed as putative markers for
pathogenic IBDV. First, amino acid alignments of vVP2
in classical or vvIBDV isolates revealed that most of the
latter shared four conserved amino acids, A222, I256,
I294 and S299 (Brown & Skinner, 1996; Eterradossi et al.,
1999). These positions have since been used as the target
of several molecular or antigenic tests aimed at the presumptive molecular or antigenic identification of putative
vvIBDVs (Eterradossi et al., 1998; Zierenberg et al., 2000).
Second, using the reverse-genetic system developed for
IBDV (Mundt & Vakharia, 1996), it was recently demonstrated that vVP2 mutations Q253H, D279N and A284T are
involved in both cell-culture adaptation and attenuation
(Lim et al., 1999; Mundt, 1999; Brandt et al., 2001; van Loon
et al., 2002). Altogether, the molecular basis for pathogenicity of IBDV is not yet fully understood. Indeed, other
reverse-genetics studies showed that mosaic classical virus
containing VP2 of a vvIBDV strain induced neither morbidity nor mortality in young chickens (Boot et al., 2000),
thus demonstrating that VP2 was not the sole determinant
for virulence. In addition, other studies have also suggested
a role for VP5 (Yao et al., 1998) or suggested, for a limited number of strains, that the B segments of vvIBDVs
might also be genetically related. This finding supports
the hypothesis that both segments may be involved in
pathogenicity (Islam et al., 2001), as also suggested by data
on laboratory-generated chimeric viruses which showed
segment B to be involved in the efficiency of virus replication (Zierenberg et al., 2004; Liu & Vakharia, 2004; Boot
et al., 2005).
The purpose of this study was to compare the molecular
epidemiology of IBDV segments A and B of a wider panel
of natural or vaccine IBDV strains that were isolated or
produced in several countries over a long period and
exhibited phenotypes ranging from attenuated to vvIBDV.
These strains were submitted to sequence and phylogenetic analysis based on partial sequences of genome segments
A and B. We showed that, although there is generally
co-evolution of the two genome segments (70 % of IBDV
strains kept the same phylogenetic relatives in both the
segment A- and segment B-derived trees), several strains
(26 %) were identified as exhibiting significantly different
genetic relatives depending on which segment was analysed.
This suggested that natural reassortment is likely to have
occurred. One of the possible naturally occurring reassortant strains (02015.1), which exhibited a segment A related
to the vvIBDV cluster whereas its segment B was not, was
sequenced more thoroughly and was submitted to a
210
standardized experimental characterization of its acute
pathogenicity.
METHODS
Virus strains in the phylogenetic study. Fifty IBDV isolates
exhibiting various degrees of virulence (vvIBDV, classical, attenuated) were collected from 17 countries and four continents from
1972 to 2002 (Supplementary Table S1, presenting references for the
geographical origin and sources of the isolates, is available in JGV
Online). Viruses were propagated in chicken embryo fibroblasts
(CEF) for the cell-culture-adapted strains or in specific-pathogenfree (SPF) chickens for the non-adapted strains.
Virus strains in the experimental study. IBDV strains 89163
(Eterradossi et al., 1992) and Faragher 52/70 (Bygrave & Faragher,
1970) were used as reference strains typifying vvIBDV and classical
IBDV strains, respectively. Strain 02015.1 (vvIBDV-like segment A,
unrelated segment B) was selected for experimental assessment of
acute pathogenicity. Virus suspensions, produced as described previously from the bursae of inoculated chickens (Eterradossi et al.,
1992), were used as inocula. Inocula were titrated by inoculating
serial tenfold dilutions (0?1 ml per egg, via the chorioallantoic membrane using seven eggs per dilution) into 9- to 10-day-old SPF eggs
(AFSSA-Ploufragan). IBDV titres were calculated according to the
method of Reed & Muench (1938) and expressed as median embryo
infectious dose (EID50). Absence of reovirus or adenovirus contaminants in the inoculated field strain was assessed by three serial passages in SPF chicken liver embryonic cells. The absence of other
avian pathogens potentially present in the inoculum was assessed by
serological testing of the sera collected 27 days post-inoculation
(p.i.) of SPF chickens. The following antigens were studied: reovirus
strain 1133, fowl adenovirus group 1 serotype 1 (CELO virus), infectious bronchitis virus, infectious laryngotracheitis virus, turkey rhinotracheitis virus, the viruses of Marek’s disease (antigen A),
Newcastle disease, egg-drop syndrome and chicken anaemia and
influenza virus.
Partial amplification and sequencing for the phylogenetic
study. Virus RNA was extracted from the bursa of Fabricius of ino-
culated chickens or from infected CEF monolayers. The sequences of
the oligonucleotides used in this study are presented in Supplementary Table S2. The antisense primers were used for reverse transcription (RT). For the PCR, pairs of conserved primers defining
approximately 600 bp PCR products were selected in genome
regions flanking either the hypervariable domain of the VP2 gene
in segment A (nt 744–1180; sequence of primers as in Eterradossi
et al., 1998) or in the 59 two-thirds of segment B (nt 297–1749).
These regions have been identified previously as the more phylogenetically representative parts of segments A and B, respectively
(Le Nouën et al., 2005). To facilitate sequencing of the RT-PCR products, chimeric oligonucleotide primers were defined by coupling
the sequence of the M13 and 21M13 standard primers to the 59 end
of the IBDV-specific sense and antisense primers, respectively. The
resulting primer pairs allowed the determination of 514 or 517 bp in
the segments A of serotype 1 or 2 strains, respectively, and of 544 bp
in the segment B of all studied strains. RT-PCRs, purification of RTPCR products and sequencing were performed according to previously published protocols (Eterradossi et al., 1998, 1999), except
for the use of the HEF proofreading enzyme (Roche) which was
used instead of Taq polymerase at the PCR step. The resulting
nucleotide sequences were submitted to the EMBL database with the
accession numbers shown in Supplementary Table S1.
Phylogenetic analysis of the A and B partial sequences. The
nucleotide sequences were aligned using CLUSTAL W and were then
analysed using the PHYLIP (version 3.57c; Felsenstein, 1993) and
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Journal of General Virology 87
Low pathogenicity in natural reassortant vvIBDV
PhyML (version 2.1b1; Guindon & Gascuel, 2003) suites of programs. The aligned sequences were submitted to bootstrap (500
replicates; SEQBOOT). The bootstrap-generated datasets were then
analysed by the neighbour-joining (NJ) method (DNADIST with
Kimura two-parameter distances, followed by NEIGHBOR), the parsimony method (P) (DNAPARS) and the maximum-likelihood (ML)
method (PhyML, using the HKY model with optimized invariant
sites and transition to transversion ratios). The Jasper strain of infectious pancreatic necrosis virus (genus Aquabirnavirus) was used as
an outgroup in all phylogenetic analysis (GenBank accession numbers M18049 and M58756 for segments A and B, respectively).
Consensus trees for both regions were determined with CONSENSE.
Only groups defined by bootstrap values greater than 80 % with all
of the NJ, P and ML methods were considered as significant groups.
Congruence between the A and B phylogenies. This was
tested with the incongruence length difference (ILD) test (Farris
et al., 1994), using the PAUP* program (Swofford, 2002). The ILD
test evaluates the null hypothesis that two datasets A and B describing two different characters of the same strains are congruent, i.e.
these datasets, when combined, produce a parsimonious tree with a
length statistically comparable to the sum of the lengths of the most
parsimonious trees obtained from the separate data. To do so, the
program calculates the lengths of the most parsimonious trees as
measured from the A and B datasets (LA and LB, respectively), as
well as LA+B from the (A+B) dataset combining the A and B characters. The test calculates ILDAB=LA+B2(LA+LB). To test the null
hypothesis of congruence, two datasets (X and Y) are drawn at
random from the combined dataset (A+B), each set having the
same size as the original A and B sets, respectively. The value of
ILDXY test is then calculated for a large (e.g. 1000) number of such
random partitions. When ILDAB exceeds a proportion of 95 or 99 %
of the ILDXY values, the null hypothesis of congruence can be
rejected at P¡0?05 or 0?01, respectively. As an iterative process,
serial removal of the same strain from the A and B datasets (and
studying how the ILDAB index varies) allows the determination of
how critically this strain contributes to the incongruence of the
phylogenies derived from A and B (Lecointre et al., 1998).
In the present study, the parsimonious trees were obtained using PAUP*
(Swofford, 2002) through heuristic procedures with random sequence
addition and tree bisection–reconnection (TBR) branch-swapping
algorithms. The distribution of ILDXY was assessed on 1000 resampled
datasets. The 0?05 probability threshold was taken as significant
incongruence. One or a minimal combination of the studied IBDV
strains were iteratively removed from the A and B datasets, until the
probability of the ILDAB index was greater than 0?05, hence allowing
the null hypothesis of congruence between the A and B datasets to be
accepted.
Full-length amplification and sequencing of genome segments A and B of a selected strain. Strain 02015.1 was selected
following the phylogenetic study as having significantly different
phylogenetic relatives in segments A and B. In order to test the
hypotheses of (i) a recombination event occurring within segment A
or B of 02015.1 and (ii) other specific genetic changes affecting this
strain, we sequenced the full coding sequence of both genome segments of strain 02015.1 from overlapping RT-PCR products (same
protocol as above; primers as shown in Supplementary Table S2).
The full-length coding sequences of segments A and B (nt 188–3103
and 131–2752; GenBank accession numbers AJ879932 and AJ880090,
respectively) of strain 02015.1 were used to determine their genetic
relatives, as described above, by NJ, P and ML phylogenetic analysis,
based on a panel of homologous IBDV sequences retrieved from
databases that had been used to analyse the same regions in a previous
study (Le Nouën et al., 2005).
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Experimental study of the pathogenicity of the potential
reassortant virus. One hundred 6-week-old SPF white leghorn
chickens (AFSSA-Ploufragan) were housed in four groups of 25 birds
of comparable sex and weight, in separate filtered-air, negativepressure isolation units. Chickens in each group were blood sampled
for serological testing on the first day. On the same day, each bird
in three of the four groups was inoculated by the intranasal route
with 105 EID50 of either strain 89163, strain Faragher 52/70 or strain
02015.1; the remaining group was kept as a mock-inoculated control
receiving PBS only. Cumulative mortality rates at 5 days p.i. were
measured. At 4, 13 and 27 days p.i., at least five birds per group
were weighed, humanely killed and necropsied and their bursae were
weighed for calculation of the bursa to body weight (b/B) ratio.
Each bursa was then cut in two halves; one half was kept for histological examination and the other was processed for preparation of a
virus suspension (see below). All surviving birds at 27 days p.i. were
blood sampled for final serological examination.
Histological examination. Histological examination was per-
formed by Dr M. Lagadic (Maisons-Alfort, France). Sixty-one bursae
of Fabricius from the four different groups (mock-inoculated group
and the three inoculated groups) and from three sampling times (4,
13 and 27 days p.i.) were examined individually. The severity of the
IBD-induced lesions was quantified according to Skeeles’ scale
(Skeeles et al., 1979), which is based on five degrees: 0, no lesions; 1,
mild scattered cell depletion in a few follicles; 2, moderate, one-third
to half of the follicles having atrophy or depletion of cells; 3, diffuse,
atrophy of all follicles; 4, acute inflammation and acute necrosis
typical of IBD.
IBDV neutralization test. Virus neutralization (VN) tests to detect
antibodies induced by IBDV were performed in CEF cells, using 100
median tissue culture infective doses (TCID50) per well of the CT
(serotype 1) IBDV strain, as described previously (Eterradossi et al.,
1997a). The VN titre was expressed as log2 of the last dilution resulting in 100 % neutralization of the cytopathogenic effect. VN titres
greater than 3?3 log2 were considered as positive.
Virus titres in bursae collected at 4 days p.i. Virus suspensions
were prepared separately from each half-bursa collected at 4 days
p.i., according to previously published protocols (Eterradossi et al.,
1992) and based on a standardized organ-to-buffer dilution ratio
(1 g to 2 ml). For each of the three challenge viruses, the five virus
suspensions obtained after processing of the five birds studied at
4 days p.i. were pooled in equal amounts and mixed. The three
pools (one per inoculated virus) were titrated as described above in
10-day-old embryonated SPF eggs.
Amounts of IBDV antigen in bursae collected at 4 days p.i.
Antigen capture- (AC-) ELISA was performed as described previously (Eterradossi et al., 1997a, b). Briefly, 96-well polystyrene
ELISA plates were coated with a chicken monospecific polyclonal
anti-F52/70 serum diluted in PBS. Blocking was performed overnight in blocking buffer. Serial twofold dilutions of the individual
virus suspensions prepared at 4 days p.i. were incubated in the
coated plates and captured IBDV particles were detected with a
reference mouse anti-F52/70 antiserum, followed by an anti-mouse
alkaline phosphatase conjugate. Absorbances measured for the serially diluted individual virus suspensions were plotted as dose–effect
curves.
Statistical analysis of results. The x2 test was used to compare
percentage mortality measured for 5 days p.i. The Kruskal–Wallis
one-way analysis of variance was used to compare body weights
measured at day 0, b/B ratios, histological scores, levels of virusneutralizing antibodies and amounts of IBDV antigen detected by
AC-ELISA in the bursae of inoculated chickens, as measured in the
four groups.
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C. Le Nouën and others
RESULTS
Molecular epidemiology of IBDV segments A
and B
The phylogenetic study of segment A revealed nine significant clusters (bootstrap value ¢80 % in NJ, P and ML)
(Fig. 1a). Cluster (i) grouped Ivorian-like pathogenic
IBDVs (four isolates), which proved significantly related
to cluster (ii), grouping European-like vvIBDVs (23 isolates). Cluster (iii) contained European classical pathogenic
viruses (two isolates), whereas clusters (iv) and (v) grouped
most vaccine strains (six strains). Cluster (vi) grouped US
antigenic variants (three isolates), cluster (vii) grouped
vaccine strains (two strains), cluster (viii) grouped the early
European strains (two isolates) and cluster (ix) grouped
four Australian isolates. The phylogenetic study of segment
B proved highly congruent with that of segment A, as six out
of nine segment-A-defined significant clusters, clusters (i),
(ii), (iii), (v), (vii) and (viii), also proved significant in the
segment-B-defined consensus tree (Fig. 1b). Of 46 IBDV
isolates exhibiting significant segment-A-defined genetic
relationships (all strains except 228E, 05/5, 23/82 and TY89),
32 isolates (70 %) kept the same genetic relatives in the
segment-B-defined consensus tree. Moreover, segments B
of four Australian strains [group (ix) defined in segment A
phylogeny] proved phylogenetically related with the NJ and
P methods (bootstrap value >80 %) but not with the ML
method (66 % bootstrap value) (data not shown).
The ILD test was used to identify potential naturally occurring reassortant strains. The ILD test revealed that the two trees
were not congruent (P¡0?05) unless strains GX, Henan,
02015.1, 02015.3, 02015.2, TY89, 23/82, 05/5, Cu1WT,
78GSZ, 80GA, Bursine2 and Bursine [13 strains (26 %);
four groups] were removed. When all of these strains were
Fig. 1. Phylogenies obtained by the parsimony method of IBDV segments A (a) and B (b). Groups of strains with bootstrap
values greater than 80 % with the P, NJ and ML methods and present in both segment A and B phylogenies are represented
by shading. Arrows indicate movements of strains or group of strains which exhibited different genetic relatives for segments A
and B. Asterisks indicate nodes with bootstrap values greater than 80 % with the P, NJ and ML methods but possibly not in
both phylogenies.
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Journal of General Virology 87
Low pathogenicity in natural reassortant vvIBDV
removed from both phylogenies, the congruence hypothesis
could not be rejected (P=0?37), and removing more strains
did not change this result. Among the 13 incongruent
strains, five were especially interesting with regard to the
hypothesis that possible reassortment involving vvIBDV
had occurred. Indeed, strains GX, Henan, 02015.1 and
02015.3 exhibited a segment A significantly related to the
vvIBDV cluster (bootstrap value of 100 %), whereas their
B segments, although they exhibited some differences, all
proved significantly different from the viruses belonging to
the segment-B-defined clusters (i) or (ii) (vvIBDV-related
strains) or to the viruses belonging to clusters (iii), (v), (vii)
and (viii) (classical, vaccine, early European and serotype
2 strains). Similarly, strain 02015.2 exhibited a segment A
belonging to the vvIBDV cluster but a segment B more significantly related to classical, vaccine or early European
viruses [clusters (iii), (v), (vii) and (viii), respectively].
(I61V, D146E, N147S, T236S, E242D, M274V, M390L,
V557I, Q561H, P562S, T576A, R695K and T859I). Phylogenetic analysis of the 02015.1 sequences with their counterparts in a panel of vvIBDV and classical strains retrieved
from databases (Le Nouën et al., 2005) produced results
that were highly consistent with the phylogenies previously
obtained for the partial sequences of the studied segments
(data not shown). Indeed, the coding region of the A segment of 02015.1 proved significantly related to the vvIBDV
cluster (100 % bootstrap value with the NJ, P and ML
methods), whereas the coding region of the B segment of
the same strain branched out of the vvIBDV group in 95, 99
and 89 % of the bootstrap-generated trees with the NJ, P
and ML methods, respectively.
Pathogenicity of the possible naturally
reassortant 02015.1 isolate
On the first day of the experiment, the mean weight of
the chickens in the four groups ranged from 440 to 463 g
and did not prove to be significantly different (Table 1).
Following inoculation, neither disease signs nor mortality
were observed in the mock-inoculated control group. In the
groups receiving the challenge viruses, morbidity and mortality was observed on days 3 to 5 p.i. The typical vvIBDV
strain 89163, the classical strain F52/70 and isolate 02015.1
induced 100, 32 and 24 % morbidity and 52, 24 and 8 %
mortality, respectively. The x2 test showed that mortality
induced by the 89163 virus was significantly higher than
that observed following challenge with F52/70 or 02015.1,
which were not significantly different (Table 1).
Amplification, sequencing and phylogenetic
analysis of the full-length coding regions of
segments A and B of the 02015.1 isolate
The coding regions of segments A and B (nt 188–3103
and 131–2752, respectively) of the 02015.1 isolate were
sequenced and compared with the corresponding consensus sequence of the typical vvIBDV strains HK46 (GenBank
accession numbers AF092943 and AF092944), BD3 (AF362776 and AF362770), D6948 (AF240686 and AF240687) and
UK661 (X92760 and X92761). This revealed two segmentA-encoded amino acid changes, one in VP5 (position 135; T
in vvIBDV and S in 02015.1, abbreviated as T135S) and one
in VP4 (P613S). In genome segment B, mutations encoding
13 amino acid changes were spread along the VP1 gene
The study of b/B ratios at 13 days p.i. revealed comparable
bursal atrophy in the three challenged groups (with mean
Table 1. Pathogenicity of IBDV isolate 02015.1 compared with the reference vvIBDV strain 89163 and classical strain F52/
70 and mock-inoculated controls
Data are presented as means ± SD (minimum–maximum) [number of individuals]. Within a row, values indicated by the same superscript
letter do not differ significantly according to the x2 test (mortality) or the Kruskal–Wallis test (other parameters) at the P¡0?05 confidence
level.
Parameter
Initial weight (g)
Mortality at 5 days p.i. (%)
b/B ratio (%)
4 days p.i.
13 days p.i.
27 days p.i.
Bursal lesion score*
4 days p.i.
13 days p.i.
27 days p.i.
VN titre (log2) at 27 days p.i.
Final weight (g) at 27 days p.i.
Mock-inoculated
02015.1
89163
F52/70
458±61 (376–574) [25]
0
440±47 (355–530) [25]
8a
463±48 (394–559) [25]
52b
451±45 (389–558) [25]
24a
4?2±0?4a (3?8–4?7) [4]
3?7±0?7a (3–4?8) [5]
3?2±0?9a (1?5–5?1) [15]
2?9±0?8b (1?9–3?9) [5]
0?9±0?2b (0?7–1?1) [5]
0?6±0?2b (0?1–0?9) [15]
3?1±0?3b (2?7–3?5) [6]
1±0?2b (0?8–1?2) [5]
0?6±0?1b (0?4–0?7) [7]
4?4±1?2a (2?6–5?8) [7]
1?1±0?3b (0?7–1?4) [5]
0?6±0?2b (0?3–1) [13]
0±0a (0–0) [5]
0±0a (0–0) [5]
0±0a (0–0) [5]
2?3±0a (2?3–2?3) [15]
1116±155a (914–1321)
[15]
4±0b (4–4) [5]
3±0b (3–3) [5]
2?8±0?5b (2–3) [5]
12?3±1?5b (10–15) [15]
983±130b (742–1199)
[15]
4±0b (4–4) [5]
3±0b (3–3) [5]
3±0b (3–3) [4]
12?5±1?4b (11–15) [7]
980±78b (871–1078)
[7]
4±0b (4–4) [6]
3±0b (3–3) [4]
3±0b (3–3) [5]
12?4±1?0b (11–4) [13]
979±107b (850–1178)
[13]
a
a
a
a
*Determined by histological grading according to Skeeles et al. (1979) as outlined in Methods.
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C. Le Nouën and others
b/B ratios ranging from 0?9 to 1?1 %), which proved
statistically significant compared with mock-inoculated
controls (mean b/B ratio 3?7 %; P¡0?05). The same level
of atrophy was still apparent in the three challenged groups
at 24 days p.i. (mean b/B ratio in the three inoculated
groups 0?6 % versus 3?2 % for the controls). Histological
examination of the bursae revealed no lesions in the mockinoculated control group and extensive lesions at 4, 13 and
24 days p.i. in the three inoculated groups. The mean lesion
scores of the three latter groups did not differ significantly,
irrespective of the examination date (Table 1).
VN titres were measured at 24 days p.i. and revealed high
anti-IBDV antibody titres in the three inoculated groups,
whereas no neutralizing antibodies were detected in the
SPF controls. No significant difference in antibody levels
was observed in birds inoculated with 02015.1, 89163 or
F52/70 (geometric mean VN titres of 12?3 log2 ± 1?5, 12?5
log2 ± 1?4 and 12?4 log2 ± 1?0, respectively).
On the last day of the experiment, the mean weight of the
chickens revealed a statistically lower mean weight in the
three challenged groups (mean weights of 979–983 g)
compared with the mock inoculated group (mean weight
1116 g; P¡0?05). However, the weights of the three
inoculated groups did not differ significantly (Table 1).
Virus production in the bursae of infected
chickens
Virus titres in the pools of bursal homogenates collected
at 4 days p.i. were 105?88, 105?64 and 105?67 EID50 ml21 for
viruses 02015.1, 89163 and F52/70, values not considered as
different. Similarly, the amounts of IBDV antigen in the
bursae at 4 days p.i. were determined after 02015.1, 89163
and F52/70 inoculation. Dose–effect curves obtained in
AC-ELISA from serially diluted individual virus suspensions did not exhibit any differences, as tested with the
Kruskal–Wallis test (data not shown).
DISCUSSION
Our phylogenetic studies have shown strong sequence conservation of both segments A and B in different genetic clusters of IBDV and not only in vvIBDVs. These results suggest
co-evolution of the two genome segments. This could be due
partly to the geographical clustering of some of the studied
isolates: Australian IBDV have already been shown to evolve
‘on their own’, probably due to the geographical isolation
of the Australian continent (Ignjatovic & Sapats, 2002),
as already described for Australian strains of infectious
bronchitis virus (Sapats et al., 1996). Similarly, the coevolution could stem from the temporal clustering of some
isolates, such as the early European strains 78GSz and 80GA,
which have been suggested to represent a European IBDV
lineage possibly already established prior to the emergence
of the ‘F52/70-like’ classical IBDVs (Domanska et al., 2004).
However, co-evolution of genome segments as a major
evolutionary feature may still appear surprising in IBDV, as
214
co-infection (and hence reassortment) is likely to be
encouraged in the field when some flocks infected by
pathogenic IBDVs possibly receive live classical vaccines
while incubating the disease. The observed co-evolution
therefore suggests that some mechanisms reduce the generation or successful transmission of reassortant viruses:
possible mechanisms controlling segment compatibility
could include interactions between the segment-A-encoded
VP3 and segment-B-encoded VP1 (Tacken et al., 2000,
2002) or between VP1 or VPg and the genome segments
(Müller & Nitschke, 1987). Our results also confirmed
previous reports that the vvIBDV cluster can be defined
based on both genome segments (Islam et al., 2001), which
suggests a worldwide spread of a single vvIBDV clone and/
or an essential contribution of both genome segments to
virus replication and virulence.
Although most significant clusters were maintained in the
A- and B-derived phylogenies, 13 strains (26 %) were identified as exhibiting a significantly different phylogenetic
position depending on which segment was analysed. Most
strikingly, some strains exhibited markedly different genetic
relatives for segments A and B. Sequencing the full coding
sequence of both genome segments of strain 02015.1 confirmed that its segment A was phylogenetically related to
the vvIBDV cluster, whereas its segment B was not. The
demonstration of such a strain could suggest the existence
of previously unrecognized segment B lineages that can be
associated with a vvIBDV-like segment A and/or a possible
reassortment between a vvIBDV (segment A) and a yet-tobe-identified parent with a different genetic background. In
IBDV, several reports have proposed that natural reassortment is possible (Brown & Skinner, 1996; Yamaguchi et al.,
1997; Sun et al., 2003; Kong et al., 2004); however, reassortment has not been assessed statistically on such a wide panel
of strains as used in the present study. Chimeric IBDVs have
been constructed in the laboratory, thus demonstrating the
feasibility of reassortment of virus RNAs (Boot et al., 2000;
Brandt et al., 2001; Liu & Vakharia, 2004; Zierenberg et al.,
2004), but the nucleotide signals that control the compatibility of genome segments from different IBDV lineages
(and thus acceptance of these segments by reassortant
viruses) have not yet been identified. These mechanisms
have been recently characterized in other dsRNA viruses
with a segmented genome, the mammalian reoviruses. In
these viruses, the incorporation of an engineered ssRNA
into the genome depends critically on three consecutive
nucleotides of the 59 UTR, and mutations at these positions
most likely modify the secondary structure of the 59 UTR
(Roner et al., 2004). The reverse-genetics system developed
for IBDV (Mundt & Vakharia, 1996) provides a unique tool
to perform a similar investigation in IBDV, and the UTRs of
the genome segments of strain 02015.1 are currently under
study.
The pathogenicity of the possibly reassortant strain 02015.1
was shown experimentally to be significantly lower than that
of a typical vvIBDV, a result which might suggest that both
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Journal of General Virology 87
Low pathogenicity in natural reassortant vvIBDV
segments might be involved in the pathogenicity of IBDV.
The published literature regarding the influence of segment B on IBDV phenotype produces somewhat conflicting
results: one study suggests that segment B has a limited
influence on pathogenicity (Boot et al., 2000), whereas
others have suggested that segment B could influence
replication efficiency and modulate bursal lesions (Liu &
Vakharia, 2004; Zierenberg et al., 2004; Boot et al., 2005). In
the present study, the three pathogenic viruses induced the
same virus titres and amounts of antigen in the bursae of
challenged chickens. Hence, segments A and B of strain
02015.1 seem to be compatible, and the reduced mortality
caused by 02015.1 does not seem to be due to inefficient
replication of this virus. However, segment A of 02015.1 still
has specific mutations (two amino acid mutations were
identified in the coding region and the UTRs have not yet
been sequenced). We therefore cannot be sure that segment
A of the reassortant has retained the potential for hypervirulence. To answer this question, the recovery in vitro of
a pathogenic virus generated by reverse genetics from segment A of 02015.1 combined with segment B of one or
more vvIBDVs would be required to demonstrate that the
atypical segment B of strain 02015.1 was indeed responsible
for its reduced pathogenicity.
To conclude, the present study provides interesting insights
into the evolution of vvIBDV. Indeed, there are increasing
reports of vvIBDV-related strains that differ genetically
and/or phenotypically from the vvIBDV reference strains
isolated in the late 1980s. van den Berg et al. (2004) described the Henan strain, which exhibited a vVP2 phylogenetically related to the vvIBDV cluster but had reduced
pathogenicity compared with typical vvIBDVs. The present
study suggests that the Henan strain resembles 02015.1 with
respect to reduced pathogenicity and possible reassortment.
Others reports describe vvIBDV isolates from Indonesia and
Malaysia with amino acid substitutions in the vVP2 region
and which did not cause clinical disease or mortality (Hoque
et al., 2001; Parede et al., 2003), whereas another report
described a pathogenic vvIBDV strain with extensive antigenic changes due to amino acid substitutions in the vVP2
region (Eterradossi et al., 2004). Taken together, these
observations suggest that genetic evolution from the initial
vvIBDV clone has led to the emergence of new and diversified vvIBDV-related virus strains. The characterization of
such emerging viruses calls for the definition of validated
pathogenicity markers and for the development of diagnostic assays targeted at both genome segments.
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