Download GENETIC CHARACTERIZATION OF POLISH INFECTIOUS BURSAL

Bull. Vet. Inst. Pulawy 47, 61-69, 2003
GENETIC CHARACTERIZATION OF POLISH INFECTIOUS
BURSAL DISEASE VIRUS STRAINS
KATARZYNA DOMAŃSKA, GAËLLE RIVALLAN*,
KRZYSZTOF ŚMIETANKA, DIDIER TOQUIN*,
CLAIRE DE BOISSESON*, ZENON MINTA
AND NICOLAS ETERRADOSSI*
Department of Poultry Diseases, National Veterinary Research Institute,
24-100 Pulawy, Poland
*Laboratoire d’Etudes et de Recherches Avicoles et Porcines – Unité de Virologie
Immunologie et Parasitologie Aviaires et Cunicoles, Agence Française de Securité
Sanitaire des Aliments (AFSSA), BP 53, 22 440 Ploufragan, France
e-mail: [email protected]
Received for publication February 19, 2003.
Five Polish infectious bursal disease viruses (IBDVs) isolated from two epidemics on
the turn of 70/80s (early IBDV) and in the 90s (recent IBDV) were characterized by nucleotide
sequencing of VP2 variable domain (vVP2). They were compared to the Faragher 52/70 (F52/70)
reference strain of European classical serotype 1 IBDV and to the 89/163 (antigenically typical)
and 91/168 (antigenically atypical) French very virulent (vv) IBDV isolates. All Polish viruses,
irrespectively of the isolation date, are different from the reference strain F52/70. The early
Polish strains exhibit several amino acid changes that make them markedly different from recent
vvIBDV. The recent Polish strains are closely related to other European vvIBDV. The 93/35
virus has the same amino acid sequence as the French atypical 91/168. Phylogenetic analysis
suggests that the Polish isolates belong to two genetically distinct clusters of IBDV: the recent
are significantly related to vvIBDV and the older form cluster unrelated to other groups of IBDV.
Key words: infectious bursal disease virus, nucleotide sequences,
epidemiology.
Infectious bursal disease virus (IBDV) is a member of the genus Avibirnavirus
within the family Birnaviridae (24). Due to its immunosuppressive effect, even in
subclinical infections, and high rate of mortality in acute infections, infectious bursal
disease (IBD) is of major importance to the poultry industry. Since the first report in
the USA, the disease became widespread all over the world (5). In Europe, it appeared
for the first time in the middle of 1960s. Until the 80s, the strains of the virus were of
low virulence, they caused low mortality, and were satisfactorily controlled by
vaccination but at the end of the 1980s vaccination failures were observed and a very
severe form of the disease caused by very virulent IBDV (vvIBDV) was described first
in Europe (4, 11, 23, 26) and later in the early 1990s in Japan (14, 21). In the USA, it
was demonstrated that the new isolates had been affected by an antigenic drift against
which classical IBD virus (IBDV) vaccines were not satisfactorily protective (13, 25).
During the 63rd General Session of the Office International des Epizooties (OIE, Paris,
15-19 May 1995), it was estimated that IBD has a considerable socio-economic
importance at the international level, as the disease is present in more than 95% of
Member Countries (7).
The major immunogenic component of IBDV is the viral capsid protein VP2,
encoded by segment A, one of the two segments of IBDV genome which consists of
double stranded RNA (12, 25). VP2 amino acid (aa) positions 206 to 353 are
recognized as an important neutralizing antigenic site (1). This part is named “VP2
variable domain” (vVP2) because most of the aa changes between antigenically
different IBDVs are clustered in this area. This region includes two major sets of
hydrophilic aa, termed peak A and B, from aa positions 210 to 225 and 312 to 324,
respectively, with smaller hydrophilic peaks present in between (2). So far, the four aa
changes in vVP2 at positions 222A, 256I, 294I, and 299S have been shown to be
present in all European-like vvIBDVs and to be differentiated from the classical viruses
(3, 10, 27, 28). Although there is no experimental evidence that these aa changes play a
role in the increased virulence of vvIBDVs, they have been generally considered as a
putative marker for vvIBDV-related strains.
In Poland, the first real epidemic of IBD occurred at the end of the seventies
(15). Despite of the wide spread of IBDV, the clinical form of the disease was rarely
observed and the field strains isolated in 1978 and 1980 from broiler chickens exhibited
a low pathogenicity for SPF chickens (16, 17). However, these strains caused economic
losses due to impaired growth and acquired immunodeficiency (18).
The first cases of the acute form of IBD were diagnosed at the end of 1991.
During the next year, the disease spread rapidly throughout the whole country and
affected broilers and laying pullets flocks with mortality rates up to 50% and 70%,
respectively (19). Pathogenic characterization of the IBDVs isolated in 1991, 1992 and
1993 confirmed their high virulence, as mortality rates in experimentally infected 4week-old SPF chickens ranged from 40 to 100% (20).
In previous studies of Polish IBDV strains with the panel of monoclonal
antibodies (Mabs) it was shown that they all had antigenic characterization different
from the F52/70 reference strain. The early Polish strains isolated between 1978 and
1980 were also different from vvIBDVs. The recent Polish IBDV strains isolated after
1990 were similar to the French antigenically typical or atypical vvIBDV.
Antigenically typical strains reacted with all studied Mabs but Mab 3 and Mab 4, and
additionally antigenically atypical did not react with Mab 8 (6).
The aim of the present study was to characterize genetically the Polish IBDV
isolates obtained from field outbreaks between 197 and 1981 and after 1990 and to
compare them with other IBDV strains. The genetic relationships between these viruses
were investigated by using reverse transcription, amplification and direct sequencing of
a genome fragment encoding vVP2.
Material and Methods
Viruses. Two early Polish isolates 78/GSz and 80/GA were collected during
the first epidemic of IBD in Poland between 1978 and 1980. Three recent IBDV strains
were isolated between 1990 and 2000 from chickens with clinical symptoms of acute
form of IBD. The reference IBDV strains included in this study were the Faragher
52/70 (F52/70) strain as reference for the classical European serotype 1 strains, the
French 89/163 strain as reference for the very virulent (vv) IBDV, and the 91/168 strain
as an atypical vvIBDV (8). The viruses (isolates and strains) were propagated in 4-to-6week-old SPF chickens, with sampling of the infected bursae 4 d p.i. Bursal
homogenates were prepared by blending the bursae w/v in phosphate buffered saline
(PBS), then clarified v/v in Freon. All the viruses were stored at -70 0C.
Genome analysis strategy. The genetic characterization of the Polish isolates
was achieved by reverse transcription and amplification using chimeric oligonucleotide
primers (coupling the sequence of M13 and 21M13 standard primers to IBDV-specific
L2 and U2 sequences, respectively) according to Eterradossi et al. (10). The resulting
primer pair generated a 604 base pair (bp)-long genome fragment, 516 bp of which
were IBDV-specific and span the region encoding vVP2 (11). The RT-PCR products
were purified with the Qiaquick Gel purification kits (Qiagen, Chatsworth, CA, USA).
Sequencing of the purified RT-PCR products was performed using M13 and 21M13
specific primers with an automated 373 ABI DNA sequencer (Applied Biosystems
Inc.).
Phylogenetic analysis. The phylogenetic tree was generated as follows: the
nucleotide sequences (nt positions 762 to 1151 according to Bayliss et al. (2) were
aligned using the Clustal W programme. A set of 500 aligned sequences was then
generated by the bootstrap method (Seqboot programme). Genetic distances were
calculated for each set of aligned sequence with the DNADIST programme, and the
trees were calculated by the neighbor joining method (Neighbour programme) using
the OH strain (serotype 2) as an extra group. Finally, the consensus tree for 22
representative isolates was generated using the Consense programme (all programmes
by J. Felsenstein, Department of Genetics, University of Washington, Seattle,
Washington, USA, 1993).
Results
The comparison of the 390 nt-long sequences that have been determined in all
studied viruses revealed two groups with a high level of genetic identity. The first
group included two early isolates (97.2 to 99.0% identity), the second group included
three recent Polish isolates and the 89/163 and 91/168 vvIBDV strains (97.2 to 99.7%
identity). Fig. 1 presents amino acid sequence of VP2 variable domain, positions 183 to
353 (according to Bayliss et al., 1990) in the studied IBDV strains. The early 78/GSz
and 80/GA exhibited several amino acid changes, the first of these conserved changes
was from proline to serine at aa position 222 (P 222→S). The other aa changes
conserved in the early isolates were I 242→V, E 245→G, A 270→T, I 272→T, D
279→N, L 289→P, M 290→I, and I 296→F. There was a difference at the amino acid
in position 254 between the 78/GSz (glicine) and 80/GA (aspartic acid) strains. All
three recent Polish strains exhibited four amino acids: 222 A, 256 I, 294 I, and 299 S
which differ vvIBDVs from F52/70 and have been found so far in all European – like
vvIBDV. The 93/35 virus had the same amino acid sequence as the 91/168 French
atypical vvIBDV. The changes relay on substitution of leucine in the place of glutamine
in amino acid position 324.
190
F52/70
80/GA
78/GSz
89/163
94/48
00/40
91/168
93/35
210
220
230
240
250
260
270
PIPAIGLDPKMVATCDSSDRPRVYTITAADDYQFSSQYQPGGVTITLFSANIDAITSLSIGGELVFQTSVQGLVLGATIYLIGFDGTAVI
.......................................S...................V..G........D.............. T.T
.......................................S...................V..G........................T.T
.......................................A.................................I................
.......................................A.................................I................
.......................................A.................................I................
.......................................A.................................I................
.......................................A.................................I................
280
F52/70
80/GA
78/GSz
89/163
94/48
00/40
91/168
93/35
200
290
300
310
320
330
340
350
TRAVAADNGLTAGTDNLMPFNLVIPTNEITQPITSIKLEIVTSKSGGQAGDQMSWSASGSLAVTIHGGNYPGALRPVTLVA
......N.........PI.....F.........................................................
......N.........PI.....F.........................................................
.....................I....S......................................................
.....................I....S......................................................
.....................I....S......................................................
.....................I....S........................L.............................
.....................I....S........................L.............................
Fig. 1. Amino acid sequence of VP2 variable domain (aa positions 183 to 353 according to Bayliss et al., 1990) in IBDV strains Faragher 52/70 (classical),
89 163 (French vvIBDV with typical antigenicity), 91168 (French vvIBDV with atypical antigenicity) and Polish strains 78/GSz, 80/GA, 93/35, 94/48, and
00/40. Dots indicate aa positions where the sequence is identical to F52/70.
93
91168/ France 1991
93/35 (Poland 1993)
89 163 / France 1989
Very virulent virus group
00/40 (Poland 2000)
HK46 / China 1998
98
OKYM / JAPAN 1991
95
94/48 (Poland 1994)
93
Wade3 / Senegal 1998
100
Civ/88/109 / Ivory Coast 1988
Civ/88/180 / Ivory Coast 1988
Classical
Virus
F52/70 / United Kingdom 1970
Sen/98/1552 / Senegal 1998
VarE / USA 1984
US
Variants
80
VarA / USA 1984
93
GLS5 / USA 1988
99
Cell
cultured
Vaccines
100
D78 / nr
Cu1 / Germany 1976
CT / nr
Early
viruses
100
80/GA (Poland 1980)
78/GSz (Poland 1978)
002/73 / Australia 1974
OH / USA 1982
Serotype 2
Fig. 2. Phylogenetic consensus tree of the studied IBDV strains.
The nucleotide (nt) sequences encoding VP2 variable domain in Polish strains (underlined)
were compared with several very virulent, classical, variant or cell culture adapted viruses of
various geographical origin. The OH strain was used as an extra group. Branch length has no
special meaning. The figures at the forks are bootstrap values, i.e. they measure the likelihood
that viruses which are to the right of that fork are indeed genetically related.
Fig. 2 presents the consensus phylogenetic tree constructed following analysis
by the neighbor joining method of the previously aligned nucleotide sequences
encoding VP2 variable domain of the studied IBDV strains: Polish strains (underlined)
and several very virulent, classical, variant or cell culture adapted viruses of various
geographical origin. The OH strain was used as an extra group. All serotype 1 strains
appear genetically related (bootstrap 100%) however the Australian strain differs
significantly (it branches out of other serotype 1 strains in 93% of the bootstrap
generated trees). Polish early strains 80/GA and 78/GSz (100% bootstrap) form a
separate genetic cluster (cluster i). There were four other remarkable genetic clusters
with a bootstrap exceeding 75%, namely the US variant viruses (cluster ii), bootstrap
76.6%, the cell culture adapted viruses (cluster iii), bootstrap 99.2%, the classical
viruses including the F52/70 isolate (cluster iv), bootstrap 99.8%, and the very virulent
group (cluster v), bootstrap 88.2%, which included recent Polish strains 93/35, 94/48,
00/40 as well as French 89/163 and 91/168. The genetic relationships between early
strains (cluster i) and clusters ii, iii, and iv are unclear. Significant subclustering was
apparent within group v: the Polish 93/35 and French 91/168 antigenically atypical
strains appeared genetically related (bootstrap value 87.8%), as could be expected since
they differed only by one nucleotide over the sequenced region.
Discussion
Recent Polish isolates had the aa sequence of their VP2 variable domain as
typical vvIBDV represented by the 89/163 French isolate containing four aa (222A,
256I, 294I, and 299S) different in comparison to classical virulent F52/70 (222P, 256V,
294L, and 299N). These changes have so far been demonstrated in most vvIBDVs,
especially those of European origin. The phylogenetic analysis of the nucleotide
sequences confirmed these relationships, as the three recent Polish IBDVs appeared
significantly genetically related to other vvIBDVs, with which they were associated in
95% of the bootstrap generated trees. Hence, the present study confirms previous
pathotypic studies and demonstrates that vvIBDVs are now present in Poland.
Interestingly, one of the recent Polish viruses (93/35) appeared similar to the French
strain 91/168. These both isolates were found to share 99.8% identical nucleotide
sequences, and the same deduced aa sequence (one aa change in VP2 hydrophilic peak
B). Because these atypical French and Polish vvIBDV-related isolates have been
isolated over a relatively short period of time (1991-1993), share a very high nucleotide
identity although they originate from geographically distant areas, and have not so far
been isolated elsewhere, it could be speculated that the same IBDV strains were
exchanged between the two countries or that the outbreaks in the two countries might
have a common source. However, the epidemiological links between the outbreaks in
the two countries are not known, and a survey of the French vvIBDV isolates from
1989 to 1997 suggested that the 91/168 strain had caused only a sporadic outbreak in
France (one farm) and did not replace the more typical 89/163-like vvIBDVs (9).
The aa sequence of two early Polish strains (78/GSz and 80/GA) revealed that
these strains exhibit 9 to 10 aa changes as compared with the classical F52/70 strain,
and 12 to 13 as compared with typical vvIBDVs.
The present results with early Polish isolates could give new insights into the
epidemiology of the early outbreaks of IBD infection in Europe. The current idea of
IBD epidemiology in Europe is that strains antigenically and genetically similar to the
classical virulent strains (i.e F52/70-like) had been prevalent worldwide and they were
replaced in Europe at the end of the eighties by the so called very virulent viruses (4,
26), which in turn seem to have now spread worldwide with the exception of North
America and Australia (7, 21, 23). This theory stems from the antigenic and genetic
studies first of a limited number of early pathogenic viruses which have been
maintained as reference strains (e.g. the 002/73, STC, Cu1 and F52/70 strains), and
second of a limited number of some IBD attenuated vaccine strains which have been
developed from several early IBDV isolates (Lukert strain, D78). However, the extent
to which early IBDVs were actually antigenically and genetically homogeneous is not
known precisely because of a limited number of available early IBDV strains.
Similarly, it is not known if other IBDV strains, possibly involved in subclinical
infections, were present in Europe before the F52/70-like viruses were isolated. These
points might be important to investigate, as the epidemiological origin of the IBDV
strains that have recently emerged in Europe is still unknown. The present report
showing that early Polish isolates share unclear genetic relationships with the classical
IBDVs (bootstrap value ranging 26-52%) and the previous one concerning antigenic
characterization of these isolates suggest that more diversity than currently admitted
might have existed in Europe prior to the emergence of the vvIBDV. Similar strains
described Palya et al. (22). The early Hungarian IBDV isolates collected in the late 70s
and early 80s belonged to a genetic cluster significantly different from the “classical”
viruses.
The first cases of acute form of IBD were recorded in 1970. Even though the
early IBDV strains were isolated from 1978 to 1980, so much later than classical
F52/70 strain, it could be suggested that early IBDV strains were present in Poland
before the F52/70-like viruses. The early viruses might have been replaced in the midseventies to the late eighties by F52/70-like viruses and process of this replacement
might have lasted for some time when both types of the viruses coexisted. Similar
phenomenon was noted at the end of 1980s when the classical viruses were replaced by
the vvIBDVs. On the other hand, it might be possible that the early and F52/70-like
viruses stem independently from one common ancestor. They might expand putting
pressure one on another thus easier acquiring new characteristics (antigenicity and
virulence). Such effect might be also reason for raising very virulent form of IBDV.
However, such hypothesis should be confirmed by testing more of early viruses, which
unfortunately may not be available unless fixed infected material is investigated.
Acknowledgments: This work was supported by COST Action 839
“Immunosuppressive viral diseases in poultry” from UE.
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