Download antigenic characterization of polish infectious bursal disease virus

Bull. Vet. Inst. Pulawy 46, 45-52, 2002
ANTIGENIC CHARACTERIZATION
OF POLISH INFECTIOUS BURSAL DISEASE VIRUS STRAINS
KATARZYNA DOMAŃSKA, GAËLLE RIVALLAN*,
KRZYSZTOF ŚMIETANKA, DIDIER TOQUIN*, 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 53-22440 Ploufragan, France
Antigen Capture-ELISA based on eight different neutralizing mouse monoclonal
antibodies (Mabs) was used to study Polish infectious bursal disease viruses (IBDVs) isolated
from two epidemics on the turn of 70/80s (early IBDV) and in the 90s (recent IBDV). They were
compared to the Faragher 52/70 (F52/70) reference strain of European classical serotype 1 IBDV
and to the 89/163 (typical) and 91/168 (atypical) French very virulent (vv) IBDV isolates. All
Polish viruses, irrespective of the isolation date, exhibited antigenic profile which was different
from the F52/70 reference strain (no binding of Mabs 3 and 4). Two of the early IBDVs did not
react with Mab 5 and two of six recent IBDV did not react with Mab 8. The other recent Polish
viruses had an antigenic reactivity similar to that of typical vvIBDV.
Key words: infectious bursal disease virus, IBDV, Antigen Capture–ELISA,
monoclonal antibody.
Infectious bursal disease (IBD) is an important virus-induced, highly
contagious disease of chickens (31). The disease causes heavy economical losses due to
immunosuppression in subclinical cases or to acute cases associated with mortality,
haemorrhages and bursal damage. Since the first report in the USA (3) the disease
became widespread all over the world. In Europe it appeared for the first time in the
middle of 1960s. Until 1987 the strains of the virus were of low virulence, they caused
low mortality, and were satisfactorily controlled by vaccination. However, in 1987
some vaccination failures were described in different parts of the world (2, 10, 24, 28).
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 (11, 27). In Europe, the first cases of acute IBDV were described at the same
time (2, 28). The acute forms of the disease, caused by very virulent strains, were then
described in Japan in the early 1990s (12, 23) and they have rapidly spread all over the
world (30). 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-
46
economic importance at the international level, as the disease is present in more than
95% of Member Countries (6).
IBDV is a small, non-enveloped virus belonging to the family Birnaviridae.
The genomes of the virus consist of two segments of double-stranded RNA contained
within a single-shelled icosahedral capsid composed of four structural proteins (4).
Two of these proteins, VP2 and VP3, are major immunogens. VP2 (MW 40-45 kDa)
encoded by the larger genome segment, has major neutralizing, conformational
epitopes within a central variable region. This part is named “VP2 variable domain”
because most of the amino acids (aa) changes between antigenically different IBDVs
are clustered in this area. Major differences in the reactivity of IBDV strains with
neutralizing Mabs may be referred to aa changes in two hydrophilic aa sets termed
peak A and B which flank VP2 variable domain (25, 29).
Antigenic diversity among IBDV isolates has been recognized since 1981
when serotypes 1 and 2 were defined on the basis of their lack of in vitro crossneutralization (15). So far, virus strains that are pathogenic for chicken have all been
shown to belong to serotype 1. Further antigenic differences have been demonstrated
within this serotype since 1984, and the study of North-American IBDV isolates
causing little mortality but significant immunosuppression has led to dividing the
serotype into six subtypes which were originally differentiated by cross-neutralization
assays using polyclonal sera (11). Studies based on Mabs subsequently demonstrated a
growing number of modified neutralizing epitopes in the recent serotype 1 isolates
from the United States, which were designated as “variant viruses”. It was hence
suggested that North-American IBDV isolates might have been affected by an
antigenic drift and the epidemiology of IBDV in the US has been defined since then by
the natural occurrence of neutralizing monoclonal antibodies escape variants (26, 27).
In Poland first cases of IBD were diagnosed at the end of 60s but the real
epidemic of IBD occurred at the end of 70s (1, 14, 16). Despite of a wide spreading of
IBDV, the clinical form of the disease was rarely observed. The field strains isolated in
1978 and 1980 revealed low pathogenicity for SPF chickens (17, 18). However, they
caused economic losses due to impaired growth and acquired immunodeficiency (19).
At the end of 1991 the first cases of acute form of IBD were diagnosed in
western and central Poland. During the next year the disease spread rapidly throughout
the whole country and affected broilers and laying pullets flocks with mortality up to
50% and 70%, respectively (20, 21). Pathotypic characterization of the IBDVs isolated
in 1991-93 confirmed their high virulence (22). Mortality rates in challenged 4-weekold SPF chickens ranged from 40 to 100%. The aim of the present study was to
compare antigenically early and recent Polish IBDV isolates in a Mab-based Antigen
Capture-ELISA (AC-ELISA).
Materials and Methods
Viruses. Three early Polish isolates 78/GSi, 78/GSz and 80/GA were collected
during the first epidemic of IBDV in Poland between 1978-80. Six recent IBDV strains
were isolated in 1990-2000 from chickens with clinical symptoms of acute form of
IBDV. 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 91168 strain as an
atypical vvIBDV (7). The viruses (isolates and strains) were propagated in 4-to-6-
47
week-old SPF chickens, with sampling of the infected bursae 4 days p.i. Bursal
homogenates were prepared by blending the bursae w/v in phosphate buffered saline
(PBS), then clarified v/v in Freon. All viruses were stored at -70 0C.
AC-ELISA. The antigenic characterization was performed using a Mab based
AC-ELISA according to Eterradossi (7). The study was performed in two steps, the
first aimed at standardizing the amount of captured antigens (with polyclonal ascitic
fluid), the second one at measuring Mab binding in a Mab-based AC-ELISA (7).
Briefly, 96-well polystyrene plates were coated with a chicken anti-F52/70 polyclonal
antiserum, diluted in PBS. Blocking was then achieved by incubating the coated plates
overnight at 370C with a blotto-tween solution containing 10% foetal calf serum and
0.02% sodium azide. Bursal homogenates were then incubated with the coated plates
for 1 h at 370C. After incubation the captured IBDV antigens were detected with mouse
polyclonal anti-IBDV antibodies or with a panel of eight mouse anti-IBDV neutralizing
Mabs 1, 3, 4, 5, 6, 7, 8 and 9 (ascitic fluids) followed by a goat anti-mouse alkalinephosphatase conjugate (KPL, USA), both incubated for 1 h at 370C. Optical densities
(OD) were read at 405 nm after incubating an alkaline-phosphatase substrate (pNPP,
Sigma, USA) for 1 h at 370C. All immunological reagents used in each step of the
assay were first equilibrated so that the capture of an undiluted infected bursal
homogenate would yield an OD averaging the maximum reading value of the reader
(3.000). In the next step the equilibration of the captured antigen was assayed; dilutions
yielding 50% of the maximum OD were used in the antigenic typing. These dilutions
ensure that differences in the intensity of binding of Mabs originate from true antigenic
differences of the tested viruses, and not from the testing of different quantities of the
same virus that may be captured at various level resulting from variation of the
antigenic content in bursae infected by the same virus. Additionally, a few controls
were included in each assay: the evaluation of the unspecific binding of mouse
antibody (an equilibrated dose of the infected homogenates with a negative mouse
ascitic fluid containing no anti-IBDV Mab), the control of the 50% effect that was to be
yielded by the equilibrated antigens (an equilibrated dose of both the infected and the
SPF homogenates with a mouse polyclonal ascitic fluid) and the control of the
maximum effect (the undiluted infected and SPF homogenates with polyclonal ascitic
fluid). The reactivity of the different Mabs was calculated as a percentage, according to
the formula below (4, 6, 9).
Percentage of reactivity =
OD Mab _ OD Negative ascitic fluid
x100
OD Reference polyclonal _ OD Negative ascitic fluid
Results
In AC-ELISA, all bursal homogenates containing the recent Polish IBDVs
proved to contain high amounts of IBDV antigens, and compared well with the
reference F52/70-, 89/163- and 91/168-containing homogenates (optical densities
ranging 2.2 to 2.9). On the contrary, all homogenates prepared from bursae infected
with the early Polish isolates contained low amounts of antigens (optical densities
ranging from 0.5 to 1.0).
48
Table 1 presents the reactivity of the eight anti-IBDV Mabs in paired ACELISAs performed with 12 viruses (the following marks are used: differences in the
Mab reactivity of the tested viruses from F52/70 are evidenced as gray areas of the
table, reduced binding (<25%) of the Mab with the tested viruses are evidenced as pale
shading, lack of binding (<15%) as darker shading of the table). None of the tested
Polish IBDV strains allowed significant binding of Mab 3 and 4 (mean reactivities of
these Mabs ranging from 0-2% and 0-14%, respectively). This result suggests that the
studied viruses, irrespective of their date of isolation, might have aa changes in the first
major hydrophilic peak (peak A) of VP2 variable domain. This was a very interesting
finding, for the early IBDV strains isolated before the emergence of the acute form of
IBD all had shown a classical antigenicity (high reactivity versus all Mabs as a
reference F52/70). Moreover, two early Polish viruses (78/GSi and 80/Ga) did not react
with Mab 5 and also exhibited a two- to three-fold reduction in the binding of Mab 8
(mean reactivities were 31% and 21% for 78/GSi and 80/Ga, respectively, versus 2-3%
in atypical vv IBDV or 82-88% in typical isolates). From previous studies, the lack of
or a low reactivity with Mab 5 was considered as an indication of aa changes in the first
minor hydrophilic peak of VP2 variable domain. Two of the recent Polish viruses
(92/111 and 93/35) exhibited an antigenic profile similar to the atypical French 91/168
isolate as they did not react with Mab 8. This result suggested that aa changes may
exist in their second major hydrophilic peak B. The 91/272, 94/48, 99/150 and 00/40
viruses had an antigenic reactivity similar to that of typical vvIBDV.
Discussion
Regarding recent Polish viruses, four of them had exactly the same antigenic
profile as typical vvIBDVs (no binding of Mabs 3 and 4). The present study could
confirm previous pathotypic studies and demonstrate that vvIBDVs are now present in
Poland. Interestingly, two of the recent Polish viruses (isolates 92/11 and 93/35)
appeared to be antigenically different from other vvIBDVs, as did the 91/168 French
atypical vvIBDV strain, because they all had a reduced ability to bind Mab 8. As
shown for the French strain 91/168 responsible for this are aa differences located at the
C-terminal end of peak B (Q324L in 91/168 in comparison to other typical vvIBDV)
(8). As these atypical French and Polish vvIBDV-related isolates have been isolated
during a relatively short period (1991-1993) and so far have not been isolated
elsewhere, it is tempting to speculate that the same IBDV strains were exchanged
between the two countries. However, the epidemiological links between the outbreaks
in the two countries are not known and more isolates collected in the same areas /
periods would be necessary to precise where the virus was originally prevalent. A
1989-1997 survey of French vvIBDV isolates suggested that the 91/168 strain caused
only a sporadic outbreak in France and did not replace in this country the more typical
89163-like vvIBDVs.
49
Table 1
Antigenic characterization of Polish IBDV strains in a Mab-based AC-ELISA
IBDV
strain
Reactivity in AC-ELISA with
Mab1
Mab3
Mab4
Mab5
Mab6
Mab7
Mab8
Mab9
72a
71
58
59
88
96
91
76
71-72
68-73
51-65
52-65
76-100
90-102
86-96
74-78
89/163
typical
vvIBD
V
58
50-66
1
0-1
6
3-8
27
17-37
95
101-88
86
78-94
76
60-92
73
76-69
91/168
atypica
l
vvIBD
V
62
61-62
1
1-1
15
10-19
46
32-60
96
82-109
50
30-69
3
2-4
68
57-79
31
26-35
61
52-70
17
16-18
68
45-91
82
66-98
72
66-78
65
62-67
89
97-80
72
71-72
0
0-0
2
1-2
2
1-2
2
1-2
1
1-1
2
1-3
1
0-2
1
0-1
2
1-3
1
0-1
6
5-7
0
0-0
11
8-13
13
10-15
14
14-14
8
6-10
9
7-11
12
7-16
0
0-0
58
45-70
2
0-4
60
51-68
46
36-55
44
45-43
39
31-46
62
56-68
53
47-58
115
118
106-123 119-117
119
117
106-131 99-135
88
94
94-82
93-95
96
104
89-102 97-111
123
51
99-146
34-68
113
56
106-120 42-69
109
112
113-104 107-117
104
107
114-94 118-95
129
118
108-149 110-125
31
30-32
88
85-91
21
17-25
82
63-101
2
2-2
3
2-4
84
75-93
86
85-87
88
84-92
121
118-124
79
73-84
98
102-94
68
67-69
77
69-85
76
73-79
76
73-78
72
71-73
81
80-82
F52/70
b
78/GSi
78/GSz
80/GA
91/272
92/111
93/35
94/48
99/150
00/40
a
b
: Mean result from two repeated tests.
: Range in two repeated tests.
50
Unexpected results were also obtained with the 78/GSz, 78/GSi and 80/GA
early Polish viruses. Indeed, these isolates did not react with Mabs 3 and 4 and the two
latter viruses also reacted poorly, if at all, with Mabs 5 and 8. Hence, the neutralizing
epitopes present on the early Polish IBDVs were clearly different in comparison with
both the European F52/70 reference strain and the recent vvIBDV isolates from Poland.
With respect to strain identification in the laboratory, the results concerning
early Polish viruses are interesting as several previous studies had suggested that the
lack of reactivity to Mabs 3 and 4 in AC-ELISA might be a character of very virulent
and variant IBDVs and help in differentiation between classical and recent isolates in
addition to pathogenicity testing (9). The present results indicate that early Polish
IBDVs isolated between 1978-80, hence before the identification of variant and very
virulent strains, may also not react with Mab 3 and 4, in spite of being early strains
with a low pathogenicity. It should be remembered that bursal homogenates
contaminated with the early Polish isolates contained low amounts of IBDV antigens,
as compared with homogenates containing pathogenic strains such as F52/70 or the
vvIBDVs. Hence, as precised previously (6, 9), both the high antigen content in bursal
homogenates and the lack of binding of Mabs 3 and 4 should be considered as relevant
in the presumptive identification of vvIBDVs. The identification should always be
definitively assessed by pathogenicity testing (8).
With respect to IBDV epidemiology, the present results of testing early Polish
isolates are also interesting. Indeed, the current idea is that early IBDVs, prevalent
worldwide until the emergence of the US variants and vvIBDVs, were “classical” ones
with more or less the same antigenic profile as strain F52/70. This theory stems from
the antigenic and genetic study, firstly 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 secondly of some IBD attenuated vaccine strains which have been
developed from several early IBDV isolates (Lukert strain, D78, etc). However, the
extent to which early IBDVs were actually antigenically and genetically homogeneous
is not known precisely, as the number of available early IBDV strains is, unfortunately,
limited. Similarly, it is not known whether 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 appear antigenically original suggests that
more diversity than currently admitted might have existed in Europe prior to the
emergence of the vvIBDV. Such an assertion is in accordance with a recent report by
Mato et al. (12) showing that several early Hungarian IBDV isolates collected in the
late 70s and early 80s belong to a genetic cluster significantly different from the
“classical” viruses. Analysis of more early IBDV isolates is necessary to get a more
precise insight into the actual epidemiology of IBD.
Acknowledgement: This work was supported by COST
“Immunosuppressive viral diseases in poultry” from the EU.
Action
839
51
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