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Applied and Environmental Microbiology, February 2000, p. 839-843, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Diversity of Infectious Pancreatic Necrosis Virus
Strains Isolated from Fish, Shellfish, and Other Reservoirs in
Northwestern Spain
J. M.
Cutrin,*
J. G.
Olveira,
J. L.
Barja, and
C. P.
Dopazo
Departamento de Microbioloxia e
Parasitoloxia, Instituto de Acuicultura-Facultade de Bioloxia,
Universidade de Santiago de Compostela, Santiago de Compostela 15706, Spain
Received 21 July 1999/Accepted 2 November 1999
 |
ABSTRACT |
A comparison was done of 231 strains of birnavirus isolated from
fish, shellfish, and other reservoirs in a survey study that began in
1986 in Galicia (northwestern Spain). Reference strains from all of the
infectious pancreatic necrosis virus serotypes were included in the
comparison, which was done by neutralization tests and agarose and
polyacrylamide gel electrophoresis of the viral genome. The
neutralization tests with antisera against the West Buxton, Spajarup
(Sp), and Abild (Ab) strains showed that most of the Galician isolates
were European types Sp and Ab; however, many isolates (30%) could not
be typed. Results from agarose gels did not provided information for
grouping of the strains, since all were found to have genomic segments
of similar sizes. Analysis of polyacrylamide gels, however, allowed six
electropherogroups (EGs) to be differentiated on the basis of genome
mobility and separation among segments, and a certain relationship
between EGs and serotypes was observed. A wide diversity of
electropherotypes was observed among the Galician isolates, and as
neutralization tests showed, most of the isolates were included in EGs
corresponding to European types Ab and Sp. Only 6.5% of the isolates
had the electropherotype characteristic of American strains.
| |
TEXT |
In the last 2 decades, the culture
of molluscs and fresh- and saltwater fish has become very important in
Galicia (northwestern Spain) in terms of the economy and in providing
employment. Since the middle of the 1980s, our laboratory has conducted
viral surveys of turbot, trout, and salmon from farms located in the
area, as well as other reservoirs of viruses, including molluscs, wild fish and sediments around fish farms. During this study, a great number
of viruses have been isolated from fish farms (17) and reservoirs (27) and most have been identified as belonging
to the family Birnaviridae and being related to the
infectious pancreatic necrosis virus (IPNV).
IPNV is the etiological agent of a highly infectious disease of several
species of wild and cultured aquatic organisms (11). Birnaviruses have a bisegmented double-stranded RNA genome, and segments A and B, respectively, have molecular masses of 2.5 and 2.3 MDa. Although mainly affecting young salmonids, birnaviruses have also
been detected in nonsalmonid fishes (3, 13, 29), as well as
in molluscs (20, 22, 27) and crustaceans (2). However, most of the strains isolated from invertebrates were nonpathogenic to the host.
It may be that the existence of a wide range of hosts for this virus is
the reason for the large number of strains described. Classification of
IPNV strains has traditionally been established by means of serological
techniques. Throughout the last decade, only three serological types of
strains were recognized, of which two are considered typically European
(serotypes Sp and Ab) and the other is considered typically North
American (serotype VR-299) (14, 19, 23, 30). Due to the
large number and diversity of new viral strains reported, many
discrepancies in that serological classification came out
(11). Recently, Hill and Way (11; B. J. Hill and K. Way, Abstr. 1st Int. Conf. EAFP, p. 10, 1983; B. J. Hill and K. Way, Abstr. Int. Fish Health Conf., p. 151, 1988) have
demonstrated that the aquatic birnaviruses are distributed worldwide in
two serogroups (A and B), with up to 10 serotypes (9 and 1, respectively). Other authors, employing monoclonal antibodies, found a
pattern of serological relationships similar to that shown by Hill and
Way (11; Hill and Way, abstr. IFHC), who used
polyclonal antibodies, as recently reviewed by Reno (26).
Furthermore, some authors have compared the electropherotypes of viral
polypeptides and genomes for typing of IPNV strains (9, 10,
12) and have observed a certain relationship with serotyping. In
the present study, a large number of IPNV strains isolated from several
fish farms and viral reservoirs in Galicia were compared in order to study the diversity of strains of this virus in the area.
Cell lines and viral strains.
Monolayers of the CHSE-214 cell
line were grown at 15°C in minimum essential medium supplemented with
10% fetal bovine serum, penicillin at 100 U/ml, and streptomycin at
100 µg/ml. Viral strains were propagated in confluent monolayers at
15°C in minimum essential medium without fetal bovine serum.
For comparison, the following 25 reference strains belonging to both
serogroups of IPNV (11) were analyzed: strain TV-1 of
serotype B1; 10 strains of serotype A1,
including the representative WB (West Buxton) strain; 4 strains of
serotype A2, including the representative Sp (Spajarup)
strain; Ab (Abild), EVE, and CVHB-1 of serotype A3; He
(Hecht) of serotype A4; Te-2 (tellina virus) of serotype
A5; C1 (Canada 1) and AS (Atlantic salmon) of
serotype A6; C2 of serotype A7;
C3 of serotype A8; and Ja (Jasper) of serotype A9.
The Galician IPNV isolates (a total of 231 isolates) were obtained from
two main sources: (i) 176 strains isolated from fish
farms from both
epizootics and routine surveys (28 from rainbow
trout
[
Oncorhynchus mykiss], 6 from Atlantic salmon
[
Salmo salar],
and 12 from turbot [
Scophthalmus
maximus]) and (ii) 55 IPNV-like
strains isolated from
environmental reservoirs sampled near the
affected fish farms. These
environmental reservoirs comprised
(i) molluscs, including mussels
(
Mytilus galloprovincialis; 32
isolates), oysters
(
Crassostrea gigas; 5 isolates), and periwinkles
(
Littorina littorea; 4 isolates); (ii) wild fish, such as
sand
eels (
Ammodytes sp.; 6 isolates), blue whiting
(
Micromesistius poutasou, 1 isolate), and sprat
(
Sprattus sprattus; 1 isolate),
which are used as food for
cultured turbot; and (iii) marine sediments
(5 isolates) and moist food
pellets (1 isolate). Part of these
isolates were previously reported by
Ledo et al. (
17) and Rivas
et al. (
27). However,
most of them correspond to unpublished
isolations performed as
described by those
authors.
Virus purification and antisera.
The Sp, Ab, and WB viral
strains were purified in order to obtain specific antisera. For this
purpose, 20 flasks of 150 cm2 were inoculated at a
multiplicity of infection of 0.1 to 0.01 and when cytopathic effects
were extensive, cells were scraped into the medium and cell debris was
pelleted at 1,500 × g for 30 min. The supernatant was
supplemented with 8% polyethylene glycol (PEG; molecular weight,
20,000). Pelleted cell debris was resuspended in 10 ml of 1× SSC (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) mixed with 1 volume of Freon,
and the mixture was shaken vigorously for 5 min. The resultant emulsion
was separated into Freon and aqueous phases by centrifugation at
10,000 × g for 20 min, and the supernatant was
transferred to the former PEG suspension. After incubation overnight at
4°C, the PEG-virus complex was pelleted at 8,000 × g
for 20 min and resuspended in 10 ml of 50 mM Tris-HCl (pH 9.0)
supplemented with 0.2% Tween 20. An equal volume of Freon was added,
the mixture was shaken vigorously for 5 min, and virions were collected
from the aqueous phase as described above and pelleted by
centrifugation for 90 min at 10,000 × g, resuspended
in 0.5 ml of 1× SSC, layered onto a 10 to 50% continuous sucrose
gradient, and centrifuged at 150,000 × g for 1 h.
The visible virus band was collected and concentrated by centrifugation
(150,000 × g for 90 min), and the purified virus was
resuspended in 500 µl of 1× SSC and stored at 4°C until use.
The antisera were obtained by inoculating young New Zealand White
rabbits with purified virus in accordance with the schedule
previously
reported by Dopazo et al. (
5) for
aquareovirus.
Neutralization test.
For serological characterization of the
Galician IPNV isolates, the neutralization test was applied with
antisera against the Sp, Ab, and WB serotypes. The neutralization test
was carried out basically as described by Okamoto et al.
(23). The viruses were diluted in Earle's buffer to a titer
of approximately 103 50% tissue culture-infective
doses/ml. The antisera were diluted from 1/10 to 1/100,000 in Earle's
buffer, and virus and antiserum dilutions were mixed (1:1). After
1 h of incubation at room temperature, the mixtures were
inoculated into three wells (100 µl/well) of confluent CHSE-214 cells
in 96-well plates. The plates were incubated at 15°C for a maximum
period of 15 days. The neutralization antibody titers were calculated
by the method of Reed and Muench (25) and expressed as the
reciprocal of the highest antiserum dilution protecting 50% of the
inoculated wells. The antigenic relatedness of the Galician isolates
with the three serotypes (Sp, Ab, and WB) was determined as the ratio
of the homologous to the heterologous titers. The closer the ratio was
to 1, the higher the relatedness with the corresponding serotype.
When reciprocal cross-neutralization was possible (i.e., among the
three reference strains [Sp, Ab, and WB]), the degree of
antigenic
relatedness was calculated and interpreted as suggested
by Hill and Way
(
11). As expected, the three antisera showed
higher
neutralization titers with the corresponding homologous
type strain
than with a heterologous strain (data not shown).
Furthermore,
cross-neutralization ratios were higher than 10 among
the three
antisera: ratios of 32 and 80 were obtained when WB
was compared with
Ab and Sp, respectively, and between Sp and
Ab, the ratio was
11.
Most of the isolates showed higher relatedness with the European
serotypes. As shown in Table
1, 128 isolates (55%) showed
a ratio of antigenic relatedness of 1 to 2 with
European serotype
Sp, and more than 30% (71 strains) showed antigenic
relatedness
with serotype Ab. However, it must be pointed out that some
strains
(29%; data not shown) gave ratios of 1 to 2 with both
serotypes.
Only one isolate gave a ratio of 1 to 2 with serotype WB. A
large
number of isolates (30%) gave low antigenic relationships (ratio
higher than 4) with all three antisera. Therefore, use of
neutralization
has demonstrated that most of the Galician isolates were
closely
related to the European strains and we were not able to
demonstrate
a great diversity among them. Perhaps the use of antisera
against
the 10 serotypes would be more appropriate to determine the
serotype
of those strains not typed with any of the three antisera.
Furthermore,
as pointed out by other authors (
24), a
complete analysis of
the serological relationships among viruses
requires cross-neutralization
tests. However, although these
possibilities were taken into consideration,
due to the large number of
isolates included in this study, they
were not applied.
Comparison of electropherotypes.
For agarose and sodium
dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, each viral
strain was inoculated into a 75-cm2 flask of confluent
CHSE-214 cells and when the cytopathic effect was extensive, the viral
suspension was clarified by centrifugation at 2,500 × g for 10 min at 4°C. Supernatants were pelleted for 1 h at
78,000 × g, and the pellet was resuspended in 1× SSC
buffer and sonicated for 30 s (at 20 kHz). Virions were cleaned by
centrifugation through a 30% sucrose cushion for 1 h at
100,000 × g, and the pellet was resuspended in 1×
SSC. The final concentration was achieved by centrifugation at
150,000 × g for 90 min at 4°C, and the virus was
resuspended in 50 µl of 1× SSC buffer and stored at 
20°C until
use. Prior to electrophoresis, concentrated virus was subjected to
digestion with proteinase K at 2 mg/ml in the presence of 0.5% SDS at
65°C for 2 h. The viral genome was extracted by
phenol-chloroform-isoamyl alcohol (25:24:1) and ethanol precipitated overnight at 20°C. The pellets were vacuum dried in a Speedvac (Savant) for about 5 min and resuspended in TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA).
Horizontal agarose gel electrophoresis was performed on 0.5% agarose
gels containing 0.5 µg of ethidium bromide per ml at
100 V for 2 h. SDS-polyacrylamide gel electrophoresis was carried
out in 6%
polyacrylamide vertical slab gels (
16) at 150 V for
16 h and revealed by staining with ethidium bromide. In both cases,
molecular weights were determined by comparison with the turbot
aquareovirus TRV RNA segments which were used as
markers.
Different percentages of agarose gels were tested, from 0.5 to 1.2%,
and the best results, in terms of separation of the bands,
were
achieved with the 0.5% agarose gels (results not shown).
The molecular
masses of A and B segments were around 1.98 × 10
6 and
1.82 × 10
6 Da, respectively. As expected, no
variability in the genome mobility
of the Galician IPNV isolates and
reference strains was observed
in agarose gels. This is easily
explained, as mobility basically
depends on the sizes of the
double-stranded RNAs, which do not
differ by large numbers of base
pairs.
One interesting result was the difference in the molecular masses
observed with the different electrophoretic techniques:
polyacrylamide
gels gave molecular masses 0.1 to 0.2 MDa higher
than those obtained
with agarose gels. These differences are due
to the different ways that
nucleic acid migrates in the two types
of gel (
1). The sizes
of the genomic segments in polyacrylamide
gels ranged from 1.99 × 10
6 to 2.14 × 10
6 Da for segment A and
from 1.92 × 10
6 to 2.02 × 10
6 Da
for segment B, which also differed from the values reported
by other
authors (
4,
9,
10,
12,
28). This was probably
due to the
different percentages of acrylamide-bisacrylamide and/or
the different
molecular weight standards employed. As shown at
Fig.
1, using polyacrylamide gels produced a
noticeable divergence
among the genomic profiles of the IPNV strains
assayed. Those
differences included the mobility of the segments and
also their
separation: narrow patterns (with relatively small
differences
in molecular weight between segments), such as the one
shown by
the Ja strain, could be easily differentiated from the wide
patterns
(with larger differences between segments) shown, e.g., by
strains
WB and Ab. Among these, differences were also observed in the
sizes of the segments, i.e., in their mobility (the Ab genome
clearly
showed higher mobility than the WB genome).
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FIG. 1.
Comparison of RNA patterns of the genomes of IPNV
isolates from fish and other reservoirs with those of reference type
strains using SDS-6% polyacrylamide gels. The values on the left are
the molecular masses of the genomic segments of the turbot aquareovirus
TRV, expressed in megadaltons.
|
|
Electrophoresis of the genome in polyacrylamide gels has been found by
us to be an easy, fast, and powerful technique for
comparison of IPNV
strains. Based on the mobility of segments
and the separation among
them, six different electropherotypes,
which we named
electropherogroups (EGs), were established (Fig.
2). It is important to point out that a
certain relationship was
observed between the EGs and the serotypes of
serogroup A. EG3,
-5, and -6 were represented by serotypes
A
3 (including strains
Ab, EVE, and CVHB-1), A
4
(strain Hecht), and A
9 (strain Jasper),
respectively (Table
2). Serotypes A
2 (including
strains Sp, d'Honnincthum,
Bonamy, and N1) and B
1
(represented by tellina virus strain TV-1)
were included in EG4. EG2
was even more complex, since it included
several serotypes, comprising
the American WB type strain (serotype
A
1); Canadian strains
C
1, C
2, and C
3 (serotypes
A
6, A
7, and A
8);
and strain Te-2,
isolated in the United Kingdom from a mollusc
(serotype
A
5). Moreover, it must be noted that the two reference
strains isolated from the
Tellina mollusc (Te-2 and TV-1,
corresponding
to serotypes A
5 and B
1,
respectively) showed distinctive electropherotypes
(EG2 and -4, respectively). None of the reference strains was
included in EG1.
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FIG. 2.
Schematic representation of the electropherotypes
obtained from reference strains and Galician isolates and the EGs
established.
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|
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TABLE 2.
Distribution of the Galician IPNV isolates and reference
strains among the six EGs established by polyacrylamide gel
electrophoresis of the viral genome
|
|
Based on our results, it seems clear that analysis of IPNV genome
profiles can help in the differentiation of serotypes, although
the
technique must be complemented by others in order to distinguish
serotypes with the same electropherotype. Hedrick et al. (
7,
9,
10) reported that a combined analysis of the electrophoretic
profile of RNA genome segments and virion polypeptides can distinguish
different IPNV strains. However, they suggested that mobility
of the
virion polypeptides is a more suitable basis for grouping
of new
isolates. Other authors (
6) also reported the use of
genomic
electropherotypes as a means by which new strains of IPNV
could be
identified.
Among the Galician isolates, a wide diversity of electropherotypes was
observed. They were distributed among EG1 to -5 (Table
2), and none of
them showed the narrowest electropherotype (EG6).
Although most of the
isolates belonged to EG4 (63.6%), the remaining
EGs (except EG6)
contained a variable number of isolates: 0.9%
in EG1, 6.5% in EG2,
18.6% in EG3, and 10.4% in EG5. As regards
the isolates from fish
farms, most of them (115 strains, i.e.,
more than 65%) belonged to
EG4. Around 12.5% (22 strains) of the
isolates from fish were included
in EG3, and 13.5% were in EG5
(24 strains). Thirteen strains showed
the wide pattern typical
of EG2, and only two strains were included in
the widest EG (EG1).
Of the strains isolated from different types of
reservoirs, the
majority were in EG3 and -4. Only two isolates, one
from
L. littorea (periwinkles) and a strain isolated from
moist fish pellets (prepared
with raw fish), showed the pattern
characteristic of EG2. It is
interesting that none of the reservoir
strains were included in
the group with the widest pattern (EG1) or in
the groups with
the narrowest patterns (EG5 and -6). This wide
diversity among
the strains isolated in our area can be explained by
the fact
that when our laboratory began its work in diagnosis, the
importation
of eggs or fish from other European countries, as well as
from
North America and Australia, was a common practice among fish
farms.
Other authors have published similar studies of comparisons of IPNV
strains by neutralization and polyacrylamide gel electrophoresis,
although they reported lower levels of divergence. Hedrick et
al.
(
9) reported the analysis of four birnaviruses isolated
from
fish in Taiwan; three of them were closely related to the
Ab strain,
and the fourth was related to serotype VR-299. Hsu
et al. (
12,
13) studied a larger number of strains, also from
Taiwan, all of
them related to the Ab or the VR-299 serotype.
Moreover, another four
viruses isolated from fish in Korea were
found to be closely related to
reference strain VR-299 (
8).
In another report, Kusuda et
al. (
15) compared six Japanese
IPNV isolates with the Ab,
Sp, and VR-299 type strains, concluding
that the Japanese strains
constituted a completely separate group.
However, all of these reports
have in common the lack of use of
reference strains from the 10 serotypes. Other authors, such as
Lee et al. (
18), used
other molecular techniques to compare
a relatively large number of
strains, but even then the study
did not include strains from the 10
serotypes.
To our knowledge, this is the first time that such a large number of
isolates of IPNV from a specific area has been included
in a study and
compared with reference strains of all of the IPNV
serotypes.
Nevertheless, further experiments are being carried
out to compare
these strains using other techniques (such as restriction
fragment
length polymorphism and sequencing) in order to confirm
the diversity
of the strains and establish groups of similarity
among
them.
| |
ACKNOWLEDGMENTS |
This study was supported by grant AGF93-0769-C02-01 from the
Ministerio de Educación y Ciencia and grant XUGA 20009B96 from the Xunta de Galicia, Spain.
We thank B. L. Nicholson, C.-F. Lo, and R. P. Hedrick for
supplying viral strains. J. M. Cutrin acknowledges the Ministerio de Educación y Ciencia (Spain) for research fellowships.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Microbioloxia e Parasitoloxia, Instituto de Acuicultura, Facultade de
Bioloxia, Universidade de Santiago de Compostela, Santiago de
Compostela 15706, Spain. Phone: 34-981-563100, ext. 16051 or 16052. Fax: 34-981-547165. E-mail: mpdopazo{at}uscmail.usc.es.
Scientific contribution 0003/99 of the Instituto de Acuicultura.
| |
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Applied and Environmental Microbiology, February 2000, p. 839-843, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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