Download Antigenic properties and experimental transmission to several fish

Veterinary Microbiology 82 (2001) 11±25
Antigenic properties and experimental transmission
to several ®sh species of a marine birnavirus
isolated from sole (Solea senegalensis)
S. Perez-Prietoa, E. Garcia-Rosadob, S. Rodrigueza,
D. Castrob, J.J. Borregob,*
a
Centro de Investigaciones BioloÂgicas, Consejo Superior de Investigaciones Cientõ®cas,
VelaÂzquez 144, 28006 Madrid, Spain
b
Department of Microbiology, Faculty of Sciences, University of Malaga,
Campus of Teatinos, 29071 Malaga, Spain
Received 2 July 2000; received in revised form 20 February 2001; accepted 3 March 2001
Abstract
A cross-neutralization test was used to study the antigenic relationship of an aquabirnavirus
isolated from sole (Solea senegalensis), named solevirus, and several infectious pancreatic necrosis
virus (IPNV) strains. Solevirus was antigenically similar to IPNV strain Sp. Transmission of the
solevirus to other ®sh species has been determined by inoculation to freshwater and marine ®sh
species (two salmonids and gilt-head seabream). A higher pathogenicity was obtained for the
marine ®sh species, although solevirus caused an asymptomatic infection in all species tested, as
demonstrated by the detection of viral RNA and of viral antigens in ®sh leucocytes, respectively,
using polymerase chain reaction (PCR) and ¯ow cytometry (FC). # 2001 Elsevier Science B.V. All
rights reserved.
Keywords: Solea senegalensis; Aquabirnavirus; Solevirus; IPNV; Antigenic characterization; Experimental
transmission; PCR; Flow cytometry
1. Introduction
Infectious pancreatic necrosis virus (IPNV) is the type species of the genus
Aquabirnavirus that comprises viruses affecting ®sh and other aquatic animals, and
which represents the largest and most antigenically diverse group within the family
*
Corresponding author. Tel.: ‡34-5-2131893; fax: ‡34-5-2132000.
E-mail address: [email protected] (J.J. Borrego).
0378-1135/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 1 3 5 ( 0 1 ) 0 0 3 5 5 - 8
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S. Perez-Prieto et al. / Veterinary Microbiology 82 (2001) 11±25
Birnaviridae (Murphy et al., 1995). IPNV is the etiological agent of a well-characterized
acute disease of young hatchery-reared salmon ®sh, infectious pancreatic necrosis (IPN)
(Wolf et al., 1960). Although, three major serotypes, VR-299 strain (the prototype of the
USA isolates), Sp and Ab strains (both representative of the European isolates) have been
well established, several strains with different virulence and serological characteristics
have been continuously reported (McMichael et al., 1975; Christie et al., 1988; Wolf,
1988; Hill and Way, 1995). However, all IPNV serotypes show some degree of crossreaction in reciprocal neutralization tests with rabbit antiserum (McMichael et al., 1975;
Caswell-Reno et al., 1989; Kusuda et al., 1993).
Birnaviruses have also been isolated from marine ®sh such as sea bass (Dicentrarchus
labrax), turbot (Scophthalmus maximus), halibut fry (Hippoglossus hippoglossus), and
Dover sole (Solea solea) (Hill, 1982; Bonami et al., 1983; Castric et al., 1987; Mortensen
et al., 1990). These viruses present a broad host range comprising more than 30 different
®sh families, 11 species of molluscs, and four species of crustacea (Hill, 1982; Wolf,
1988). Hill and Way (1995) proposed the term ``aquatic birnavirus'' for those viruses
isolated from non-salmonid ®sh for which there is no information on their ability to
produce IPN disease in salmonids.
A disease with high mortality affecting senegalense sole (S. senegalensis)
characterized by dark coloration, hyperactivity, erratic swimming, and abnormal behavior
appeared suddenly in a culture facility in southwestern Spain in 1993. Later, Rodriguez
et al. (1997) isolated and characterized a birnavirus, named solevirus, from the skin and
internal organs of moribund and dead soles.
In this work, we have determined the virulence of this birnavirus for salmonids such as
rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta), and for a marine
®sh of interest for Spanish aquaculture, such as gilt-head seabream (Sparus aurata). In
addition, antigenic relationships of several selected strains of IPNV and solevirus were
also compared.
2. Materials and methods
2.1. Virus growth and cell culture
Chinook salmon embryo (CHSE-214) and Bluegill fry (BF-2) epitheloid cell lines were
used to isolate a strain of aquatic birnavirus (named solevirus) closely related to IPNV
during a natural epizootic outbreak affecting soles (Rodriguez et al., 1997). BF-2 cells
were cultured for subsequent serial virus passages and stock virus. Cell monolayers were
grown at 188C in Eagle's minimal essential medium with Earle's salts (MEM; Gibco,
Renfrewshire, Scotland, UK) containing 10% fetal bovine serum (FBS; Gibco), 100 IU/
ml penicillin (Gibco), and 100 mg/ml streptomycin (Gibco) (P ‡ E). MEM supplemented
with 2% FBS was used as a maintenance medium (MM). For virus titer assays, the cells
were seeded into 96-well cell culture plates (Falcon, Becton-Dickinson). Viral 10-fold
serial dilutions were added onto drained BF-2 cells (six wells per dilution) and incubated
for 1 h at 158C to allow adsorption of the virus to the cells. Then, the monolayers were
overlaid with MM and observed daily for the development of cytopathic effect (CPE).
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The dilution of the virus infecting 50% of the cell cultures was considered the end point
dilution (TCID50/ml).
2.2. Virus puri®cation and antisera production
Infected cells were incubated at 208C in Leibovitz L 15 medium (Gibco) containing
2% FBS until the maximal development of CPE. Cultures were frozen and thawed three
times and centrifuged at 900 g for 5 min. Birnaviruses from clari®ed supernatants were
concentrated by polyethylene glycol (PEG)±NaCl precipitation (7% PEG±2.2% NaCl,
both supplied by Sigma Chemical Co., St. Louis, MO, USA), stirred at 48C for 2±4 h, and
centrifuged at 10,000 g for 75 min. Then, the supernatant was discarded and the pelletcontaining virus was resuspended in 1 ml of TNE buffer (0.025 M Tris, 0.1 M NaCl,
1 mM EDTA, pH 7.3). The samples were puri®ed by isopynic CsCl gradient
centrifugation (1.5 ml of 40% CsCl, 1.0 ml of 30% CsCl, and 0.5 ml of 20% CsCl in a
nitrocellulose centrifuge tube) at 114,300 g for 16 h (SW 50.1 rotor, Beckman L2-65B
ultracentrifuge). UV absorbance (at 280 nm) was spectrophotometrically determined in
each fraction (1 ml) of the centrifuged gradient. The highest UV absorbance corresponded
to a white band containing the virus fraction. This fraction was overlaid onto 3 ml of 20%
sucrose solution in TNE buffer. The virus was collected by centrifugation at 100,000 g
for 2 h and the pellet resuspended in 1 ml of 0.15 M phosphate-buffered saline (PBS,
pH 7.2), and referred as puri®ed virus.
New Zealand white rabbits were intramuscularly injected with puri®ed virus (about
50 mg) in PBS containing 50% Freund's complete adjuvant (Difco Laboratories, Detroit,
MI, USA). A second booster without adjuvant was intravenously inoculated 3 weeks
later. Third and fourth booster doses were repeated 2 and 3 weeks later. After 1 week, the
rabbit was exsanguinated and the immune serum was decontaminated by membrane
®ltration, inactivated at 568C for 30 min and stored at 708C. Pre-immune and antiFreund's adjuvant sera were used as negative controls.
2.3. Cross-neutralization test
The serological relatedness of solevirus and several IPNV reference strains (VR-299,
Sp, Ab, Jasper and Tellina) was conducted by cross-neutralization test using polyclonal
rabbit sera prepared against each of the ®ve IPNV strains mentioned above. The
neutralization tests were performed following the technique described by Okamoto et al.
(1983). The serological similarity among the tested viruses was calculated applying the
formula r ˆ …r1 r2 †1=2 (Archetti and Horsfall, 1950), where r1 ˆ ‰reciprocal of dilution
of antiserum to virus (1) neutralizing virus (2)]/[reciprocal of serum dilution of virus (1)
neutralizing virus (1)], and r2 ˆ ‰reciprocal of dilution of antiserum to virus (2) neutralizing
virus (1)]/[reciprocal of dilution of antiserum to virus (2) neutralizing virus (2)].
2.4. Fish infection and maintenance
IPNV-free ®ngerlings of brown trout (S. trutta) and rainbow trout (O. mykiss) (mean
weight 6 g; 4 months old) were kindly provided by the farm of UnÄa (ConsejerõÂa de
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Agricultura y Medio Ambiente de la Comunidad de Castilla La Mancha, Cuenca, Spain).
Fish were divided into groups of 20, acclimated for 2 weeks and maintained in separate
35 l aquaria with aerated, dechlorined water, exterior carbon ®lters and temperature
controlled (158C) by water chiller units. The room lighting was controlled to correspond
to the natural photoperiod. Fish were fed twice per day with dry trout pellets. Prior to any
experimental procedure, ®sh were individually anaesthetized in 100 ppm MS222 (tricane
methanesulfonate) at 158C until loss of the righting re¯ex. To perform the virus infection
of ®sh, two groups of 20 specimens of each ®sh species were intraperitoneally injected
with 0.2 ml of a dilution in PBS of solevirus (104 TCID50). Twenty control ®sh of each
species were also injected with 0.2 ml of PBS. The ®sh were placed in 35 l aquaria and
observed daily for ®sh deaths. Dead ®sh were stored at 48C and assayed for virus
recovery within 24 h, and ®ve ®sh survivors of each group, including the control group,
were sampled for viral assay in BF-2 cells and ¯ow cytometry (FC) and polymerase chain
reaction (PCR) analysis at the end of the experiment (about 4 weeks). As positive control,
a group of 20 specimens of brown trout were intraperitoneally injected with 0.2 ml of a
dilution in PBS of IPNV Sp (ATCC VR1318) (104 TCID50) obtained from two successive
reisolations from inoculated brown trouts.
To determine the pathogenic potential of solevirus in other marine ®sh species, groups
of 20±25 seabream (mean weight 10 g; 5 months old) were intraperitoneally injected with
0.1 ml (104 TCID50) of a dilution in PBS of the virus. As controls, two other groups of
20±25 ®sh were injected with 0.1 ml PBS. Fish were held in 60 l aquarium receiving
®ltered marine water in a recirculating system with carbon ®lters, and temperature
maintained at 208C. Fish were fed ad libitum with cephalopoda. Dead ®sh were stored at
208C until assayed for virus by inoculation onto cell monolayers and posterior
identi®cation by seroneutralization.
A lot of dead ®sh were processed for histopathological examination. Whole ®sh were
®xed in 10% (v/v) buffered formaldehyde (pH 7.2). Tissue samples (skin, digestive tract,
kidney, and liver) were dissected and embedded in paraf®n. Thin sections (5 mm) were
stained with hematoxylin±eosin and hematoxylin±VOF (Sarasquete et al., 1998).
At the end of the experimental infection (45 days post-infection), ®ve survivor ®sh
were individually processed for viral assay in BF-2 cells, FC, and PCR analysis. Survivors
were maintained for 120 days and later processed for virus detection after inoculation on
BF-2 cells by PCR or directly by ¯ow cytometry analysis of the leucocytes.
2.5. Virological examination
Pools of organs from ®sh were diluted in MEM with P ‡ E to a ®nal concentration
(w/v) of 1/5, homogenized with a stomacher, and centrifuged (1200 g, 10 min at 48C).
Supernatants were ®ltered through 0.45 mm pore size and stored at 48C for tests carried
out within 24 h (Amos, 1985).
The IPNV titer was determined by serial dilution end point assays, in which 10-fold
dilutions of samples were incubated with BF-2 cells at 188C. Cell monolayers were daily
examined for CPE for 7 days, then all negative samples were blind-passaged.
Neutralization assays were used to con®rm that CPE observed in passages 1 and 2 was
caused by solevirus.
S. Perez-Prieto et al. / Veterinary Microbiology 82 (2001) 11±25
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2.6. RNA extraction and cDNA synthesis
Cell monolayers were infected with the homogenates of ®sh infected with solevirus
(1:100 dilution). After 5 days, total RNA was extracted following guanidinium
thiocyanate±phenol±chloroform method (Chomcynski and Sacchi, 1987). RNA pellets
were washed with 70% ethanol, dried and resuspended in diethyl pyrocarbonate (DEPC)treated water. Single-stranded cDNA was synthesized from 5 mg of total RNA using the
®rst strand synthesis kit for RT-PCR as described by the manufacturer (Amersham
Pharmacia Biotech, Essex, UK). Brie¯y, RNA was incubated at 428C for 1 h in reverse
transcription reaction mixture (50 mM Tris±HCl, pH 8.3; 50 mM KCl; 10 mM MgCl2;
80 mM sodium pyrophosphate; 10 mM each dATP, dGTP, dTTP, and 5 mM dCTP; 20
units of human placental ribonuclease inhibitor; 70 mM of random hexanucleotide primer,
and 5 U of reverse transcriptase). RNA±DNA hybrids were denatured at 1008C for 5 min.
Volumes of 2 ml of this reaction were used in PCR.
2.7. Polymerase chain reaction (PCR) ampli®cation assay
The oligonucleotide primers used in this study were selected according to Pryde et al.
(1993) on the basis of the nucleotide sequence of IPNV VP2 gene and synthesized at the
Laboratorio de QuõÂmica de ProteõÂnas (C.I.B., CSIC, Madrid, Spain). The ®rst primer (50 GAACCCCCAGGACAAAGT-30 ) hybridized with positions 568±585 (sense orientation)
in the open reading frame of the VP2 gene, and the second (50 -TGATTGGTCTGAGCACGC-30 ) with positions 1164±1181 (antisense orientation).
Ampli®cation of DNA was carried out in a 100 ml reaction volume containing
2.5 mM MgCl2, 10 mM Tris±HCl (pH 8), 50 mM KCl, 50 pM of each primer, 2 mM
of deoxinucleoside triphosphate mix dNTPs, 1 U Taq-polymerase and 2 ml of the
cDNA template. Nucleic acids from uninfected cell cultures were used as negative
controls, and 50 ml of mineral oil (Sigma) was overlaid on the surface of the reaction
mixtures.
Ampli®cation was performed in an automatic thermal cycler (Perkin-Elmer Cetus)
programmed under the following conditions: 35 cycles of 1 min at 948C, 1 min at 538C,
and 1 min at 728C. The ampli®ed products (a 613 bp region of the IPNV VP2 gene)
were analyzed by electrophoresis in a 1.2% agarose gels stained with ethidium
bromide. Only cDNA from solevirus-infected cells produced an ampli®ed DNA
product. To validate the PCR assay, ampli®ed DNA product was sequenced and
compared with the nucleotide sequence deposited in EMBL (accession number
AJ011774).
2.8. Flow cytometry (FC) assay of cells from infected ®sh
The anterior kidney (pronephros) from the infected ®sh were aseptically removed and
transferred to MEM medium. The cells were dispersed by gently teasing the tissue
samples over a metal mesh in a Petri dish. Leucocytes were isolated from the cell
suspension using the lymphocyte separation medium (Flow Laboratories, Scotland, UK).
After centrifugation at 300 g for 20 min at 48C, the buffy coat of leucocytes located at
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the plasma/Ficoll interface (Blaxhall, 1985) was carefully recovered and the cells were
washed twice and resuspended in PBS. Cell concentration was determined with a
hemocytometer and adjusted at about 1 106 cells/ml.
To analyze the cells by FC, indirect immuno¯uorescence stain was used, considering
the ¯uorescent cells as IPNV-cell carriers. The FC analysis was conducted according to
the methodology previously described by us (Perez et al., 1994). Brie¯y, the leucocytes
were resuspended in a small volume of PBS, gently mixed with 1 ml cold formaldehyde
(3.7%) at 48C for 15 min, and centrifuged at 300 g. Afterwards, the pellet was mixed
with a PBS-Triton X-100 (0.1%) solution for a few seconds and immediately washed by
centrifugation (twice for 5 min in PBS). Then, the primary antibody anti-solevirus (1:100
diluted in PBS) was added to the pelleted cells and incubated for 30 min at room
temperature. After washing three times by centrifugation in PBS, the second antibody,
500 ml goat anti-rabbit IgG±FITC conjugate (Sigma) (1:40 in PBS) was added and
incubated at room temperature for 30 min. Two further centrifugation cycles in PBS were
carried out to eliminate nonspeci®c binding, and the pellet was resuspended in 0.5 ml of
PBS to determine the ¯uorescence of the cellular suspensions by FC using a cytometer
(EPICS XL, Coulter, Madrid, Spain) equipped with an argon ion laser (200 mW at
488 nm excitation).
3. Results
3.1. Cross-neutralization tests
Serological relatedness were de®ned by titer-ratio analysis of the cross-neutralization
values. Values of 1/r higher than 20 establish the boundary among antigenically distinct
groups (Jorgensen, 1972). A value of 1/r ˆ 1:0 indicates complete homology and
increasing values indicate higher antigenic unrelatedness. In this study, solevirus
presented a lower relationship to INPV serotype Ab (1=r ˆ 45:5) than to serotype Sp
(1=r ˆ 1:4), and was serologically distinct from serotype VR, Tellina and Jasper
(Table 1).
Table 1
Antigenic relationship based on 1/r between ®ve strains of infectious pancreatic necrosis virus and the solevirus
isolate
Virus
Sp
Ab
VR-299
Jasper
Tellina
Solevirus
Antiserum
Sp
Ab
VR-299
Jasper
Tellina
Solevirus
1
64.1
81.3
90.0
275.1
1.4
1
114.6
16.1
64.0
45.5
1
22.6
31.8
64.0
1
22.6
72.4
1
256.5
1
Days
post-infection
Number of
dead fisha
Cumulative
mortality
Rainbow trout
9
33
3
0
Brown trout
9
33
IPNV Spe
11
33
a
Virus detection (and titer) in cell culturesb
Dead fish
Visceral poolsc of fish survivors
3/20
3/20
3/3 (1 106 TCID50/ml)
±
NDd
4/4 (1 104 TCID50/ml)
0
0
0/20
0/20
±
±
ND
1/4 (1 103.2 TCID50/ml)
5
0
5/20
5/20
5/5 (1 106.5 TCID50/ml)
±
ND
ND
No fish died in the control group.
Number of fish or visceral pools positive for virus/number of fish or visceral pools processed; mean of the infective titer expressed as tissue culture infective dose
50 (TCID50)/ml.
c
Pools of viscera from three to four fish were processed.
d
ND: not done.
e
Used as control.
b
S. Perez-Prieto et al. / Veterinary Microbiology 82 (2001) 11±25
Table 2
Infection of two species of salmonid ®sh following intraperitoneal injection of the solevirus isolate
17
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3.2. Virus isolation from experimentally infected salmonids
Visceral pools of dead rainbow trout at 9 days post-infection (PI) caused cytopathic
effects (CPE) on the second passage on BF-2 cells, and the solevirus was recovered and
identi®ed by seroneutralization, achieving infective titers of 1 106 TCID50/ml. No
deaths occurred among either the salmonid ®sh injected with solevirus or the control
group injected with PBS maintained in aquaria from 9 to 33 days PI. Four pools of viscera
(three to four ®sh each) from the survivors at 33 days PI were also homogeneized and
inoculated on cell monolayers. Blind passages were necessary to recover virus from some
of the ®sh samples. Virus titer from infected ®sh survivors at the end of the experiment
ranged between 1 103:2 and 1 104 TCID50/ml (Table 2).
INP Sp virus was used as positive control to assess the viral susceptibility of salmonid
®sh. Dead ®sh were recorded between 7 and 11 days PI, with a cumulative mortality of
25%. Infective titers reached 1 106:5 TCID50/ml in BF-2 cells (Table 2).
3.3. Virus detection from experimentally infected seabream
Seabream fry showed high cumulative mortalities when infected with solevirus. The
®sh died at 17 days PI and the percentages of cumulative mortalities increased from 16.7
to 66.7% at the end of the experiment (45 days) (Table 3). The dead ®sh between 23 and
28 days were negative for virus detection onto BF-2 cell cultures, but solevirus was
isolated from all of the other ®sh samples tested, although blind passages were necessary
to recover virus from most of them, with viral infective titers of 1 104 TCID50/ml. The
maximum cumulative mortality in the non-infected control group was 10% and no virus
was recovered from any of the control ®sh assayed. Detection of virus was positive in BF2 cells from survivor ®sh at 45 days PI, with a mean titer of 1 104 TCID50/ml (Table 3).
Histological studies were performed in dead seabream at 17 and 20 days PI. All the
organs examined (skin, digestive tract, kidney, and liver) showed important histological
Table 3
Virus detection in seabream following intraperitoneal injection of the solevirus isolate
Days
post-infection
Number of
dead fish
Cumulative
mortalitya
Virus detection (and titer)
in cell culturesb
17
20
23
24
28
31
36
40
45
7
5
2
1
2
2
2
7
0
7/42
12/42
14/42
15/42
17/42
19/42
21/42
28/42
28/42
NDc
ND
0/2
0/1
0/2
2/2 (1 104.1 TCID50/ml)
2/2 (1 104 TCID50/ml)
7/7 (1 104 TCID50/ml)
4/4 (1 104 TCID50/ml)
a
Cumulative mortality in the control group was 10% and no virus was recovered.
Number of fish or visceral pools positive for virus/number of fish or visceral pools processed; mean of the
infective titer expressed as tissue culture infective dose 50 (TCID50)/ml.
c
ND: not done (fish processed for histology).
b
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Table 4
Virus detection by PCR and ¯ow cytometry in rainbow trout, brown trout, and seabream survivors after infection
by the solevirus isolate
Fish
Days post-infection
Virus detection
PCRa
Flow cytometryb
Rainbow trout
33
‡
‡
Brown trout
33
‡
‡
45
60
120
‡
‡
‡
‡
Seabream
a
b
Visceral pools of five fish were inoculated on cell cultures and supernatants were assayed.
Kidneys from five fish were individually processed and the purified leucocytes were analyzed.
alterations, similar to those described previously for IPNV infection in rainbow trout by
McKnight and Roberts (1976).
3.4. Solevirus detection by PCR assay
Visceral pools from ®sh survivors infected with solevirus were sampled at different
times in each one of the experiments performed with the different ®sh species tested.
Homogenates (1:100 diluted) were inoculated onto BF-2 cells, and after 3 days,
Fig. 1. Analysis of PCR products on agarose gels: (A) ampli®cation of IPNV-VP2 gene fragment from
supernatants of BF-2 cells inoculated with visceral pools from rainbow trouts infected with solevirus after 33
days (lane 1) or BF-2 cells inoculated with the reference strain IPNV Sp (lane 2); (B) ampli®cation of IPNV-VP2
gene fragment from supernatants of BF-2 cells inoculated with visceral pools from seabream mock-infected with
solevirus (lane 1), infected with solevirus after 45 days (lanes 2 and 5), 60 days (lanes 3 and 6), and 120 days
(lanes 4 and 7); M: molecular marker.
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supernatants were analyzed to determine if the virus was present in low amounts in these
apparently healthy ®sh by PCR (Table 4).
The RT-PCR assay was able to amplify ef®ciently solevirus-RNA from all the samples
assayed, except those corresponding to survivors of seabream at 120 days PI (Fig. 1A). A
613 bp PCR product for solevirus was obtained as expected for IPNV. No PCR product
was detected in any of the control ®sh tested (Fig. 1B).
3.5. Flow cytometry analysis of leucocytes
Flow cytometry analysis of leucocytes stained by indirect immuno¯uorescence was
applied to detect viral antigen and to determine the asymptomatic infection of different
Fig. 2. Fluorescence histograms of leucocyte samples from rainbow trout kidneys after 33 days of
intraperitoneal injection with solevirus. The cells were labelled with rabbit anti-solevirus serum and antirabbit IgG±HTC conjugate. Uninfected control leucocytes were included in the analysis to allow determination
of the reaction speci®city and the overlap between positive and negative cells. Each histogram corresponds to a
different ®sh. Percentages of ¯uorescent cells are indicated.
S. Perez-Prieto et al. / Veterinary Microbiology 82 (2001) 11±25
21
Fig. 3. Fluorescence histograms of leucocyte samples from the peripheral blood from two rainbow trouts after
33 days of intraperitoneal injection with solevirus. The leucocytes were stained by indirect immuno¯uorescence
assay with rabbit anti-solevirus serum and anti-rabbit IgG±FITC conjugate. Prede®ned background of
¯uorescence was delimited by the negative control of cells from uninfected ®sh. Percentages of ¯uorescent cells
are indicated.
®sh populations injected with the solevirus isolate (Figs. 2±4). Three types of samples
were analyzed: (i) leucocytes from kidney of rainbow trout at 33 days PI; (ii) leucocytes
from kidneys and peripheral blood of brown trout at 33 days PI; (iii) leucocytes from
kidneys of seabream survivors at 45, 60, and 120 days PI.
In the rainbow trout population, solevirus antigen was present between 66.4 and 91.4%
of the leucocytes examined from kidneys individually processed at 33 days PI (Fig. 2). It
was also possible to analyze leucocytes from peripheral blood in some ®sh (Fig. 3). These
samples showed a pike in the histograms corresponding to a similar intensity of
¯uorescence in the cell population and in addition, very high percentages of viral-antigen
carrier cells were obtained (>95%).
The results obtained with brown trout population were not as clear, since some samples
of ®sh kidney leucocytes were positive, with lower (31 2%) percentages of ¯uorescent
cells (data not shown).
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Fig. 4. Flow cytometry histograms for samples of seabream kidney leucocytes from different solevirus injected
®sh after (A) 45 days, (B) 60 days, and (C) 120 days. The leucocytes were labelled with anti-solevirus serum and
anti-rabbit IgG±HTC conjugate. Prede®ned background of ¯uorescence was delimited by the negative control of
cells in each one of the experiments. Percentages of ¯uorescent cells are indicated.
From the seabream survivors, ®ve ®sh were sacri®ced at 45, 60, and 120 days PI to
analyze the kidney leucocytes. The histograms for quantitative analysis of ¯uorescence in
leucocyte samples from ®sh at 45 and 60 days PI were homogenous and clearly positive,
with percentages of ¯uorescent cells about 60% (Fig. 4A and B). However, at 120 days
PI, the samples analyzed were negative for virus detection (Fig. 4C).
The ¯ow cytometry analyses of leucocytes for the three species tested showed similar
results. From the fourth week PI, all the ®sh populations could be considered as potential
virus carriers, although brown trout ®sh seems to be less susceptible. Since the in vivo
experiments with salmonids were ®nished at this time (33rd day), we did not know how
transient or permanent this state could be for solevirus strain.
4. Discussion
The serological comparison of solevirus with reference strains of IPNV showed that the
virus was clearly related to the Sp serotype (1=r ˆ 1:4), which is the most frequently
isolated in Spain (Rodriguez et al., 1994). The 1/r value is a convenient index to quantify
the antigenic relatedness of viruses in cross-neutralization studies and it has usually been
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23
applied by different authors for serotyping and in characterization studies of new
birnavirus isolations from different ®sh species (Okamoto et al., 1983; Hill and Way,
1995).
The results obtained in this study demonstrate that solevirus present a low virulence for
the salmonid species tested, since their mortality was very low (15%) or absent (Table 2).
However, the inoculated virus was detected directly by the recovery of infective viruses
from several pools of viscera from survivors at 33 days PI, or indirectly by the presence of
viral RNA (PCR test) and viral antigens (FC test). McAllister et al. (1984) have reported
that adult Atlantic salmon exposed to IPNV rarely suffer mortalities due to IPN and that
their antibody response is variable. Disease seemed to occur only under conditions of
virus exposure accompanied by additional stress, such as might be found when the ®sh
were undergoing a concurrent bacterial infection (Poppe et al., 1989; Bootland et al.,
1991; Smail et al., 1992). On the other hand, McAllister and McAllister (1988)
demonstrated that IPNV can be transmitted from striped bass to a salmonid and even with
low level of virus-positive mortality, and they concluded that the infected striped bass
pose a potential threat to salmonids via water route.
Experimental infections of gilt-head seabream provoked a high cumulative mortality
(66.7%). The presence of solevirus in this ®sh species was clearly established by
detection of viral RNA and antigens from the leucocytes of ®sh survivors at 45 and 60
days PI. However, at 120 days PI, all the samples tested were negative for virus detection
using PCR and FC tests, which suggests that virus±leucocyte association had ®nished in
this period or that the ®sh had been able to eliminate the virus.
The detection of IPNV from leucoytes and the establishment of an asymptomatic
infection have been previously demonstrated in salmonid ®sh by several authors
(Swanson and Gillespie, 1982; Yu et al., 1982; Ahne and Thomsen, 1986). More recently,
we reported the recovery of infective IPNV from peripheral leucocytes collected from
virus-carrier trouts, and that the percentage of viral antigen-expressed cells increased
from 30 to 90% in only 7 days (Rodriguez et al., 1991). Similar results were reported by
Johansen and Sommer (1995) working with kidney macrophages of Atlantic salmon.
In the present study, we have detected speci®c ¯uorescence for solevirus in a high
proportion of kidney leucocytes from ®sh survivors of the three ®sh species tested.
Previously, the isolation of IPNV from kidney has been described in several salmonid
®sh, but the fact that this organ could be the target for the viral replication has never been
demonstrated (Swanson and Gillespie, 1982). The kidney in salmonids and other ®sh
species possesses a blood ®ltration function, as well as being a hematopoietic organ.
In recent years, the isolation of aquatic birnaviruses from marine environment has
increased in zones with extensive ®sh and shell®sh culture. In addition, the detection of
these viruses from wild ®sh population and from shell®sh has been frequently reported
(Wolf, 1988; Castric, 1997). In Spain, Rivas et al. (1993) demonstrated that the wild
shell®sh and ®sh populations, and the marine sediment next to a ®sh farm acted as
reservoirs of birnaviruses and reoviruses in the marine ecosystem. In this sense, the
asymptomatic infection of ®sh may be very important for the dissemination of solevirus
to other ®sh species and to the environment.
In this paper, the higher susceptibility of gilt-head seabream to solevirus compared to
salmonid ®sh has been demonstrated. In addition, the PCR assay and the FC analysis have
24
S. Perez-Prieto et al. / Veterinary Microbiology 82 (2001) 11±25
been proved to be sensitive methods to detect solevirus, and may be considered as a
useful diagnostic tool in ®sh survey and monitoring studies.
Acknowledgements
This work was supported by grant AGF98-0837 and MAR95-1949-C02-01/02, both
from the Comision Interministerial de Ciencia y TecnologõÂa (CICYT) and a grant
CAM07B-9. We thank CUPIMAR S.A. and ConsejerõÂa de Agricultura y Medio Ambiente
de la Comunidad de Castilla La Mancha for supply of ®sh specimens, and M. SaÂnchez
Carmona and M. Jose Navarrete for their excellent technical assistance.
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