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HELGOL~NDER MEERESUNTERSUCHUNGEN
Helgol~inder Meeresunters. 37, 317-328 (1984)
Isolation of infectious pancreatic necrosis virus
(serotype Ab) from diverse species of estuarine fish
P. E. M c A l l i s t e r 1, M. W. N e w m a n 2, J. H. S a u b e r 3, W. J. O w e n s 1
1 U.S. Fish and Wildlife Service, National Fish Health Research Laboratory;
Box 700, Kearneysville, West Virginia 25430, USA
2 National Marine Fisheries Service, Northeast Fisheries Center;
Oxford, Maryland 21654, USA
3 North Carolina Department of Natural Resources, and Community Development,
Division of Natural Resources; Raleigh, North Carolina 27611, USA
ABSTRACT: Two significant fish kills occurred in the Pamlico River estuary (North Carolina, USA),
one in D e c e m b e r 1981 a n d J a n u a r y 1982, a n d the other in J u n e 1982. T h e first involved only the
southern flounder (Parallchthys lethostigma). Histopathologic e x a m i n a t i o n of morbid a n d m o r i b u n d
flounder revealed extensive s l o u g h i n g a n d necrosis of the mucosa of the pyloric caeca a n d intestine,
and inflammation of the submucosa of the pyloric caeca. Brain a n d internal o r g a n h o m o g e n a t e s
from morbid a n d m o r i b u n d flounder were assayed on CHSE-214 cells, a n d a virus was isolated.
Virus titers r a n g e d from _< 8.4 . 102 to 6,3 • 107 TCID50 per gram of tissue, Cross-plaque neutralization assays indicated that the southern flounder virus was infectious pancreatic necrosis virus
serotype Ab. Immersion c h a l l e n g e showed the isolate is only slightly virulent for fry of brook trout
(Satvelinus fontinalis]. The second fish kill involved the southern flounder a n d six other species:
hogchoker (Trinectes maculatus), Atlantic silverside (Menidia menidia), spot (Leiostomus xanthurus), Atlantic croaker (Micropogon undulatus), silver p e r c h (Bairdiella chrysura), a n d striped
mullet (Mugil cephalus). Virus was isolated from southern founder, hogchoker, Atlantic silverside,
a n d spot. Neutralization assays indicated that the four isolates were nearly identical; however, the
diversity of species affected suggests that the virus m i g h t not have b e e n the specific cause of
mortality.
INTRODUCTION
I n f e c t i o u s p a n c r e a t i c n e c r o s i s (IPN) is p r i n c i p a l l y k n o w n a s a v i r a l d i s e a s e t h a t
c a u s e s a c u t e d i s e a s e a n d h i g h m o r t a l i t y i n 1- to 4 - m o n t h - o l d trout• A l t h o u g h n o n s a l m o n i d s w e r e i n i t i a l l y r e g a r d e d a s r e f r a c t o r y to i n f e c t i o n b y I P N v i r u s , t h e v i r u s h a s s i n c e
b e e n i s o l a t e d f r o m m a n y f r e s h w a t e r s p e c i e s ( D o r s o n , 1983; M c A l l i s t e r , 1983). G e n e r a l l y
t h e s e i s o l a t i o n s h a v e b e e n m a d e i n t h e a b s e n c e of c l i n i c a l s i g n s of d i s e a s e (Hill, 1982).
H i l l (1976) a n d U n d e r w o o d e t al. 0 9 7 7 ) e x t e n d e d t h e h o s t r a n g e of I P N - l i k e v i r u s e s to
marine mollusks and crustaceans, and more recently the virus has been isolated from
m a r i n e fish. S t e p h e n s e t al. (1980) a n d M. N e w m a n ( p e t s . c o m m . ) i s o l a t e d I P N v i r u s f r o m
A t l a n t i c m e n h a d e n (Brevoortia tyrannus) a n d b l u e b a c k h e r r i n g (Alosa aestivMis). H i l l
(1982) i s o l a t e d t h e v i r u s f r o m y o u n g h a t c h e r y - r e a r e d s e a b a s s (Dicentrarchus labrax)and
f r o m a b n o r m a l e g g s of t u r b o t (Scophthalmus maximus) a n d D o v e r s o l e (Solea solea).
© Biologische Anstalt Helgoland, H a m b u r g
]
318
P . E . McAllister et al.
We report here the isolation of IPN virus from four species of m a r i n e fish involved in
two fish kills i n the Pamlico River e s t u a r y i n North C a r o l i n a (USA). The first kill, in
D e c e m b e r 1981 a n d J a n u a r y 1982, i n v o l v e d almost exclusively southern flounder (Paralichthys lethostigma); the second, i n J u n e 1982, i n v o l v e d at least s e v e n species: southern
flounder, h o g c h o k e r (Trinectes maculatus), A t l a n t i c silverside (Menidia menidia), spot
(Leiostomus xanthurus), Atlantic croaker (Micropogon undulatus), silver perch (BairdieHa chrysura), a n d striped m u l l e t (Mugil cephalus). Virus was isolated from southern
flounder, hogchoker, spot, a n d Atlantic silverside.
MATERIALS AND M E T H O D S
Histology
Tissues from five m o r i b u n d s o u t h e r n f l o u n d e r collected d u r i n g the first fish kill were
sent to the Oxford (Maryland) Laboratory, N a t i o n a l M a r i n e Fisheries Service, for histological e x a m i n a t i o n . The s p e c i m e n s consisted of the formalin-fixed visceral mass from
each fish, a n d the rest of each carcass was p a c k e d o n ice. O n arrival at the laboratory,
k i d n e y a n d gill tissues were excised from carcasses. K i d n e y imprints were p r e p a r e d a n d
e x a m i n e d for the p r e s e n c e of Trypanoplasma, a n d the r e m a i n i n g k i d n e y a l o n g with the
gills were fixed in 10 % seawater-formalin. The fixed tissues were d e h y d r a t e d in
ethanol, cleared i n xylene, a n d e m b e d d e d i n Paraplast. The e m b e d d e d tissues were
sectioned at 6 ~m a n d s t a i n e d with h e m a t o x y l i n - e o s i n .
Cells, media, and viruses
M o n o l a y e r cell cultures of chinook s a l m o n e m b r y o (CHSE-214), r a i n b o w trout
g o n a d (RTG-2), b l u e g i l l fry (BF-2), b r o w n b u l l h e a d (BB), a n d fathead m i n n o w (FHM)
were m a i n t a i n e d u s i n g E a g l e ' s m i n i m u m e s s e n t i a l m e d i u m s u p p l e m e n t e d with 10 %
fetal b o v i n e s e r u m (MEM-10). The CHSE-214 a n d RTG-2 cells were p r o p a g a t e d at 18 °C
a n d the BF-2, BB, a n d F H M cells at 25 °C.
P l a q u e c l o n e d stocks of IPN virus serotypes Ab, Sp, a n d VR-299 a n d isolates from
s o u t h e r n flounder, hogchoker, spot, a n d Atlantic silverside were p r e p a r e d i n CHSE-214
cells infected at a m u l t i p l i c i t y of infection of 0.01-0.001 PFU/cell. The stocks were stored
at - 7 0 ° C , a n d infectivity was d e t e r m i n e d b y p l a q u e assay as described by Wolf &
Q u i m b y (1973).
Electron microscopy
Ultrathin sections of CHSE-214 cells infected with the s o u t h e r n flounder virus
isolate were p r e p a r e d for electron microscopic e x a m i n a t i o n . M o n o l a y e r cultures were
infected as p r e v i o u s l y described. After 12 h i n c u b a t i o n at 18°C, cells were scraped from
the flask, fixed in 2 % g l u t a r a l d e h y d e (pH 7.2), processed, a n d e m b e d d e d in Spurt lowviscosity epoxy resin (Polysciences, Inc., W a r r i n g t o n , P e n n s y l v a n i a ) , sectioned, a n d
s t a i n e d with OsO 4.
For n e g a t i v e l y s t a i n e d preparations, i n f e c t e d CHSE-214 cells were i n c u b a t e d u n t i l
CPE was e v i d e n t in over 90 % of the m o n o l a y e r (about 72 h post-infection). The culture
m e d i u m was d e c a n t e d a n d c e n t r i f u g e d for 15 m i n at 2000 × g t o pellet cell debris, a n d
Infectious p a n c r e a t i c necrosis
319
the s u p e r n a t a n t c e n t r i f u g e d for 60 m i n at 100 000 x g. The r e s u l t a n t virus c o n c e n t r a t e
was mixed with 4 % p h o s p h o t u n g s t i c acid (pH 6.0), a p p l i e d to a c a r b o n - c o a t e d grid, a n d
e x a m i n e d with a Zeiss EM-5 electron microscope.
Virus purification and antiserum production
Virus was c o n c e n t r a t e d b y p o l y e t h y l e n e glycol p r e c i p i t a t i o n ( M a c d o n a l d &
Yamamoto, 1977) a n d purified on l i n e a r 10-50 % sucrose g r a d i e n t s b y c e n t r i f u g a t i o n at
97 000 x g for 45 m i n at 4 °C. The virus b a n d was r e m o v e d b y side p u n c t u r e , d i a l y z e d
overnight against TNE buffer (0.2 M tris, 0.15 M NaC1, 0.001 M Na2EDTA; p H 7.2), a n d
frozen at - 7 0 °C.
New Z e a l a n d white rabbits were i n j e c t e d i n t r a v e n o u s l y with purified virus a n d both
i n t r a m u s c u l a r l y a n d s u b c u t a n e o u s l y with a n e m u l s i o n of p u r i f i e d virus a n d F r e u n d ' s
incomplete a d j u v a n t (1:1). The i n t r a m u s c u l a r a n d s u b c u t a n e o u s i n j e c t i o n s were repeated 2 w e e k s after p r i m a r y i n o c u l a t i o n . Rabbits w e r e b l e d 10 to 14 days after the last
injection. Sera were collected, d e c o n t a m i n a t e d b y m e m b r a n e filtration, i n a c t i v a t e d at
56°C for 30 rain, a n d stored at --70°C.
Virus neutralization assays
Cross-plaque n e u t r a l i z a t i o n assays were performed b e t w e e n IPN virus serotypes
Ab, Sp, a n d VR-299 a n d the s o u t h e r n f l o u n d e r isolate. We also performed p l a q u e
n e u t r a l i z a t i o n assays u s i n g a n t i s e r a to the s o u t h e r n f l o u n d e r isolate a n d viruses isolated
from hogchoker, spot, a n d Atlantic silverside. Dilutions of a n t i s e r u m a n d virus were
made in tris buffered M E M (pH 7.8) s u p p l e m e n t e d with 2 % fetal b o v i n e s e r u m (MEM-2
tris). Virus s a m p l e s were d i l u t e d to give 80 PFU p e r 3 5 - m m assay dish. E q u a l v o l u m e s
of a n t i s e r u m a n d virus were c o m b i n e d , a l l o w e d to react for 1 h at 18°C with m i x i n g at
15-min intervals, a n d assayed for r e s i d u a l infectivity b y p l a q u e assay. The s e r u m
dilution g i v i n g 50 % p l a q u e r e d u c t i o n was c a l c u l a t e d (K~irber, 1931), a n d the serological
relationships b e t w e e n virus isolates were d e t e r m i n e d by u s i n g the titer-ratio m e t h o d of
Archetti & Horsfall (1959} as d e s c r i b e d b y O k a m o t o et al. (1983).
Virus isolation
Morbid a n d m o r i b u n d s p e c i m e n s were r e c e i v e d frozen or on ice. S a m p l e s of liver,
kidney, spleen, caecum, a n d b r a i n were r e m o v e d a n d assayed i n d i v i d u a l l y or pooled.
For small fish the entire visceral mass was r e m o v e d a n d assayed. S p e c i m e n s were
homogenized, d i l u t e d i n 0.1 M p h o s p h a t e buffered s a l i n e (pH 7.2) c o n t a i n i n g 100 IU/ml
p e n i c i l l i n a n d 100 ~g/ml streptomycin, a n d a s s a y e d on CHSE-214 cells i n 96-well
microculture plates (McDaniel, 1979). Plates w e r e i n c u b a t e d at 18 °C a n d e x a m i n e d for
10 days for cytopathic effects (CPE).
Virus replication - cell line specificity
Drained monolayers of CHSE-214, RTG-2, BF-2, BB, a n d F I t M cells were i n o c u l a t e d
with the southern flounder isolate to d e t e r m i n e the cell culture specificity. Stock virus
320
P . E . M c A l l i s t e r et al.
w a s d i l u t e d 10 -1 to 10 -9 in M E M - 2 tris a n d 0.05 m l of e a c h d i l u t i o n i n o c u l a t e d into each
of e i g h t w e l l s on a 9 6 - w e l l m i c r o c u l t u r e plate; M E M - 2 tris w a s u s e d as control inoculum.
After 1 h a d s o r p t i o n at 18°C, 0.1 ml of M E M - 1 0 w a s a d d e d to e a c h w e l l a n d i n c u b a t i o n
c o n t i n u e d at 18°C. C u l t u r e s w e r e e x a m i n e d d a i l y for CPE. W h e n no further CPE
d e v e l o p e d , or 10 days h a d e l a p s e d , t h e cell c u l t u r e fluid w a s h a r v e s t e d from the h i g h e s t
d i l u t i o n of virus i n o c u l u m s h o w i n g CPE in all e i g h t w e l l s a n d titrated on CHSE-214 cells
a n d on t h e o r i g i n a l host cells.
Virulence assay in salmonid fish
To assay the s o u t h e r n f l o u n d e r virus isolate for v i r u l e n c e in s a l m o n i d fish, w e used
t h e c h a l l e n g e protocol d e s c r i b e d by M c A l l i s t e r et al. (1983). S i x t y - d a y - o l d (T° = 12.5 °C)
b r o o k trout (Salvelinus fontinalis] w e r e p l a c e d in 1.5-1 a q u a r i a at a stocking density of
1 g fish p e r 25 ml water. A q u a r i a w e r e s u p p l i e d w i t h 12.5 °C s p r i n g water. Fish w e r e
e x p o s e d for 5 h to 105 P F U / m l w a t e r of IPN virus (VR-299 serotype) or the southern
f l o u n d e r isolate. C o n t r o l fish w e r e e x p o s e d to PBS. M o r t a l i t y w a s m o n i t o r e d for 21 days.
RESULTS
Histopathology
Superficially, t h e f l o u n d e r e x a m i n e d a p p e a r e d normal. No e v i d e n c e of Trypanoi n f e c t i o n w a s found. Sections of t h e l i v e r i n d i c a t e d that the p a r e n c h y m a l cells
c o n t a i n e d l i p i d or g l y c o g e n i n d i c a t i n g that t h e fish h a d b e e n f e e d i n g well. T h e h e a r t and
k i d n e y a p p e a r e d normal. E x t e n s i v e s l o u g h i n g of t h e m u c o s a was e v i d e n t in the intestine, b u t s o m e of this w a s p r o b a b l y a t t r i b u t a b l e to p o s t - m o r t e m c h a n g e . T h e d u o d e n u m
a n d caeca, h o w e v e r , s h o w e d necrosis as w e l l as s l o u g h i n g of the m u c o s a (Fig. 1) a n d a
diffuse l y m p h o c y t i c infiltrate into t h e s u b m u c o s a (Fig. 2). B e c a u s e of t h e s e lesions, a
d i a g n o s i s of viral e n t e r i t i s w a s c o n s i d e r e d .
plasma
Virus isolation
When brain and internal organ homogenates from morbid and moribund southern
flounder collected in December 1981 were assayed on CHSE-214 cells at 18°C, virus was
isolated from 8 of 15 fish. Virus titer ranged from --<8.4 x 102 to 6.3 × I0~TCIDs0/g of
tissue (Table I). In the fish kill, in June 1982, IPN virus was recovered from visceral
homogenates of four species: southern flounder,hogchoker, spot, and Atlanticsilverside
(Table I). Preliminary characterization of virus isolates by standard procedures indicated that they were ether-stable but sensitive to heating at 56 °C for 60 rain (99.94 %
inactivation; data not shown).
Electron microscopy
Electron micrographs of thin sections of CHSE-214 cells infected with the southern
flounder isolate showed cytoplasmicaccumulations of singly encapsidated icosahedral
virus particles. Individual or small aggregates of virus were randomly distributed
throughout the cytoplasm; there was no evidence of large crystallinearrays, and no virus
Infectious pancreatic necrosis
321
Fig. 1, Section through pyloric caeca of a m o r i b u n d southern flounder showing sloughing of
mucosal epithelium a n d inflamed submucosa (SM)
w a s e v i d e n t i n t h e n u c l e u s , N e g a t i v e l y s t a i n e d p r e p a r a t i o n s of c o n c e n t r a t e d v i r u s
s h o w e d t h e u n e n v e l o p e d i c o s a h e d r a to b e a b o u t 57 n m i n d i a m e t e r (Fig. 3). T h e d i a m e t e r of i n t r a c e l l u l a r a n d r e l e a s e d v i r u s w a s t h e s a m e .
322
P.E.
McAllister
et al.
T a b l e 1. Q u a n t i t a t i o n of v i r u s i s o l a t e d from v a r i o u s t i s s u e s of m a r i n e f i s h e s c o l l e c t e d in P a m l i c o
River
Species and tissue
Southern flounder
P o o l e d liver, k i d n e y s , s p l e e n , a n d c a e c u m
Kidneys
Spleen
Caecum
Brain
Hogchoker
Viscera
Spot
Viscera
Atlantic silverside
Viscera
V i r u s t i t e r (range) *
6.3 X 103- 6.3 x 107
1.1 × 104---~6.3 × 104
~
-_~6.3 × 104
_~2.7 × 1 0 3 - _~ 6.3 × 104
~
---~ 8.4 × 102
--~6.3 × 103
--~6.3 × 103
_~ 6.3 × 103
* V i r u s t i t e r e x p r e s s e d as TCIDs0/g of t i s s u e
No v i r a l i n f e c t i v i t y d e t e c t e d
Fig. 2. S e c t i o n t h r o u g h p y l o r i c c a e c a of a m o r i b u n d s o u t h e r n f l o u n d e r s h o w i n g e x t e n s i v e l y m p h o c y t i c i n f i l t r a t i o n in t h e s u b m u c o s a (SM) a n d n o r m a l a p p e a r a n c e of e x o c r i n e p a n c r e a s (EP)
Infectious p a n c r e a t i c necrosis
323
Fig. 3. Electron micrograph of negatively stained virus isolated from morbid southern flounder. Bar
represents 100 nm
Cross-neutralization
assays
Preliminary n e u t r a l i z a t i o n assays s h o w e d the s o u t h e r n f l o u n d e r isolate was slightly
n e u t r a l i z e d (Log NI ---- 0.8) by a p o l y v a l e n t a n t i s e r u m to s e v e n North A m e r i c a n s a l m o n i d
IPN virus isolates, s u g g e s t i n g a p o s s i b l e s e r o l o g i c a l r e l a t i o n s h i p to IPN virus. Crossp l a q u e assays i n d i c a t e d a h i g h d e g r e e of cross-reactivity b e t w e e n the s o u t h e r n f l o u n d e r
isolate and the s a l m o n i d IPN virus serotypes Ab, Sp, a n d VR-299 (Table 2). S e r o l o g i c a l
relationships w e r e d e f i n e d by titer-ratio analysis of the c r o s s - n e u t r a l i z a t i o n data. The
southern flounder isolate was found to be closely r e l a t e d to both serotypes Ab and Sp
(Table 3).
P l a q u e - n e u t r a l i z a t i o n assays of isolates from the h o g c h o k e r , spot, and A t l a n t i c
silverside s h o w e d t h e m to b e closely related, if not identical, to that of the southern
flounder (data not shown).
324
P . E . M c A l l i s t e r e t al.
T a b l e 2. Cross-plaque neutralization assays of IPN virus (serotypes Ab, Sp, a n d VR-299) a n d the
s o u t h e r n flounder virus isolate
Virus
Ab
IPN virus serotype
Ab
Sp
VR-299
Southern flounder isolate
4.82
3.12
2.39
4.06
Antiserum *
IPN virus serotype
Sp
VR-229
4.22
5.26
4.61
4.36
3,92
4.21
6.39
4.07
Southern flounder
isolate
4.74
4.66
3.80
5.72
* A n t i s e r u m titer is expressed as the reciprocal of the logarithm of the antiserum dilution giving
50 % p l a q u e reduction
T a b l e 3. Serological relationship (l/r) b e t w e e n IPN virus (serotypes Ab, Sp, a n d VR-299) and the
southern flounder virus isolate
Virus
Antiserum"
IPN virus serotype
IPN virus serotype
Ab
Sp
VR-299
Southern flounder isolate
Southern flounder
Ab
Sp
VR-229
1.0
23.5
1.0
282.8
26.0
1,0
isolate
7.4
9.6
132.5
1.0
• The serological relationship (l/r) b e t w e e n virus isolates was d e t e r m i n e d from analysis of crossp l a q u e neutralization assays by the titer-ratios m e t h o d of Archetti & Horsfall (1950) as described
by Okamoto et al. (1983)
Cell line specificity
T h e s o u t h e r n f l o u n d e r I P N v i r u s w a s o r i g i n a l l y i s o l a t e d b y u s i n g C H S E - 2 1 4 cells.
F o u r o t h e r c e l l l i n e s R T G - 2 , BF-2, BB, a n d F H M w e r e a l s o e v a l u a t e d for v i r u s s u s c e p t i b i l i t y ( T a b l e 4). T h e v i r u s w a s p a s s a g e d t w i c e i n e a c h c e l l l i n e . A s j u d g e d b y v i r u s y i e l d
(2.7 × 109 T C I D s 0 / m l ) a n d t i m e for m a n i f e s t C P E ( 4 8 - 7 2 h), C H S E - 2 1 4 c e l l s w e r e t h e
m o s t s u s c e p t i b l e c e l l l i n e . A n i n c u b a t i o n t i m e of 1 2 0 - 1 4 4 h w a s r e q u i r e d t o m a n i f e s t
C P E i n R T G - 2 a n d B F - 2 c e l l s a n d t h e v i r u s y i e l d s w e r e l o w e r - 6.3 × 106 a n d 1.5 × 10 ~
TCIDso/ml, r e s p e c t i v e l y . N o C P E d e v e l o p e d i n F H M a n d BB c e l l s a f t e r 10 d a y s i n c u b a t i o n at 18°C.
H i g h e r v i r u s t i t e r s w e r e r e c o r d e d for v i r u s h a r v e s t e d f r o m R T G - 2 a n d BF-2 c e l l s w h e n
a s s a y e d o n C H S E - 2 1 4 cells, a n d a s s a y s of h a r v e s t s f r o m F H M a n d BB c e l l s s h o w e d c o m p l e t e
r e c o v e r y of i n p u t v i r u s . T h i s o b s e r v a t i o n f u r t h e r d e m o n s t r a t e d t h a t t h e C H S E - 2 1 4 c e l l l i n e
w a s t h e m o s t s u s c e p t i b l e of t h o s e t e s t e d .
Infectious p a n c r e a t i c necrosis
325
Table 4. Comparative response of five fish cell lines to infection with the southern flounder IPN
virus isolate
Cell line
Cytopathic effect"
Virus yield (TCIDs0/mt)
CHSE-214
RTG-2
BF-2
FHM
+
+
+
--
2.7 × 109
6.3 x 1 0 6
1.5 × l0 T
BB
- -
" Cytopathic effect was characterized by rounding of the cell, pyknosis, lysis, and detachment
from the flask
No viral activity detected when virus assayed in test cell line
S u s c e p t i b i l i t y of b r o o k t r o u t t o I P N v i r u s i s o l a t e d f r o m s o u t h e r n f l o u n d e r
Specific p a t h o g e n free b r o o k trout o b t a i n e d from the W h i t e S u l p h u r S p r i n g s (West
Virginia) N a t i o n a l Fish H a t c h e r y w e r e c h a l l e n g e d by i m m e r s i o n w i t h t h e s o u t h e r n
flounder IPN virus isolate a n d w i t h IPN v i r u s (serotype VR-299) r e c o v e r e d from a n a t u r a l
epizootic in b r o o k trout. Both isolates w e r e in t h e s e c o n d p a s s a g e after p r i m a r y isolation.
Fish w e r e 60 days old at c h a l l e n g e a n d w e r e h e l d in s p r i n g w a t e r at 12.5 °C. E x p o s u r e for 5 h
to l 0 s PFU of v i r u s / m l of w a t e r c a u s e d 89 % mortality in fish c h a l l e n g e d w i t h s e r o t y p e VR299 but only 5 % m o r t a l i t y in t h o s e e x p o s e d to t h e s o u t h e r n f l o u n d e r i s o l a t e (Table 5). No
control fish died. All d e a t h s o c c u r r e d 6 to 14 days after c h a l l e n g e ; m o r t a l i t y w a s m o n i t o r e d
for 21 days.
Table 5. Susceptibility of brook trout to the southern flounder IPN virus isolate and to IPN virus
{serotype VR-299)
Vires*
Fish (died/total)
Mortality {%}
33/37
89
IPN virus (serotype VR-299}
Southern flounder isolate
Control
2/41
5
0/40
0
* Fish were challenged by immersion for 5 h in water containing 105 PFU/ml of virus. Control
fish were exposed to PBS. Mortality was monitored for 21 days
DISCUSSION
W h e n m o r b i d a n d m o r i b u n d s o u t h e r n flounder, c o l l e c t e d from the P a m l i c o River
estuary in D e c e m b e r 1982, w e r e e x a m i n e d for b a c t e r i a l i n f e c t i o n a n d parasites, a n d for
toxic r e s i d u e s from industrial a n d a g r i c u l t u r a l c h e m i c a l s , n o n e of t h e s e factors c o u l d b e
a s s i g n e d a causal role in the fish kill. W a t e r c h e m i s t r y studies a n d e x a m i n a t i o n of
nekton, phytoplankton, a n d b e n t h i c m i c r o i n v e r t e b r a t e s i n d i c a t e d no a b n o r m a l i t i e s
(Helms, 1981). H o w e v e r , w h e n h o m o g e n a t e s of b r a i n a n d i n t e r n a l o r g a n s from f l o u n d e r
326
P . E . M c A l l i s t e r et al.
w e r e a s s a y e d on CHSE-214 cells, a virus w a s i s o l a t e d from 8 of 15 fish. M a n y fish that
w e r e n e g a t i v e for virus w e r e o n l y partly intact a n d h a d b e e n s c a v e n g e d . H i g h e s t
c o n c e n t r a t i o n s of virus (> l 0 T TCIDs0/g tissue) w e r e r e c o v e r e d from visceral organs, and
virus w a s also r e c o v e r e d from b r a i n tissue (< 103 TCIDso/g tissue). H i s t o l o g i c a l e x a m i n a tion r e v e a l e d e x t e n s i v e s l o u g h i n g a n d necrosis of the m u c o s a of the pyloric c a e c a and
i n t e s t i n e a n d diffuse l y m p h o c y t i c infiltration in the s u b m u c o s a of the pyloric caeca.
K i d n e y tissue a p p e a r e d normal. Viral e n t e r i t i s w a s c o n s i d e r e d as a c o n t r i b u t i n g cause of
the f l o u n d e r mortality.
T h e s o u t h e r n f l o u n d e r virus isolate s h o w e d characteristics similar to those of IPN
virus from s a l m o n i d fish. T h e virus w a s e t h e r - s t a b l e , i n a c t i v a t e d by h e a t i n g at 56 °C for
1 h, a n d h a d a n o n e n v e l o p e d , singly e n c a p s i d a t e d , i c o s a h e d r a l m o r p h o l o g y and a
d i a m e t e r of 57 nm. A n a l y s i s of c r o s s - p l a q u e n e u t r a l i z a t i o n assays by the titer-ratio
m e t h o d e s t a b l i s h e d that t h e s o u t h e r n f l o u n d e r i s o l a t e w a s closely r e l a t e d to IPN virus
s e r o t y p e s Ab a n d Sp. T h e 1/r v a l u e d e l i n e a t e d b y t h e titer-ratio analysis i n d i c a t e s the
e x t e n t of a n t i g e n i c d i f f e r e n c e or, conversely, r e l a t e d n e s s b e t w e e n two viruses w h e n both
v i r u s e s a n d t h e i r h o m o l o g o u s antisera are u s e d in c r o s s - n e u t r a l i z a t i o n assays. T h e
s m a l l e r the I/r, t h e g r e a t e r the a n t i g e n i c r e l a t e d n e s s . If 1/r ---- 1, the viruses are serologically i d e n t i c a l ; v a l u e s of 1/r > 20 i n d i c a t e distinct serotypes. C o m p a r i s o n of 1/r values
i n d i c a t e d that t h e s o u t h e r n f l o u n d e r isolate w a s m o r e closely r e l a t e d to serotype Ab (1/
r = 7.4) t h a n to s e r o t y p e Sp (1/r = 9.6), a n d w a s s e r o l o g i c a l l y distinct from serotype VR299 (1/r ---- 132.5). In cell culture susceptibility studies the southern f l o u n d e r isolate
s h o w e d a c h a r a c t e r i s t i c of s e r o t y p e Ab in not r e p l i c a t i n g in F H M cells. T h e most
s e n s i t i v e cell line for virus d e t e c t i o n was CHSE-214; the virus r e p l i c a t e d in RTG-2 and
BF-2 cells, b u t not in BB cells.
W h e n 6 0 - d a y - o l d b r o o k trout, u s e d to assay for v i r u l e n c e to s a l m o n i d fish, w e r e
c h a l l e n g e d by i m m e r s i o n , only 5 % of the fish e x p o s e d to the s o u t h e r n f l o u n d e r isolate
died, c o m p a r e d with 89 % of those e x p o s e d to serotype VR-299. T h e southern flounder
i s o l a t e w a s c l e a r l y avirulent. In s a l m o n i d fish, IPN virus isolates in serotype A b are n o t e d
for a v i r u l e n c e , w h e r e a s those in serotype Sp are virulent. S e e m i n g l y , such consistency
m a y not o c c u r w i t h IPN virus isolates from m a r i n e fish. Hill (1982), w h o isolated IPN
virus (serotype Sp) from sea bass, found t h e isolate to b e infectious but a v i r u l e n t for
r a i n b o w trout fry. T h e s o u t h e m f l o u n d e r isolate w a s i n d e e d infectious for salmonids, and
the b r o o k trout e x p o s e d to the virus b e c a m e carriers. W h e n the virus w a s r e c o v e r e d
1 y e a r after e x p o s u r e a n d u s e d to c h a l l e n g e 4 2 - d a y - o l d b r o o k trout, less t h a n 5 % of the
fish d i e d - i n d i c a t i n g that the isolate r e t a i n e d its a v i r u l e n c e .
W e also p e r f o r m e d v i r u l e n c e assays w i t h p o s t - m e t a m o r p h o s e s u m m e r flounder
(Paralichthys dentatus} 3 - 5 cm long, and 1- to 2 - y e a r - o l d s o u t h e r n flounder. Fish w e r e
i n o c u l a t e d by i n t r a p e r i t o n e a l injection. Unfortunately, results w e r e i n c o n c l u s i v e and the
s o u t h e r n f l o u n d e r w e r e f o u n d to be virus carriers. Virus w a s r e c o v e r e d from e a c h of the
e i g h t control fish e x a m i n e d . Sera c o l l e c t e d from s o u t h e r n f l o u n d e r n e u t r a l i z e d the virus
isolate.
In fish from the s e c o n d kill, in J u n e 1982, virus w a s i s o l a t e d from h o m o g e n a t e s of
v i s c e r a l o r g a n s of s o u t h e r n flounder, h o g c h o k e r , spot, a n d A t l a n t i c silverside. P l a q u e
n e u t r a l i z a t i o n assays s h o w e d that virus isolates w e r e n e u t r a l i z e d strongly by antisera
to s o u t h e r n flounder. IPN isolate a n d o n l y w e a k l y by a n t i s e r a to IPN virus (serotype
VR-299). A l t h o u g h c r o s s - n e u t r a l i z a t i o n assays are n e e d e d for confirmation, isolates from
Infectious pancreatic necrosis
327
h o g c h o k e r , spot, a n d A t l a n t i c s i l v e r s i d e a p p e a r e d to b e v e r y c l o s e l y r e l a t e d , if n o t
i d e n t i c a l , to s o u t h e r n f l o u n d e r I P N v i r u s , T h e d i v e r s i t y of s p e c i e s a f f e c t e d i n t h e s e c o n d
f i s h k i l l m i g h t i n d i c a t e t h a t v i r u s or v i r u s e s , a l t h o u g h r e c o v e r e d a t s i g n i f i c a n t t i t e r s ,
might be merely adventitious agents.
Infectious pancreatic necrosis and IPN-like viruses have been isolated from an
i n c r e a s i n g d i v e r s i t y of f i s h a n d i n v e r t e b r a t e s p e c i e s , a n d a d d i t i o n a l i s o l a t i o n s w i l l
undoubtedly be made. Defining serological relationships and virulence characteristics
of i s o l a t e s is c o m p l e x , f o r m i d a b l e , a n d a c o n t i n u i n g t a s k . T r a d i t i o n a l l y , b y v i r t u e of
antigenic character and geographical distribution, the salmonid IPN isolates are
categorized into three basic serotypes: the European Ab and Sp and the North American
VR-299. T h i s r u d i m e n t a r y c l a s s i f i c a t i o n c o n t i n u e s as t h e b a s i c f r a m e w o r k a g a i n s t w h i c h
n e w i s o l a t e s a r e m e a s u r e d - t h e p r e s e n t s t u d y i n c l u d e d . T h u s , t h i s r e p o r t of t h e first
i s o l a t i o n i n N o r t h A m e r i c a of a n a t u r a l l y o c c u r r i n g I P N v i r u s h a v i n g a n t i g e n i c c h a r a c t e r i s t i c s of I P N v i r u s e s f o u n d i n E u r o p e is of p a r t i c u l a r s i g n i f i c a n c e . I n d e e d , t h e I P N
i s o l a t e f r o m t h e s o u t h e r n f l o u n d e r is r e l a t e d to b o t h s e r o t y p e s A b a n d Sp. A s s i g n m e n t to
s e r o t y p e A b w a s b a s e d o n c e l l c u l t u r e s p e c i f i c i t y a n d r e l a t i v e a v i r u l e n c e t o a s a l m o n i d . It
must yet be determined whether this serologic character represents an intermediate
a n t i g e n i c s t r u c t u r e w i t h b i v a l e n t r e a c t i o n s i t e s or m u l t i p l e d i s c r e t e a n t i g e n s . S u c h
d e t e r m i n a t i o n m a y offer i n s i g h t n o t o n l y i n t o t h e e v o l u t i o n a r y d e v e l o p m e n t of I P N v i r u s
b u t a l s o i n t o a l t e r n a t i v e r e a g e n t s for v i r a l d i a g n o s t i c s a n d d i s e a s e c o n t r o l .
Acknowledgements. Antisera to IPN virus serotypes Ab a n d Sp a n d homologous virus were
provided by B. J. Hill, Ministry of Agriculture, Fisheries, a n d Food, Fish Disease Laboratory, the
Nothe, Weymouth, England. C, A. Farley p r e p a r e d n e g a t i v e l y - s t a i n e d specimens for electron
microscopy. We t h a n k P. H. Eschmeyer, B. C, Lidgerding, C. L Schultz, R. C. Simon, a n d K, E. Wolf
for reviewing the manuscript, B.D. Lawson for editorial a n d secretarial assistance, a n d H. M.
Stuckey for the photography.
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