Download Interferon Production in Rainbow Trout (Salmo gairdneri

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J. gen. Virol. ( I 9 7 3 ) , x9, I 2 5 - I 2 7
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Interferon Production in Rainbow Trout (Salmo gairdneri)
Experimentally infected with Egtved Virus
(Accepted I3 December I972)
Egtved virus (Zwillenberg, Jensen & Zwillenberg, I965), a rhabdovirus, is the causative
agent of virus haemorrhagic septicemia of trout (V.H.S.), a disease which causes important
losses in European hatcheries. This disease occurs mainly when the water temperature is
between 6 and I2 °C, and spontaneously disappears when the temperature rises over 14 to
15 °C, in spite of the fact that the latter temperature is the thermal optimum for in vitro
virus growth (de Kinkelin & Scherrer, I97o).
Although the trout is able to produce circulating antibodies against a variety of antigens
at I5 °C (Post 1966, Hodgins, Weiser & Ridgway, 1967; Dorson I972), there is no evidence
of neutralizing antibodies in the serum of trout recovering from an epizootic of V.H.S., and
several injections of the virus have been necessary to obtain a low titre of neutralization
(Vestergfird-Jorgensen, 197 0. Amend (197o) succeeded in preventing infectious haematopoietic necrosis (I.H.N., another virus disease of trout caused by a rhabdovirus) by elevating
the water temperature. The rapidity of appearance and disappearance of the protective effect
led Amend to suggest the synthesis of an interferon. In vitro production of interferon by fish
cells has been reported by Gravell & Malsberger (I965), Beasley, Sigel & Clem (I966),
Oie & Lob (I97I) and also observed by P. de Kinkelin (unpublished observations), but such
production following a virus infection has never been demonstrated in vivo. The experiments
reported here provide for interferon synthesis in rainbow trout inoculated with egtved virus.
Twenty 8o g trouts were injected intraperitoneally with 5 x Io 6 p.f.u, of egtved virus,
then kept at 15 °C, and bled 3, 9 and ~4 days after infection. Interferon assays were made
as follows: the sera were first heated at 37 °C for 2 h. We had previously determined that
this treatment inactivated egtved virus without affecting interferon activity of the preparation: furthermore, virus thus inactivated did not induce interferon in the titration system
and could not therefore influence the results of the interferon titration. Rainbow trout
gonad (R.T.G. 2) cells (Wolf & Quimby I962 ) grown for 24 h in Petri dishes were treated for
I6 h by fish sera dilutions ranging from 1:50 to 1:2ooo0. Thereafter, the cells were washed
twice and challenged with lOO p.f.u, of infectious pancreatic necrosis (1.P.N.) virus (Wolf
et al. 196o), which belongs to the reovirus group (Moss & Gravell, 1969; Cohen & Scherrer,
~972). The plaque number reduction percentage was converted into interferon units according to Wagner (I 96 ~). R.T.G. 2 cell protecti on was also checked for two other salmonid viruses:
I.H.N. and V.H.S. viruses. Cell specificity of the protective effect was demonstrated in both
R.T.G.2 (salmonid cells) and Fathead Minnow line (cyprinid cells, Gravell & Malsberger,
1965), challenged with the same input of I.H.N. virus. Interferon characterization was performed with pooled sera collected on the third day after infection; details are given in
Table 1.
R.T.G.2 cells treatedwithserial dilutions of individual sera revealed a significant protective
effect, mostly with the third-day sera, while sera from non-injected fishes did not exhibit any
protective activity. The relationship between serum dilution logarithm and plaque number
reduction percentage was linear between 15 and 75 %, with a slope of o'or7 log unit[I
plaque number reduction.
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T a b l e i. Properties of the inhibitory factor from pooled sera of trouts
injected with V.H.S. virus (5 x 1oSp . f u . at day o) and bled at day 3
Broad antiviral activity
Cell specificity
Non-sedimentable, non-dialysable
Heat-stable
Stable at pH 2
Trypsin-labile
RNase-resistant
Similar protection of R.T.G.2 cells respectively challenged with
3 viruses: V.H.S. virus, I.H.N. virus and I.P.N. virus
I5 times better (as expressed in interferon units) for R.T.G.2 than
for F.H.M. cells, both challenged with I.H.N. virus
No loss of activity after sedimentation 0ooooog, 3 h), followed
by dialysis against phosphate buffer at pH 7"5 for 24 h
No loss of activity after heating (56 °C, 30 rain)
No loss of activity when held at pH 2.o for z4 h at 4 °C
(pH lowered by adding dilute HC1, then neutralized with NaOH
and dialysed against Earle's solution for 24 h)
Complete inactivation of one 11 interferon units/ml preparation
after enzymatic treatment. Crystallized trypsin A Grade (Calbiochem) o.2 mg/ml; 6 h at 37 °C, pH 8; followed by treatment
with soybean inhibitor (o'4 mg/ml)
No loss of activity after enzymatic treatment - RNase (Boehringer)
25/~g/ml, 3o rain at 37 °C of a 3 interferon units/ml preparation
T a b l e 2. Interferon titres in sera from five trout infected at day 0 by intraperitoneal
injection of 5 x Io6 p.fu. of V.H.S. virus, and bled at day 3, 9 and I4
Trout
number
,
Day 3
2
3
4
5
485
23oo
IOO
2750
38o
Interferon units
~
,
Day 9
Day I4
92
o
4"5
240
Dead
3
o
4"5
Dead
T h e i n d i v i d u a l interferon p r o d u c t i o n o f five t r o u t is given in T a b l e 2.
Since it was obvious t h a t the t h i r d - d a y bleedings p r o v i d e d the best interferon titres, the
s u b s e q u e n t c h a r a c t e r i z a t i o n experiments were p e r f o r m e d using p o o l e d sera f r o m these
samples. T h e experiments a n d results d e m o n s t r a t i n g the interferon n a t u r e o f the seric inh i b i t o r are r e p o r t e d in T a b l e I.
I t is evident f r o m the p r o p e r t i e s described in T a b l e I t h a t the t r o u t serum i n h i b i t o r
exhibits the m a i n characteristics o f a n interferon (Isaacs, I963). S u p p r e s s i o n o f the protective effect b y a c t i n o m y c i n D, a n i m p o r t a n t characteristic o f interferon, c o u l d n o t be checked
with I . P . N . virus because the d r u g p r o d u c e s a 9o ~ r e d u c t i o n o f I.P.N. virus g r o w t h in
R . T . G . 2 cells.
P r i o r to these experiments, the same interferon c h a r a c t e r i z a t i o n tests h a d been p e r f o r m e d
with the virus i n h i b i t o r synthetized b y F . H . M . cells following V.H.S. virus infection. T h e y
p r o v i d e d results similar to those i n d i c a t e d in T a b l e I a n d to those r e p o r t e d b y Oie & L o h
( I 9 7 I ) , w h o d e m o n s t r a t e d the i n h i b i t o r y fluid to be an interferon.
The results in T a b l e 2 suggest t h a t significant individual variations in interferon p r o d u c tion can be exhibited b y r a i n b o w trout. Possibly this is due to genetic heterogeneity o f t r o u t
p o p u l a t i o n s ; a difficulty which will be o v e r c o m e only when w o r k on genetic selection in
t r o u t proves m o r e successful t h a n t o date.
I n o r d e r to establish the p a r t p l a y e d b y interferon in t r o u t resistance to viral infection
further investigations are to be carried out. I t w o u l d be interesting to investigate the
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possibility that the apparent relationship between the water temperature and the appearance
of V.H.S. and I.H.N. diseases is a result of the temperature dependence of interferon
production by the trout.
From a practical view point, such investigations are justified by the wide spread of fish
culture activities and the heavy losses caused by viral diseases among fish populations.
We wish to thank Miss B6atrice Galimard, Miss Monique Le Berre and Miss Annie Barde
for excellent technical assistance and E. De Maeyer for valuable suggestions in our work
and helpful criticisms in the redaction of the manuscript.
Institut National de la Recherche Agronomique
Station de Recherches de Virologic et d'Immunologie
Laboratoire d' Ichtyopathologie
7885o Thiverval-Grignon, France
P.
DE K I N K E L I N
M . DORSON
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(Received 9 October 1972)
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