Archives of Virology 4L8, 181--185 (1975) © by Springer-Verlag 1975

A Rhabdovirus Isolated from Grass Carp

(Ctenophargngodon idella Val.) Brief Report By W. A ~ E Institute of Zoology and I-IydrobiologTg, Ludwig Maximilian's University of Munich, Federal Republic of Germany With 1 Figure Accepted February 27, 1975 During recent years the production of East-Asiatic cyprinids especially the grass carp (Ctenopharyngodon idella Val.) has steadily increased in Europe. The grass carp is mainly kept together with the common carp (Cyprinus carpio L.). Carp fish farmers are faced with two serious infectious diseases: the infectious dropsy (IDC) and the swim bladder inflammation (S]3I). Both diseases have been diagnosed in fish species other than carp (2, 13). An outbreak of a disease among grass carp similar to IDC has been observed and the authors pointed out that although the symptoms clearly indicated IDC, common carps living in the same pond were not infected (12). Since l~habdovirus earpio (RVC) causes spring viremia of carp (SVC) (5) and a similar if n.ot identical virus is associated with 8131 (1, 3, 4), the importance of infectious virus diseases in carp hatcheries is emphasized. In April 197zi a moribund three year old grass carp with ventral hemorrhagic inflammation, bleedings in the scale bases, necrotic fins, inflammation of the stomach, serous liquid in the abdomen, swoilen spleen, pale liver with swollen capillary vessels, opaque internM wall of the swim bladder with petechial hemorrhages and widened blood vessels was sent to this laboratory. Tissue samples from kidney, liver, spleen and intestine were collected aseptically, homogenized, diluted 1 : 20 in P]3S and centrifuged at 2000 g for 20 minutes (4 ° C). The supernatant was then filtered through a 450 nm membrane filter and the filtrate inoculated in amounts of 0,5 ml onto FgM-(fathead minnow)-eell cultures (CCL-42) grown in Leighton tubes using Eagle's MEM (EMEM) supplemented with 5 per cent fetal calf serum (FCS) and penicillin 100 IU/ml, streptomycin 100 ~g/ml and nistatin 50 IU/ml. RTG-(rainbow trout)-2-cells (CCL-55) grown at 20°C in EMEM, B]3-cells (CCL-59) grown at 30°C in EMEM and RF-(rainbow trout gonad)-28-cells (initiated 1971 by the author from trypsinized ovaries of Salmo gairdneri and continuously subcultivated to the 52th subculture) grown at. 20°C in TC199,

182

W. AHNE :

were also used for virus p r o p a g a t i o n . The m e d i a c o n t a i n e d 10 per cent FCS a n d antibiotics. The infected cells were i n c u b a t e d a t 1 7 ° C (4-1) a n d checked d a i l y for cytop a t h i c effects (CPE). The first isolation of a eytopaghic a g e n t was m a d e in F H M cells. Cells showed g r a n u l a t i o n a n d r o u n d i n g up a b o u t 2 0 - - 3 0 hours p o s t infection (p.i.). A f t e r staining w i t h h e m a t o x y l i n - e o s i n m a r g i n a t i o n of chromagin was o b s e r v e d in infected cells. Similar C P E was also d e t e c t e d in all o t h e r cell cultures used. Results of the i n f e c t i v i t y tigers (TCIDa0/0.1 ml) c a l c u l a t e d according to t h e m e t h o d of KXRBSR (6) are shown in Table 1. Table 1. Replication o/ Isolate /rom Grass Carp in Di//erent JFish Cell Cultures at 23 ° C ( ~=1). Samples Were Collected Three Days p.i., Frozen at - - 7 4 ° C and a/ter Thawing they Were Titrated in F H M Cells

Cell cultures

Infectivity three days p.i. (loglo TCIDso/0. t ml)

BB-cells FHM-cells RTG-2-cells R F - 28 -cells

5.75 7.20 6.25 6.25

TCID50/0,1 m[

107. /"

106

105

\ ' \

lO4

\.

- ....

4°C 16 (-+I):C

- -

23 ( + - I ) ° C

....

-......

\-%

28°C 37

°C

\.

\ \. \ 6 Fig.

t. R e p l i c a t i o n

6

12

"~4

3'6

of the grass carp rhabdovirus temperatures

4'8 hours p.int isolate in FI-tM-cells at different

Samples were collected 6 12--24--36 and 48 hours p.i. then frozen at - - 7 4 ° C and after thawing they were t i t r a t e d in FI-I~?¢I cells

A Rhabdovirus Isolated from Grass Carp

183

O p t i m a l v i r u s r e p l i c a t i o n in F H M - c e l l s was o b s e r v e d a t i n c u b a t i o n t e m p e r a t a r e s b e t w e e n 16 ° (~:1) a n d 23 ° C (4-1) (Fig. 1). The isolate showed o n l y little g r o w t h a t 28 ° a n d 4 ° C; a t 37 ° C t h e a g e n t d i d n o t m u l t i p l y . The highest virus i n f e c t i v i t y titers (107.5 TCIDs0/0.1 mI) were o b s e r v e d a f t e r 36 hours a t 23 ° C ( ± l ) (Fig. 1). S i m i l a r results were d e s c r i b e d for R V C (5, 10) a n d S B I - v i r u s (1, 3). T h e isolate from grass c a r p was labile to lipid s o l v e n t a n d h e a t t r e a t m e n t (56 ° C). Table 2. In/ectivity o] the Isolate/tom Grass Carp be/ore a~wl aJter Treatment with Lipid Solvent (CHCl3) and Heat (56 ° C) Infectivity (log10 TCID~0/0.1 in/) before after

Treatment Lipid solvent (CHC13) Heat(56 ° C) 30 minutes 60 minutes 120 minutes

6.8

0

7.5 7.5 7.5

5.2 4.2 1.8

Electronmicrogra.phs of t h e grass c a r p isolate r e v e a l e d a t y p i c a l r h a b d o v i r u s m o r p h o l o g y . The b u l l e t s h a p e d virus p a r t i c l e s m e a s u r e d 70 n m (:j:10) in d i a m e t e r , 120 n m ( ± 1 0 ) in l e n g t h a n d t h e y consisted of an o u t e r c o a t a n d a core w i t h a d i a m e t e r of a p p r o x i m a t e l y 25 n m (4-10). N e u t r a l i z a t i o n tests ( i m m u n e s e r u m p r o d u c t i o n has been described elsewhere; 4) were p e r f o r m e d w i t h a n t i s e r u m a g a i n s t S B I - v i r u s . Serial twofold dilutions of a n t i s e r u m were m i x e d w i t h 100 TCIDs0/0.1 ml of RVC, S B I - v i r n s a n d t h e grass carp isolate. S B I - v i r u s - a n t i s e r u m d i d n o t neutralize t h e i n f e c t i v i t y of t h e grass carp isolate whereas RVC a n d S B I - v i r u s were r e g u l a r l y n e u t r a l i z e d (Table 3). Table 3. Neutralization Tests o/ R VC-, S B I - and the Isolated Virus ]rom Grass Carp with Rabbit Antiserum against S B I - Virus and Normal Rabbit Serum in 2 Fold Dilutions and a Constant Quantity (100 TCIDbo/O.1 ml) o/Each Virus Neutralization Virus

rabbit a n t i - S B I serum

normal r a b b i t serum

SBI-virus l~VC-virus Isolate from grass carp

1 : 16 1 : 16 < 1: 2

< 1:2 < 1:2 < 1:2

A n i m a l e x p e r i m e n t s h a v e d e m o n s t r a t e d t h a t t h e isolate was infectious to grass carp. F i f t e e n y o u n g grass carp (100--170 mg) were held in a b e a k e r containing 2 liters of w a t e r (23 ° C) (4-1) infected w i t h 3 ml of virus from t h e 6th F H M passage (infectivity t i t e r 107 TCID50/0.1 ml). All infected grass carp d i e d b e t w e e n 8 a n d 9 d a y s p . i . while t h e 10 controls ( k e p t in 2 liters of w a t e r w i t h 3 ml of noni n f e c t e d tissue culture m e d i u m ) r e m a i n e d unafflicted. Virus could be re-isolated f r o m t h e infected grass carps w i t h a v e r a g e v i r u s t i t e r s of 103 TCIDbo/100 m g fish.

184

W. AR~E :

The grass carp isolate can be differentiated from the sMmonid rhabdoviruses E g t v e d (7) and infectious hematopoietic necrosis virus (IHN) (11, 14) b y its optimal temperature for replication. The cyprinid rhabdoviruses RVC (5, 10) and S B I (1, 3) and the isolated virus from grass carp display similar optimal replication temperatures. I n this s t u d y it was of interest, t h a t the S B L a n t i s e r u m did n o t neutralize the isolate from grass earp. The lack of a n y serological relationship to I~VC and SBI-virus as determined in these investigations m a y be in accordance with the observation t h a t an infection in grass carp similar to I D C did not spread to the c o m m o n carp held in the same p o n d (12). The results indicate t h a t the isolated virus from grass carp is a new serotype of eyprinid rhabdoviruses. W h e t h e r this virus shares antigenic properties with the pike fry rhabdovirus (8, 9) has to be determined in further investigations.

Acknowledgments I am indebted to Prof. Dr. Mahnel, Institute of Microbiology and Infectious Diseases of Animals, University Munieh, for the electron microscopy and to Dr. Baehmann for the criticism in the preparation of this manuscript.

Referenees 1. AR~E, W. : Zellkulturen aus verschiedenen Sfil3wasserteleosteergeweben und Untersuehungen fiber die Atiologie der Sehwimmblasenentzfindung der Karpfen. Ph. D. Thesis, University Munich, 1973. 2. ANTALFI, A., TSLG, I.: Graskarpfen --pflanzenfressende Fisehe. Gfinzburg: Donau-Verlag, 1971. 3. BACR~AN~¢, P.A., A~NE, W.: Isolation and characterization of agent causing swim bladder inflammation in carp. Nature (London) 244, 235 (1973), 4. BAOas~A~c>r,P. A., ATONE,W. : Biological properties and identification of the agent causing swim bladder inflammation in carp. Areh. ges. Virusforsc:h. 44, 261 (1974). 5. FIJAN, N. N., PET~IIgEC, Z., SULII~IANOVIC, D., ZWILLENBERG, L. O. : Isolation of viral causative agent from the acute form of infectious dropsy in carp. Vet. Archly (Zagreb) 41, 125 (1971). 6. K31XBEI~, G. : Beitrag zur kollektiven Behasadlung pharmakologischer Reihenversuche. Arch. exp. Pathol. Pharmakol. 162, 480 (1931). 7. KINKELIN DE, P., SCHE~I~ER, 1~. : Lo virus d'Egtved. I. Stabilit6 developpement et structure du virus de la souche Danoise Fi. Ann. l%ech, refer. I, 17 (1970). 8. KINKELIN DE, P., BOOTSMA, R., GALIMAI~D, B. : Isolation and identification of Lhe causative agent of pike (Esox lucius L.) "Read disease". Nature (London) 241, 465 (1973). 9. KINKELIN DE, P., LE BERIIE, M., LElgOl]a, G.: l~habdovirus des poissons. I. proprietes in vitro du virus de la maladie rouge de l'alevin de brother. Ann. Microbiol. 125A, 93 (1974). I0. KINKELIN DE, P., LE BEma]~, M. : Rhabdovirus des poissons. II. proprietes in vitro du virus de la viremie printaniere de la carpe. Ann. Microbiol. 125A, 113 (1974). II. McALLIST]~IL P.E., FI~YEI~, J.L., PILCHER, K.S.: Further characterization of Infectious Hematopoietic Necrosis Virus of salmonid fish (Oregon Strain). Arch. ges. Virusforseh. 44, 270 (1974). 12. SZA!ZOI~CZAI, J., Mo]55rir, K.: Veterin/~rmedizinisehe Untersuehungen an den in Ungarn eingebfirgerten pflanzenfressenden Fischarten. Ztschr. Fischerei, N. F. 14, 139 (1966).

A l~habdovirus

Isolated from Grass Carp

185

13. TOi~[ASEC,I. : Infectious dropsy of carp (IDC). I n : Symposium on the major communicable fish diseases in Europe and their control. F A 0 - s y m p o s i a , Aviemore (Scotland), E I F A C / T 17, 107 (1974). 14. WINGFIELD, W. I:L, FRYER, J. L., PILCHER, K. S. : Properties of the Sockeye Salmon Virus (Oregon strain). Proe. Soc. exp. Biol. Med. 30, 1055 (1969). Author's address: Dr. W. Ahne, Institute of Zoology and Hydrobiology, Veterinary Faculty, University of Munich, I~:aulbachstraBe 37, D-800O Miinehen 22, Federal Republic of Germany. Received December 12, 1974