Archives of Virology
Archives of Virology 61, 255--259 (1979)
© by Springer-Verlag 1979
A N e w Calieivirus Isolated F r o m a M a r i n e M a m m a l Brief Report By A. W. SMITH1, T. G. AKERS2, A. B. LATHAM3, D. E. SKrLLI~G4, and t L L . BRAY3 Naval Biosciences Laboratory, University of California., School of Public ~Iealth, Berkeley, California, U.S.A. Accepted February 28, 1979
Summary A new serotype of ealicivirus, designated as San 3iiguel sea lion virus type 6 (SMSV-6), was isolated from vesicular lesions on the flipper of a California sea lion pup. Serologic studies show that SMSV-6 neutralizing antibodies (SN) occur frequently among California sea lions and occasionally among northern fur seals. Feral swine, 1- to 6-week elephant seal pups and grey whales tested negative for SMSV-6 antibody. Since 1972, four serotypes of caliciviruses, San Miguel sea lion virus (SMSV1-2-4-5) have been isolated from pinnipeds. I t has been suggested that all are antigenic variants of vesicular exanthema of swine virus (VESV) (9, 10, 13, 14), an exotic virus thought in 1959 to have been eradicated from domestic swine in the United States (5). SMSV-4 and SMSV-5 have been shown experimentally to infect one primate species (Cercopithecus aethiops) and researchers exposed to both agents have developed virus neutralizing (VN) antibodies (15). SMSV-5 was first isolated from vesicular lesions on the flippers of fur seals (Callorhinusursinus) (14). Since then, two 4-month-old California sea lions (Zalophus cali/ornianus) examined on San Miguel Island, California, were found to have on the foreflippers vesicles containing clear fluid. A virus, the subject of this report, was isolated from these lesions. 1 Present address: NavM Ocean Systems Center, Code 5103, San Diego, CA 92152, U.S.A. 2 Present address: Tulane University, School of Public Health and Tropical Medicine, 1430 Tulane Avenue, New OEeans, LA 70011, U.S.A. 3 Present address: Naval Biosciences Laboratory, University of California, School of Public Health, Berkeley, CA 94720, U.S.A. 4 Present address: Zoological Society of San Diego, P.O. Box 551, San Diego, CA 92112, U.S.A.
0304.8608/79/0061/0255/$ 01.00
256
A. ),V. SMIT~ et aL :
F l u i d was a s p i r a t e d from the vesicles a n d t r a n s f e r r e d to vials containing 1 ml of p h o s p h a t e - b u f f e r e d glycerol (pH 7.2). These -were i m m e d i a t e l y frozen on d r y ice a n d s t o r e d a t - - 7 0 ° C. T h e s a m p l e s were s u b s e q u e n t l y t h a w e d a n d p a s s a g e d in p r i m a r y rabbit, k i d n e y ( P R K ) cells a n d Veto eells (African green m o n k e y k i d n e y , Cercopithecus aethiops). A f t e r 24 hours on second passage in b o t h P R K a n d Vero cells, t h e eytop a t h o l o g y seen was almost t o t a l d i s r u p t i o n of t h e cell monolayers. The cells were s h r u n k e n , r o u n d a n d d e t a c h e d from t h e glass surface. The same o b s e r v a t i o n s were n o t e d on m a t e r i a l t h a t h a d been p a s s a g e d once on Vero cells a n d t h e n t r a n s f e r r e d for one passage to pig k i d n e y cells (PK-15). A f t e r 11 passages in Veto cells, virus titers were found to be 10 s.5 TCIDh0/ml. The isolate was classified as a ealicivirus b y t h e same p h y s i c o - c h e m i c a l criteria used to i d e n t i f y p r e v i o u s l y r e p o r t e d calieiviruses (10, 3, 4, 7, 17, 18). E l e c t r o n m i c r o s c o p y of n e g a t i v e s t a i n e d p r e p a r a t i o n s , including those for i m m u n o - e l e c t r o n m i c r o s c o p y was carried out as p r e v i o u s l y described (8, 16). Virus stocks c o n t a i n i n g 100 tissue culture infecting doses (TCID50) of virus isolate 4391 were m i x e d with e q u a l volumes of 12 different calicivirus a n t i s e r u m s k n o w n t,o contain 20 u n i t s of a n t i b o d y when t e s t e d a g a i n s t t h e i r homologous virus types. The a n t i s e r u m s used were V E S V t y p e s A4s, C ~ , D53, F54, G55, Iah, J56, K56, SMSV t y p e s SMSV-1, SMSV-2, SMSV-4, SNSV-5, as well as homologous s e r u m to isolate 349. I n a d d i t i o n , sera from a v a r i e t y of a n i m a l species i n h a b i t i n g t h e California coastal zones were e x a m i n e d for virus n e u t r a l i z i n g (VN) a n t i b o d i e s using previously- r e p o r t e d p r o c e d u r e s (12). I s o l a t e 439 was n e u t r a l i z e d only b y homologous serum a n d in t h e reciprocal reactions 100 TCIDh0 of t h e 12 test viruses were n o t n e u t r a l i z e d b y 20 a n t i b o d y Table 1. Immuno-electron microscopic comparisons o/ isolate 439 with other calicivirus typesa Serum Virus
Pooled SMSV b
SMSV-4
F-9
Pooled VESV c
439 ~ a
~-
--
+
--
-~
--
±
VESV-A4s
~-
--
--
~c
439
+
--
+
+
FCV.F
9
( + ) = only- virus-antibody complexes seen, some containing 15 or more virus particles. ( -- ) =:: only single virus particles observed b Pooled serum containing 20 serum neutralizing antibody units of SMSV-1, SMSV-2, SMSV-4, and SMSV-5 antibody c Pooled serum eont, aining 20 serum neutralizing a n t i b o d y units of VESV types A4s, C~, Fhe, Ihs, J ~ , K56 antibody a (:}:) = aggregates of virus-antibody containing 15 or more particles were seen and some single virus particles were also seen. When the three viruses (SMSV-4, FCV-F 9 and VESV-Aas) were diluted 1 : 100 and retested against 439 antiserum, only virusantibody aggregates were seen indicating t h a t the 4- reaction had occurred because excess antibody specific for these viruses was not present 439 = tag number of the 4-month-old sea lion pup yielding the virus isolate
Marine Calicivirus
257
units of 439 a n t i s e r u m . These results show this n e w virus to be d i s t i n c t f r o m t h e o t h e r calicivirus types t e s t e d a n d are the basis for classifying the a g e n t as a n e w s e r o t y p e d e s i g n a t e d SMSV-6. I m m u n o - e ] e c t r o n m i c r o s c o p y results are shown in T a b l e I. I s o l a t e 439 f o r m e d a n t i g e n - a n t i b o d y complexes w i t h h o m o l o g o u s antis e r u m a n d p o o l e d V E S V a n d SMSV a n t i s e r u m s b u t was n o t b o u n d b y feline ealicivirus (FCV) a n t i s e r u m . T h e reciprocal r e a c t i o n s where 439 a n t i s e r u m wast e s t e d against each virus showed the f o r m a t i o n of a n t i g e n - a n t i b o d y complexes. H o w e v e r e v e n in the case of F C V - F 9 , these reactions were n o t c o m p l e t e a n d i n d i v i d u a l virus particles were present. This suggests t h a t SMSV-6 m a y h a v e a r a t h e r b r o a d antigenic relatedness to t h e o t h e r caliciviruses including F C V - F 9 . A l t h o u g h we h a v e p r e v i o u s l y r e p o r t e d (16) on the possible existence of such a~ r e l a t i o n s h i p b e t w e e n V E S V and FCV, this in the first t i m e we h a v e seen a n y m a r i n e caliciviruses react in this way. Table 2. Serologic survey o/ marine and ]eral terrestrial mammals ]or antibodies to S M S V isolate 639
Animal speeies
No. positive/ No.tested 1:10
1:20
Serum dilution~ t:40 t:80 I:1601:320
California sea lion adult female (1972)
6/20
4
0
1
1
0
0
California sea lion adult male (1970)
1/13
1
0
0
0
0
0
California sea lion 9 months old pups (1975)
3/5
0
1
1
0
0
1
California sea lion 4 months old pups b (1975)
23/47
4
3
11
3
0
2
California sea lion 4 months old pups (1977)
0/30
0
0
0
0
0
0
Fur seal, Bering Sea adult female (1974)
0/38
0
0
0
0
0
0
F u r seal, San Miguel Is. 4 months old pups (1975)
3/36
0
1
2
0
0
0
F u r seal, San Miguel Is. 4 months old pups (1977)
4/47
0
1
2
I
0
0
Elephant seal 1--6 weeks old (1977)
0/46
0
0
0
0
0
0
Gray whale
0/16
0
0
0
0
0
0
Feral swine
0/17
0
0
0
0
0
0
a The number of animals whose serum neutralized 100 TCIDs0 of virus at the terminal dilution indicated b Isolate 439 was first recovered from an individual which tested negative within thisgroup
258
A.W. S~ITH et
al. :
The results of our serologic survey for antibodies to isolate 439 (Table 2) ~uggest that this agent was present along the California coast in 1970 when our first sea lion serum samples were collected, but perhaps more important, this was the first time we had observed vesicular lesions on the flippers of infected sea lions. This finding is consistent with reports of similar lesions occurring on northern fur seals infected with SMSV-5 (14) and swine naturally infected with VESV (5) or experimentally exposed to SMSV types 1, 2, 4 and 5 (2). In 1975, judging by the incidence of VN antibodies against isolate 439, the involvement of sea lion pups on San Miguel Island was quite extensive, but in 1977, all sea lion pups tested negative whereas 10 percent of the fur seal pups on the same island had VN antibodies in both 1975 and 1977. We have previously reported on the geographic and interspeeies distribution of neutralizing antibodies to 14 seretypes of calicivirus and speculated that there exists for this group of viruses an ocean reservoir unique in some ways to California (6, 11, 12). Two additional questions have arisen. First, how do the many serotypes of calicivirus remain active among marine mammals such as California sea lions, especially when each type is thought to produce strong, type-specific immunity (1, 5, 12), and second, why do we so infrequently re-isolate viruses of the same antigenic type ? In answer, we have postulated that some of the fishes eaten by marine mammals form large populations but have a geographic range limited to southern California. Such population could maintain many active virus types and exert pressures resulting in new types (9). We also recognize that the probability ~of isolating previously known serotypes is inversely proportional to the total number of serotypes :in existence. We are now suggesting that there are a great m a n y more serotypes than are presently known. Because some of the known calicivirus types have proven to be pathogenic for important domestic species and infectious for primates and possibly man (16), new and as yet undiscovered serotypes may have the potential for again spreading through herds of domestic swine or perhaps producing disease in man under ~eonditions of natural exposure. Acknowledgments The authors wish to thank William Gilmartin and 1%obert Gunnels, Naval Undersea -Center, San Diego, California; l~obert DeLong and Clifford Fiscus, National Marine Fisheries Service for their assistance in collecting California sea lion sera and northern fur seal sera on San Miguel Islanc~, California ; and i. lVi. S. Watkins, Naval Bioseiences Laboratory for providing whale sera. This research was supported by the Office of
Naval Research.
Referenees 1. BANKOWSKI,R. A. : Vesicular Exanthema. In: Advances in Veterinary Science, Chapt. 10, 23--64. New York: ~ a d e m i c Press Inc. 1965. 2. CALLIS, J. J. : Plum Island Animal Disease Laboratory, New York, personal communication (1977). 3. DULBECCO,1%., VOGT, M. : Plaque formation and isolation of pure lines of poliomyelitis viruses. J. exp. Med. 99, 167--182 (1953). 4. HownTso~, A. F., W~IT~O~E, G. F.: Development and structure of vesicular stomatitis virus. Virology 16, 466--478 (1973).
Marine Calicivirus
259
5. MADII~, S. H. : Diseases of Swine, 3rd ed. DIYlWNE,]:L W. (ed.). Ames, Iowa: Iowa State University Press 1970. 6. PRATO, C. M., AKERS, T. G., SMITH, A. W. : Serologic evidence of calicivirus transmission between marine mammals and terrestrial mammals. Nature 249, 255---25~ (1974). 7. RE~ozzo, G. C., BURKE, C. N. : A Manual of Basic Virological Techniques, 1 5 0 - 151. Englewood Cliffs, N.J. : Prentice Hall 1973. 8. RITCmE, A. E., FE~rELIUS, A. L.: Characterization of bovine viral diarrhea.viruses. V. Morphoh)gy of characteristic particles by electron microscopy. Arch. ges. Virusforsch. 28, 369--389 (1969). 9. SMIT~, A. W., AKERS, T. G. : Vesicular exanthema of swine. JA V MA 169, 700--703 (1976). 10. SMrr]% A. W., AKERS, T. G., MAt)IN, S. H., VEDROS, N. A. : San Miguel sea lion virus isolation, preliminary characterization and relationship to vesicular e x a n t h e m a of swine virus. ]N'ature 244, 108--110 (1973). 11. SMITH, A. W., LATHAM, A. B.: Prevalence of vesicular exanthema of swine antibodies among feral mammals associated with the Southern California coastal zones. AMS Vet. Res. 39, 291---296 (1978). 12. SMITH, A. W., PR-aTO, C. M., BRAY, H. L. : Prevalence and distribution of f o u r serotypes of SMSV serum neutralizing antibodies in wild animal populations. J . ~Vitdl. Dis. 12, 326--334 (1976). 13. S)~ITH, A. W., PRATO, C. M., SKILLI~G, D. E. : Characterization of two new serotypes of San Miguel sea lion virus (SMSV). Intervirology 8, 30--35, (1976). 14. SMIT~, A. W., PRATO, C. M., GILM~TI~, W. G., BROW~, R. J., KEYES, M. C. : A preliminary report on potentially pathogenic microbial agents recently isolated from pinnipeds. J . Wildl. Dis. 19, 34--59 (1974). 15. S)~ITH, A. W., PRATE, C. M., SKILLING, D. E. : Caliciviruses infecting monkeys and_ possibly man. Amer. J. vet. Res. 39, 287--289 (1978). 16. SMITH, A. W., S~,ILL~NG, D. E., RITCHIE, A. E. : Immuno-electron microscopi~ comparisons of ealiciviruses. Amer. J. vet. Res. 39, 1531--1533 (1978). 17. WALL,S, C., MEL~CK, J. L.: Cation s t a b i l i z a t i o n - - a new property of enteroviruses. Virology 16, 504--506 (1962). 18. ZEE, Y. C., HACKETT, A. J. : The influence of cations on the thermal inactivation of vesicular exanthema of swine virus. Arch, ges. Virusforsch. 20, 473 ~-476 (I967). Authors' address: Dr. A. W. SMITH, Naval Ocean Systems Center, Code 5103,. San Diego, CA 92152, U.S.A. Received February 20, 1979
Herausgeber, Eigentiimer und Verleger: Springer~Verlag, MSlkerbastei 5, A-1011 Wien. Fiir den Textteil verant~ wortlich: Dr. Wilhelm Schwabl, MSlkerbastei 5, A-1011 Wien. Fftr den Anzeigenteil verantwortlich: Mag~ Bruno Schweder, M~lkerbastei 5, A-1011 Wien. Druck: R. Spies & Co., StrauQengasse 16, A-1050 Wien. Printed in Austria.
Archives of Virology 61, 255--259 (1979)
© by Springer-Verlag 1979
A N e w Calieivirus Isolated F r o m a M a r i n e M a m m a l Brief Report By A. W. SMITH1, T. G. AKERS2, A. B. LATHAM3, D. E. SKrLLI~G4, and t L L . BRAY3 Naval Biosciences Laboratory, University of California., School of Public ~Iealth, Berkeley, California, U.S.A. Accepted February 28, 1979
Summary A new serotype of ealicivirus, designated as San 3iiguel sea lion virus type 6 (SMSV-6), was isolated from vesicular lesions on the flipper of a California sea lion pup. Serologic studies show that SMSV-6 neutralizing antibodies (SN) occur frequently among California sea lions and occasionally among northern fur seals. Feral swine, 1- to 6-week elephant seal pups and grey whales tested negative for SMSV-6 antibody. Since 1972, four serotypes of caliciviruses, San Miguel sea lion virus (SMSV1-2-4-5) have been isolated from pinnipeds. I t has been suggested that all are antigenic variants of vesicular exanthema of swine virus (VESV) (9, 10, 13, 14), an exotic virus thought in 1959 to have been eradicated from domestic swine in the United States (5). SMSV-4 and SMSV-5 have been shown experimentally to infect one primate species (Cercopithecus aethiops) and researchers exposed to both agents have developed virus neutralizing (VN) antibodies (15). SMSV-5 was first isolated from vesicular lesions on the flippers of fur seals (Callorhinusursinus) (14). Since then, two 4-month-old California sea lions (Zalophus cali/ornianus) examined on San Miguel Island, California, were found to have on the foreflippers vesicles containing clear fluid. A virus, the subject of this report, was isolated from these lesions. 1 Present address: NavM Ocean Systems Center, Code 5103, San Diego, CA 92152, U.S.A. 2 Present address: Tulane University, School of Public Health and Tropical Medicine, 1430 Tulane Avenue, New OEeans, LA 70011, U.S.A. 3 Present address: Naval Biosciences Laboratory, University of California, School of Public Health, Berkeley, CA 94720, U.S.A. 4 Present address: Zoological Society of San Diego, P.O. Box 551, San Diego, CA 92112, U.S.A.
0304.8608/79/0061/0255/$ 01.00
256
A. ),V. SMIT~ et aL :
F l u i d was a s p i r a t e d from the vesicles a n d t r a n s f e r r e d to vials containing 1 ml of p h o s p h a t e - b u f f e r e d glycerol (pH 7.2). These -were i m m e d i a t e l y frozen on d r y ice a n d s t o r e d a t - - 7 0 ° C. T h e s a m p l e s were s u b s e q u e n t l y t h a w e d a n d p a s s a g e d in p r i m a r y rabbit, k i d n e y ( P R K ) cells a n d Veto eells (African green m o n k e y k i d n e y , Cercopithecus aethiops). A f t e r 24 hours on second passage in b o t h P R K a n d Vero cells, t h e eytop a t h o l o g y seen was almost t o t a l d i s r u p t i o n of t h e cell monolayers. The cells were s h r u n k e n , r o u n d a n d d e t a c h e d from t h e glass surface. The same o b s e r v a t i o n s were n o t e d on m a t e r i a l t h a t h a d been p a s s a g e d once on Vero cells a n d t h e n t r a n s f e r r e d for one passage to pig k i d n e y cells (PK-15). A f t e r 11 passages in Veto cells, virus titers were found to be 10 s.5 TCIDh0/ml. The isolate was classified as a ealicivirus b y t h e same p h y s i c o - c h e m i c a l criteria used to i d e n t i f y p r e v i o u s l y r e p o r t e d calieiviruses (10, 3, 4, 7, 17, 18). E l e c t r o n m i c r o s c o p y of n e g a t i v e s t a i n e d p r e p a r a t i o n s , including those for i m m u n o - e l e c t r o n m i c r o s c o p y was carried out as p r e v i o u s l y described (8, 16). Virus stocks c o n t a i n i n g 100 tissue culture infecting doses (TCID50) of virus isolate 4391 were m i x e d with e q u a l volumes of 12 different calicivirus a n t i s e r u m s k n o w n t,o contain 20 u n i t s of a n t i b o d y when t e s t e d a g a i n s t t h e i r homologous virus types. The a n t i s e r u m s used were V E S V t y p e s A4s, C ~ , D53, F54, G55, Iah, J56, K56, SMSV t y p e s SMSV-1, SMSV-2, SMSV-4, SNSV-5, as well as homologous s e r u m to isolate 349. I n a d d i t i o n , sera from a v a r i e t y of a n i m a l species i n h a b i t i n g t h e California coastal zones were e x a m i n e d for virus n e u t r a l i z i n g (VN) a n t i b o d i e s using previously- r e p o r t e d p r o c e d u r e s (12). I s o l a t e 439 was n e u t r a l i z e d only b y homologous serum a n d in t h e reciprocal reactions 100 TCIDh0 of t h e 12 test viruses were n o t n e u t r a l i z e d b y 20 a n t i b o d y Table 1. Immuno-electron microscopic comparisons o/ isolate 439 with other calicivirus typesa Serum Virus
Pooled SMSV b
SMSV-4
F-9
Pooled VESV c
439 ~ a
~-
--
+
--
-~
--
±
VESV-A4s
~-
--
--
~c
439
+
--
+
+
FCV.F
9
( + ) = only- virus-antibody complexes seen, some containing 15 or more virus particles. ( -- ) =:: only single virus particles observed b Pooled serum containing 20 serum neutralizing antibody units of SMSV-1, SMSV-2, SMSV-4, and SMSV-5 antibody c Pooled serum eont, aining 20 serum neutralizing a n t i b o d y units of VESV types A4s, C~, Fhe, Ihs, J ~ , K56 antibody a (:}:) = aggregates of virus-antibody containing 15 or more particles were seen and some single virus particles were also seen. When the three viruses (SMSV-4, FCV-F 9 and VESV-Aas) were diluted 1 : 100 and retested against 439 antiserum, only virusantibody aggregates were seen indicating t h a t the 4- reaction had occurred because excess antibody specific for these viruses was not present 439 = tag number of the 4-month-old sea lion pup yielding the virus isolate
Marine Calicivirus
257
units of 439 a n t i s e r u m . These results show this n e w virus to be d i s t i n c t f r o m t h e o t h e r calicivirus types t e s t e d a n d are the basis for classifying the a g e n t as a n e w s e r o t y p e d e s i g n a t e d SMSV-6. I m m u n o - e ] e c t r o n m i c r o s c o p y results are shown in T a b l e I. I s o l a t e 439 f o r m e d a n t i g e n - a n t i b o d y complexes w i t h h o m o l o g o u s antis e r u m a n d p o o l e d V E S V a n d SMSV a n t i s e r u m s b u t was n o t b o u n d b y feline ealicivirus (FCV) a n t i s e r u m . T h e reciprocal r e a c t i o n s where 439 a n t i s e r u m wast e s t e d against each virus showed the f o r m a t i o n of a n t i g e n - a n t i b o d y complexes. H o w e v e r e v e n in the case of F C V - F 9 , these reactions were n o t c o m p l e t e a n d i n d i v i d u a l virus particles were present. This suggests t h a t SMSV-6 m a y h a v e a r a t h e r b r o a d antigenic relatedness to t h e o t h e r caliciviruses including F C V - F 9 . A l t h o u g h we h a v e p r e v i o u s l y r e p o r t e d (16) on the possible existence of such a~ r e l a t i o n s h i p b e t w e e n V E S V and FCV, this in the first t i m e we h a v e seen a n y m a r i n e caliciviruses react in this way. Table 2. Serologic survey o/ marine and ]eral terrestrial mammals ]or antibodies to S M S V isolate 639
Animal speeies
No. positive/ No.tested 1:10
1:20
Serum dilution~ t:40 t:80 I:1601:320
California sea lion adult female (1972)
6/20
4
0
1
1
0
0
California sea lion adult male (1970)
1/13
1
0
0
0
0
0
California sea lion 9 months old pups (1975)
3/5
0
1
1
0
0
1
California sea lion 4 months old pups b (1975)
23/47
4
3
11
3
0
2
California sea lion 4 months old pups (1977)
0/30
0
0
0
0
0
0
Fur seal, Bering Sea adult female (1974)
0/38
0
0
0
0
0
0
F u r seal, San Miguel Is. 4 months old pups (1975)
3/36
0
1
2
0
0
0
F u r seal, San Miguel Is. 4 months old pups (1977)
4/47
0
1
2
I
0
0
Elephant seal 1--6 weeks old (1977)
0/46
0
0
0
0
0
0
Gray whale
0/16
0
0
0
0
0
0
Feral swine
0/17
0
0
0
0
0
0
a The number of animals whose serum neutralized 100 TCIDs0 of virus at the terminal dilution indicated b Isolate 439 was first recovered from an individual which tested negative within thisgroup
258
A.W. S~ITH et
al. :
The results of our serologic survey for antibodies to isolate 439 (Table 2) ~uggest that this agent was present along the California coast in 1970 when our first sea lion serum samples were collected, but perhaps more important, this was the first time we had observed vesicular lesions on the flippers of infected sea lions. This finding is consistent with reports of similar lesions occurring on northern fur seals infected with SMSV-5 (14) and swine naturally infected with VESV (5) or experimentally exposed to SMSV types 1, 2, 4 and 5 (2). In 1975, judging by the incidence of VN antibodies against isolate 439, the involvement of sea lion pups on San Miguel Island was quite extensive, but in 1977, all sea lion pups tested negative whereas 10 percent of the fur seal pups on the same island had VN antibodies in both 1975 and 1977. We have previously reported on the geographic and interspeeies distribution of neutralizing antibodies to 14 seretypes of calicivirus and speculated that there exists for this group of viruses an ocean reservoir unique in some ways to California (6, 11, 12). Two additional questions have arisen. First, how do the many serotypes of calicivirus remain active among marine mammals such as California sea lions, especially when each type is thought to produce strong, type-specific immunity (1, 5, 12), and second, why do we so infrequently re-isolate viruses of the same antigenic type ? In answer, we have postulated that some of the fishes eaten by marine mammals form large populations but have a geographic range limited to southern California. Such population could maintain many active virus types and exert pressures resulting in new types (9). We also recognize that the probability ~of isolating previously known serotypes is inversely proportional to the total number of serotypes :in existence. We are now suggesting that there are a great m a n y more serotypes than are presently known. Because some of the known calicivirus types have proven to be pathogenic for important domestic species and infectious for primates and possibly man (16), new and as yet undiscovered serotypes may have the potential for again spreading through herds of domestic swine or perhaps producing disease in man under ~eonditions of natural exposure. Acknowledgments The authors wish to thank William Gilmartin and 1%obert Gunnels, Naval Undersea -Center, San Diego, California; l~obert DeLong and Clifford Fiscus, National Marine Fisheries Service for their assistance in collecting California sea lion sera and northern fur seal sera on San Miguel Islanc~, California ; and i. lVi. S. Watkins, Naval Bioseiences Laboratory for providing whale sera. This research was supported by the Office of
Naval Research.
Referenees 1. BANKOWSKI,R. A. : Vesicular Exanthema. In: Advances in Veterinary Science, Chapt. 10, 23--64. New York: ~ a d e m i c Press Inc. 1965. 2. CALLIS, J. J. : Plum Island Animal Disease Laboratory, New York, personal communication (1977). 3. DULBECCO,1%., VOGT, M. : Plaque formation and isolation of pure lines of poliomyelitis viruses. J. exp. Med. 99, 167--182 (1953). 4. HownTso~, A. F., W~IT~O~E, G. F.: Development and structure of vesicular stomatitis virus. Virology 16, 466--478 (1973).
Marine Calicivirus
259
5. MADII~, S. H. : Diseases of Swine, 3rd ed. DIYlWNE,]:L W. (ed.). Ames, Iowa: Iowa State University Press 1970. 6. PRATO, C. M., AKERS, T. G., SMITH, A. W. : Serologic evidence of calicivirus transmission between marine mammals and terrestrial mammals. Nature 249, 255---25~ (1974). 7. RE~ozzo, G. C., BURKE, C. N. : A Manual of Basic Virological Techniques, 1 5 0 - 151. Englewood Cliffs, N.J. : Prentice Hall 1973. 8. RITCmE, A. E., FE~rELIUS, A. L.: Characterization of bovine viral diarrhea.viruses. V. Morphoh)gy of characteristic particles by electron microscopy. Arch. ges. Virusforsch. 28, 369--389 (1969). 9. SMIT~, A. W., AKERS, T. G. : Vesicular exanthema of swine. JA V MA 169, 700--703 (1976). 10. SMrr]% A. W., AKERS, T. G., MAt)IN, S. H., VEDROS, N. A. : San Miguel sea lion virus isolation, preliminary characterization and relationship to vesicular e x a n t h e m a of swine virus. ]N'ature 244, 108--110 (1973). 11. SMITH, A. W., LATHAM, A. B.: Prevalence of vesicular exanthema of swine antibodies among feral mammals associated with the Southern California coastal zones. AMS Vet. Res. 39, 291---296 (1978). 12. SMITH, A. W., PR-aTO, C. M., BRAY, H. L. : Prevalence and distribution of f o u r serotypes of SMSV serum neutralizing antibodies in wild animal populations. J . ~Vitdl. Dis. 12, 326--334 (1976). 13. S)~ITH, A. W., PRATO, C. M., SKILLI~G, D. E. : Characterization of two new serotypes of San Miguel sea lion virus (SMSV). Intervirology 8, 30--35, (1976). 14. SMIT~, A. W., PRATO, C. M., GILM~TI~, W. G., BROW~, R. J., KEYES, M. C. : A preliminary report on potentially pathogenic microbial agents recently isolated from pinnipeds. J . Wildl. Dis. 19, 34--59 (1974). 15. S)~ITH, A. W., PRATE, C. M., SKILLING, D. E. : Caliciviruses infecting monkeys and_ possibly man. Amer. J. vet. Res. 39, 287--289 (1978). 16. SMITH, A. W., S~,ILL~NG, D. E., RITCHIE, A. E. : Immuno-electron microscopi~ comparisons of ealiciviruses. Amer. J. vet. Res. 39, 1531--1533 (1978). 17. WALL,S, C., MEL~CK, J. L.: Cation s t a b i l i z a t i o n - - a new property of enteroviruses. Virology 16, 504--506 (1962). 18. ZEE, Y. C., HACKETT, A. J. : The influence of cations on the thermal inactivation of vesicular exanthema of swine virus. Arch, ges. Virusforsch. 20, 473 ~-476 (I967). Authors' address: Dr. A. W. SMITH, Naval Ocean Systems Center, Code 5103,. San Diego, CA 92152, U.S.A. Received February 20, 1979
Herausgeber, Eigentiimer und Verleger: Springer~Verlag, MSlkerbastei 5, A-1011 Wien. Fiir den Textteil verant~ wortlich: Dr. Wilhelm Schwabl, MSlkerbastei 5, A-1011 Wien. Fftr den Anzeigenteil verantwortlich: Mag~ Bruno Schweder, M~lkerbastei 5, A-1011 Wien. Druck: R. Spies & Co., StrauQengasse 16, A-1050 Wien. Printed in Austria.
