_Archives
Vi rology
Arch Virol (1992) 123:267-277
© Springer-Verlag 1992 Printed in Austria
Monocional antibodies for the identification of herpesvirus simiae (B virus) L. M. Cropper l, D. N. Lees u*, R. Patt a, I. R. Sharp 2, and D. Brown l Virus Reference Laboratory and 2 Division of Microbiological Reagents and Quality Control, Central Public Health Laboratory, London, U.K. Accepted September 12, 1991
Summary. To differentiate between B virus and HSV isolates from monkeys and man monoclonal antibodies (mabs) were produced to herpesvirus simiae (B virus) and herpes simplex type 1 and 2 (HSV-1 and HSV-2). Mabs were tested by indirect immunofluorescence (IFAT) for reactivity against herpesviruses from Asiatic monkeys (B virus), African monkeys (SA 8 virus), and man (HSV-1, HSV-2, varicella-zoster virus, cytomegalovirus, and Epstein-Barr virus). Mabs could be divided into groups A-E displaying specific reactivity for B virus (A); reactivity with both B virus and SA 8 but not HSV (B); reactivity with B virus, SA 8 virus and HSV strains (C); specific reactivity with HSV-1 (D); and specific reactivity with HSV-2 (E). Two of the B virus specific mabs were able to differentiate between cynomolgus and rhesus strains of B virus. None of the mabs reacted with human varicella-zoster virus, cytomegalovirus, or Epstein-Barr virus. A panel of mabs for the unequivocal identification of B virus isolates from monkey or man is proposed. Introduction Herpesvirus simiae (B virus) is an alphaherpesvirus of macaque monkeys closely related to herpes simplex virus (HSV) of man. B virus infection in naturally infected macaques resembles human HSV infection, with primary oral or genital infection followed by seroconversion and virus latency in nerve ganglia [1, 15]. The medical significance of B virus lies in the lethal ascending encephalomyelitis that may follow transmission of B virus from monkey to man [ 15, 18]. Awareness of this rare but devastating human disease has been heightened by recent cases in Pensacola, Florida [-6] and Kalamozoo, Michigan [3]. The apparent success of early acyclovir therapy in controlling its lethal consequences [-3, 6] * Present address: Fish Disease Laboratory, The Nothe, Weymouth, Dorset, U.K.
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emphasises the need for rapid and unequivocal laboratory diagnosis of B virus infection. Unfortunately identification of virus isolates and serological investigations are confounded by common antigenic determinants among alphaherpesviruses from Old World primates and man [4, 7]. In suspected human B virus infections it is particularly important to distinguish B virus from HSV. This report describes the production of monoclonal antibodies (mabs) to B virus and their characterisation by immunofluorescence against a range of alphaherpesviruses from Old World primates and man. Materials and methods
Virus strains The B virus strain used for mouse immunisation (cyno 2) was an oral isolate from a cynomolgus monkey (Macaca fascicularis) [20]. Virus strains used to characterise monoclonal antibodies were as follows. Cynomolgus B virus strains 1/375 m, 3/C 20, 5/C9, 8/ C 18, 10/A 28 m, 14/332 and rhesus (M. mulatta) strains 12/268 and 13/559 are all monkey isolates described previously [21] and typed as B virus by serum neutralisation. Strain HZ 9 was isolated from a genital lesion in a baboon (Papio sp.) by Professor K. McCarthy, Department of Medical Microbiology, University of Liverpool, and typed as B virus by serum neutralisation [2t]. The SA 8 virus prototype strain (B 264) was isolated from the central nervous system of an African vervet (Cercopithecus aethiops) monkey [14]. The above strains were obtained from Dr. H. Zwartouw, Chemical Defence Establishment, Porton Down, Salisbury, U.K. Prototype B virus (Sabin strain), isolated from the spleen and CNS of a patient with ascending myelitis following a bite from a rhesus monkey [18], was obtained from the American Type Culture Collection (ATCC VR-126). A further B virus strain (130-65) was an isolate from a rhesus monkey which died of cerebral infarction [2] and was obtained from Professor H. Ludwig, Institut fiir Virotogie, Freie Universit/it Berlin, Berlin, Federal Republic of Germany. SA 8 strain 0430 was isolated from the lung of a fatally-infected perinatal baboon [5] and SA 8 strains X 313 and 1401 were genital isolates from baboons housed in an outdoor corral [12]. These strains were obtained from Dr. J. Hilliard, Southwest Foundation for Biochemical Research, San Antonio, Texas. HSV-1 strains KOS, VR3 (MacIntyre) (ATCC VR-539), SC 16, HFEM, S y n l 7 + , and HSV-2 clinical isolates Ve 70 and Vt 5392 were obtained from the North Regional Virus Reference Laboratory, Manchester, U.K. An additional HSV-2 strain 186 was obtained from Dr. H. Zwartouw, Chemical Defence Establishment, Porton Down, Salisbury, U.K. HSV-1 strain VRL 10711/54 was obtained from the PHLS Virus Reference Laboratory, HSV-1 strain Stoker, HSV-2 strain Parker, VZV (Marsden strain), and CMV (Rawles strain) from the PHLS Division of Microbiological Reagents and Quality Control, Central Public Health Laboratory, Colindale, London. EBV was obtained as virus-infected EB3 cells from Dr. M. Hambling, Virology Department, Public Health Laboratory, Leeds, U.K.
Gamma irradiation The radiation source was a gamma-cell model 220 (Atomic Energy Commission of Canada, Ontario, Canada) containing cobalt 60. Antigen preparations for immunisation received 2 x 166 rads: Virus-infected cells for immunofluorescence studies received 3 x 106 rads. All preparations were irradiated on ice. Samples were tested post-irradiation for residual infectious virus by inoculation onto 25 cm 2 flats of Vero cells. Virus-induced cytopathic effects (CPE) were not observed in any irradiated samples.
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Production of monoclonal antibodies Hybridoma cell lines were produced by intramuscular immunisation of 6-10 week old Balb/ c mice with 0.1 ml B virus antigen (strain cyno 2). The antigen preparation consisted of an emulsion of 0.4ml gamma cell inactivated B virus (titre before irradiation 108.5 TCIDs0/ ml, 0.4 ml Freund's incomplete adjuvent and 300 gg muramyl dipeptide (Sigma Ltd., Poole, Dorset). The mice received intravenous booster inoculations of 0.1 ml antigen without adjuvent at 2 weeks, 7 weeks, 'and 13 weeks. Three days after each booster inoculation two mice were sacrificed and their spleens removed for fusion. Spleen cells were fused with myeloma cells JK Ag 8-653 [19] according to the method of Kohler and Milstein [11] with modifications as described by Russell etal. [17]. Hybridoma supernatants were screened for B virus antibody by indirect immunofluorescence. Positive hybridomas were subcultured and cloned twice by limiting dilution on a thymocyte feeder layer following the methods of Lostrom et al. [13]. Clarified hybridoma supernatants were used for all studies. Monoclonal antibodies to HSV-1 and HSV-2 were prepared using a similar regime. Mice were immunised with HSV-1 strain Stoker and HSV-2 strain Parker. Antigen preparations consisted of an emulsion of 1.0 ml purified virus (formaldehyde inactivated; 0.05 % final concentration), 1.0 ml Freund's incomplete adjuvent and 750 gg muramyl dipeptide (Sigma Ltd., Poole, Dorset). The virus was purified following the method of Powell and Watson [16] from 6 x 108 infected Vero cells. Positive hybridomas were identified by indirect immunofluorescence. Six positive hybridomas were selected for further study on the basis of their reactivity with a small panel of B virus and HSV strains.
Indirect immunoJluorescence test Virus-infected cells were prepared by infection of Veto cell monolayers at a multiplicity of infection of 0.01 PFU/cell. Cells were collected by trypsinisation when virus-induced CPE was maximal. Cells were washed in phosphate buffered saline (PBS), resuspended in foetal calf serum and irradiated. Post-irradiation cells were washed 3 times in PBS, dotted on to multiwell slides (Flow Laboratories, Irvine, Scotland) and fixed in cold acetone for 10 rain. Slides were stored at - 70 °C until use. Hybridomas were tested by adding approximately 15 gl of supernatants per well and incubating at room temperature for 20 rain. After washing three times in PBS, bound antibody was detected with FITC-conjugated goat anti-mouse IgG diluted in PBS containing 0.005% Evans Blue counterstain (Sigma Ltd.).
Antibody subclass Monoclonal antibody subclass was determined by Ouchterlony double diffusion. Hybridoma supernatants and goat antisera to specific mouse immunoglobulin subclasses (IgG 1, IgG 2 a, IgG 2 b, and IgG 3) (Sigma Ltd.) were added to adjacent wells on an agarose gel slide and allowed to diffuse overnight in a humidified chamber. Subclasses were identified by the precipitin reactions.
Mouse antisera Polyclonal mouse ascitic fluid was prepared in Balb/c mice against gamma cell irradiated HSV-1 strain (VR 3) and B virus strain (cyno 2), titre before irradiation 10 7.5 TCIDs0/ml and 108.5 TCIDs0/ml respectively. The mice were inoculated intraperitoneally with 0.1 ml virus suspension at day 0, followed by intravenous inoculation of a further 0.1 ml virus suspension at day 10 and day 20. Three days later 106 Ehrliche ascitic tumour cells were inoculated intraperitoneally into each mouse. The mice were observed daily for the production of tumour growth and when this was evident, ascitic fluid was tapped from the mice and centrifuged to remove cells.
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Plaque reduction neutralisation assay Neutralising activity of the mabs and mouse polyclonal sera was tested in a plaque reduction assay. 0.1 ml of hybridoma supernatants were mixed with an equal volume of clarified virus suspension (approx. 100 PFU virus) containing 2.5 HAU of guinea complement (TCS) and incubated for 1 h at room temperature. 0.1 ml volumes were added to duplicate 25 cmz flats of Veto cells, adsorbed for 1 h at room temperature, and overlayed with 5 ml of 1% carboxymethylcellulose in Eagles minimal essential medium (Flow Laboratories). The overlay was removed after 3 days incubation and the cells fixed by immersion in 10% formal saline for 30 rain. Plaques were visualized by staining with 1% Coomassie blue (Sigma) in 45% methanol: 10% acetic acid. Results were expressed as percent plaque reduction compared to control. Results
Preliminary characterisation of monoclonal antibodies to B virus Hybridoma supernatants were screened by immunofluorescence against the homologous virus (B virus strain cyno 2). Twenty four positive hybridomas were identified and cloned. Preliminary characterisation included immunoglobulin subclass, ability to neutralise B virus and type of fluorescence pattern (Table 1). Mabs of subclasses IgG 1, I g G 2 a , I g G 2 b , and IgG3 were isolated. Four mabs B7.3, C30.2, C34.1, and C39.5, demonstrated significant (>f 50%) neutralisation in the plaque reduction assay. Immunofluorescence staining was generally observed in the cytoplasm and on the membrane of infected cells, however, mabs C 21.6 and C 51.2 demonstrated exclusively nuclear staining.
Reactivity against primate and human herpesviruses All of the B virus derived mabs and six selected HSV derived mabs were tested by immunofluorescence for reactivity against a range of primate and human alphaherpesviruses. The alphaherpesviruses were chosen on the basis of their known antigenic cross-reactivity with B virus and included cynomolgus and rhesus strains of B virus, vervet and baboon strains of SA 8 virus and human HSV-1 and HSV-2 strains. Reactivity against human varicella-zoster virus (VZV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV) was also examined. The monoclonal antibodies could be separated into 5 groups (Table2). Group A consisted of 6 mabs reacting exclusively with some or all of the B virus strains (except strain HZ 9 from a baboon). This group could be subdivided into a subgroup (A69.11, B 15.3, and B 13.1) reactive with all B virus strains except HZ9; a subgroup (C30.2 and C34.1) reactive exclusively with cynomolgus strains of B virus, and a single monoclonal antibody, C 18.7, which reacted with all of the cynomolgus strains and gave equivocal reactions with two of the rhesus strains. Group B consisted of 9 mabs reacting with all of the B virus and SA 8 strains tested but with none of the HSV strains. Group C comprised 11 mabs reactive with all B virus, SA 8 and HSV strains tested. Of these mabs
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Table 1. Preliminary characterisation of monoclonal antibodies to B virus Monoclonal antibody
Immunoglobulin subclass
Neutralisation of B virus a
Immunofluorescence type
A 40.5 A 69.11 A 84.4 B 1.1 B 3.2 B 6.1 B 7.3 B9.1 B t3.1 B 15.3 B 19.2 C2.1 C 8.4 C 9.2 C 16.2 C 18.7 C 19.3 C21.6 C 30.2 C 34.1 C 36.2 C 39.5 C 40.2 C51.2
IgG t IgG 1 IgG 1 IgG2a IgG 3 IgG 1 IgG 2 b IgG 1 IgG3 ND IgG 1 IgG3 IgG 3 IgG 1 IgG 3 IgG 1 IgG2a IgG2a IgG 2 b IgG 2 a IgG 1 IgG 2 b IgG 1 IgG2a
0 0 0 15 12 17 50 11 0
cytoplasmic cytoplasmm cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic nuclear cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic nuclear
0
28 0 0 27 0 0 0 0 73 69 22 66 0 0
a Percentage reduction of B virus inoculum by plaque assay
9 were produced to B virus and two (H21.1 and H32.2) were produced to HSV. Mabs in groups D and E were produced to HSV-1 and HSV-2. G r o u p D (H20.7 and H 11.25) reacted exclusively with HSV-1 and group E (H23.4 and H 12.3) with HSV-2. N o n e of the monoclonal antibodies reacted with h u m a n varicella-zoster virus, cytomegalovirus of Epstein-Barr virus. F r o m these results a panel of mabs (Table 3) was designed for the identification of primate alphaherpesvirus isolates.
Baboon strain H Z 9 The unexcepted non-reactivity of baboon strain H Z 9 with mabs A 69.11, B 15.3, and B 13.1, which were reactive with all other B virus strains, prompted us to re-examine the typing of this isolate. Neutralisation by the sensitive plaque reduction assay (Table 4) showed that H Z 9 was neutralised by both anti-B virus and anti-SA 8 mouse polyclonal ascitic fluids. In contrast, prototype B virus (strain cyno 2) was not neutralised by anti-SA 8 ascitic fluid, suggesting that H Z 9 is related to SA 8 virus and is not B virus.
BV BV BV BV BV BV BV BV BV BV BV BV SA 8 SA 8 SA 8 SA 8 BV a HSV- 1 HSV-1 HSV- 1 HSV- 1 HSV- 1 HSV-1
+ + + + + + + + + + + + . . . . . . . . . . . . . . . . . . . . . .
+ + + + + + + + + + + + . . . . . . . . . . .
+ + + + + + + + + + + + . . . . . . . . . . .
B13.1
cyno 2 1/375 m 3 / C 20 5/C 9 6/C 6 8 / C 18 1 0 / A 28 m 14/332 12/268 13/559 Sabin 130-65 B 264 0403 X 313 1401 HZ 9 KOS S C 16 HFEM VR 3 s y n 17 + 10711/54
B15.3
Cynomolgus Cynomolgus Cynomolgus Cynomolgus Cynomolgus Cynomolgus Cynomolgus Cynomolgus Rhesus Rhesus R h e s u s (via m a n ) Rhesus Vervet Baboon Baboon Baboon Baboon Human Human Human Human Human Human
A69.11
Group A
strain designation
species
typed as
Monoclonal antibodies
Herpesvirus strains
+ + + + + + + + . . . . . . . . . . .
C30.2
. . . . . . . . . . .
+ + + + + + + + -
C34.1
. . . . . .
+ + + + + + + + 4+ -
C18.7 + + + + + + + + + + + + + + + + +
Group B
+ + + + + + + + + + + + + + + + + + + + + + +
Group C
+ + + + + +
-
Group D
Group E
T a b l e 2. R e a c t i v i t y o f m o n o c l o n a l a n t i b o d i e s b y i n d i r e c t i m m u n o f l u o r e s c e n c e t o h e r p e s v i r u s e s i s o l a t e d f r o m A s i a t i c m o n k e y s , A f r i c a n m o n k e y s , and man
o
t~
Ve 70 Vt 5392 186 Parker Marsden Rawles EB 3
HSV-2 HSV-2 HSV-2 HSV-2 VZV CMV EBV
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . .
. . .
. . .
+ + + +
-
+ + + +
Group A Mabs reacted specifially with B virus by IF Group B Mabs reacted with B virus and SA 8 but not HSV, and comprised clones A 40.5, A 84.4, B 3.2, B 1.1, B 9.1, C 19.3, C 40.2 Group C Mabs reacted with B virus, S A 8 and HSV, and comprised clones B6.1, B7.3, B 19.2, C2.1, C9.2,C21.6, C36.2, C39.5, C51.2, produced to B virus clone H 21.1 produced to HSV-1 and clone H 32.2 produced to HSV-2 Group D Mabs reacted specifically with HSV-1, and comprised clones H 20.7 and H 11.25 produced to HSV-1 Group E Mabs reacted specifically with HSV-2, and comprised clones H 23.4 and H 12.3 produced to HSV-2 a Typed by Wall et al. [21] as B virus by serum neutralisation
Human Human Human Human Human Human Human
O
0 o 0
0
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Table 3. A monoclonal antibody panel for differential identification of alphaherpesvirus isolates
Virus
B virus SA 8 HSV-1 HSV-2
Monoclonal antibody A69.11
A40.5
B 19.2
H 11.25
H23.4
+ -
+ + -
+ + + +
+ -
+
Table 4. Neutralisation of baboon strain HZ 9
Virus
B virus (cyno 2) SA 8 (B 264) Isolate HZ 9
Antiseraa anti B virus
SA 8
98b 99 99
10 t 00 85
a Mouse ascitic fluid produced by immunisation of mice with inactivated virus antigen b Percentage reduction of virus inoculum by plaque assay
Discussion
Several methods have been described for the identification of B virus isolates including serum neutralisation [10], restriction endonuclease analysis [9] and protein profiles by P A G E [8]. Identification of B virus by serum neutralisation is critically dependent upon the quality of serum used, and antigenic cross reactions observed may lead to ambiguous results [10]. Restriction analysis has been used extensively for typing HSV isolates and is a powerful epidemiologicat tool. However, its suitability is restricted by its complexity and the time that it takes. The promising results presented here suggest that monoclonal antibodies will prove useful for typing B virus and that through their use, by direct or indirect fluorescence, they may become the favoured typing method as is already the case for HSV-1 and HSV-2. The mabs could be divided into 5 groups based on their reactivity patterns. Group A was B virus specific, group B was "primate" specific reacting with both B virus and SA 8, and group C was cross-reactive,
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reacting with B virus, SA 8 and HSV-1 and HSV-2. The HSV mabs were either cross-reactive (Group C) or specific for HSV-1 (Group D) or HSV-2 (Group E). Of the twenty seven previously characterised herpes virus strains examined in this study, twenty six gave similar typing results with the monoclonal antibody panel. One strain, HZ 9, previously typed as B virus, gave results identical to the prototype SA 8 virus (Table 2). B virus could be excluded on the basis of these results, although the isolate could not be unequivocally typed as SA 8. This strain was isolated from a genital lesion in a baboon, and had been previously typed as B virus using polyclonal sera. The same authors had shown by restriction endonuclease profiles that this isolate differed significantly from the other B virus strains examined [21]. HZ 9 is the only putative B virus isolate reported from a baboon. These anomalies, its isolation from an African monkey, the lack of reactivity with B virus specific mabs A69.11, B 15.3, and B 13.1 and our serum neutralisation data (Table 4) all strongly suggest that HZ 9 has been mistyped and is in fact an SA 8 strain. These results serve to illustrate the ambiguous results of neutralisation with polyclonal sera in contrast to the unequivocal results obtained by monoclonal antibody analysis. Two B virus specific mabs, C 30.2 and C 34.1, reacted with the cynomolgus but not the rhesus strains of B virus. It may be significant that these two mabs also showed the strongest neutralisation activity. This differentation of cynomologus and rhesus strains by monoclonal antibody analysis is a novel finding and substantiates the differences already observed in restriction endonuclease profiles [21]. The ability to distinguish cynomolgus strains may have important applications in more closely defining the virulence of B virus. It is striking that only rhesus monkeys have been unambiguously linked to human B virus disease [15-] despite the many thousands of cynomolgus monkeys housed in biomedical institutions. It would be interesting to type virus isolates from human B virus infections where there was contact with more than one monkey species or where the aetiological link with monkeys was obscure. The 9 "primate" specific mabs (Group B) reacted with all B virus and SA 8 strains but not with HSV-1 or HSV-2. We did not find any monoclonal antibodies with the reciprocal pattern, i.e., reactive with B virus and HSV but not with SA 8. This suggests that B virus and SA 8 are antigenically more closely related to each other than to either HSV-1 or HSV-2. It would be interesting to raise monoclonal antibodies to SA 8 which may prove useful in defining the evolutionary relationship among Old World primate alphaherpesviruses. This is the first description of mabs specific for B virus. Mabs were chosen for their strong reactivity in the indirect immunofluorescence test, and Table 3 shows the panel of antibodies we propose for the rapid (2-3 h) unequivocal typing of isolates in culture. Although these antibodies have been evaluated on isolates in culture, they may also react with virus infected cells in clinical material, providing the basis for a "same day" assay. This panel of monoclonal antibodies was successfully used to confirm the identity B virus isolates in the 1987 outbreak in Pensacola, Florida (J. K. Hillard, pers. comm., 1988).
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Acknowledgements This work was supported by a special project grant from Medical Research Council. We are grateful to Mr J. Norcott for excellent technical assistance.
References 1. Boulter EA (1975) The isolation of monkey B virus (herpesvirus simiae) from the trigeminal ganglia of a healthy seropositive rhesus monkey. J Biol Stand 3:279-280 2. Daniel MD, Garcia FG, Melendez RD, Hunt RD, O'Connor J, Silva D (1975) Multiple Herpesvirus simiae isolation from a rhesus monkey which died of cerebral infarction. Lab Anim Sci 25:303-308 3. Davenport DS et al (1989) B virus infections in humans-Michigan. MMWR 38: 453454 4. Eberle R, Black D, Hilliard JK (1989) Relatedness of glycoproteins expressed on the surface of simian herpesvirus virions and infected cells to specific HSV glycoproteins. Arch Virol 109:233-252 5. Eichberg JW, McCaltough B, Kalter SS, Thor DE, Rodriguez AR (1975) Clinical, virological and pathological features of herpesvirus SA 8 infection in conventional and gnotobiotic infant baboons (Papio cynocephalus). Arch Virol 50:255-270 6. Griffin DG et al (1987) B virus infection in h u m a n s - Pensacola, Florida. MMWR 36: 289-296 7. Hillard JK, Black D, Eberle R (1989) Simian alphaherpesviruses and their relation to the human herpes simplex viruses. Arch Virol 109:83-102 8. Hillard JK, Eberle R, Lipper SL, Munoz RM, Weiss SA (1987) Herpesvirus simiae (B virus): replication of the virus and identification of viral polypeptides in infected cells. Arch Virol 93:185-194 9. Hillard JK, Munoz RM, Lipper SL, Eberle R (1986) Rapid identification ofherpesvirus simiae (B virus) DNA from clinical isolates in non human primate colonies. J Virol Methods 13:55-62 10. Hull RN (1973) The simian herpesviruses. In: Kaplan AS (ed) The herpesviruses. Academic Press, New York, pp 389.426 11. Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495-497 12. Levine JL, Hilliard JK, Lipper SL, Butler TM, Goodwin WJ (1988) A naturally occurring epizootic of simian agent 8 in the baboon. Lab Anita Sci 38:394-396 13. Lostrom ME, Stone MR, Tam M, Burnette WN, Pinter A, Nowinski RC (1979) Monoclonal antibodies against murine leukemia viruses: identification of six antigenic determinants of the p 15 (E) and gp 70 envelope proteins. Virology 98:336-350 14. Malherbe H, Harwin R (1958) Neurotropic virus in African monkeys. Lancet ii: 530 15. Palmer AE (1987) B virus, herpesvirus simiae: historical perspective. J Med Primatol 16:99-130 16. Powell KL, Watson DH (t975) Some structural antigens of herpes simplex type 1. J Gen Virol 29:167-178 17. Russell WC, Patel G, Precious B, Sharp I, Gardner PS (1981) Monoclonal antibodies against adenovirus type 5. Preparation and preliminary characterisation. J Gen Virol 56:393-408 18. Sabin AB, Wright AM (1934) Acute ascending myelitis following a monkey bite, with the isolation of a virus capable of reproducing the disease. J Exp Med 59:115-136 19. Tedder RS, Yao JL (1982) The production of monoclonat antibodies to rubella haemagg!utin and their use in an antibody capture assay for rubella specific IgM. J Hyg Camb 88:335-350
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20. Vizoso AD (1974) Heterogeneity in herpes simiae (B virus) and some antigenic relationships in the herpes group. Br J Exp Path 55:471~477 21. Wall LV, Zwartouw HT, Kelly DC (1989) Discrimination between twenty isolates of herpesvirus simiae (B virus) by restriction enzyme analysis of the viral genome. Virus Res 12:283-296 Authors' address: Dr. D. Brown, Virus Reference Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5 HT, U.K. Received June 10, 1991
Vi rology
Arch Virol (1992) 123:267-277
© Springer-Verlag 1992 Printed in Austria
Monocional antibodies for the identification of herpesvirus simiae (B virus) L. M. Cropper l, D. N. Lees u*, R. Patt a, I. R. Sharp 2, and D. Brown l Virus Reference Laboratory and 2 Division of Microbiological Reagents and Quality Control, Central Public Health Laboratory, London, U.K. Accepted September 12, 1991
Summary. To differentiate between B virus and HSV isolates from monkeys and man monoclonal antibodies (mabs) were produced to herpesvirus simiae (B virus) and herpes simplex type 1 and 2 (HSV-1 and HSV-2). Mabs were tested by indirect immunofluorescence (IFAT) for reactivity against herpesviruses from Asiatic monkeys (B virus), African monkeys (SA 8 virus), and man (HSV-1, HSV-2, varicella-zoster virus, cytomegalovirus, and Epstein-Barr virus). Mabs could be divided into groups A-E displaying specific reactivity for B virus (A); reactivity with both B virus and SA 8 but not HSV (B); reactivity with B virus, SA 8 virus and HSV strains (C); specific reactivity with HSV-1 (D); and specific reactivity with HSV-2 (E). Two of the B virus specific mabs were able to differentiate between cynomolgus and rhesus strains of B virus. None of the mabs reacted with human varicella-zoster virus, cytomegalovirus, or Epstein-Barr virus. A panel of mabs for the unequivocal identification of B virus isolates from monkey or man is proposed. Introduction Herpesvirus simiae (B virus) is an alphaherpesvirus of macaque monkeys closely related to herpes simplex virus (HSV) of man. B virus infection in naturally infected macaques resembles human HSV infection, with primary oral or genital infection followed by seroconversion and virus latency in nerve ganglia [1, 15]. The medical significance of B virus lies in the lethal ascending encephalomyelitis that may follow transmission of B virus from monkey to man [ 15, 18]. Awareness of this rare but devastating human disease has been heightened by recent cases in Pensacola, Florida [-6] and Kalamozoo, Michigan [3]. The apparent success of early acyclovir therapy in controlling its lethal consequences [-3, 6] * Present address: Fish Disease Laboratory, The Nothe, Weymouth, Dorset, U.K.
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emphasises the need for rapid and unequivocal laboratory diagnosis of B virus infection. Unfortunately identification of virus isolates and serological investigations are confounded by common antigenic determinants among alphaherpesviruses from Old World primates and man [4, 7]. In suspected human B virus infections it is particularly important to distinguish B virus from HSV. This report describes the production of monoclonal antibodies (mabs) to B virus and their characterisation by immunofluorescence against a range of alphaherpesviruses from Old World primates and man. Materials and methods
Virus strains The B virus strain used for mouse immunisation (cyno 2) was an oral isolate from a cynomolgus monkey (Macaca fascicularis) [20]. Virus strains used to characterise monoclonal antibodies were as follows. Cynomolgus B virus strains 1/375 m, 3/C 20, 5/C9, 8/ C 18, 10/A 28 m, 14/332 and rhesus (M. mulatta) strains 12/268 and 13/559 are all monkey isolates described previously [21] and typed as B virus by serum neutralisation. Strain HZ 9 was isolated from a genital lesion in a baboon (Papio sp.) by Professor K. McCarthy, Department of Medical Microbiology, University of Liverpool, and typed as B virus by serum neutralisation [2t]. The SA 8 virus prototype strain (B 264) was isolated from the central nervous system of an African vervet (Cercopithecus aethiops) monkey [14]. The above strains were obtained from Dr. H. Zwartouw, Chemical Defence Establishment, Porton Down, Salisbury, U.K. Prototype B virus (Sabin strain), isolated from the spleen and CNS of a patient with ascending myelitis following a bite from a rhesus monkey [18], was obtained from the American Type Culture Collection (ATCC VR-126). A further B virus strain (130-65) was an isolate from a rhesus monkey which died of cerebral infarction [2] and was obtained from Professor H. Ludwig, Institut fiir Virotogie, Freie Universit/it Berlin, Berlin, Federal Republic of Germany. SA 8 strain 0430 was isolated from the lung of a fatally-infected perinatal baboon [5] and SA 8 strains X 313 and 1401 were genital isolates from baboons housed in an outdoor corral [12]. These strains were obtained from Dr. J. Hilliard, Southwest Foundation for Biochemical Research, San Antonio, Texas. HSV-1 strains KOS, VR3 (MacIntyre) (ATCC VR-539), SC 16, HFEM, S y n l 7 + , and HSV-2 clinical isolates Ve 70 and Vt 5392 were obtained from the North Regional Virus Reference Laboratory, Manchester, U.K. An additional HSV-2 strain 186 was obtained from Dr. H. Zwartouw, Chemical Defence Establishment, Porton Down, Salisbury, U.K. HSV-1 strain VRL 10711/54 was obtained from the PHLS Virus Reference Laboratory, HSV-1 strain Stoker, HSV-2 strain Parker, VZV (Marsden strain), and CMV (Rawles strain) from the PHLS Division of Microbiological Reagents and Quality Control, Central Public Health Laboratory, Colindale, London. EBV was obtained as virus-infected EB3 cells from Dr. M. Hambling, Virology Department, Public Health Laboratory, Leeds, U.K.
Gamma irradiation The radiation source was a gamma-cell model 220 (Atomic Energy Commission of Canada, Ontario, Canada) containing cobalt 60. Antigen preparations for immunisation received 2 x 166 rads: Virus-infected cells for immunofluorescence studies received 3 x 106 rads. All preparations were irradiated on ice. Samples were tested post-irradiation for residual infectious virus by inoculation onto 25 cm 2 flats of Vero cells. Virus-induced cytopathic effects (CPE) were not observed in any irradiated samples.
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Production of monoclonal antibodies Hybridoma cell lines were produced by intramuscular immunisation of 6-10 week old Balb/ c mice with 0.1 ml B virus antigen (strain cyno 2). The antigen preparation consisted of an emulsion of 0.4ml gamma cell inactivated B virus (titre before irradiation 108.5 TCIDs0/ ml, 0.4 ml Freund's incomplete adjuvent and 300 gg muramyl dipeptide (Sigma Ltd., Poole, Dorset). The mice received intravenous booster inoculations of 0.1 ml antigen without adjuvent at 2 weeks, 7 weeks, 'and 13 weeks. Three days after each booster inoculation two mice were sacrificed and their spleens removed for fusion. Spleen cells were fused with myeloma cells JK Ag 8-653 [19] according to the method of Kohler and Milstein [11] with modifications as described by Russell etal. [17]. Hybridoma supernatants were screened for B virus antibody by indirect immunofluorescence. Positive hybridomas were subcultured and cloned twice by limiting dilution on a thymocyte feeder layer following the methods of Lostrom et al. [13]. Clarified hybridoma supernatants were used for all studies. Monoclonal antibodies to HSV-1 and HSV-2 were prepared using a similar regime. Mice were immunised with HSV-1 strain Stoker and HSV-2 strain Parker. Antigen preparations consisted of an emulsion of 1.0 ml purified virus (formaldehyde inactivated; 0.05 % final concentration), 1.0 ml Freund's incomplete adjuvent and 750 gg muramyl dipeptide (Sigma Ltd., Poole, Dorset). The virus was purified following the method of Powell and Watson [16] from 6 x 108 infected Vero cells. Positive hybridomas were identified by indirect immunofluorescence. Six positive hybridomas were selected for further study on the basis of their reactivity with a small panel of B virus and HSV strains.
Indirect immunoJluorescence test Virus-infected cells were prepared by infection of Veto cell monolayers at a multiplicity of infection of 0.01 PFU/cell. Cells were collected by trypsinisation when virus-induced CPE was maximal. Cells were washed in phosphate buffered saline (PBS), resuspended in foetal calf serum and irradiated. Post-irradiation cells were washed 3 times in PBS, dotted on to multiwell slides (Flow Laboratories, Irvine, Scotland) and fixed in cold acetone for 10 rain. Slides were stored at - 70 °C until use. Hybridomas were tested by adding approximately 15 gl of supernatants per well and incubating at room temperature for 20 rain. After washing three times in PBS, bound antibody was detected with FITC-conjugated goat anti-mouse IgG diluted in PBS containing 0.005% Evans Blue counterstain (Sigma Ltd.).
Antibody subclass Monoclonal antibody subclass was determined by Ouchterlony double diffusion. Hybridoma supernatants and goat antisera to specific mouse immunoglobulin subclasses (IgG 1, IgG 2 a, IgG 2 b, and IgG 3) (Sigma Ltd.) were added to adjacent wells on an agarose gel slide and allowed to diffuse overnight in a humidified chamber. Subclasses were identified by the precipitin reactions.
Mouse antisera Polyclonal mouse ascitic fluid was prepared in Balb/c mice against gamma cell irradiated HSV-1 strain (VR 3) and B virus strain (cyno 2), titre before irradiation 10 7.5 TCIDs0/ml and 108.5 TCIDs0/ml respectively. The mice were inoculated intraperitoneally with 0.1 ml virus suspension at day 0, followed by intravenous inoculation of a further 0.1 ml virus suspension at day 10 and day 20. Three days later 106 Ehrliche ascitic tumour cells were inoculated intraperitoneally into each mouse. The mice were observed daily for the production of tumour growth and when this was evident, ascitic fluid was tapped from the mice and centrifuged to remove cells.
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Plaque reduction neutralisation assay Neutralising activity of the mabs and mouse polyclonal sera was tested in a plaque reduction assay. 0.1 ml of hybridoma supernatants were mixed with an equal volume of clarified virus suspension (approx. 100 PFU virus) containing 2.5 HAU of guinea complement (TCS) and incubated for 1 h at room temperature. 0.1 ml volumes were added to duplicate 25 cmz flats of Veto cells, adsorbed for 1 h at room temperature, and overlayed with 5 ml of 1% carboxymethylcellulose in Eagles minimal essential medium (Flow Laboratories). The overlay was removed after 3 days incubation and the cells fixed by immersion in 10% formal saline for 30 rain. Plaques were visualized by staining with 1% Coomassie blue (Sigma) in 45% methanol: 10% acetic acid. Results were expressed as percent plaque reduction compared to control. Results
Preliminary characterisation of monoclonal antibodies to B virus Hybridoma supernatants were screened by immunofluorescence against the homologous virus (B virus strain cyno 2). Twenty four positive hybridomas were identified and cloned. Preliminary characterisation included immunoglobulin subclass, ability to neutralise B virus and type of fluorescence pattern (Table 1). Mabs of subclasses IgG 1, I g G 2 a , I g G 2 b , and IgG3 were isolated. Four mabs B7.3, C30.2, C34.1, and C39.5, demonstrated significant (>f 50%) neutralisation in the plaque reduction assay. Immunofluorescence staining was generally observed in the cytoplasm and on the membrane of infected cells, however, mabs C 21.6 and C 51.2 demonstrated exclusively nuclear staining.
Reactivity against primate and human herpesviruses All of the B virus derived mabs and six selected HSV derived mabs were tested by immunofluorescence for reactivity against a range of primate and human alphaherpesviruses. The alphaherpesviruses were chosen on the basis of their known antigenic cross-reactivity with B virus and included cynomolgus and rhesus strains of B virus, vervet and baboon strains of SA 8 virus and human HSV-1 and HSV-2 strains. Reactivity against human varicella-zoster virus (VZV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV) was also examined. The monoclonal antibodies could be separated into 5 groups (Table2). Group A consisted of 6 mabs reacting exclusively with some or all of the B virus strains (except strain HZ 9 from a baboon). This group could be subdivided into a subgroup (A69.11, B 15.3, and B 13.1) reactive with all B virus strains except HZ9; a subgroup (C30.2 and C34.1) reactive exclusively with cynomolgus strains of B virus, and a single monoclonal antibody, C 18.7, which reacted with all of the cynomolgus strains and gave equivocal reactions with two of the rhesus strains. Group B consisted of 9 mabs reacting with all of the B virus and SA 8 strains tested but with none of the HSV strains. Group C comprised 11 mabs reactive with all B virus, SA 8 and HSV strains tested. Of these mabs
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Table 1. Preliminary characterisation of monoclonal antibodies to B virus Monoclonal antibody
Immunoglobulin subclass
Neutralisation of B virus a
Immunofluorescence type
A 40.5 A 69.11 A 84.4 B 1.1 B 3.2 B 6.1 B 7.3 B9.1 B t3.1 B 15.3 B 19.2 C2.1 C 8.4 C 9.2 C 16.2 C 18.7 C 19.3 C21.6 C 30.2 C 34.1 C 36.2 C 39.5 C 40.2 C51.2
IgG t IgG 1 IgG 1 IgG2a IgG 3 IgG 1 IgG 2 b IgG 1 IgG3 ND IgG 1 IgG3 IgG 3 IgG 1 IgG 3 IgG 1 IgG2a IgG2a IgG 2 b IgG 2 a IgG 1 IgG 2 b IgG 1 IgG2a
0 0 0 15 12 17 50 11 0
cytoplasmic cytoplasmm cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic nuclear cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic nuclear
0
28 0 0 27 0 0 0 0 73 69 22 66 0 0
a Percentage reduction of B virus inoculum by plaque assay
9 were produced to B virus and two (H21.1 and H32.2) were produced to HSV. Mabs in groups D and E were produced to HSV-1 and HSV-2. G r o u p D (H20.7 and H 11.25) reacted exclusively with HSV-1 and group E (H23.4 and H 12.3) with HSV-2. N o n e of the monoclonal antibodies reacted with h u m a n varicella-zoster virus, cytomegalovirus of Epstein-Barr virus. F r o m these results a panel of mabs (Table 3) was designed for the identification of primate alphaherpesvirus isolates.
Baboon strain H Z 9 The unexcepted non-reactivity of baboon strain H Z 9 with mabs A 69.11, B 15.3, and B 13.1, which were reactive with all other B virus strains, prompted us to re-examine the typing of this isolate. Neutralisation by the sensitive plaque reduction assay (Table 4) showed that H Z 9 was neutralised by both anti-B virus and anti-SA 8 mouse polyclonal ascitic fluids. In contrast, prototype B virus (strain cyno 2) was not neutralised by anti-SA 8 ascitic fluid, suggesting that H Z 9 is related to SA 8 virus and is not B virus.
BV BV BV BV BV BV BV BV BV BV BV BV SA 8 SA 8 SA 8 SA 8 BV a HSV- 1 HSV-1 HSV- 1 HSV- 1 HSV- 1 HSV-1
+ + + + + + + + + + + + . . . . . . . . . . . . . . . . . . . . . .
+ + + + + + + + + + + + . . . . . . . . . . .
+ + + + + + + + + + + + . . . . . . . . . . .
B13.1
cyno 2 1/375 m 3 / C 20 5/C 9 6/C 6 8 / C 18 1 0 / A 28 m 14/332 12/268 13/559 Sabin 130-65 B 264 0403 X 313 1401 HZ 9 KOS S C 16 HFEM VR 3 s y n 17 + 10711/54
B15.3
Cynomolgus Cynomolgus Cynomolgus Cynomolgus Cynomolgus Cynomolgus Cynomolgus Cynomolgus Rhesus Rhesus R h e s u s (via m a n ) Rhesus Vervet Baboon Baboon Baboon Baboon Human Human Human Human Human Human
A69.11
Group A
strain designation
species
typed as
Monoclonal antibodies
Herpesvirus strains
+ + + + + + + + . . . . . . . . . . .
C30.2
. . . . . . . . . . .
+ + + + + + + + -
C34.1
. . . . . .
+ + + + + + + + 4+ -
C18.7 + + + + + + + + + + + + + + + + +
Group B
+ + + + + + + + + + + + + + + + + + + + + + +
Group C
+ + + + + +
-
Group D
Group E
T a b l e 2. R e a c t i v i t y o f m o n o c l o n a l a n t i b o d i e s b y i n d i r e c t i m m u n o f l u o r e s c e n c e t o h e r p e s v i r u s e s i s o l a t e d f r o m A s i a t i c m o n k e y s , A f r i c a n m o n k e y s , and man
o
t~
Ve 70 Vt 5392 186 Parker Marsden Rawles EB 3
HSV-2 HSV-2 HSV-2 HSV-2 VZV CMV EBV
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . .
. . .
. . .
+ + + +
-
+ + + +
Group A Mabs reacted specifially with B virus by IF Group B Mabs reacted with B virus and SA 8 but not HSV, and comprised clones A 40.5, A 84.4, B 3.2, B 1.1, B 9.1, C 19.3, C 40.2 Group C Mabs reacted with B virus, S A 8 and HSV, and comprised clones B6.1, B7.3, B 19.2, C2.1, C9.2,C21.6, C36.2, C39.5, C51.2, produced to B virus clone H 21.1 produced to HSV-1 and clone H 32.2 produced to HSV-2 Group D Mabs reacted specifically with HSV-1, and comprised clones H 20.7 and H 11.25 produced to HSV-1 Group E Mabs reacted specifically with HSV-2, and comprised clones H 23.4 and H 12.3 produced to HSV-2 a Typed by Wall et al. [21] as B virus by serum neutralisation
Human Human Human Human Human Human Human
O
0 o 0
0
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Table 3. A monoclonal antibody panel for differential identification of alphaherpesvirus isolates
Virus
B virus SA 8 HSV-1 HSV-2
Monoclonal antibody A69.11
A40.5
B 19.2
H 11.25
H23.4
+ -
+ + -
+ + + +
+ -
+
Table 4. Neutralisation of baboon strain HZ 9
Virus
B virus (cyno 2) SA 8 (B 264) Isolate HZ 9
Antiseraa anti B virus
SA 8
98b 99 99
10 t 00 85
a Mouse ascitic fluid produced by immunisation of mice with inactivated virus antigen b Percentage reduction of virus inoculum by plaque assay
Discussion
Several methods have been described for the identification of B virus isolates including serum neutralisation [10], restriction endonuclease analysis [9] and protein profiles by P A G E [8]. Identification of B virus by serum neutralisation is critically dependent upon the quality of serum used, and antigenic cross reactions observed may lead to ambiguous results [10]. Restriction analysis has been used extensively for typing HSV isolates and is a powerful epidemiologicat tool. However, its suitability is restricted by its complexity and the time that it takes. The promising results presented here suggest that monoclonal antibodies will prove useful for typing B virus and that through their use, by direct or indirect fluorescence, they may become the favoured typing method as is already the case for HSV-1 and HSV-2. The mabs could be divided into 5 groups based on their reactivity patterns. Group A was B virus specific, group B was "primate" specific reacting with both B virus and SA 8, and group C was cross-reactive,
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reacting with B virus, SA 8 and HSV-1 and HSV-2. The HSV mabs were either cross-reactive (Group C) or specific for HSV-1 (Group D) or HSV-2 (Group E). Of the twenty seven previously characterised herpes virus strains examined in this study, twenty six gave similar typing results with the monoclonal antibody panel. One strain, HZ 9, previously typed as B virus, gave results identical to the prototype SA 8 virus (Table 2). B virus could be excluded on the basis of these results, although the isolate could not be unequivocally typed as SA 8. This strain was isolated from a genital lesion in a baboon, and had been previously typed as B virus using polyclonal sera. The same authors had shown by restriction endonuclease profiles that this isolate differed significantly from the other B virus strains examined [21]. HZ 9 is the only putative B virus isolate reported from a baboon. These anomalies, its isolation from an African monkey, the lack of reactivity with B virus specific mabs A69.11, B 15.3, and B 13.1 and our serum neutralisation data (Table 4) all strongly suggest that HZ 9 has been mistyped and is in fact an SA 8 strain. These results serve to illustrate the ambiguous results of neutralisation with polyclonal sera in contrast to the unequivocal results obtained by monoclonal antibody analysis. Two B virus specific mabs, C 30.2 and C 34.1, reacted with the cynomolgus but not the rhesus strains of B virus. It may be significant that these two mabs also showed the strongest neutralisation activity. This differentation of cynomologus and rhesus strains by monoclonal antibody analysis is a novel finding and substantiates the differences already observed in restriction endonuclease profiles [21]. The ability to distinguish cynomolgus strains may have important applications in more closely defining the virulence of B virus. It is striking that only rhesus monkeys have been unambiguously linked to human B virus disease [15-] despite the many thousands of cynomolgus monkeys housed in biomedical institutions. It would be interesting to type virus isolates from human B virus infections where there was contact with more than one monkey species or where the aetiological link with monkeys was obscure. The 9 "primate" specific mabs (Group B) reacted with all B virus and SA 8 strains but not with HSV-1 or HSV-2. We did not find any monoclonal antibodies with the reciprocal pattern, i.e., reactive with B virus and HSV but not with SA 8. This suggests that B virus and SA 8 are antigenically more closely related to each other than to either HSV-1 or HSV-2. It would be interesting to raise monoclonal antibodies to SA 8 which may prove useful in defining the evolutionary relationship among Old World primate alphaherpesviruses. This is the first description of mabs specific for B virus. Mabs were chosen for their strong reactivity in the indirect immunofluorescence test, and Table 3 shows the panel of antibodies we propose for the rapid (2-3 h) unequivocal typing of isolates in culture. Although these antibodies have been evaluated on isolates in culture, they may also react with virus infected cells in clinical material, providing the basis for a "same day" assay. This panel of monoclonal antibodies was successfully used to confirm the identity B virus isolates in the 1987 outbreak in Pensacola, Florida (J. K. Hillard, pers. comm., 1988).
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Acknowledgements This work was supported by a special project grant from Medical Research Council. We are grateful to Mr J. Norcott for excellent technical assistance.
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20. Vizoso AD (1974) Heterogeneity in herpes simiae (B virus) and some antigenic relationships in the herpes group. Br J Exp Path 55:471~477 21. Wall LV, Zwartouw HT, Kelly DC (1989) Discrimination between twenty isolates of herpesvirus simiae (B virus) by restriction enzyme analysis of the viral genome. Virus Res 12:283-296 Authors' address: Dr. D. Brown, Virus Reference Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5 HT, U.K. Received June 10, 1991
