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Analysis of antibody response in goats to caprine herpesvirus 1
Biologicals 33 (2005) 283e287 www.elsevier.com/locate/biologicals
Analysis of antibody response in goats to caprine herpesvirus 1 Maria Tempesta a,*, Grazia Greco a, Elvira Tarsitano a, Julien Thiry a,b, Michele Camero a, Nicola Decaro a, Vito Martella a, Etienne Thiry b, Canio Buonavoglia a a
Department of Animal Health and Well-being, Faculty of Veterinary Medicine, University of Bari, Strada provinciale per Casamassima km. 3, 70010 Valenzano, Italy b Virology, Department of Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, University of Lie`ge, B-4000 Lie`ge, Belgium Received 31 May 2005; received in revised form 7 July 2005; accepted 18 July 2005
Abstract Serum samples of goats experiencing natural and experimental infections and/or reactivation of caprine herpesvirus 1 (CpHV.1) were analysed with neutralization and Western blotting (WB) tests. WB immunological patterns resulted differently and related to neutralizing titers. In serum samples having neutralizing titer 1:2e1:4, antibodies to two proteins of Mw of 150 and 34 kDa were present. Antibodies against several proteins, two of those being characterized by monoclonal antibodies as gB and gC, were visualized by WB in sera having titer R 1:8. The neutralizing antibody titers and the pattern of antibody reactivity were hypothesized to modulate the reactivation and re-excretion process of CpHV.1. Ó 2005 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved. Keywords: Caprine herpesvirus 1; Goat; Antibodies; Western blotting; Reactivation
1. Introduction Caprine herpesvirus 1 (CpHV.1) is responsible for lethal systemic infections in 1- to 2-week-old kids [1,2] and for subclinical infections of the respiratory and genital tracts in adults. CpHV.1 may also cause infertility, abortions, returns to service, and genital lesions [3e5]. In natural conditions, as well as most herpesviruses, CpHV.1 induces a primary infection evolving in latent infection characterized by reactivation [6]. Virus can naturally be reactivated during mating season probably as a result of stress due to hormonal changes associated with oestrus [7,8]. Experimental reactivation occurs following administration of high dosage of desamethasone
* Corresponding author. Tel.: C39 804679838; fax: C39 804679843. E-mail address: [email protected] (M. Tempesta).
sulphate [7,9e11]. Nevertheless, even if in very few occasions it was possible to reactivate CpHV.1 in goats with antibody titers higher than 1:8, usually the CpHV.1 reactivation and re-excretion have been experimentally observed in animals having low virus neutralizing (VN) titers [12] and it is easier to be induced when the antibody value is %1:4. Following a primary infection, neutralizing antibodies are detectable after 1e2 weeks from infection; they generally reach a peak in the following 3rd or 4th week and decrease slowly up to 1:4e1:8 about 6e10 months later; in this stage, when a reactivation stimulus is applied, CpHV.1 can reactivate and be shed by goats. After that, a booster effect will happen inducing an increase in humoral and, above of all, cellular immunity and then, consequently, a state of viral latency will follow. It is well known that inactivated vaccines mainly stimulate humoral immunity. However, in a previous study, an experimental CpHV.1 inactivated vaccine was
1045-1056/05/$30.00 Ó 2005 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biologicals.2005.07.003
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able to provide clinical genital protection and a reduction or absence of viral shedding after challenge in goats immunized with one or two doses of vaccine, respectively [14]. These observations were already made with bovine herpesvirus 1 (BoHV.1), a ruminant alphaherpesvirus closely related to CpHV.1 [13]. Based on the relationship between antibody titers and reactivation, serum samples with different VN titers from CpHV.1 infected goats were analysed by means of Western blotting (WB) in order to single out to which viral protein specific antibodies are produced and whether different reactivity patterns exist based on the serum titers.
apart [14]. Two negative field sera (no. 40 and no. 41) coming from a CpHV.1 free goat flock were used as negative control. Serum having a titer of 1:256, collected from a goat 10 months after an experimental reactivation, was used as positive control [6]. 2.2. Monoclonal antibodies Monoclonal antibody (MAb) 3A4C8G8 is directed against CpHV.1 glycoprotein B and MAb 2E5G5G1 against glycoprotein C [15]. Both MAbs were used at the dilution of 1:5. 2.3. Western blotting (WB) test
2. Materials and methods 2.1. Goat serum samples Forty-two sera were analysed in this study (Table 1). Two serum samples (no. 1 and no. 2) had VN titer of 1:2 and they belonged to naturally infected animals; 12 serum samples (no. 3e14) had a VN titer of 1:4 and were collected from naturally infected animals (no. 3e10) or from animals before the pharmacological reactivation of the virus (no. 11 and no. 12) [6], or from goats before natural reactivation at oestrus (no. 13 and no. 14) [8]; 15 serum samples (no. 15e29) belonged to naturally infected goats and had a VN titer of 1:8; three serum samples (no. 30e32) came from naturally infected animals and had a VN titer of 1:16; two serum samples had a VN titer of 1:32, one of which was collected from a naturally infected goat (no. 33) and the other from a goat 30 days after experimental intravaginal infection (no. 34) [12]; three serum samples (no. 35e37) with a VN titer of 1:64 were collected from goats 30 days after experimental intranasal infection [12]; two serum samples (no. 38 and no. 39) with a VN titer of 1:128 were collected from goats 80 days after the inoculation of an inactivated vaccine against CpHV.1 given twice a month Table 1 Goat serum samples tested for antibodies to CpHV.1 Serum samples
VN titersa Sources/[references]
1e2 3e10 11e12 13e14 15e29 30e32 33 34 35e37 38e39 40e41 Positive control
1:2 1:4 1:4 1:4 1:8 1:16 1:32 1:32 1:64 1:128 Negative 1:256
a
Virus neutralizing titers.
Field sera Field sera Before experimental reactivation/[6] Before natural reactivation/[8] Field sera Field sera Field serum After experimental vaginal infection/[12] After experimental nasal infection/[12] 80 Days after immunization/[14] Field sera 10 Months after experimental reactivation/[8]
Ba.1 strain [9] of CpHV.1 was cultured in Madin Darby bovine kidney (MDBK) cells grown in Dulbecco’s minimal essential medium (D-MEM) containing 10% bovine foetal serum. MDBK cells’ monolayer infected with CpHV.1 was incubated for 3 days at 37 C. When the cytopathic effect (cpe) was complete, the supernatant was collected and spun at 3000 ! g for 20 min at 4 C to remove cell debris. Then the supernatant was centrifuged for 18 h at 10,000 ! g. and the pellet was suspended in 50 mM Tris/HCl pH 6.8 and left at 4 C for 3e4 h to disperse clumps. Total protein concentration was measured with the Bio-Rad protein assay (Biorad Laboratories S.r.l., USA), based on the Bradford dye-binding procedure and then subjected to SDS-PAGE according to Laemmli [16] on 4e22% resolving gels and 4% stacking gels. Disruption of viral proteins was carried out by the addition of an equal volume of ‘‘sample buffer’’ (Tris/HCl, 0.05 M (pH 6.8); SDS 2.5%; 2-b-mercaptoethanol 5%; glycerol 15%; bromophenol blue 0.05%). This was placed in a sealed vial and incubated in a water bath at 100 C for 6 min. One hundred and forty milligrams of proteins was loaded per gel. Separation was carried out by using a 20-cm vertical electrophoresis apparatus (Biorad Laboratories S.r.l., USA), at 20 mA/ gel in 25 mM Trise192 mM glycine buffer, pH 8.3, containing 0.1% SDS. The molecular weights of the bands were determined by comparing the relative front (Rf) values of these bands with the Rf values of markers of known molecular weights (Prestained Precision Plus ProteinÔ standard, Biorad Laboratories S.r.l., USA) using the Quantity One Image analysis software (Biorad Laboratories S.r.l., USA). After electrophoresis, the fractionated components were electrophoretically (Biorad Transblot cell apparatus) transferred onto polyvinylidene difluoride membrane (PVDF) (Immobilon P, pore size 0.45 mm, Biorad Laboratories S.r.l., USA) with a current of 50 V for 18 h at 10 C (electrode buffer: 25 mM Trise 192 mM glycine buffer (pH 8.3), 10% methanol). Once the proteins had been transferred onto PVDF membrane, it was cut into strips and they were blocked
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by incubating with 1% bovine seroalbumin (Sigma Chemicals, St. Louis, MO) diluted in 0.01 M Tris-buffered saline (TBS) (pH 7.5) overnight at 4 C with gentle shaking. The strips were then washed three times with TBS containing 0.1% bovine seroalbumin, each wash lasting for 10 min. Strips were then incubated for 2 h at 25 C with field sera diluted 1:100, or monoclonal antibodies diluted 1:5 in 0.1% bovine seroalbumin in TBS. The strips were then washed exactly as above and incubated for 2 h at 25 C with 1:6000 peroxidase labelled rabbit IgG anti-goat IgG or with 1:4000 peroxidase labelled rabbit IgG anti-mouse IgG (Sigma Chemicals, St. Louis, MO). Once again the strips were washed as above, and treated with developing solution [3,3#diaminobenzidine tetrahydrochloride (Sigma Chemicals, St. Louis, MO) in TBS (pH 7.8), 0.08% hydrogen peroxide]. When the color reaction was complete, further reaction was stopped by washing with distilled water. These incubation steps were carried out with gentle shaking at room temperature.
of MAbs 3A4C8G8 and 2E5G5G1, respectively, against CpHV.1 glycoproteins B and C. Row A shows the pattern of positive serum (titer 1:256) in which it is possible to observe reactivity to more than 30 viral proteins. Sera having titers 1:2 and 1:4 (rows B and C) have similar patterns sharing antibodies against two viral proteins with molecular weights of 150 and 34 kDa, respectively. All sera with a titer of 1:8 (row D) have antibodies raised against viral proteins of about 150, 92, 53, 46, 40 and 34 kDa. Sera with titers O 1:8 show reactivity to more than 30 viral proteins. In row E the pattern of a serum having VN titer of 1:32 is shown. MAb 3A4C8G8 specific for glycoprotein B has reacted with a protein of about 53 kDa and MAb 2E5G5G1 specific for glycoprotein C reached with a protein of about 92 kDa. Table 2 Western blotting reactivity of sera from CpHV.1 infected goats having different VN titers A, Mw B, 1:2a C, 1:4 (kDa)
3. Results and discussion Results are reported in Fig. 1 and Table 2. Molecular weights of proteins recognized by positive sera are listed. Reactive patterns of positive sera having VN titers of 1:2, 1:4, 1:8, and O1:8 (1:32) are reported with those
Fig. 1. Western blotting reactivity of sera from CpHV.1 infected goats having different VN titers. M: Marker e Prestained Precision Plus ProteinÔ standard (Biorad Laboratories S.r.l., USA); A: CpHV.1 positive control serum (titer 1:256); BeE: CpHV.1 positive sera with titers of 1:2, 1:4, 1:8 and 1:32, respectively; F and G: CpHV.1 monoclonal antibodies 3A4C8G8 and 2E5G5G1 to glycoproteins B (anti-gB) and C (anti-gC), respectively; H: negative control serum. *Seroneutralizing titer of sera.
1 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.
150.14 113.85 94.98 92.78 88.01 85.47 80.60 76.46 68.41 61.99 58.55 56.46 53.88 49.45 48.62 46.41 40.02 38.52 36.93 35.92 34.67 34.26 32.80 30.55 28.90 27.45 25.87 20.27 19.22 18.05 15.55 15.19 12.60 11.04
150.14
34.26
D, 1:8 E, O1:8 F, MAb G, MAb anti-gB anti-gC
150.14 150.14 150.14 113.85 94.98 92.78 92.78 88.01 85.47 80.60 76.46 68.41 61.99 58.55 56.46 53.88 53.88 49.45 48.62 46.41 46.41 40.02 40.02 38.52 36.93 35.92 34.67 34.26 34.26 34.26 32.80 30.55 28.90 27.45 25.87 20.27 19.22 18.05 15.55 15.19 12.60 11.04
92.78
53.88
Mw: Molecular weight; A: CpHV.1 positive control serum; BeE: Reactivity of CpHV.1 positive sera with titer of 1:2, 1:4, 1:8 and O1:8, respectively; F and G: Reactivity of anti-CpHV.1 monoclonal antibodies 3A4C8G8 and 2E5G5G1 to glycoproteins B (anti-gB) and C (anti-gC), respectively. a Neutralizing titer of sera.
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CpHV.1 is closely related to BoHV.1 based on clinical signs and genomic and antigenic features [17,18]. WB and radioimmunoassay analyses of monoclonal and polyclonal antibodies to BoHV.1 and CpHV.1 have revealed that cross reacting antibodies are induced by glycoprotein B, by a major capsidic protein and by non-structural proteins whereas glycoproteins C and D induce virus-specific antibodies [19,20]. Mab 3A4C8G8 reactivity to 53.88 kDa protein (p50) supports the identity of p50 with glycoprotein B according to Ros and Belak [21], who proved the strict homology between gB of CpHV.1 and BoHV.1. BoHV.1 gB is an heterodimer of 130 kDa separated by SDS-PAGE into two proteins of 74 and 55 kDa [22]. In the same way Mab 2E5G5G1 reactivity to 92.78 kDa protein supports the identity of this protein with glycoprotein C [15]. In fact, gC is a homodimer of 180 kDa that in SDS-PAGE undergoes a disulphuric bound rupture originating two proteins of about 91 kDa each. Sera with titers of 1:2 and 1:4 show identical patterns having antibodies to 150.14 and 34.26 kDa proteins; 150.14 kDa protein has a molecular weight equivalent to that of VP4 which is the major capsidic protein of BoHV.1, whereas 34.26 kDa protein seems to be glycoprotein K on the basis of the molecular weight [23]. Antibodies to gB, gC, p46, and p40 detected in the serum samples with a titer of 1:8, are absent in sera with titers of 1:4 independently from the history of the infection (i.e. reactivation and latent infection). It could be hypothesized that these antibodies together with other factors are to be able to modulate the viral reactivation and excretion. These data are in agreement with the pathogenic role of viral surface glycoproteins against which antibodies in sera higher than 1:4 were arisen. Glycoproteins B and gC together with gD are responsible for viral pathogenicity: gC and gB allow the adsorption to the target cell surface binding to a heparinlike receptor [24,25]; after that gD binds to a second cell receptor leading to viral penetration into the cell. Glycoprotein B is also involved in fusion mechanisms between viral envelope and cell membrane [26]. Therefore, it seems that antibodies raised against these glycoproteins are able to affect viral pathobiology blocking one or several steps during the reactivation and re-excretion process. In view of control of the spreading of infection within the flocks, these data are interesting because such immune response prevents the epidemiological consequences, i.e. the re-excretion and the transmission of the virus among a flock. It is well known that cell-mediated immunity plays a major role against herpesviral infections. Nevertheless, high levels of neutralizing antibody seem to modulate the process of CpHV.1 infectionereactivation. In this view subunit or recombinant vaccines expressing the gB and/ or gC glycoproteins could provide a good clinical and virological protection against CpHV.1 infection in goats.
Acknowledgements The authors thank Dr. Veronique Keuser for stimulating discussions. The work was supported by grants from MIUR ex 40% 2002.
References [1] Berrios PE, McKercher DG. Characterization of a caprine herpesvirus. Am J Vet Res 1975;36:1755e62. [2] Tempesta M, Pratelli A, Buonavoglia D, Greco G. Infezione da caprine herpesvirus 1 e mortalita` neonatale in capretti. Large Anim Rev 1998;4:59e62. [3] Horner GW, Hunter R, Day AM. An outbreak of vulvovaginitis in goats caused by a caprine herpesvirus. N Z Vet J 1982;30:150e2. [4] Tempesta M, Camero M, Sciorsci RL, Greco G, Minoia R, Martella V, et al. Experimental infection of goats at different stages of pregnancy with caprine herpesvirus 1. Comp Immunol Microbiol Infect Dis 2004;27:25e32. [5] Williams NM, Vickers ML, Tramontin RR, PetritesMurphy MB, Allen GP. Multiple abortions associated with caprine herpesvirus infection in goat herd. J Am Vet Med Assoc 1997;211:89e91. [6] Tempesta M, Buonavoglia D, Greco G, Pratelli A, Normanno G, Carelli G, et al. A study on the infection and reactivation of caprine herpesvirus 1 in goats. In: Proceedings of seventh international conference on goat, Tours. p. 288e9. [7] Koptopoulos G, Papanastasopoulou M, Papadopoulos O, Ludwig H. The epizootology of caprine herpesvirus (BHV-6) infections in goat populations in Greece. Comp Immunol Microbiol Infect Dis 1988;11:199e205. [8] Tempesta M, Buonavoglia D, Sagazio P, Pratelli A, Buonavoglia C. Natural reactivation of caprine herpesvirus 1 in latently infected goats. Vet Rec 1998;143:200. [9] Buonavoglia C, Tempesta M, Cavalli A, Voigt V, Buonavoglia D, Conserva A, et al. Reactivation of caprine herpesvirus 1 in latently infected goats. Comp Immunol Microbiol Infect Dis 1996;19: 275e81. [10] Papanastasopoulou M, Koptopoulos G, Lekkas S, Georgakopoulou E, Papadopoulos O. A preliminary investigation on the latency of the goat herpesvirus BHV-6. Zentralbl Veterinarmed B 1990;37:781e4. [11] Plebani GF, Engels M, Metzler AE, Wyler R. Caprine herpesvirus in der Schweiz: verbreitung, ha¨ufigkeit und latenz der infektion. Schweiz Arch Tierheilkd 1983;125:395e411. [12] Tempesta M, Greco G, Pratelli A, Buonavoglia D, Camero M, Martella V, et al. Reactivation of caprine herpesvirus 1 in experimentally infected goats. Vet Rec 2002;4:116e7. [13] Pastoret PP, Thiry E, Brochier B, Derboven G, Vindevogel H. The role of the epizootiology of infectious bovine rhinotracheitis. In: Wittmann G, Gaskell RM, Rziha HJ, editors. Latent herpesvirus infections in veterinary medicine. Dordecht: Martinus Nijhoff Publishers; 1984. p. 211e27. [14] Tempesta M, Camero M, Greco G, Pratelli A, Martella V, Buonavoglia C. A classical inactivated vaccine induces protection against caprine herpesvirus 1 infection in goats. Vaccine 2001; 19:3860e4. [15] Keuser V, Schynts F, Detry B, Collard A, Robert B, Vanderplasschen A, et al. Improved antigenic methods for the differential diagnosis of bovine, caprine and cervine alphaherpesviruses related to bovine herpesvirus 1. J Clin Microbiol 2004; 42:1228e35. [16] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 1970;227:680e5.
M. Tempesta et al. / Biologicals 33 (2005) 283e287 [17] Engels M, Palatini M, Metzler AE, Probst U, Kihm U, Ackermann M. Interactions of bovine and caprine herpesviruses with the natural and the foreign hosts. Vet Microbiol 1992;33:69e78. [18] Ros C, Belak S. Studies of genetic relationships between bovine, caprine, cervine, and rangiferine alphaherpesviruses and improved molecular methods for virus detection and identification. J Clin Microbiol 1999;37:1247e53. [19] Ackermann M, Metzler AE, McDonaugh H, Bruckner L, Muller HK, Kihm U. Stellen nichtbovine paarhufer ein IBRVirus-reservoir dar? I. BHV-1 und CapHV-1 infektions und reaktivierungsversuche an ziegen, virustyp-spezifitat der humoralen antikorper und charakterisierung der viralen antigene. Schweiz Arch Tierheilkd 1986;128:557e73. [20] Friedli K, Metzler AE. Reactivity of monoclonal antibodies to proteins of a neurotropic bovine herpesvirus (BHV.1) strain and to proteins of representative BHV-1 strains. Arch Virol 1987; 94:109e12.
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[21] Ros C, Belak S. Characterization of the glycoprotein B gene from ruminant alphaherpesviruses. Virus Genes 2002;24: 99e105. [22] Tikoo SK, Campos M, Babiuk LA. Bovine herpesvirus 1 (BHV-1): biology, pathogenesis, and control. Adv Virus Res 1995;45: 191e223. [23] Khadr A, Tikoo SK, Babiuk LA, van Drunen Littel-van den Hurk S. Sequence and expression of a bovine herpesvirus-1 gene homologous to the glycoprotein K-encoding gene of herpes simplex virus-1. Gene 1996;168:189e93. [24] Byrne KM, Horohov DW, Kousoulas KG. Glycoprotein B of bovine herpesvirus-1 binds heparin. Virology 1995;209:230e5. [25] Okazaki K, Honda E, Kono Y. Heparin-binding domain of bovid herpesvirus 1 glycoprotein gIII. Arch Virol 1994;134:413e9. [26] Fitzpatrick DR, Zamb TJ, Babiuk LA. Expression of bovine herpesvirus type 1 glycoprotein gI in transfected bovine cells induces spontaneous cell fusion. J Gen Virol 1990;71:1215e9.
Analysis of antibody response in goats to caprine herpesvirus 1 Maria Tempesta a,*, Grazia Greco a, Elvira Tarsitano a, Julien Thiry a,b, Michele Camero a, Nicola Decaro a, Vito Martella a, Etienne Thiry b, Canio Buonavoglia a a
Department of Animal Health and Well-being, Faculty of Veterinary Medicine, University of Bari, Strada provinciale per Casamassima km. 3, 70010 Valenzano, Italy b Virology, Department of Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, University of Lie`ge, B-4000 Lie`ge, Belgium Received 31 May 2005; received in revised form 7 July 2005; accepted 18 July 2005
Abstract Serum samples of goats experiencing natural and experimental infections and/or reactivation of caprine herpesvirus 1 (CpHV.1) were analysed with neutralization and Western blotting (WB) tests. WB immunological patterns resulted differently and related to neutralizing titers. In serum samples having neutralizing titer 1:2e1:4, antibodies to two proteins of Mw of 150 and 34 kDa were present. Antibodies against several proteins, two of those being characterized by monoclonal antibodies as gB and gC, were visualized by WB in sera having titer R 1:8. The neutralizing antibody titers and the pattern of antibody reactivity were hypothesized to modulate the reactivation and re-excretion process of CpHV.1. Ó 2005 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved. Keywords: Caprine herpesvirus 1; Goat; Antibodies; Western blotting; Reactivation
1. Introduction Caprine herpesvirus 1 (CpHV.1) is responsible for lethal systemic infections in 1- to 2-week-old kids [1,2] and for subclinical infections of the respiratory and genital tracts in adults. CpHV.1 may also cause infertility, abortions, returns to service, and genital lesions [3e5]. In natural conditions, as well as most herpesviruses, CpHV.1 induces a primary infection evolving in latent infection characterized by reactivation [6]. Virus can naturally be reactivated during mating season probably as a result of stress due to hormonal changes associated with oestrus [7,8]. Experimental reactivation occurs following administration of high dosage of desamethasone
* Corresponding author. Tel.: C39 804679838; fax: C39 804679843. E-mail address: [email protected] (M. Tempesta).
sulphate [7,9e11]. Nevertheless, even if in very few occasions it was possible to reactivate CpHV.1 in goats with antibody titers higher than 1:8, usually the CpHV.1 reactivation and re-excretion have been experimentally observed in animals having low virus neutralizing (VN) titers [12] and it is easier to be induced when the antibody value is %1:4. Following a primary infection, neutralizing antibodies are detectable after 1e2 weeks from infection; they generally reach a peak in the following 3rd or 4th week and decrease slowly up to 1:4e1:8 about 6e10 months later; in this stage, when a reactivation stimulus is applied, CpHV.1 can reactivate and be shed by goats. After that, a booster effect will happen inducing an increase in humoral and, above of all, cellular immunity and then, consequently, a state of viral latency will follow. It is well known that inactivated vaccines mainly stimulate humoral immunity. However, in a previous study, an experimental CpHV.1 inactivated vaccine was
1045-1056/05/$30.00 Ó 2005 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biologicals.2005.07.003
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able to provide clinical genital protection and a reduction or absence of viral shedding after challenge in goats immunized with one or two doses of vaccine, respectively [14]. These observations were already made with bovine herpesvirus 1 (BoHV.1), a ruminant alphaherpesvirus closely related to CpHV.1 [13]. Based on the relationship between antibody titers and reactivation, serum samples with different VN titers from CpHV.1 infected goats were analysed by means of Western blotting (WB) in order to single out to which viral protein specific antibodies are produced and whether different reactivity patterns exist based on the serum titers.
apart [14]. Two negative field sera (no. 40 and no. 41) coming from a CpHV.1 free goat flock were used as negative control. Serum having a titer of 1:256, collected from a goat 10 months after an experimental reactivation, was used as positive control [6]. 2.2. Monoclonal antibodies Monoclonal antibody (MAb) 3A4C8G8 is directed against CpHV.1 glycoprotein B and MAb 2E5G5G1 against glycoprotein C [15]. Both MAbs were used at the dilution of 1:5. 2.3. Western blotting (WB) test
2. Materials and methods 2.1. Goat serum samples Forty-two sera were analysed in this study (Table 1). Two serum samples (no. 1 and no. 2) had VN titer of 1:2 and they belonged to naturally infected animals; 12 serum samples (no. 3e14) had a VN titer of 1:4 and were collected from naturally infected animals (no. 3e10) or from animals before the pharmacological reactivation of the virus (no. 11 and no. 12) [6], or from goats before natural reactivation at oestrus (no. 13 and no. 14) [8]; 15 serum samples (no. 15e29) belonged to naturally infected goats and had a VN titer of 1:8; three serum samples (no. 30e32) came from naturally infected animals and had a VN titer of 1:16; two serum samples had a VN titer of 1:32, one of which was collected from a naturally infected goat (no. 33) and the other from a goat 30 days after experimental intravaginal infection (no. 34) [12]; three serum samples (no. 35e37) with a VN titer of 1:64 were collected from goats 30 days after experimental intranasal infection [12]; two serum samples (no. 38 and no. 39) with a VN titer of 1:128 were collected from goats 80 days after the inoculation of an inactivated vaccine against CpHV.1 given twice a month Table 1 Goat serum samples tested for antibodies to CpHV.1 Serum samples
VN titersa Sources/[references]
1e2 3e10 11e12 13e14 15e29 30e32 33 34 35e37 38e39 40e41 Positive control
1:2 1:4 1:4 1:4 1:8 1:16 1:32 1:32 1:64 1:128 Negative 1:256
a
Virus neutralizing titers.
Field sera Field sera Before experimental reactivation/[6] Before natural reactivation/[8] Field sera Field sera Field serum After experimental vaginal infection/[12] After experimental nasal infection/[12] 80 Days after immunization/[14] Field sera 10 Months after experimental reactivation/[8]
Ba.1 strain [9] of CpHV.1 was cultured in Madin Darby bovine kidney (MDBK) cells grown in Dulbecco’s minimal essential medium (D-MEM) containing 10% bovine foetal serum. MDBK cells’ monolayer infected with CpHV.1 was incubated for 3 days at 37 C. When the cytopathic effect (cpe) was complete, the supernatant was collected and spun at 3000 ! g for 20 min at 4 C to remove cell debris. Then the supernatant was centrifuged for 18 h at 10,000 ! g. and the pellet was suspended in 50 mM Tris/HCl pH 6.8 and left at 4 C for 3e4 h to disperse clumps. Total protein concentration was measured with the Bio-Rad protein assay (Biorad Laboratories S.r.l., USA), based on the Bradford dye-binding procedure and then subjected to SDS-PAGE according to Laemmli [16] on 4e22% resolving gels and 4% stacking gels. Disruption of viral proteins was carried out by the addition of an equal volume of ‘‘sample buffer’’ (Tris/HCl, 0.05 M (pH 6.8); SDS 2.5%; 2-b-mercaptoethanol 5%; glycerol 15%; bromophenol blue 0.05%). This was placed in a sealed vial and incubated in a water bath at 100 C for 6 min. One hundred and forty milligrams of proteins was loaded per gel. Separation was carried out by using a 20-cm vertical electrophoresis apparatus (Biorad Laboratories S.r.l., USA), at 20 mA/ gel in 25 mM Trise192 mM glycine buffer, pH 8.3, containing 0.1% SDS. The molecular weights of the bands were determined by comparing the relative front (Rf) values of these bands with the Rf values of markers of known molecular weights (Prestained Precision Plus ProteinÔ standard, Biorad Laboratories S.r.l., USA) using the Quantity One Image analysis software (Biorad Laboratories S.r.l., USA). After electrophoresis, the fractionated components were electrophoretically (Biorad Transblot cell apparatus) transferred onto polyvinylidene difluoride membrane (PVDF) (Immobilon P, pore size 0.45 mm, Biorad Laboratories S.r.l., USA) with a current of 50 V for 18 h at 10 C (electrode buffer: 25 mM Trise 192 mM glycine buffer (pH 8.3), 10% methanol). Once the proteins had been transferred onto PVDF membrane, it was cut into strips and they were blocked
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by incubating with 1% bovine seroalbumin (Sigma Chemicals, St. Louis, MO) diluted in 0.01 M Tris-buffered saline (TBS) (pH 7.5) overnight at 4 C with gentle shaking. The strips were then washed three times with TBS containing 0.1% bovine seroalbumin, each wash lasting for 10 min. Strips were then incubated for 2 h at 25 C with field sera diluted 1:100, or monoclonal antibodies diluted 1:5 in 0.1% bovine seroalbumin in TBS. The strips were then washed exactly as above and incubated for 2 h at 25 C with 1:6000 peroxidase labelled rabbit IgG anti-goat IgG or with 1:4000 peroxidase labelled rabbit IgG anti-mouse IgG (Sigma Chemicals, St. Louis, MO). Once again the strips were washed as above, and treated with developing solution [3,3#diaminobenzidine tetrahydrochloride (Sigma Chemicals, St. Louis, MO) in TBS (pH 7.8), 0.08% hydrogen peroxide]. When the color reaction was complete, further reaction was stopped by washing with distilled water. These incubation steps were carried out with gentle shaking at room temperature.
of MAbs 3A4C8G8 and 2E5G5G1, respectively, against CpHV.1 glycoproteins B and C. Row A shows the pattern of positive serum (titer 1:256) in which it is possible to observe reactivity to more than 30 viral proteins. Sera having titers 1:2 and 1:4 (rows B and C) have similar patterns sharing antibodies against two viral proteins with molecular weights of 150 and 34 kDa, respectively. All sera with a titer of 1:8 (row D) have antibodies raised against viral proteins of about 150, 92, 53, 46, 40 and 34 kDa. Sera with titers O 1:8 show reactivity to more than 30 viral proteins. In row E the pattern of a serum having VN titer of 1:32 is shown. MAb 3A4C8G8 specific for glycoprotein B has reacted with a protein of about 53 kDa and MAb 2E5G5G1 specific for glycoprotein C reached with a protein of about 92 kDa. Table 2 Western blotting reactivity of sera from CpHV.1 infected goats having different VN titers A, Mw B, 1:2a C, 1:4 (kDa)
3. Results and discussion Results are reported in Fig. 1 and Table 2. Molecular weights of proteins recognized by positive sera are listed. Reactive patterns of positive sera having VN titers of 1:2, 1:4, 1:8, and O1:8 (1:32) are reported with those
Fig. 1. Western blotting reactivity of sera from CpHV.1 infected goats having different VN titers. M: Marker e Prestained Precision Plus ProteinÔ standard (Biorad Laboratories S.r.l., USA); A: CpHV.1 positive control serum (titer 1:256); BeE: CpHV.1 positive sera with titers of 1:2, 1:4, 1:8 and 1:32, respectively; F and G: CpHV.1 monoclonal antibodies 3A4C8G8 and 2E5G5G1 to glycoproteins B (anti-gB) and C (anti-gC), respectively; H: negative control serum. *Seroneutralizing titer of sera.
1 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.
150.14 113.85 94.98 92.78 88.01 85.47 80.60 76.46 68.41 61.99 58.55 56.46 53.88 49.45 48.62 46.41 40.02 38.52 36.93 35.92 34.67 34.26 32.80 30.55 28.90 27.45 25.87 20.27 19.22 18.05 15.55 15.19 12.60 11.04
150.14
34.26
D, 1:8 E, O1:8 F, MAb G, MAb anti-gB anti-gC
150.14 150.14 150.14 113.85 94.98 92.78 92.78 88.01 85.47 80.60 76.46 68.41 61.99 58.55 56.46 53.88 53.88 49.45 48.62 46.41 46.41 40.02 40.02 38.52 36.93 35.92 34.67 34.26 34.26 34.26 32.80 30.55 28.90 27.45 25.87 20.27 19.22 18.05 15.55 15.19 12.60 11.04
92.78
53.88
Mw: Molecular weight; A: CpHV.1 positive control serum; BeE: Reactivity of CpHV.1 positive sera with titer of 1:2, 1:4, 1:8 and O1:8, respectively; F and G: Reactivity of anti-CpHV.1 monoclonal antibodies 3A4C8G8 and 2E5G5G1 to glycoproteins B (anti-gB) and C (anti-gC), respectively. a Neutralizing titer of sera.
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CpHV.1 is closely related to BoHV.1 based on clinical signs and genomic and antigenic features [17,18]. WB and radioimmunoassay analyses of monoclonal and polyclonal antibodies to BoHV.1 and CpHV.1 have revealed that cross reacting antibodies are induced by glycoprotein B, by a major capsidic protein and by non-structural proteins whereas glycoproteins C and D induce virus-specific antibodies [19,20]. Mab 3A4C8G8 reactivity to 53.88 kDa protein (p50) supports the identity of p50 with glycoprotein B according to Ros and Belak [21], who proved the strict homology between gB of CpHV.1 and BoHV.1. BoHV.1 gB is an heterodimer of 130 kDa separated by SDS-PAGE into two proteins of 74 and 55 kDa [22]. In the same way Mab 2E5G5G1 reactivity to 92.78 kDa protein supports the identity of this protein with glycoprotein C [15]. In fact, gC is a homodimer of 180 kDa that in SDS-PAGE undergoes a disulphuric bound rupture originating two proteins of about 91 kDa each. Sera with titers of 1:2 and 1:4 show identical patterns having antibodies to 150.14 and 34.26 kDa proteins; 150.14 kDa protein has a molecular weight equivalent to that of VP4 which is the major capsidic protein of BoHV.1, whereas 34.26 kDa protein seems to be glycoprotein K on the basis of the molecular weight [23]. Antibodies to gB, gC, p46, and p40 detected in the serum samples with a titer of 1:8, are absent in sera with titers of 1:4 independently from the history of the infection (i.e. reactivation and latent infection). It could be hypothesized that these antibodies together with other factors are to be able to modulate the viral reactivation and excretion. These data are in agreement with the pathogenic role of viral surface glycoproteins against which antibodies in sera higher than 1:4 were arisen. Glycoproteins B and gC together with gD are responsible for viral pathogenicity: gC and gB allow the adsorption to the target cell surface binding to a heparinlike receptor [24,25]; after that gD binds to a second cell receptor leading to viral penetration into the cell. Glycoprotein B is also involved in fusion mechanisms between viral envelope and cell membrane [26]. Therefore, it seems that antibodies raised against these glycoproteins are able to affect viral pathobiology blocking one or several steps during the reactivation and re-excretion process. In view of control of the spreading of infection within the flocks, these data are interesting because such immune response prevents the epidemiological consequences, i.e. the re-excretion and the transmission of the virus among a flock. It is well known that cell-mediated immunity plays a major role against herpesviral infections. Nevertheless, high levels of neutralizing antibody seem to modulate the process of CpHV.1 infectionereactivation. In this view subunit or recombinant vaccines expressing the gB and/ or gC glycoproteins could provide a good clinical and virological protection against CpHV.1 infection in goats.
Acknowledgements The authors thank Dr. Veronique Keuser for stimulating discussions. The work was supported by grants from MIUR ex 40% 2002.
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