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Interactions of bovine and caprine herpesviruses with the natural and the foreign hosts
Veterinary Microbiology, 33 (1992) 69-78 Elsevier Science Publishers B.V., Amsterdam
69
Interactions of bovine and caprine herpesviruses with the natural and the foreign hosts M o n i k a Engels a, M o n i c a P a l a t i n i a, A l f r e d E. M e t z l e r ~, U r s P r o b s t u, U e l i K i h m c and Mathias Ackermann a alnstitute of Virology, Veterinary Medical Facuhy, University of Ziirich, Switzerland bVeterinaria AG, Ziirich, Switzerland Clnstitute of Viral Diseases, Basel, Switzerland (Accepted 26 June 1992)
ABSTRACT Engels, M., Palatini, M., Metzler, A.E., Probst, U., Kihm, U. and Ackermann, M., 1992. Interactions of bovine and caprine herpesviruses with the natural and the foreign hosts. Vet. Microbiol., 33: 6978. Bovine herpesvirus 1 (BHV1) and caprine herpesvirus 1 (CapHVI) are useful models to study virus-host interactions, as well as pathogenicity and latency, when comparing the outcome of infection in the natural and the foreign hosts, Molecular seroepidemiological analyses revealed that cross-reacting antibodies were mainly induced by glycoprotein gl (gB analogue), by the major capsid protein and by nonstructural proteins, whereas the most virus-specific antibodies were elicited by glycoproteins gill and glV. These glycoproteins, especially gill (gC analogue), might therefore play an important role in the virus-hostinteractions. As a basis for further studies, we re-evaluated observations concerning experimental infections with BHVI and CapHVI in the natural and the foreign hosts. All parameters indicated that both viruses were able to infect either host, but that the pathogenicity was restricted to the natural host. Latent virus could be reactivated exclusively from cows infected with BHV 1. It was possible neither to reactivate BHVI from goats, nor to reactivate CapHVI from either species. The experiments indicated that the outcome ofinfection in the natural and the foreign host is dependent on host and viral factors, whereby gill is only one important virus component involved. Further investigations in the host and host cell range of BHV 1 and CapHV 1 will help to clarify the role of factors responsible for virus-hostinteractions.
INTRODUCTION Bovine herpesvirus 1 (BHVI) and caprine herpesvirus I (CapHV1) are useful models to study virus-host interactions, as well as pathogenicity and l a t e n c y , w h e n c o m p a r i n g t h e o u t c o m e o f i n f e c t i o n in t h e n a t u r a l a n d t h e f o r Correspondence to." M. Engels, Institute of Virology, Vet. Med. Faculty, University of Zurich, Winterthurerstrasse 266a, CH-8057, Zurich, Switzerland.
0378-1135/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
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M. ENGELS ET AL.
eign hosts. BHVI and CapHVI are closely related viruses, exhibiting common and specific properties. They exhibit related clinical patterns in the natural host. BHVI causes a variety of clinical diseases, such as infectious bovine rhinotracheitis (IBR), infectious pustular vulvovaginitis/balanoposthitis (IPV/IPB), abortion and encephalitis. The course of the infection may also remain subclinical (Wyler et al., 1989 ). CapHV 1 causes a severe generalized infection in newborn kids, mainly affecting the digestive tract and often leading to death (Saito et al., 1974, Mettler et al., 1979). Infection in adult goats takes either a subclinical course or results in an IPV/IPB-like disease (Tarigan et al., 1987 ). There are indications that CapHV l also can provoke abortion (Waldvogel et al., 1981 ). Both viruses are able to establish latent infections, which can be reactivated by dexamethasone treatment (Plebani et al., 1983; Wyler et al., 1989). The behaviour of BHV1 and CapHVl in cell culture is characterized by a rapid replication cycle and a broad host cell range (Engels et al., 1983). Host range. Besides cattle as principal host, natural BHV 1 infections occur in goats, sheep, swine, a number of wild ruminants and possibly in cats, as evidenced by serological data and/or virus isolations (Herbst et al., 1988; Wyler et al., 1989). Natural CapHVI infections have hitherto only been recognized in goats. Sheep and calves, however, are susceptible to experimental CapHV 1 infection (Berrios et al., 1975, Papanastopoulou et al., 1991 ). Despite the antigenic relationship and the susceptibility of cattle and goats to both viruses, there is no evidence for the existence of related virus reservoirs. Genome relatedness. This is manifested by their classification as having group D DNA and by almost identical physico-chemical properties. The restriction patterns of the DNAs, however, differ clearly (Engels et al., 1983, Engels et ai., 1987). BHVI strains can be differentiated into "subtypes" BHVI.1, BHVI.2 and BHVI.3. BHVI.3 strains are proposed to be variants of BHVI with neuropathogenic potential (Metzler et al., 1986). In contrast, different CapHV1 strains showed only minor differences in their restriction patterns (Engels et al., 1983 and 1987, Tisdall et al., 1984). Estimations of the base sequence homology of the BHVI and CapHVI DNA revealed a 50 to 80% homology (Engels et al., 1987; Bulach and Studdert, 1990). An antigenic relationship between BHVI and CapHV1 has been demonstrated by various tests. Differentiation, however, is possible, based on nonreciprocal reactions of neutralizing and ELISA antibodies (Berrios et al., 1975; Mettler et al., 1979; Engels et al., 1983; Ackermann et al., 1986; Hasler and Engels, 1986). Western blot immunoassays and radioimmunoprecipitation analyses, using monoclonal and polyclonal antibodies, revealed that glycoprotein gI (the gB analogue), the major capsid protein and nonstructural proteins are mainly responsible for the cross-reactivity, whereas glycoproteins
>4 >4 > 100 > 100
-
BHVI
(1-12) 4 n.d.6 n.d. (6-10)
n.d. n.d. n.d. n.d.
n.d.
CapHVI
Severe IBR, with high fever and milk loss (3_14) 3
4 20 27
-
BHVI
(3-10) _5 -
none
4 35 4
.
CapHVI .
-
BHVI .
(2-5) (3-5) -
none
.
3 4 13 II
CapHV1
C a p H V 1 infected calves (n=2) 2
3 4 6.8 6.8
BHVI
(1-6) -
>4 >4 >2187 >2187
3
CapHVI
Fever, mild depression, loss o f appetite (3-7)
C a p H V I infected goats (n=2)
t Dexamethasone treatment: 6 m o n t h s p.i. B H V 1 / c o w s (reaction see text), 3 weeks p.i. B H V l / g o a t s , C a p H V 1 / c a l v e s , 4 weeks p.i. C a p H V 1 / g o a t s ; 20nly one of two calves reacted to the CapHV I inoculation; 3duration: days post infection (d p.i. ); 4virus isolated: d p.i.; Sno virus isolated/no antibodies detectable; 6n.d.: not done; 7antibody titers did not change significantly until the end o f the experiments.
Virus isolated from: nasal swabs ocular swabs rectal swabs vaginal/prepucial swabs Serological reaction: neutralizing antibodies to I week p.i. 2 weeks p.i. 3 weeks p.i. 4 weeks p.i. 5 weeks p.i. 7
Clinical signs
BHVI infected goats (n=2)
C
t~
N Z m -r.
Z (7
o<
O
5 z
,e"3
Clinical, virological and serological reactions o f cattle and goats, experimentally infected with BHVI and C a p H V I
BHVI infected cows ~ (n=8)
.q
TABLE 1
72
M. ENGELS ETAL.
glII and gIV seem to be the most virus-specific proteins (Ackermann et al., 1986; Friedli and Metzler, 1987 ). Apparently, glycoprotein gIII, the gC analogue, is the most type-specific viral glycoprotein, and might therefore direct the outcome of the infections in the natural and the foreign host. Observations, based on limited numbers of experimental infections, confirmed this presumption, but showed the influence of host factors as well. In this paper, we evaluate our previously published observations (Probst et al., 1985; Ackermann et al., 1986) together with new data concerning BHVI and CapHVI infections in the natural and the foreign host. MATERIALS AND METHODS
Viruses and cell cultures Low passage Swiss field isolates of CapHVI (Mettler et al., 1979) and BHV1 (Probst et al., 1985) were used for animal inoculation and serologic tests. They had been propagated on either fetal bovine nasal epithelium or lung cells.
Experimental design The experimental design, described by Probst et al. (1985) for the infection of cows with BHVI and by Ackermann et al. (1986) for the infection of goats with BHVI or CapHVI, was adopted for the infection of calves with CapHV1. Briefly: The animals were inoculated intranasally with BHVI or CapHV1 (107TCIDso) and observed daily for clinical signs. Swab samples were taken daily for various time periods, and serum samples were collected twice a week throughout the duration of the experiments. The animals were treated with dexamethasone on 5 consecutive days at times post infection (p.i.) as indicated in Table 1, and sentinel calves or goats were placed with the treated animals, except the cows infected with BHV 1. Trigeminal ganglia
Fig. I. Reactivity of caprine and bovine field sera with antigens of BHVI and CapHV1 as revealed by western blot immunoassay. The upper part represents the reactions of three representative field sera from goats, one seronegative (a), two with moderate neutralizing antibody titres against CapHV1 (b and c) and one high titred CapHV 1 reference serum (d). The lower part represents the reactions of three representative field sera from cattle, one seronegative (a), one with low (b) and one with moderate (c) neutralizing antibody titres against BHV 1, and one high titred BHV l reference serum (d). The sera were analysed in the presence of 35S-methionine labeled proteins (label: 7 to 22 hours post infection) from mock-infected (~,), BHVl-infected (B) and CapHVl-infected (C) MDBK cells. Fig. l a' represent the autoradiographic pictures of the labeled, membrane bound proteins in lanes A, B and C. Reacting structural proteins are designated by numbers, nonstrucrural proteins by lower case letters.
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ue
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74
M. ENGELS ET AL.
and brain sections were collected from calves inoculated with CapHVI and stored for further examinations.
Virus isolation, serological and antigenic examinations Virus isolation procedures and antibody determination by serum neutralization test and ELISA were carried out as described (Ackermann et al., 1986). Western blot immunoassays were done according to Friedli and Metzler (1987). RESULTS
Molecular seroepidemiology In order to characterize the antigenic relationship between BHVI and CapHV1, the reactivity of field sera from goats and cattle was analysed in western blot immunoassays, using 35S-methionine labeled proteins from BHVI-, CapHVI- and mock-infected cells. Fig. 1 (upper part) shows the results obtained with three representative field sera of goats (one seronegative and two seropositive) in comparison with one CapHV 1 reference serum, and Fig. 1 (lower part) those with three representative field sera of cattle (one seronegative and two seropositive) in comparison with one BHVI reference serum. The neutralizing antibody titres of the cattle and goat field sera against the homologous virus were low to moderate, whereas those of the reference sera were high. The neutralizing antibody titres correlated with the intensity of the reactions in western blot analyses. CapHVl-specific antisera reacted mainly with CapHV1 proteins corresponding to the major capsid protein (VP4), the major tegument protein (VP11 ), glycoproteins gI (gVP 7/17/23; gB analogue) and gill (gVP3/12; gC analogue), as well as with several nonstructural proteins. The same sera reacted with BHV1 proteins corresponding to glycoprotein gI, to VP4 and nonstructural proteins. Interestingly, the seronegative field serum reacted with VP4 of CapHV1 and BHVI. BHVl-specific antisera reacted mainly with BHVI proteins VP4, several nonstructural proteins and glycoproteins gI, glII and glV (gVPI7A; gD analogue), and with CapHV 1 proteins corresponding to glycoprotein gI, to VP4 and nonstructural proteins. In summary, western blot immunoassays revealed BHV1 and CapHVI proteins VP4 and VPI 1, several nonstructural proteins and glycoproteins gI, gill and glV to be the major immunogens. Among these, glycoprotein gI, VP4, and nonstructural proteins represent common antigens, and glycoproteins gill and glV virusspecific antigens.
Experimental infections of the natural and the foreign hosts As already proposed by Ackermann et al. (1986) and Friedli and Metzler (1987), gIII seems to be the most type-specific glycoprotein, and it is there-
INTERACTIONS OF BOVINE AND CAPRINE HERPESVIRUSES
75
fore of special interest to evaluate its role in virus-host-interrelationships. In a first approach to this aim, we compared the outcome of experimental intranasal infections with identical doses of the same strains of BHVI and CapHVl in the natural and the foreign host. The results are summarized in Table 1. Clinical signs were observed in BHVI infected cows and CapHVl infected goats. Virus shedding and seroconversion, however, were observed in all animals, except for one calf inoculated with CapHVl. This calf shed virus with nasal discharge on day 1 p.i. This was probably residual inoculated virus, since no further responses could be observed. Reactivation of latent infection was observed in the cows infected with BHV l, as evidenced by clinical symptoms, such as nasal discharge, fever and milk reduction, and by virus isolation from different sites. In contrast, no reactivation occurred in the other animals, including sentinel animals, as shown by the lack of clinical signs and virus shedding. As measured by the neutralizing antibody production, the immune response was strong in the respective natural host and weak in the foreign host. Reactivation of latent infection only induced a serological reaction in BHVI infected cows with low neutralizing antibody titers. The serological reactions, as revealed by serum neutralization test (see Table 1 ), ELISA and western blot (not shown), confirmed the known antigenic cross-reactivity between BHV l and CapHV1. DISCUSSION
BHV 1 and CapHV 1 are useful models to study virus-host-interactions. They exhibit a close relationship, which is manifested by the clinical patterns in the natural host, and by related genomes and antigens. Both viruses, however, exhibit specific properties as well. Molecular seroepidemiological data suggested that glycoprotein glII plays a major role in the outcome of infection in the natural and the foreign host. Analyses of monoclonal and polyclonal BHVI and CapHVI antibodies in western blot and radioimmunoassays revealed that cross-reacting antibodies were mainly induced by glycoprotein gI (gB analogue), by the major capsid protein and by nonstructural proteins, whereas glycoproteins gill (gC analogue ) and glV (gD analogue) were found to induce virus-specific antibodies (Ackermann et al., 1986; Friedli and Metzler, 1987). This could be confirmed by testing the reactivity of cattle and goat field sera with BHV 1 and CapHV 1 antigens in western blot immunoassay. Glycoprotein gI (gB) is known to be the most conserved within the whole herpesvirus group (Hammerschmidt et al., 1988), and cross-reacting antibodies were to be expected. Other common antigenic sites were restricted to non-glycosylated or nonstructural proteins, which generally are less susceptible to selective pressure. Interestingly, several seronegative goat sera have been
76
M. ENGELS ET AL.
found, which reacted with the major capsid protein of CapHVI and BHVI (see example in Fig. I a, upper part), indicating that infections with CapHV1 or a related virus may occur more often than demonstrable by serum neutralization tests. Glycoproteins gIII (gC) and gIV (gD) are known to be less conserved within the herpesvirus group, whereby gC analogues exhibit the most virus and strain specific properties (Ben-Porat et al., 1986; Friedli and Metzler, 1987; Allen and Coogle, 1988). Although gC analogues are involved in the adsorption process, they are not essential for virus replication. Their function, therefore, can be replaced by other factors, but it has been shown that the infectivity ofgIII/gC-minus mutants is significantly reduced (Schreurs et al., 1988; Hidaka et al., 1990; Liang et al., 1991 ). Thus, gIII/gC might play an important role for individual herpesviruses in their pathogenicity and virulence, as well as in their ability to interact with the host in general. As a basis for further studies of the virus-host-interaction, we have evaluated both published and new observations concerning experimental infections with BHV l and CapHV 1 in the natural and the foreign host, carried out under identical conditions. All parameters indicated that both viruses are able to infect either host, but that the pathogenicity is restricted to the natural host. A reduced susceptibility was observed in the heterologous hosts. Thus, only one of two calves inoculated with CapHV 1 became infected, as indicated by virus shedding and seroconversion. Moreover, the serological reaction of all animals infected with the heterologous virus remained weak. The serological investigations, addressed to the antigenic relationship between BHV1 and CapHV1, confirmed earlier results. Latent virus could only be reactivated in cows infected with BHV1. The reason why latent virus could not be reactivated from CapHV 1 infected goats is not clear. Plebani et al. (1983) were able to reactivate CapHVl from naturally infected goats, whereas Pirak et al. ( 1983 ) and Papanastopoulou et al. (1990) failed to reactivate virus from experimentally infected goats, too. Failure of virus reactivation in the heterologous host either means that latency has not been established, or that latent virus could not be reactivated. Experiments with different herpesviruses demonstrated that reactivation of latent infection is dependent on the dose and kind ofcorticosteroid used (Plebani et al., 1983; Wittmann et al., 1984; Straub and Lorenz, 1991 ), as well as on host factors and on the time period between infection and corticosteroid treatment (Pastoret et al., 1986; Sears and Roizman, 1990). In situ hybridization and PCR are being established in order to look for viral DNA or RNA in sections of trigeminal ganglia and brain, collected from the CapHVl inoculated calves. CONCLUSIONS
The results presented indicate that glycoprotein gIII (gC analogue) might be only one important virus component involved in the outcome of BHVI
INTERACTIONS OF BOVINE AND CAPRINE HERPESVIRUSES
77
a n d C a p H V l i n f e c t i o n s in the n a t u r a l a n d the foreign host. T h e i n t e r a c t i o n o f o t h e r viral, as well as h o s t f a c t o r s m u s t n o t be neglected. F u r t h e r investig a t i o n s in t h e h o s t a n d t h e h o s t cell r a n g e o f B H V 1 a n d C a p H V 1 are p l a n n e d to e v a l u a t e m o d e l s for m o r e d e t a i l e d a n a l y s e s o f the v i r u s - h o s t - i n t e r a c t i o n s .
REFERENCES Ackermann, M., Metzler, A.E., McDonagh, H., Bruckner, L., Miiller, H.K. and Kihm, U., 1986. Stellen nichtbovine Paarhufer ein IBR-Virus-Reservoir dar? I. BHV-I und CapHV-l-lnfektions- und Reaktivierungsversuche an Ziegen, Virustyp-Spezifit~it der humoralen AnlikiSrper und Charakterisierung der viralen Antigene. Schweiz. Arch. Tierheilk., 128: 557-573. Allen, G.P. and Coogle, L.G., 1988. Characterization of an equine herpesvirus type 1 gene encoding a glycoprotein (gpl 3) with homology to herpes simplex virus glycoprotein C. J. Virol., 62: 2850-2858. Ben-Porat, T., DeMarchi, J.M., Lomniczi, B. and Kaptan, A.S., 1986. Role of glycoproteins of pseudorabies virus in eliciting neutralizing antibodies. Virology, 154: 325-334. Berrios, P.E., McKercher, D.G. and Knight, H.D., 1975. Pathogenicity ofa caprine herpesvirus. Am. J. Vet. Res., 36: 1763-1769. Bulach, D.M. and Studdert, M.J., 1990. Comparative genome mapping of bovine encephalitis herpesvirus, bovine herpesvirus 1, and buffalo herpesvirus. Arch. Virol., 113:17-34. Engels, M., Gelderblom, H., Darai, G. and Ludwig, H., 1983. Goat herpesviruses: Biological and physico-chemical properties. J. Gen. Virol., 64: 2237-2247. Engels, M., Loepfe, E., Wild, P., Schraner, E. and Wyler, R., 1987. The genome ofcaprine herpesvirus 1: Genome structure and relatedness to bovine herpesvirus 1. J. Gen. Virol., 68: 2019-2023. Friedli, K. and Metzler, A.E., 1987. Reactivity of monoclonal antibodies to proteins of a neurotropic bovine herpesvirus 1 (BHV-I) strain and to proteins of representative BHV-1 strains. Arch. Virol., 94: 109-122. Hammerschmidt, W., Conraths, F., Mankertz, J., Buhk, H.-J., Pauli, G. and Ludwig, H., 1988. Common epitopes of glycoprotein B map within the major DNA-binding proteins of bovine herpesvirus type 2 (BHV-2) and herpes simplex virus type 1 (HSV-l ). Virology, 165: 406418. Hasler, J, and Engels, M., 1986. Stellen nichtbovine Paarhufer ein IBR-Virus-Reservoir dar? lI. Seroepidemiologische Untersuchungen an Ziegen, Schafen, Schweinen und Wildpaarhufern in der Schweiz. Schweiz. Arch. Tierheilk., 128: 575-585. Herbst, W., Lange, H., Danner, K. and Schliesser, T., 1988. Virologisch-diagnostische Untersuchungen beim "Katzenschnupfen". Kleintierpraxis, 33: 433-434. Hidaka, Y., Sakuma, S., Kumano, Y., Minagawa, H. and Mori, R., 1990. Characterization of glycoprotein C-negative mutants of herpes simplex virus type 1 isolated from a patient with keratitis. Arch. Virol., 113:195-207. Liang, X., Babiuk, L.A., Van Drunen Littel-Van den Hurk, S., Fitzpatrick, D.R. and Zamb, T.J., 199 I. Bovine herpesvirus 1 attachment to permissive cells is mediated by its major glycoproteins gl, gIII, and gIV. J. Virol., 65:1124-1 i 32. Mcttler, F., Engels, M., Wild, P. and Bivetti, A., 1979. Herpesvirus-lnfektion bei Zicklein in der Schweiz. Schweiz. Arch. Tierheilk., 121 : 655-662. Metzler, A.E., Schudel, A.A. and Engels, M., 1986. Bovine herpesvirus l: Molecular and antigenic characteristics of variant viruses isolated from calves with neurological disease. Arch. Virol., 87:205-217. Papanastasopoulou, M., Koptopoulos, G., Lekkas, S., Georgakopoulou, E. and Papadopoulos,
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O., 1990. A preliminary investigation on the latency of the goat herpesvirus BHV-6. J. Vet. Med. B, 37:781-784. Papanastasopoulou, M., Koptopoulos, G., Lekkas, S., Papadopoulos, O. and Ludwig, H., 1991. An experimental study on the pathogenicity of the caprine herpesvirus type 1 (CHV-I). Comp. Immun. Microbiol. Infect. Dis., 14: 47-53. Pastoret, P.P., Thiry, E. and Thomas, R., 1986. Logical description of bovine herpesvirus 1 latent infection. J. Gen. Virol., 67: 885-897. Pirak, M., Thiry, E., Brochier, B. and Pastoret, P.P., 1983. Infection experimentale de la chevre par le virus de la rhinotracheite infectieuse bovine (Bovine herpesvirus 1 ) et tentative de reactivation virale. Rec. Med. Vet., 159:1103-1106. Plcbani, G.F., Engels, M., Metzler, A.E. and Wyler, R., 1983. Caprines Herpesvirus in der Schweiz: Verbreitung, H~iufigkeit und Latenz der Infektion. Schweiz. Arch. Tierheilk., 125: 395-411. Probst, U., Wyler, R., Kihm, U., Ackermann, M., Bruckner, L., MUller, H.K. and Ehrensperger, F., 1985. Zur IBR-Virus-Ausscheidung experimentell infizierter Kiihe insbesondere in der Milch. Schweiz. Arch. Tierheilk., 127: 723-733. Saito, J.K., Gribble, D.H., Berrios, P.E., Knight, H.D. and McKercher, D.G., 1974. New Herpesvirus isolate from goats: Preliminary report. Am. J. Vet. Res., 35: 847-848. Schreurs, C., Mettenleiter, T.C., Zuckermann, F., Sugg, N. and Ben-Porat, T., 1988. Glycoprotein gill of pseudorabies virus is multifunctional. J. Virol., 62:2251-2257. Sears, A.E. and Roizman, B., 1990. Amplification by host cell factors of a sequence contained within the herpes simplex virus 1 genome. Proc. Natl. Acad. Sci. USA, 87: 9441-9444. Straub, O.C. and Lorenz, R.J., 1991. Behandlung von im Atmungstrakt latent BHV 1-infizierten Rindern mit verschiedenen Immunsuppressiva. Tier~irztl. Umschau, 6: 344-354. Tarigan, S., Webb, R.F. and Kirkland, D., 1987. Caprine herpesvirus from balanoposthitis. Austral. Vet. J., 64: 321. Tisdall, D.J., Bentley, C.B. and Collins, D.M., 1984. New Zealand caprine herpes virus: Comparison with an Australian isolate and with bovine herpes virus type 1 by restriction endonuclease analysis. N.Z. Vet. J., 32: 99-100. Waldvogel, A., Engels, M., Wild, P., Stiinzi, H. and Wyler, R., 1981. Caprine herpesvirus infection in Switzerland: Some aspects of its pathogenicity. Zbl. Vet. Med. B, 28:612-623. Wittmann, G., Ohlinger, V. and Rziha, H.J, 1984. Latency of Aujeszky's disease virus (ADV) following challenge in previously vaccinated pigs. In: G. Wittman, R.M. Gaskell and H.J. Rziha (Editors), Latent herpesvirus infections in veterinary medicine, Martinus Nijhoff Publishers, Boston/The Hague/Dordrecht/Lancaster, pp. 445-455. Wyler, R., Engels, M. and Schwyzer, M., 1989. Infectious bovine rhinotracheitis/vulvovaginitis (BHVI). In: G. Wittmann (Editor), Herpesvirus diseases of cattle, horses, and pigs, Kluwer Academic Publishers, Boston/Dordrecht/London, pp. 1-72.
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Interactions of bovine and caprine herpesviruses with the natural and the foreign hosts M o n i k a Engels a, M o n i c a P a l a t i n i a, A l f r e d E. M e t z l e r ~, U r s P r o b s t u, U e l i K i h m c and Mathias Ackermann a alnstitute of Virology, Veterinary Medical Facuhy, University of Ziirich, Switzerland bVeterinaria AG, Ziirich, Switzerland Clnstitute of Viral Diseases, Basel, Switzerland (Accepted 26 June 1992)
ABSTRACT Engels, M., Palatini, M., Metzler, A.E., Probst, U., Kihm, U. and Ackermann, M., 1992. Interactions of bovine and caprine herpesviruses with the natural and the foreign hosts. Vet. Microbiol., 33: 6978. Bovine herpesvirus 1 (BHV1) and caprine herpesvirus 1 (CapHVI) are useful models to study virus-host interactions, as well as pathogenicity and latency, when comparing the outcome of infection in the natural and the foreign hosts, Molecular seroepidemiological analyses revealed that cross-reacting antibodies were mainly induced by glycoprotein gl (gB analogue), by the major capsid protein and by nonstructural proteins, whereas the most virus-specific antibodies were elicited by glycoproteins gill and glV. These glycoproteins, especially gill (gC analogue), might therefore play an important role in the virus-hostinteractions. As a basis for further studies, we re-evaluated observations concerning experimental infections with BHVI and CapHVI in the natural and the foreign hosts. All parameters indicated that both viruses were able to infect either host, but that the pathogenicity was restricted to the natural host. Latent virus could be reactivated exclusively from cows infected with BHV 1. It was possible neither to reactivate BHVI from goats, nor to reactivate CapHVI from either species. The experiments indicated that the outcome ofinfection in the natural and the foreign host is dependent on host and viral factors, whereby gill is only one important virus component involved. Further investigations in the host and host cell range of BHV 1 and CapHV 1 will help to clarify the role of factors responsible for virus-hostinteractions.
INTRODUCTION Bovine herpesvirus 1 (BHVI) and caprine herpesvirus I (CapHV1) are useful models to study virus-host interactions, as well as pathogenicity and l a t e n c y , w h e n c o m p a r i n g t h e o u t c o m e o f i n f e c t i o n in t h e n a t u r a l a n d t h e f o r Correspondence to." M. Engels, Institute of Virology, Vet. Med. Faculty, University of Zurich, Winterthurerstrasse 266a, CH-8057, Zurich, Switzerland.
0378-1135/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
70
M. ENGELS ET AL.
eign hosts. BHVI and CapHVI are closely related viruses, exhibiting common and specific properties. They exhibit related clinical patterns in the natural host. BHVI causes a variety of clinical diseases, such as infectious bovine rhinotracheitis (IBR), infectious pustular vulvovaginitis/balanoposthitis (IPV/IPB), abortion and encephalitis. The course of the infection may also remain subclinical (Wyler et al., 1989 ). CapHV 1 causes a severe generalized infection in newborn kids, mainly affecting the digestive tract and often leading to death (Saito et al., 1974, Mettler et al., 1979). Infection in adult goats takes either a subclinical course or results in an IPV/IPB-like disease (Tarigan et al., 1987 ). There are indications that CapHV l also can provoke abortion (Waldvogel et al., 1981 ). Both viruses are able to establish latent infections, which can be reactivated by dexamethasone treatment (Plebani et al., 1983; Wyler et al., 1989). The behaviour of BHV1 and CapHVl in cell culture is characterized by a rapid replication cycle and a broad host cell range (Engels et al., 1983). Host range. Besides cattle as principal host, natural BHV 1 infections occur in goats, sheep, swine, a number of wild ruminants and possibly in cats, as evidenced by serological data and/or virus isolations (Herbst et al., 1988; Wyler et al., 1989). Natural CapHVI infections have hitherto only been recognized in goats. Sheep and calves, however, are susceptible to experimental CapHV 1 infection (Berrios et al., 1975, Papanastopoulou et al., 1991 ). Despite the antigenic relationship and the susceptibility of cattle and goats to both viruses, there is no evidence for the existence of related virus reservoirs. Genome relatedness. This is manifested by their classification as having group D DNA and by almost identical physico-chemical properties. The restriction patterns of the DNAs, however, differ clearly (Engels et al., 1983, Engels et ai., 1987). BHVI strains can be differentiated into "subtypes" BHVI.1, BHVI.2 and BHVI.3. BHVI.3 strains are proposed to be variants of BHVI with neuropathogenic potential (Metzler et al., 1986). In contrast, different CapHV1 strains showed only minor differences in their restriction patterns (Engels et al., 1983 and 1987, Tisdall et al., 1984). Estimations of the base sequence homology of the BHVI and CapHVI DNA revealed a 50 to 80% homology (Engels et al., 1987; Bulach and Studdert, 1990). An antigenic relationship between BHVI and CapHV1 has been demonstrated by various tests. Differentiation, however, is possible, based on nonreciprocal reactions of neutralizing and ELISA antibodies (Berrios et al., 1975; Mettler et al., 1979; Engels et al., 1983; Ackermann et al., 1986; Hasler and Engels, 1986). Western blot immunoassays and radioimmunoprecipitation analyses, using monoclonal and polyclonal antibodies, revealed that glycoprotein gI (the gB analogue), the major capsid protein and nonstructural proteins are mainly responsible for the cross-reactivity, whereas glycoproteins
>4 >4 > 100 > 100
-
BHVI
(1-12) 4 n.d.6 n.d. (6-10)
n.d. n.d. n.d. n.d.
n.d.
CapHVI
Severe IBR, with high fever and milk loss (3_14) 3
4 20 27
-
BHVI
(3-10) _5 -
none
4 35 4
.
CapHVI .
-
BHVI .
(2-5) (3-5) -
none
.
3 4 13 II
CapHV1
C a p H V 1 infected calves (n=2) 2
3 4 6.8 6.8
BHVI
(1-6) -
>4 >4 >2187 >2187
3
CapHVI
Fever, mild depression, loss o f appetite (3-7)
C a p H V I infected goats (n=2)
t Dexamethasone treatment: 6 m o n t h s p.i. B H V 1 / c o w s (reaction see text), 3 weeks p.i. B H V l / g o a t s , C a p H V 1 / c a l v e s , 4 weeks p.i. C a p H V 1 / g o a t s ; 20nly one of two calves reacted to the CapHV I inoculation; 3duration: days post infection (d p.i. ); 4virus isolated: d p.i.; Sno virus isolated/no antibodies detectable; 6n.d.: not done; 7antibody titers did not change significantly until the end o f the experiments.
Virus isolated from: nasal swabs ocular swabs rectal swabs vaginal/prepucial swabs Serological reaction: neutralizing antibodies to I week p.i. 2 weeks p.i. 3 weeks p.i. 4 weeks p.i. 5 weeks p.i. 7
Clinical signs
BHVI infected goats (n=2)
C
t~
N Z m -r.
Z (7
o<
O
5 z
,e"3
Clinical, virological and serological reactions o f cattle and goats, experimentally infected with BHVI and C a p H V I
BHVI infected cows ~ (n=8)
.q
TABLE 1
72
M. ENGELS ETAL.
glII and gIV seem to be the most virus-specific proteins (Ackermann et al., 1986; Friedli and Metzler, 1987 ). Apparently, glycoprotein gIII, the gC analogue, is the most type-specific viral glycoprotein, and might therefore direct the outcome of the infections in the natural and the foreign host. Observations, based on limited numbers of experimental infections, confirmed this presumption, but showed the influence of host factors as well. In this paper, we evaluate our previously published observations (Probst et al., 1985; Ackermann et al., 1986) together with new data concerning BHVI and CapHVI infections in the natural and the foreign host. MATERIALS AND METHODS
Viruses and cell cultures Low passage Swiss field isolates of CapHVI (Mettler et al., 1979) and BHV1 (Probst et al., 1985) were used for animal inoculation and serologic tests. They had been propagated on either fetal bovine nasal epithelium or lung cells.
Experimental design The experimental design, described by Probst et al. (1985) for the infection of cows with BHVI and by Ackermann et al. (1986) for the infection of goats with BHVI or CapHVI, was adopted for the infection of calves with CapHV1. Briefly: The animals were inoculated intranasally with BHVI or CapHV1 (107TCIDso) and observed daily for clinical signs. Swab samples were taken daily for various time periods, and serum samples were collected twice a week throughout the duration of the experiments. The animals were treated with dexamethasone on 5 consecutive days at times post infection (p.i.) as indicated in Table 1, and sentinel calves or goats were placed with the treated animals, except the cows infected with BHV 1. Trigeminal ganglia
Fig. I. Reactivity of caprine and bovine field sera with antigens of BHVI and CapHV1 as revealed by western blot immunoassay. The upper part represents the reactions of three representative field sera from goats, one seronegative (a), two with moderate neutralizing antibody titres against CapHV1 (b and c) and one high titred CapHV 1 reference serum (d). The lower part represents the reactions of three representative field sera from cattle, one seronegative (a), one with low (b) and one with moderate (c) neutralizing antibody titres against BHV 1, and one high titred BHV l reference serum (d). The sera were analysed in the presence of 35S-methionine labeled proteins (label: 7 to 22 hours post infection) from mock-infected (~,), BHVl-infected (B) and CapHVl-infected (C) MDBK cells. Fig. l a' represent the autoradiographic pictures of the labeled, membrane bound proteins in lanes A, B and C. Reacting structural proteins are designated by numbers, nonstrucrural proteins by lower case letters.
.:~
..
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¢)
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o
,
,
,
ue
ot
°
°
,
o
3=-
~,
...4
,.~ ~
"
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......
~.!.I~~ ~...~..~~,~...,;~I
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Z
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D
74
M. ENGELS ET AL.
and brain sections were collected from calves inoculated with CapHVI and stored for further examinations.
Virus isolation, serological and antigenic examinations Virus isolation procedures and antibody determination by serum neutralization test and ELISA were carried out as described (Ackermann et al., 1986). Western blot immunoassays were done according to Friedli and Metzler (1987). RESULTS
Molecular seroepidemiology In order to characterize the antigenic relationship between BHVI and CapHV1, the reactivity of field sera from goats and cattle was analysed in western blot immunoassays, using 35S-methionine labeled proteins from BHVI-, CapHVI- and mock-infected cells. Fig. 1 (upper part) shows the results obtained with three representative field sera of goats (one seronegative and two seropositive) in comparison with one CapHV 1 reference serum, and Fig. 1 (lower part) those with three representative field sera of cattle (one seronegative and two seropositive) in comparison with one BHVI reference serum. The neutralizing antibody titres of the cattle and goat field sera against the homologous virus were low to moderate, whereas those of the reference sera were high. The neutralizing antibody titres correlated with the intensity of the reactions in western blot analyses. CapHVl-specific antisera reacted mainly with CapHV1 proteins corresponding to the major capsid protein (VP4), the major tegument protein (VP11 ), glycoproteins gI (gVP 7/17/23; gB analogue) and gill (gVP3/12; gC analogue), as well as with several nonstructural proteins. The same sera reacted with BHV1 proteins corresponding to glycoprotein gI, to VP4 and nonstructural proteins. Interestingly, the seronegative field serum reacted with VP4 of CapHV1 and BHVI. BHVl-specific antisera reacted mainly with BHVI proteins VP4, several nonstructural proteins and glycoproteins gI, glII and glV (gVPI7A; gD analogue), and with CapHV 1 proteins corresponding to glycoprotein gI, to VP4 and nonstructural proteins. In summary, western blot immunoassays revealed BHV1 and CapHVI proteins VP4 and VPI 1, several nonstructural proteins and glycoproteins gI, gill and glV to be the major immunogens. Among these, glycoprotein gI, VP4, and nonstructural proteins represent common antigens, and glycoproteins gill and glV virusspecific antigens.
Experimental infections of the natural and the foreign hosts As already proposed by Ackermann et al. (1986) and Friedli and Metzler (1987), gIII seems to be the most type-specific glycoprotein, and it is there-
INTERACTIONS OF BOVINE AND CAPRINE HERPESVIRUSES
75
fore of special interest to evaluate its role in virus-host-interrelationships. In a first approach to this aim, we compared the outcome of experimental intranasal infections with identical doses of the same strains of BHVI and CapHVl in the natural and the foreign host. The results are summarized in Table 1. Clinical signs were observed in BHVI infected cows and CapHVl infected goats. Virus shedding and seroconversion, however, were observed in all animals, except for one calf inoculated with CapHVl. This calf shed virus with nasal discharge on day 1 p.i. This was probably residual inoculated virus, since no further responses could be observed. Reactivation of latent infection was observed in the cows infected with BHV l, as evidenced by clinical symptoms, such as nasal discharge, fever and milk reduction, and by virus isolation from different sites. In contrast, no reactivation occurred in the other animals, including sentinel animals, as shown by the lack of clinical signs and virus shedding. As measured by the neutralizing antibody production, the immune response was strong in the respective natural host and weak in the foreign host. Reactivation of latent infection only induced a serological reaction in BHVI infected cows with low neutralizing antibody titers. The serological reactions, as revealed by serum neutralization test (see Table 1 ), ELISA and western blot (not shown), confirmed the known antigenic cross-reactivity between BHV l and CapHV1. DISCUSSION
BHV 1 and CapHV 1 are useful models to study virus-host-interactions. They exhibit a close relationship, which is manifested by the clinical patterns in the natural host, and by related genomes and antigens. Both viruses, however, exhibit specific properties as well. Molecular seroepidemiological data suggested that glycoprotein glII plays a major role in the outcome of infection in the natural and the foreign host. Analyses of monoclonal and polyclonal BHVI and CapHVI antibodies in western blot and radioimmunoassays revealed that cross-reacting antibodies were mainly induced by glycoprotein gI (gB analogue), by the major capsid protein and by nonstructural proteins, whereas glycoproteins gill (gC analogue ) and glV (gD analogue) were found to induce virus-specific antibodies (Ackermann et al., 1986; Friedli and Metzler, 1987). This could be confirmed by testing the reactivity of cattle and goat field sera with BHV 1 and CapHV 1 antigens in western blot immunoassay. Glycoprotein gI (gB) is known to be the most conserved within the whole herpesvirus group (Hammerschmidt et al., 1988), and cross-reacting antibodies were to be expected. Other common antigenic sites were restricted to non-glycosylated or nonstructural proteins, which generally are less susceptible to selective pressure. Interestingly, several seronegative goat sera have been
76
M. ENGELS ET AL.
found, which reacted with the major capsid protein of CapHVI and BHVI (see example in Fig. I a, upper part), indicating that infections with CapHV1 or a related virus may occur more often than demonstrable by serum neutralization tests. Glycoproteins gIII (gC) and gIV (gD) are known to be less conserved within the herpesvirus group, whereby gC analogues exhibit the most virus and strain specific properties (Ben-Porat et al., 1986; Friedli and Metzler, 1987; Allen and Coogle, 1988). Although gC analogues are involved in the adsorption process, they are not essential for virus replication. Their function, therefore, can be replaced by other factors, but it has been shown that the infectivity ofgIII/gC-minus mutants is significantly reduced (Schreurs et al., 1988; Hidaka et al., 1990; Liang et al., 1991 ). Thus, gIII/gC might play an important role for individual herpesviruses in their pathogenicity and virulence, as well as in their ability to interact with the host in general. As a basis for further studies of the virus-host-interaction, we have evaluated both published and new observations concerning experimental infections with BHV l and CapHV 1 in the natural and the foreign host, carried out under identical conditions. All parameters indicated that both viruses are able to infect either host, but that the pathogenicity is restricted to the natural host. A reduced susceptibility was observed in the heterologous hosts. Thus, only one of two calves inoculated with CapHV 1 became infected, as indicated by virus shedding and seroconversion. Moreover, the serological reaction of all animals infected with the heterologous virus remained weak. The serological investigations, addressed to the antigenic relationship between BHV1 and CapHV1, confirmed earlier results. Latent virus could only be reactivated in cows infected with BHV1. The reason why latent virus could not be reactivated from CapHV 1 infected goats is not clear. Plebani et al. (1983) were able to reactivate CapHVl from naturally infected goats, whereas Pirak et al. ( 1983 ) and Papanastopoulou et al. (1990) failed to reactivate virus from experimentally infected goats, too. Failure of virus reactivation in the heterologous host either means that latency has not been established, or that latent virus could not be reactivated. Experiments with different herpesviruses demonstrated that reactivation of latent infection is dependent on the dose and kind ofcorticosteroid used (Plebani et al., 1983; Wittmann et al., 1984; Straub and Lorenz, 1991 ), as well as on host factors and on the time period between infection and corticosteroid treatment (Pastoret et al., 1986; Sears and Roizman, 1990). In situ hybridization and PCR are being established in order to look for viral DNA or RNA in sections of trigeminal ganglia and brain, collected from the CapHVl inoculated calves. CONCLUSIONS
The results presented indicate that glycoprotein gIII (gC analogue) might be only one important virus component involved in the outcome of BHVI
INTERACTIONS OF BOVINE AND CAPRINE HERPESVIRUSES
77
a n d C a p H V l i n f e c t i o n s in the n a t u r a l a n d the foreign host. T h e i n t e r a c t i o n o f o t h e r viral, as well as h o s t f a c t o r s m u s t n o t be neglected. F u r t h e r investig a t i o n s in t h e h o s t a n d t h e h o s t cell r a n g e o f B H V 1 a n d C a p H V 1 are p l a n n e d to e v a l u a t e m o d e l s for m o r e d e t a i l e d a n a l y s e s o f the v i r u s - h o s t - i n t e r a c t i o n s .
REFERENCES Ackermann, M., Metzler, A.E., McDonagh, H., Bruckner, L., Miiller, H.K. and Kihm, U., 1986. Stellen nichtbovine Paarhufer ein IBR-Virus-Reservoir dar? I. BHV-I und CapHV-l-lnfektions- und Reaktivierungsversuche an Ziegen, Virustyp-Spezifit~it der humoralen AnlikiSrper und Charakterisierung der viralen Antigene. Schweiz. Arch. Tierheilk., 128: 557-573. Allen, G.P. and Coogle, L.G., 1988. Characterization of an equine herpesvirus type 1 gene encoding a glycoprotein (gpl 3) with homology to herpes simplex virus glycoprotein C. J. Virol., 62: 2850-2858. Ben-Porat, T., DeMarchi, J.M., Lomniczi, B. and Kaptan, A.S., 1986. Role of glycoproteins of pseudorabies virus in eliciting neutralizing antibodies. Virology, 154: 325-334. Berrios, P.E., McKercher, D.G. and Knight, H.D., 1975. Pathogenicity ofa caprine herpesvirus. Am. J. Vet. Res., 36: 1763-1769. Bulach, D.M. and Studdert, M.J., 1990. Comparative genome mapping of bovine encephalitis herpesvirus, bovine herpesvirus 1, and buffalo herpesvirus. Arch. Virol., 113:17-34. Engels, M., Gelderblom, H., Darai, G. and Ludwig, H., 1983. Goat herpesviruses: Biological and physico-chemical properties. J. Gen. Virol., 64: 2237-2247. Engels, M., Loepfe, E., Wild, P., Schraner, E. and Wyler, R., 1987. The genome ofcaprine herpesvirus 1: Genome structure and relatedness to bovine herpesvirus 1. J. Gen. Virol., 68: 2019-2023. Friedli, K. and Metzler, A.E., 1987. Reactivity of monoclonal antibodies to proteins of a neurotropic bovine herpesvirus 1 (BHV-I) strain and to proteins of representative BHV-1 strains. Arch. Virol., 94: 109-122. Hammerschmidt, W., Conraths, F., Mankertz, J., Buhk, H.-J., Pauli, G. and Ludwig, H., 1988. Common epitopes of glycoprotein B map within the major DNA-binding proteins of bovine herpesvirus type 2 (BHV-2) and herpes simplex virus type 1 (HSV-l ). Virology, 165: 406418. Hasler, J, and Engels, M., 1986. Stellen nichtbovine Paarhufer ein IBR-Virus-Reservoir dar? lI. Seroepidemiologische Untersuchungen an Ziegen, Schafen, Schweinen und Wildpaarhufern in der Schweiz. Schweiz. Arch. Tierheilk., 128: 575-585. Herbst, W., Lange, H., Danner, K. and Schliesser, T., 1988. Virologisch-diagnostische Untersuchungen beim "Katzenschnupfen". Kleintierpraxis, 33: 433-434. Hidaka, Y., Sakuma, S., Kumano, Y., Minagawa, H. and Mori, R., 1990. Characterization of glycoprotein C-negative mutants of herpes simplex virus type 1 isolated from a patient with keratitis. Arch. Virol., 113:195-207. Liang, X., Babiuk, L.A., Van Drunen Littel-Van den Hurk, S., Fitzpatrick, D.R. and Zamb, T.J., 199 I. Bovine herpesvirus 1 attachment to permissive cells is mediated by its major glycoproteins gl, gIII, and gIV. J. Virol., 65:1124-1 i 32. Mcttler, F., Engels, M., Wild, P. and Bivetti, A., 1979. Herpesvirus-lnfektion bei Zicklein in der Schweiz. Schweiz. Arch. Tierheilk., 121 : 655-662. Metzler, A.E., Schudel, A.A. and Engels, M., 1986. Bovine herpesvirus l: Molecular and antigenic characteristics of variant viruses isolated from calves with neurological disease. Arch. Virol., 87:205-217. Papanastasopoulou, M., Koptopoulos, G., Lekkas, S., Georgakopoulou, E. and Papadopoulos,
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O., 1990. A preliminary investigation on the latency of the goat herpesvirus BHV-6. J. Vet. Med. B, 37:781-784. Papanastasopoulou, M., Koptopoulos, G., Lekkas, S., Papadopoulos, O. and Ludwig, H., 1991. An experimental study on the pathogenicity of the caprine herpesvirus type 1 (CHV-I). Comp. Immun. Microbiol. Infect. Dis., 14: 47-53. Pastoret, P.P., Thiry, E. and Thomas, R., 1986. Logical description of bovine herpesvirus 1 latent infection. J. Gen. Virol., 67: 885-897. Pirak, M., Thiry, E., Brochier, B. and Pastoret, P.P., 1983. Infection experimentale de la chevre par le virus de la rhinotracheite infectieuse bovine (Bovine herpesvirus 1 ) et tentative de reactivation virale. Rec. Med. Vet., 159:1103-1106. Plcbani, G.F., Engels, M., Metzler, A.E. and Wyler, R., 1983. Caprines Herpesvirus in der Schweiz: Verbreitung, H~iufigkeit und Latenz der Infektion. Schweiz. Arch. Tierheilk., 125: 395-411. Probst, U., Wyler, R., Kihm, U., Ackermann, M., Bruckner, L., MUller, H.K. and Ehrensperger, F., 1985. Zur IBR-Virus-Ausscheidung experimentell infizierter Kiihe insbesondere in der Milch. Schweiz. Arch. Tierheilk., 127: 723-733. Saito, J.K., Gribble, D.H., Berrios, P.E., Knight, H.D. and McKercher, D.G., 1974. New Herpesvirus isolate from goats: Preliminary report. Am. J. Vet. Res., 35: 847-848. Schreurs, C., Mettenleiter, T.C., Zuckermann, F., Sugg, N. and Ben-Porat, T., 1988. Glycoprotein gill of pseudorabies virus is multifunctional. J. Virol., 62:2251-2257. Sears, A.E. and Roizman, B., 1990. Amplification by host cell factors of a sequence contained within the herpes simplex virus 1 genome. Proc. Natl. Acad. Sci. USA, 87: 9441-9444. Straub, O.C. and Lorenz, R.J., 1991. Behandlung von im Atmungstrakt latent BHV 1-infizierten Rindern mit verschiedenen Immunsuppressiva. Tier~irztl. Umschau, 6: 344-354. Tarigan, S., Webb, R.F. and Kirkland, D., 1987. Caprine herpesvirus from balanoposthitis. Austral. Vet. J., 64: 321. Tisdall, D.J., Bentley, C.B. and Collins, D.M., 1984. New Zealand caprine herpes virus: Comparison with an Australian isolate and with bovine herpes virus type 1 by restriction endonuclease analysis. N.Z. Vet. J., 32: 99-100. Waldvogel, A., Engels, M., Wild, P., Stiinzi, H. and Wyler, R., 1981. Caprine herpesvirus infection in Switzerland: Some aspects of its pathogenicity. Zbl. Vet. Med. B, 28:612-623. Wittmann, G., Ohlinger, V. and Rziha, H.J, 1984. Latency of Aujeszky's disease virus (ADV) following challenge in previously vaccinated pigs. In: G. Wittman, R.M. Gaskell and H.J. Rziha (Editors), Latent herpesvirus infections in veterinary medicine, Martinus Nijhoff Publishers, Boston/The Hague/Dordrecht/Lancaster, pp. 445-455. Wyler, R., Engels, M. and Schwyzer, M., 1989. Infectious bovine rhinotracheitis/vulvovaginitis (BHVI). In: G. Wittmann (Editor), Herpesvirus diseases of cattle, horses, and pigs, Kluwer Academic Publishers, Boston/Dordrecht/London, pp. 1-72.