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infections in . Role of viral proteins in virulence, pathogenesis and transmission Wam Mulder, Jma Pol, E Gruys, L Jacobs, Mcm de Jong, Bph Peeters, Tg Kimman

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Wam Mulder, Jma Pol, E Gruys, L Jacobs, Mcm de Jong, et al.. Pseudorabies virus infections in pigs. Role of viral proteins in virulence, pathogenesis and transmission. Veterinary Research, BioMed Central, 1997, 28 (1), pp.1-17. ￿hal-00902454￿

HAL Id: hal-00902454 https://hal.archives-ouvertes.fr/hal-00902454 Submitted on 1 Jan 1997

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Pseudorabies virus infections in pigs. Role of viral proteins in virulence, pathogenesis and transmission

WAM Mulder JMA Pol E Gruys L Jacobs1 MCM De Jong BPH Peeters TG Kimman

J Deportment qf Pathnbiology «rtd Epidemiology. Department ofPorcille and Erotic Viml Diseases. ln.ctitute for Alima Science and Health (ID-I)LO), PO Box 365. 8200 AJ Gelo.rtud; 2 Deportment ofllarB’ Veteri fnf/)o/f!t,’v. t7;?n’

(Received 2 May 1996; accepted 23 July 1996)

Summary ― This paper reviews new findings on the biological functions of pseudorabies virus (PRV) proteins. It focuses on the role of PRV proteins in the pathogenicity, immunogenicity and transmission of PRV strains in pigs. Furthermore, it evaluates potential risks that are connected with the use of PRV vector strains. Special emphasis is placed upon the spread of genetically engineered vaccine strains within pigs or between pigs. pseudorabies virus protein f pseudorabies virus vector f virulence f pathogenesis f transmission

Résumé ― Rôle des protéines du virus de la maladie d’Aujeszky dans la virulence, la pathogenèse et la transmission chez le porc. Cet article est une revue de nouveaux résultats concernant les fonctions biologiques des protéines du virus de la maladie d’Aujeszky. Le rôle de ces protéines est étudié dans le pouvoir pathogène, la réponse immunitaire et la transmission de souches vaccinales entre porcs. De plus les risques liés à l’utilisation de souches de virus de maladie d’Aujeszky comme vecteurs sont évalués, et particulièrement le risque de dissémination de souches de virus de maladie d’Aujeszky génétiquement modifiées chez le porc et entre porcs. protéine du virus de la maladie d’Aujeszky f vecteur de maladie d’Aujeszky f virulence / pouvoir pathogène f transmission

* Correspondence and reprints Tel: (31) 320 238 238: fax: (31 ) 320 238 668: e-mail: W [email protected] Introduction: PRV infection and vaccination ...... 2

Viral genome and encoded proteins ...... 3

Functions of PRV proteins in virulence and pathogenesis ...... 4

Envelope glycoproteins ...... 4

Virus-encoded enzymes ...... 6 Proteins involved in virion assembly...... 7

PRV proteins in immunity ...... 7

PRV proteins in transmission...... 8

PRV as a vector vaccine ...... 9

Prospects and new developments ...... 11I

Conclusions ...... 1I

INTRODUCTION: PRV INFECTION causing the infection (reviewed in Gustafson, AND VACCINATION 1986; Pensaert and Kluge, 1989; Wittmann and Rziha, 1989). Natural infections mainly occur Pseudorabies virus (PRV) (synonyms: by the respiratory route. Young pigs may Aujeszky’s disease virus and suid herpesvirus develop neurological signs such as vomiting, type 1) is a member of the subfamily alpha- scratching, trembling, ataxia, paralysis and con- herpesvirinae, which also includes the human vulsions, and they may die of severe (HSV) types I and 2, vari- encephalomyelitis. When the central nervous cella-zoster virus, bovine herpesvirus type l, system (CNS) is infected, PRV generally goes and equine herpesvirus type I (Roizman and latent (Rziha et al, 1986). Older pigs usually Baines, 1991; Roizman, 1992). PRV is a highly survive the infection, but may develop fever and that causes neurological dis- respiratory signs, for example, sneezing and orders in pigs, which are the natural host, as well pneumonia, and they may grow more slowly. as a wide range of domestic and wild animals In addition, infection of the respiratory tract usu- (Gustafson, 1986; Pensaert and Kluge, 1989). ally causes the draining lymph nodes in the The infection is usually fatal to susceptible ani- oropharynx region to become infected. A low mals, and only pigs and horses may survive number of cell-associated and cell-free virus can infection (Kimman et al, 1991 ). Clinical signs of be found in viremic pigs (Wittmann et al, 1980; Aujeszky’s disease in pigs vary widely, from Nauwynck and Pensaert, 1995). PRV-infected subclinical symptoms to death. The outcome of mononuclear blood cells may damage internal the disease depends upon the age of the pigs, organs, and may cause reproductive disorders the level of passive or active immunity, the vir- in pregnant sows (Pensaert et al, 1991; ulence of the virus strain, and the dose of virus Nauwynck and Pensaert, 1992). Pigs are the sole reservoir of PRV and the kinase (TK), which supports viral DNA repli- only source of virus transmission. PRV is preva- cation, has been deleted from these virus strains. lent in most parts of the world and causes severe In most eradication campaigns, gE-negative economic losses in the swine Because industry. vaccine strains are used in conjunction with a the presence of the disease can lead to trade bar- serological test that specifically detects anti- riers set between countries being up (Van bodies against gE. The use of these so-called Oirschot, 1994), campaigns have been mounted marker makes it possible to identify to eradicate PRV from swine with or populations pigs infected with wild-type virus within vac- without the use of vaccines (reviewed in Stege- cinated populations (Van Oirschot et al, 1988, man, 1995). Several types of PRV vaccines are 1990; Kit, 1990). available, namely conventionally or genetically However, there is still the concern that live engineered live attenuated vaccines, killed whole PRV vaccines may still be virulent, that they virus vaccines, and subunit vaccines (Wittmann, may be able to go latent, and that they may be 1991; Kimman, 1992a; Pensaert et al, 1992; transmitted to unvaccinated pigs. Subsequent Kimman et al, However, in 1995). general, live recombination with related vaccine strains or vaccines are more effective than dead vaccines wild-type PRV strains could lead to recombi- and the efficacy of live vaccines can be further nant PRV strains that go wild. Furthermore, enhanced by (Pensaert et al, 1992). adjuvants there remains a need for more potent vaccines The of PRV vaccines also protective efficacy that induce complete protection (Pensaert et al, on the virus strain, the of virus, depends quantity 1992). This review summarizes recent findings and the vaccination scheme (Kimman, 1992b). on the role of PRV proteins in virulence, patho- Conventionally attenuated live vaccine genesis, immunogenicity and transmission. strains, however, contain several characterized and uncharacterized mutations that may reduce the immunogenicity. Furthermore, the possibil- VIRAL GENOME AND ENCODED ity cannot be excluded that these strains may PROTEINS revert to virulence. Consequently, several groups have developed genetically engineered vaccine The genome of PRV is a linear double-stranded strains with defined deletions in the genes encod- DNA molecule of approximately 140 kb (fig 1). ing glycoproteins E (gE), C (gC), or G (gG) (Kit It is divided in a unique long sequence of 95 kb et al, 1987; Marchioli et al, 1987; Moormann et and a unique short region of 9 kb, which is al, 1990). To enhance their safety, thymidine flanked by inverted repeat sequences of 15 kb. The viral genome encodes at least 70 proteins, branes of infected cells (Rauh and Mettenleiter, and the nucleotide sequence of more than 40 1991; Peeters et at, 1992a,b; Spear, 1993b). genes has been obtained (TC Mettenleiter, per- Because PRV glycoproteins show similarities sonal communication). The gene arrangement with their respective glycoproteins in other her- in PRV is collinear with that of highly herpes pesviruses, it was agreed at the l8th lnternn- virus I the simplex type (HSV-1), prototype tional Herpesviru.s Work.shop (Pittsburgh, Penn- And because amino acid alphaherpesvirus. the sylvania, July 1993) that the nomenclature for the of PRV and HSV- sequences many proteins envelope glycoproteins of all herpesviruses are these have iden- homologous, proteins may should follow the alphabetic designation of tical or similar functions in both biological HSV- glycoproteins. Ten PRV glycoproteins . Much has been made in the progress have been identified thus far: gB, gC, gD, gE, structural and functional of the viral analysis gG, gH, gl, gK, gL and gN (Mettenleiter, 1994a; glycoproteins and other gene products involved Jons et at, 1995). Four of these (gC, gE, gI and in virulence (Mettenleiter, 1991, 1994a; De gG) are not necessary for virus replication in Wind, 1992; Kimman et al, 1992d; De Wind et tissue culture, and are therefore considered al, 1994; Gielkens and Peeters, 1994). nonessential. However, none of the field virus strains isolated from pigs lack gl or gC (Kit, 1990), (Van Oirschot, 1989) or (Marchi- FUNCTIONS OF PRV PROTEINS gE gG oli et at, 1987), which that are IN VIRULENCE AND PATHOGENESIS suggests they probably essential for the virus to survive in the populations. In contrast, glycoproteins gB, The PRV proteins that determine virulence of gD, gH and probably gL are essential for the the virus can be roughly categorized into three infectivity of the virus, since virus particles lack- groups: I) envelope glycoproteins that mediate ing one of these glycoproteins cannot penetrate virus entry and virus spread in the host; 2) virus- host cells (Rauh and Mettenleiter, 1991; Peeters encoded enzymes involved in DNA metabolism et al, 1992a,b). or phosphorylation; and 3) proteins involved in virion assembly. Glycoprotein C (gC) mediates the attachment of PRV, HSV and BHV-1 to host cells (Spear, 1993a). This glycoprotein contains a heparin- Envelope glycoproteins binding domain that is involved in the primary attachment of PRV to heparan sulphate proteo- The entry of PRV into host cells is mediated by glycans on the cell surface (Sawitzky et at, 1990; glycoprotein spikes that project from the sur- Mettenleiter, 1990). This low affinity binding face of the virus particles. These are involved is heparin-sensitive. Stable attachment of both in several important steps during the infection, PRV and HSV to host cells is subsequently such as attachment to the host cell, fusion with mediated by the binding of gD to an unidentified the cellular membrane, and entry of the nucleo- cellular receptor and results in a heparin-resistant capsid (reviewed in Spear, 1993a). They also binding (Johnson and Ligas, 1988; Karger and mediate viral spread from infected to uninfected Mettenleiter, 1993). Cells expressing gD cells. Viral spread in the host can occur in two encoded by HSV-I, HSV-2, PRV and BHV-II ways, either by virus particles being released can be resistant to viral infection (Spear, 1993a). from infected cells and the subsequent infection For HSV, it has been shown that the virus can of uninfected cells, or by direct cell-to-cell trans- bind, but cannot penetrate cells expressing gD mission. Although the exact mechanism for the (Campadelli-Fiume et at, 1988; Johnson and latter is unknown, it has been suggested that Spear, 1989). Surprisingly, however, gC is not PRV-induced cell-cell fusion requires the essential for virus infectivity, indicating either expression of viral glycoproteins in the mem- that other viral glycoproteins also recognize hep- aran sulphate or that there exists a pathway for reinfection of postsynaptic neurons. Our find- virus attachment that does not require gC. After ing that PRV can spread across neurons with- attachment, the virion envelope and the cyto- out the presence of gD suggests that the mecha- plasmic membrane fuse, although a principal nism of neuronal transfer may resemble the fusion protein has not yet been identified. The cell-to-cell spread of PRV in tissue culture. We current opinion is that several glycoproteins act speculate that the nature of the cell surface may together to cause this fusion (Mettenleiter, determine whether gD is required for penetration 1994b). Glycoproteins B and H (and probably or not. It has been reported that whereas apical gL), which mediate membrane fusion, are essen- attachment of HSV-1 to polarized Madin-Darby tial for free virions to be infective (Rauh and canine kidney cells depends on gC, but basal Mettenleiter, 199 1; Peeters et al, 1992a,b; Klupp attachment does not (Sears et al, 1991). Simi- et al, 1994). Recently, Klupp et al (1994) showed larly, it may be that gD is only required for PRV that gH and gL form a noncovalently linked to enter the cell at its apex, but not required for complex that functions as an entity. The glyco- lateral or basal entry. Different proteoglycans proteins B and H are present in the membrane of present on the cell surface may be responsible for infected cells and are also required for direct different interactions with the virus (Kjellen and cell-to-cell transmission of the virus in tissue Lindahl, 1991 As mentioned above, heparan culture. It has been suggested that they induce a sulphate proteoglycan has been shown to play a transient fusion of infected and uninfected cells, major role in the gC-mediated attachment of allowing virus particles to spread (Peeters et al, HSV-I and PRV to cells (Spear, 1993a). 1993). In contrast to gD, gE is an essential protein in Interestingly, and in contrast to gD of HSV transneuronal spread of PRV. Studies have and BHV-1, gD of PRV is not necessary for shown that gE-negative PRV replicates in cell-to-cell transmission. After gD is phenotyp- peripheral tissues, infects first-order neurons, ically complemented by propagating gD mutants and spreads towards the CNS via both the olfac- on cell lines that express gD, gD-negative PRV tory and trigeminal routes. However, gE-nega- is able to infect primary target cells and spread tive PRV is much less able to infect second- and directly from cell-to-cell both in vitro and in third-order neurons in the porcine CNS (Jacobs vivo in mice (Babic et al, 1993; Heffner et al, et al, 1993a; Mulder et al, 1994b; Kritas et al, 1993; Peeters et al, 1993). Moreover, we recently 1994a,b; 1995). Enquist et al (1994) used genetic found that phenotypically complemented gD- complementation to demonstrate that gE or gl negative PRV can also spread transneuronally, enable the transneuronal spread of PRV into the ie, axonal spread across synapses into and within visual centres of the rat after the virus enters the the CNS of pigs (Mulder et al, 1996). Immuno- first-order neurons in the retina. In addition, gE histochemical examination of infected pigs and gI appear to function during the anterograde showed that PRV gD mutants had infected sec- transport of PRV, as demonstrated by studying ond- and third-order neurons in the olfactory the dissemination of PRV strains in the maxillary bulb, brain stem, and medulla oblongata. Thus, nerve and the trigeminal ganglion of intranasally not only in rodents, but also in pigs, the natural inoculated pigs (Kritas et al, 1995). In vitro the host of PRV, the transneuronal spread of the gE-gI complex promotes cell-to-cell spread and virus can occur independent of gD. Although it is involved in release of the virus from cells and is not known how herpesviruses spread through in cell fusion (Zsak et al, 1992; Jacobs et al, neurons and across synapses, Card et al (1993) 1993b; Jacobs, 1994). These findings suggest suggested that transneuronal transfer of PRV that the gE-gI complex may promote the neuron- requires the fusion of viral envelopes with the to-neuron transmission of PRV by a mechanism synaptic membranes, which leads to the release that is similar to cell-to-cell spread in tissue cul- of virions in the synaptic cleft and subsequent ture. Thus, the gE-gI complex may promote the release of virus from first-order neurons, or may 1995a). Moreover, although viral TK and RR promote the fusion of synaptically linked neu- activity were required in order for PRV to repli- rons. The possibility cannot be excluded, how- cate in vitro in resting peripheral blood lym- ever, that the gE-gI complex has other func- phocytes and monocytes, the glycoproteins E tions in the transneuronal transfer of PRV, such and G and the US3-encoded protein kinase (PK) as promoting the intraneuronal transport of the were not. And while Con A stimulation of lym- virus. phocytes restored the viral TK defect, it did not Like gE-negative PRV, gl-negative PRV has restore the viral RR defect (Mulder et al, 1995a). In with this, virus mutants a reduced transneuronal spread in pigs, but to a agreement lacking RR or TK no severe of dis- lesser extent than gE-negative PRV (Kritas et caused clinical signs al, 1994a,b, 1995). This neuronal spread might ease in pigs, and the replication of an RR mutant be restricted because the interaction with gE is was more severely retarded (100- to 1 000-fold) disturbed. Results from pig and rodents studies in the nasal and oropharyngeal tissues than the of a TK mutant Wind et are similar. Both gE and gI proteins are required replication (De al, 1993; et in for the infection of second- and third-order neu- Kimman al, 1994). Thus, results of both vitro and in vivo indicate that viral rons in some circuits of the rat eye and heart, experiments RR is more for efficient viral but not in other circuits (Card et al, 1992; Ter activity important DNA than viral TK Horst et al, 1993; Whealy et al, 1993; Standish synthesis activity. et al, 1994). Another virus-encoded enzyme, alkaline nuclease, which is encoded by the UL12 gene, has not been found essential for growth of PRV Virus-encoded enzymes in tissue culture, although it is important for vir- ulence of PRV in mice (De Wind et al, 1992a, The UL23-encoded thymidine kinase (TK) and 1994). It has been reported that this alkaline the UL39- and the UL40-encoded ribonucleotide nuclease of HSV plays a role in processing or reductase subunits (RRI, RR2) support viral packaging viral DNA into viral particles (Weller DNA replication. TK and RR function in the et al, 1990; Shao et al, 1993). Another enzyme salvage and de novo pathways of deoxyribo- encoded by PRV that functions in the repair nucleotide synthesis, respectively (Reichard, pathway of nucleic acid metabolism is uracil 1988). Although viral RR and TK are not essen- DNA glycosylase (UL2; Dean and Cheung, tial for the virus to grow in dividing cells (De 1993; Klupp et al, 1993). How and to what Wind et al, 1993), they are required to produce extent this enzyme effects the virulence of PRV, infectious virus particles in non-dividing cells, however, is unknown. Finally, the dUTPase such as neurons and resting peripheral blood (UL50) of HSV-1 is reportedly involved in mononuclear cells (Mulder et al, 1995a). A low neurovirulence in mice (Pyles et al, 1992). The number of cell-associated and cell-free PRV can corresponding PRV homolog, however, has not be found in viraemic pigs and may damage inter- been identified yet. nal and cause abortion, even in vacci- organs Another group of viral enzymes are the pro- nated animals et al, 1980; (Wittmann Nauwynck tein kinases (PK), which comprise a large fam- and Pensaert, 1992, It has been 1995). reported ily of enzymes. These PK regulate the protein that cells, in addition to tonsils and lymphoid phosphorylation that plays a major role in signal various neural cells, may harbour latent virus transduction, regulation of growth and differ- and (Wittmann Rziha, 1989). entiation of cells (Hanks et al, 1988). Both the We found that replication of PRV in porcine US3 and UL13 gene encode a serine/threonine peripheral blood monocytes and lymphocytes kinase that is nonessential for growth of PRV depends on the cell type, the viral genotype, and in cell culture (Van Zijl et al, 1990; De Wind et on the state of cellular activation (Mulder et al, al, 1992a). UL13 mutants grow as efficiently as wild-type virus in cell culture, and their viru- for virus replication and could play an essential lence for mice is not diminished (De Wind et role in the formation of mature virions. al, 1992b, 1994). In contrast, the growth of US3- encoded PK mutants is retarded compared to wild-type virus and depends on cell type (Kim- PRV PROTEINS IN IMMUNITY man et al, 1994; Mulder et al, 1995a). More- over, US3 mutants are less virulent than UL133 When infected with a virulent PRV strain, pigs mutants in both mice and pigs (Kimman et al, develop an immune response that can completely 1992a; 1994; De Wind et al, 1994). Wagenaar et or almost completely prevent the virus from al (1995) found that a defect in the morphogen- replicating after the pig becomes reinfected. esis of US3 mutants is responsible for their However, vaccination seldom produces this level reduced replication in vitro and their reduced of immunity. Most vaccines in fact offer only virulence in vivo. They found that PK-negative partial protection against viral replication (Pen- virus particles were not debudded at the outer saert et al, 1992). In fact, serum antibodies, nuclear membrane, both in epithelial cells of whether actively or passively acquired, as well porcine nasal mucosa explants and in cells of as mucosal IgA antibodies appear to induce only the porcine kidney cell line SK6 (Kasza et al, partial protection (Kimman et al, 1992e). More- 1971 ). As a result, US3-mutant virions accu- over, pigs that are fully immune after a first mulated in the perinuclear membrane. Little is infection do not develop a secondary B cell known about the substrates of the PRV kinases. response upon a second infection, although they There is one in vitro report showing phospho- do develop a strong secondary T cell response. PK of a rylation by US3 major virion phospho- It is possible that this T cell response may induce l22 kDa protein of (Zhang et al, 1990). the emergence of cytolytic cells that quickly eliminate the challenge virus, and thus prevent- ing a secondary B cell response. However, the Proteins involved in virion assembly lymphoproliferative response probably also trig- gers antiviral activity, such as the release of The of is an icosahedral capsid herpesviruses interferons and tumour necrosis factor (Pol, protein structure that encloses the core contain- 1990; Schijns et al, 199 1).). ing the viral DNA. Seven capsid proteins have It is known that the envelope glycoproteins B, been identified for HSV-1 (encoded by UL18, C and D are for immune UL19, UL26, UL26,5, UL35 and UL38) (Rixon, major targets responses. Cells that express these glycoproteins can be 1993; Haarr and Skulstad, 1994). The sequences recognized and killed by various immune mech- encoding two major PRV capsid proteins of 142 anisms, such as and medi- and 32 kDa have recently been elucidated antibody complement ated cell lysis, antibody-dependent cell-medi- (Yamada et al, 1991; Klupp et al, 1992), and are ated cytotoxity, and cytotoxic T-lymphocytes the homologues of genes UL19 and UL18 of (reviewed in Chinsakchai and Molitor, 1994). HSV-1. In addition, the PRV gene that is the homolog of the HSV-1 UL21 gene has been One immune mechanism, the induction of identified (De Wind et al, 1992b). The PRV neutralizing antibodies, peaks within 2-3 weeks UL21 gene encodes a 62 kDa protein, and is after infection with PRV (Wittmann et al, 1976; probably closely associated with capsids during Martin et al, 1986). Antibodies in pigs that are capsid assembly and plays a role in processing or infected by PRV field strains, are directed against packaging viral DNA (De Wind et al, 1992b). gB, gC, gD, gE, gH, gG and gl (Kit, 1990), The function of the 79 kDa PRV protein that iss immediate early (IE) and nucleocapsid proteins homologous to infected cell protein (ICP)18.5 (Cheung, 1990; McGinley et a], 1992). Of these (UL28) of HSV-1 was studied by Mettenleiter et glycoproteins, gB, gC and gD appear to be the al (1993). This protein appears to be essential major targets for neutralizing antibodies, whereas gG and gE are not (Ben Porat et al, 1986; Zuck- These results indicate that lymphokine activated ermann et al, 1988). Furthermore, gC is a tar- killer (Lak) cells kill the PRV-infected L14 cells. get antigen for cytotoxic T cells in both mice The Lak cells appear to recognize different viral and pigs (Zuckermann et al, 1990). However, it proteins. L14 cells that were transfected with also binds to the complement C3 component and stably expressed gB or gC were killed to from pigs and cows, but not from humans the same extent as PRV-infected target cells. In (Heumer et al, 1993), as a result of which the contrast, L14 cells that were transfected with complement is less able to neutralize the virus. gD or the immediate early protein were not lysed This phenomenon is interesting, because the above background values (Kimman et al, 1996b). interaction between gC and C3 is specific to cer- Surprisingly, PRV proteins that apparently tain animal species and not to others, and it may do not have particular epitopes that trigger the therefore attribute to the host range of alpha- immune system, such as those involved in virus herpesviruses. replication or phosphorylation, may still con- The role of PRV glycoproteins B, C and D in tribute to the immunogenicity of live PRV inducing protection has been derived from vac- strains. Kimman et al (1994) and De Wind et al cination experiments with purified glycopro- (1993) found that inactivating TK, RR or the teins (Iglesias et al, 1990; Mukamoto et al, 1991), US-3-encoded PK affected the immunogenic- because these virus mutants with glycoproteins expressed by adenovirus vec- ity of PRV, possibly tor (Eloit et al, 1990), and with vaccinia virus replicated less efficiently in vivo. In addition, vectors (Riviere et al, 1992; Brockmeier et al, the effect of inactivating gE and PK or gE and 1993; Mengeling et al, 1994). In addition, their TK appeared to be synergistic (Kimman et al, that these viral protecting properties were studied using the bac- 1994). They hypothesized ulovirus expression system (Xuan-XueNan et enzymes help induce protective immunity by the viral mass to al, 1995), and an immune stimulating complex increasing antigenic presented (ISCOM) (Tsudi et al, 1991).). the immune system. In general terms, PRV strains that replicate strongly, not only appear Little is known about how PRV proteins to be more virulent, but also more interact with effector cells the cell-medi- immunogenic during and protective. ated immune response. It has been shown that gB and gC can induce the proliferation of T cells et In not the (Kimman al, 1996a). HSV, only PRV PROTEINS IN TRANSMISSION structural proteins, but also the immediate early are for T proteins target antigens cytotoxic lym- There has been some concern voiced about the and natural killer cells et al, phocytes (Martin possible transmission of live PRV strains to 1988; Banks et al, 1991; Fitzgerald-Bocarsly et unvaccinated animals, and the possibility that al, blood of 1991 ). When peripheral lymphocytes these might then recombine with related vac- PRV-immune miniature of SLAd/d pigs haplo- cine or wild-type strains, leading to the spread or type were antigenically stimulated in vitro, cyto- survival of recombinant PRV. Recently, in fact, toxic cells that were able to lyse PRV-infected Christensen et al (1992) suggested that certain L14 cells were L]4 cells are immor- generated. field isolates of PRV may have been derived talized B-cells of SLAdld haplotype. The PRV- from attenuated vaccine strains. When attenu- infected L14 cells were killed in the presence ated vaccine strains are administered intramus- of CD2+CD4-CD8hright-cells, did not appear to cularly, virus is generally not excreted through be was MHC-restricted, strongly augmented by the mucosa or transmitted to susceptible ani- in vitro antigenic stimulation. Moreover, killing mals (Kimman et al, 1995). However, the pos- did not appear to be virus-specific, that is, the sibility cannot be excluded that vaccine strains target cell line K562 for natural killer cells was might, by error or deliberately, be administered also lysed by effector cells of immune pigs. intranasally. In order to develop safe PRV live vaccines In theory, live PRV vaccines could become that are not transmitted to susceptible pigs, cer- transmissible through recombining with related tain PRV genes should be deleted. To determine vaccine strains or wild-type PRV. Coinfection of the genes of PRV to which the transmission of a host with two different herpesvirus strains may the virus should be attributed, we used a small- result in the generation of recombinant strains scale experimental model (De Jong and Kim- (Henderson et al, 1991; Dangler et al, 1993; man, 1994) to analyze PRV mutant strains and Glazenburg et al, 1995). The question thus arose PRV vector strains under experimental condi- as to whether a gE-negative, TK-positive PRV tions et The basic (Mulder al, 1995b). repro- strain could be transmitted in a population which duction ratio or R, was used to predict the spread is also infected with wild-type PRV. When pigs of virus from infected animals to contact- were coinoc:ulated with a gE-negative PRV strain animals. In the has a threshold exposed model, R and a gE-positive (wild-type) PRV strain to property: when R > 1, the infection can spread; determine whether they competed in transmis- when R < I, the infection will (Ander- disappear sion (Mulder et al, 1995b), both strains were son and 1982, 1985; De and Diek- May, Jong transmitted to contact pigs and no severe com- mann, 1992). The transmission of PRV strains petition was detected. However, less gE-negative that lacked gE or TK and the transmission of a virus was excreted in oropharyngeal fluid in co- PRV TK-, vector strain that (gE-, gG-) expresses inoculated pigs than in pigs inoculated with only E of HCV were studied. The value of R for a gE-negative virus. Furthermore, co-inoculated gE-negative strain was found to be 10.1, and for pigs excreted gE-positive virus longer than gE- a TK-negative strain it was 5. The R value for the negative virus. This finding could mean that gE- vector strain expressing E I was 0. I 8, which did negative viruses will have less chance to sur- not differ significantly from the R value of con- vive in pigs as gE-positive viruses. trol strain, which did not express E I . Only the R of the gE-negative strain was significantly Although gE-/TK- PRV strains do not appear greater than 1 (P = 0.0005). to spread, the risks of transmission of PRV vac- cine strains can be further reduced The R values in populations larger than those by deleting essential genes or genes in used in the experiments will be the same as esti- inserting foreign essential PRV so that if mated from these experiments. The supposition genes (for example gD), that R is independent of the population size fol- recombination were to occur, the resulting strains would be nonviable. lows logically from the mass-action argument (De Jong et al, 1995) and was also shown to be true for PRV in pigs (Bouma et al, 1995). Thus our studies indicated that PRV strains that lack PRV AS A VECTOR VACCINE only gE or TK may be transmitted from intranasally inoculated pigs to contact pigs, but PRV strains can be used as vectors to express one or more of other a strain that lacks gE, TK and gG appears unable foreign genes microorgan- to spread to susceptible contact pigs. However, isms. For example, Van Zijl et al (1991 ) showed vaccination reduces transmission of PRV (De that immunizing pigs with PRV recombinants Jong and Kimman, 1994). Therefore, PRV that expressed the envelope glycoprotein E1I strains are less likely to be transmitted in vacci- (E I ) of hog cholera virus (HCV) protected them nated populations. These studies show that in against both pseudorabies and hog cholera. So order to develop nonspreading PRV strains, not far, different proteins have been expressed by only TK or gE, but both or others should be recombinant PRV to: 1 ) induce immunity against deleted. Moreover, the results show that a small- foreign pathogens; 2) monitor virus infection scale experimental model can be used to esti- more easily; or 3) demonstrate functional homol- mate and predict the transmission of PRV mutant ogy between viral proteins (Thomsen et al, 1987; strains. Whealy et al, 1988, 1989; Mettenleiter et al, 1990; Mettenleiter, 1991; Kopp and Metten- field virus would probably result in a less viru- leiter, 1992; Sedegah et al, 1992). Like vac- lent virus. However, if it was inserted in a viral cinia virus, which is the best known and most gene that does not contribute to virulence (for widely studied vector (Bostock, 1990; Moss, example gG of PRV), recombination with the 1991 PRV has a large genome (± 140 kb) in fieldvirus would probably result in a strain that which large fragments of foreign DNA can be is as virulent or more virulent than the wild-type stably integrated. Non-essential genes can be strain. Recently we constructed just such a worst- deleted to generate additional space. Foreign case recombinant strain, with the envelope glyco- genes have been stably inserted in the TK, gG, protein El of HCV inserted into the gG locus gE and gD loci of PRV (Mettenleiter et al, 1990; of PRV, and tested it in pigs. The (gG-,E+) I Van Zijl et al, 1991; Peeters et al, unpublished vector strain was indeed virulent for pigs, but results). the incorporation of EI did not essentially Inserting foreign genes into live vector vac- change the pathogenesis of the PRV vector (Mul- cines demands extra safety precautions, how- der et al, 1994a). ever, because the expression of foreign genes Several experimental studies have demon- may alter the biological properties of the vec- strated that coinoculated modified live PRV tor virus. Three potential risks should always be (vaccine) strains could recombine in vivo to cre- examined (Kimman, 1992c). First, the tropism of ate virulent recombinant strains (Henderson et at, the vector virus for particular cells, tissues or 1990, 1991; Katz et al, 1990; Dangler et al, hosts could change as a result of which the vir- 1993). However, part of the increased virulence ulence of the vector virus for different hosts may have been caused by complementation could change. Second, through homologous or (Glazenburg et at, 1994; Visser and Rziha, the illegitimate recombination, foreign gene(s) 1994). A prerequisite for a recombinational event could be transferred from the vector virus to between PRV strains is that these viruses have to other vaccine or wild-type strains. Third, the enter the same cell. However, vaccine viruses transmission of the vector virus could properties are generally administered intramuscularly and change, as a result of which recombinant virus replication is restricted to the site of inoculation could spread. Little is known about the proper- (Kimman et al, 1995), whereas after natural ties that determine cell or host tropism. Some infection the virus replicates primarily in the results have also been on in published changes nasopharyngeal mucosa, tonsils, regional lymph cell or host tropism in poxvirus vector strains nodes and lungs. Recombination has been et and on that deter- (Taylor al, 1991), genes observed only in a worst-case scenario: in mice mine host tropism for poxviruses (Perkus et al, and pigs in vivo recombination of PRV strains 1989). Unfortunately, however, it is unknown only occurred when high doses of two virus which genes determine these properties of a strains were coinoculated at the same location. gi.ven microorganism. (Glazenburg et at, 1995). Two separately inoc- Perhaps the greatest concern about the use ulated mutant virus strains still recombined in of PRV vector vaccines is that a recombinant mice even after a lapse of 2 h (Glazenburg et at, pathogen goes wild. This could occur if recom- 1994). The frequency of recombination appeared bination causes the foreign gene to be trans- to depend on the capacity of the virus strains to ferred from the vector vaccine to a virulent field replicate and on the sequence or the genomic strain. However, the virulence of such a recom- location of the genetic markers. Although the binant would not only depend on the foreign possibility of recombination between vaccine gene inserted, but also on the site where it was and PRV field strains cannot completely be inserted. If a foreign gene was inserted in a viral excluded, the risks can be further greatly less- gene that contributed to virulence (for example, ened if (several) virulent genes are deleted from TK and gE of PRV), recombination with the live vaccines, if the coinoculation of PRV vac- cines with complementing gene deletions is pathogens could be used for vaccination pur- avoided, and if the pig population is thoroughly poses in pigs or other animal species. vaccinated.

CONCLUSION PROSPECTS AND NEW DEVELOPMENTS The deletion of two virulence-determining genes, such as TK and gE, is probably necessary to pre- Many researchers are trying to develop non- vent PRV from spreading. A drawback is that transmissible PRV strains or strains that cannot deleting several genes renders PRV strains less be transmitted after recombination with wild- replicating, thus less immunogenic. In addition, type strains. Essential PRV genes, such as the thorough vaccination of the pig population essential glycoproteins involved in viral entry reduces the transmission of PRV vaccine strains and them from or to and spread, are inactivated by deleting these may prevent spreading with other PRV strains. genes or by inserting a foreign gene. Vaccination recombining with phenotypically complemented gD mutants Recombining live PRV (vector) vaccine seems promising because the gD protein has a strains will probably cause no real problems, unique biological property. The gD-negative because it was only demonstrated under worst- mutants, however, can still spread in the host case scenarios. Moreover, inserting foreign via direct cell-to-cell transmission, and there- genes in essential genes of PRV (for example fore probably induce a solid immune response. gD, gH, gB) will render recombinant strains Moreover, progeny virions that are shed by vac- non-viable. cinated animals are noninfectious because they lack gD, and the vector vaccine strains cannot be transmitted to other susceptible animals. (Heffner REFERENCES et al, 1993; Mettenleiter et al, 1994; Peeters et al, 1994). The gD mutants, however, could still be Anderson RM, May RM (1982) Directly transmitted problematic, because they are able to infect and infectious diseases: control by vaccination. Sci- replicate within the CNS of pigs (Mulder et al, ence 215, 1053-1060 1996). Nonetheless, by deleting extra viral pro- Anderson RM, May RM ( 1985) Vaccination and herd to infectious diseases. Nature 318, 323- teins, such as the gE/gI complex, the transneu- immunity 329 ronal spread of gD-negative PRV can be pre- vented. The immunogenicity of gD mutants with Babic N, Mettenlciter TC, Flammand A, Ugolini G ( 1993) Role of essential glycoproteins gII and gp50 additional mutations in gE and/or gI must still be in transneuronal transfer of pseudorabies virus assessed. from the hypoglossal nerve of mice. J Virol 67, Fortunately, area-wide eradication of PRV 4421-4426 is feasible (Stegeman et al, 1994; Stegeman, Banks TA, Allen EM, Dasgupta S, Sandri-Goldin R, 1995). If it is achieved, the need to vaccinate Rouse BT ( 1991 ) Herpes simplex virus type-I spe- cific T immedi- against PRV will come to an end. However, in cytotoxic lymphocytes recognize ate-early protcin ICP27. J Virol 65, 3185-31911 border areas where different countries adjoin, vaccination against PRV will probably continue Ben Porat T, DeMarchi J, Lomniczi B, Kaplan AS ( 1986) Role of pseudorabies virus to be for some time in order to control of glycoproteins necessary in eliciting neutralizing antibodies. Vir-ology 154, new small outbreaks. These may be caused by 325-334 importing from other where PRV is pigs regions Bouma A, De Jong MCM, Kimman TG (1995) still or the of virus prevalent by spread through Transmission of pseudorabies virus within pig pop- the air. Besides vaccination against PRV, PRV ulations is independent of the size of the population. vector strains expressing foreign gene(s) of other Prev Vet Med 23, 163-172 Bostock CB (1990) Viruses as vectors. Vet Microbiol De Jong MCM, Diekmann 0, Heesterbeek JAP ( 1995) 23, 55-711 How does transmission of infection depend on Brockmeier SL, Lager KL, Tartaglia J, Riviere M, population size? In: Epidemic Models: Their Struc- Paoletti E, Mengeling WL (1993) Vaccination of ture and Relntion to Data (D Mollison, ed), Cam- pigs against pseudorabies with highly attenuated bridge University Press, 84-94 vaccinia (NYVAC) recombinant viruses. Vet De Wind N ( 1992) Mutagenesis and characterization Microbiol 38. 41-58 of large subgenomic regions of pseudorabies virus. Campadelli-Fiumc G, Arsenakis M, Farabegoli F, PhD Thesis, University of Amsterdam, The Nether- Roizman B ( 1988) Entry of herpes simplex virusI lands in BJ cells that constitutively express viral glyco- De Wind N, Doomen J, Berns A (1992a) Her- protein D is by endocytosis and results in degra- pesviruses encode an unusual protein-serine/thre- dation of the virus. J Virol 62, I 59-167 onine kinase which is nonessential for growth in Card JP, Whealy ME, Robbins AK, Enquist LW culture cells. J Virol 66, 5200-5209 (1992) Pseudorabies virus envelope glycoprotein De Wind N, Wagenaar F, Gielkens ALJ, Kirnman TG, gI influences both neurotropism and virulence dur- Berns A (1992b) The pseudorabies virus homolog ing infection of the rat visual system. J Nmrnsci 66, of the herpes simplex virus UL21 gene product is 3032-3041 a capsid protein which is involved in capsid mat- Card JP, Rinaman L, Lynn RB, Lee BH, Meade RP, uration. J Virol 66, 7096-5209 Miselis RR, Enquist LW (1993) Pseudorabies virus De Wind N, Berns A, Gielkens A, Kimrnan T ( 1993) infection of the rat central nervous system: ultra- Ribonucleotide reductase-deficient mutants of structural characterization of viral replication, trans- pseudorabies virus are avirulent for pigs and induce port, and pathogenesis. J Neurosci 13, 2515-2539 partial protective immunity. J Gen Virol74, 351- Cheung AK (1990) Humoral response to immedi- 359 ate-early protein of pseudorabies virus in swine De Wind N, Peeters BPH, Zijderveld A, Gielkens with induced or naturally acquired infection. Am J ALJ, Berns AJM, Kimman TG (1994) Mutagene- Vet Re.r 51, 222-226 sis and characterization of a 41-kilobase-pair region Chinsakchai S, Molitor TW (1994) lminunobiology of the pseudorabies virus genome: transcription of pseudorabies virus infection in swine. Vet map search for virulence genes, and comparison Iiiiiiiiitiol Iiiiiiiiitiop(ithol 43, 107-I 166 with homologs of herpes simplex virus type 1. 200, 784-790 Christensen LS, Medveczky I, Strandbygaard BS, Pejsak Z (1992) Characterization of field isolates Eloit M, Gilardi-Hebcstreit P, Toma B, Perricaudet of suid herpesvirus I (Aujeszky’s disease virus) M ( 1990) Construction of a defective adenovirus as deratives of attenuated vaccine strains. Arch vector expressing the pseudorabies virus glyco- Virol 124, 225-234 protein gp50 and its use as a live vaccine. J Gen 1 Dangler CA, Henderson LM, Bowman LA, Deaver Virol 71, 2425-2431 RE (1993) Direct isolation and identification of Enquist LW, Dubin J. Whealy ME, Card JP (1994) recombinant pseudorabies virus strains from tis- Complementation analysis of pseudorabies virus sues of experimentally co-infected swine. Am J gE and gl mutants in retinal ganglion cell neu- Vet Re.s 54, 540-545 rotropism. J Virol 68, 5275-5279 Dean H, Cheung A (1993) Nucleotide sequence and Fitzgerald-Bocarsly P, Howell DM, Pettera L, Tehrani transcriptional analysis of pseudorabies virus genes S, Lopez C (1991) Immediate-early gene expres- corresponding to HSV-1 ULl to UL5 and a unique sion is sufficient for induction of natural killer cell- UL3.5 gene. Abstracts 18th Int Herpesvirus Work- mediated lysis of herpes siinplex virus type-1- shop Pittsburgh, USA, A-38 infected fibroblasts. J Viro165, 3151-3160 De Jong MCM, Diekmann 0 ( 1992) A method to cal- Gielkens ALJ, Peeters BP (1994) Functions of culate - for computer-simulated-infections - the Aujeszky’s disease virus proteins in virus repli- threshold value, Ro, that predicts whether or not cation and virulence. Actn Vet Hungar 42, 227- the infection will spread. Prev Vet Med 12, 269- 241l 285 Glazenburg KL, Moormann RJM, Kimman TG, De Jong MCD, Kimman TG (1994) Experimental Gielkens ALJ, Peeters BPH ( 1994) In vivo recom- quantification of vaccine-induced reduction in bination of pseudorabies virus strains in mice. virus transmission. Vaccine 12, 76l-766 Virus Res 34, 1 15-126 Glazenhurg KL, Moormann RJM, Kimman TG, Johnson DC, Ligas MW ( 1988) Herpes simplex viruses Gielkens ALJ, Peeters BPH ( 1995) Genetic recom- lacking glycoprotein gD are unable to inhibit virus bination of pscudorabies virus: evidence that penetration: quantitative evidence for virus-spe- homologous recombination between insert cific cell surface receptors. ,I Virol 62, 4605- 46122 sequences is less frequent than between autolo- Johnson RM, Spear PG ( 1989) Herpes simplex virus gous sequences. Arch Virnl 140, 671-685 mediates interference with herpes simplex virus Gustafson DP (1986) Pseudorabies.In: Disecises nf’ infection. J Virol 63, 8l9-827 Swine Leman et Iowa State (AD al, eds), Ames, J6tis A, Granzow H, Kuchling R, Mettenlciter TC 274-289 University Press, (1995) UL49.5 of pseudorabies virus (PRV) codes Haarr L, Skulstad S (1994) The herpes siinplex virus for an O-glycosylated structural protein of the viral type I particle: structure and molecular functions. envelope. 20th Int Herpesvirus Workshop Gronin- Acra Pathol Microbiot Inununol Scand 102, 321-1 - gen, The Netherlands, Abstract 69 346 Karger A, Mettenleiter TC (1993) Glycoprotein glll Hanks SK, Quin AM, Hunter T ( 1988) The protein and gp50 play dominant roles in the biphasic kinase family: conserved features and deduced phy- attachment of pseudorabies virus. Virolog!- 194, logeny of the catalytic domains. Science 241, 42-52 654-664 Heffner S, Kovacs F, Klupp BG, Mettenleiter TC Kasza L, Shadduck JA, Christofinis GJ (1971) Estab- (1993) Glycoprotein gp50-negative pseudorahics lishmcnt, viral susceptibility, and biological char- virus: a novel toward a approach nonspreading acteristics of a swine kidney cell line SK-6. Re.s live herpesvirus vaccine. J Mw/67. 1529-1537 Vet Sci 13, 46-51 Henderson LM, Katz JB, Erickson GA, Mayfield JE Katz JB, Henderson LM, Erickson GA ( 1990) Recom- (1990) In vivo and in vitro genetic recombination bination in vivo of pseudorabies vaccine strains between conventional and vaccine gene-deleted to produce new virus strains. Vaccine 8, 286-288 strains of pseudorabies virus. Am J Vet Res 51,1, Kimman TG, Binkhorst Van den 1656-1662 GJ, Ingh TSGAM, Pol JMA, Roelvind ME (1991) Aujeszky’s dis- Henderson LM, RL, Davies AJ, Sturtz DR Levings ease in horses fulfils Koch’s postulates. Vet Rec ( 1991 ) Recombination of pseudorabies virus vac- I 28, 103- I Ob cine strains in swine. Am J Vm Re.s 52, 820-825 Kimman TG ( 1992a) Acceptability of Aujeszky’s dis- Heumcr HP, Larcher C, Van Drunen Littel-Van Den ease vaccines. Dei, Biol Stand 79, 129-136 Hurk S, Babiuk LA ( 1993) Species selective inter- action of with the ’unspecific’ Kimman TG ( 19926) Comparative efficacy of three doses of the immune system of the host. Arch Virol 130, 353- genetically engineered Aujeszky’s 364 disease virus vaccine strain 783 in pigs with mater- nal antibodies. Vaccine 10, 363-365 Iglesias G, Molitor T, Reed D, L’italien J (1990) Anti- Kimman TG Risks connected with the use of bodies to Aujeszky’s disease virus in pigs immu- ( 1992c) nized with purified virus glycoproteins. Vet Micro- conventional and genetically engineered vaccines. biol24, 1 -10 VeiQuarl 15, 110-1188 Jacobs L, Mulder WAM, Van Oirschot JT, Gielkens Kimman TG, De Wind N, Oei-Lie N, Pol JMA, Berns ALJ, Kimman TG (1993a) Deleting two amino AJM, Gielkens ALJ ( 1992a) Contribution of single acids in glycoprotein gi of pseudorabies virus genes within the unique short region of Aujeszky’ss decreases virulence and neurotropism for pigs, but disease virus (suid herpesvirus type I)to virulence, does not affect immunogenicity. J Gen Virol 74, pathogenesis and immunogenicity. J Gen Virol 2201-2206 73, 243-2511 Jacobs L, Rziha HJ, Kimman TG, Gielkens ALJ, Van Kimman TG, Brouwers RAM, Daus FJ, Van Oirschot Oirschot JT ( 1993b) Deleting valine-125 and cys- JT, Van Zaane D (1992b) Measurement of iso- teine-126 in glycoprotein gl of pseudorabies virus type-specific antibody responses to Aujeszky’ss strain NIA-3 decreases plaque size and reduces disease virus in sera and mucosal secretions of virulence for mice. Arch Virol 131, 251-264 pigs. Vet Immunot Inittiunopathol 31, 95-1133 Jacobs L ( 1994) Glycoprotein I of pseudorabies virus Kimman TG, De Wind N, De Bruin T, De Visser Y, (Aujeszky’s disease virus; suid herpesvirus-1) and Voermans J (1994) Inactivation of glycoprotein homologous proteins in other alphaherpesvirinae: gE and thymidine kinase or the US3-encoded pro- a brief review. Arch Virnl 137, 209-228 tein kinase synergistically decreases in vivo repli- cation of pseudorabies virus and the induction of Kritas SK, Pensaert MB, Mettenleiter TC (1994b) protective immunity. Virology 205, 51 1-5188 Role of envelope glycoproteins gl, gp63 and gIll in Kimman TG, Gielkens AJL, Glazenburg K, Jacobs the invasion and spread of Aujeszky’s disease in L, De Jong MCM, Mulder WAM, Peeters BPH the olfactory nervous pathways of the pig. J Gen ( 1995) Characterization of live pseudorabies virus Virol75, 2319-2327 vaccines. Den Biol Stand 84, 89-96 Kritas SK, Nauwynck HJ, Pensaert MB (1995) Dis- Kimman TG, De Bruin TGM, Voermans JJM, Peeters semination of wild-type and gC-, gE- and gl- BPH, Bianchi ATJ ( 1996a) Development and anti- deleted mutants of Aujeszky’s disease virus in the nerve and of gen specificity of lymphoproliferation response of maxillary trigeminal ganglion pigs after intranasal inoculation. J Gen Virol76, 2063- pigs to pseudorabies virus: dichotomy between 2066 secondary B- and T-cell responses. Imml/llOlogy 86, 372-378 Marchioli CC, Yancey RY, Wardley RC, Thomsen Kimman TG, Dc Bruin TGM, Voennans JJM, Bianchi BS, Post LE (1987) A vaccine strain of pseudora- bies virus with deletions in the kinase ATJ ( 19966) Cell-mediated immunity to pseudo- thymidine virus: cytolytic effector cells with charac- and glycoprotein X genes. Alii J Vet Res 48, 1577- teristics of lyinphokine-activated killer cells lyse 1583 virus-infected cells and glycoprotein gB- and gC- Martin S, Wardley RC, Donaldson Al (1986) Func- transfected L 14 cells. J Geii Virol77, 987-990 tional antibody responses in pigs vaccinated with Kit S, Shepard M, Ichimura H, Kit M (1987) Second live and inactivated Aujeszky’s disease virus. Res generation of pseudorabies vaccines with deletions Vet Sci 41, 331-335 in thymidinc kinase and glycoprotein genes. Am Martin S, Courtney RJ, Fowler G, Rouse B (1988) J Vet Re 48. 780-793 Herpes simplex virus type I-specific cytotoxic T virus non structural Kit S ( 1990) Genctically engineered vaccines for con- lymphocytes recognize pro- trol of Aujeszky’s disease virus (pseudorabies teins. J Virol 62, 2265-2273 virus). Vaccine 8, 420-424 McGinley MJ, Todd DL, Hill HT, Platt KB (1992) Detection virus infection in sub- Kjell6n L, Lindahl U (1991) Proteoglycans: structures of pseudorabies and interactions. Annu Rer Biochem 60, 443- 475 unit-vaccinated and nonvaccinated pigs using a nucleocapsid-based enzyme-linked immunosor- Kern Mettenleiter TC The viru- Klupp BG, H, (1992) bent assay..l Vet Diagtiost lnve.stig 4, 164-169 lence determining genomic BamHI fragment 4 of WL, Brockmeier SL, KM ( 1994) pseudorabies virus contains genes corresponding to Mengeling Lager the UL I (partia!), UL 18. ULI 9, UL20 and UL211 Evaluation of a recombinant vaccina virus con- virus as genes of herpes simplex virus and a putative origin taining (PR) glycoproteins gp50, gll, gIII of replication. Virology 191, 900-908 a PR vaccine for pigs. Arch Virol 134, 259-269 Klupp B Baumcister J Visser N and Mettenleitcr TC Mettenleiter TC (1990) Interaction of glycoprotein ( 1993) Identification and characterization of two gUt with a cellular hcparinlike substance medi- novel glycoproteins in pseudorabies virus, gK and ates adsorption of pseudorabies virus. J Vir-ol 64, gL. Abstracts l8th Int Herpesvirus Workshop, 278-286 Pittsburgh, USA, C-94 Mettenleiter TC, Kern H, Rauh 1 (1990) A glycopro- Klupp BP, Baumeister J, Karger A, Visser N, Met- tein gX-f3-galaclosidase fusion gene as insertional tenleiter TC ( 1994) Identification and characteri- marker for rapid identification of pseudorabies zation of a novel structural glycoprotcin in pseudo- virus mutants. J Virol Methods 30, 55-66 , gL. J Virol 68, 3868-3878 Mettenleiter TC ( 1991 ) Molecular biology of pseudo- rabies virus disease Kopp A, Mettenlciter TC (1992) Stable rescue of a (Aujeszky’s virus). Conrp 1111111111101 Dis 151-163 glycoprotein gif deletion mutant of pscudorabies MicrobiolIl/fect 14, virus by glycoproteiii gl of bovine herpesvirus I. Mettenleiter TC, Saalmuller A, Weiland F (1993) J Virol 66, 2754-2762 Pseudorabies virus protein homologous to herpes Kritas SK, Pensaert MB, Mettenleiter TC (1994a) simplex virus type I ICPI 8.5 is necessary for cap- Invasion and spread of single glycoprotein deleted sid maturation. J Virol 67, 1236-1245 mutants of Aujeszky’s disease virus (ADV) in the Mettenlciter TC (1994a) Pseudorabies (Aujeszky’s trigeminal nervous pathway of pigs after intranasal disease) virus: state of the art, August 1993. Actn inoculation. VotMicrnbiol 40, 323-334 Vet Hung 42, 153-177 Mettenleiter TC (1994b) Initiation and spread of a- tion with Aujeszky’s disease virus. Vet Microbiol herpesvirus infections. Trends Microbiol2, 2-4 43,307-3144 Mettenleiter TC, Klupp BG, Weiland F, Visser N Peeters B, De Wind N, Hooisma M, Wagenaar F, (1994) Characterization of a quadruple glycopro- Gielkens A, Moormann R (1992a) Pseudorabies tein-delete pseudorabies virus mutant for use as a virus envelope glycoproteins gp50 and gII are biologically safe live virus vaccine. J Gen Virol essential for virus penetration, but only gll is 75,1723-1733 involved in membrane fusion. J Virol 66, 894-905 Moormann RJM, De Rover T, Briaire J, Peeters BPH, Peeters B. De Wind N, Broer R, Gielkens A, Moor- Gielkens ALJ, Van Oirschot JT ( 1990) Inactivation mann R (l992b) Glycoprotein H of pseudorabies of the thymidine kinase gene of a gl deletion virus is essential for virus entry and cell-to-cell mutant of pseudorabies virus generates a safe but spread of the virus. J Virol 66, 3888-3892 highly immunogenic vaccine strain. J Gen Virol Peeters B, Pol J, Gielkens A, Moormann R (1993) 71,5191-1595 Envelope glycoprotein gp50 of pseudorabies virus Moss B (1991) Vaccinia virus: A tool for research is essential for virus entry but is not required for and vaccine development. Science 252, 1662- 1667 viral spread in mice. J Virol 67, 170-177 Mukamoto M, Watanabe I, Kobayashi Y, Icatlo- Jr Peeters B, Bouma A, de Bruin T, Moormann R, FC, Ishii H, Kodama Y (1991) Immunogenicity Gielkens A, Kimman T (1994) Non-transmissible of Aujeszky’s disease virus structural glycopro- pseudorabies virus: a new generation of safe live tein gVI (gp50) in swine. Vet Microbiol 29, 109- vaccines. Vaccine t2, 375-380 121 Pensaert MB, Kluge JP (1989) Pseudorabies virus Mulder WAM, Priem J, Glazenburg KL, Wagenaar (Aujeszky’s disease). In: Virus Infections of F, Gruys E, Gielkens ALJ, Pol JMA, Kimman TG Porcines (MB Penseart, ed), Elsevier Science Pub- (1994a) Virulence and pathogenesis of non-virulent lishers, Amsterdam, The Netherlands, 39-64 and virulent strains of pseudorabies virus express- Pensaert MB, Nauwynck H, De Smet K (1991) ing envelope glycoprotein E1 of hog cholera virus. Pathogenic features of pseudorabies virus infec- JGenVirol75, 117-124 tions in swine with implications to control. In: Mulder WAM, Jacobs L, Priem J, Kok GL, Wage- First International Symposium on Eradication of Pseudorabie.s Virus naar F, Kimman TG, Pol JMA ( 1994b) Glycopro- (Aujeszky’s) (RB Morrison, of Medicine, tein gE-negative pseudorabies virus has a reduced ed), College Veterinary University of Minnesota, MN, USA, I-166 capacity to infect second- and third-order neurons of the olfactory and trigeminal routes in the porcine Pensaert MB, Gielkens ALJ, Lomniczi B, Kimman central nervous system. J Gen Virol75, 3095-3106 TG, Vannier P, Eloit M (1992) Round table on Mulder WAM, Priem J, Pol JMA, Kimman TG control of Aujeszky’s disease and vaccine devel- (1995a) Role of viral proteins and concanavalin opment based on molecular biology. Vet Micro- A in in vitro replication of pseudorabies virus in biol 33, 53-67 porcine blood mononuclear cells. J Gen Virol 76, Perkus ME, Limbach K, Paoletti E (1989) Cloning 1433-1442 and expression of foreign genes in vaccinia virus, Mulder WAM, De Jong MCM, Priem J, Bouma A, using a host range selection system. J Virol 63, Pol JMA, Kimman TG (1995b) Experimental 3829-3836 quantification of transmission of genetically engi- Pol JMA ( 1990) Interferons affect the morphogenesis neered pseudorabies virus. Vaccine 13, 1770-1778 and virulence of pseudorabies virus. PhD Thesis, Mulder WAM, Pol JMA, Kimman TG, Kok GL, Priem University of Utrecht, The Netherlands J, Peeters BPH (1996) Glycoprotein D-negative Pyles RB, Sawtell NM, Thompson RL (1992) Herpes pseudorabies virus can spread transneuronally via simplex virus type I dUTPase mutants are attenu- direct neuron-to-neuron transmission in its natural ated for neurovirulence, neuroinvasiveness, and host, the pig, but not after additonal inactivation of reactivation from latency. J Virol 66, 6706- 67133 gE or gl. J Viro170, 2191-2200 Rauh I, Mettenleiter TC (1991). Pseudorabies virus Nauwynck HJ, Pensaert MB (1992) Abortion induced glycoproteins gII and gp50 are essential for virus by cell-associated pseudorabies virus in vaccinated penetration. J Virol 65, 5348-5356 sows. Am J Vet Res 53, 489-493 Reichard P (1988) Interactions between deoxy- Nauwynck HJ, Penseart MB (1995) Cell-free and cell- ribonucleotide and DNA synthesis. Annu Rev associated viraemia in pigs after oronasal infec- Biochem 57,349-374 Riviere M, Tartaglia J. Perkus ME. Norton EK, medullary origin demonstrated by viral tracing. Bongermino CM, Lacostc F, Duret C, Desmettre P, Science 263, 232-234 Paoletti C Protection E, Molnar-Borgennino ( 1992) Stegcman JA, Tielen MJM, Kimman TG. Van of mice and swine from virus con- pseudorabies Oirschot JT, Hunneman WA, Berndsen FW ( 1994) ferred vaccinia virus-based recombinants. by Intensive regional vaccination with a gl-deleted J Virol 66. 3424-3434 vaccine markedly reduces pseudorabies virus infec- Rixon FJ ( 1993) Structure and assembly of hcr- tions. Vaccine 12, 527-5311 Seniin 135-144 pesviruses. Virol 4, Stegeman JA ( 1995) Pseudorabies virus eradication Roizman B, Baines J (1991) The diversity and the by area-wide vaccination is feasible. PhDThesis, unity of . Comp lmnnmol Microbiol University of Utrecht, The Netherlands Di.s 63-79 InFect 14, Taylor G, Scott EJ, Wertz G, Ball A ( 1991 ) Compar- Roizman B ( 1992) The family Herpcsviridae: an ison of the virulence of wild-type thymidine kinase update. Arch Viro1123. 425-449 (tk)-deficient and tk+ phenotypes of vaccinia virus Rziha H-J, Mettenlciter TC, Ohlingcr V, Wittmann recombinants after intranasal inoculation of mice. G (1986) Herpesviruses (pseudorabies virus) ,I Gen Virol 72, 125-130 latency in swine: occurrence and physical state of Ter Horst GJ, Van den Brink A, Homminga SA, Haut- viral DNA in neural tissues. Virology 155, 600- vast RWM, Rakhorst G, Mettenleiter TC, De Jong- 6133 stc MJ, Lic KI, Korf J ( 1993) Transneuronal viral Sawitzky D, Hampl H, Haberiiiehl KO (1990) Com- labclling of rat heart left ventricle controlling path- 4, 1307-13100 parison of heparin-sensitive attachment of pseu- ways. Ntitrortport dorabies virus (PRV) and herpes simplex virus Thomsen DR, Marotti KR, Palermo DP, Post LE type I and identification of heparin binding PRV ( 1987) Pseudorabies virus as alive vector for glycoproteins.ll J Ce Virol 7 1, 1221-1225 expression of foreign genes. Gene 57, 261-265 Schijns VECJ, van der Neut R, Haagmans BL, Bar Tsudi T, Sugimura T, Murakami Y (1991) Evaluation DR, Schellekens H, Horzinek MC (1991) Tumour of glycoprotein gli ISCOMS subunit vaccine for necrosis factor-alpha, interferon-alpha, interferon- pseudorabies in pigs. Vaccine 9, 648-652 exert antiviral gamma, and interferon-beta activity Van Oirschot JT, Houwers DJ, Rziha HJ, Van Zaane in nervous tissue cells. J Gen Virol 72, 809-8155 ( 1988) Development of an ELISA for detection of Sears AE, McGwire BS, Roizman B ( 1991) Infcction antibodies to glycoprotcin gI of Aujeszky’s dis- of polarized MDCK cells with herpes sirnplex virus ease virus: a method for the serological ditieren- I: two asymmetrically disturbed cell receptors tiation between infected pigs and vaccinated pigs. interact with different viral proteins. Proc Natl J Virol Methods 22, 191-206 Acncl Sci USA 88, 5078-50911 Van Oirschot JT ( 1989) The antibody response to gly- Sedegah C, Chiang C, Weiss W, Mellouk S, Cochran coprotein gl and the control of Aujeszky’s disease M, Houghten R, Beaudoin R, Smith D, Hoffman S virus. In: CEC Seminar on Vaccination and Con- ( 1992) Recombinant pseudorabies virus carrying a rrol oIAl/ies!kv’s Disease (JT Van Oirschot, ed),), plasmodium gene: herpesvirus as a new live viral Kluwer Academic Publishers, Dordrecht, The vector for inducing T- and B-cell immunity. Vac- Netherlands, 129-138 cine 10, 578-584 Van Oirschot JT, Gielkens ALJ, Moormann RJM, Shao L, Rapp LM, Weller SK ( 1993) Herpes simplex Berns AJM (1990) Marker vaccines virus-specific virus I alkaline nuclease is required for efficient antibody assays and the control of Aujeszky’s dis- egress of capsids from the nucleus. Virology 196, ease. Vet Microlinl 23, 85- I OI 146-162 Van Oirschot JT (1994) Vaccination in food animals. Spear PG (1993a) Entry of alphahcrpesviruses into Vaccine 12, 415-4188 cells. Semin 167-180 Virol4, Van Zijl M, Van Der Gulden H, De Wind N, Gielkens Spear PG (1993b) Membrane fusion induced by her- A, Berns A (1990) Identification of two genes pes simplex virus. In: Viral Fu.sion A4ech(iiiisi7is within the unique short region of pseudorabies (J Bentz, ed), CRC press Boca Raton, FL, USA, virus: comparison with herpes simplex virus and 201-232 varicella-zostcr virus. J Gcn Virol 7 1747-1755 Standish A, Enquist LW, Schwaber JS ( 1994) Vagal Van Zijl M, Wensvoort G, De Kluyver E, Hulst M, cardiac ventricular innervation and its central Van Der Gulden H. Gielkens A, Berns A, Moor- mann R ( 1991 ) Livc attenuated pseudorabies virus rabies) virus in vaccinated and nonvaccinated pigs expressing envelope glycoprotein El of hog after intranasal infection. Arch Virol 66, 227-240 cholera virus protects swine both against pseudo- Wittmann G. Rziha H-J ( 1989) Aujcszky’s disease rabies and hog cholera. J Virol 65, 2761-2765 (pseudorabies) in pigs. In: Herpesvirus Diseases n/- Visser N, Rziha HJ (1994) Is recombination of PRVV Ctittle, Horses alld Pigs (G Wittmann, ed), Kluwer vaccine strains a real problem’? Actu Vet HLiliq 42, Academic Publishers, Norwell, Massachusetts, 183-193 230-325 Wagenaar F. Pol JMA, Peeters BPH, Gielkens ALJ, De Wittmann G ( 1991 ) Sprcad and control of Aujeszky’s Wind N, Kimman TG (1995) The US3-encodcd disease (AD). Cony> Iiiiiiiiiiiol Mi(-t-obiol hif!(-t I)is protein kinase from pseudorabics virus affects 14, 165-173 egress of virions from the nucleus. J Gon Vir-o176, Xuan-XueNan Nakamura T, Ihara T, Sato I, Tuschiya 1851-1859 K. Nosetto E, Ishihama A, Ucda S, Xuan XN Weller SK, Steghatoleslami MR, Shao L, Rowse D, (1995) Characterization of pseudorabies virus Carmichael EP (1990) The herpes simple virus glycoprotein gil expressed by recombinant bac- type I alkaline nuclease is not essential for viral utovirus. Virus Res 36, I51-1611 DNA synthesis: isolation and characterization of Yamada S. Imada T, Watanabe W, Honda Y, Naka- a lacZ insertion mutant. J Gen Virnl 71, 2941-- jima-lijima S, Shitnuzu Y. Sekikawa K (1991) 2195 Nucleotide sequence and transcriptional mappingg Whealy ME, Baumeister K, Robbins AK, Enquist LW of the major capsid protein gene of pseudorabies (1988) A herpesvirus vector for expression of gly- virus. Virologr 185, 56-66 cosylated membrane antigens: fusion proteins of Zhang G. Stevens R, Leader DP ( 1990) The protein pseudorabies virus gill and human immunodefi- kinase encoded in the short region of pseudorabies virus J Virol ciency type I envclope glycoproteins. virus: dcscription of the gene and identification of 62, 4185-4194 its products in virions and infected cells. J Gen Whealy ME, Robbins AK, Enquist LW ( 1989) A her- Virol 71, 1757-1765 pesvirus vector for expression of glycosylated Zsak L, Zuckermann F, Stugg N, Bcn-Porat T ( 1992) membrane fusion of antigens: proteins pseudo- Glycoprotein gI of pseudorabies virus promotes rabies virus gIll and human simplex virus type 1. J cell fusion and virus spread via direct cell-to-cell Virol 63, 4055-4059 transmission. J Gen Virol 66, 2316-2325 Whealy ME, Card JP, Robbins AK, Dubbin JR, Rziha Zuckermann FA, Mettenlciter TC, Ben-Porat T ( 1988) HJ, Enquist LW ( 1993) Specific pseudorabies Role of pseudorabies virus glycoproteins in virus infection of the rat visual system requires immune response. In: CEC Seminar on Vaccilla- both gl and gp63 glycoproteins. J Virol 67, 3786- lioll and Control n/’Aujeszky’.s disease (JT Van 3797 Oirsehot, ed), Kluwcr Academic Publishers, Dor- Wittmann G, Bartcnbach G, Jakubik J (1976) Cell- drecht. The Netherlands, 107-I 177 mediated immunity in Aujeszky’s disease virus- Zuckermann F, Zsak L, Mettenleiter TC, Ben-Porat infected pigs. 1. Lymphocyte stimulation. Arch T (1990) Pseudorabies virus glycoprotein glll is Virol 50, 215-222 a major target antigen for murine and swine virus- Wittmann G, Jakubik J, Ahl R ( 1980) Multiplication spccif ic cytotoxic T-lymphocytes. J Virol64, 802- and distribution of Aujeszky’s disease (pseudo- 912?