Comp. Immun. Microbiol. infect. Dis. Vol. 15, No. 3, pp. 189 201, 1992 Printed in Great Britain. All rights reserved

THE

HOG

CHOLERA

0147-9571/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd

VIRUS

VOLKER MOENNIG Institute of Virology, Hannover Veterinary School, Bfinteweg 17, D-3000 Hannover 1, Fed. Rep. Germany A ~ t r a c t - - H o g cholera virus (HCV) is a spherical enveloped particle of about 40450 nm dia. The viral genome is a single strand RNA of about 12,000 bases with positive polarity. One single large open reading frame codes for presumably four structural, i.e. three glycoproteins and a core protein, and about three to five nonstructural proteins. The functional role is not yet fully clear for all viral proteins. HCV belongs to the pestivirus group and it is closely related to bovine viral diarrhoea and border disease viruses. The relationship extends to morphology, antigenicity, host spectrum and molecular properties. Pestiviruses hold generic status in the family Flaviviridae. Key words: Classical swine fever, hog cholera, proteins, antigens, genome.

LE VIRUS CHOLI~RIQUE DU PORC R t s u m t - - L e virus choltrique du porc (HCV) est une particule sphtrique recouverte d'une enveloppe, et d'environ 4 0 ~ 0 nm de diamttre. Le gtnome viral est un A R N fi une seule fibre, d'environ 12,000 bases, fi polarit6 positive. Un seul cadre de lecture grand ouvert code vraisemblablement quatre prottines structurelles, c'est-fi-dire trois glycoprottines et une nucltoprottine, et de trois fi cinq prottines non-structurelles environ. Le r61e fonctionnel n'est pas encore trts clair pour toutes les prottines virales. Le HCV appartient au groupe des pestivirus et est apparent6 fi la diarrhte bovine virale et aux virus des maladies voisines. Ce rapport s'ttend ~ la morphologie, au pouvoir antigtne, au spectre de l'h6te et aux proprittts moltculaires. Les pestivirus posstdent un status gtntrique dans la famille des flaviviridae. Mots-clefs: Peste porcine classique, choltra du porc, prottines, antigtnes, genoma.

INTRODUCTION

Pestiviruses are a small group of single-stranded RNA viruses comprising hog cholera virus (HCV), bovine viral diarrhoea virus (BVDV) and border disease virus (BDV) of sheep. Based on their morphology and type of nucleic acid they held generic status in the family Togaviridae [1-3]. Recently--based on novel insights in the replication strategy and genome organization--pestiviruses were allocated to the family Flaviviridae [4]. HCV has been a favourite subject of research ever since it was shown to be a filtrable agent early this century [5]. The considerable impetus for research was mainly derived from the devastating economical impact of hog cholera (HC) on the pig industry. However, like other pestiviruses HCV was a difficult virus to work with. Therefore many details concerning the viron remained obscure for a long time. A great leap forward was achieved during the last few years when modern biotechnological techniques were applied for the analysis of HCV. However, despite all recent progress, there are still open questions concerning, e.g. the composition of the virion, molecular markers of virulence and function of viral nonstructural proteins. 189

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In vivo During initial stages of HCV research serial passages of the virus in susceptible pigs was the only method of virus propagation. Thus viral isolates retained their original properties with respect to antigenicity and pathogenicity. For diagnostic purposes this method proved to be useful and with respect to sensitivity inoculation of animals is still considered superior to cell culture isolation protocols [6]. Several attempts were made to adapt HCV to heterologous hosts and today we know that the virus can be transmitted to probably all ruminants, but with certainty to goats, sheep, calves and deer [7-10]. Wild boar (sus scrofa fetus) was also shown to be susceptible. These data confirm that pestiviruses in general share ruminants and pigs as common hosts. Apart from ruminants HCV was also transmitted to rabbits and peccaries. It failed to propagate in mice, rats, racoons and birds tested so far, i.e. sparrows and pigeons [11]. Of all heterologous hosts the rabbit became the most important. Like other mammalian viruses, e.g. poliomyelitis, dengue and rinderpest virus, HCV was successful adapted to the rabbit ultimately resulting in an efficient and relatively innocuous live virus vaccine, the Chinese (C) strain ([12, 13], for review see [14]). In vitro The costs and difficulties associated with the serial passage of HCV in pigs prompted early attempts to cultivate the virus in vitro. Hecke succeeded first in growing the virus in organ explants, e.g. lymphnodes, bone marrow, spleen and plexus chorioideus [15]. Later attempts were focused on the use of suspended blood cells derived from spleen, bone marrow and peripheral blood [16-22]. Minced porcine testicle tissue was first used by Ten Broeck [23]. The rapid progress in cell culture technology in the 1940s and '50s provided the tools for growing HCV in primary monolayer cells of trypsinized, mostly foetal organs [24-26]. Among primary cells supporting HCV growth, porcine kidney and testicle cells were widely used. With the establishment of permanent porcine monolayer cell lines an additional option became available and today HCV is mostly grown on permanent kidney (e.g. PK [15]) or testicle cells. Despite all progress in cultivation techniques virus yield in cell culture remained relatively low compared to other mammalian viruses, namely model viruses like polio- and some alphaviruses. In order to overcome this impediment a permanent suspension cell derived from a porcine lymphoma was used yielding more virus than conventional monolayers [27, 28]. The in vitro host spectrum of HCV was shown to include ruminant cells, guinea pig, squirrel, rabbit, skunk and American badger [29]. In contrast to BVDV and BDV, the vast majority of HCV strains and isolates is of the noncytopathogenic (ncp) biotype, i.e. infected cultured cells are not lysed. This notion is strengthened by the observations that in HCV-infected cells only the nonstructural protein p125 can be detected using radioimmunoprecipitation analysis with monoclonal antibodies. The expression of p80, a degradation product of p125 which is characteristic for the cytopathogenic (cp) biotype of BVDV [30] has not been found [31]. Nothing is known about molecular properties of the few cpHCV strains which have been described [32-34]. It would be interesting to analyse their genomes for host cell-derived insertions as described for some BVDV strains [35] or to look for the expression of the above mentioned p80 in infected cells.

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Cell culture has become a convenient tool for research and diagnostic work. However, hopes to adapt HCV to cultured cells and use it for vaccine production did not materialise fully. It was possible to adapt HCV to cell culture in a way that it lost its pathogenicity [19, 36, 37] but it was also observed that in parallel this might lead to the loss of the virus' original antigenicity. The latter virus failed to induce protective immunity in pigs. Apparently this does not apply for all HCV strains and culture conditions used, since the use of ovine, rabbit, and guinea pig kidney cells for, e.g. C-strain vaccine production was also reported [38-42]. The use of permanent cell cultures for the propagation of HCV has greatly reduced the risk of contamination with heterologous viruses picked up from the organ donor. However, the possibility of fortituous contamination with ncp ruminant pestiviruses, namely BVDV still persists. BVDV is efficiently spread to tissue cultures by the use of foetal calf sera. With around 1% of persistently infected cattle it is not surprising that many lots of foetal calf sera are infected [43-47]. In recent years the awareness for this source of contamination has grown and with the development of HCV and BVDV specific monoclonal antibodies (MoAbs) the diagnosis of impurities was greatly facilitated (see below). MORPHOLOGY Ever since the technical means were available, attempts were made to investigate the morphology of the virion. However, like all other pestiviruses HCV is difficult to work with. Virus yield in cell culture is relatively poor, viral particles are fragile, membraneassociated and therefore almost impossible to purify using classical protocols including density gradient centrifugation [48]. Accordingly, early reports about the morphology of the virion were conflicting (for review see [49]). After initial setbacks enveloped, spherical particles with diameters ranging between 40-60 nm have been consistently reported in preparations of purified virus (Fig. 1). The particles are covered by irregularly shaped projections [50-52]. The observation that the projections are not consistently found and that they seem to be sensitive to shearing forces indicate that they are somewhat loosely attached to the viral envelope [53]. The envelope surrounds an electron dense inner core structure with a diameter of about 30 nm. The core seems to be hexagonally shaped [50]. The morphogenesis of HCV is difficult to study due to the low production rate of infected cells. Hence little information is available on that subject. Budding was observed in rare instances in HCV infected cells [54], whereas no such observation was made with ruminant pestiviruses. Instead it was suggested that BVDV is assembled in a condensationlike procedure within smooth membrane vesicles in which viral particles gather. From there, mature virus is probably released from the cells by exocytosis [55-57]. This assumption is supported by the observation that there are no viral proteins expressed on the surface of cells infected with BVDV [58]. Whether HCV is assembled in a similar fashion is not yet clear, but with all the analogies between pestiviruses it may be assumed that HCV morphogenesis is not significantly different from that of BVDV. STABILITY The stability of HCV in the environment is of particular practical importance, since experience has shown that many outbreaks of the disease may b e caused by

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vector-mediated spread of the virus (for review see [59]). Infectivity of HCV is inactivated by elevated temperature, e.g. 10min, 60°C, and u.v. radiation [60]. Compared to non-enveloped viruses like foot-and-mouth disease virus proteolytic enzymes had only a moderately inactivating effect [60, 61]. Due to HCV's lipid envelope, detergents and lipid solvents, e.g. deoxycholate, Nonidet 40 (NP40), saponin, chloroform and ether, destroy viral infectivity with ease [61-63]. But the virus remains infective in a relatively broad pH range (for review see [64]), and a low pH at low temperature does not show a marked effect. The average half-life of a virus suspension at pH = 4.0 and 4°C is 260 hr. A shift in temperature to 20-22°C dramatically reduces the half-life to 11 hr [65], thereby illustrating the effect of temperature. The relative pH stability enables the virus to survive in unheated meat products like ham and salami for weeks and months thereby contributing to the uncontrolled spread of the virus via the feeding of garbage and causing disease outbreaks in HC-free areas [6, 66, 67]. ANTIGENICITY The first description of antigenic relationship between HCV and BVDV indicated a high degree of homology [68]. This had been confirmed by numerous other investigators and the relationship was extended to BDV of sheep (for review see [69]). Within the group of HCV strains and isolates on one side and ruminant pestiviruses on the other side there are no major antigenic variations. Polyclonal antisera against pestiviruses generally fail to distinguish species, strains or isolates when techniques such as immunofluorescence or immunodiffusion are used. Although cross-neutralization assays have the potential of differentiating between HCV and pestiviruses of ruminants, no serotypes or subgroups can be discriminated within these two complexes [70, 71] (for review see [72]). Instead, a continuous spectrum of overlapping antigenic variations is noticed. Differences among HCV strains and isolates seem to be less divergent compared to those in ruminant pestiviruses (for reviews see [49, 73]). The viral envelope appears to be the major site for antigenic variation and the development of MoABs directed against pestiviruses provided tools for a more detailed analysis. Most MoAbs raised against HCV were directed against the large viral envelope glycoprotein (gp53) and the majority of them was shown to neutralize virus infectivity, indicating that gp53 constitutes a crucial part of the virion membrane. Most MoAbs reacted with more than one isolate of the homologous species, but binding was not always paralleled by neutralization [74, 75]. This observation indicates that the functional role of conserved epitopes for HCV neutralization may vary from virus to virus as described for BVDV [76]. MoAbs against both, variable and conserved epitopes of the viral envelope glycoprotein, were generated. Notably the latter did not cross-react with ruminant pestiviruses and they proved useful for the differential diagnosis of HCV and BVDV/BDV (see following text). The MoAbs against the major viral glycoprotein were suitable tools for the analysis of the antigenic composition of the viral surface. Like flaviviruses, pestiviruses display several antigenic domains on their surface each including a number of single individual epitopes [77]. Using various techniques Wensvoort [74] designed a topographical and functional map of the HCV gp53 using 13 MoAbs recognizing different epitopes of the homologous Brescia strain. Four distinct domains (AI_3, B, C, D) were identified by competitive binding studies, antigen capture assays, neutralization and isolation of neutralization escape

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l

I

lOOnm Fig. 1. Electronmicrograph of density gradient purified hog cholera virus. With the permission of B. Liess.

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mutants. Domains Aj and A2 were shown to be highly conserved on all HCV strains and isolates tested, whereas domains m3, B, C, and D displayed some variability. Domains A~, B and C were shown to be important for the induction of neutralizing antibodies. A synergistic neutralization was observed when MoAbs against A~ and B or A 1 and C were used. Cross-neutralization between HCV and ruminant pestiviruses using MoAbs so far has not been described. This is in contrast to results obtained with polyclonal antisera with cross-neutralizing properties. A tentative explanation is that cross-neutralization is effectively achieved by the binding of more than one antibody species directed to different epitopes rather than the binding of a single antibody species (MoAb) to a single antigenic site on the major viral glycoprotein. Within pestiviral species some epitopes are rather conserved and the respective Moabs were shown to react panpestivirus-specific. In general MoAbs with that broad reactivity are directed against the nonstructural protein p125 [78, 79]. These results are in agreement with genomic sequence data available so far indicating strong homologies ( > 80%) in the respective parts of pestiviral genomes ([80, 81], see following text). The analysis of pestiviruses with MoAbs has led to considerable practical progress in laboratory diagnostic methods. First, advances were made in the diagnosis and differentiation of pestiviral antigens. An international workshop at the Hannover Veterinary School in 1987 under the auspices of the Commission of the European Communities (CEC) was held in order to analyse the reactivity of 50 MoAbs with some 43 pestiviral strains and isolates [82]. Apart from antibodies with a somewhat heterogeneous reactivity three groups of MoAbs relevant for diagnostic work were recognized: (a) reactivity with all pestiviruses (panpestivirus-specific), (b) reactivity with HCV only, and (c) reactivity with ruminant pestiviruses. Today MoAbs with defined specificity are used for the identification of pestiviruses in organ tissue sections and in cell culture [75, 83-85]. This method has proved to be suitable to discriminate HCV- and BVD-infected cells in organ tissue sections of animals which were simultaneously infected with both viruses [86]. An international reference panel of MoAbs for the differentiation of pestiviruses is available and the use of defined MoAbs against pestiviruses will officially be recommended for laboratory diagnostic methods by the CEC [87]. The serodiagnosis of HCV was greatly improved by the use of MoAbs too. In the past the development of conventional indirect enzyme-linked immunosorbent assays (ELISA) was hampered by both, the lack of purified HCV serving as diagnostic antigen and the extensive cross-reactions of antibodies directed against ruminant pestiviruses with HCV. A discrimination of cross-reacting antibodies in pig sera was achieved by Wensvoort and coworkers [88]. Two HCV specific MoAbs discriminating ruminant pestiviruses and reacting with two conserved and separate epitopes on the major viral glycoprotein of HCV were utilized for the development of the "complex-trapping-blocking" ELISA (CTBELISA). The wells of a microtitre test plate are coated with one of the above MoAbs as a capture antibody. After washing, the second MoAb conjugated to peroxidase is added to the wells. In parallel unpurified HCV antigen from lysed infected cells is incubated with the field serum dilution. Subsequently the antigen-serum mixture is added to the conjugate in the MoAb-coated well. After incubation, reaction wells are washed and chromogenic substrate is added. Colouration of the fluid indicates that both MoAbs have bound to the test antigen and no HCV specific antibodies were present in the sample. In case one or both epitopes of the test antigen is blocked by HCV specific antibodies in the serum, the test well will stay colourless indicating a positive outcome. The design of the assay

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ensures that antibodies against ruminant pestiviruses are discriminated and HCV specific antibodies are detected only [88]. GENOME AND PROTEINS As mentioned previously, pestiviruses and HCV in particular are difficult to work with. Since HC is a notifiable disease exotic to many countries, HCV research is further complicated and it is restricted to relatively few laboratories with adequate safety levels. This might explain why the bulk of information concerning pestiviral genomes and proteins so far was obtained from work with BVDV. The respective data have been reviewed elsewhere and will not be the subject of this paper [89]. However, recent results on molecular properties of HCV confirm that there is a close resemblance with ruminant pestiviruses. The genome of pestiviruses is a single strand of RNA with positive polarity. Using electrophoretic analysis the size of RNAs of strains Brescia and Alfort was shown to be about 12 kilobases [28, 90]. No size differences were observed among virion RNAs of different HCV strains. Infected cells only contain one species of viral RNA with the size of the viral RNA. Subviral RNAs, replicative forms or replicative intermediates, respectively, have not been detected [90]. Synthesis of HCV RNA is not impaired by the presence of actinomycin D [28]. This suggests that the viral polymerase essential for replication is directly translated from the positive strand RNA. However, the identity of a putative viral RNA-directed RNA polymerase is unclear. After the cloning and sequencing of almost all of the genomes of strains Osloss and N A D L of BVDV [91,92], corresponding data for HCV strains Alfort and Brescia were published [28, 81, 93]. The poor yield of virus necessary for RNA extraction was overcome by the use of suspension cells (see above) and subsequent concentration and purification of the virus from cell supernatants [28]. Alternatively, as described for BVDV viral RNA was extracted directly from infected cells [81]. One of the common complications for the sequencing of complete pestiviral genomes is the lack of polyadenylation at the Y-end of the RNA preventing an oligo(dT)-primed cDNA synthesis [94, 95]. The difficulty was overcome when synthetic random hexanucleotides or calf thymus oligonucleotides were used as primers [28, 81]. Another problem was the sequencing of the respective termini of the viral genome. Meyers and coworkers [93] suggest that--compared to the N A D L sequence [92]--the 12,284 nucleotides sequenced from the HCV strain Alfort RNA comprise almost the entire genome. Moormann and coworkers [81] succeeded in polyadenylating the Y-end of HCV RNA using E. coli poly(A) polymerase. This allowed the oligo(dT)-primed transcription and sequencing of the complete Y-end of the RNA of HCV strain Brescia. As described for BVDV there is one significant large open reading frame (ORF) spanning almost the entire length of the HCV genome coding for 3898 amino acids [81, 93]. The presence of one large ORF explains the above mentioned absence of subgenomic viral RNAs in infected cells and suggests that a large polyprotein precursor is translated and processed by either co- or post-translational proteolytic cleavage. The number, the molecular weights and the nomenclature of pestiviral proteins have long been, and to a certain extent still are, subject to controversy. In order to avoid confusion, the provisional nomenclature for viral proteins proposed by Collett and coworkers [80] will be used in this paper.

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Recent progress in the exploration of the molecular biology of pestiviruses has provided new insights concerning viral proteins. Initial interest was focused on the putative structural proteins, namely the viral glycoproteins which are thought to be components of the virion envelope. The characteristics of the deduced amino acid sequence, i.e. the clustering of potential glycosylation sites and the location of potential signalase cleavage sites, suggested that viral glycoproteins are coded for at the Y-terminus of the genome [81,93]. Viral glycoproteins were identified by radioimmunoprecipitation using either antisera against sequence specific oligopeptides or a polyclonal antiserum against HCV. The latter was used to identify bacterial fusion proteins. Three glycoproteins were identified in the following order: NH2-gp48-gp25-gp53-COOH [96]. The largest one, gp53, binds neutralizing antibodies and seems to be the major viral envelope glycoprotein [75, 97]. In infected cells and possibly in mature virions it forms a disulphide-bridged heterodimer with the hydrophobic gp25, the latter possibly anchoring gp53 in the viral membrane [98]. This is a feature commonly seen in enveloped viruses. It is still unclear what the functional role of gp48 is and whether it is a constituent of the viral membrane. In contrast to flavivirus E protein, pestiviral glycoproteins are heavily glycosylated. The major envelope glycoprotein is of considerable practical interest, since it might be the basis of a genetically engineered vaccine lacking the risks of conventional live H C V vaccines. First attempts to express H C V structural proteins have been made using vaccinia and pseudorabies viruses [99, 100]. In both systems it was demonstrated that the immune response against gp53 conferred full protection against challenge infection with virulent HCV. Upstream the viral glycoprotein complex p20, the putative core protein is located. It was first visualized using an antiserum directed against a bacterial fusion protein. It has no larger precursor suggesting cotranslationai cleavage by an autoproteolytic activity as described for other viral systems [99, 101]. Downstream the viral structural proteins the nonstructural proteins are encoded with p125 being adjacent to the glycoproteins. Panpestivirus-specific MoAbs do precipitate the H C V nonstructural p125. No p80--the cleavage product of cytopathogenic ruminant pestiviruses--could be demonstrated [31]. With respect to nonstructural pestiviral proteins even less is known about H C V compared with BVDV [89]. However, it is a safe assumption that there are no fundamental functional differences between H C V and BVDV nonstructural proteins. Acknowledgement--The author gratefully acknowledgesthe experience and knowledge contributed by Professor

B. Liess during stimulating discussions.

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