Rev. sci. tech. Off. int. Epiz., 1990, 9 (1), 13-23.

Bovine virus diarrhoea virus: an introduction M.C. HORZINEK *

Summary: In view of the recently established genome organisation of pestiviruses, their classification as members of the togavirus family is no longer tenable. They should rather be provisionally considered as a new genus of the Flaviviridae, irrespective of differences in the nonstructural genes. Like other positive-stranded RNA viruses, pestiviruses are highly variable; apart from point mutations, recombinations are expected to contribute to their capricious behaviour. One trait of expected pathogenetic significance in infections with bovine virus diarrhoea virus is a change from the non-cytopathogenic to a cytopathogenic biotype. Cooperation of both variants in an animal to produce the severe disease picture known as mucosal disease is unique in virology; elucidation of this mechanism may shed light on the pathogenesis of other sporadic diseases with suspected viral origin. KEYWORDS: Antigenic variation - Biotypes - Bovine virus diarrhoea virus Flaviviridae - Monoclonal antibodies - Mucosal disease - Pestivirus Polypeptides - Taxonomy.

M o r e t h a n forty years have passed since Olafson et al. (42) and Childs (9) simultaneously published descriptions of an acute enteric disease in cattle which was later found to be caused by a virus (46, 52). T h e disease was designated bovine viral diarrhoea (BVD), and the causative agent was given the graceless and redundant name bovine viral diarrhoea virus (BVDV). Some years later, the same virus was also implicated in causing " m u c o s a l disease" (MD), a fatal condition first described by Ramsey and Chivers (49). Again a n ominous and confusing terminology emerged, when aetiologically diverse but clinically similar syndromes were a c c o m m o d a t e d in the " m u c o s a l disease c o m p l e x " : foot and m o u t h disease, rinderpest, malignant catarrhal fever and others. Fortunately, this designation is now obsolete. The M D which follows BVDV infection occurs in cattle that are persistently infected and immunotolerant to the virus; tolerance is established in the fetus during early pregnancy following transplacental infection. This hypothesis (34) has subsequently been supported by both experimental and epidemiological findings, and its molecular basis is about to be unraveled. A review on BVD has recently been published by Liess (32).

* Department of Virology, Institute of Infectious Diseases and Immunology, Veterinary Faculty, Yalelaan 1 (de Uithof), 3508 TD Utrecht, The Netherlands.

14 T H E N E W T A X O N O M I C POSITION OF B V D V

For a long time, viral t a x o n o m y was based exclusively o n structural features of the infectious particle. Three alternative criteria were used: the type of nucleic acid ( D N A or R N A ) , the lipoprotein envelope (present or absent) and the nucleocapsid symmetry type (icosahedral or helical). While the first two features of a virus are easily established — using nucleic acid inhibitors and organic solvents or detergents, respectively - identification of the symmetry type in enveloped virions is less straightforward. This requires electron microscopic analysis after selective removal of the virion m e m b r a n e , an a p p r o a c h that led to the identification of icosahedral capsids in the arthropod-borne alphaviruses (25) and in other, then unclassified, small enveloped viruses without conspicuous a r t h r o p o d transmission (26). In recent years, there has been a tendency to a b a n d o n strictly structural criteria of classification and to m a k e use of other properties, e.g. the replication strategy of viruses. This is a rational approach if t a x o n o m y is to reflect evolutionary relationships. After all, more constraints and more selective pressure may be expected to operate at the level of the extracellular virus particle, its proteins and antigenic determinants t h a n , e.g., at the level of m R N A transcription; the m o d e of replication is probably better conserved during evolution. In 1973, the present author coined the term "pestiviruses" to group two antigenically related enveloped R N A viruses: hog cholera virus (HCV) and BVDV (22). A third animal pathogen, the Border disease virus of sheep, was later found to be a close relative of BVDV. In its fourth report published in 1982 for the Virology Division of the International U n i o n of Microbiological Societies (IUMS), the International Committee on Taxonomy of Viruses (ICTV) adopted this nomenclature and assigned generic status to the pestiviruses, with BVDV as the prototype (36). Pestiviruses are amongst the smallest enveloped animal R N A viruses (measuring about 40 n m in diameter) and possess a nucleocapsid of non-helical, probably icosahedral symmetry (26). They share these traits with numerous flaviviruses, of which the mosquito-transmitted yellow fever virus is the prototype (the pestiviruses are nonarthropod-borne). Previously, the flaviviruses also held generic status in this family, b u t when details of their molecular structure, replication strategy and gene sequence became known in the early 1980's, the Togavirus Study G r o u p acknowledged the differences as fundamental and established the new family Flaviviridae - with flavivirus as its only genus (56). In view of recent progress in the description of the molecular features of pestiviruses, the discussion of virus classification must now be re-opened. The first molecular data which suggested that pestiviruses are distinct from members of the Togaviridae family relate to characteristics of the virus-specific R N A . In infected cells, only a single high molecular weight species was found which lacked a 3' poly(A) tract (47, 5 1 ; M o o r m a n n , personal communication). N o subgenomic R N A was detected at any time after infection. These properties distinguish pestiviruses from togaviruses - of which b o t h the alpha- and rubiviruses possess one subgenomic R N A - a n d suggest a similarity to flaviviruses. Much of the nucleotide sequence and genetic organisation of BVDV is known, and further comparisons with flaviviruses have been m a d e (10). With the exception of several short but significant stretches of identical amino acids within two of the putative nonstructural proteins, n o extended regions of homology exist between BVDV and representatives of the three antigenic subgroups

15 of mosquito-borne flaviviruses. Nevertheless, the molecular layout of the BVDV a n d flavivirus genomes is strikingly similar. Comparison of the arrangement of the proteincoding domains along b o t h genomes and the hydropathic features of their amino acid sequences revealed pronounced similarities. Based o n these comparisons, it was proposed that the Pestiviruses n o longer be grouped in the Togaviridae family, b u t rather be considered a genus within the Flaviviridae (10). F o r such a proposal t o be accepted, additional molecular d a t a for other BVDV isolates (and other pestiviruses) will be required. However, the implications of this proposition are immediately provocative. Considering the parallel organisation of their genomes, analogous polypeptides m a y be predicted to possess similar biologic functions. Examples corroborating this hypothesis have been given in our recent review on molecular advances in pestivirus research (12). Drawing analogies to the flaviviruses m a y help in experimental designs to resolve the issue of structural vs. nonstructural proteins for pestiviruses. Certainly insights beyond those in molecular biology m a y be gained from further comparisons between these two groups of viruses. N o n - a r t h r o p o d - b o r n e togaviruses were last reviewed in 1981, when only limited molecular d a t a were available (23). Meanwhile, expanding knowledge has m a d e another earlier classification untenable: equine arteritis virus (EAV) which has been listed as a possible member of the Togaviridae family (36) replicates via multiple subgenomic m R N A ' s (54); they form a 3'-coterminal nested set, not unlike t h a t in coronaviruses (Spaan and Horzinek, unpublished observations) and toroviruses (53). However, by possessing an enveloped icosahedral nucleocapsid, E A V meets the structural criteria of a togavirus. The presence of two " i n c o m p a t i b l e " taxonomic elements in one virus indicates that our concept of classification is certainly too narrow; however, it m a y also indicate convergent evolution (24).

T H E RECENT HISTORY OF RESEARCH ON B V D V

It was an old and enigmatic finding that fatal M D , one of the consequences of BVDV infection of cattle, could not be reproduced experimentally, thereby defying Koch's postulates. The conditions which must be met for M D t o develop have now been defined. Cows infected during the first four m o n t h s of gestation with BVDV can give birth to healthy, persistently viraemic calves (7, 33). W h e n this virus is of a non-cytopathogenic (ncp) biotype, superinfection with a " m a t c h i n g " cytopathogenic (cp) strain of BVDV will result in the severe M D condition (3, 4, 6). It has been suggested that M D m a y be a consequence of the in vivo conversion of the ncp strain (the one that causes persistent infection) to cytopathogenicity (7, 27), which would explain the erratic and sporadic occurrence of M D in a cattle population. As will be discussed below, the cp and ncp strains differ in the expression of at least one polypeptide. Antigenic variation and epitope mapping Since the historic observation by Darbyshire (15) that H C V and BVDV are antigenically related, n u m e r o u s attempts have been m a d e to elucidate the degree and the basis of serological cross-reactions. A l t h o u g h most workers agree t h a t each pestivirus is antigenically homogeneous, i.e. that serotypes of H C V , BVDV or Border

16 disease virus do not exist (8), analyses using conventional antisera have shown strain variations within each pestivirus detectable by cross-neutralisation tests (2, 41). Neutralisation assays are also able to distinguish between H C V and BVDV, but not between BVDV and Border disease virus. This latter distinction must be based o n biological and epidemiological data (30). Monoclonal antibodies (MAB's) now provide the tools for a more detailed analysis; they have been characterised as to their spectrum of reactivity with different pestivirus strains and isolates, their virus neutralising capacity and their protein specificity. Preparation of the first M A B ' s to BVDV was reported by Peters et al. (44) and to H C V by Wensvoort et al. (55). The former antibodies were found to be specific for p l 2 5 of ncpBVDV. W h e n analysed with cpBVDV, they reacted with b o t h the p125 and p80 found in cells infected with this biotype (Bolin and Moennig, unpublished observations). When tested against other pestiviruses using indirect immunofluorescence and peroxidase-linked antibody tests, some of these M A B ' s recognised only cpBVDV, while others reacted broadly with all BVDV isolates a n d / o r with b o t h Border disease virus and H C V (20). One antibody (BVD/C16) was pestivirus-specific, reacting with all fifty pestivirus isolates tested at this point. These results suggest that sequences within the p125 are well conserved among pestiviruses. The limited genomic sequence d a t a available indicate that the conserved component is p80 (see below). This protein probably represents the soluble ( " S " ) antigen forming the "single line of identity" observed by Darbyshire (15) in agar gel immunodiffusion tests with H C V and BVDV specific antisera. Dubovi (personal communication) has characterised a M A B specific for a minor glycoprotein (gp48) of BVDV which reacted with all pestiviruses tested, including one strain of H C V . Additional M A B ' s with b r o a d anti-pestiviral activity have been generated (Chappuis, Edwards, Nettleton, unpublished results); their protein specificity is not yet k n o w n . A number of M A B ' s directed against the major glycoprotein of BVDV and H C V (gp50-59, referred to hereafter as gp53; see Table I in ref. 12) were shown to possess virus neutralising activity. M A B ' s specific to gp53 of BVDV lacking neutralising activity have also been described (17; Moennig and Bolin, unpublished observations). Of the former M A B ' s , most neutralised several isolates of the homologous virus, but not of other pestiviruses (5, 17; Coulibaly, unpublished observations; Wensvoort, unpublished observations). Interestingly, however, some M A B ' s which neutralised one isolate did bind to another virus without neutralising its infectivity (Mateo and Moennig, unpublished observations). A similar p h e n o m e n o n was recently described for Sindbis virus and some of its variants (43). T h u s , the significance of conserved epitopes for virus neutralisation differs among pestiviruses. Using neutralising M A B ' s , Wensvoort and co-workers (unpublished observations) have identified three antigenic domains with a total of eight epitopes on the major glycoprotein of H C V . Three additional domains on the same glycoprotein comprising five epitopes were not involved in neutralisation. Extending the results of Bolin et al. (5) by using 47 pesti­ viruses in competitive binding studies, Mateo and Moennig (unpublished observations) identified ten epitopes on gp53 of BVDV relevant for neutralisation. Eight of them were clustered in one domain, whereas one epitope - which so far could be identified only on the homologous virus - was located outside this domain. In these studies, binding of a single M A B was sufficient for virus neutralisation. However, a synergistic effect of M A B ' s directed against different domains was observed with H C V (Wensvoort, unpublished observations); in contrast, there was no such effect with anti-gp53 M A B ' s of BVDV (Mateo and Moennig, unpublished observations).

17 In 1987, a workshop was held at the Hanover Veterinary School (FRG) to compare 50 M A B ' s against 43 pestivirus isolates. Materials were contributed by thirteen E u r o p e a n laboratories (38; tables summarising the data are provided u p o n request by Prof. V. Moennig). It soon became clear that the ability of M A B ' s to discriminate between antigenic variants was very powerful: numerous differences were found among strains bearing the same n a m e b u t coming from different laboratories a n d having distinct passage histories. These results emphasise the need for cautious interpretation when comparing results obtained with the " s a m e " viruses in different laboratories (38). Interactions between BVDV and the host cell All pestiviruses possess a similar host spectrum. BVDV naturally infects pigs, sheep, goats a n d a wide range of wild ruminants whereas H C V is transmissible to cattle and small ruminants (14, 18, 39, 40). T h e pestivirus host range for cultured cells is even broader (23). However, despite their ability t o cross species barriers, pestiviruses in general replicate inefficiently in heterologous hosts. In most cases they cause neither cytopathology in culture n o r clinical disease. BVDV-induced disease in sheep seems t o be an exception. In Prof. Moennig's laboratory in Hanover, a M A B directed against a bovine cell surface protein was shown to interfere specifically with the infectivity of a n u m b e r of cpBVDV strains while leaving infection unimpaired with H C V a n d Border disease virus, as well as with parainfluenza 3 virus and infectious bovine rhinotracheitis virus (38). These findings suggest that a specific cell surface receptor mediates entry of BVDV into bovine cells. F u r t h e r m o r e , it appears that different pestiviruses m a y n o t share the same receptor, at least not in bovine cells. However, when studied more closely using immunoperoxidase techniques, the inhibition of infection by the Hanover M A B was not always complete; a few foci of infected cells were detectable in monolayers pretreated with this M A B . Therefore, either multiple receptors for BVDV m a y exist on cells or a less efficient, receptor-independent mechanism of virus internalisation m a y be operative, as has been described for other viruses (35, 37). The biological significance of receptor molecules for the histotropism of pestiviruses is n o t yet understood. It has been shown that the receptor specificity of viruses can be altered u p o n passage in cultured cells (50). T h e ability of pestiviruses to adapt to heterologous hosts by expressing new attachment sites on the virion needs to be investigated. T h u s , pestiviruses infecting bovine, ovine a n d porcine cells alike have been identified, b u t so have strains infecting two or only one of the above species (Moennig, unpublished observation). T h e variation may even be greater when wild ruminants are t a k e n into account (18, 40). The biotypes of BVDV Cytopathogenicity of pestiviruses is a property which depends on genetic factors of the virus as well as on the type of cell culture used (23). In general, H C V does not produce cytopathology in porcine cell cultures; only a few exceptions have been reported (19, 29). Border disease virus a n d BVDV strains behave differently with respect to cytopathogenicity, and numerous cp isolates exist. The recent appreciation of the significance of ncp and cp biotypes for the pathogenicity of BVDV has focussed attention on the determinants of cytopathogenicity. T h e fact that pairs of ncp a n d cpBVDV isolates from MD-affected animals are serologically indistinguishable (but

18 distinct from other pairs) suggests that only minor alterations have occurred. Comparison of the viral polypeptides of the two BVDV biotypes has indeed revealed a difference: whereas cpBVDV isolates possess b o t h the p80 and p l 2 5 (amongst other proteins), ncp viruses display only p l 2 5 ( 1 , 16, 45). Earlier peptide mapping data have shown the p80 and p l 2 5 to be structurally related (48). More recently, the genetic relationship between these polypeptides has been proven, and a 54kd protein related t o p l 2 5 b u t unrelated to p80 has been identified (11). With this difference between the cp and ncp biotypes of BVDV identified, questions arise concerning the function of the p l 2 5 and its cleavage products p54 and p80, during viral replication and in the origin of biotypes. It has been suggested that cp biotypes arise by m u t a t i o n from ncpBVDV in persistently infected animals (27). It is tempting to speculate that a mutation affecting a virus-encoded protease or a protein cleavage site might be responsible for the conversion. W h e n various unrelated BVDV field isolates were screened using M A B ' s directed against the gp53 and gp48 envelope glycoproteins (for protein nomenclature see ref. 12), a wide spectrum of divergent reactivities was found that appeared independent of the biotype. T h e same analysis, but now including pairs of cp and ncp viruses isolated from fatal M D cases, showed a high degree of antigenic similarity within a pair. This observation underlines the concept that cp biotypes of BVDV m a y arise by mutation from ncp strains (13). Mutations in envelope genes can be easily demonstrated. In collaboration with the Regional Veterinary L a b o r a t o r y in G o u d a (The Netherlands) and the Institute of Virology in Hanover, we have selected a panel of ten M A B ' s directed against four domains and multiple subdomains of the gp53 (20, 44) and tested it against m o r e t h a n 500 isolates of BVDV. Again, a wide variety of reactivities was seen, with every conceivable combination of one to eight M A B ' s binding t o cell monolayers infected with a given isolate. Interestingly, however, there were m a n y isolates in which a peroxidase-linked antibody staining pattern indicative of mixed viral populations was observed. Thus with a given isolate, one or several MAB's led to homogeneous staining of the monolayer, whereas other M A B ' s stained only patches or single infected cells (Kreeft and Horzinek, unpublished observations). Since the homogeneously stained cultures establish the presence of BVDV in every cell, the anomalous antigen distribution evidenced by some M A B ' s must b e interpreted in terms of emergence of m u t a n t s which have acquired (or lost) the corresponding epitope.

THE OUTLOOK FOR PESTIVIROLOGY

The diagnosis of pestivirus infections is faced with several problems. Current routine procedures are time-consuming and often inaccurate. Discrimination between pestivirus strains by classical virological and serological means is difficult (8, 31). However, all this is now expected to change as M A B ' s , nucleic acid probes and synthetic peptides become available. Broadly reactive M A B ' s to H C V are currently used in routine diagnostic procedures for the differentiation of H C V and BVDV infections (21, 55). BVDVspecific M A B ' s offer the opportunity for development of an antigen-capture E L I S A .

19 Such an assay will be valuable for the detection of persistently infected animals within herds. Although yet to be explored, nucleic acid probes offer the potential for highly specific and sensitive detection of virus directly in blood cells. A problem of economic significance is the lack of simple procedures t o detect antibodies against pestiviruses in field sera. Present serological techniques d o not discriminate between antibodies produced in pigs after infection with H C V and BVDV. Competition E L I S A procedures using M A B ' s as described by Juntti et al. (28) can be implemented to approach this problem. Synthetic peptides reactive exclusively with antibodies to specific pestiviruses offer another diagnostic possibility. Understanding the pathogenesis of the M D syndrome is a major objective of future work. The elucidation of the molecular differences between biotype pairs of BVD viruses will be important in attaining this goal. Currently, two cp virus genomes (Osloss and NADL) have been largely cloned and sequenced, and others will follow. However, if we are t o gain insight into the cooperation between cytopathogenic and n o n cytopathogenic BVDV, it will be essential to analyse naturally occurring virus pairs capable of inducing M D and identify the mutations relevant t o pathogenesis.

ACKNOWLEDGMENTS The author should like to t h a n k M . S . Collett and V. Moennig for their contributions to this chapter, m a n y of which have been included in a recent review article on pestiviruses in general (12). The collaboration of H . Kreeft in screening BVDV field isolates is gratefully acknowledged. T h a n k s are also due t o the authors whose unpublished observations are mentioned in the text. * * INTRODUCTION SUR LE VIRUS DE LA DIARRHÉE VIRALE BOVINE. - M.C. Horzinek. Résumé : Etant donné l'organisation du génome des pestivirus, telle qu'elle a été récemment déterminée, il n'est plus possible de les classer dans la famille des Togaviridae. A titre provisoire, il faudrait plutôt les considérer comme un nouveau genre des Flaviviridae, indépendamment des différences dans les gènes non structurels. Comme les autres virus à ARN positif, les pestivirus sont extrêmement variables ; outre des mutations ponctuelles, des recombinaisons peuvent favoriser leur comportement capricieux. Une caractéristique pouvant être importante dans la pathogénie des infections par le virus de la diarrhée virale bovine, est le fait qu'un biotype non cytopathogène peut devenir cytopathogène. L'action conjointe des deux variantes chez un même animal, produisant le tableau clinique grave connu sous le nom de «maladie des muqueuses», est un cas unique en virologie ; l'élucidation de ce mécanisme peut éclairer d'un jour nouveau la pathogénie d'autres maladies sporadiques dont on soupçonne qu'elles sont dues à des virus. MOTS-CLÉS : Anticorps monoclonaux - Biotypes - Flaviviridae - Maladie des muqueuses - Pestivirus - Polypeptides - Taxonomie - Variation antigénique Virus de la diarrhée virale bovine.

*

* *

20 INTRODUCCIÓN AL VIRUS DE LA DIARREA VIRAL BOVINA. - M.C. Horzinek. Resumen: Dada la organización del genoma de los pestivirus tal como se ha establecido recientemente, su clasificación como miembros de la familia Togaviridae ya no es sostenible. Convendría, provisoriamente, considerarlos más bien como un nuevo género de Flaviviridae, independientemente de las diferencias en los genes no estructurales. Como ocurre con los demás virus de ARN positivo, los pestivirus son extremadamente variables; además de las mutaciones puntuales, las recombinaciones pueden favorecer su comportamiento caprichoso. Una característica que puede ser importante en la patogenia de las infecciones por virus de la diarrea viral bovina es que un biotipo no citopatógeno puede hacerse citopatógeno. La acción conjunta de las dos variantes en un mismo animal, que produce el cuadro clínico grave conocido como enfermedad mucosa, es un caso único en virología; la elucidación de tal mecanismo puede contribuir a aclarar la patogenia de otras enfermedades esporádicas que se sospecha causadas por un virus. P A L A B R A S CLAVE: Anticuerpos monoclonales - Biotipos - Enfermedad mucosa - Flaviviridae - Pestivirus - Polipéptidos - Taxonomía - Variación antigénica - Virus de la diarrea viral bovina. * * *

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