Comp. lmmun. Microbiol. infect. Dis. Vol. 14, No, 2, pp. 187-195, 1991 Printed in Great Britain. All rights reserved

0147-9571/91 $3.00+0.00 Copyright © 1991 Pergamon Press plc

BIOLOGICAL A N D MOLECULAR ASPECTS OF BOVINE HERPESVIRUS 4 (BHV-4) MICHAEL GOLTZ I a n d HANNS LUDWIG 2. ~Fachgebiet Molekularbiologie und Genetik, Robert Koch-Institut des BGA, und 21nstitut f/Jr Virologie, Freie Universit~it Berlin, Nordufer 20, 1000 Berlin 65, Deutschland A~traet--This review summarizes most recent information on the bovine cytomegalovirus BHV-4. The virus is not associated with clearly defined clinical entities in cattle. It can easily be isolated in tissue culture and has a broad host range. BHV-4 strains are rather similar in restriction enzyme analysis of their DNAs, the size of the pr DNAs, however, differs. The genome represents a 7-herpesvirus. Because of its uniqueness BHV-4 is discussed as an appropriate vector. Key words: BHV-4, cytomegalovirus, molecular biology.

DES ASPECTS

BIOLOGIQUES ET MOLI~CULAIRES DE BHV-4

Rrsumr---Cette revue rrsume rinformation la plus rrcente sur cytomrgalovirus bovin. Le virus n'est pas associ6 avec des entitrs cliniques drfinies des bovins. L'isolement dans les cultures cellulaires est facile et le virus prrsente un large 6ventail d'h6tes. Par rapport ~i ranalyse d'enzyme de restriction de leurs grnomes les souches de BHV-4 sont relativement similaires, par contre la taille de I'ADNpr est diffrrente. Le grnome reprrsente un 7-herpesvirus. A cause de sa particularit6 BHV-4, le caractere vecteur du virus est discutr. Mots-clefs: BHV-4, cytomegalovirus, biologie molrculaire.

INTRODUCTION B o v i n e h e r p e s v i r u s 4 ( B H V - 4 ) is a c o m m o n p a t h o g e n in cattle. T h e virus has b e e n i s o l a t e d f r o m d i f f e r e n t clinical entities as well as f r o m tissue c u l t u r e cells. O u r g r o u p has first c h a r a c t e r i z e d t h e d i f f e r e n t strains a n d s h o w n t h a t these viruses h a v e c o m m o n clearly d e f i n e d f e a t u r e s a n d c a n all be g r o u p e d as B H V - 4 [1-3]: --they are widely spread under ruminants; --isolates were obtained from diseased animals; ~efined v i r u s strains h a v e a r a t h e r b r o a d tissue c u l t u r e s p e c t r u m [4]; - - i n f e c t i o n s o f tissue c u l t u r e cells r e s u l t e d in i n c l u s i o n b o d y f o r m a t i o n [5]; this p o i n t e s p e c i a l l y led to the c l a s s i f i c a t i o n o f B H V - 4 as a b o v i n e c y t o m e g a l o v i r u s a n d w o u l d g r o u p it to t h e f l - h e r p e s v i r u s e s ; - - t h e g e n o m e c a r r i e s p o l y r e p e t i t i v e D N A e l e m e n t s o n b o t h t e r m i n i [2], w h i c h w o u l d g r o u p it to the 7 - h e r p e s v i r u s e s .

*To whom all correspondence should be addressed. 187

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MICHAEL GOLTZ and HANNS LUDWIG

In this short review we want to summarize the most important biological and molecular biological features of BHV-4 strains. Furthermore we give an outlook on the potency of this virus to function as a vector for recombinant DNA vaccines.

BIOLOGY

Isolates and their derivation At the end of the 1950s and the beginning of the 1960s several herpesviruses were isolated from bovine animals (for review see Ref. [6]). Some of the viruses showed the same cytomegalovirus-like features (formation of electron-dense cytoplasmic inclusions, morphogenesis, ultrastructure) and were grouped as bovine cytomegaloviruses [5]. They have been designated later as bovine herpesvirus type 4 (BHV-4) [3]. The first isolate of this group came from cattle with respiratory distress and conjunctivitis [7]. Searching for the ethiologic agent of malignant catarrahl fever (MCF) further isolates were made and characterized biologically [8-11]. Then a variety of viruses were obtained either from diseased cattle, from abortions or even from healthy animals. In ruminants this virus has also been isolated from tumors. However, its status as a potential passenger in such cattle tumors can not be excluded [12]. Besides the isolation from infected animals, BHV-4 can be recovered from normal tissue culture cells [6, 13]. Our group has worked with an isolate from an animal with sign of MCF [9]. This isolate (strain 66-p-347) served for a variety of molecular biological studies and was the first isolate shown to have the group B genome [2]. Besides that, studies of the literature have shown that the virus obviously causes a clinical entity in cats associated with urinary stones; first a cat herpesvirus had been made responsible for this symptomatology. These isolates were reported as feline herpesvirus 2 (FHV-2) or BHV-4, strain FeCAHV. Different working groups are investigating details of this clinical problem [14--17]. Furthermore, first evidence exists that also wild living carnivores can be infected with BHV-4. Bartha (1989, personal communication) recovered a BHV-4-1ike isolate from a deceased lion. Biological properties as well as preliminary restriction enzyme analysis pointed to the fact that this agent can be grouped to BHV-4 (Bartha and Egelhof, unpublished data). This information suggests that the virus might easily be transmitted from cattle or from ruminant meat to carnivores causing undefined clinical sympthomatology which can even be fatal.

Cytopathology BHV-4 isolates cause a typical cytopathic effect. Starting from one center the cells round up and diffusely are eliberated from the tissue culture monolayer (see Fig. 1). None of the isolates is known to cause syncycial formation. As in other herpesvirus infection, the typical antigens accumulate first in the nucleus. Fine antigenic granules or diffuse nuclear staining can be observed when defined reconvalescent sera for staining are used. It is of interest that some of the infected cattle sera only mark nuclear antigens reminiscent to EBNA fluorescence caused by EBV. There is no clear evidence, whether in the infectious cycle antigens are incorporated into the plasma membrane. This would also correlate with the morphogenesis discussed next.

(A)

(B)

Fig. 2. BHV-4 infected GBK cells in transmission electromicroscopy. Intracytoplasmic accumulation of virions typical for cytomegalovirus infections; scale bar = 100 nm. Photograph kindly provided by H. Gelderblom. 190

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Fig. 4. Restriction analysis of seven BHV-4 strains with BamH I. The restriction patterns of the different strains show great similarities. Pay attention to the size variation of the prDNA elements (hypermolar bands in the + 2 kb range). Sizes of the 2/Hind III marker are given in kb. WSM--isolate from a cow in Mecklenburg; HB 450--reference strain Bartha; HB 420--reference strain Bartha; LVR 140 (Ref, [11]); Movar 33/63--reference strain Bartha; 75-P-2756 (Ref. [5]); 66-P-347 (Ref. [9]); (analysis and photograph kindly provided by Stephan Egelhof).

Biological and molecular properties of BHV-4

193

BHV-4 multiplies in a typical one-step growth curve when adapted to bovine cells. Relatively high virus titers (~< 107 PFU/ml) are found in the tissue culture supernatant approx. 4 0 h p.i. [1,4, 18]. Although BHV-4 follows in general the morphogenesis of other herpesvirus some pecularities have been reported: after nucleocapsid formation and envelopement most of the virions assemble in cytoplasmic vacuoles [5, 6] (Fig. 2). This feature is known also for human cytomegalovirus [19] which reinforced our suggestion to classify BHV-4 as a fl-herpesvirus although its genome is representative for -herpesviruses. A variety of antibody studies have been carried out. It is of considerable pathogenic interest that under natural conditions almost no neutralizing antibodies are formed in cattle. This phenomenon has recently been investigated in more detail by the production of monoclonal antibodies against the different infected cell proteins [20]. The results show that neutralizing domains are hidden and can be detected by a limited number of antibodies. However, there is no explanation yet why the virus in nature does not cause a neutralizing response. Most probably it hides itself by masking these protectionally important antigens. There is no information on the cellular immune response during the BHV-4 infection in bovines. Further details on epidemiology and pathogenesis are refered by Castrucci et al. in this Special Issue.

MOLECULAR BIOLOGY G e n o m e structure

According to Roizmans [21] classification of herpesvirus genomes BHV-4 is a group B virus. The structure of BHV-4 has first been reported by our group [2]. The genome consists of 140-150 kbp linear double-stranded DNA. A central unique part of the genome (about 110 kbp) is flanked by two stretches of tandemly arranged repetitive elements (Fig. 3), which are characterized by their high G C content. Herpesvirus saimiri and herpesvirus ateles [22, 23] are two well described examples for other members of the group B. The tandem repeats are designated as polyrepetitive DNA (prDNA) in order to differentiate them from high, middle and low repetitive DNA of cellular origin. The total number of elements per genome is 15 on average but the number of these elements varies at the genomic ends. The size of these elements differs from strain to strain. They differ in

prDNA

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110 kb

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Fig. 3. Genome organization of BHV-4. The unique central part of the genome is flanked by stretches of tandemly arranged repeats. These stretches are called polyrepetitiveDNA (prDNA). The number of prDNA elementsat each genomicend varies but the total number of elements per genome is 15 on average.

194

MICHAEL GOLTZ and HANNS LUDWIG

200 bp steps due to the fact that each p r D N A element contains 200 bp internal repeats [2]. Up to now, eight different sizes have been reported (1450-2850 bp). Southern blotting of restriction enzyme digested viral D N A demonstrates that one or two p r D N A sizes predominate (hypermolar fragments) and some further prDNA sizes are present in lower quantity in each strain, even if the virus has been plaque-purified. These facts may indicate a mixed population of prDNAs within one genome even if one size dominates [24]. Within the scope of investigations in the cleavage-packaging process and recombination events our group determined the nucleotide sequence of a p r D N A element, the genomic termini and the junctions between p r D N A and unique DNA (Goltz et al., paper in preparation). Beside the size variation of the p r D N A there are slight but distinct differences in the restriction pattern of BHV-4 strains. They allow to define two groups: the DN-like (reference strain DN 599) [10] and MOV-like (reference strain movar 33/66) [7] strains [25]. Restriction maps of four BHV-4 strains have been published [24]. Figure 4 shows the BamH I restriction patterns of different BHV-4 isolates. Currently investigations to further characterize BHV-4 proteins with the help of monoclonal antibodies are under way. Ten glycoproteins which are part of the viral envelope and a potential major nucleocapsid protein were recently identified using monoclonal antibodies [26].

OUTLOOK

Since the original isolation [7] and molecular biological characterization of BHV-4, more and more working groups pay attention to this virus. Its broad hostrange, the unsettled pathogenical role in diseases of cattle and other animals, and the possible interaction with other viruses to support certain clinical entities are the reasons for this interest. Due to our knowledge about this virus we have recently postulated BHV-4 to be a passenger virus in cattle [12]. Thus this virus could serve as a basis for the development of recombinant life vaccines for use in bovine animals. But further investigations have to be carried out to solve the questions whether BHV-4 is causing diseases directly or via transactivation events with other viruses not only in cattle but also in carnivores and other animals. Other most interesting points to be studied are molecular aspects of replication and the cleavage-packaging process during virus maturation. We believe BHV-4 to be a good substrate for such work due to its uncritical handling in tissue cultures and its plain genome structure.

Acknowledgements--We are grateful to Hans-J6rg Buhk and Kerstin Borchers for many helpful discussions and Hans Gelderblom for supply of the electromicrograph. We thank Wolfgang Weigelt for typing the manuscript. Some of the data are drawn from the ongoing Ph.D. work of Michael Goltz and DVM work of Stefan Egelhof. The work was supported by a NAF(~G scholarship to Michael Goltz and by grants of the BMFT (No. 07010660) to Hanns Ludwig and (No. BCT 0363) to Hans-J6rg Buhk.

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