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Arch Virol (1992) 124:379-387

© Springer-Verlag 1992 Printed in Austria

Cloning and restriction endonuclease mapping of the genome of an equine herpesvirus 4 (equine rhinopneumonitis virus), strain 405/76 Brief R e p o r t

H. S. Nagesha, J. R. McNeil, N. Ficorilli and M. J. Studdert School of Veterinary Science, The University of Melbourne, Parkville, Victoria, Australia Accepted October 31, 1991

Summary. Purified virion DNA of an Australian isolate of equine herpesvirus 4 (EHV 4.405/76) was digested with restriction enzymes and the DNA fragments were cloned into pUC 19. The resulting recombinant plasmid library, representing 92% of the virus genome, was used in hybridization analyses to construct restriction maps for Bam HI, Eco RI, and SalI for the EHV4 genome. The results show that the genome of EHV 4.405/76 was approximately 145 kb and comprised a unique long (UL) region of I 12 kb and a unique short (Us) region of 12.4kb. Us is flanked by an internal and terminal repetitive sequence (IR s and TRs) of about 10.3 kb. The Bam HI and Eco RI restriction maps are similar to those previously published for an English isolate EHV4.1942 strain [4] although some differences such as location of an additional fragment and changes in positions of two other small fragments were found.

Equine herpesvirus 4 (EHV4) and 1 (EHV 1) are members of the subfamily Alphaherpesvirinae. These two viruses cause serious diseases in horses and are responsible for considerable economic loss to the equine industry world wide. EVH 4 is the major cause of rhinopneumonitis and it is occasionally isolated from aborted foetuses. Conversely EHV 1 is mainly recognised as a cause of abortion, less commonly of neurological disease and occasionally of respiratory disease [1, 13]. The genomes of the two viruses are characteristic and have distinct restriction endonuclease DNA patterns I-8, 13]. The restriction endonuclease fingerprints of 20 epidemiologicallyunrelated EHV 4 isolates from the respiratory tract of horses in Australasia showed some heterogeneity [12]. Thus far only a single English isolate (EHV4, strain 1942) grown in equine

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dermal cells has been mapped by restriction endonucleases [4]. It was of interest to map and compare the genome of an epidemiologically different EHV 4 isolate which has been grown in equine foetal (EFK) cells. EHV 4.405/76 strain was isolated from the respiratory tract of a horse with rhinopneumonitis [-10]. A plaque purified stock of this virus was propagated in EFK cells as previously described [14]. Cell lysates from virus infected cell culture were clarified and virus from the supernate was pelleted at 50,000 g for 1.5 h in Beckman ultracentrifuge and further purified on 5 to 15% Ficoll-400 (Pharmacia) gradients [3]. Virus DNA digested with a number of restriction enzymes was electrophoresed on 0.7% agarose gels, stained with ethidium bromide and photographed. Fragment sizes were estimated using Gel Frag Sizer program developed by D. G. Gilbert (1989; Biology Department, Bloomington, Indiana University, Indiana, U.S.A.). The restriction enzymes Bam HI, Eco RI, and S a l I produced a relatively small number of fragments, hence they where chosen for mapping the virus genome (Fig. 1). The sizes of the individual restriction fragments are listed in Table 1 and the approximate size of the genome was 145kb. This is in agreement with the previously published values for EHV 4.1942 strain [4]. To identify fragments at or near each terminus of the genome the DNA was incubated with )~ exonuclease, then digested with Bam HI. Exonuclease

Fig. 1. Restriction endonuclease profiles of EHV 4.405/76 DNA electrophoresed through 0.7% (w/v) agarose gel. B is BamHI, E is Eco RI, S is SalI. )~ is phage )~ DNA digested with Hind III used as a kilobase (kb) marker

Restriction maps for EHV4

381

Table 1. Designation, size, and relative molarities of equine herpesvirus 4.405/76 restriction endonuclease DNA fragments Restriction enzyme Barn HI

Eco RI

Sal I

fragment size designation kb

molarity fragment size designation kb

molarity fragment size designation kb

molarity

A BCD E F G H I JKL MNNO P Q R S

18.7 13.7 12.3 t 1.2 9.4 8.7 6.5 4.6 4.3 3.5 1.2 0.8 0.6

1 3 I 1 1 1 1 3 4 1 1 1 1

2 1 1 1 1 1 1 1 1 1 1 I 1 1 1

1 1 1 2 1 1 1 1 1

Total

145.0

AB C D E F G H I J K L M N 0 P

27.5 18.7 14.1 12.0 9.4 7.0 6.4 6.1 5.8 3.4 2.8 2.0 1.0 0.9 0.6 145.3

A B C DE F G H I J

42.0 27.6 17.4 14.0 10.6 9.7 7.0 1.9 0.8

145.0

treatment resulted in the disappearance of 3.5 kb fragment P and reduced molarity of 13.7kb triplet fragments B, C, D (Fig. 2) indicating that fragment P and one of the 13.7kb triplet are terminal. Subsequent hybridization results confirmed that fragment P is at the right terminus and fragment B is at the left terminus for Barn HI map of this genome. Termini for Eco RI and SalI were deduced from the hybridization results using Barn HI P fragment and a clone carrying Eco RI I insert. Hybrid bands formed by these probes are shown in Fig. 3. Based on hybrid bands detected in Southern blots and also based on the relative position of other fragments as revealed by hybridization results (see below ) it was possible to identify that the fragments I (6.1 kb) and F (9.4 kb) are the termini for the Eco RI map. Similarly results indicated that the termini for the SalI map were A (42 kb) and H (7 kb). The plasmid vector p U C 19 was used for construction of genomic library of the viral D N A fragments using E. coli D H 5 ~ (BRL) as the host cell as described by Sambrook etal. [9]. Where it was difficult to clone an entire fragment it was subcloned using a combination of restriction enzymes such as Barn HI/Eco RI, Eco RI/Hind III, or Hind III alone. Eco RI terminal fragment I was blunt ended with Klenow fragment and cloned into the SrnaI site of p U C 19, however, a similar approach was not successful for cloning another

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Fig. 2. EHV4.405/76 termini as determined by exonuclease digestion. Both lanes show Bam HI restriction patterns. DNA in the left lane was treated with )~exonucleaseprior to Barn HI digestion.The fragmentswith reduced molarity or which disappeared are indicated by arrows Fig.3. Southern blots showing termini for EHV4.405/76. B is BamHI, E is EcoRI, S is SalI. )~ phage )~ DNA digested with Hind III used as a kilobase (kb) marker. 1 Eeo RI fragment I cloned into pUC 19 was used as probe. 2 BamHIP fragment isolated from gel was used as probe

terminal fragment Bam HI P. A total of 24 clones and a fragment isolated from the gel (Barn HI P) were used for construction of the three complete maps. Southern blot hybridization was performed essentially as described by Sambrook et al. [-9]. Briefly, after electrophoresis, restriction fragments generated by single and double digestions with Barn HI, Eco RI, and S a l I were transferred and fixed to Hybond-N membranes (Amersham). Recombinant plasmids were labelled with [a 32p]dATP or [a 32p]dCTP using a random priming kit (Amersham). The radiolabelled probes were hybridized to the Southern blots (1MNa +, 68 °C), then washed under highly stringent conditions (16.5mM, 68 °C), wrapped in plastic film and autoradiographed. Figure 4 shows the results of hybridization between clones and virion DNA fragments. The results of hybridization are summarized in Table 2. Repeat structures in the genome were identified as follows. Probe L (Bam HI clone) hybridized to Barn HI fragments L and D, N to N and P, P to N and P. Eco RI B clone hybridized to Eco RI fragments A and B as well as F. Similarly the presence of repetitive sequence was also revealed by a subclone of Eco R I G fragment and an isolated fragment Barn HI P. From the data summarized in the Table 2, restriction endonuclease

Restriction maps for EHV4

383

Fig. 4. Hybridization of Barn HI, Eco RI, or Sal I cloned fragments of EHV 4.405/76 with digests of whole virai genomic DNA (E4 in each panel) digested with BarnHI (I), Eco RI (II), or SalI (III).)~ is phage ~ DNA digested with Hind III used as a kilobase (kb) marker. The clones used as probes are shown above each lane and are as listed in Table 2. In lanes A and C additional bands that hybridized with the probes were considered partial digests

maps were constructed (Fig. 5) and these maps were further confirmed by double digestion of some of the clones. The genome is approximately 145 kb comprising a long region (L) of 112 kb and a short region (S) of about 33 kb. The short region comprises a unique short (Us) region bounded by repetitive sequences at the terminus (TRs) and internally (IRs). Sizes of the IRs and TRs were estimated from fragment sizes in hybridization analysis to be of 10.3 kb. Barn HI clone L hybridized to Eco RI fragments F (9.5 kb) and also to G and J indicating that the size of the repeat structure is more than 9.5 kb. To determine the size of the repeat structure more accurately, Eco R I G fragment was subcloned (Fig. 6 a) and the subclones were used in hybridizations. One of the subclones G 19 hybridized only to Eco R I G indicating that this component of Eco R I G is not in the repeat structure. However, the other subclone G41 hybridized to Eco R I G as expected and also to Eco R I J but not to Eco RI F. This suggested that the parts of Eco R I G and Eco RI J are in the repeat structure and hence the size of the repeat structure is more than 9.5 kb. Furthermore, when subclone G41 was digested with Sma I to delete 0.8 kb fragment and the resulting subclone HS i used in hybridization, it was found that this subclone hybridized to Eco R I G as expected, and also to Eco RI J but not to Eco RI F. These results indicated that the size of the repeat structure is at least 10.3 kb (0.8 kb + 9.5kb). Therefore the size of the

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Table 2. Cross-hybridization of EHV 4.405/76 restriction endonuclease cloned fragments Clone

Restriction enzymes Barn HI

Eco RI

SalI

Barn HI/Eco RI

Barn HI/SalI

18.7a 13.7 12.3 11.2 9.4 8.7 6.5 4.6 4.6; 13.7 4.3 4.3; 3.5 4.3 4.3; 3.5 1.2 0.8 0.6

18.7, 6.4 27.5, 12.0, 0.6 27.5 27.5 14.1, 6.4 18.7, 3.4, 0.9 27.5 12.0, 0.9, 0.6 27.5; 9.4; 7.0, 5.8 12.0 27.5; 9.4 27.5 27.5, 9.4 27.5 6.4 12.0

27.6, 14.0 10.6 17.4 14.0, 27.6 46.0 27.6 46.0 27.6, 46.0 46.0 46.0 17.4, 14.0 27.6

13.5, 4.3 10.1, 3.0, 0.6 4.8 11.2 9.0, 0.4 5.0, 3.4, 0.3 6.5 3.2; 0.9; 0.5 2.8; 1.3 4.3 4.3, 3.5 4.3 4.3; 3.5 1.2 0.8 0.6

13.5, 5.5 9.4, 3.2 10.6, 0.8 11.2 7.4, 1.2, 0.8 8.7 6.5 4.6 14.0; 4.3 3.08 4.3, 3.5 4.3 4.3; 3.5 0.6, 0.6 0.8 0.6

t3.7, 12.3, 6.5, 4.6, 4.3, 3.5 13.7, 4.6 13.7 13.7 13.7 13.7 13.7

27.5, 9.4

46.0, 10.6 46.0 46.0 7.0 46.0 9.7, 7.0 46.0

8.0, 4.3, 7.0, 7.0 6.1 2.5, 2.8 2.0

6.5, 4.6, 4.3, 3.5 13.7, 4.6 13.7 7.0 13.7 7.0, 7.0 13.7

7.0, 5.8 7.0 6.1 5.8 2.8 2.0

13.7, 9.4

14.1, 2.8

9.7

5.0

Barn HI

A C E F G H I J L M N O pb Q R

S

14.0

9.7, 0.8

14.0

1.2

Eco RI

A G41 G 191 I

J L M

6.5, 4.6, 3.5, 1.5 2.5 2.5

SalI

G

7.0, 2.0

a Numbers indicate size of DNA fragments in kitobase pairs b DNA fragment isolated from agarose gel used as probe

unique short region was estimated to be a b o u t 12.4kb, i.e., {S - (IRs + TRs)} = {33 - (10.3 kb + 10.3 kb)}. Cullinane et al. [-4] described similar g e n o m e structure for EHV4.1942. The g e n o m i c architecture o f these two E H V 4 isolates is consistent with the g e n o m e structure o f E H V 1 [5, 7, 15]. A l t h o u g h these two viruses have a similar g e n o m e structure the location o f restriction e n z y m e cleavage sites was different [4]. W h e n restriction endonuclease m a p s o f epidemiologically different E H V 4 isolates, i.e., strains 405/76 a n d 1942, are c o m p a r e d , the locations o f Barn H I a n d Eco RI cleavage sites a p p e a r similar. However, there are some differences

Restriction maps for EHV 4

385

L

~

UL

S

mR

US

TR

140 145

I

Bam HI

B

~

R

'I L DI"HI' H G ~l E

EcoRI

Sal I kilobase pairs

0

10

20

30

40

50

60

70

80

I

'

I

I

I

I

I

I

I

map units 0

0!1

0.2

0.3

0!4

0.5

100

110

120

130

I

90

II

,

I

l

0.6

0.7

0!8

I

~

01.9

1.

Fig. 5. Restriction endonuclease maps of the EHV 4.405/76 genome for BarnHI, Eco RI, and SalI. Terminal (TR) and internal (IR) repeat structures that bracket unique short (Us) region are shown as black boxes

Eco RI fragment G

(a) 0.8

l

Clone G19

E

2.9

3.6

t

0.5

1

S

t

H

H

I

E

Clone G41 Clone HS1

UL

(b)

US

TR IIIIIIIIII

EHV4.405/76 I

B

I G R,,

A

i H IJSMI

I G

A

I H IJQMt

! EHV4.1942

IR

1R B

C

I

F Q,, E

i I OIKIN I

D

ILINIPI

C

I

F

t I OIKIN {

D

ILINIPI

i I

)

E

Fig. 6. a Restriction map of Eco RI (E) fragment G. Fragments generated by Hind III (H) and Sma I (S) were cloned into pUC 19. b Comparison of BarnHI restriction endonuclease map of EHV 4.405/76 with that of EHV 4.1942. The sequence arrangements in EHV 4.1942 DNA is according to that described by Cullinane et al. I-4] and Riggio et al. [6]. Arrows indicate differences in the two restriction maps

in the unique long region (Fig. 6b). Bam H I R fragment (0.8kb) is located at the left terminus on E H V 4.1942 strain whereas it is found in between Barn HI G and A in strain 405. Bam HI Q (1.2 kb) mapped between F and E in strain 405 while it mapped between M and C in strain 1942. In addition, a small fragment Barn HI S (0.6 kb) which was not identified in strain 1942, is located between J and M in strain 405. These additions and deletions of small restriction fragments may be due to the gain or loss of restriction sites associated with growth of the two viruses in different cells: 405 in E F K and 1942 in equine dermal cells. In previous studies differences in restriction patterns of epidemiologically unrelated E H V 4 after passage in BHK-21 cells were observed [12-]. Nevertheless, the

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minor variations observed in the present study for the locations of small restriction fragments do not appear to alter the over-all genome structure of E F K grown 405 and equine dermal grown 1942 strains. In addition to comparative mapping as described above, this study also records additional mapping using SalI restriction endonuclease. A large fragment (SalI A) is located at the right terminus covering both terminal and the internal repeat structure as well as a part of unique long region. SalI H fragment (7 kb) forms the left terminus of the genome. Other fragments lie in the UL region. It will be of interest to see whether the SalI restriction map of English isolate (i.e., strain 1942) is similar to our Australian isolate (i.e., strain 405). The construction of recombinant library and derivation of three restriction maps for EHV 4 will help future work on molecular biology of equine herpesviruses, particularly in understanding the genetic relationships of equine herpesviruses. The physical maps are also useful aids for molecular epidemiology of equine herpesviruses [2, 11, 12].

Acknowledgements We are grateful to J. M. Whalley and B. S. Crabb for helpful discussions. This study was funded by the Melbourne University Equine Research Fund, and Ministry for Sports and Recreation, Victoria, Australia.

References 1. Allen GP, Bryans JT (1986) Molecular epizootiology, pathogenesis, and prophylaxis of equine herpesvirus-1 infections. Prog Vet Microbiol Immunol 2:78-144 2. Allen GP, Yeargan MR, Turtinen LW, Bryans JT (1985) A new field strain of equine abortion virus (equine herpesvirus-1) among Kentucky horses. Am J Vet Res 46: 138140 3. Crabb BS, Studdert MJ (1990) Comparative studies of the proteins ofequine herpesvirus 4 and 1 and asinine herpesvirus 3: antibody reponse of the natural hosts. J Gen Virol 71:2033-2041 4. Cullinane AA, Rixon F J, Davison AJ (1988) Characterization of the genome of equine herpesvirus 1 subtype 2. J Gen Virol 69:1575-1590 5. Henry BE, Robinson RA, Dauenhauer SA, Atherton SS, Hayward GS, O'Callaghan DJ (1981) Structure of the genome of equine herpesvirus type 1. Virology 115:97-114 6. Riggio MP, Cullinane AA, Onions DE (1989) Identification and nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4. J Virol 63:1123-1133 7. Ruyechan WT, Dauenhauer SA, O'Callaghan DJ (1982) Electron microscopic study of equine herpesvirus type 1 DNA. J Gen Virol 42:297-299 8. Sabine M, Robertson GR, Whalley JM (198 t) Differentiation of the subtypes of equine herpesvirus 1 by restriction endonuclease analysis. Aust Vet J 57:148-149 9. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 10. Studdert MJ, Blackney MH (1979) Equine herpesviruses: On the differentiation of respiratory from foetal strains of type 1. Aust Vet J 55:488-492 11. Studdert MJ, Crabb BS, Ficorilli N (1991) The molecular epidemiology of equine herpesvirus 1 abortion in Australasia from 1975 to 1989. Aust Vet J (in press) 12. Studdert MJ, Fitzpatrick DR, Browning GF, Cullinane AA, Whalley JM (1986) Equine

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herpesvirus genomes: heterogeneity of naturally occurring type 4 isolates and of a type 1 isolate after heterologous cell passage. Arch Virol 91:375-381 13. Studdert MJ, Simpson T, Roizman B (1981) Differentiation of respiratory and abortigenic isolates of equine herpesvirus 1 by restriction endonucleases. Science 214: 562564 14. Studdert MJ, Turner AJ, Peterson JE (1970) Equine herpesvirus 1. Isolation and characterization of equine rhinopneumonitis virus and other equine herpesviruses from horses. Aust Vet J 46:83-89 15. Whalley JM, Robertson GR, Davison AJ (1981) Analysis of the genome of equine herpesvirus type 1: arrangement of cleavage sites for restriction endonucleases Eco RI, Bgl II and Barn HI. J Gen Virol 57:307-323 Authors' address: M. J. Studdert, School of Veterinary Science, The University of Melbourne, Parkville, Vic. 3052, Australia. Received August 23, 1991