Analysis of genomic rearrangement and subsequent gene deletion of the attenuated Orf virus strain D1701

Virus Research 56 (1998) 53 – 67

Analysis of genomic rearrangement and subsequent gene deletion of the attenuated Orf virus strain D1701 Rosita Cottone, Mathias Bu¨ttner, Berthilde Bauer, Marco Henkel, Eduard Hettich, Hanns-Joachim Rziha * Federal Research Centre for Virus Diseases of Animals, Institute For Vaccines, Paul-Ehrlich-Strasse 28, D-72076 Tu¨bingen, Federal Republic of Germany Received 18 February 1998; received in revised form 3 April 1998; accepted 29 April 1998

Abstract The orf virus (OV) strain D1701 belongs to the genetically heterogenous parapoxvirus (PPV) genus of the family Pox6iridae. The attenuated OV D1701 has been licensed as a live vaccine against contagious ecthyma in sheep. Detailed knowledge on the genetic structure and organization of this PPV vaccine strain is an important prerequisite to reveal possible genetic mechanisms of PPV attenuation. The present study demonstrates a genomic map of the approximately 158 kbp DNA of OV D1701 established by hybridization studies of cloned restriction fragments covering the complete viral genome. The results show an enlargement of the inverted terminal repeats (ITR) to up to 18 kbp due to recombination between nonhomologous sequences during cell culture adaptation. DNA sequencing of the region adjacent to the ITR junction revealed the absence of one open reading frame designated E2L. In contrast to a transposition-deletion variant of the New Zealand OV strain NZ2 (Fleming et al., 1995) the two genes E3L (a homologue of dUTPase) and G1L neighbouring E2L are retained in OV D1701. DNA and RNA analyses proved the presence of E2L gene in wild-type OV isolated directly from scab material. The data presented indicate that the E2L gene is nonessential for virus replication in vitro and in vivo, and may represent one important viral gene in determining virulence and pathogenesis of OV. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Parapoxvirus; Attenuated Orf virus D1701; Genomic map; Gene deletion

1. Introduction * Corresponding author. Tel: + 49 7071 967253; fax: +49 7071 967303; e-mail: [email protected]

Contagious pustular dermatitis known as Orf is a disease of sheep and goats caused by infection

0168-1702/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0168-1702(98)00056-2

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with parapoxvirus (PPV) ovis (Orf virus; OV), which can also affect man (Robinson and Balassu, 1981). Like all poxviruses the double-stranded DNA genome of PPV contains inverted terminal repeats (ITR) and hairpin structures (Garon et al., 1978; Wittek et al., 1978; Wittek and Moss, 1980; Baroudy et al., 1982). Poxviral genes essential for viral replication are located in the central region of the genome and are highly conserved among the different poxvirus species (Mackett and Archard, 1979; Drillien et al., 1987; Binns et al., 1988; Turner and Moyer, 1990). Genes located near the genomic termini usually encode viral functions nonessential for replication, but play a role in pathogenesis, host and tissue tropism, and virulence (Wittek et al., 1977; Gillard et al., 1986; Perkus et al., 1991; Fleming et al., 1997). Genomic rearrangements between non-homologous sequences are frequently obtained during serial propagation of different poxviruses in cell culture, particularly involving the ends of the viral genome. Deletions in the terminal regions have been described for vaccinia virus (VV) (Drillien et al., 1981; Moss et al., 1981; Panicali et al., 1981; Paez et al., 1985; Dallo and Esteban, 1987; Lai and Pogo, 1989; Meyer et al., 1991), rabbitpox virus (Lake and Cooper, 1980; Moyer and Rothe, 1980), cowpox virus (Archard and Mackett, 1979) and monkeypox virus (Esposito et al., 1981). Transposition of duplicated sequences has also been reported for VV (Kotwal and Moss, 1988), cowpox virus (Archard et al., 1984; Pickup et al., 1984), rabbitpox virus (Moyer et al., 1980) and monkeypox virus (Dumbell and Archard, 1980). The OV is the prototype species of the genetically heterogeneous parapoxvirus genus. Until now, detailed information about the genetic structure and transcriptional organization is exclusively available for the OV strain NZ2 (Mercer et al., 1987; Robinson et al., 1987; Fraser et al., 1990; Fleming et al., 1993; Mercer et al., 1995). So far, other OV isolates have been characterized only by comparison of restriction enzyme pattern and DNA hybridization (Robinson et al., 1982; Gassmann et al., 1985; Rafii and Burger, 1985). The strain D1701 represents a highly attenuated OV originally isolated from sheep. After serial cell

culture passages the resulting avirulent D1701 strain was successfully used for the development of a live vaccine against contagious ecthyma in sheep (Mayr et al., 1981). Scarification of sheep with OV D1701 causes only mild skin lesions and induces immunity against OV infection lasting for about 4–6 months. PPV, particularly OV, is considered as a promising candidate for gene vectors (Robinson and Lyttle, 1992). The perspective of a potential vector lacking pathogenicity, however demands a detailed study of its genomic organisation and on the influence of viral genes in OV attenuation. Therefore, we cloned almost the entire genome and for the first time established a detailed genomic map of D1701. The results show that sequences of the right end of the genome were duplicated and translocated to the left end resulting in an increase of the size of the ITRs to approximately 18 kbp. DNA sequencing results located the junction between the ITR and the unique part of the D1701 genome. Furthermore, in comparison to both a wild-type OV isolated directly from scab material and the prototype NZ-2 strain the deletion of one open reading frame designated E2L (Fleming et al., 1995) was detected in D1701. The presence of the E2L gene in wildtype OV could be demonstrated by DNA and RNA analyses. The data presented indicate that the E2L gene is nonessential for virus replication in vitro and in vivo, and might play some role in determining virulence and pathogenesis of OV.

2. Material and methods

2.1. Cells and 6irus The OV strain D1701 originally isolated from sheep was adapted to cell culture (Mayr et al., 1981). The virus was plaque-purified and propagated on bovine kidney cell line (BK-KL3A) in minimal essential medium supplemented with 5–7% fetal bovine serum (Costar, FRO). The isolate BO15 was obtained from a lesion derived from a severe case of Orf in sheep. Scab virus was used for propagation in BK-KL3A cells. MRIscab and Orf11 virus was kindly provided by Colin McInnes, Moredun Research Institute, Edin-

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burgh, UK. Virions were purified for DNA extraction as described (Esposito et al., 1978).

2.2. Manipulation of 6irus DNA Restriction endonucleases and other modifying enzymes were purchased from New England Biolabs (FRG), and used as recommended by the manufacturer. Viral DNA fragments were cloned by standard procedure into the vector plasmids pSPT18 or pSPT19 (Boehringer Mannheim, FRG).

2.3. Gel electrophoresis and Southern blot hybridisation The digested DNA was separated in 0.4 – 0.8% agarose gels and transferred onto nylon membrane (HybondN+, Amersham Life Science) as described (Reed and Mann, 1985). Hybridisation probes were prepared by random priming using the Rediprime labelling system (Amersham Life Science, FRG) and [a-32P]-dCTP ( \ 3000 Ci/ mM, ICN Biomedicals, FRG). Oligonucleotide probes were labelled with the T4-Polynucleotide kinase (New England Biolabs, FRG) and [g-32P]ATP (\ 3000 Ci/mM, ICN Biomedicals, FRG). Hybridisation was performed for 4 – 18 h at 54°C in 4× SSPE (1×SSPE: 0.18 M sodium-chloride, 0.01 M phosphate buffer, 0.001 M EDTA, pH 7.4), 0.5% (w/v) non-fat dry milk, 1.0% (w/v) SDS, 0.5 mg/ml denatured calf thymus DNA, and 50% (v/v) deionized formamide.

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SDS, 0.5%(v/v) DEPC (Diethyl-pyrocarbonate, Serva, FRG), 7.0% (w/v) dextransulfate, and 60% deionized formamide (Life Technologies GIBCOBRL, FRG).

2.5. DNA sequencing For standard DNA sequencing the T7 sequencing kit (Pharmacia Biotech., FRG) and [ot-35S]dATP ( \650 Ci/mM, ICN Biomedicals, FRG) was used as recommended by the manufacturer. In addition, an automatic DNA sequencing machine (Applied Biosystems Division PE/ABI, Foster City, ABI model 377 sequencing system) and the ABI PRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems GmbH, Perkin Elmer Corporation, USA) was used. Oligonucleotides were synthesised on a DNA synthesizer (Biosearch 8700, New Brunswick, UK) and subsequently purlfied by denaturing gel electrophoresis and reversed phase chromatography on SePack columns (Millipore Waters, FRO). Nucleotide and amino acid sequences were compiled and analysed using the Wisconsin Package Version 9.1, Genetics Computer Group (GCG) Madison, Wisc. USA and the Basic Alignment Search Tool (BLAST) provided by the National Center For Biotechnology Information (http:¯¯www.ncbi.nhm.nih.gov).

3. Results

3.1. The genomic ends of D1701 are duplicated 2.4. RNA isolation and Northern blot hybridisation Total RNA was isolated from BK-KL3A cells at 2 –28 h after infection (mod 10) by extraction with acidic guanidinium thiocyanate (Chomczynski and Sacchi, 1987). Early viral RNA was extracted from cells infected in the presence of cycloheximide (100 mg/ml). The RNA was separated in agarose gels containing formaldehyde (Kroczek and Siebert, 1990) and transferred onto nylon membranes (HybondN+, Amersham Life Science). Hybridisation was performed overnight at 54°C in 3 × SSPE, 1.0% non-fat dry milk, 2.0%

The restriction patterns of D1701 DNA were found slightly different from those of the strain NZ2 representing the only OV intensively studied since many years (Robinson and Lyttle, 1992). To establish the genomic map of OV D1701 viral DNA was single and double digested with the restriction endonucleases HindIII, EcoRI, BamHI and KpnI. The HindIII-, EcoRI- and KpnI- fragments of D1701 were plasmid cloned, partially sequenced, and individually used as hybridisation probes (data not shown). The resulting map location of the D1701 fragments is depicted in Fig. 1A, the individual restriction fragments and the

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Fig. 1. Genomic map of D1701. (A) Map location of the BamHI-, EcoRI-, HindIII- and KpnI-fragments of D1701. Identical sized fragments mapping in the ITR (terminal inverted repeat) region are designated with identical letters, but differentiated by an apostrophe. (B) The left hand and the right hand end (fragment HindIII-D) are enlarged and the respective EcoRI- (upper letters) and KpnI- (lower letters) fragments denoted. The thick bars indicate those subfragments used as hybridisation probes (see also text - Section 3.1).

calculated molecular weights are listed in Table 1. So far, the map location of some of the smaller KpnI fragments (O, P, T, and U) could not yet be determined and therefore, are not illustrated in Fig. 1. The total genomic size of D1701 was found to be enlarged to 158 kbp in comparison to the smaller size of 139 kbp for OV strain NZ2 (Mercer et al., 1987). During the mapping experiments it became evident that the terminal EcoRI fragments -C and -F, the HindIII fragments -H and -J as well as the KpnI fragments -F and -J were present in a double molar quantity. Therefore, these fragments were designated as EcoRI-C/C% and F/F%, HindIII-H/ H% and J/J%, and KpnI F/F% and J/J%. Blot hybridisation with the cloned fragments HindIII-H, KpnI-J and EcoRI-F as probes proved the identity of each of the two molar DNA fragments (data not shown). The obvious symmetrical distribution of the restriction sites at the genomic ends indicated the possibility that the terminal regions of Dl701 DNA are duplicated and translocated to the opposite end of the genome

resulting in an expansion of the ITR region. Hybridisation of digested DNA of plaque-purlfied D1701 with radioactively labelled EcoRI-subfragments of HindIII-D and KpnI-I as probes allowed a more exact mapping of the ITR junction. To this end, DNA prepared from D1701 after 20 cell culture passages was compared with that obtained from virus after 135 cell culture passages, which constitutes the attenuated Dl701 vaccine strain. As can be seen in Fig. 2, the genome of both virus preparations did not exhibit detectable differences. Fig. 2A shows that the KpnI-I/EcoRI-F subfragment (2.85 kbp; designated as probe IEa in Fig. 1B) hybridised with the genomic fragments KpnI-B and -I, EcoRI-F/F%, BamHI-A and -C, and HindIII-C and -D due to its localisation within the ITR. The identity of the EcoRI-F and EcoRI-F% was also confirmed by positive hybridisation of probe Ea with the subfragments HindIII-D/EcoRI-F% (Fig. 1B and 2A, DEb) and the HindIII-D/KpnI-B (Fig. 1B and Fig. 2A, DKa, approximately 7.12 kbp), respectively.

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Fig. 2. Viral DNA was prepared from Dl701 obtained after 20 cell culture passages (lane 1) and after 135 cell culture passages (lane 2), and restriction digested as indicated. Digestion products of the isolated fragment HindIII-D are separated in lanes H-D. As size marker the 1 kbp ladder (GIBCO BRL Life Technologies) was used (lane M). (A) The ethidiumbromide-stained gels (right) were blot hybridised with 32P-labelled fragment EcoRI-F (probe IEa, Fig. 1B) and (B) fragment EcoRI-G (probe DEd). The detected restriction fragments are indicated to the left. To the right, the EcoRI subfragments (DE) and KpnI subfragments (DK) of the isolated fragment HindIII-D are designated according to Fig. 1B.

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Using EcoRI-G (Fig. 1B, probe DEd) as a probe, strong signals were found with the fragments KpnI-B, EcoRI-G, BamHI-A, HindIII-D fragments and the DKa and DEd subfragments (Fig. 2B). The relatively weak hybridisation signals of the labelled Eco-G with fragments KpnI-I, EcoRI-B, BamHI-C and HindIII-C can be explained by a small portion of sequence homology. Using the IEb probe (Fig. 1B, approximately 6 kbp), strong signals were obtained with the fragments KpnI-I, EcoRI-B, BamHI-B and -C, HindIII-C, and weak signals with fragments KpnI-B, HindIII-D, BamHI-G (data not shown). The results of the blot hybridisation experiments allowed the assignment of the end of the ITR region near the junctions between the EcoRI-F and EcoRI-B fragments and between the EcoRIF% and the EcoRI-G fragments. In conclusion, not only identical restriction profiles but particularly the use of ITR-specific probes in blot hybridisation indicated the occurrence of the described

Table 1 Size of restriction fragments of D1701 DNA in kilobase pairs (kbp) HindIII

KpnI

EcoRI

BamHI

A 37.50 B 33.00 C 25.50 D 15.60 E 12.50 F 8.80 G 7.30 H/H% 5.52 I 5.20 J/J% 0.95

A 24.50 B 22.00 C 18.50 D 12.30 E 11.30 F/F% 11.0 G 9.30 H 9.20 I 8.90 J/J% 3.95 K 2.90 L 2.80 M 2.70 N 1.15 O 0.98 P 0.64 Q 0.54 R 0.425 S 0.123 T 0.117 U

A 62.00 B 48.50 C/C% 15.00 D 6.40 E 4.25 F/F% 2.85 G 1.75

A 57.00 B 45.00 C 19.00 D 17.50 E 12.30 F 3.50 G 2.15 H 1.80

158.275

158.60

158.25

Totals 158.34

genomic rearrangement already after 20 cell culture passages of D1701.

3.2. DNA sequencing In order to map the ITR junction in D1701 precisely, the cloned IEb and DEd (EcoRI-G) fragments were sequenced. The DNA sequence of both fragments was identical over a stretch of 352 bp (Fig. 3A) downstream of the EcoRI site separating fragments F and B, or F% and G, and diverged thereafter. Therefore, the breakpoint of sequence identity defined the limit of the ITR region at both genomic termini. According to the size of the terminal restriction fragments covering the duplicated sequences the size of the ITR in D1701 can be calculated to approximately 18 kbp. Homology search of the sequenced part of IEb (1081 nt) revealed that 729 bases of D1701 were 94.9% identical to NZ2. The homology starts at the three last bases of the D1701 ITR and proceeds into the unique part of the genome. In Fig. 3B, the sequence of the complementary strand is depicted and compared with the NZ2 sequence (upper line). It can be seen that the C-terminal part of the G1L gene and the complete E3L gene was detected in D1701. In the sequenced part of D1701 G1L gene seven base changes compared to NZ2 were found, leading to an alteration of five amino acids (Fig. 3B). The G1L gene of D1701 is followed by the E3L gene (Fig. 3B), a counterpart of dUTPase as reported for NZ2, too (Mercer et al., 1989; Sullivan et al., 1995). The E3L DNA sequence demonstrates 93.75% identical nucleotides (nt) encoding 160 amino acids in D1701. In the C terminal part of the D1701 E3L gene, one additional amino acid asparagine was found as compared to NZ2 E3L (159 amino acids). The overall amino acid homology between both dUTPase-like genes accounts to 94.34% with only nine differing amino acids (Fig. 3B), which do not influence the characteristics of the polypeptide like hydrophobicity and secondary structure as predicted by computer (not shown). As marked in Fig. 3B, five nt downstream of the translational stop codon of D1701 E3L the ITR region begins, and the sequence homology with NZ2 terminates. Therefore, the early transcriptional stop motif (T5NT), which is

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Fig. 3. DNA sequence around the ITR junction of D1701. (A) Bestfit of the sequence obtained with subfragment IEb (upper sequence) and EcoRI-G (lower sequence) revealed 352 identical nucleotides downstream of the EcoRI site. The arrow marks the ITR junction. (B) Sequenced part of subfragment IEb. The nucleotide sequence of NZ2 (upper sequence; accession no. U34774 and M30023) is compared with the complementary strand of D1701 (lower sequence; 1081 bases downstream of the EcoRI site; accession no. AF056304). The amino acid sequence of D1701 is given below the DNA sequence, the identified G1L and E3L genes indicated. For NZ2 only the amino acids differing to D1701 are written in each last line. Homology between NZ2 and D1701 ends are at position 729, three bases after the beginning of D1701 ITR (open arrow). Asterisks mark translational stop codons, the arrows indicate the T5NT motifs for transcriptional termination.

present in NZ2 23 nt downstream of the translational stop, is absent in D1701. Instead of that two TsNT motifs are found 194 nt and 233 nt downstream of the stop codon of D1701 E3L (Fig. 3B, arrows).

3.3. G1L and E3L are transcribed in D1701 To analyse whether the E3L gene is transcribed in D1701, we synthesised an oligonucleotide (30 mer, E3L probe) complementary to the N-terminal region of E3L of D1701 and NZ2 (Fig. 3B, nt 283 to 313), and used it as a probe for Northern blot hybridisation. As shown in Fig. 4, the E3L probe specifically hybridised with a RNA of approximately 700 bases in size. This RNA was clearly detectable from 4 h after infection on, and accumulated in the presence of cycloheximide (Fig. 4, lane CH), which demonstrated that the E3L gene is transcribed as an early RNA in D1701. The size of the detected RNA is nearly identical to that of the E3L-specific mRNA described for NZ2 (Fleming et al., 1992). Assuming

the use of the next available T5NT motif (as described in Section 3.2) the addition of 194 bases to the size of the E3L open reading frame (483 bp) nicely correlates with the size of E3L-specific transcript detected in D1701. Expression of the G1L gene in D1701 also could be shown by Northern blot hybridisation with a GlL-specific probe (derived from fragment BamHI-G). The detected transcript of 1.55 kb in size (data not shown) correlates with the described size (1551 bp) of the G1L gene of NZ2 (Sullivan et al., 1995). Thus, D1701 seems also to contain a G1L very similar to NZ2, although the complete DNA sequence of this region has not been determined.

3.4. Absence of E2L in D7101 To confirm the absence of the E2L gene in D1701 a specific oligonucleotide (35 nt, E2L probe) according to the sequence of the N-terminal part of the NZ2 E2L was used for Southern blot hybridisation. No signals were detectable after hybridisation of the E2L-probe with digested

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Fig. 3. (Continued)

D1701 DNA (Fig. 5A). Because of the lack of neither a parental scab virus nor a very early D1701 cell culture passage, we cannot prove un-

equivocally the presence of the E2L in the original D1701. Therefore, we analysed DNA prepared from virulent scab virus detected in sheep lesions.

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Fig. 3. (Continued)

HindIII-, EcoRI-, BamHI-, and KpnI-digested DNA of the OV field isolates BO15 and MRIscab was hybridised with the E2L-probe. Specific

hybridisation signals were obtained with single fragments of MRI-scab DNA (Fig. 5B) and BO15-scab DNA (Fig. 5C). Furthermore, E2L-

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Fig. 3. (Continued)

specific sequences were also detected in DNA isolated from BO15 virus, which had been propagated three times in BK-KL3A cells (Fig. 5D). Blot hybridisation of BO15-scab or MRI-scab DNA with labelled probes representing different regions of the ITR of D1701 did not indicate translocation-duplication of the genomic termini (data not shown). In contrast, analysis of another cell culture adapted OV, strain Orf11 also missed E2L-specific sequences (data not shown), and exhibited a similar genomic rearrangement as D1701 (data not shown).

Fig. 4. Northern blot hybridisation to detect E3L-specific RNA in D1701 infected cells. Total RNA was isolated at the indicated hours after infection, or from cycloheximide treated cells (CH) and blot hybridised with 32P-labelled E3L-specific oligonucleotide (35 mer) as probe. The detected RNA with a size of approximately 700 bases is indicated.

4. Discussion The present study represents the first detailed analysis of the genome of the highly attenuated OV strain D1701 and enables us now a more precise comparison with the virulent OV strain NZ2 studied since many years (Mercer et al., 1987; Fraser et al., 1990; Fleming et al., 1992; Robinson and Lyttle, 1992). Comparative restriction analysis shows different size of the respective restriction fragments, however, when compared to other PPV strains and isolates (Robinson et al., 1982; Gassmann et al., 1985; Mercer et al., 1995; Rziha et al., unpublished) NZ2 and D1701 look quite similar. The close relationship between NZ2 and D1701 is also reflected by a high degree of DNA sequence homologies and analogous gene arrangement. This is also evident from preliminary sequence information of cloned DNA fragments of D1701 (Rziha et al., unpublished data). Interestingly we found an enlargement of the D1701 genome up to about 158 kbp compared to 139 kbp of NZ2 (Mercer et al., 1987). This can be partly explained by the increased size of the ITR region (18 kbp). Southern blot hybridisation and DNA sequencing demonstrated that approximately 18 kbp of the right terminus had been duplicated and translocated to the opposite end of the viral genome. Thus, for the first time rearrangement of OV sequences has been demonstrated in an attenuated European OV. A similar but not identical genomic alteration was described recently in a variant of NZ2 (Fleming et al., 1995). As a consequence of the genomic recombination event in D1701, all genes encoded in the elarged

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Fig. 5. Detection of the E2L gene in the genome of (A) D1701, (B) MRI-scab, (C) BO15-scab, and (D) BO15 after the third cell culture passage. The DNA was separated in 0.7% agarose gel (M: 1 kbp ladder as size standard) and blot hybridised with 32 P-labelled E2L-specific oligonucleotide as probe. Whereas no signals could be detected with D1701 DNA (even after 14 days exposure), single restriction fragments of all other OV exhibited specific hybridisation.

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Fig. 6. Comparison of the left hand terminus of D1701 DNA with wild-type OV NZ2 (NZ2) and the described NZ2 variant (NZ2 var; Fleming et al., 1995) demonstrates different gene deletions (brackets).

ITR region have been duplicated (like VEGF and IL-10, Fig. 1). In the NZ2 variant the original ITR comprising 3.1 kbp (Fraser et al., 1990) is increased to 19.3 kbp, and after duplicationtranslocation 3.3 kbp of the left end of the genome are deleted. Assuming a comparative size of the original ITR region in D1701, which is corroborated by the extensive DNA sequence homology, we suppose that in D1701 approximately 4.3 kbp of the left terminus have been replaced by a sequence duplication of approximately 18 kbp in size, which is derived from the right end of the genome. Therefore, the net loss of unique DNA sequences of D1701 can be calculated to approximately 1 kbp. Notably the terminal duplication along with the deletion expanded the total size of the D1701 genome to approximately 158 kbp, in contrast to the approximately 150 kbp sized DNA of the NZ2 variant. This indicates the presence of additional sequences in the Dl701 genome not present in NZ2. The terminal deletion of the NZ2 variant is associated with the loss of the three early genes E2L, E3L, and G1L (Fig. 6; Fleming et al., 1995). In contrast, the analysis of D1701 demonstrates the presence of two of these genes: E3L and G1L, respectively (Fig. 6). Determination of the sequence of the D1701 E3L gene revealed that the proposed early transcriptional termination signal T5NT (Fleming et al., 1992) is missing. Northern blot hybridisation with an E3L-specific probe,

however, showed that the E3L gene is transcribed giving rise to a distinct early mRNA of approximately 700 bases in size. Usually early poxviral transcripts possess well defined 3%ends due to termination of the mRNA 20–30 bp downstream of the T5NT motif (Yuen and Moss, 1987; Moss, 1990). Calculating from the size of the E3L encoding region (483 bp) it is reasonable to assume that the TsNT signal, which is found 194 bp downstream of the E3L gene, functions as a transcriptional termination signal in D1701. Most probably this termination signal was transferred from the opposite end of the D1701 genome during the rearrangement event. As a consequence the E3L-specific mRNA of D1701 seems to contain a 3% non-coding region of approximately 200 bases. In NZ2 the size discrepancy between the E3L encoding region and the mRNA of approximately 700 bases is explained by a poly(A) tract of about 200 bases (Fleming et al., 1992). The E3L gene of OV encodes a potential polypeptide which is homologous to the dUTPase of e.g. herpes simplex virus (Mercer et al., 1989; McGeoch, 1990), in which it is involved in neurovirulence and neuroinvasiveness (Pyles et al., 1992). It is tempting to speculate about the biological role of E3L in OV virulence, further experiments for analysing the expression and identification of functional domains of E3L are needed. Adjacent to the E3L gene we found a gene designated as G1L in NZ2, which encodes a protein of unknown function containing ankyrin repeat motifs (Sullivan et al., 1995). This gene is transcribed in D1701 also as an early RNA of 1.55 kb in size, and the sequenced C-terminal exhibits high homology to NZ2, as it was also found for the E3L gene. Homology searches did not detect any potential counterpart in gene data bank neither for the ITR sequence nor for the sequenced part (790 bp) of the fragment EcoRI-G of D1701. The hybridisation results and the sequence data showed that compared to NZ2 only the 216 bp sized E2L gene is missing in the analysed part of the D1701 genome. E2L counterparts are also found in cowpox virus, ectromelia virus, and in the Western Reserve strain, but not in strain Copenhagen of vaccinia virus (Goebel et al., 1990;

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Mercer et al., 1996). In addition, the E2L gene (71 amino acids) with a coding capacity for a 6-kDa protein exhibits homology to a DNA binding protein of African swine fever virus (Mercer et al., 1996). As for VV the E2L gene can be regarded as nonessential for OV replication in cell culture. Fleming et al. (1995) suspected that the loss of the three genes E2L, E3L and G1L might be associated with an attenuated character of the described NZ2-variant. The attenuated, avirulent strain D1701 was obtained after 135 passages of the parental scab virus isolate in cell culture (Mayr et al., 1981). Although no direct comparison with the original D1701 scab virus is possible, there are several reasons to assume a preceding deletion of at least the E2L gene during an early cell culture passage (earlier than 20 passages) of D1701 as summarised in Fig. 6. The close relationship of OV D1701 to NZ2 became also evident after preliminary spot sequencing of the cloned D1701 DNA restriction fragments (H.-J. Rziha, unpublished data), which revealed the existence of at least 30 poxviral gene homologues, similar to NZ2 (Mercer et al., 1995). This also includes OV-specific genes like the VEGF- (Lyttle et al., 1994) and the IL-10 homologues (Fleming et al., 1997) or ORF 3 (Fleming et al., 1991). Blot hybridisation studies with DNA isolated from virulent virus derived directly from Orf lesions of sheep (BO15-scab and MRI-scab) clearly demonstrated the absence of the described duplication-translocation event (data not shown), and the existence of the E2L gene (Figs. 5 and 6). Therefore, we would predict that the original D1701 virus also contained this gene. Since only this gene is missing in that part of the D1701 genome, a potential function of the E2L gene in OV virulence appears conceivable. Further functional studies are now underway to elucidate the expression and biological role of the E2L protein in vitro and in vivo. However, it can be suggested that OV attenuation is mediated by the rearrangement of limited terminal regions of the genome, which is accompanied by gene deletion. Similar genomic recombination events initiated by terminal deletions are described for

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various poxviruses (Dumbell and Archard, 1980; Esposito et al., 1981; Archard et al., 1984; Pickup et al., 1984; Kotwal and Moss, 1988; Robinson and Lyttle, 1992). Those poxvirus variants display altered genomic size and changed characteristics in virulence, pathogenicity or host specificity, which are associated with the terminal localisation of the corresponding genes (Moyer et al., 1980; Dallo and Esteban, 1987; Bloom et al., 1991; Buller and Palumbo, 1991; Perkus et al., 1990; Turner and Moyer, 1990). As a consequence of the duplication-translocation event all genes mapping in the enlarged ITRs of D1701 are duplicated. It is unknown whether an increased synthesis of these gene products does occur, which might also affect the viral phenotype. The possibility of cell culture propagation of the OV isolate BO15 now enables a detailed investigation of genomic alterations occurring during the different passages in cell culture. Finally, the described results represent a first important step towards characterisation and improvement of PPV vaccines, and the potential use of D1701 as a viral vector. The identification of non-essential OV genes, which interfere in virus pathogenesis and virulence, is of importance to understand virus-host interactions. The construction of defined D1701 and BO15 mutants can also be envisaged by deleting or inserting specific genes, not only to analyse possible alterations in virulence of OV, but also to express foreign DNA.

Acknowledgements The excellent technical assistance of Angelika Braun and the help in automatic DNA sequencing of Karl-Heinz Adam is gratefully acknowledged. We thank Dr. Gisela Du¨hrsen (Schleswig-Holstein, FRG) for providing scab material. This study was supported by BAYER AG, FRG. The D1701 sequence presented in this manuscript has been deposited to the GenBank under the accession number AF056304.

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