Comparative Sequence Analysis of the South African Vaccine Strain and Two Virulent ﬁeld Isolates of Lumpy Skin Disease Virus

Arch Virol (2003) 148: 1335–1356 DOI 10.1007/s00705-003-0102-0

Comparative sequence analysis of the South African vaccine strain and two virulent ﬁeld isolates of Lumpy skin disease virus

P. D. Kara1, C. L. Afonso2, D. B. Wallace1, G. F. Kutish2, C. Abolnik1, Z. Lu2, F. T. Vreede3, L. C. F. Taljaard1, A. Zsak2, G. J. Viljoen1, and D. L. Rock2 1Biotechnology Division, Onderstepoort Veterinary Institute, Onderstepoort, South Africa 2Plum Island Animal Disease Centre, Agricultural Research Service, U.S. Department of Agriculture, Greenport, New York, U.S.A. 3IGBMC, Illkirch, France

Received November 25, 2002; accepted February 17, 2003 Published online May 5, 2003 c Springer-Verlag 2003

Summary.The genomic sequences of 3 strains of Lumpy skin disease virus (LSDV) (Neethling type) were compared to determine molecular differences, viz. the South African vaccine strain (LW), a virulent field-strain from a recent outbreak in South Africa (LD), and the virulent Kenyan 2490 strain (LK). A comparison between the virulent field isolates indicates that in 29 of the 156 putative genes, only 38 encoded amino acid differences were found, mostly in the variable terminal regions. When the attenuated vaccine strain (LW) was compared with field isolate LD, a total of 438 amino acid substitutions were observed. These were also mainly in the terminal regions, but with notably more frameshifts leading to truncated ORFs as well as deletions and insertions. These modified ORFs encode proteins involved in the regulation of host immune responses, gene expression, DNA repair, host-range specificity and proteins with unassigned functions. We suggest that these differences could lead to restricted immuno-evasive mechanisms and virulence factors present in attenuated LSDV strains. Further studies to determine the functions of the relevant encoded gene products will hopefully confirm this assumption. The molecular design of an improved LSDV vaccine is likely to be based on the strategic manipulation of such genes.

Introduction Lumpy skin disease (LSD) is an important infectious disease of cattle, probably insect-borne and occurring epidemically or sporadically in southern and eastern 1336 P. D. Kara et al.

Africa and more recently in northern Africa and the Middle East [21, 31, 68]. The etiological agent is a capripoxvirus related to sheeppox and goatpox viruses. Vac- cination with an attenuated strain of Lumpy skin disease virus (LSDV) is presently the only viable means of control in endemic areas such as South Africa. The South African vaccine was developed by passage of a field isolate in tissue culture and on the chorio-allantoic membranes of embryonated chicken eggs [70]. Although the vaccine has proven safe and effective [69] apparent “vaccine breakdown” was observed during the 1990–1991 LSD outbreak [31]. Further investigations however, failed to prove that actual vaccine breakdown had occurred (H.Aitchison, unpublished data, 1997). A possible explanation could be improper vaccination programs. Other limitations include loss in milk production post-vaccination and appearance of local reactions lasting up to a month in animals of show quality or of high value [15]. The molecular characterisation of the virus is an important step in the process of developing more effective diagnostic reagents and vaccines, as well as in the understanding of mechanisms of viral pathogenesis and epidemiology. A suitably tailored vaccine could also allow for the rapid determination of vaccine status and virus presence in cattle during LSD outbreaks. This would be of considerable importance in the timely control of disease outbreaks. We have cloned and sequenced the genomes of both the attenuated South African vaccine strain of LSDV (LW) and a virulent field isolate from a recent outbreak in the Warmbaths region of South Africa (LD) to define genomic differences between these two viral strains. The virulent South African isolate was also compared to the previously sequenced virulent Kenyan 2490 strain (LK) [63].

Materials and methods LSDV DNA isolation, cloning, sequencing and sequence analysis The LSDV Neethling strain 2490 (LK) was originally isolated in Kenya in 1958, passed 16 times in lamb testes (LT) cells, and was subsequently reisolated in 1987 from lesions of an experimentally infected cow [63]. The South African LSDV (type SA-Neethling) vaccine strain was developed from a field isolate by 61 serial passages in monolayers of lamb kidney tissue cultures followed by 20 passages in the chorio-allantoic membranes (CAMs) of embryonated chicken eggs. It was then passaged 3 times in lamb kidney cell monolayers. At this stage the virus was shown to be sufficiently attenuated for use in cattle as a vaccine. The virus then underwent a further 10 passages in Madin-Darby bovine kidney (MDBK) cells and 5 passages in foetal bovine testes (FBT) cells [65]. The LSDV Neethling Warmbaths isolate (LD) was extracted from a lesion on a severely infected calf. The virus was passaged twice in MDBK cells and twice in lamb foetal testes (LFT) cells. Purification of LSDV from cell cultures was based primarily on the method described by Esposito et al. [20]. Viral DNA was obtained according to standard DNA purification procedures as described by Sambrook et al. [48]. Random DNA fragments were generated by incomplete enzymatic digestion of the LW and LD LSDV strains with Tsp509 I endonuclease (New England Biolabs, Beverly, MA) and DNA fragments of 1 to 6 kbp were cloned and used in dideoxy sequencing reactions as previously described [2, 63]. Reaction products were run on an Applied Biosystems PRISM 3700 automated DNA sequencer (PE Biosystems, Foster City, CA). Sequencing data was assembled, and gaps were closed as Comparative sequence analysis of LSDV 1337 ) ‡ N, I, K T T F N N M, M V G continued ( V L 129 48 48 157 163 135 129 123 6 70 144 122 136 79 functional domains I I changes changes in ∗∗ (LW) B15-like protein 2 D Ring finger domain 1 D Zinc finger domain 1 D family Phospholipase D domain 0 Double-stranded RNAbinding motif domain 0 Transmembrane segment 2 I # protein protein membrane protein, A apoptosis regulator virion envelope protein inhibitor and the South African LSDV Neethling vaccine strain Position Predicted structure SMART analysis (LD) † Position LW Conservative amino acid differences occurring in functional domains between the South African LSDV Neethling Warmbaths isolate ∗ Table 1. LD neworfs (length, codons) new (length, codons) and/or function orfs Functional domain No. of aa Type of aa LD001 717-241(159) LW001 592-116(159) Hypothetical protein Signal peptide 0 LD028 21986-20877(370) LW028 21864-20755(370) Putative palmitylated Phospholipase D domain 1 A LD034 27926-27396(177) LW034 27800-27270(177) Putative PKR Z-DNA-binding domain 1 S LD067 57404-57994(197) LW067 57273-57863(197) Putative host range Pox C7 F8A domain 1 R LD010 6933-6448(162) LW010 6810-6325(162) LAP/PHD-finger Transmembrane segment 1 I LD017 11547-11020(176) LW017 11437-10910(176) Putative integral Transmembrane segment 2 V LD024 16677-16030(216) LW024 16552-15905(216) Hypothetical protein L1L/F9/C19 poxvirus orf 1 L LD021 15115-14858(86) LW021 14999-14742(86) Hypothetical protein Signal peptide 1 F 1338 P. D. Kara et al. ‡ S, K T D, F L A N, Q 93 309 54 231 258 121 191 162 607 functional domains L changes changes in ∗∗ T domain 2 E (DUF230 domain) 2 A domain mut protein F Poxvirus protein ofunknown function R complex with S-Adenosylhomocysteine Transmembrane segmentTransmembrane segment 0 0 VV Protein Vp39 in 1 A # ) continued ( S T motif small subunit,PAP regulatory subunit membrane protein protein glycosylasemut domain core protein P4b Poxvirus P4b major core 2 D Table 1 Position Predicted structure SMART analysis † Position LW ∗ LD neworfs (length, codons) newLD068 58056-59054(333) (length, codons) LW068 57923-58921(333) and/or function orfs Poly(A) polymerase Poly A polymerase 0 Functional domain No. of aa Type of aa LD108 101316-100186(377) LW108 101139-100009(377) Putative myristylated Transmembrane segment 0 LD086 79895-80533(213) LW086 79767-80396(210) LD102 97585-98535(317) LW102 97408-98358(317) Hypothetical Coiled coil domain 1 T LD094 89214-87232(661) LW094 89086-87104(661) Putative virion Signal peptide 0 LD082 74375-75028(218) LW082 74247-74900(218) Uracil DNA Uracil-DNA glycosylase 1 R Comparative sequence analysis of LSDV 1339 N D, R, K N, S, D L M I I L V T V 187 167 139 208 129 22 19 20 56 182 169 10 133 102 144 E I inhibitors (SERPIN) N Serine proteinase 2 K B15-like protein 2 D Transmembrane segment 1 V Transmembrane segment 1 V Ankyrin repeats domainAnkyrin repeats domain 0 1 I Ankyrin repeats domainAnkyrin repeats domain 0 2 N Ankyrin repeats domain 0 RK 1 protein glycoprotein host range protein, N SMART (Simple Modular Architecture Research Tool) allows for the identification and annotation of genetically mobile domains and the Function was deduced either from the degree of similarity to known genes or from the presence of Prosite signatures LD, South African LSDV Neethling Warmbaths isolate LW, South African LSDV Neethling vaccineaa, strain amino acids ∗ † ‡ # ∗∗ analysis of domain architecture LD113 103932-103588(115) LW113 103761-103411(117) Hypothetical protein Transmembrane segment 1 V LD140 132575-133294(240) LW140 132374-133093(240) Putative RING finger Ring finger domain 2 A LD146 139263-140501(413) LW146 139120-140355(412) Phospholipase D-like Phospholipase D domain 1LD153 I 148293-148565(91) LW153 148136-148408(91) Hypothetical protein Signal peptide 0 LD118 111264-110845(140) LW118 111095-110676(140) Hypothetical protein Transmembrane segment 1 I LD148 142115-143455(447) LW148 141962-143302(447) Ankyrin repeat protein Phospholipase D domain 0 LD132 119792-120319(176) LW132 119593-120123(177) Hypothetical protein Signal peptide 0 LD149 143479-144489(337) LW149 143327-144337(337) Serpin-like protein Signal peptide 0 LD122 113444-114031(196) LW122 113275-113862(196) Putative EEV Transmembrane segment 1 A LD156 150076-150552(159) LW156 149918-150394(159) Hypothetical protein Signal peptide 0 1340 P. D. Kara et al. , D, V 75 39 A ,D N, K, N T 211 V V 66 S 134 36 , 25 25 ; ; I, E I ,Q ,K 111 changes in 24 24 ; 7V D E, I , I ‡ 180 125 ,I ,I T I, 11 T 41 34 I N 112 64 91 ,I ,V ,V ,S V V ,I , 328 127 I 268 ; ; 216 I A S ; ; 24 24 S K, G R, T E A ; ; Y L L T Y V, K 123 324 186 32 40 45 66 201 125 23 23 25 264 13 61 217 16 87 113 88 179 S I I K changes functional domains ) LW ∗∗ Complex of interferon - 17 N Immunoglobulin domain 1 S Immunoglobulin-like 1 V domain Immunoglobulin domain 1 D family) M Immunoglobulin domain 2 S (overlapping with transmembrane segment) Interleukin-10 domain 0 gamma receptor domain N Transmembrane segment 2 A Ankyrin repeats 2 F Ankyrin repeatsAnkyrin repeats 0 1 M # protein Ankyrin repeats 0 gamma receptor like protein Immunoglobulin domain 2 K protein receptor-like protein receptor (rhodopsin T ) and the South African LSDV Neethling vaccine strain ( LD Position Predicted structure SMART analysis isolate ( † Non-conservative amino acid differences occurring in functional domains between the South African LSDV Neethling Warmbaths Position LW ∗ Table 2. orfsLD005 2450-2959(170) LW005 2324-2836(171) Interleukin-10-like protein orfs Signal peptideLD008 5668-4844(275) LW008 2 5544-4720(275) Putative soluble Interferon A Signal peptideLD011 8111-6981(377) LW011LD012 7999-6857(381) 8853-8221(211) 1 CC chemokine LW012 8742-8110(211) S Ankyrin Functional repeat domain 7 Transmembrane No. of aa Ankyrin repeats Type of aa 3 M 0 LD new (length, codons) new (length, codons) and/or function LD013 9918-8935(328) LW013 9809-8787(341) Interleukin-1 receptor- Transmembrane segment 1 C LD006 3668-2976(231) LW006 3544-2855(230) Interleukin-1 receptor-like Signal peptide 1 F Comparative sequence analysis of LSDV 1341 , ) D, P V L R D G, 659 374 250 56 135 128 ; V, 100 ; ; continued 57 89 S ( ; ,K N, T 136 D, N D, D 101 A L 531 114 D, E 295 ,N 648 219 L, I V 235 A, ,C 48 100 R, I ,E ,D T ,E 128 107 T 124 ; R E I I, V ; H V F I I 79 48 ; T C I, N ; ; N P, R 320 129 132 75 73 693 107 80 734 171 44 460 112 344 456 604 125 52 83 206 K M /1Y T-like domain 1 K subunit domain RNA polymerase A/beta (DEXDc3) type-B family domain V terminal domain (HELICc3) protein family G Immunoglobulin domain 5 V mut Transmembrane segment 3 T Transmembrane segmentTransmembrane segment 0 Transmembrane segment 0 Transmembrane segment 0 0 A T motif, putative gene small subunit domain subunit RPO147 subunit domain expression regulator small subunit; VITF enzyme, small subunit large subunit large subunit K transcriptionalelongation factor superfamily domain Helicase superfamily c- 1 L mut subunit RPO132 subunit domain LD018LD020 12026-11589(146) 14814-13852(321) LW018 LW020 11919-11482(146) 14699-13737(321) dUTPase Ribonucleotide reductase, ribonucleotide reductase 3 S dUTPase domain 1 G LD039 35345-32316(1010) LW039 35220-32191(1010) DNA polymerase DNA polymerase 4 T LD071 60022-63876(1285) LW071 59889-63743(1285) RNA polymerase RNA polymerase alpha 1 M LD035 28592-29797(402) LW035 28541-29671(377) Hypothetical protein E5R Poxvirus 3 Q LD089 84100-83240(287)LD110 101937-103376(480) LW089 LW110 83972-83112(287) 101760-103199(480) mRNA capping Putative enzyme, DNA helicase Poxvirus mRNA capping DEAD-like helicases 2 V 3 S LD087 80536-81294(253) LW087 80409-81008(200) LD042 38096-36045(684) LW042 37971-35920(684) Hypothetical protein Transmembrane segment 1 T LD079 70682-73207(842) LW079 70554-73079(842) mRNA capping enzyme, mRNA capping enzyme, 4 I LD128 118433-117531(301) LW128 118233-117334(300) CD47-like protein Signal peptide 0 LD116 106911-110378(1156) LW116 106741-110208(1156) RNA polymerase RNA polymerase beta 2 A 1342 P. D. Kara et al. I, , , 92 , ; , M 93 M , T P Y 86 S A Q Y ; T K 101 E, 344 108 87 272 ; E, L , 514 267 ; 103 156 99 ; ,I 94 ; 100 91 ; I ,S ,E 102 ; 109 S 95 104 97 I K, S N, T 100 T, R 92 ; V changes in I, Q ,G ,L 85 ,S 312 98 ,N ‡ ; T , ,K 446 196 ,D 129 93 263 N L 86 24 E ,L L G Q W ,A ,V F, S 100 104 R, K N, N 102 Y, G Y ; ; L N 93 88 90 98 96 L S, S ; ; ; ; ; K K ; L, N N 89 91 94 99 347 14 134 346 124 97 101 105 38 65 84 275 43 172 103 213 G G I D N Y Y Y changes functional domains ∗∗ protein domain (CCP) Complement controlprotein domain (CCP) 0 Complement control 1 E type domain Immunoglobulin domain 1 R Immunoglobulin C-2 1 I Immunoglobulin domain 3 T Transmembrane segment 0 Transmembrane segment 2 G Transmembrane segment 1 F ) continued ( # Table 2 protein protein binding protein like protein dismutase domain (SODC) H Position Predicted structure SMART analysis † Position LW ∗ LD neworfs (length, codons)LD131 new 119272-119754(161) LW131 (length, 119076-119399(108) codons) Superoxide dismutase- and/or function Copper/zinc superoxide orfs 14LD133 120352-122028(559) LW133 F 120156-121832(559) DNA ligase-like protein DNA ligase domainLD138 131027-131584(186) 6 LW138 130827-131384(186) OX-2-like proteinLD139 131626-132540(305) LW139 S 131426-132340(305) Putative Ser/Thr protein kinase Protein kinase domain Immunoglobulin domain 3 3 Functional domain S C No. of aa Type of aa LD143 134463-135368(302) LW143 134258-135163(302) Tyrosine protein kinase-like Protein kinase domain 3 K LD141 133346-134017(224) LW141 133145-133819(225) Putative EEV host range Signal peptide 0 LD135 128334-129413(360) LW135 128133-129212(360) Putative IFN-alpha/beta Signal peptide 0 Comparative sequence analysis of LSDV 1343 D, A I, 205 D, 440 269 ; ,N 341 E, I, L 442 R V V, E 349 264 202 N A, S 139 340 55 420 V, V N, D N, S S I, I H A V G S G, I P ; I, I G 261 196 273 342 336 416 47 81 133 353 167 210 422 121 309 H N T Y Kelch domainKelch domain 0 2 T Kelch domain 0 Ankyrin repeats domain 4 M Ankyrin repeats domain 1 S Ankyrin repeats domain 1 V Ankyrin repeats domainAnkyrin repeats domainAnkyrin 0 repeats domain 1 1 A E Ankyrin repeats domainAnkyrin repeats domain 0 4 G Ankyrin repeats domain 6 V Ankyrin repeats domainAnkyrin repeats domainAnkyrin 0 repeats domain 0 0 SMART (Simple Modular Architecture Research Tool) allows for the identification and annotation of genetically mobile domains and the Non-conserved amino acid substitutions using the PAM 250 matrix (27) where 0.4 is the cut-off value Function was deduced either from the degree of similarity to known genes or from the presence of Prosite signatures LD, South African LSDV Neethling Warmbaths isolate LW, South African LSDV Neethling vaccineaa, strain amino acids ∗ † ‡ # ∗∗ The substituted amino acid position between the LD and LW genomes is indicated as eg. N71K. In the instance where an insertion, deletion analysis of domain architecture LD145 137230-139131(634) LW145 137011-138918(636) Ankyrin repeat protein Ankyrin repeats domain 2 S or frameshift occurs, the aa position is indicated as eg. T374;375K. Position 374 in LD and position 375 in LW LD151 145058-146710(551) LW151 144907-146553(549) Kelch-like protein BTB domain 1 R LD152 146779-148245(489) LW152 146622-148088(489) Ankyrin repeat protein Ankyrin repeats domain 2 V 1344 P. D. Kara et al. ‡ K K, V K S F K S S 71 196 225 134 82 107 113 130 83 functional domains changes changes in ) LD ∗∗ T-like domain 1 E mut (DEXDc3) Ankyrin repeats domainAnkyrin repeats domain 0 0 Ankyrin repeats domain 1 S # T motif, protein Ankyrin repeats domain 1mut N proteinproteinputative gene expression protein family regulator Transmembrane segmentenzyme, small 1subunit; VITF enzyme, small subunit L kinase-like N protein helicasetranscriptional superfamily domain helicase ) and the South African LSDV Neethling Warmbaths isolate ( Position Predicted structure SMART analysis LK ‡ Strain 2490 ( Conservative and non-conservative amino acid differences occurring in functional domains between the Kenyan LSDV Neethling Position LW ∗ Table 3. orfsLK012 8860-8228(211) LD012 8853-8221(211) orfs Ankyrin repeatLK087 80536-81294(253) Ankyrin repeats domain LD087 0 80536-81294(253) Functional domain No. of aa Type of aa LD new (length, codons) new (length, codons) and/or function LK035 28590-29795(402) LD035 28592-29797(402) Hypothetical E5R poxvirus 1 F LK143 134456-135361(302) LD143 134463-135368(302) Tyrosine protein Protein kinase domain 2 R LK061 53928-54203(92) LD061 53929-54204(92)LK089 Hypothetical 84100-83240(287)LK110 LD089 Transmembrane 101937-103376(480) 84100-83240(287) segment LD110 0 mRNA 101937-103376(480) capping Putative DNA Poxvirus mRNA capping 1 DEAD-like A 1 P Comparative sequence analysis of LSDV 1345 K 375 F , I 55 316 374 Serine proteinase 1 L domain domain domain domain domain domain Kelch domainKelch domain 0 0 inhibitors (SERPIN) Kelch domain 1 T Ankyrin repeatsAnkyrin repeats 0 Ankyrin repeats 0 Ankyrin repeats 0 Ankyrin repeats 0 Ankyrin repeats 0 0 K. In the instance where an insertion, deletion 71 protein domain K. Position 374 in LK and postion 375 in LD 375 ; 374 SMART (Simple Modular Architecture Research Tool) allows for the identification and annotation of genetically mobile domains and the Function was deduced either from the degree of similarity to known genes or from the presence of Prosite signatures LK, Kenyan LSDV Neethling Strain 2490 LD, South African LSDV Neethling Warmbathsaa, isolate amino acids Amino acid substitutions that are non-conservative using the PAM 250 matrix (Gribskov et al., 1986) where 0,4 is the cut-off ∗ † ‡ # ∗∗ The amino acid (aa) position is indicated, occurring between the LK and LD genomes eg. N analysis of domain architecture LK149 143465-144475(337) LD149 143479-144489(337) Serpin-like protein Signal peptide 0 LK152 146764-148230(489) LD152 146779-148245(489) Ankyrin repeat Ankyrin repeats 1 N or frameshift occurs, the aa position is indicated as T LK151 145045-146694(550) LD151 145058-146710(551) Kelch-like protein BTB domain 0 1346 P. D. Kara et al. described previously [1, 63]. The final DNA consensus sequences for the LD and LW genome represented on average a 6- and 9-fold redundancy at each base position respectively. Genome DNA composition, structure and restriction enzyme patterns were analysed as previously described using the GCG v.10 software package [1, 18, 63]. Open reading frames (ORFs) longer than 30 amino acids with a methionine start codon were evaluated for coding potential as previously described [1, 2, 63]. All potential gene-encoding ORFs and ORFs greater than 55 codons were subjected to homology searches as previously described [1, 2, 63]. Based on these criteria, 156 ORFs were annotated as potential genes. Gene families were analysed and annotated as previously described [1, 2, 63]. SMART(Simple Modular Architecture Research Tool) was used for the identification and analysis of domain architecture (http://coot.embl-heidelberg.de/SMART) [50, 51]. Once domains were identified in an ORF, the region was aligned with the lalign programme (http://ch.embnet.org/cgi-cin/LALIGN) to identify polymorphisms. The PAM250 matrix [28] was used to identify whether amino acid changes occurring within the functional domains as identified by SMART (Table 1, Table 2, Table 3) were conservative or non-conservative using a cut-off value of 0.4.

Nucleotide sequence accession number The LD and LW genome sequences have been deposited in GenBank under accession numbers AF409137 and AF409138 respectively.

Results The genome sequences of the South African LSDV Neethling vaccine strain (LW) and the virulent South African LSDV Neethling Warmbaths isolate (LD) were sequenced. Sequence data from the LD and LW genomes were then assembled into contiguous sequences of 150,793 base pairs (bp) and 150,509 bp respectively. The genome sequence of the virulent LSDV Kenyan Neethling strain 2490, described here as LK, was assembled into a contiguous sequence of 150,773 bp by Tulman et al. [63]. The terminal hairpin loops were not sequenced and therefore the leftmost nucleotide of each assembled genome was arbitrarily designated as base 1. The nucleotide composition of both LD and LW is 74% A + T and is uniformly distributed. LW and LD each contain 156 open reading frames (ORFs) that have been annotated here as putative genes. These genes encode proteins containing 53 to 2,025 amino acids (aa). Major amino acid differences were observed in the comparison between the highly cell-attenuated SA vaccine strain (LW) and the virulent Warmbaths strain (LD). 114 of the 156 genes (73%) common to both viruses were found to contain between 1 to 42 amino acid differences. Although these differences are spread across the genome, the majority occurs in the terminal regions. Twelve genes are affected by insertions of between 1 and 11 aa (30 aa in total), whereas 10 genes are affected by deletions of between 1 and 9 aa (21 aa in total). Nine frameshifts occurred between the LD Warmbaths isolate and the LW vaccine strain. Of the 9 frameshifts, 4 of the frameshifts have resulted in a truncation. The 4 genes involved are ORF019 and ORF144, both kelch-like proteins, ORF026, a hypothetical protein of unknown nature and ORF134, which is similar to vaccinia virus B22R protein. The other ORFs affected by frameshift Comparative sequence analysis of LSDV 1347 mutations are ORF013, an interleukin-1 receptor-like protein truncated by 14 aa at the C-terminus, ORF035, a hypothetical protein of unknown nature truncated by 26 aa at the N-terminus, ORF086, a mutT motif protein truncated by 7 aa at the C-terminus, ORF087, a mutT motif putative gene expression regulator protein truncated by 53 aa at the C-terminus and ORF131, a superoxide dismutase-like protein truncated by 77 aa at the C-terminus. The same number of ORFs (156) are also present in the LSDV Kenyan Neethling strain 2490 (LK) [63]. Only minor amino acid differences were observed in the comparison between the two virulent wild-type strains (LD and LK). 29 (18%) of the 156 genes common to both viruses were found to contain between one to three amino acid differences located mostly in the terminal regions. In addition to these amino acid differences, 6 deletion and 8 insertion sites occurred. A total of 3 frameshifts occured in LD. ORF019, encoding a kelch-like protein and ORF026, encoding a hypothetical protein of unknown nature, were truncated by frameshift mutations in the virulent LD strain. A third open reading frame affected by a frameshift mutation, ORF013, encodes an interleukin-1 receptor-like protein, which is truncated by 14 aa at the c-terminus. Conservative and non-conservative amino acid differences occurring between the vaccine strain (LW) and the South African virulent isolate (LD) are indicated in Table 1 and Table 2 respectively, while differences occurring between the two virulent isolates (LK and LD) are indicated in Table 3. Only signiﬁcant differences occurring in functional domains will be discussed.

Discussion Transcription and mRNA biogenesis Differences have been noted in several of the transcription factors (TF) [early (E), intermediate (I) and late (L)] between LD and LW. Of these, the seemingly most signiﬁcant occurs within ORF110, where a non-conservative amino acid difference has occurred in the DEAD-like helicases superfamily (DEXDc) domain (Table 2). DexH/D proteins are essential to all aspects of cellular RNA metabolism and the replication of many viruses [34]. Their functions include the hydrolyses of nucleoside triphosphates (NTPs) and the unwinding of RNA [34]. An orthologue was identiﬁed in vaccinia virus (VV) (A18R) and this protein too contains conserved motifs belonging to the DEXH family of DNA and RNA helicases [25, 37, 55]. mRNA capping occurs by a series of three sequential enzymatic reactions in which the 5 triphosphate-terminated primary transcript is converted to a diphosphate-terminated RNA by RNA triphosphate, capped with GMP by RNA guanylyl transferase and than methylated by RNA (guanine-7-) methyltransferase [17, 29, 43]. In VV, the three steps in cap formation are catalysed by a het- erodimeric protein encoded by VV D1 and D12 genes [17]. ORF079 and ORF089 are orthologues of VV D1R and D12L respectively. Non- conservative amino acid differences occur between both mRNA capping enzyme subunits between LD and 1348 P. D. Kara et al.

LW (Table 2). Five sequence motifs within the VV D1 protein are arranged in the same order and with similar spacing in LSDV (ORF079) as well as in the capping enzymes of other DNA viruses [17, 71]. The capping enzyme plays a pivotal role in regulating viral gene expression at both the mRNA transcription and processing level [29, 43]. The non-conservative amino acid differences that do occur in both ORF079 and ORF089 are outside motifs I to V and may therefore not have an effect on the protein. ORF086 and ORF087 contain mutT-like motifs and are similar to VV D9R and to VV D10R, which is a negative regulator of viral transcription [36, 52]. Schors et al. [52] indicates that the D10R protein might bind to or hydrolyse cap structures thereby affecting the stability and translatability of mRNAs.Amino acid differences occur in both ORF086 and ORF087 between LD and LW, but only in ORF087 between LK and LD. C-terminal frameshifts in ORF086 and ORF087 lead to the absence of the last 5 and last 29 respective encoded amino acids of the mutT-like domain. A non-conservative change occurs in ORF087 between the vaccine and wild-type as well as the wild-types (Table 2, Table 3). The MutT proteins or Nudix hydrolases are a family of versatile, and widely distributed “house-cleaning” genes with the function of cleansing the cell of potentially deleterious metabolites and to modulate the accumulation of intermediates in biochemical pathways [11, 36, 52].

Nucleotide metabolism The genomes of LD and LW contain several orthologues of poxviral genes involved in nucleotide metabolism. These include a dUTP pyrophosphatase (ORF018), thymidine kinase (ORF066) and the small subunit of ribonucleotide reductase (RR) (ORF020) [41]. High levels of dUTP have been shown to be lethal to cells. dUTPase removes dUTP from the dNTP pool thereby generating dUMP, and is involved in maintaining ﬁdelity of DNA replication [47]. It is advantageous that LSDV encodes both a dUTPase (ORF018) to reduce the dUTP pool and a uracil DNA glycosylase (ORF082) that acts by excision repair to remove dUTP that has been misincorporated into the replicating DNA [9, 19, 38, 59]. The same complement of nucleotide metabolism genes found in leporipoxviruses are present in LSDV [41]. LSDV contains 7 orthologues of chordopoxvirus genes necessary for, or potentially involved in, DNA replication [63]. ORF039 is an orthologue of VV E9L, a DNA polymerase. A single non-conservative difference between LD and LW occur within the functional domain (Table 2). Poxviral polymerases can promote strand annealing and strand-transfer reactions that form an integral step in most recombination schemes [70]. ORF082 (D4R) of LD and LW encodes a uracil- DNA glycosylase protein (UNG).A single conservative amino acid difference occurred between LD and LW (Table 1). Uracil DNA glycosylases (UDGs) are major repair enzymes protecting DNA from mutational damage caused by uracil incorporation [9, 19, 38]. The uracil DNA glycosylase is essential for virus viability and functions in the initial Comparative sequence analysis of LSDV 1349 phase of the base excision repair pathway of uracil in DNA by cleaving the glycosidic bond, resulting in an apyrimidinic (AP) site [9, 19, 38, 59]. The LSDV uracil DNA glycosylase may therefore function in a similar manner. Ellison et al. [19] showed that mutations at 3 active-site amino acids of vaccinia virus resulted in proteins being defective in uracil excision but still retaining their ability to bind DNA. The 3 active-site amino acids namely Asp68, Asn120 and His181 are conserved between LD and LW. It is therefore unclear what effect the single conservative amino acid difference may have.

Virion structure and assembly Four genes of LSDV potentially encode proteins present in the extracellular enveloped virus (EEV) outer envelope which are involved in or associated with the release of EEV, and affecting virus infectivity, while 2 genes, ORF126 and ORF141, resembling VV EEV proteins are also present [12, 40, 41]. ORF141 is an orthologue of VV B5R. VV B5R contains 4 complement control protein (CCP) domains compared to the 2 CCP domains present in LSDV. The CCP modules contain approximately 60 amino acid residues and have been identiﬁed in several proteins of the complement system. Given the similarity of the domain to complement control proteins, the ORF141 protein may be involved in viral evasion from host immune responses. It is not clear what effect the presence or loss of 2 CCP domains may have on the protein.

Immune evasion functions Viral orthologues of cellular interleukin-10 (IL-10) have been shown to possess im- munosuppressive and immunostimulatory activities [22, 23, 30, 46]. The genomes of LD and LW (ORF005) were found to encode an orthologue of IL-10. Non- conservative amino acid differences between LD and LW occurred in both the amino-terminal signal peptide domain as well as the overlapping transmembrane segment (Table 2). Signal peptides target proteins for secretion in both prokary- otes and eukaryotes, taking part in an array of protein–protein and protein–lipid interactions resulting in initiation of protein translocation through a proteinaceous channel in the endoplasmic reticulum (ER) of eukaryotic cells [33, 67]. Changes within the signal peptide domain can affect protein secretion, while mutations in transmembrane domains can partially or completely disrupt the receptor surface expression and function. ORF135, ORF006 and ORF013 are similar to cellular and viral IL-1R orthologues (IL-1R) but differ from each other [63]. ORF135 encodes a protein with a signal peptide domain as well as 2 immunoglobulin (Ig) domains and an Ig C-2 type domain. Non-conservative differences occur in both the Ig domains (Table 2). The protein encoded by ORF135 is most similar to VV B18R (strain Western reserve [WR]). It is an interferon (IFN) -α/β binding protein which is both soluble and present on the cell surface where it binds to and inhibits the cellular binding and antiviral activity of mammalian α, β, and ω type I IFN [5, 16, 61]. ORF135 likely 1350 P. D. Kara et al. serves a similar function. ORF006 and ORF013, like other poxviral orthologues of VV IL-1 binding protein are most similar to mammalian IL-1R, particularly to type II IL-1R (IL-1R II) [56]. ORF006 encodes a protein with a signal peptide domain as well as 2 Ig domains. Non-conservative differences occur within both the Ig domains (Table 2). ORF013 encodes a protein with a transmembrane segment, 2 Ig domains and an Ig-like domain. Non-conservative differences occur within both the Ig domains (Table 2). The frameshift occurs outside the domains identified. ORF006 encodes a protein similar to the VV (strain Wyeth) B18R protein, (which binds interferon less efficiently than B18R from other VV strains) lacks a third Ig domain in the carboxyl-terminus and may perform a different immunomodulatory function [5]. ORF008 encodes an orthologue of the soluble VV B8R and myxoma virus (MYX) M007 IFN-γ receptors (IFN-γ R), that bind IFN-γ and influence viral virulence [3, 64]. A single as well as 9 non-conservative amino acid differences occurred in the signal peptide as well as in the IFN-γ receptor domain respectively (Table 2). The VV B8R protein is secreted, binds soluble IFN-γ and prevents binding to cellular receptors [3]. Poxviruses are therefore able to inhibit both the antiviral and immune functions of IFN-γ [4]. ORF008 may similarly inhibit the antiviral and immune functions of IFN-γ. Srollerˇ et al. [58] showed in rabbits that VV (Strain Western reserve) lacking the IFN-γ R was attenuated as skin lesions tended to disappear earlier than those caused by the wild-type virus. Verardi et al. [66] found that recombinant VV with a deleted B8R gene are attenuated for normal and nude mice without exhibiting a concomitant reduction in immunogenicity. Deletion of the VV B8R gene thus leads to attenuation of the virus in a mouse model as demonstrated above. Deletion mutants lacking a part of or the entire B8R protein have been studied. It is therefore unclear whether the non-conservative amino acid differences that have occurred have an effect on the attenuation of LSDV. The effects of these differences will have to be studied further. The amino acid differences observed in ORF008 between LD and LW may influence viral virulence while changes within the signal peptide domain may affect protein secretion. ORF011 encodes an orthologue of a CC chemokine receptor [63]. Chemokine receptors are integral membrane proteins that transduce extracellular signals to the intracellular environment through heterotrimeric guanine nucleotide-binding (G) proteins [49]. ORF011 encodes a transmembrane-domain receptor belonging to the rhodopsin family, with 2 of the 3 amino acid substitutions not being conserved between LD and LW (Table 2). ORF138 encodes an orthologue of MYX M141R protein, and is similar to other viral and cellular OX-2-like proteins which are dendritic cell surface antigens involved in co-regulation of T-cell stimulation and development of type 1/type 2 cytokine [14, 26, 27, 63]. Viral OX-2 like proteins may interfere with development of effective antiviral cellular immune responses. Two transmembrane segments as well as an Ig domain occur in ORF138, with non-conservative substitutions in LD and LW occurring in one of the transmembrane segments and the Ig domains (Table 2). Comparative sequence analysis of LSDV 1351

Other virulence and host range and cellular functions Poxviral ankyrin (ANK)-repeat genes encode proteins that have been associated with host range functions in MYX, cowpox virus (CPV), and VV, and proteins which possibly inhibit virally induced apoptosis [24, 32, 42, 44, 57, 60]. The loss of ANK genes may be associated with a narrowing of host range [8, 54]. While the mode of action of these proteins is unknown, ANK-repeat motifs of other proteins are clearly involved in mediating protein–protein interactions [53]. ORF012, ORF145, ORF147, ORF148 and ORF152 encode proteins in LSDV that contain ANK-repeat motifs. SMART analysis identiﬁed 5 to 7 ANK repeats occurring in the above ORFs. One of the ANK-repeats of both ORF012 and ORF152 contain non-conservative amino acid differences between the wild-types (Table 3), while and 1, 2 and 4 of the ANK-repeats of ORF012, ORF145 and ORF152 respectively contain non-conservative amino acid differences between LD and LW (Table 2). ORF131 is an orthologue to the cellular Cu-Zn superoxide dismutase (SODC) gene and the SOD-like genes found in leporipoxviruses and orthopoxviruses [62, 63]. It encodes a protein with a SODC domain within which substitutions occur in LD and LW. Non-conservative changes occur within the SODC domain (Table 2). The C-terminal frameshift in LW results in a truncated protein in which last 54 amino acids are absent. LSDV, like all other poxviruses except Amsacta moorei virus lacks residues in the SOD protein that would predict enzymatic activity [7, 63]. Almaz´an et al. [7] showed that a VV A45R mutant (lacking the majority of the A45R gene) replicated normally in vitro and displayed unaltered virulence in mice and rabbits. However, in a murine intranasal model, infection with this deletion mutant resulted in an earlier onset of disease symptoms, suggesting a possible function in the progression of the infection.

Gene families of unknown function Three ORFs (ORF019, ORF144 and ORF151) encoded by LSDV are similar to the Drosophila kelch protein [13, 63]. All contain a Broad-complex, Tramtrack and Bric-a-Brac (BTB) domain also known as POZ (poxvirus and zinc ﬁnger) domain. The BTB/POZ domain mediates homomeric dimerisation and in some instances heteromeric dimerisation [10]. Although the function of poxviral kelch- like proteins is unknown, they are non-essential for replication of VV in cell culture [39, 45]. Four kelch domains are present in both ORF019 and ORF144 encoded proteins while 3 kelch domains are found in that of ORF151. In LD and LW, ORF151 has a non-conservative amino acid difference in the encoded BTB domain (Table 2). A frameshift occurs in ORF144 of LW, resulting in truncation. The encoded BTB region occurs in one half of the ORF, and the 4 kelch domains occur in the other half. Furthermore, ORF019 of LD as well as LW encodes a protein that is truncated. The BTB domain occurs in the one half with the 4 kelch domains occurring in the other half. 1352 P. D. Kara et al.

Conclusion A comparison between the highly cell-attenuated South African LSDV vaccine strain (LW) and the virulent South African LSDV Warmbaths strain (LD) revealed major differences. 114 of the 156 putative genes (73%) were found to have amino acid differences. Variations were not limited to the terminal regions of the LSDV genome, with many of the affected genes occurring in the central region containing the house-keeping genes such as those involved in viral DNA replication. The impediment of viral DNA replication has been shown to play a key role in the attenuation of a pathogenic virus [35]. Several virulence factors occurring in both field and vaccine isolates have also undergone amino acid changes, as have several proteins, such as the interleukin-like proteins which are secreted and play an important role in suppressing the host immune response to infection, as well as the IFN-γ R which binds IFN-γ and influences viral virulence. A target gene for further studies in vaccine improvement is the IFN-γ R which may be one of the key virulence factors since a VV mutant lacking the IFN-γ R was shown to induce skin lesions in rabbits that tended to disappear earlier than those of the wild-type, while in a mouse model, a VV recombinant with a deleted IFN-γ R was shown to be attenuated. In contrast to the comparison between the vaccine strain (LW) and the virulent South African field isolate (LD), minor differences were observed in the comparison between the virulent Kenyan 2490 (LK) and the South African Warmbaths (LD) strains of LSDV (Neethling type). Despite the geographic distance and time separation between the origins of the isolates (South Africa [LD] and Kenya [LK]), minimal genetic variation was observed thereby suggesting that lumpy skin disease is genetically stable. We conclude that the attenuation observed in the vaccine strain of LSDV (SA-Neethling) is likely to be the sum of the altered phenotypes of the expressed proteins, however it is also likely that a few specific proteins have greater influence. The results of this comparison should at least narrow the search for the most important determinants of LSDV virulence in its bovine host, and consequently aid in the development of improved vaccines.

Acknowledgments We thank theAgricultural Research Council (ARC), OnderstepoortVeterinaryInstitute (OVI), the National Research Foundation (NRF) and Plum Island Animal Disease Centre (PIADC) for ﬁnancial assistance. Onderstepoort Biological Products (OBP) for providing the South African LSDV Neethling vaccine strain.

References 1. Afonso CL, Tulman ER, Lu Z, Oma E, Kutish GF, Rock DL (1999) The genome of Melanoplus sanguinipes entemopoxvirus. J Virol 73: 533–552 2. Afonso CL, Tulman ER, Lu Z, Zsak L, Kutish GF, Rock DL (2000) The genome of fowlpox virus. J Virol 74: 3815–3831 Comparative sequence analysis of LSDV 1353

3. Alcami A, Smith GL (1995) Vaccinia, cowpox, and camlepoxviruses encode soluble gamma interferon receptors with novel broad species specificity. J Virol 69: 4633–4639 4. AlcamiA, Smith GL (1996) Soluble Interferon-γ receptors encoded by poxviruses. Comp Immun Microbiol Infect Dis 19: 305–317 5. Alcami A, Symons JA, Smith GL (2000) The vaccinia virus soluble Alpha/beta interferon (IFN) receptor binds to the cell surface and protects cells from the antiviral effects of IFN. J Virol 74: 11230–11239 6. Alexander RA, Plowright W, Haig DA (1957) Cytopathogenic agents associated with Lumpy-skin Disease of Cattle. Bull Epizootic Dis Afr 5: 489–492 7. Almazán F, Tscharke DC, Smith GL (2001) The vaccinia virus superoxide dismutase-like protein (A45R) is a virion component that is nonessential for virus replication. J Virol 75: 7018–7029 8. Antoine G, Scheiflinger F, Dorner F, Falkner FG (1998) The complete genomic sequence of the modified vaccinia Ankara strain: comparison with other orthopoxviruses. Virology 244: 365–396 9. Aravind L, Koonin EV (2000) The α/β fold uracil DNA glycosylases: a common origin with diverse fates. Genome Biol 1: research 0007.1–0007.8 10. Bardwell VJ, Treisman R (1994) The POZ domain: a conserved protein–protein interaction motif. Genes Devel 8: 1664–1677 11. Bessman MJ, Frick DN, O’Handley SF (1996) The MutT proteins or “nudix” hydrolases, a family of versatile, widely distributed, “housecleaning” enzymes. J Biol Chem 271: 25059–25062 12. Blasco R, Moss B (1991) Extracellular vaccinia virus formation and cell-to-cell virus transmission are prevented by deletion of the gene encoding the 37,000-Dalton outer envelope protein. J Virol 65: 5910–5920 13. Bork P, Doolittle RF (1994) Drosophila kelch motif is derived from a common enzyme fold. J Mol Biol 236: 1277–1282 14. Cameron C, Hota-Mitchell S, Chen L, Barrett J, Cao JX, Macaulay C, Willer D, Evans D, McFadden G (1999) The complete DNA sequence of myxoma virus. Virology 264: 298–318 15. Carn VM (1993) Control of capripox infection. Vaccine 11: 1275–1279 16. Colamonici OR, Domanski P, Sweitzer SM, Larner A, Buller RM (1995) Vaccinia virus B18R gene encodes a type I interferon-binding protein that blocks interferon alpha transmembrane signalling. J Biol Chem 270: 15974–15978 17. Cong P,Shuman S (1995) Mutational analysis of mRNA capping enzyme identifies amino acids involved in GTP binding, enzyme-guanylate formation, and GMP transfer to RNA. Mol Cell Biol 15: 6222–6231 18. Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12: 387–395 19. Ellison KS, Peng W, McFadden G (1996) Mutations in active-site residues of the uracil- DNA glycosylase encoded by vaccinia virus are incompatible with virus viability. J Virol 70: 7965–7973 20. Esposito J, Condit R, Obijeski J (1981) The preparation of Orthopoxvirus DNA. J Virol Methods 2: 175–179 21. Fenner F (1996) Poxviruses. In: Fields BN, Knipe DM, Howley PM (eds), Fields virology third edn., vol 2. Chapter 84. Lippincott-Raven, Philadelphia, PA 22. Fleming SB, McCaughan CA, Andrews AE, Nash AD, Mercer AA (1997) A homolog of interleukin-10 is encoded by the poxvirus orf virus. J Virol 71: 4857–4861 1354 P. D. Kara et al.

23. Gesser B, Leffers H, Jinquan T, Vestergaard C, Kirstein N, Sindet-Pedersen S, Jensen SL, Thestrup-Pedersen K, Larsen CG (1997) Identification of functional domains on human interleukin 10. Proc Natl Acad Sci USA 94: 14620–14625 24. Gillard S, Spehner D, Drillien R, Kirn A (1986) Localization and sequence of a vaccinia virus gene required for multiplication in human cells. Proc Natl Acad Sci USA 83: 5573–5577 25. GorbalenyaAE, Koonin EV,DonchenkoAP,BlinovVM (1989) Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res 17: 4713–4730 26. Gorczynski L, Chen Z, Hu J, Kai Y, Lei J, Ramakrishna V, Gorczynski RM (1999) Evidence that an OX-2-positive cell can inhibit the stimulation of type I cytokine production by bone marrow-derived B7-1 (and B7-2)-positive dendritic cells. J Immunol 162: 774–781 27. Gorczynski RM, Chen Z, Clark DA, Hu J, Yu G, Li X, Tsang W, Hadidi S (2000) Regulation of gene expression of murine MD-1 regulates subsequent T cell activation and cytokine production. J Immunol 165: 1925–1932 28. Gribskov M, Burgess RR (1986) Sigma factors from E. coli, B. subtilis, phage SP01, and phage T4 are homologous proteins. Nucleic Acids Res 14: 6745–6763 29. Higman MA, Bourgeois N, Niles EG (1992) The vaccinia virus mRNA (Guanine-N7-) – methyltransferase requires both subunits of the mRNA capping enzyme for activity. J Biol Chem 267: 16430–16437 30. Hsu DH, de Waal Malefyt R, Fiorentino DF, Dang MN, Vieira P, de Vries J, Spits H, Mosmann TR, Moore KW (1990) Expression of interleukin-10 activity by Epatein-Barr virus protein BCRFI. Science 250: 830–832 31. Hunter P,Wallace D (2001) Lumpy skin disease in southernAfrica: a review of the disease and aspects of control. J S Afr Vet Assoc 72: 68–71 32. Ink BS, Gilbert CS, Evan GI (1995) Delay of vaccinia virus-induced apoptosis in non- permissive Chinese hamster ovary cells by the cowpoxvirus CHOhr and Adenovirus E1B 19K genes. J Virol 69: 661–668 33. Jain RG, Rusch SL, Kendall DA (1994) Signal peptide cleavage regions. J Biol Chem 269: 16305–16310 34. Jankowsky E, Gross CH, Shuman S, PyleAM (2001)Active disruption of an RNA-protein interaction by a DexH/D RNA helicase. Science 291: 121–124 35. Kiyotani K, Sakaguchi T, Fujii Y, Yoshida T (2001) Attenuation of a field Sendai virus isolate through egg-passages is associated with an impediment of viral genome replication in mouse respiratory cells. Arch Virol 146: 893–908 36. Koonin EV (1993) A highly conserved sequence motif defining the family of MutT- related proteins from eubacteria, eukaryotes and viruses. Nucleic Acids Res 21: 4847 37. Koonin EV,Senkevich TG (1992) Vaccinia virus encodes four putative DNA and/or RNA helicases distantly related to each other. J Gen Virol 73: 989–993 38. Krokan HE, Standal R, Slupphaug G (1997) DNA glycosylases in the base excision repair of DNA. Biochem J 325: 1–16 39. Kotwal GJ, Moss B (1988) Analysis of a large cluster of nonessential genes deleted from a vaccinia virus terminal transposition mutant. Virology 167: 524–537 40. McIntosh AAG, Smith GL (1996) Vaccinia virus glycoprotein A34R is required for infectivity of extracellular enveloped virus. J Virol 70: 272–281 41. Moss B (1996) Poxviridae: The viruses and their replication. In: Fields BN, Knipe DM, Howley PM (eds), Fields virology, vol 2. Chapter 83, p 2637–2671. Lippincott-Raven, Philadelphia, PA Comparative sequence analysis of LSDV 1355

42. Mossman K, Lee SF, Barry M, Boshkov L, McFadden G (1996) Disruption of MT-5, a novel myxoma virus gene member of poxvirus host range superfamily, results in dramatic attenuation of myxomatosis in infected European rabbits. J Virol 70: 4394–4410 43. Myette JR, Niles EG (1996) Domain structure of the vaccinia virus mRNA capping enzyme. J Biol Chem 271: 11936–11944 44. Perkus ME, Goebel SJ, Davis SW, Johnson GP, Limbach K, Norton EK, Paoletti E (1990) Vaccinia virus host range genes. Virology 179: 276–286 45. Perkus ME, Goebel SJ, Davis SW, Johnson GP, Norton EK, Paoletti E (1991) Deletion of 55 open reading frames from the termini of vaccinia virus. Virology 180: 406–410 46. Rode HJ, Janssen W, Rosen-Wolff A, Bugert JJ, Thein P, Becker Y, Darai G (1993) The genome of equine herpesvirus type 2 habors an interleukin 10 (IL-10)-like gene. Virus Genes 7: 111–116 47. Roseman NA, Evans RK, Mayer EL, Rossi MA, Slabaugh MB (1996) Purification and characterisation of the vaccinia virus deoxyuridine triphosphate expressed in Escherichia coli J Biol Chem 271: 23506–23511 48. Sambrook J, Fritcsh EF, Maniatis T (eds) (1989) Molecular Cloning – A Laboratory Manual, 2nd edn. Cold Spring Harbour Laboratory Press 49. Schoneberg T, Schultz G, Gudermann T (1999) Structural basis of G protein-coupled receptor function. Mol Cell Endocrinol 151: 181–193 50. Schultz J, Milpetz F, Bork P, Ponting CP (1998) SMART, a simple modular architecture research tool: Identification of signalling domains. Proc Natl Acad Sci USA 95: 5857–5864 51. Schultz J, Copley RR, Doerks T, Ponting CP, Bork P (2000) SMART. A web-based tool for the study of genetically mobile domains. Nucleic Acids Res 28: 231–234 52. Schors T, Keck JG, Moss B (1999) Down regulation of gene expression by the vaccinia virus D10 protein. J Virol 73: 791–796 53. Sedgwick SG, Smerdon SJ (1999) The ankyrin repeat: a diversity of interactions on a common structural framework. Trends Biochem Sci 24: 311–316 54. Shchelkunov SN, Safronov PF, Totmenin AV, Petrov NA, Ryazankina OI, Gutorov VV, Kotwal GJ (1998) The genome sequence analysis of the left and right species-specific terminal region of a cowpox virus strain reveals unique sequences and a cluster of intact ORFs for immunomodulatory and host range proteins. Virology 243: 432–460 55. Simpson DA, Condit RC (1995) Vaccinia virus gene A18R encodes an essential DNA helicase. J Virol 69: 6131–6139 56. Smith GL, Chan YS (1991) Two vaccinia virus proteins structurally related to the interleukin-1 receptor and the immunoglobin superfamily. J Gen Virol 72: 511–518 57. Spehner D, Gillard S, Drillien R, Kirn A (1988) A cowpox virus gene required for multiplication in Chinese hamster ovary cells. J Virol 62: 1297–1304 58. Srollerˇ V, Ludvíková V, Mareˇsová L, Hainz P, N˘emeˇcková Sˇ (2001) Effect of IFN-γ receptor gene deletion on vaccinia virus virulence. Arch Virol 146: 239–249 59. Stuart DT, Upton C, Higman MA, Niles EG, McFadden G (1993) A poxvirus-encoded Uracil DNA Glycosylase is essential for virus viability. J Virol 67: 2503–2512 60. Sutter G, Ramsey-Ewing A, Rosales R, Moss B (1994) Stable expression of the vaccina virus K1L gene in rabbit cells complements the host range defect of a vaccinia virus mutant. J Virol 68: 4109–4116 61. Symons JA,AlcamiA, Smith GL (1995)Vacciniavirus encodes a soluble type I interferon receptor of novel structure and broad species specificity. Cell 81: 551–560 62. Tomalski MD, Eldridge R, Miller LK (1991) A baculovirus homolog of a Cu/Zn superoxide dismutase gene. Virology 184: 149–161 1356 P. D. Kara et al.: Comparative sequence analysis of LSDV

63. Tulman ER, Afonso CL, Lu Z, Zsak L, Kutish GF, Rock DL (2001) Genome of lumpy skin disease virus. J Virol 75: 7122–7130 64. Upton C, Mossman K, McFadden G (1992) Encoding of a homolog of the IFN-gamma receptor by myxoma virus. Science 258: 1369–1372 65. Van Rooyen PJ, Munz EK,Weiss KE (1969) The optimial conditions for the multiplication of Neethling-Type Lumpy Skin Disease Virus in embryonated eggs. Onderstepoort J Vet Res 36: 165–174 66. Verardi PH, Jones LA, Aziz FH, Ahmad S, Yilma TD (2001) Vaccinia virus vectors with an inactivated gamma interferon receptor homolog gene (B8R) are attenuated in vivo without a concomitant reduction in immunogenicity. J Virol 75: 11–18 67. Von Heijne G (1998) Life and death of a signal peptide. Nature 396: 111–113 68. Weiss KE (1963) Lumpy skin disease. Emerging diseases of animals. FAO Agricultural Studies 61: 179–201 69. Weiss KE (1968) Lumpy skin disease. Virology monographs, Vol 3. Springer Wien New York 70. Willer DO, Mann MJ, Zhang W, Evans DH (1999) Vaccinia virus DNA polymerase promotes DNA pairing and strand-transfer reactions. Virology 257: 511–523 71. Yu L, Shuman S (1996) Mutational analysis of the RNA triphosphatase component of vaccinia virus mRNA capping enzyme. J Virol 70: 6162–6168 Author’s address: Gerrit J. Viljoen, Biotechnology Division, Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort, 0110, South Africa; e-mail: [email protected]