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

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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 differ- ences 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 Table 1. Conservative amino acid differences occurring in functional domains between the South African LSDV Neethling Warmbaths isolate (LD) and the South African LSDV Neethling vaccine strain (LW) LD∗ Position LW† Position Predicted structure SMART analysis∗∗ new (length, codons) new (length, codons) and/or function# orfs orfs Functional domain No. of aa Type of aa‡ changes changes in functional domains Comparative sequence analysis of LSDV 1337 LD001 717-241(159) LW001 592-116(159) Hypothetical protein Signal peptide 0 B15-like protein 2 D129N, I144M LD010 6933-6448(162) LW010 6810-6325(162) LAP/PHD-finger Transmembrane segment 1 I79V protein Transmembrane segment 2 I122M, I136V Ring finger domain 1 D48N Zinc finger domain 1 D48N LD017 11547-11020(176) LW017 11437-10910(176) Putative integral Transmembrane segment 2 V157I, membrane protein, A163T apoptosis regulator LD021 15115-14858(86) LW021 14999-14742(86) Hypothetical protein Signal peptide 1 F6L LD024 16677-16030(216) LW024 16552-15905(216) Hypothetical protein L1L/F9/C19 poxvirus orf 1 L123F family LD028 21986-20877(370) LW028 21864-20755(370) Putative palmitylated Phospholipase D domain 1 A135T virion envelope protein Phospholipase D domain 0 LD034 27926-27396(177) LW034 27800-27270(177) Putative PKR Z-DNA-binding domain 1 S70G inhibitor Double-stranded RNA 0 binding motif domain LD067 57404-57994(197) LW067 57273-57863(197) Putative host range Pox C7 F8A domain 1 R129K protein (continued) 1338 P. D. Kara et al. Table 1 (continued) LD∗ Position LW† Position Predicted structure SMART analysis∗∗ new (length, codons) new (length, codons) and/or function# orfs orfs Functional domain No. of aa Type of aa‡ changes changes in functional domains LD068 58056-59054(333) LW068 57923-58921(333) Poly(A) polymerase Poly A polymerase 0 small subunit, regulatory subunit PAPS domain VV Protein Vp39 in 1 A309T complex with S-Adenosylhomocysteine LD082 74375-75028(218) LW082 74247-74900(218) Uracil DNA Uracil-DNA glycosylase 1 R54Q glycosylase domain LD086 79895-80533(213) LW086 79767-80396(210) mutT motif mutT domain 2 E121D, L191F LD094 89214-87232(661) LW094 89086-87104(661) Putative virion Signal peptide 0 core protein P4b Poxvirus P4b major core 2 D93N, protein F607L LD102 97585-98535(317) LW102 97408-98358(317) Hypothetical Coiled coil domain 1 T162A protein Transmembrane segment 0 Transmembrane segment 0 LD108 101316-100186(377) LW108 101139-100009(377) Putative myristylated Transmembrane segment 0 membrane protein (DUF230 domain) 2 A231S, Poxvirus protein of R258K unknown function LD113 103932-103588(115) LW113 103761-103411(117) Hypothetical protein Transmembrane segment 1 V22I LD118 111264-110845(140) LW118 111095-110676(140) Hypothetical protein Transmembrane segment 1 I10V LD122 113444-114031(196) LW122 113275-113862(196) Putative EEV Transmembrane segment 1 A56T glycoprotein LD132 119792-120319(176) LW132 119593-120123(177) Hypothetical protein Signal peptide 0 Transmembrane segment 1 V19L LD140 132575-133294(240) LW140 132374-133093(240) Putative RING finger Ring finger domain 2 A182S, host range protein, N1RK187N Comparative sequence analysis of LSDV 1339 LD146 139263-140501(413) LW146 139120-140355(412) Phospholipase D-like Phospholipase D domain 1 I133V protein LD148 142115-143455(447) LW148 141962-143302(447) Ankyrin repeat protein Phospholipase D domain 0 Ankyrin repeats domain 0 Ankyrin repeats domain 1 I102L Ankyrin repeats domain 0 Ankyrin repeats domain 2 N167D, E169D Ankyrin repeats domain 0 LD149 143479-144489(337) LW149 143327-144337(337) Serpin-like protein Signal peptide 0 Serine proteinase 2 K139R, inhibitors (SERPIN) N208K LD153 148293-148565(91) LW153 148136-148408(91) Hypothetical protein Signal peptide 0 Transmembrane segment 1 V20I LD156 150076-150552(159) LW156 149918-150394(159) Hypothetical protein Signal peptide
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