179,234-246
VIROLOGY
(1990)
Genetic Variation INMACULADA Centro
de Biologia
Molecular
and Multigene DE LA VEGA,’
(CSIC-UAM),
Facultad
Received
Families in African
EL4DlO VINUELA,’ de Ciencias,
February
RAFAEL BLASC03
AND
UniversidadAuthoma,
16, 1990; accepted
Swine Fever Virus
Canto
Blanco,
28049
Madrid,
Spain
July 2, 1990
The genome of a virulent strain (LIS57) of African swine fever virus differs from that of the Vero-cell-adapted strain (BA71V) in several deletions located in the variable regions. The region which contains the most differences is located 8-20 kb from the left end. The DNA sequence of this region was obtained from LIS57 virus DNA and compared with the overlapping sequences of BA71V virus. This comparison revealed that the changes in the variable regions result in differences in the number of genes which belong to the multigene families 360 and 110. Virus isolate LIS57 contains at least 8 genes of the multigene family 360 and 12 genes of the multigene family 110, instead of the 6 and 5 genes, respectively, found in BA71V virus strain. The position of the deletions indicates that new combinations of multigene family members in African swine fever virus DNA may arise by in-frame recombination between homologous genes. These data indicate that the evolution of the multigene families 360 and 110 in African swine fever virus DNA has involved different processes, including gene duplication, divergence of duplicated genes, and gene deletion. o isso Academic
Press, Inc.
INTRODUCTION
of the dispensable sequences present in the variable regions of ASF virus DNA. To address this point, we have determined the nucleotide sequence of the dispensable DNA sequences present in the left variable region of a virulent ASF virus isolate (LIS57). We show in this paper that the number of members of the multigene families may vary greatly among different ASF virus isolates.
African swine fever (ASF) virus is a large DNA virus which infects domestic pigs and related species. Its genome is a double-stranded DNA molecule with a length of about 170 kb (for reviews, see Vifiuela, 1985, 1987). Genetic variation in ASF virus DNA takes place mainly through deletion or addition of DNA sequences in regions located close to the genome ends (Blasco et a/., 1989a). Most variation detected when comparing different virus field isolates occurs in the region located between 8 and 20 kb from the left DNA end, a region that contains several repeated sequences (Blasco et al., 1989b). The terminal regions in the DNA of the Vero-celladapted virus strain (BA71 V) contain two multigene families, termed 110 and 360 (Almendral et a/., 1990; Gonzalez et a/., 1990). The BA7 1V virus strain is known to have undergone several deletions affecting both variable regions. Some virus field isolates have about 16 kb of additional sequences relative to BA71V in the left variable DNA region. These sequences are dispensable in cell culture, as well as highly variable in vivo (Blasco et al., 1989a). However, nothing is known about the genetic content
MATERIALS
0042-6822/90
$3.00
METHODS
Cells and viruses Virus isolate LIS57 (Manso Ribeiro et a/., 1958) was originally obtained from J. Vigario and cloned in porcine macrophages as described (Blasco et a/., 1989a). The Vero-adapted strain (BA7 1V) of ASF virus, derived from the BA71 virus field isolate, has been previously described (Enjuanes et a/., 1976). Cell culture of porcine monocytes and purification of viral DNA were carried out as described (Blasco et al., 1989a). DNA cloning
and sequencing
Restriction fragments from LIS57 virus DNA were purified from agarose gels (Langridge et al., 1980) and inserted in pUC plasmids (Messing and Vieira, 1982). For DNA sequencing, a number of nested deletion subclones were obtained by an Exolll-based technique (Henikoff, 1984) in mpl9 phage (Messing, 1983). The sequences in these clones were obtained by the dideoxy chain termination procedure (Sanger et al., 1977) using the M 13 universal primers. The nucleotide
Sequence data from this article have been deposited with the EMBUGenBank Data Libraries under Accession No. M34948. ’ Present address: CEPSA, Picas de Europa, 7, Poligono Industrial de S. Fernando de Henares, Madrid, Spain. 2 To whom requests for reprints should be addressed. 3 Laboratory of Viral Diseases, National Institutes of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892.
Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.
AND
234
GENETIC 0
VARIATION
IN ASF
VIRUS 150
100
50
235 190 kb
kb
0
L
HH
LIS57
YW
BA?lV
B
MM
PR
H PR R RRRSRH
Y’
’
PR
H PR
*’ ’ llwF
i!%?
SRR
“
SH
’
t,
RRRR
Ilit
oy-
RRR .l,,
FIG. 1. Restriction site map of the left DNA end of ASF virus field isolate LIS57. The SalI restriction shown in the upper part. L, left variable region; C, central region; R, right variable regron. Numbers fragments. The left DNA end is shown enlarged below. The black bar indicates the regions which follows: S, .%/I; R, EcoRI; H. HindIll; P, f’stl; Pv, Pvul.
sequence was completed on both strands using synthetic oligonucleotides when necessary. Computer analysis DNA sequences were compiled and routinely analyzed using the software package of the University of Wisconsin Genetics Computer Group (Devereux et al., 1984). Dot matrix analysis was carried out as described (Maize1 and Lenk, 1981). Hydropathic profiles were obtained using the Kyte and Doolittle (I 982) procedure. Multiple sequence alignments were obtained with the aid of a progressive alignment algorithm (Feng and Doolittle, 1987). RESULTS Structure
of the left end of ASF virus strain LIS57
Restriction endonuclease maps of the left variable DNA regions of ASF virus isolates LIZi57 and BA71 showed the existence of several deletions located within both the terminal inverted repetitions and unique sequences (Blasco et al., 1989b). t-lindlll and SalI restriction fragments spanning the region between 2.7 and 24 kb from the left DNA end were cloned in plasmids as described under Materials and Methods, These restriction fragments were compared with the fragments of the same region from the BA71V strain of ASF virus (not shown). Figure 1 shows the differences
map of virus isolates LIS57 and BA7 1 V is indicate the size, in kilobases, of the SalI were sequenced. Restriction sites are as
found in the left terminal region between the virus isolates LIS57 and BA7 1V. In agreement with previous observations, most of the variation was easily accounted for by deletions or additions of DNA sequences. An exception to this was the region located around 20 kb from the left DNA end in the composite map in Fig. 1. In this region, several contiguous EcoRl fragments are present in both LIS57 and BA71V isolates. None of these fragments comigrated (not shown), and therefore we could not align the restriction sites in this region in the DNAs of the two viruses. Nucleotide sequence LIS57 virus
of the left variable region of
To determine the genetic content of the dispensable region in LIS57 DNA, we obtained the nucleotide sequence of the region located between 8 and 22 kb from the left DNA end (Fig. 1 1 black bar). Figure 2 shows the DNA sequence obtained. The comparison of this sequence with the overlapping sequence of BA7 1V virus DNA allowed us to determine the precise extent of the three deletions present in the BA71V virus. These were, from left to right, 684, 117, and 8704 nucleotides in length.
DE LA VEGA, VIthELA,
236
AND BLASCO
600 601
900 901
1000
1001
1100
1101
1200
1201
1300
a(R
MCCCGTATMGM2CGAAGMAlC
1301
TA~ACCTGMThGCAlTTACM~TFATAlTC
1401
TCAhkCA-AGGClTMTM’lVFAAMATMSCllT
AenmMmAATGYTr~AcAclTM~
1501
M6Tl'lVSTCA3GGACACCAG'XAA~ATAAACA
~~~~~A~~~~~A~MT~
1601
CAATPFl’Al’MTlUA‘lTACAl’lTA-
1400
e
LIS 37s
M.X3TfSACATAGGAMTl’
1500 1600
RNSLQVLTXXVLIENXAF
ATCCA’MATAAT~--TMAAMC-
1700
ml 1701
8EYHEDDIFILQQLGLHRHNGPIGFCKQCKRVT T~~ATM~AT~
AAGcAA~MM’Iw;TAAEc
1000
b 1601
1900
1901
2000
2001
VGLYNRPIDLIKQQKVLLAIDIYYDllIBILDSF lGTAGUWlCTATAATA’TVlTSA~~MTl’AMC-
ATNGCl’ATAGAThTITA’lTARlATAATA~~
2101
2201
2200 FWFKIAIEFNLIKPIKFLKKKl”PRLDYHRLKTA M~AAGNlWCCATAG~AA~ATMAACCTA~
MTMGAAA~AlCTMATI’A--
2300
2308
2401
2100
2400 QNKLGIYYCFVLGANINTALETLIGFIllHEVURE CAMATMAlTAGGTATATA~A~MGCGCMA
TAYCMT-AGAGA-T
M
2500
2501
2600
2601
2700
2701
RKIISTYEYTCTFRSF6LLRDNL CVTAMATM2TlCMCC
Trd
AGTAMAlUlTAlVATGTAYfXAElVTAT
2000
FIG. 2. DNA sequence at the left variable region in virus isolate LIS57. The sequence is shown in a 5 -f 3’ direction and from right to left according to the restriction map. Translation of ORFs is shown in single-letter amino acid code. Bent arrows indicate the exact boundaries of the segments deleted in BA7 1 V virus DNA. Arrows above the sequence indicate the presence of tandemly repeated sequences, named with Greek letters. ORFs are named according to the number of coding triplets in the reading frame.
GENETIC
VARIATION
IN ASF
VIRUS
237 2900
ATC(;TATACMTGXTA~ATCiXCATAMTACO
2601
CCAYGAATATATlTMMTAATlTEWlT
2901
l CACYITFATAATATATGCCMCATSA~TA~
3001
ATfTMmAT-A-
ACMTAYTAMlTAllTFYATATCA~#K!ATMMMMTA~lT T
MTATPA’lTATAlTCATA?ii .-
3000 3100
1
3101
CXA YGAGGGACCCCCCCCCCCCCCAGTAA~AGMAGTM~~CMACAYCA~MAC-AT-ATMGMA
320 1
~AAATITXATC-ATMAmATAGAGTACAAlT
3301
-.. ~ AATAAWCACMTMTGX-AITAGGACCCCCCC--A-
3401
-MAT-
3501
CTACAYTAYWllTICMCCA~TITI¶C~~-AmTMTMTACGAB
360 1
QVLTKKVLIETKAFSKYHEDDIFILQQLGLNIIE TmACT-TAGAAACC
3701
IGPIGFC~ AA-T-
380 1
-CAGAClEATCTMTCAAGATATP
3901
DLCIQLGADPEFLDVGLYNNFVYLIKQKKVLLA mzxxlmm A~CMCTM%ACCCCATCCAGM~~~~ATMTA~A~A~~
4001
Clr~TA~A~A~MTA~~~~~~~-~
3200 3300
~MTA~A~TA~~~~A~A~ATA~ TAAAWXAACA~AACWAM~M~AAAGfTI
3400
TITIOGWA~ZMCATCTA
AMltlCTACTITITNATMMM~hM~
TlTIllWAYGMCAT
LIS 312
w
N
3500 s
L 3600
~AYCA?CAGGA’IGATAYTI’TTA~~MC-A-
3700 LDRALV 3600
KAVKENQTDLIKIFVSYGAEINFGINCAKTKQTK -AMA~AGA~A
3900
4000
IDIYYDNISILDSFDSliDl’HVLIDFVY#RFILY AlVlTATAT
4100
LDEKEEENTRNTLVLKFYYKFAIDFKLTKPIRYL 4 101
YTAGA--TmAYTMM
4201
SKKFPHLDIYRLQTAIYLGNIDEVHHAYFQEDI lTACMMAAATllCC’KATITAGATA-
4301
RLSLNVNNFLACARPGNKLGIYYCFALGADLDR A~ATCCCMAACGlCAlCA-
4401
ALERLISFNSINRKISGETRLCIEGSYLSNVYFC GCCITAGAGAGA ~TArrC~~A~AT~~A
4501
IGLGANPYTKKIQETIKQKHSNINILLFSKKKI CTATAM;TITUX;TCCCMCCCGTATACAAAAAAGATACAAGAAACCA~AMCMAAACATAGCMTA~AT~AT~~?~A~NXTTXT~~T
4600
4601
LSPHSVLQNKILDPSDVRKNISTYENTESFYPF TITAAGCCCACATPCAD AMATMMTAlTAGATCCN2lUA-
4700
4701
FSLAVKLIQQAK ~~~AOITMrrn;JITACMC~~T-~~ATAT~~~~
4801
GATCCAAGAAATMTATAAGMATI-ITMGG
4901
KVLLGLLLGYSVLILAHELPDLPRTQHPPKSEL pm-
5001
lCATAl7GGP.X
5101
ATCAllTPATNXAMCNXA
5201
LHPRPYTCNEIFNRTWGGGG YTTACATC~AGACCATATACAGGAAATCAAATATI’CA~~
fCCA~ATC
4200
-ATACIT-TAm-2CWrA~AGGMGATAT
4300
C
T~CW
4400
T
4500
MMlCA~MCCTAlCMMTACA-AECA’ll.r
‘GGGCA
4000
LIS 121-2 VATAlTCAAAATlTAAAm
AACltC-ATlTAGACGCMCi!T
f-
4900
5000
SYWCTYVPQCDFCUDCQDGICKNKITESRFIDSN
~A~~XACCACM~CCGA~YCGCA-TAAGATMcAGAA
WXTCITKA~CTCCA
5100
HSIVNCRVFRNSNTQSCLYEISSKNPNHFSNEC
5301
GAClW7CCCfMY’ICCA~-
ATAlCAAAllTCAlCFMM~MYCACllTAGTAn%AATC
A’lCMCMTPATCTATAAAGCMTAcWlU’ITA
52oc
53oc
LIS 117 NKLFVLLSI
~A~CATICGTATDXA~~TAC~A~AAGAA
lCIUCIW-ACMCCA-ATI FIG.
2-Continued
54oc
238
DE LA VEGA, VIhJELA,
1501
YWCTYGKHCDFCWDCKRGICKNKVLDDRPLIVQ ~A~A~A~~~~~A~~~~TA~A~A
5601
AMYCATSACAlCkCCCGTPlTA~~
5701
LPHIFSLI CllXCCCACATA TITIWlTAATATAMMTA~
6101
lCAGCCAA=
6201
mATANAGFlC
W
6301
6401
D
Y
I
SK
C
S
I
TR
AND BLASCO
5600 I
F
DR
UIYHY~EPK
I
T
P
I
I
H
YUW
C
S
5700
AlWGTMTACACATAC'lGTMMTITAMTA-MTMCA-A
TMl%AXA-ACAm--
ssoo
6200
ACOGATMZA~ltTATMXAMTAT-ATA
6300
MTM~ATA~A~MT~~~~~~~~~A~
LIS 1193I4
K V L L C GACACMCAm-AClCMYCClCATEKGC
L
L
L
C
Y
S
V
L
I
L
AA&k'zu&K
EEL MccAoGAccT
PYWCTYVKNCDFCWDCQWGICKRKITWESISHN TccATATEG'lCT ACGTAlVTAAAGAATElCA C
6400
ATlTCGAW
6500
Eco
6601
SIVWCIVNRNSWGCFYEISVKUPNHHNHECSHPR TCGATlCTAMC CACA~~A Fi PYTGNEIFllEKWGGVIIGQSL GACCCYATACAGGAMlWAATATICAlKKZMM~lTA~
6701
GCAlTlCl'AGGlTACATlCTATllCCGT
6601
FGLNRPLSIFYTKQWLPRTYTPPVRELEY~CTY ActMACCGACCCCllTCCATClTlTATACOIUAC
6901
GKHCDFCWECRWGICKRKVWDDl4PLIKQNDYIS WXAAAAC'
6501
6600 I
A~AT~~~~~~~A~~A~A~~A~
LIs 119-1M K
AMAlTmc
ACCllCTATlTAGTA?T L
L
A
L
L
C
I
L
I
Y
C-AWA
QCSIARYFDRCRYFIKPKSPYIHYl#DCSQPIVYK CMlCl’AGTATECCC
TCACC
7101
G F S H AAGGGllTAGTCATI'MTAGATATATATCTMAAAAl-CCAGCATlTA-
7201
WlCAGCCTACATAITATGATTCGTATAAGM~ATACA~MAAMAM~AT-
Q
OS00
7200
TACAlTAlYXGGATTMMPCP
LIS 290
7000 7100
T
MCMMTlTlTMlCAGCCCACA~ACCGTA~~
EcoRl
S
6900
‘ICATATGC~AlTMACMM~YTATAlT~
7001
L
6700
HKVIVL
7300
7301
~A~A~CATAC~~AT~~~~AT~~~~A~
7400
7401
LLVLAVMQPVIQSQPFPGTEELPMTRRPPKGELE ClTllWXA C
7500
7501
A
YWCTYAKSCDFCWNCRHGICKWKVFEKHPLIKK
7601
WDYIQICRVSRYWNRCSYFTDSRIRRFHKKSCT AMlCA~ACATACMATAYtXCCC
7701
WPTYYDWFDELMQVKEDRVIDVENIKHTCLCMIA MTCCCACATA’XTA~ -'NiAGlTMlCC~-T--TA
7SOl
TIALIGYVRKQYSRMRMQAATRLLIFLGLYVLL ~ACCATlSCCClCATAtXlTAl!XWGCAMCAGTA~~lCC
m-m
-ATCAAMA
7600
G-i-ACAmA=AGClCTACA
'ATAG
A FIG. 2-Continued
7700 7800
7900
GENETIC
7901
C I L MT N I I HN L P M;GM~~CCMCATMTMn;MCCTAC~CAC~ATM~CM~~~~~~~~~
VARIATION
IN ASF
239
VIRUS
STDNPMQI’IRR
L
PPERDLKFWC a000
TYAKHCDFCWTCKDGllCKNKVFSOHPIITQllDYI 8001
AC~A~~CA~A~CPCTCAC~T~~~M~~T
8101
VNCTVSRWHDRCMYEAHFRIHYQHNt4NCSQPKD lTGlTAATIGTAC~CCG~A~ACC~AXTACGAACCCCA~ACGATACACTA~MCATMCA~llCl¶’C~CCAMGA
-ACICACCACCC
8100
8200
8201 8301 8401
TCCMGMTATITACITAGAlCTI’ATMTATlTA~ATMCAMTl’-TATITATAMTITGGGT
ATATCITATMGATACATATAMTATI’MTA
as01
TMTMlCClCATl’Ml-ZITMTATM~ACCATAAAGATATMTITMCGClllTM~
8601
TCGTCATA-MTAAAtXMCll’CCCCCAGGAACATl7l-CTAGCAAACGATATA~AGACMCM~
8701
TlYTMGCTCTKCGTAATl7n7TK(?lYiGAlllTACGAGACCTACGATAAAATATACATll.TAlCGGA
8801
TMClXAGGATAGTITCCCAGTCATF3XAATMACl-IT
ATCCACCGMTCC
1500 e600
YCATAMAATITMNXT
TIWXMATMCCTMCCATCllT-
8700
eeoo
AlTACAAAATATAlTM-ATAAAA’ITAmAAAATCAlXZAT
8900
8901 9001
TWXCGETNXAT-
CAC-
GTATl’AlTCIltM
ATACCTAMTIy;AfcTA
9100
3101 9201 9301
CAGAAACCATACAGATCA~AAA~c?ClCCAAATCCMCATcCCATAClTATCCACATAGAGAATAcAnXhGATCTA~
9401
ATpAGAlTCITATAMmA
lY%CCTAYGA
ATPAMGATAGlCMXC
-ATATCCGCCAMMCGGGATCAMA~AM~
CTAACATIT
9400 9500
9501 9601 9701
AGGATITCATAGATATCAGTATGACCATCCTC
9801
cpMcATpcCA~ATcACAGM;rrrMCA~M~~T~~A
CCll’TCACACMClTATPCATCTACCAU;CACPCITOPAI
CACA-emC
~ATACACCAT~A~AGCP~TITFA
9800 9900
liizii 9901
10000
CCTTATACCCTl-GCCAMAATl’AAACCCTPAGTMCGCAMTC
10001
TFIltMltC
MMAAATCl-MTCGAATllTMA~
ATAAAGAGTACACTCTMGCCACTA
10101
--AG~~-CACM
10201
TSTRVCCHLPLTFDPPENELGYWCTYVESCRFCW m~A~CA~~C~~~~M~M~TA~~A~
10301
O
10401
G
LIS137
-AGCCA~AGCTA~~A
HLVIILGVIGLLASSNLVS8S
10100
10200
-lTAl’C-AGmATICCC-AGTm-A-
10300
OCCOCICTSRVWCNNSTSIIEnDYVIICEVSRW
10400
-MCIWACMGTA~AlTGAGMCGACTAWl%MM’AGOLCRYOVEEHIYHSMllCSDPKPWNPYKIANKC ACATlTACCATAGCAlCAA-AMGC--ATMAA-
10500
4 lOSO
WKKOKHFRKELKKOEF PXMMWGATAAACATI-IK-AAAMAAG
10600
-AITAGA~CAGATACA~~-
TMlP
ECOR I 10601
AGCATCAGMTC
CTAoCAMCYAl%AAAMA
M
-TcItMAluA~~ xii
FIG.
P-Continued
10700
DE LA VEGA, VItiUELA, AND BLASCO
240
v11 10701
TACAGXtXATK2fXAATNXXXAGCTATI'CAGTC
HLVIFLGILGLLASQ
-AGcCAGccGTAGcM~A--AGlxA
lOSO EtoRl
lOSO
VSSQLVGQLRPTEDPPEEELEYWCAYbtESCQFC GGmrCmxcAAmG2TcGAc AAcITcGACCMCAGNXA~CPCCACACCM
10901
WDCQDGTCINKIDGSA lUXAClCCCMGA'lfXC~ATAMCAMATAGA
11001
C H Y 0 L D K G M~A~A~~~T~A~~CAT~CA~~~CA~
I
11201
TTAAAAATACCTl-llTCCTGTAAAAC
GTNXMMATGTI'GGACTCAAT~MATCATCATATAMGMT
11301
GtTAGfXATCl'AGC
GMClYXAAT~~ACA~~
10900
IYKNEYVKACLVSRWLDK TCCDPCCCCCAlTI'ATMGMSAGTA-PTA Y IiTt!
N C
Lis121-1 H AGCPCTPCACCCACCC~M~~~~CC~
L
V
S Q
I
F
PW
L
G
11000
S W N;&Mh;&R
I
L
K EWKK AGGm2GMluGMMA
11100
FAAlTA G
L
L
A
N Q
11300 V
S S
Q
L
V
G
11400
EcoRl 11401
QLHSTENPSENELEYWCTYMECCQFCWDCQNGL GACMCrPCA~MCCr.l~~AGM:MTC,AACPP
11501
CVHKLCNTTILENEYVHPCIVSRWLNKC~YDLG TEWXECAT-TACAACMVEAGTA-AXCAWAT-
11500 11600
C 4
11601
QGIDHVblVCSQPKHWNPYKILKKEWKENNSQNI CAAGGCATIYXKAWM ~MCC~~A~~~ATAAMTC
11700 4
11701
MTlTAm
Mn;n;CCGMCCATCrCCATPCATACACTAAAMTA~~C~~~
11001
CATA~MGAGTAGAGGGGCGA~A-
ltGMTcTrAMm
L&124-2 M
NLVIFLG
llSO0
11900
AGCCA-
. 11901
ILGLLANQVLGLPTQAGCHLRSTDllPPQEELGYW Al-PXlWC-CMX-AGGACTACCCACICAGGCAGGMGGCA
TCITCDITCMCGGATAA~CAC-Tm
12000
GilI 1200 1
CTYMESCKFCWACAHGICKNKVNnSnPLIIEWS GGTGCAfXTACATGGMAGCMG ~A~ACA~~~MCAMX;T
12101
YLTSCEVSRWYNQCTYSEGNGHYHVHDCSDPVP TA'A'ITIEACATCITDPCA GGllTCKGCTCCTATAACCAGlCCACATATAOn;MCGA
12100
TGGGCATIACCAE'ITATGGA-
lv3xGTm
12200
4
12301
-CMMTITMCA-MTATMCAC
AOITACCCCGGCl'AGCCMMTTAAATAA~A~MT
12401
GKXGGMCCATVlCCATPCATMAATMMATATCTRTC
12501
AGCGCCADPCA~A~AGCAGCI'ATITAGCCA
CC
12400
CCGMTCTl'AM~ATAlT~~
L&124-1 GCTGTICAGCCAGCCATMCM~A~
12500
MLVIFLGILCLLA
12600 Etdl
NQVLGLPTQAEGHLRSTDNPPQEELGYNCTYBIE AGGACTACCPAC AGAAGUXATCTPCOrrCMCGGATAAGCATMTCCPCCACMGMGMCllGGATA~
12601
CCM’X-
12701
SCKFCWECEHGICKNKVNRSHPWIIENSYLTSC MAlCGAA~MGMC-
12SOl
EVSRWYWQCTYDEGNGHYHVnDCSWPVPHWRPHR ~ATMCCU3I'CTACATAlCAlCMGGAM
12901
GA-TlTA-mMmAGAmmCWmATPM
13001
AlTlTMCATA~
TAGGAGCATGCCAlCGA~A~C!AGlTATln2MTA~
l6GACATl'ACCAlCTMlCGATlCTICT
LGRKIYCKEDL GTMTATMCATACMT
AcrrwMuGA
MlWXfXACCICACM~ACACC
12100
12soo
12900 13000
13036 FIG. 2-Continued
GENETIC
R
VARIATION
RRRSRH
SR
I Ilufl
IN ASF
VIRUS
S
R
II
241
H
R
I I
I
,”
.
h_
RR
I ;
.,’
.
‘.
:
_: I
..
’
‘. .
.
: */‘I
.’
. ‘.
:.
R
II 1
.;:y
: /
‘/. ‘2,
.
1;’
‘:
. .
.I, ,‘,“.
,‘.’