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, ,‘,“.

,‘.’