JOURNAL OF VIROLOGY, Feb. 1991, p. 1046-10520022-538X/91/021046-07$02.00/0Copyright C) 1991, American Society for Microbiology

Expression and Characterization of the Thymidine KinaseGene of African Swine Fever Virus

A. M. MARTIN HERNANDEZ AND E. TABARES*

Departamento de Microbiologia, Facultad de Medicina, Universidad Autonoma de Madrid,Arzobispo Morcillo 4, 28029 Madrid, SpainReceived 5 July 1990/Accepted 26 October 1990

The thymidine kinase (TK) gene of African swine fever virus (ASFV) was located within the viral genome byusing two degenerate oligonucleotide probes derived from sequences of the vaccinia virus and cellular TKgenes. The TK gene was mapped within a 0.72-kbp Bglll-XhoI fragment (0.242 to 0.246 map units) derivedfrom a 23.9-kbp Sal-B fragment of the ASFV genome. Identification of this region as the ASFV TK gene wasconfirmed by expression of TK in Escherichia coli and by the synthesis of active TK in a cell-free systemprogrammed with RNA synthesized in vitro. The sequenced gene for TK includes an open reading frame of 588nucleotides encoding a protein of 196 amino acids. The deduced amino acid sequence shows 32.4% identity withthe TK of vaccinia virus.

The thymidine kinase (TK) marker has proven valuable inthe establishment of vaccinia virus and herpes simplex viruscloning vectors and for the development of surrogate genet-ics in these viruses (18, 21). Therefore, it is of particularinterest to identify and characterize the African swine fevervirus (ASFV) TK gene as a potential target site for geneticmanipulations. ASFV is an icosahedral cytoplasmic DNAvirus with properties common to iridoviruses and poxviruses(30, 34). It causes a highly contagious and generally fataldisease of pigs (8, 9, 16, 23, 34, 35). Productive infection isaccompanied by a weak shutoff of host protein synthesis andthe appearance of 44 virus-specific polypeptides ranging inmolecular weight from 9,500 to 243,000 (5, 27, 29, 30). Thesepolypeptides have been classified as immediate early, early,and late (5, 33). Increases in the levels of two enzymaticactivities which may be involved in DNA replication, thoseof a TK (19) and a DNA polymerase (20), have been detectedfollowing ASFV infection. ASFV infection of baby hamsterkidney (BHK) cells may induce the formation of a TKdifferent from that found in normal cells (19). This activitycan be induced in BHK cells deficient in TK as an immedi-ate-early protein, and we have generated ASFV TK- mu-tants by bromodeoxyuridine mutagenesis (data not shown).These results strongly suggested that the TK induced afterinfection is encoded by ASFV. Here we report the identifi-cation, characterization, and expression of the ASFV TKgene as an initial step toward the genetic manipulation of thisvirus.Mapping of the ASFV TK gene. The use of degenerate

oligonucleotides to locate the TK genes of Shope fibromavirus and avipoxvirus (26, 32) suggested that such oligonu-cleotides could also be used for ASFV, given the phyloge-netic proximity between ASFV and the poxviruses. Oligo-nucleotide pools 1 [GG(A/G/T/C)CCCATGTT(T/C)TC(A/G/T/C)GG] and 2 [GA(T/C)GA(G/A)GG(G/A)CA(G/A)TT(T/C)TT], representing conserved regions in the 3' portion of thevaccinia virus, human, and mouse TK genes (32), were

synthesized by F. Barahona in Centro de Biologia Molecu-lar, Madrid, Spain. ASFV DNA isolated from MS cells

* Corresponding author.

infected with ASFV strain E70MS44 (31) was digested withClaI, SalI, and SmaI and electrophoresed in an agarose gel.The DNA fragments were denatured in the gel, transferredto a nitrocellulose filter, and hybridized to the 32P-oligonu-cleotide probes (32). Probe 1 annealed with the Sall-A, -B,-H', and -E fragments (see Fig. 4). This indicated possibleviral sequences related to proteins that contain an ATP-binding domain, since probe 1 represents a consensus se-

quence for such a nucleotide-binding site. Probe 2 annealedonly with the SalI-B and SalI-E fragments (data not shown).We studied the Sall-B fragment because SalI-E encodes lateproteins (22) and the TK of ASFV is an immediate-earlyprotein. ASFV DNA isolated from strain E70MS14 was usedin the construction of the recombinant plasmid pRPEM513,and the EcoRI-K fragment was subcloned in pUC18 (Fig. 1)by standard techniques (15). These plasmids were subjectedto restriction enzyme analysis, and the DNA fragments were

hybridized to 32P-labeled oligonucleotides in order to mapthe TK gene more precisely. A region of homology with theTK gene was localized in a 390-bp PstI-HindIII fragment ofpRPEM204 (Fig. 2, lanes 2). Sail-B represents unique se-

quences on the viral genome because this segment hybrid-izes only with fragments SmaI-A, ClaI-B, -F, -P, and -R, andSall-B (reference 31 and data not shown), indicating that theTK gene is a single-copy gene.

Identification of the ASFV TK gene. The TK gene and itsflanking regions were sequenced by the dideoxynucleotidechain termination method (24), with recombinant pUC18 or

pGEM3Z as the template. The nucleotide and deducedprotein sequences are presented in Fig. 3. The TK genemaps at coordinates 0.242 to 0.246 (Fig. 4) in E70MS14 DNA(31). The predicted ASFV TK protein comprises 196 aminoacids, with a calculated molecular weight of 22,394. Thetranslation initiation codon of the open reading frame hasbeen assigned on the basis of homology of the amino-terminal domain to that of other TK proteins. Also, theproposed initiator AUG is flanked by nucleotides character-istic of preferred eucaryotic initiation sites, whereas the nextATG codon, located 93 nucleotides downstream, is notflanked by consensus translation initiation sequences (25).

Characterization and expression of the TK gene in Esche-

1046

Vol. 65, No. 2

NOTES 1047

E

B

p

x

H BEn/Ea/ Bom HI

FIG. 1. Physical map of plasmids containing the TK gene. Viral DNA was digested with SalI and cloned in plasmid pUC18 (2,686 bp), andrecombinant plasmids were obtained upon transformation of E. coli NM522. One of these plasmids, pRPEM513, contained the SalI-B(23.9-kbp) and SaII-J (0.9-kbp) fragments (31). After digestion with EcoRI, the EcoRI-K fragment (1) was subcloned in pUC18 to obtainplasmid pRPEM204. The larger BgIII fragment and the HindIII-BglII fragment of this plasmid were cloned in pGEM3Z (2,743 bp) (PromegaBiotec, Madison, Wis.) and pINIIIAl (7.4 kbp) (kindly supplied by E. Garcia, Madrid, Spain) (4, 12), respectively, to yield pGEM204a andpINTK14, respectively. Restriction enzyme sites BglII (B), ClaI (C), EcoRI (E), HindlIl (H), PstI (P), PvuI (Pv), and Xhol (X) are indicated.

richia coli cells. The HindIII-BglII fragment was cloned intothe HindIll and BamHI sites of pINIIIAl (4, 12) to yieldplasmid pINTK14 (Fig. 1). In this clone, the N-terminal fiveamino acids of ASFV TK protein are replaced by the five

A B

1 23456789 1 2345 6789

amino acids encoded by linker DNA from pINIIIAL. Plas-mid pINTK14 was used to transform E. coli KY893 (TK-).Untransformed E. coli KY893 TK- is unable to grow onselection medium containing 5-fluorouracil (11). Only clonescontaining viral TK from pINTK14, which complementedthe cellular TK defect, survived on the drug selection plates.It was important to confirm the TK complementation ofKY893 TK- cells harboring the plasmid pINTK14 by exam-ining incorporation of [3H]thymidine into the bacterial cells.The KY893 mutant of E. coli transformed with the plasmidswas cultured overnight in peptone-glucose medium (10)containing (per ml) 50 ,ug of ampicillin, 25 ,ug of 5-fluorou-

FIG. 2. Mapping of the ASFV TK gene within plasmid pRPEM204. (A) Lanes 1 to 8, pRPEM204 digested with HindlIl plus EcoRI,HindlIl plus PstI, XhoI plus PstI, BgIIH plus PstI, BglII plusHindIII, BglII plus HindlIl plus EcoRI, EcoRI, and BamHI, respec-tively; lane 9, plasmid pUC18 digested with RsaI. The gel was 0.75%agarose (31), and the DNA was stained with ethidium bromide. (B)Southern blot of the gel shown in panel A probed with end-labeledoligonucleotide pool (map positions of the various restriction en-zymes are shown in Fig. 1). The asterisk indicates the 390-bpPstI-HindIII fragment of pRPEM204.

a&1769bp

676 bp

241 bp

VOL. 65, 1991

1048 NOTES

MAp x

GCRARRRGTR CCGCRRRTRR RRRRRCRRCG RRGGGCTCCT CCRRRTCTOG TTCCTCCRGRGGCCRCRCCG OCRRRRCCCR TGCTTCTTCG TCCRTGCRTT CCGGGRTGCT CTRTRRRGRT

Bgl URTGGTRRRTR TTGCTRfiRTCTRGRGGCRTT CCGRTTTRCC RGRRTGGRTC GCGTCTTRCT

RBS fidmRRRRGTGRRT TGGRGRRRRR RRTRRRCffILCRM RTG RRT RTR RTT RGG fiflCtL

M N I I R K LRCR RTT RGC CTTT I S L

RTT CRT TGC RTTI H C I

RRR TCT RCC RRRK S T K

CAG CTR CGR CCCo L R PGTG GGR TCT CTCV G S L

TTT GRC GRT TTRF D D L

RTT CTT GCG GGRI L A G

GTT COT RTT TTTV R I FRTG RRR TGT RRCM K C NRRG RCO CTT RTCK T L I

RRC TGT CTR RRRN C L K

GTG CTO GGR CCCV L G P

TRC RTG CTC ORRY M L E

RRC RCC CGR GRCN T R D

RRG CRR TGT RRRK Q C K

RCC GRT RTC CRTT D I H

RTR RRR TGC CGCI K C R

CTC RRT OCT TCCL N A S

PsICCT TRC TGCRACP. y C SCRR CRT RRT OCRQ H N ACTT GCG GOR GORL A G G

RRT RCR TTT RTTN T F I

RTG TTT 0CC GOCM F A G

CGT TTG GRR RRRR L E KRRR ACT RTT RRRK T I K

RTC RTR GRR RGCI I E S

GCR GTT GTC GTRA V V VRCC TGG GCR GRGT W A E

TTC GRG CRG RRRF E Q K

RRR RCTK T

RRR GTRK V

RCR CRCT HRCA CR0T O

GRT GRRD EGRR GRRE ERTO TTTM F

TOG OTT RRG TRT RTT GOCW V K Y I GTGC TTT RRT GTG COT RRGC F N V R KRGT GRR CTO TRC OTR RCRS E L Y V T

RRG CR0 TTG CRR CCT RTTK Q L Q P I

ARRTCTTRTR CRRTRRTOGR TCRTTRTCTT RRRRRRTTRC RRGRTRTTTR

XhOITRCGRRGALfLQGGGGCCRTC CCTTTCTTTT TROCCCGTCG RRRRCCRRTO RRRRRGRGTTTRTTRCTCTO CTRRRCCROG CCTTGGCCTC RRCGCROCTT TRCCOCRGCR TRCRRCRACTGTTTTTRRCG RTOTATRROC TRGRTCCCRT TGOGTTTRTT RRCTRTRTTR RRRCGOGTRRRCRROROTRT TTATGCCTGT TORTTARTCC TRRACTCGTT RCTRRGTTTT TARRRATAAC

FIG. 3. Nucleotide sequence of the ASFV TK gene and flanking regions. (A) The PstI-EcoRI 1.4-kbp TK gene fragment from pRPEM204 was

inserted in pUC18 to yield pRPEM207. Then the PstI-HindIII 390-bp, HindIII-EcoRI 1.1-kb, and PstI 1.1-kbp TK gene fragments were subclonedto yield plasmids pRPEM211, pRPEM210, and pRPEM213, respectively. These plasmids were sequenced, and the open reading frames of bothstrands were determined. Arrows show the direction and extent of sequence determination from each restriction site. Restriction enzyme cleavagesites BglII (B), HindlIl (H), PstI (P), and XhoI (X) are indicated. (B) Nucleotide sequence and predicted amino acid sequence of the TK protein(single-letter amino acid code). Ter, Termination codon. Locations of cleavage sites BgII, HindIII, PstI, and XhoI and the ribosome-binding siteare underlined.

B HII1IiT

T K

B

RRG CCT GGRK P G

RCG TTT CTTT F L

OTC TTC RTRV F I

TCC GGT RTRS G I

TTR TCT GRCL S D

GCG CRT TTTA H F

RRR ATT RTTK I I

CCG CCC RTCp p I

CGC RCC TGTR T C

RRC GCR GRCN A D

TGT TGT RRCC C N

RRR TRT TRRK Y Ter

J. VIROL.

NOTES 1049

.2B I

.4l l

.6 .a4 156 Kbp

KC II C I H'i B F'I A

.I IHI E IGI~

E C C CE ESI I a-I . .I 1

E BH P X

a Ip a E

I I aWa

TKFIG. 4. Summary of the mapping and precise location of the TK gene in the ASFV genome. The size of the DNA from ASFV growing

in MS cells is 156 kbp (31). Sall fragments are shown (boxed). The location of the . K gene within the SalI-B fragment is indicated. Restrictionenzymes BglII (B), ClaI (C), EcoRI (E), HindIll (H), PstI (P), Sall (S), and Xhol (X) are indicated.

racil, 2.5 ,ug of uridine, 5 ,ug of thymidine, 250 ,ug ofdeoxyadenosine, and 1 ,uCi of [3H]thymidine (82.2 Ci/mmol;New England Nuclear Corp., Boston, Mass.). The cellswere lysed with 2% sodium dodecyl sulfate-0.2 N NaOH

ABC D

P73

1P2 3.5

IP16 ._

FIG. 5. Sodium dodecyl sulfate-polyacrylamide gel electropho-resis of polypeptides translated from different RNA preparations.The RNA resulting from in vitro transcription by T7 RNA polymer-ase from plasmid pGEM204a linearized with XhoI (pGEM204aX)(lane C) was translated in a rabbit reticulocyte lysate system(Promega), with [35S]methionine as the labeled amino acid. Labeledproteins were analyzed by 12% polyacrylamide-sodium dodecylsulfate gel electrophoresis, and the labeled polypeptides were visu-alized by autoradiography of the dried gel. Lane D, No RNA; laneA, polypeptides from uninfected MS cells; lane B, infected cellslabeled with [35S]methionine from 20 to 22 h postinfection (28). IP,Polypeptides from infected cells; numbers are molecular masses inkilodaltons.

and precipitated with ice-cold 10% trichloroacetic acid.Precipitates were collected by filtration on glass fiber filtersand counted in toluene-based scintillation fluid. When thesecells were grown in the presence of isopropyl-p3-D-thiogalac-topyranoside (IPTG), the incorporation of [3H]thymidinewas increased about 25 times in relation to that in parallelcultures in the absence of IPTG (Table 1). Since the pINI-IIAl vector contains the lac promoter-operator region, theexpression of the viral TK gene can be induced by a lacinducer such as IPTG. The increase of [3H]thymidine indi-cates that the expression of the ASFV TK gene is regulatedby a lac promoter-operator region.

Cell-free translation of synthetic RNA. Additional evidencethat the BglII-XhoI fragment contains the TK gene wasobtained by the synthesis of active TK protein in a cell-freesystem programmed with RNA synthesized in vitro, usingplasmid pGEM204a (Fig. 1) and T7 or SP6 RNA polymerasein the presence of 0.5 mM m7G(5')ppp(5')G. The RNA wastranslated in a reticulocyte cell-free system, and samplesfrom the translation mixture were then assayed for TKactivity. Positive controls were early RNAs from BHK TK-cells (kindly supplied by B. Roizman, University of Chicago)at 14 h after infection with ASFV (strain SpainM0/MS44 [31])in the presence of 0.1 mM cycloheximide. Total RNA wasisolated from infected and mock-infected cells by guanidineisothiocyanate-CsCl extraction (15). Active TK was ob-tained upon translation ofRNA from infected cells (Table 2).

TABLE 1. Incorporation of [3H]thymidine into DNA of plasmid-transformed TK- E. coli (KY893 mutant strain)a

Bacterial strain . cpm/ml of(culture supplementb) asma bacterial culture

KY893 37NH522 1,877KY893 (FUdr) pINIIIAl 37KY893 (FUdr + IPTG) pINIIIAl 58KY893 (FUdr) pINTK14 166KY893 (FUdr + IPTG) pINTK14 4,110

a For selection ofTK expression, the TK-deficient (TK-) strain KY893 (10)of E. coli was used. TK activity was assayed by measuring the uptake of[3H]thymidine as acid-precipitable radioactivity.

b FUdr, Fluorodeoxyuridine.

PRPEM 513

pRPEM 204_" . . V

VOL. 65, 1991

1050 NOTES

fHIIRKLKP6TISLULGPfFRGKTTFLIHCIYMLERLEKKUUFIKSTKHTR- OKMSC INLPTULP6SPSKTRGQIQUILGPMFSGKSTELIRRURRFQIROYKCLUIKYRKOTRY----SSMSYIHLPTULPSSPSKTRGQIQUILGPnFSGKSTELIRRURRFQIRQYKCLUIKYRKDTRY----SKIHCLTUPGUHPGSPGRPRGQIQUIFGPfFSGKSTELIRRURRFQLRQYRCLLUKYRKDTRYC---TT

1SSGSIHUITGPnFSGKTSELURRIKRFnLSNFKCIIIKHCGONRYNDEDINnHGGHIOLIIGPnFSGKSTELIRRURRYQ1RQYKCUTIKYSNDNRYG----TINGGHIQLIIGPMFSGKSTELIRRURRYQIRQYKCUTIKYSHDHRYG----TIHGGHIQLIIGPIFSGKSTELIRRURRYQIRQYKCUTIKYSHDHRYG----Tn1IGHIHLIIGP1FRGKSTELIRLURRYQIRKHKCLUUKYEKDIRYG----N

G I + GPfF+GK+++L +++ + + ++ +K+ + R+

TIKTHSGIQLRPKQCKIIESTQLSDU--GSLTO--IHRUU-UDERHFFODLIK-CRTUREEEKIIILSFCTHD.-----RHTERLPRCLLRDURQERL----6URUIGIDEGQFFPDInEFCERnRHRGKTUIUSFSTHD-----RHT1DRLPRCfLRDUTQELL----GURUIGIOEGQFFPDIUDFCE1IRHEGKTUIUGUSTHD-----RHTMERRPRCRLQDUYQERL----GSRUIGIDEGQFFPDIUEFCEKIRHTGKTUIUKUYTHD-----LLFMERTRSSHLS-ULUPTLLHD-GUQUIGIDERQFFLDIUEFSESMRHLGKTUIUGLUTHD-----KHHFRRLEUTKLCDU----LERITDFSUIGIDE6QFFPDUUEFCERMRHEGKIUIUGLUTHD-----KHHFERLERTKLCDU----LESITDFSUIGIDEGQFFPDIUEFCERMRNEGKIUIUGLUTHD-----KNHFERLERTKLCDU----LERITDFSUIGIDEGQFFPOUUEFCERMRHEGKIUIUGUCTHD-----NS I TRUCTPSLDK I ----DSURENREUII6OEGQFFPNIRTFCERRtHRGKUL I U

TH+ + L ++ + U+++DE++FF++ +++ oR++K +1+

AGLHRSFEQKMFPPIURIFPYCSIUKYIGRTCIKCHQHHRCFHURKHRDKTLILRGGSELYUTCCHHRARLDGTFQRKPFGRILHLUFLRESUUKLTRUCIECF-RERRYTKRLGTEKEUEUIGGRDKYHSUCRLRRLDGTFQRKRFGSILHLUPLRESUUKLTRUCMECF-RERRYTKRLGLEKEUEUIGGRDKYHSUCRLRRLOGTFQRKRFGSILHLUPLRESUUKLHRUCfECY-RERSYTKRLGREREUEUIGGRDKYHSUCRRRRLHGDFKRELFGHUYKLLSLRETUSSLTRICUKCY-CDRSFSKRUTENKEUMDIGGKDKYIRUCRKRRLDGTFQRRPFHHILHLIPLSEMUUKLTRUCMKCF-KERSFSKRLGTETEIElI6GHODYQSUCRKRRLOGTFQRKPFN ILHLIPLSEIUUKLTRUCMKCF-KERSFSKRLGEETEIElIGGIDIYQSUCRKRRLOGTFQRKPFHHILOLIPLSEfUUKLTRUCMKCF-KERSFSKRLGTETKIEIIGGHDIYQSUCRKRRLDGTFORKPFSHISELIPLRENUTKLNRUCfYCY-KHGSFSKRLGDKMEIEUIGGSDKYKSUCRKR+L+++F+++ F + + ++ + + +++C+ C + *R++ + + GG + Y ++C+

ASFV CLKHTFIKQLQPIKYHuman CYFKKRSGQPRPDONKEICPUPOKPGERURRRKLFRPQQILQCSPRHMouse CYFKKSSRQTRGSONKN-CLULGQPGERLUURKLFRSQQULQYHSRHChicken CYFQKRPQQL-GSEHKENUPIGUKQLDfPRSRKIFRSFPV CFFSHMPV CYIDSVV CYIDSVar V CYIDSSFV CYEEHomology C+

FIG. 6. Alignment of amino acid sequences of TK from ASFV, humans (H) (3), mice (M) (13), chickens (Ch) (17), vaccinia virus (VV) (37),variola virus (Var V) (6), monkeypox virus (MPV) (6), fowlpox virus (FPV) (2), and Shope fibroma virus (SFV) (32). The consensus sequence andthe positions at which identical residues are observed in seven or more of the nine aligned sequences are indicated (+) below the alignment. Themaximum identity with vaccinia virus TK was located in the nucleotide-binding site at residues 17 to 25 in the ASFV TK gene, which correspondto residues 11 to 19 in the vaccinia virus TK gene (7), and in the possible nucleoside-binding site at residues 88 to 122 in ASFV TK, whichcorrespond to residues 77 to 116 in the vaccinia virus TK gene.

The polypeptide synthesized had a molecular weight ofabout 22,400 (Fig. 5, lane C), in good agreement with the sizepredicted from the open reading frame gene sequence. Inconclusion, we have identified and expressed, both in bac-

terial cells and by cell-free translation, the TK gene ofASFV.Comparison of the ASFV TK gene with poxvirus and

cellular TK genes. In addition to providing a tool for the

TKASFVHumanMouseChickenFPVMPVvVVar V

Homology

ASFVHumanMouseChickenFPVMPVVVVar VSFVHomology

ASFVHumanMouseChickenFPVMPVVVVar VSFH lHomology

J. VIROL.

NOTES 1051

TABLE 2. TK activity in cell extract after translationsa

Source of Plasmid TK activityRNA (cpm)

BHK (TK-) 10,046BHK 17,629BHK (TK-) with cycloheximideb 18,126In vitro transcription pGEM204aS 90,737In vitro transcription pGEM204aX 97,308In vitro transcription pGEM204aE 10,133

a The level ofTK activity was assayed by conversion of [3H]thymidine into[3H]thymidylate. Plasmid pGEM204a (Fig. 1) was linearized with Sail(pGEM204aS) or XhoI (pGEM204aX), transcribed with T7 RNA polymeraseor further linearized with EcoRI (pGEM204aE), and then transcribed with SP6RNA polymerawe. About 3 S.g of RNA transcribed in vitro and about 35 ,ug oftotal RNA from infected or uninfected BHK cells were translated for 4 h in amicrococcal nuclease-treated reticulocyte lysate, and the TK activity wasassayed in the presence of 50 p.M thymidine to stabilize nascent TK activity(36).

b Cells infected with ASFV.

development of ASFV genetics, the TK gene is of interestfor further defining phylogenetic relationships among largeDNA viruses. The nucleotide sequence of the ASFV TKgene includes an open reading frame of 196 codons, with acalculated molecular weight for ASFV TK of 22,394, onlyslightly higher than that of the vaccinia virus TK, which is20,102 (37). Homology analysis of the deduced ASFV TKgene protein sequence indicates a relationship with bothpoxvirus and cellular TK proteins (Fig. 6). The percentagesof identity in TK sequences aligned by the FASTP program(14) were shown to be 28% for human, 29.5% for mouse,26.1% for chicken, 26.2% for fowlpox virus, 31.3% formonkeypox virus, 32.4% for vaccinia virus, 31.8% for vari-ola virus, and 29% for Shope fibroma virus TK polypeptides.The identification of the ASFV TK gene will now permit

direct construction, by genetic manipulation, of TK- mu-tants to serve as recipients for reinsertion of foreign genes.One important change that occurs in the adaptation ofASFVstrain E70 to monkey cells is the loss of pathogenicity (31).Consequently, ASFV could be used as a model in studies ofpathogenesis of persistent infections by inserting genespresumably involved in pathogenesis into the genome of theavirulent E70MS14 strain (31).

We are grateful to S. Fernandez and J. Alvarez for excellenttechnical assistance.

This work was supported by a grant from Comision Asesora parael Desarrollo de la Investigaci6n Cientifica y Tdcnica (Spain).

REFERENCES

1. Almendral, J. M., R. Blasco, V. Ley, A. Blasco, A. Talavera, andE. Vihuela. 1984. Restriction site map of African swine fevervirus DNA. Virology 133:258-270.

2. Boyle, D. B., B. E. H. Coupar, A. J. Gibbs, L. J. Seigman, andG. W. Both. 1987. Fowlpox virus thymidine kinase: nucleotidesequence and relationships to other thymidine kinases. Virology156:355-365.

3. Bradshaw, H. D., Jr., and P. L. Deininger. 1984. Humanthymidine kinase gene: molecular cloning and nucleotide se-quence of a cDNA expressible in mammalian cells. Mol. Cell.Biol. 4:2316-2320.

4. Duffand, G. D., P. E. March, and M. Inouye. 1987. Expressionand secretion of foreign proteins in Escherichia coli. MethodsEnzymol. 153:492-507.

5. Escribano, J. M., and E. Tabaires. 1987. Proteins specified byAfrican swine fever virus. V. Identification of immediate early,

early and late proteins. Arch. Virol. 92:221-232.6. Esposito, J. J., and J. C. Knight. 1984. Nucleotide sequence of

the thymidine kinase gene region of monkeypox and variolavirus. Virology 135:561-567.

7. Gentry, G. A. 1985. Locating a nucleotide-binding site in thethymidine kinase of vaccinia virus and of herpes simplex virusby scoring triply aligned protein sequences. Proc. Natl. Acad.Sci. USA 82:6815-6819.

8. Hess, W. R. 1971. African swine fever virus. Virol. Monogr.9:1-33.

9. Hess, W. R. 1981. African swine fever: a reassessment. Adv.Vet. Sci. Comp. Med. 25:39-69.

10. Hiraga, S., K. Igarashi, and T. Yura. 1967. Deoxythymidinekinase-deficient mutant of Escherichia coli. I. Isolation andsome properties. Biochim. Biophys. Acta 145:41-51.

11. Igarashi, K., S. Hiraga, and T. Yura. 1967. A deoxythymidinekinase-deficient mutant of Escherichia coli. II. Mapping andtransduction studies with phage 480. Genetics 37:643-654.

12. Inouye, S., and M. Inouye. 1985. Up-promoter mutations in thelpp gene of Escherichia coli. Nucleic Acids Res. 13:3101-3110.

13. Lin, P.-F., H. B. Lieberman, D.-B. Yeh, T. Xu, S.-Y. Zhao, andF. H. Ruddle. 1985. Molecular cloning and structural analysis ofmurine thymidine kinase genomic and cDNA sequences. Mol.Cell. Biol. 5:3149-3156.

14. Lipman, D. J., and W. R. Pearson. 1985. Rapid and sensitiveprotein similarity searches. Science 227:1435-1441.

15. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

16. Mebus, C. A. 1988. African swine fever. Adv. Virus Res.35:251-269.

17. Merrill, G. F., R. M. Harland, M. Groudine, and S. L.McKnight. 1984. Genetic and physical analysis of the chicken tkgene. Mol. Cell. Biol. 4:1769-1776.

18. Piccini, A., and E. Paoletti. 1988. Vaccinia: virus, vector,vaccine. Adv. Virus Res. 34:43-64.

19. Polatnick, J., and W. Hess. 1970. Altered thymidine kinaseactivity in culture cells inoculated with African swine fevervirus. Am. J. Vet. Res. 31:1609-1613.

20. Polatnick, J., and W. Hess. 1972. Increased deoxyribonucleicacid polymerase activity in African swine fever virus-infectedculture cells. Arch. Gesamte Virusforsch. 38:383-385.

21. Roizman, B., and F. J. Jenkins. 1985. Genetic engineeringof novel genomes of large DNA viruses. Science 229:1208-1214.

22. Salas, M. L., J. Rey-Campos, J. M. Almendral, A. Talavera, andE. Vinuela. 1986. Transcription maps of African swine fevervirus. Virology 152:228-240.

23. Sanchez Botia, C. 1982. Peste porcina africana. Nuevos de-sarrollos. Rev. Sci. Tech. O.I.E. 1:991-1029.

24. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc-ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci.USA 74:5463-5467.

25. Sargan, D. R., S. P. Gregory, and P. H. W. Butterworth. 1982.A possible novel interaction between the 3'-end of 18 S ribo-somal RNA and the 5'-leader sequence of many eukaryoticmessenger RNAs. FEBS Lett. 147:133-136.

26. Schnitzlein, W. M., N. Ghildyal, and D. N. Tripatly. 1988. Arapid method for identifying the thymidine kinase genes ofavipoxvirus. J. Virol. Methods 20:341-352.

27. Tabares, E. 1987. Characterization of African swine fever virusproteins, p. 51-61. In Y. Becker (ed.), African swine fever.Martinus Nijhoff Publishing, Boston.

28. Tabares, E., M. A. Marcotegui, M. Fernandez, and C. SanchezBotija. 1980. Proteins specified by African swine fever virus. I.Analysis of the structural proteins and antigenic properties.Arch. Virol. 66:49-59.

29. Tabares, E., J. Martinez, F. R. Gonzalvo, and C. Sanchez Botja.1980. Proteins specified by African swine fever virus. II. Anal-ysis of proteins in infected cells and antigenic properties. Arch.Virol. 66:119-132.

30. Tabares, E., J. Martinez, E. Martin, and J. M. Escribano. 1983.

VOL. 65, 1991

1052 NOTES

Proteins specified by African swine fever virus. IV. Glycopro-teins and phosphoproteins. Arch. Virol. 77:167-180.

31. Tabares, E., I. Olivares, G. Santurde, M. J. Garcia, E. Martin,and M. E. Carnero. 1987. African swine fever virus DNA:deletions and additions during adaptation to growth in monkeykidney cells. Arch. Virol. 97:333-346.

32. Upton, C., and G. McFadden. 1986. Identification and nucleo-tide sequence of the thymidine kinase gene of Shope fibromavirus. J. Virol. 60:920-927.

33. Urzainki, A., E. Tabares, and L. Carrasco. 1987. Proteinssynthesized in African swine fever virus infected cells analyzedby two dimensional gel electrophoresis. Virology 160:286-291.

34. Vifiuela, E. 1985. African swine fever virus. Curr. Top. Micro-

biol. Immunol. 116:151-170.35. Wardley, R. C., C. M. Andrade, D. N. Black, F. L. Castro

Portugal, L. Enjuanes, W. R. Hess, C. Mebus, A. Ordas, D.Rutili, J. M. Sanchez Vizcaino, J. D. Vigario, and P. J. Wil-kinson. 1983. African swine fever virus. Arch. Virol. 76:73-90.

36. Weir, J. P., G. Bajszar, and B. Moss. 1982. Mapping of thevaccinia virus thymidine kinase gene by marker rescue and bycell-free translation of selected mRNA. Proc. Natl. Acad. Sci.USA 79:1210-1214.

37. Weir, J. P., and B. Moss. 1983. Nucleotide sequence of thevaccinia virus thymidine kinase gene and the nature of sponta-neous frameshift mutations. J. Virol. 46:530-537.

J. VIROL.