Jul 5, 1990 - The thymidine kinase (TK) gene of African swine fever virus (ASFV) was .... 1. Physical map of plasmids containing the TK gene. Viral DNA was ...
Vol. 65, No. 2
JOURNAL OF VIROLOGY, Feb. 1991, p. 1046-1052
0022-538X/91/021046-07$02.00/0 Copyright C) 1991, American Society for Microbiology
Expression and Characterization of the Thymidine Kinase Gene 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, Spain Received 5 July 1990/Accepted 26 October 1990
The thymidine kinase (TK) gene of African swine fever virus (ASFV) was located within the viral genome by using two degenerate oligonucleotide probes derived from sequences of the vaccinia virus and cellular TK genes. The TK gene was mapped within a 0.72-kbp Bglll-XhoI fragment (0.242 to 0.246 map units) derived from a 23.9-kbp Sal-B fragment of the ASFV genome. Identification of this region as the ASFV TK gene was confirmed by expression of TK in Escherichia coli and by the synthesis of active TK in a cell-free system programmed with RNA synthesized in vitro. The sequenced gene for TK includes an open reading frame of 588 nucleotides encoding a protein of 196 amino acids. The deduced amino acid sequence shows 32.4% identity with the TK of vaccinia virus.
infected with ASFV strain E70MS44 (31) was digested with ClaI, SalI, and SmaI and electrophoresed in an agarose gel. The DNA fragments were denatured in the gel, transferred to a nitrocellulose filter, and hybridized to the 32P-oligonucleotide probes (32). Probe 1 annealed with the Sall-A, -B, -H', and -E fragments (see Fig. 4). This indicated possible viral sequences related to proteins that contain an ATPbinding domain, since probe 1 represents a consensus sequence for such a nucleotide-binding site. Probe 2 annealed only with the SalI-B and SalI-E fragments (data not shown). We studied the Sall-B fragment because SalI-E encodes late proteins (22) and the TK of ASFV is an immediate-early protein. ASFV DNA isolated from strain E70MS14 was used in 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 subjected to restriction enzyme analysis, and the DNA fragments were hybridized to 32P-labeled oligonucleotides in order to map the TK gene more precisely. A region of homology with the TK gene was localized in a 390-bp PstI-HindIII fragment of pRPEM204 (Fig. 2, lanes 2). Sail-B represents unique sequences on the viral genome because this segment hybridizes only with fragments SmaI-A, ClaI-B, -F, -P, and -R, and Sall-B (reference 31 and data not shown), indicating that the TK gene is a single-copy gene. Identification of the ASFV TK gene. The TK gene and its flanking regions were sequenced by the dideoxynucleotide chain termination method (24), with recombinant pUC18 or pGEM3Z as the template. The nucleotide and deduced protein sequences are presented in Fig. 3. The TK gene maps at coordinates 0.242 to 0.246 (Fig. 4) in E70MS14 DNA (31). The predicted ASFV TK protein comprises 196 amino acids, with a calculated molecular weight of 22,394. The translation initiation codon of the open reading frame has been assigned on the basis of homology of the aminoterminal domain to that of other TK proteins. Also, the proposed initiator AUG is flanked by nucleotides characteristic of preferred eucaryotic initiation sites, whereas the next ATG codon, located 93 nucleotides downstream, is not flanked by consensus translation initiation sequences (25). Characterization and expression of the TK gene in Esche-
The thymidine kinase (TK) marker has proven valuable in the establishment of vaccinia virus and herpes simplex virus cloning vectors and for the development of surrogate genetics in these viruses (18, 21). Therefore, it is of particular interest to identify and characterize the African swine fever virus (ASFV) TK gene as a potential target site for genetic manipulations. ASFV is an icosahedral cytoplasmic DNA virus with properties common to iridoviruses and poxviruses (30, 34). It causes a highly contagious and generally fatal disease of pigs (8, 9, 16, 23, 34, 35). Productive infection is accompanied by a weak shutoff of host protein synthesis and the appearance of 44 virus-specific polypeptides ranging in molecular weight from 9,500 to 243,000 (5, 27, 29, 30). These polypeptides have been classified as immediate early, early, and late (5, 33). Increases in the levels of two enzymatic activities which may be involved in DNA replication, those of a TK (19) and a DNA polymerase (20), have been detected following ASFV infection. ASFV infection of baby hamster kidney (BHK) cells may induce the formation of a TK different from that found in normal cells (19). This activity can be induced in BHK cells deficient in TK as an immediate-early protein, and we have generated ASFV TK- mutants by bromodeoxyuridine mutagenesis (data not shown). These results strongly suggested that the TK induced after infection is encoded by ASFV. Here we report the identification, characterization, and expression of the ASFV TK gene as an initial step toward the genetic manipulation of this virus.
Mapping of the ASFV TK gene. The use of degenerate oligonucleotides to locate the TK genes of Shope fibroma virus and avipoxvirus (26, 32) suggested that such oligonucleotides could also be used for ASFV, given the phylogenetic proximity between ASFV and the poxviruses. Oligonucleotide 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 the vaccinia virus, human, and mouse TK genes (32), were synthesized by F. Barahona in Centro de Biologia Molecular, Madrid, Spain. ASFV DNA isolated from MS cells *
Corresponding author. 1046
VOL. 65, 1991
NOTES
1047
E
B
p
x
H /Ea BEn / 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), and recombinant 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 obtain plasmid pRPEM204. The larger BgIII fragment and the HindIII-BglII fragment of this plasmid were cloned in pGEM3Z (2,743 bp) (Promega Biotec, Madison, Wis.) and pINIIIAl (7.4 kbp) (kindly supplied by E. Garcia, Madrid, Spain) (4, 12), respectively, to yield pGEM204a and pINTK14, 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 into the HindIll and BamHI sites of pINIIIAl (4, 12) to yield plasmid pINTK14 (Fig. 1). In this clone, the N-terminal five amino acids of ASFV TK protein are replaced by the five
A
B
1 23456789 1 2345 6789
1769bp 676 bp 241 bp
a&
amino acids encoded by linker DNA from pINIIIAL. Plasmid pINTK14 was used to transform E. coli KY893 (TK-). Untransformed E. coli KY893 TK- is unable to grow on selection medium containing 5-fluorouracil (11). Only clones containing viral TK from pINTK14, which complemented the cellular TK defect, survived on the drug selection plates. It was important to confirm the TK complementation of KY893 TK- cells harboring the plasmid pINTK14 by examining incorporation of [3H]thymidine into the bacterial cells. The KY893 mutant of E. coli transformed with the plasmids was 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 pRPEM 204. (A) Lanes 1 to 8, pRPEM204 digested with HindlIl plus EcoRI, HindlIl plus PstI, XhoI plus PstI, BgIIH plus PstI, BglII plus HindIII, BglII plus HindlIl plus EcoRI, EcoRI, and BamHI, respectively; 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-labeled oligonucleotide pool (map positions of the various restriction enzymes are shown in Fig. 1). The asterisk indicates the 390-bp PstI-HindIII fragment of pRPEM204.
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J. VIROL.
NOTES
MA B
x
p
H I
I1I iT TK
B GCRARRRGTR CCGCRRRTRR RRRRRCRRCG RRGGGCTCCT CCRRRTCTOG TTCCTCCRGR GGCCRCRCCG OCRRRRCCCR TGCTTCTTCG TCCRTGCRTT CCGGGRTGCT CTRTRRRGRT Bgl U RTGGTRRRTR TTGCTRfiRTC TRGRGGCRTT CCGRTTTRCC RGRRTGGRTC GCGTCTTRCT fid m RBS RRRRGTGRRT TGGRGRRRRR RRTRRRCffILCRM RTG RRT RTR RTT RGG fiflCtL
M
CCT GGR P G TTT CTT F L OTC TTC RTR
RCR T RTT I RRR V F I K TCC GGT RTR CAG S G I o TTR TCT GRC GTG L S D V GCG CRT TTT TTT A H F F RRR ATT RTT RTT K I I I RRG K RCG T
RGC S TGC C TCT RCC S T CTR CGR
RTT I CRT H
L
GGR G GRC D CTT L
R TCT S GRT D GCG A
CTT L RTT I RRR K CCC P CTC L TTR L GGR G
I
N
R
I
L
K
GTG V TRC Y RRC N
CTO GGR CCC RTG TTT 0CC GOC RRR RCT
RRG
RCA CR0
L RTG M RCC T CRR Q GRT D RRR K
G CTC L CGR R TGT C RTC I TGC C
K RCC T RTR I CTC RRT OCT L N A
P
M
F
A
G
K T
ORR CGT TTG GRR RRR RRR GTR R L E K RRR ACT RTT RRR K T I K RTC RTR GRR RGC K I I E S CRT GCR GTT GTC GTR
E GRC D RRR H
CGC R TCC S
A RCC T TTC F
K V RCR CRC T H
T GRT V V V D TGG GCR GRG GRR W A E E GRG CRG RRR RTO E Q K M
O GRR E GRR E
TTT F
PsI
CCG p CGC R RRC
CCC p RCC T GCR N A
TGT TGT C C RRR TRT K
Y
TOG OTT RRG RAC TGC RTC GTT COT RTT TTT CCT TRC I V R I F P. y C S W V K TGT RTG RRR TGT RRC CRR CRT RRT OCR TGC TTT RRT C M K C N Q H N A C F N GRC RRG RCO CTT RTC CTT GCG GOR GOR RGT GRR CTO D K T L I L A G G S E L RRC RRC TGT CTR RRR RRT RCR TTT RTT RRG CR0 TTG N N C L K N T F I K Q L TRR ARRTCTTRTR CRRTRRTOGR TCRTTRTCTT RRRRRRTTRC Ter
TRT RTT GOC Y I G GTG COT RRG V
R
K
TRC OTR RCR Y
V
T
CRR CCT RTT Q P I RRGRTRTTTR
XhOI
TRCGRRGALfLQGGGGCCRTC TRTTRCTCTO CTRRRCCROG GTTTTTRRCG RTOTATRROC RCRROROTRT TTATGCCTGT
CCTTTCTTTT CCTTGGCCTC TRGRTCCCRT TORTTARTCC
TROCCCGTCG RRCGCROCTT TGOGTTTRTT TRRACTCGTT
RRRRCCRRTO TRCCOCRGCR RRCTRTRTTR RCTRRGTTTT
RRRRRGRGTT TRCRRCRACT RRRCGOGTRR 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 subcloned to yield plasmids pRPEM211, pRPEM210, and pRPEM213, respectively. These plasmids were sequenced, and the open reading frames of both strands were determined. Arrows show the direction and extent of sequence determination from each restriction site. Restriction enzyme cleavage sites 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 site are underlined.
VOL. 65, 1991 .2 I
B
I
C
l
K C
B
I H'i
E I
_"
l
4 156 Kbp A
C
I IHI
I
.
I
..
BH
P X
p
a
I
I
I
.
.
E
IGI~
ES 1
CE
C
I-I a
a
.a
.6
.4
F'I
E
1049
NOTES
PRPEM
513
E
aWa
V
pRPEM 204
TK
FIG. 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. Restriction enzymes 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 of deoxyadenosine, and 1 ,uCi of [3H]thymidine (82.2 Ci/mmol; New England Nuclear Corp., Boston, Mass.). The cells were lysed with 2% sodium dodecyl sulfate-0.2 N NaOH
ABC D
P73
1P2 3.5
IP16
._
FIG. 5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of polypeptides translated from different RNA preparations. The RNA resulting from in vitro transcription by T7 RNA polymerase 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. Labeled proteins were analyzed by 12% polyacrylamide-sodium dodecyl sulfate gel electrophoresis, and the labeled polypeptides were visualized by autoradiography of the dried gel. Lane D, No RNA; lane A, polypeptides from uninfected MS cells; lane B, infected cells labeled with [35S]methionine from 20 to 22 h postinfection (28). IP, Polypeptides from infected cells; numbers are molecular masses in kilodaltons.
and precipitated with ice-cold 10% trichloroacetic acid. Precipitates were collected by filtration on glass fiber filters and counted in toluene-based scintillation fluid. When these cells were grown in the presence of isopropyl-p3-D-thiogalactopyranoside (IPTG), the incorporation of [3H]thymidine was increased about 25 times in relation to that in parallel cultures in the absence of IPTG (Table 1). Since the pINIIIAl vector contains the lac promoter-operator region, the expression of the viral TK gene can be induced by a lac inducer such as IPTG. The increase of [3H]thymidine indicates that the expression of the ASFV TK gene is regulated by a lac promoter-operator region. Cell-free translation of synthetic RNA. Additional evidence that the BglII-XhoI fragment contains the TK gene was obtained by the synthesis of active TK protein in a cell-free system programmed with RNA synthesized in vitro, using plasmid pGEM204a (Fig. 1) and T7 or SP6 RNA polymerase in the presence of 0.5 mM m7G(5')ppp(5')G. The RNA was translated in a reticulocyte cell-free system, and samples from the translation mixture were then assayed for TK activity. Positive controls were early RNAs from BHK TKcells (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 was isolated from infected and mock-infected cells by guanidine isothiocyanate-CsCl extraction (15). Active TK was obtained upon translation of RNA from infected cells (Table 2).
TABLE 1. Incorporation of [3H]thymidine into DNA of plasmidtransformed TK- E. coli (KY893 mutant strain)a
Bacterial strain (culture supplementb)
KY893 NH522 KY893 (FUdr) KY893 (FUdr + IPTG) KY893 (FUdr) KY893 (FUdr + IPTG)
.
asma
cpm/ml of bacterial culture 37
1,877 pINIIIAl pINIIIAl pINTK14 pINTK14
37 58 166
4,110
For selection of TK expression, the TK-deficient (TK-) strain KY893 (10) of E. coli was used. TK activity was assayed by measuring the uptake of a
as acid-precipitable radioactivity. [3H]thymidine b
FUdr,
Fluorodeoxyuridine.
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TK ASFV Human Mouse Chicken FPV MPV
fHIIRKLKP6TISLULGPfFRGKTTFLIHCIYMLERLEKKUUFIKSTKHTR- OK MSC INLPTULP6SPSKTRGQIQUILGPMFSGKSTELIRRURRFQIROYKCLUIKYRKOTRY----SS
MSYIHLPTULPSSPSKTRGQIQUILGPnFSGKSTELIRRURRFQIRQYKCLUIKYRKDTRY----SK IHCLTUPGUHPGSPGRPRGQIQUIFGPfFSGKSTELIRRURRFQLRQYRCLLUKYRKDTRYC---TT 1SSGSIHUITGPnFSGKTSELURRIKRFnLSNFKCIIIKHCGONRYNDEDIN nHGGHIOLIIGPnFSGKSTELIRRURRYQ1RQYKCUTIKYSNDNRYG----T INGGHIQLIIGPMFSGKSTELIRRURRYQIRQYKCUTIKYSHDHRYG----T
vV Var V
IHGGHIQLIIGPIFSGKSTELIRRURRYQIRQYKCUTIKYSHDHRYG----T n1IGHIHLIIGP1FRGKSTELIRLURRYQIRKHKCLUUKYEKDIRYG----N G I + GPfF+GK+++L +++ + + ++ +K+ + R+
Homology
ASFV Human Mouse Chicken FPV MPV VV Var V
SFV Homology
ASFV Human Mouse Chicken FPV MPV VV Var V SFH l Homology ASFV Human Mouse Chicken FPV
TIKTHSGIQLRPKQCKIIESTQLSDU--GSLTO--IHRUU-UDERHFFODLIK-CRTUREEEKIIIL
SFCTHD.-----RHTERLPRCLLRDURQERL----6URUIGIDEGQFFPDInEFCERnRHRGKTUIU SFSTHD-----RHT1DRLPRCfLRDUTQELL----GURUIGIOEGQFFPDIUDFCE1IRHEGKTUIU GUSTHD-----RHTMERRPRCRLQDUYQERL----GSRUIGIDEGQFFPDIUEFCEKIRHTGKTUIU KUYTHD-----LLFMERTRSSHLS-ULUPTLLHD-GUQUIGIDERQFFLDIUEFSESMRHLGKTUIU GLUTHD-----KHHFRRLEUTKLCDU----LERITDFSUIGIDE6QFFPDUUEFCERMRHEGKIUIU GLUTHD-----KHHFERLERTKLCDU----LESITDFSUIGIDEGQFFPDIUEFCERMRNEGKIUIU GLUTHD-----KNHFERLERTKLCDU----LERITDFSUIGIDEGQFFPOUUEFCERMRHEGKIUIU GUCTHD-----NS I TRUCTPSLDK I ----DSURENREUII6OEGQFFPNIRTFCERRtHRGKUL I U TH+
U+++DE++FF++
+++
oR++K +1+
RRLDGTFQRKRFGSILHLUPLRESUUKLTRUCMECF-RERRYTKRLGLEKEUEUIGGRDKYHSUCRL
RRLHGDFKRELFGHUYKLLSLRETUSSLTRICUKCY-CDRSFSKRUTENKEUMDIGGKDKYIRUCRK RRLDGTFQRRPFHHILHLIPLSEMUUKLTRUCMKCF-KERSFSKRLGTETEIElI6GHODYQSUCRK RRLOGTFQRKPFN ILHLIPLSEIUUKLTRUCMKCF-KERSFSKRLGEETEIElIGGIDIYQSUCRK
RRLOGTFQRKPFHHILOLIPLSEfUUKLTRUCMKCF-KERSFSKRLGTETKIEIIGGHDIYQSUCRK RRLDGTFORKPFSHISELIPLRENUTKLNRUCfYCY-KHGSFSKRLGDKMEIEUIGGSDKYKSUCRK R+L+++F+++ F + + ++ + + +++C+ C
+
*R++
+ + GG + Y ++C+
CLKHTFIKQLQPIKY
CYFKKRSGQPRPDONKEICPUPOKPGERURRRKLFRPQQILQCSPRH CYFKKSSRQTRGSONKN-CLULGQPGERLUURKLFRSQQULQYHSRH CYFQKRPQQL-GSEHKENUPIGUKQLDfPRSRKIFRS
VV
CYIDS
Homology
+
RRLOGTFQRKRFGSILHLUPLRESUUKLHRUCfECY-RERSYTKRLGREREUEUIGGRDKYHSUCRR
CFFSH CYIDS
SFV
L ++
AGLHRSFEQKMFPPIURIFPYCSIUKYIGRTCIKCHQHHRCFHURKHRDKTLILRGGSELYUTCCHH RARLDGTFQRKPFGRILHLUFLRESUUKLTRUCIECF-RERRYTKRLGTEKEUEUIGGRDKYHSUCRL
MPV
Var V
+
CYIDS CYEE 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 and the positions at which identical residues are observed in seven or more of the nine aligned sequences are indicated (+) below the alignment. The maximum identity with vaccinia virus TK was located in the nucleotide-binding site at residues 17 to 25 in the ASFV TK gene, which correspond to 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, which correspond to residues 77 to 116 in the vaccinia virus TK gene.
The polypeptide synthesized had a molecular weight of about 22,400 (Fig. 5, lane C), in good agreement with the size predicted from the open reading frame gene sequence. In conclusion, we have identified and expressed, both in bac-
terial cells and by cell-free translation, the TK gene of ASFV. Comparison of the ASFV TK gene with poxvirus and cellular TK genes. In addition to providing a tool for the
NOTES
VOL. 65, 1991
TABLE 2. TK activity in cell extract after translationsa Source of RNA
BHK (TK-) BHK BHK (TK-) with cycloheximideb In vitro transcription In vitro transcription In vitro transcription
Plasmid
TK activity
pGEM204aS pGEM204aX pGEM204aE
(cpm) 10,046 17,629 18,126 90,737 97,308 10,133
a The level of TK 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 polymerase or further linearized with EcoRI (pGEM204aE), and then transcribed with SP6 RNA polymerawe. About 3 S.g of RNA transcribed in vitro and about 35 ,ug of total RNA from infected or uninfected BHK cells were translated for 4 h in a micrococcal nuclease-treated reticulocyte lysate, and the TK activity was assayed 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 interest for further defining phylogenetic relationships among large DNA viruses. The nucleotide sequence of the ASFV TK gene includes an open reading frame of 196 codons, with a calculated molecular weight for ASFV TK of 22,394, only slightly higher than that of the vaccinia virus TK, which is 20,102 (37). Homology analysis of the deduced ASFV TK gene protein sequence indicates a relationship with both poxvirus and cellular TK proteins (Fig. 6). The percentages of 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% for monkeypox virus, 32.4% for vaccinia virus, 31.8% for variola 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- mutants to serve as recipients for reinsertion of foreign genes. One important change that occurs in the adaptation of ASFV strain E70 to monkey cells is the loss of pathogenicity (31). Consequently, ASFV could be used as a model in studies of pathogenesis of persistent infections by inserting genes presumably involved in pathogenesis into the genome of the avirulent E70MS14 strain (31). We are grateful to S. Fernandez and J. Alvarez for excellent technical assistance. This work was supported by a grant from Comision Asesora para el Desarrollo de la Investigaci6n Cientifica y Tdcnica (Spain).
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