Patent Application: US-47506395-A

Abstract:
what is described is a recombinant poxvirus , such as vaccinia virus or fowlpox virus , containing foreign dna from marek &# 39 ; s disease virus . what is also described is a vaccine containing the recombinant poxvirus for inducing an immunological response in a host animal inoculated with the vaccine .

Description:
the invention is directed to recombinant poxviruses containing therein a dna sequence from mdv in a nonessential region of the poxvirus genome . the recombinant poxviruses express gene products of the foreign mdv gene . in particular , mdv genes encoding mdv structural proteins were isolated , characterized and inserted into nyvac ( vaccinia virus ) and trovac ( fowlpox virus ) recombinants . cell lines and virus strains . the strain of fpv designated fp - 1 has been previously described ( taylor et al ., 1988a , b ). it is an attenuated vaccine strain useful in vaccination of day old chickens . the parental virus , duvette strain , was obtained from rhone merieux , lyon , france . the virus received by virogenetics was subjected to four successive plaque purifications . one plaque isolate was further amplified in primary cef cells , and a stock virus , designated as trovac , established . trovac was deposited feb . 6 , 1997 under the terms of the budapest treaty with the american type culture collection ( atcc ), 12301 parklawn drive , rockville , md ., 20852 , usa , atcc accession number vr - 2553 . all recombination tests , plaque assays and amplifications with trovac or trovac based recombinants were performed in primary cef monolayers made from 10 - 11 day old embryonated eggs of spf origin . the vaccinia virus strain used as a rescue for mdv sequences was nyvac ( vp866 ). nyvac is a highly attenuated strain of vaccinia virus derived from the copenhagen strain by deletion of 18 open reading frames which have been implicated in determining viral virulence and host range restriction . nyvac was deposited on mar . 6 , 1997 under the terms of the budapest treaty with the atcc , accession number vr - 2559 . recombinant plaque selection and virus amplifications were performed on rabbit kidney cells ( rk13 , atcc ccl37 ). plasmids pmdv517 and puc13gb contain dna sequences encoding mdv gd and gb glycoproteins from strain rb1b . plasmid puc13gb contains a 3 . 9 kb dna fragment of genomic dna of mdv ( strain rb1b ). the fragment which contains the mdvgb gene is inserted into puc13 as an ecori - sali fragment . the sequence of the inserted fragment is described in ross et al . ( 1989 ). plasmid pmdv517 contains a 5 . 2 kb dna fragment of genomic dna of mdv ( strain rb1b ). the fragment which contains the mdvgd gene is inserted at the ecori site of puc13 . the sequence of the fragment is described in ross et al . ( 1991 ). to develop a new vaccinia vaccine strain , the copenhagen vaccine strain of vaccinia virus was modified by the deletion of six nonessential regions of the genome encoding known or potential virulence factors . the sequential deletions are detailed below . all designations of vaccinia restriction fragments , open reading frames and nucleotide positions are based on the terminology reported in goebel et al . ( 1990a , b ). the deletion loci were also engineered as recipient loci for the insertion of foreign genes . the regions sequentially deleted in nyvac are listed below . also listed are the abbreviations and open reading frame designations for the deleted regions ( goebel et al ., 1990a , b ) and the designation of the vaccinia recombinant ( vp ) containing all deletions through the deletion specified : dna cloning and synthesis . plasmids were constructed , screened and grown by standard procedures ( maniatis et al ., 1982 ; perkus et al ., 1985 ; piccini et al ., 1987 ). restriction endonucleases were obtained from gibco / brl , gaithersburg , md . ; new england biolabs , beverly , mass . ; and boehringer mannheim biochemicals , indianapolis , ind . klenow fragment of e . coli polymerase was obtained from boehringer mannheim biochemicals . bal - 31 exonuclease and phage t4 dna ligase were obtained from new england biolabs . the reagents were used as specified by the various suppliers . synthetic oligodeoxyribonucleotides were prepared on a biosearch 8750 or applied biosystems 380b dna synthesizer as previously described ( perkus et al ., 1989 ). dna sequencing was performed by the dideoxy - chain termination method ( sanger et al ., 1977 ) using sequenase ( tabor et al ., 1987 ) as previously described ( guo et al ., 1989 ). dna amplification by polymerase chain reaction ( pcr ) for sequence verification ( engelke et al ., 1988 ) was performed using custom synthesized oligonucleotide primers and geneamp dna amplification reagent kit ( perkin elmer cetus , norwalk , conn .) in an automated perkin elmer cetus dna thermal cycler . excess dna sequences were deleted from plasmids by restriction endonuclease digestion followed by limited digestion by bal - 31 exonuclease and mutagenesis ( mandecki , 1986 ) using synthetic oligonucleotides . cells , virus , and transfection . the origins and conditions of cultivation of the copenhagen strain of vaccinia virus has been previously described ( guo et al ., 1989 ). generation of recombinant virus by recombination , in situ hybridization of nitrocellulose filters and screening for b - galactosidase activity are as previously described ( panicali et al ., 1982 ; perkus et al ., 1989 ). construction of plasmid psd460 for deletion of thymidine kinase gene ( j2r ). referring now to fig1 plasmid psd406 contains vaccinia hindiii j ( pos . 83359 - 88377 ) cloned into puc8 . psd406 was cut with hindiii and pvuii , and the 1 . 7 kb fragment from the left side of hindiii j cloned into puc8 cut with hindiii / smai , forming psd447 . psd447 contains the entire gene for j2r ( pos . 83855 - 84385 ). the initiation codon is contained within an nlaiii site and the termination codon is contained within an sspi site . direction of transcription is indicated by an arrow in fig1 . to obtain a left flanking arm , a 0 . 8 kb hindiii / ecori fragment was isolated from psd447 , then digested with nlaiii and a 0 . 5 kb hindiii / nlaiii fragment isolated . annealed synthetic oligonucleotides mpsyn43 / mpsyn44 ( seq id no : 1 / seq id no : 2 ) ## str1 ## were ligated with the 0 . 5 kb hindiii / nlaiii fragment into puc18 vector plasmid cut with hindiii / ecori , generating plasmid psd449 . to obtain a restriction fragment containing a vaccinia right flanking arm and puc vector sequences , psd447 was cut with sspi ( partial ) within vaccinia sequences and hindiii at the puc / vaccinia junction , and a 2 . 9 kb vector fragment isolated . this vector fragment was ligated with annealed synthetic oligonucleotides mpsyn45 / mpsyn46 ( seq id no : 3 / seq id no : 4 ) ## str2 ## generating psd459 . to combine the left and right flanking arms into one plasmid , a 0 . 5 kb hindiii / smai fragment was isolated from psd449 and ligated with psd459 vector plasmid cut with hindiii / smai , generating plasmid psd460 . psd460 was used as donor plasmid for recombination with wild type parental vaccinia virus copenhagen strain vc - 2 . a 32 p labelled probe was synthesized by primer extension using mpsyn45 ( seq id no : 3 ) as template and the complementary 20 mer oligonucleotide mpsyn47 ( seq id no : 5 ) ( 5 &# 39 ; ttagttaattaggcggccgc 3 &# 39 ;) as primer . recombinant virus vp410 was identified by plaque hybridization . construction of plasmid psd486 for deletion of hemorrhagic region ( b13r + b14r ). referring now to fig2 plasmid psd419 contains vaccinia sali g ( pos . 160 , 744 - 173 , 351 ) cloned into puc8 . psd422 contains the contiguous vaccinia sali fragment to the right , sali j ( pos . 173 , 351 - 182 , 746 ) cloned into puc8 . to construct a plasmid deleted for the hemorrhagic region , u , b13r - b14r ( pos . 172 , 549 - 173 , 552 ), psd419 was used as the source for the left flanking arm and psd422 was used as the source of the right flanking arm . the direction of transcription for the u region is indicated by an arrow in fig2 . to remove unwanted sequences from psd419 , sequences to the left of the ncoi site ( pos . 172 , 253 ) were removed by digestion of psd419 with ncoi / smai followed by blunt ending with klenow fragment of e . coli polymerase and ligation generating plasmid psd476 . a vaccinia right flanking arm was obtained by digestion of psd422 with hpai at the termination codon of b14r and by digestion with nrui 0 . 3 kb to the right . this 0 . 3 kb fragment was isolated and ligated with a 3 . 4 kb hincii vector fragment isolated from psd476 , generating plasmid psd477 . the location of the partial deletion of the vaccinia u region in psd477 is indicated by a triangle . the remaining b13r coding sequences in psd477 were removed by digestion with clai / hpai , and the resulting vector fragment was ligated with annealed synthetic oligonucleotides sd22mer / sd20mer ( seq id no : 6 / seq id no : 7 ) ## str3 ## generating psd479 . psd479 contains an initiation codon ( underlined ) followed by a bamhi site . to place e . coli beta - galactosidase in the b13 - b14 ( u ) deletion locus under the control of the u promoter , a 3 . 2 kb bamhi fragment containing the beta - galactosidase gene ( shapira et al ., 1983 ) was inserted into the bamhi site of psd479 , generating psd479bg . psd479bg was used as donor plasmid for recombination with vaccinia virus vp410 . recombinant vaccinia virus vp533 was isolated as a blue plaque in the presence of chromogenic substrate x - gal . in vp533 the b13r - b14r region is deleted and is replaced by beta - galactosidase . to remove beta - galactosidase sequences from vp533 , plasmid psd486 , a derivative of psd477 containing a polylinker region but no initiation codon at the u deletion junction , was utilized . first the clai / hpai vector fragment from psd477 referred to above was ligated with annealed synthetic oligonucleotides sd42mer / sd40mer ( seq id no : 8 / seq id no : 9 ) ## str4 ## generating plasmid psd478 . next the ecori site at the puc / vaccinia junction was destroyed by digestion of psd478 with ecori followed by blunt ending with klenow fragment of e . coli polymerase and ligation , generating plasmid psd478e - . psd478e - was digested with bamhi and hpai and ligated with annealed synthetic oligonucleotides hem5 / hem6 ( seq id no : 10 / seq id no : 11 ) ## str5 ## generating plasmid psd486 . psd486 was used as donor plasmid for recombination with recombinant vaccinia virus vp533 , generating vp553 , which was isolated as a clear plaque in the presence of x - gal . construction of plasmid pmp494δ for deletion of ati region ( a26l ). referring now to fig3 psd414 contains sali b cloned into puc8 . to remove unwanted dna sequences to the left of the a26l region , psd414 was cut with xbai within vaccinia sequences ( pos . 137 , 079 ) and with hindiii at the puc / vaccinia junction , then blunt ended with klenow fragment of e . coli polymerase and ligated , resulting in plasmid psd483 . to remove unwanted vaccinia dna sequences to the right of the a26l region , psd483 was cut with ecori ( pos . 140 , 665 and at the puc / vaccinia junction ) and ligated , forming plasmid psd484 . to remove the a26l coding region , psd484 was cut with ndei ( partial ) slightly upstream from the a26l orf ( pos . 139 , 004 ) and with hpai ( pos . 137 , 889 ) slightly downstream from the a26l orf . the 5 . 2 kb vector fragment was isolated and ligated with annealed synthetic oligonucleotides ati3 / ati4 ( seq id no : 12 / seq id no : 13 ) ## str6 ## reconstructing the region upstream from a26l and replacing the a26l orf with a short polylinker region containing the restriction sites bglii , ecori and hpai , as indicated above . the resulting plasmid was designated psd485 . since the bglii and ecori sites in the polylinker region of psd465 are not unique , unwanted bglii and ecori sites were removed from plasmid psd483 ( described above ) by digestion with bglii ( pos . 140 , 136 ) and with ecori at the puc / vaccinia junction , followed by blunt ending with klenow fragment of e . coli polymerase and ligation . the resulting plasmid was designated psd489 . the 1 . 8 kb clai ( pos . 137 , 198 )/ ecorv ( pos . 139 , 048 ) fragment from psd489 containing the a26l orf was replaced with the corresponding 0 . 7 kb polylinker - containing clai / ecorv fragment from psd485 , generating psd492 . the bglii and ecori sites in the polylinker region of psd492 are unique . a 3 . 3 kb bglii cassette containing the e . coli beta - galactosidase gene ( shapira et al ., 1983 ) under the control of the vaccinia 11 kda promoter ( bertholet et al ., 1985 ; perkus et al ., 1990 ) was inserted into the bglii site of psd492 , forming psd493kbg . plasmid psd493kbg was used in recombination with rescuing virus vp553 . recombinant vaccinia virus , vp581 , containing beta - galactosidase in the a26l deletion region , was isolated as a blue plaque in the presence of x - gal . to generate a plasmid for the removal of beta - galactosidase sequences from vaccinia recombinant virus vp581 , the polylinker region of plasmid psd492 was deleted by mutagenesis ( mandecki , 1986 ) using synthetic oligonucleotide mpsyn177 ( seq id no : 14 ) ( 5 &# 39 ; aaaatgggcgtggattgttaactttatataacttattttttgaatatac 3 &# 39 ;). in the resulting plasmid , pmp494δ , vaccinia dna encompassing positions 137 , 889 - 138 , 937 !, including the entire a26l orf is deleted . recombination between the pmp494δ and the beta - galactosidase containing vaccinia recombinant , vp581 , resulted in vaccinia deletion mutant vp618 , which was isolated as a clear plaque in the presence of x - gal . construction of plasmid psd467 for deletion of hemagglutinin gene ( a56r ). referring now to fig4 vaccinia sali g restriction fragment ( pos . 160 , 744 - 173 , 351 ) crosses the hindiii a / b junction ( pos . 162 , 539 ). psd419 contains vaccinia sali g cloned into puc8 . the direction of transcription for the hemagglutinin ( ha ) gene is indicated by an arrow in fig4 . vaccinia sequences derived from hindiii b were removed by digestion of psd419 with hindiii within vaccinia sequences and at the puc / vaccinia junction followed by ligation . the resulting plasmid , psd456 , contains the ha gene , a56r , flanked by 0 . 4 kb of vaccinia sequences to the left and 0 . 4 kb of vaccinia sequences to the right . a56r coding sequences were removed by cutting psd456 with rsai ( partial ; pos . 161 , 090 ) upstream from a56r coding sequences , and with eagi ( pos . 162 , 054 ) near the end of the gene . the 3 . 6 kb rsai / eagi vector fragment from psd456 was isolated and ligated with annealed synthetic oligonucleotides mpsyn59 ( seq id no : 15 ), mpsyn62 ( seq id no : 16 ), mpsyn60 ( seq id no : 17 ), and mpsyn 61 ( seq id no : 18 ) ## str7 ## reconstructing the dna sequences upstream from the a56r orf and replacing the a56r orf with a polylinker region as indicated above . the resulting plasmid is psd466 . the vaccinia deletion in psd466 encompasses positions 161 , 185 - 162 , 053 !. the site of the deletion in psd466 is indicated by a triangle in fig4 . a 3 . 2 kb bglii / bamhi ( partial ) cassette containing the e . coli beta - galactosidase gene ( shapira et al ., 1983 ) under the control of the vaccinia 11 kda promoter ( bertholet et al ., 1985 ; guo et al ., 1989 ) was inserted into the bglii site of psd466 , forming psd466kbg . plasmid psd466kbg was used in recombination with rescuing virus vp618 . recombinant vaccinia virus , vp708 , containing beta - galactosidase in the a56r deletion , was isolated as a blue plaque in the presence of x - gal . beta - galactosidase sequences were deleted from vp708 using donor plasmid psd467 . psd467 is identical to psd466 , except that ecori , smai and bamhi sites were removed from the puc / vaccinia junction by digestion of psd466 with ecori / bamhi followed by blunt ending with klenow fragment of e . coli polymerase and ligation . recombination between vp708 and psd467 resulted in recombinant vaccinia deletion mutant , vp723 , which was isolated as a clear plaque in the presence of x - gal . construction of plasmid pmpcsk1δ for deletion of open reading frames c7l - k1l !. referring now to fig5 the following vaccinia clones were utilized in the construction of pmpcsk1δ . psd420 is sali h cloned into puc8 . psd435 is kpni f cloned into puc18 . psd435 was cut with sphi and religated , forming psd451 . in psd451 , dna sequences to the left of the sphi site ( pos . 27 , 416 ) in hindiii m are removed ( perkus et al ., 1990 ). psd409 is hindiii m cloned into puc8 . to provide a substrate for the deletion of the c7l - k1l ! gene cluster from vaccinia , e . coli beta - galactosidase was first inserted into the vaccinia m2l deletion locus ( guo et al ., 1990 ) as follows . to eliminate the bglii site in psd409 , the plasmid was cut with bglii in vaccinia sequences ( pos . 28 , 212 ) and with bamhi at the puc / vaccinia junction , then ligated to form plasmid pmp409b . pmp409b was cut at the unique sphi site ( pos . 27 , 416 ). m2l coding sequences were removed by mutagenesis ( guo et al ., 1990 ; mandecki , 1986 ) using synthetic oligonucleotide . ## str8 ## the resulting plasmid , pmp409d , contains a unique bglii site inserted into the m2l deletion locus as indicated above . a 3 . 2 kb bamhi ( partial )/ bglii cassette containing the e . coli beta - galactosidase gene ( shapira et al ., 1983 ) under the control of the 11 kda promoter ( bertholet et al ., 1985 ) was inserted into pmp409d cut with bglii . the resulting plasmid , pmp409dbg ( guo et al ., 1990 ), was used as donor plasmid for recombination with rescuing vaccinia virus vp723 . recombinant vaccinia virus , vp784 , containing beta - galactosidase inserted into the m2l deletion locus , was isolated as a blue plaque in the presence of x - gal . a plasmid deleted for vaccinia genes c7l - k1l ! was assembled in puc8 cut with smai , hindiii and blunt ended with klenow fragment of e . coli polymerase . the left flanking arm consisting of vaccinia hindiii c sequences was obtained by digestion of psd420 with xbai ( pos . 18 , 628 ) followed by blunt ending with klenow fragment of e . coli polymerase and digestion with bglii ( pos . 19 , 706 ). the right flanking arm consisting of vaccinia hindiii k sequences was obtained by digestion of psd451 with bglii ( pos . 29 , 062 ) and ecorv ( pos . 29 , 778 ). the resulting plasmid , pmp581ck is deleted for vaccinia sequences between the bglii site ( pos . 19 , 706 ) in hindiii c and the bglii site ( pos . 29 , 062 ) in hindiii k . the site of the deletion of vaccinia sequences in plasmid pmp581ck is indicated by a triangle in fig5 . to remove excess dna at the vaccinia deletion junction , plasmid pmp581ck , was cut at the ncoi sites within vaccinia sequences ( pos . 18 , 811 ; 19 , 655 ), treated with bal - 31 exonuclease and subjected to mutagenesis ( mandecki , 1986 ) using synthetic oligonucleotide mpsyn233 ( seq id no : 20 ) 5 &# 39 ; tgtcatttaacactatactcatattaataaaaataatatttatt 3 &# 39 ;. the resulting plasmid , pmpcsk1δ , is deleted for vaccinia sequences positions 18 , 805 - 29 , 108 , encompassing 12 vaccinia open reading frames c7l - k1l !. recombination between pmpcsk1δ and the beta - galactosidase containing vaccinia recombinant , vp784 , resulted in vaccinia deletion mutant , vp804 , which was isolated as a clear plaque in the presence of x - gal . construction of plasmid psd548 for deletion of large subunit , ribonucleotide reductase ( i4l ). referring now to fig6 plasmid psd405 contains vaccinia hindiii i ( pos . 63 , 875 - 70 , 367 ) cloned in puc8 . psd405 was digested with ecorv within vaccinia sequences ( pos . 67 , 933 ) and with smai at the puc / vaccinia junction , and ligated , forming plasmid psd518 . psd518 was used as the source of all the vaccinia restriction fragments used in the construction of psd548 . the vaccinia i4l gene extends from position 67 , 371 - 65 , 059 . direction of transcription for i4l is indicated by an arrow in fig6 . to obtain a vector plasmid fragment deleted for a portion of the i4l coding sequences , psd518 was digested with bamhi ( pos . 65 , 381 ) and hpai ( pos . 67 , 001 ) and blunt ended using klenow fragment of e . coli polymerase . this 4 . 8 kb vector fragment was ligated with a 3 . 2 kb smai cassette containing the e . coli beta - galactosidase gene ( shapira et al ., 1983 ) under the control of the vaccinia 11 kda promoter ( bertholet et al ., 1985 ; perkus et al ., 1990 ), resulting in plasmid psd524kbg . psd524kbg was used as donor plasmid for recombination with vaccinia virus vp804 . recombinant vaccinia virus , vp855 , containing beta - galactosidase in a partial deletion of the i4l gene , was isolated as a blue plaque in the presence of x - gal . to delete beta - galactosidase and the remainder of the i4l orf from vp855 , deletion plasmid psd548 was constructed . the left and right vaccinia flanking arms were assembled separately in puc8 as detailed below and presented schematically in fig6 . to construct a vector plasmid to accept the left vaccinia flanking arm , puc8 was cut with bamhi / ecori and ligated with annealed synthetic oligonucleotides 518a1 / 518a2 ( seq id no : 21 / seq id no : 22 ) ## str9 ## forming plasmid psd531 . psd531 was cut with rsai ( partial ) and bamhi and a 2 . 7 kb vector fragment isolated . psd518 was cut with bglii ( pos . 64 , 459 )/ rsai ( pos . 64 , 994 ) and a 0 . 5 kb fragment isolated . the two fragments were ligated together , forming psd537 , which contains the complete vaccinia flanking arm left of the i4l coding sequences . to construct a vector plasmid to accept the right vaccinia flanking arm , puc8 was cut with bamhi / ecori and ligated with annealed synthetic oligonucleotides 518b1 / 518b2 ( seq id no : 23 / seq id no : 24 ) ## str10 ## forming plasmid psd532 . psd532 was cut with rsai ( partial )/ ecori and a 2 . 7 kb vector fragment isolated . psd518 was cut with rsai within vaccinia sequences ( pos . 67 , 436 ) and ecori at the vaccinia / puc junction , and a 0 . 6 kb fragment isolated . the two fragments were ligated together , forming psd538 , which contains the complete vaccinia flanking arm to the right of i4l coding sequences . the right vaccinia flanking arm was isolated as a 0 . 6 kb ecori / bglii fragment from psd538 and ligated into psd537 vector plasmid cut with ecori / bglii . in the resulting plasmid , psd539 , the i4l orf ( pos . 65 , 047 - 67 , 386 ) is replaced by a polylinker region , which is flanked by 0 . 6 kb vaccinia dna to the left and 0 . 6 kb vaccinia dna to the right , all in a puc background . the site of deletion within vaccinia sequences is indicated by a triangle in fig6 . to avoid possible recombination of beta - galactosidase sequences in the puc - derived portion of psd539 with beta - galactosidase sequences in recombinant vaccinia virus vp855 , the vaccinia i4l deletion cassette was moved from psd539 into prc11 , a puc derivative from which all beta - galactosidase sequences have been removed and replaced with a polylinker region ( colinas et al ., 1990 ). psd539 was cut with ecori / psti and the 1 . 2 kb fragment isolated . this fragment was ligated into prc11 cut with ecori / psti ( 2 . 35 kb ), forming psd548 . recombination between psd548 and the beta - galactosidase containing vaccinia recombinant , vp855 , resulted in vaccinia deletion mutant vp866 , which was isolated as a clear plaque in the presence of x - gal . dna from recombinant vaccinia virus vp866 was analyzed by restriction digests followed by electrophoresis on an agarose gel . the restriction patterns were as expected . polymerase chain reactions ( pcr ) ( engelke et al ., 1988 ) using vp866 as template and primers flanking the six deletion loci detailed above produced dna fragments of the expected sizes . sequence analysis of the pcr generated fragments around the areas of the deletion junctions confirmed that the junctions were as expected . recombinant vaccinia virus vp866 , containing the six engineered deletions as described above , was designated vaccinia vaccine strain &# 34 ; nyvac .&# 34 ; construction of fowlpox insertion plasmid at f8 locus . plasmid prw731 . 15 contains a 10 kbp pvuii - pvuii fragment cloned from trovac genomic dna . the nucleotide sequence was determined on both strands for a 3660 bp pvuii - ecorv fragment . this sequence is as follows ( seq id no : 25 ): __________________________________________________________________________ 1 gatatctgtg gtctatatat actacaccct accgatatta accaacgagt ttctcacaag 61 aaaacttgtt tagtagatag agattctttg attgtgttta aaagaagtac cagtaaaaag 121 tgtggcatat gcatagaaga aataaacaaa aaacatattt ccgaacagta ttttggaatt 181 ctcccaagtt gtaaacatat tttttgccta tcatgtataa gacgttgggc agatactacc 241 agaaatacag atactgaaaa tacgtgtcct gaatgtagaa tagtttttcc tttcataata 301 cccagtaggt attggataga taataaatat gataaaaaaa tattatataa tagatataag 361 aaaatgattt ttacaaaaat aacctataag aacaataaaa atataattac atttacggaa 421 aatagctggt tttagtttac caacttagag taattatcat attgaatcta tattgttttt 481 tagttatata aaaacatgat tagcccccaa tcggatgaaa atataaaaga tgttgagaat 541 ttcgaataca acaaaaagag gaatcgtacg ttgtccatat ccaaacatat aaataaaaat 601 tcaaaagtag tattatactg gatgtttaga gatcaacgtg tacaagataa ttgggcttta 661 atttacgcac aacgattagc gttaaaactc aaaatacctc taagaatatg cttttgtgtc 721 gtgccaaaat ttcacactac tacttctaga cactttatgt ttttaatatc cggtcttaaa 781 gaagtcgcgg aagaatgtaa aagactatgt atagggtttt cattgatata tggcgtacca 841 aaagtaataa ttccgtgtat agtaaaaaaa tacagagtcg gagtaatcat aacggatttc 901 tttccattac gtgttcccga aagattaatg aaacagactg taatatctct tccagatacc 961 atacctttta tacaagtaga cgctcataat atagtacctt gttgggaagc ttctgataaa1021 gaagaatacg gtgcacgaac tttaagaaaa aagatatttg ataaattata tgaatatatg1081 acagaatttc ctgttgttcg taaacatcca tacggtccat tttctatatc tattgcaaaa1141 cccaaaaata tatcattaga caagacggta ttacccgtaa aatgggcaac gcctggaaca1201 aaagctggaa taattgtttt aaaagaattt ataaaaaaca gattaccgtc atacgacgcg1261 gatcataaca atcctacgtg tgacgctttg agtaacttat ctccgtggct acattttggt1321 catgtatccg cacaacgtgt tgccttagaa gtattaaaat gtatacgaga aagcaaaaaa1381 aacgttgaaa cgtttataga tgaaataatt gtaagaagag aactatcgga taatttttgt1441 tactataaca aacattatga tagtatccag tctactcatt catgggttag aaaaacatta1501 gaagatcaca ttaatgatcc tagaaagtat atatattcca ttaaacaact cgaaaaagcg1561 gaaactcatg atcctctatg gaacgcgtca caaatgcaga tggtgagaga aggaaaaatg1621 catagttttt tacgaatgta ttgggctaag aagatacttg aatggactag aacacctgaa1681 gacgctttga gttatagtat ctatttgaac aacaagtacg aactagacgg cacggatcct1741 aacggatacg taggttgtat gtggtctatt tgcggattac acgatagagc gtggaaagca1801 agaccgatat ttggaaagat aagatatatg aattatgaga gttctaagaa gaaatttgat1861 gttgctgtat ttatacagaa atacaattaa gataaataat atacagcatt gtaaccatcg1921 tcatccgtta tacggggaat aatattacca tacagtatta ttaaattttc ttacgaagaa1981 tatagatcgg tatttatcgt tagtttattt tacatttatt aattaaacat gtctactatt2041 acctgttatg gaaatgacaa atttagttat ataatttatg ataaaattaa gataataata2101 atgaaatcaa ataattatgt aaatgctact agattatgtg aattacgagg aagaaagttt2161 acgaactgga aaaaattaag tgaatctaaa atattagtcg ataatgtaaa aaaaataaat2221 gataaaacta accagttaaa aacggatatg attatatacg ttaaggatat tgatcataaa2281 ggaagagata cttgcggtta ctatgtacac caagatctgg tatcttctat atcaaattgg2341 atatctccgt tattcgccgt taaggtaaat aaaattatta actattatat atgtaatgaa2401 tatgatatac gacttagcga aatggaatct gatatgacag aagtaataga tgtagttgat2461 aaattagtag gaggatacaa tgatgaaata gcagaaataa tatatttgtt taataaattt2521 atagaaaaat atattgctaa catatcgtta tcaactgaat tatctagtat attaaataat2581 tttataaatt ttataaattt taataaaaaa tacaataacg acataaagat atttaatctt2641 taattcttga tctgaaaaac acatctataa aactagataa aaagttattc gataaagata2701 ataatgaatc gaacgatgaa aaattggaaa cagaagttga taagctaatt tttttcatct2761 aaatagtatt attttattga agtacgaagt tttacgttag ataaataata aaggtcgatt2821 tttactttgt taaatatcaa atatgtcatt atctgataaa gatacaaaaa cacacggtga2881 ttatcaacca tctaacgaac agatattaca aaaaatacgt cggactatgg aaaacgaagc2941 tgatagcctc aatagaagaa gcattaaaga aattgttgta gatgttatga agaattggga3001 tcatcctcaa cgaagaaata gataaagttc taaactggaa aaatgataca ttaaacgatt3061 tagatcatct aaatacagat gataatatta aggaaatcat acaatgtctg attagagaat3121 ttgcgtttaa aaagatcaat tctattatgt atagttatgc tatggtaaaa ctcaattcag3181 ataacgaaca ttgaaagata aaattaagga ttattttata gaaactattc ttaaagacaa3241 acgtggttat aaacaaaagc cattacccgg attggaaact aaaatactag atagtattat3301 aagattttaa aaacataaaa ttaataggtt tttatagatt gacttattat atacaatatg3361 gataaaagat atatatcaac tagaaagttg aatgacggat tcttaatttt atattatgat3421 tcaatagaaa ttattgtcat gtcgtgtaat cattttataa atatatcagc gttactacgt3481 aagaaaaaca aggactttaa tgaatggcta aagatagaat catttagaga aataatagat3541 actttagata aaattaatta cgatctagga caacgatatt gtgaagaact tacggcgcat3601 cacattccag tgtaattatt gaggtcaaag ctagtaactt aatagatgac aggacagctg__________________________________________________________________________ the limits of an open reading frame designated as f8 were determined within this sequence . the open reading frame is initiated at position 496 and terminates at position 1887 . the engineered deletion was made from position 780 to position 1927 , as described below . plasmid prw761 is a sub - clone of prw731 . 15 containing a 2429 bp ecorv - ecorv fragment . plasmid prw761 was completely digested with xbai and partially digested with sspi . a 3700 bp xbai - sspi band was isolated and ligated with the annealed double - stranded oligonucleotides jca017 ( seq id no : 26 ) and jca018 ( seq no : 27 ). ## str11 ## additional cloning sites were incorporated into pjca002 by inserting the annealed and kinased oligonucleotides ce205 ( seq id no : 28 ) and ce206 ( seq id no : 29 ) into the bamhi and hindiii sites of pjca002 to form pce72 . ## str12 ## in order to increase the length of the fpv flanking arms in the insertion plasmid , plasmid pjca021 was constructed . plasmid pjca021 was obtained by inserting a 4900bp pvuii - hindii fragment from prw 731 . 15 ( previously described ) into the smai and hindii sites of pbluescript ss k + ( stratagene , la jolla , calif .). a bglii to ecori fragment from pce72 was then ligated into the bglii and ecori sites of pjca021 to generate pcen100 . construction of a plasmid for insertion of mdv gb sequence into trovac . three fragments generated by the polymerase chain reaction ( pcr ) were necessary for construction of an insertion plasmid . reaction 1 created a fragment which contained the vaccinia virus h6 promoter fused in a precise atg : atg configuration with the 5 &# 39 ; end of the mdv gb gene . for this reaction , plasmid prw825 containing the h6 promoter previously described ( taylor et al ., 1988a , b ; guo et al ., 1989 ) was used as a template and oligonucleotides rw297 ( seq id no : 30 ) and rw298 ( seq id no : 31 ) as primers . ## str13 ## reaction 2 generated a fragment containing the 5 &# 39 ; end of the mdv gb gene in which a ttttttt sequence was changed to tattctt to eliminate the possibility of early termination ( yuen and moss , 1987 ). the 3 &# 39 ; end of the fragment generated , overlapped the 5 &# 39 ; end of the fragment generated in reaction 3 . in reaction 2 , plasmid puc13gb containing the mdv gb coding sequence was used as a template and oligonucleotides rw299 ( seq id no : 32 ) and rw300 ( seq id no : 33 ) as primers . ## str14 ## reaction 3 generated a fragment defining the 3 &# 39 ; end of the mdv gb gene and removed non - coding sequences contained in puc13gb . plasmid puc13gb was used as a template for this reaction and rw 301 ( seq id no : 34 ) and rw302 ( seq id no : 35 ) as primers . ## str15 ## products of these three pcr reactions were pooled and used as a template for primers rw297 ( seq id no : 30 ) and rw302 ( seq id no : 35 ) in a fourth pcr . the final 1250 bp pcr product was isolated , cut with hincii and inserted into plasmid pcen100 which had been cut with hincii to derive prw871 . derivation of plasmid pcen100 which contains trovac genomic dna directing insertion to the f8 locus is described above . plasmid prw871 was partially cut with xbai , the linear product isolated , recut with aflii and the 7 . 7 kbp fragment was isolated . plasmid puc13gb was digested with aflii and xbai and the resulting 2140 bp fragment containing the gb coding region was inserted into the 7 . 7 kbp fragment derived from prw871 . the resulting plasmid , prw878 , was used in in vitro recombination with trovac as the rescuing virus to derive recombinant vfp108 . construction of a plasmid for insertion of mdv gb into vaccinia virus . plasmid prw878 , previously described , was digested with hincii and the 2 . 8 kbp fragment containing the mdv gb coding sequence linked to the vaccinia virus h6 promoter was inserted at the smai site of vaccinia insertion plasmid psd553vc to derive plasmid prw879 . plasmid psd553vc is an insertion plasmid utilizing the host range selection system described in perkus et al . ( 1989 ). in this plasmid , the vaccinia virus k1l gene and polylinker regions are located within flanking copenhagen vaccinia arms , replacing the ati region ( open reading frames a25l and a26l ) described in goebel et al . ( 1990a , b ). the entire region is in a puc8 vector and insertion sites are flanked by translational stop codons and transcriptional stop signals . plasmid prw879 was used in in vitro recombination with nyvac ( vp866 ) as the rescuing virus to derive recombinant vp935 expressing the marek &# 39 ; s gb gene . construction of a plasmid for insertion of mdv gd into vaccinia virus . four pcr reactions were used to create an insertion plasmid . in reaction 1 , plasmid prw880 was used as a template to derive a fragment containing the vaccinia virus h6 promoter sequence linked to the mdv gd 5 &# 39 ; sequence with the promoter &# 39 ; s atg overlapping the initiating atg of the mdv gd gene . plasmid prw880 contains the previously described h6 promoter sequence linked to a non - pertinent gene in the f16 insertion locus . the primers used in this reaction were rw389 ( seq id no : 36 ) and rw390 ( seq id no : 37 ). ## str16 ## in the second and third pcr reactions , pmdv517 was used as a template . plasmid pmdv517 contains a 5 . 2 kb dna fragment containing the mdv gd gene inserted at the ecori site of puc13 . the object of the reactions was to change two internal tttttnt signals to eliminate the possibility of premature termination ( yuen and moss , 1987 ). in reaction two , oligonucleotides rw386 ( seq id no : 38 ) and rw 391 ( seq id no : 39 ) were used as primers to change ttttttttt to ttcttcttt . ## str17 ## in reaction three , oligonucleotides rw387 ( seq id no : 40 ) and rw388 ( seq id no : 41 ) were used to alter the sequence tttttttgt to cttcttcgt . ## str18 ## the products of the three pcr reactions were pooled and primed with rw390 ( seq id no : 37 ) and rw391 ( seq id no : 39 ) for a fourth pcr reaction . the product of the final pcr reaction was cut with nrui and hindiii , resulting in a 1 . 2 kbp fragment containing the 3 &# 39 ; end of the h6 promoter and the mdv gd coding sequence . plasmid prw880 ( derivation described below ) was digested with nrui and hindiii ( which eliminates the non - pertinent gene leaving the 5 &# 39 ; end of the h6 promoter ), and ligated with the 1 . 2 kp fragment obtained after digestion of the final pcr product . the resulting plasmid prw893 was then digested with noti , releasing a 1 . 4 kbp fragment containing the h6 promoted mdv gd gene which was inserted into the smai site of psd533vc previously described to generate prw894 . plasmid prw894 was used in in vitro recombination with nyvac ( vp866 ) as the rescuing virus to generate recombinant vp1005 expressing the marek &# 39 ; s gd gene . construction of a fowlpox insertion plasmid at the f16 locus . plasmid pfp23k - 1 contains the 10 . 5 kbp hindiii fowlpox dna fragment described in tartaglia et al . ( 1990 ). plasmid pfp23k - 1 was used as a template for a pcr reaction using the following primers rw264 ( seq id no : 42 ), rw265 ( seq id no : 43 ), rw266 ( seq id no : 44 ) and rw267 ( seq id no : 45 ). ## str19 ## primers rw264 and rw267 primed the first reaction . primers rw265 and rw266 primed the second reaction . rw266 , rw267 and the two products from reactions 1 and 2 were combined for a final pcr . orientation of the primers on the template is as follows : ## str20 ## primer rw266 begins at position 9883 within the 10 . 5 kbp sequence with an ndei site . the 5 &# 39 ; ends of rw265 and rw264 overlap and are reverse complements . rw265 and rw264 replace the a at position 10054 bp with 61 bp containing the insertion sites smai , bamhi and hindiii . translation termination codons and transcription stop signals flank the insertion sites . the 5 &# 39 ; end of rw267 is at position 10229 bp beginning with an ecori site . the third and final pcr product was cut with ndei and ecori and inserted between the ndei and ecori sites of prw715 , resulting in prw864 . plasmid prw715 is plasmid puc9 cut with pvuii and an ecori linker was inserted in place of the 300 bp pvuii fragment . in order to insert the e . coli lacz gene plasmid pamibg was utilized . plasmid pamibg contains the lacz bamhi fragment from pmc1871 ( casadaban et al ., 1983 ) inserted in the previously described bamhi site 3 &# 39 ; of the 11k vaccinia virus promoter ( paoletti et al ., 1984 ). plasmid pamibg was partially cut with bamhi , the linear product isolated and cut with psti . the psti - bamhi fragment containing the 11k promoted lacz gene was blunt - ended and ligated into the smai site of prw864 ( described above ). the resulting plasmid was designated prw867a . plasmid prw866 is a subclone of plasmid pfp23k - 1 which contains the 10 . 5 kbp fowlpox dna fragment described in tartaglia et al . ( 1990 ). plasmid prw866 was constructed by insertion of a 7 . 3 kpb naei to ndei fowlpox fragment from pfp23k - 1 between the pvuii and ndei sites of puc9 . plasmid prw866 contains two fspi sites ; one is in puc and the second at position 1955 bp defines the intergenic insertion site designated f16 . the fspi insertion locus does not interrupt any open reading frame containing an atg . the linear product of a partial digestion of prw866 with fspi was isolated and ligated to a 3 . 3 kpb noti fragment from prw867a containing the 11k promoted lacz gene . this allowed insertion of the blunt ended lacz gene fragment into the fspi intergenic insertion site , creating plasmid prw868 . the lacz gene in prw868 was then replaced with the 61 bp fragment ( previously described ) containing smai , bamhi and hindiii sites and flanked by transcription termination and translation stop sequences used in development of prw864 . this replacement resulted in plasmid prw813 . plasmid prw880 used as a template for the initial construction of plasmid prw894 contains a non - pertinent gene linked to the h6 promoter in the smai site of the f16 insertion locus . plasmids previously described were transfected into nyvac or trovac infected cells by using the calcium phosphate precipitation method previously described ( panicali and paoletti , 1982 ; piccini et al ., 1987 ). positive plaques were selected on the basis of hybridization to specific mdv radiolabelled probes and subjected to sequential rounds of plaque purification until a pure population was achieved . representative plaques from each ivr were then amplified and a stock virus established . indirect immunofluorescence was performed as described in taylor et al . ( 1990 ) using a polyclonal chicken anti - mdv serum designated chicken # 392 . immunoprecipitation reactions were performed as described in taylor et al . ( 1990 ) using the reagent described above . nyvac recombinants expressing mdv glycoproteins . transfection of plasmid prw879 led to the development of nyvac recombinant vp935 . immunofluorescence analysis indicated that a protein recognized by mdv immune serum was expressed on the infected cell surface . immunoprecipitation analysis using the same immune serum from chicken detected the presence of three major expression products with the approximate molecular weights of 110 kda , 64 kda and 48 kda . these sizes correspond to expected products of the mdv gb gene . transfection of plasmid prw894 led to the development of nyvac recombinant vp1005 . a plaque immunoscreen assay indicated that a protein recognized by mdv immune serum was expressed on the infected cell surface . immunoprecipitation analysis indicated the production of two products with molecular weights of approximately 45 kda ( corresponding to the precursor form of the protein ) and 65 kda which represents the processed glycoprotein . trovac recombinant expressing the gb glycoprotein . transfection of plasmid prw878 led to the development of recombinant vfp108 . immunofluorescence analysis indicated that a protein recognized by mdv immune serum was expressed on the infected cell surface . immunoprecipitation analysis using the same immune serum from chickens demonstrated the presence of three major expression products with approximate molecular weights of 110 kda , 64 kda and 48 kda . a group of 25 one day old spf chickens were inoculated by the subcutaneous route with one dose of 4 . 0 log 10 pfu of trovac - mdv gb ( vfp108 ). ten spf birds remained uninoculated . at 14 days , the birds including 10 non - vaccinated controls , were challenged by intraperitoneal inoculation of a dilution of the jmv tumor cell line ( nazerian et al ., 1977 ) previously determined to cause 100 % mortality , and survivors were assessed . table 1______________________________________protective efficacy of trovac - mdv gb ( vfp108 ) in spf chickens protection ratiotreatment group survivors / total % survival______________________________________vfp108 . sup . a 11 / 25 44non - vaccinated controls 0 / 10 0______________________________________ . sup . a birds inoculated with 4 . 0 log . sub . 10 pfu of vfp108 . birds challenged at 14 days by inoculation with jmv tumor cell line . in a second experiment , 20 rhode island red chicks hatched in isolators and free from maternal antibodies to mdv , herpesvirus of turkeys ( hvt ) and other avian pathogens were vaccinated intramuscularly at one day of age with 6 . 3 log 10 pfu of fowlpox recombinant vfp108 . seven days later the chicks were challenged with mdv by contact infection . this was achieved by mixing the vaccinated chicks with 8 chickens inoculated 15 days previously with 3 . 0 log 10 pfu of the rb1 - b strain of mdv . a second group of 20 unvaccinated chicks derived from the same hatch served as controls . these were also challenged with mdv by contact as above . the two groups were kept in separate cages in a high security containment room . a third group of 20 unvaccinated chicks were challenged by inoculation with 3 . 01 log 10 pfu of rb1 - b and kept in a separate room . this group was included in the experiment to compare the efficiency of challenge by the two methods . the chicks were observed daily and those that died were examined for gross marek &# 39 ; s disease lesions in visceral organs and peripheral nerves . tissue samples were taken for histological examination in cases where gross marek &# 39 ; s disease lesions were not obvious . two chicks in the group vaccinated with fowlpox recombinant showed symptoms of eye infection , probably due to fowlpox , and died within 2 days after contact with mdv - infected chicks . they were eliminated from the experiment and do not appear in the mortality results shown in table 2 and fig7 . the results show that vaccination with the fowlpox recombinant delayed mortality significantly . the mean time to death in the vaccinated and unvaccinated group ( contact challenge ) was 56 days and 35 days respectively . the difference was significant ( p & lt ; 0 . 005 ) as shown by analysis of the log transformation of the data using student &# 39 ; s t test . the total mortality in the two groups after a prolonged period of 19 weeks did not differ significantly as shown by a chi - square test . however at 6 to 7 weeks post - vaccination , mortality rates differed significantly being almost 100 % in the controls and 10 % in the vaccinated birds . it should be noted that broiler chickens are normally sent to market at 6 to 7 weeks of age . it is clear from fig7 that the challenge by contact infection was efficient compared to challenge by inoculation . the total mortality in the two groups was similar and the slopes of the cumulative mortality curves were also similar after a delay of about 2 weeks ( contact infection ), which probably represented the time required to establish infection . it should be noted that the vaccinated group was continuously exposed to mdv shed by the unvaccinated group which was kept in the same room . in conclusion , the importance of mdv gb as a protective immunogen has been demonstrated under rigorous conditions which involved the use of genetically susceptible chickens vaccinated at one day old , and challenged with mdv by two different methods . table 2______________________________________time to death ( days ) fowlpox vaccinated unvaccinated unvaccinated contact challenge contact challenge inoc . challenge______________________________________ 46 36 10 44 17 26 110 32 39 77 48 26 97 64 42 53 47 28 41 31 32 35 45 24 56 33 24 52 32 33 57 13 10 51 33 34 48 22 50 10 27 42 45 9 35 29 40 24 38number 12 19 18mean 56 35 25 . 7total chicks 18 20 19per group______________________________________ the results indicate the potential of trovac - mdv recombinants for vaccination against mdv in the poultry industry . the restricted host range of fowlpox virus provides an inherent safety barrier to transmission of recombinant to non - avian species . use of antigenic regions of mdv rather than the whole virus eliminates the need to introduce live herpes virus into the environment . the ability of trovac to incorporate large amounts of foreign genetic information should allow for inclusion of multiple antigenic determinants from a range of serotypes . in previous experiments , the ability of trovac - mdv ( vfp108 ) to protect against mdv challenge was assessed in two ways , in the first experiment , day of age spf chickens were vaccinated with 40 log 10 pfu of vfp108 by the subcutaneous route in the nape of the neck . when birds were challenged at 14 days by intraperitoneal inoculation of the jmv tumor cell line , 44 % of birds survived challenge . in the second experiment , day old spf birds were vaccinated by intramuscular inoculation of 6 . 3 log 10 pfu of vfp108 . seven days later , vaccinated and unvaccinated birds were challenged by contact infection with birds infected with mdv strain rb18 . ninety percent of vaccinated birds had survived challenge at 6 - 7 weeks post - vaccination . the most commonly used mdv vaccines are the turkey herpes virus ( hvt ) vaccines which are seriologically related to mdv . this example shows the comparative efficacy of hvt and trovac - mdv ( vfp108 ). twenty day old spf birds were inoculated with 3 . 8 log 10 eid 50 of trovac - mdv ( vfp108 ) by the subcutaneous route in the nape of the neck . twenty chicks were inoculated with 3 . 0 log 10 pfu of a cell associated hvt vaccine by the subcutaneous route . ten birds remained uninoculated . five days post - inoculation , vaccinates and controls were challenged by intraperitoneal inoculation of the rb1b challenge virus . birds were observed for 49 days at which time they were necropsied and examined for lesions typical of marek &# 39 ; s disease . the results of challenge are shown in table 3 . the results indicate that 90 % of non - vaccinated challenge controls succumbed to infection . birds vaccinated with trovac - mdv ( vfp108 ) showed a 75 % survival rate while birds vaccinated with the hvt vaccine showed an 85 % survival rate . the result indicates that protection afforded by the trovac - mdv ( vfp108 ) vaccine was comparable to the cell associated hvt . thus , trovac - mdv is an effective vaccine . table 3______________________________________comparative efficacy of trovac - mdv ( vfp108 ) and hvtvaccine protection ratio . sup . a % protection______________________________________trovac - mdv ( vfp108 ) 15 / 20 75hvt 17 / 20 85none 1 / 10 10______________________________________ . sup . a ratio of birds protected to total number challenged 1 . bertholet , c ., r . drillien , and r . wittek , proc . natl . acad . sci . usa 82 , 2096 - 2100 ( 1985 ). 2 . boursnell , m . e . g ., p . f . green , a . c . r . samson , j . i . a . campbell , a . deuter , r . w . peters , n . s . millar , p . t . emmerson , and m . m . binns , virology 178 , 297 - 300 ( 1990a ). 3 . boursnell , m . e . g ., p . f . green , j . i . a . campbell , a . deuter , r . w . peters , f . m . tomley , a . c . r . samson , p . chambers , p . t . emmerson , and m . m . binns , j . gen . virol . 71 , 621 - 628 ( 1990b ). 4 . calnek , b . w . and r . l . witter , in diseases of poultry 9th edition , eds . b . w . calnek , h . j . barnes , c . w . beard , w . m . reid and h . w . yoder , jr . 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kawaoka , r . g . webster , and e . paoletti , vaccine 6 , 504 - 508 ( 1988a ). 45 . taylor , j ., r . weinberg , b . languet , p . desmettre , and e . paoletti , vaccine 6 , 497 - 503 ( 1988b ). 46 . taylor , j ., c . edbauer , a . rey - senelonge , j . f . bouquet , e . norton , s . goebel , p . desmettre , and e . paoletti , j . virol . 64 , 1441 - 1450 ( 1990 ). 47 . taylor , j ., s . pincus , j . tartaglia , c . richardson , g . alkhatib , d . briedis , m . appel , e . norton , and e . paoletti , j . virol . 65 , 4263 - 4272 ( 1991 ). 48 . yuen , l ., and b . moss , proc . natl . acad . sci . usa 84 , 6417 - 6421 ( 1987 ). __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 45 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 1 : taattaactagctacccggg20 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 28 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 2 : aattcccgggtagctagttaattacatg28 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 73 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 3 : agcttcccgggtaagtaatacgtcaaggagaaaacgaaacgatctgtagttagcggccgc60ctaattaactaat73 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 69 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 4 : attagttaattaggcggccgctaactacagatcgtttcgttttctccttgacgtattact60tacccggga69 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 5 : ttagttaattaggcggccgc20 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 22 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 6 : cgattactatgaaggatccgtt22 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 7 : aacggatccttcatagtaat20 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 41 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 8 : cgattactagatctgagctccccgggctcgagggatccgtt41 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 39 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 9 : aacggatccctcgagcccggggagctcagatctagtaat39 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 16 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 10 : gatccgaattctagct16 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 12 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 11 : agctagaattcg12 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 75 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 12 : tatgagtaacttaactcttttgttaattaaaagtatattcaaaaaataagttatataaat60agatctgaattcgtt75 ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 73 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 13 : aacgaattcagatctatttatataacttattttttgaatatacttttaattaacaaaaga60gttaagttactca73 ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 49 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 14 : aaaatgggcgtggattgttaactttatataacttattttttgaatatac49 ( 2 ) information for seq id no : 15 :( i ) sequence characteristics :( a ) length : 67 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 15 : acacgaatgattttctaaagtatttggaaagttttataggtagttgatagaacaaaatac60ataattt67 ( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 51 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 16 : tctatcaactacctataaaactttccaaatactttagaaaatcattcgtgt51 ( 2 ) information for seq id no : 17 :( i ) sequence characteristics :( a ) length : 46 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 17 : tgtaaaaataaatcactttttatactaagatctcccgggctgcagc46 ( 2 ) information for seq id no : 18 :( i ) sequence characteristics :( a ) length : 66 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 18 : ggccgctgcagcccgggagatcttagtataaaaagtgatttatttttacaaaattatgta60ttttgt66 ( 2 ) information for seq id no : 19 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 19 : tttctgtatatttgcaccaatttagatcttactcaaaatatgtaacaata50 ( 2 ) information for seq id no : 20 :( i ) sequence characteristics :( a ) length : 44 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 20 : tgtcatttaacactatactcatattaataaaaataatatttatt44 ( 2 ) information for seq id no : 21 :( i ) sequence characteristics :( a ) length : 72 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 21 : gatcctgagtactttgtaatataatgatatatattttcactttatctcatttgagaataa60aaagatcttagg72 ( 2 ) information for seq id no : 22 :( i ) sequence characteristics :( a ) length : 72 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 22 : aattcctaagatctttttattctcaaatgagataaagtgaaaatatatatcattatatta60caaagtactcag72 ( 2 ) information for seq id no : 23 :( i ) sequence characteristics :( a ) length : 72 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 23 : gatccagatctcccgggaaaaaaattatttaacttttcattaatagggatttgacgtatg60tagcgtactagg72 ( 2 ) information for seq id no : 24 :( i ) sequence characteristics :( a ) length : 72 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 24 : aattcctagtacgcatcatacgtcaaatccctattaatgaaaagttaaataatttttttc60ccgggagatctg72 ( 2 ) information for seq id no : 25 :( i ) sequence characteristics :( a ) length : 3660 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 25 : gatatctgtggtctatatatactacaccctaccgatattaaccaacgagtttctcacaag60aaaacttgtttagtagatagagattctttgattgtgtttaaaagaagtaccagtaaaaag120tgtggcatatgcatagaagaaataaacaaaaaacatatttccgaacagtattttggaatt180ctcccaagttgtaaacatattttttgcctatcatgtataagacgttgggcagatactacc240agaaatacagatactgaaaatacgtgtcctgaatgtagaatagtttttcctttcataata300cccagtaggtattggatagataataaatatgataaaaaaatattatataatagatataag360aaaatgatttttacaaaaataacctataagaacaataaaaatataattacatttacggaa420aatagctggttttagtttaccaacttagagtaattatcatattgaatctatattgttttt480tagttatataaaaacatgattagcccccaatcggatgaaaatataaaagatgttgagaat540ttcgaatacaacaaaaagaggaatcgtacgttgtccatatccaaacatataaataaaaat600tcaaaagtagtattatactggatgtttagagatcaacgtgtacaagataattgggcttta660atttacgcacaacgattagcgttaaaactcaaaatacctctaagaatatgcttttgtgtc720gtgccaaaatttcacactactacttctagacactttatgtttttaatatccggtcttaaa780gaagtcgcggaagaatgtaaaagactatgtatagggttttcattgatatatggcgtacca840aaagtaataattccgtgtatagtaaaaaaatacagagtcggagtaatcataacggatttc900tttccattacgtgttcccgaaagattaatgaaacagactgtaatatctcttccagataac960ataccttttatacaagtagacgctcataatatagtaccttgttgggaagcttctgataaa1020gaagaatacggtgcacgaactttaagaaaaaagatatttgataaattatatgaatatatg1080acagaatttcctgttgttcgtaaacatccatacggtccattttctatatctattgcaaaa1140cccaaaaatatatcattagacaagacggtattacccgtaaaatgggcaacgcctggaaca1200aaagctggaataattgttttaaaagaatttataaaaaacagattaccgtcatacgacgcg1260gatcataacaatcctacgtgtgacgctttgagtaacttatctccgtggctacattttggt1320catgtatccgcacaacgtgttgccttagaagtattaaaatgtatacgagaaagcaaaaaa1380aacgttgaaacgtttatagatgaaataattgtaagaagagaactatcggataatttttgt1440tactataacaaacattatgatagtatccagtctactcattcatgggttagaaaaacatta1500gaagatcacattaatgatcctagaaagtatatatattccattaaacaactcgaaaaagcg1560gaaactcatgatcctctatggaacgcgtcacaaatgcagatggtgagagaaggaaaaatg1620catagttttttacgaatgtattgggctaagaagatacttgaatggactagaacacctgaa1680gacgctttgagttatagtatctatttgaacaacaagtacgaactagacggcacggatcct1740aacggatacgtaggttgtatgtggtctatttgcggattacacgatagagcgtggaaagca1800agaccgatatttggaaagataagatatatgaattatgagagttctaagaagaaatttgat1860gttgctgtatttatacagaaatacaattaagataaataatatacagcattgtaaccatcg1920tcatccgttatacggggaataatattaccatacagtattattaaattttcttacgaagaa1980tatagatcggtatttatcgttagtttattttacatttattaattaaacatgtctactatt2040acctgttatggaaatgacaaatttagttatataatttatgataaaattaagataataata2100atgaaatcaaataattatgtaaatgctactagattatgtgaattacgaggaagaaagttt2160acgaactggaaaaaattaagtgaatctaaaatattagtcgataatgtaaaaaaaataaat2220gataaaactaaccagttaaaaacggatatgattatatacgttaaggatattgatcataaa2280ggaagagatacttgcggttactatgtacaccaagatctggtatcttctatatcaaattgg2340atatctccgttattcgccgttaaggtaaataaaattattaactattatatatgtaatgaa2400tatgatatacgacttagcgaaatggaatctgatatgacagaagtaatagatgtagttgat2460aaattagtaggaggatacaatgatgaaatagcagaaataatatatttgtttaataaattt2520atagaaaaatatattgctaacatatcgttatcaactgaattatctagtatattaaataat2580tttataaattttataaattttaataaaaaatacaataacgacataaagatatttaatctt2640taattcttgatctgaaaaacacatctataaaactagataaaaagttattcgataaagata2700ataatgaatcgaacgatgaaaaattggaaacagaagttgataagctaatttttttcatct2760aaatagtattattttattgaagtacgaagttttacgttagataaataataaaggtcgatt2820tttactttgttaaatatcaaatatgtcattatctgataaagatacaaaaacacacggtga2880ttatcaaccatctaacgaacagatattacaaaaaatacgtcggactatggaaaacgaagc2940tgatagcctcaatagaagaagcattaaagaaattgttgtagatgttatgaagaattggga3000tcatcctcaacgaagaaatagataaagttctaaactggaaaaatgatacattaaacgatt3060tagatcatctaaatacagatgataatattaaggaaatcatacaatgtctgattagagaat3120ttgcgtttaaaaagatcaattctattatgtatagttatgctatggtaaaactcaattcag3180ataacgaacattgaaagataaaattaaggattattttatagaaactattcttaaagacaa3240acgtggttataaacaaaagccattacccggattggaaactaaaatactagatagtattat3300aagattttaaaaacataaaattaataggtttttatagattgacttattatatacaatatg3360gataaaagatatatatcaactagaaagttgaatgacggattcttaattttatattatgat3420tcaatagaaattattgtcatgtcgtgtaatcattttataaatatatcagcgttactagct3480aagaaaaacaaggactttaatgaatggctaaagatagaatcatttagagaaataatagat3540actttagataaaattaattacgatctaggacaacgatattgtgaagaacttacggcgcat3600cacattccagtgtaattattgaggtcaaagctagtaacttaatagatgacaggacagctg3660 ( 2 ) information for seq id no : 26 :( i ) sequence characteristics :( a ) length : 68 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 26 : ctagacactttatgttttttaatatccggtcttaaaagcttcccggggatccttatacgg60ggaataat68 ( 2 ) information for seq id no : 27 :( i ) sequence characteristics :( a ) length : 65 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 27 : attattccccgtataaggatcccccgggaagcttttaagaccggatattaaaaaacataa60agtgt65 ( 2 ) information for seq id no : 28 :( i ) sequence characteristics :( a ) length : 72 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 28 : gatcagaaaaactagctagctagtacgtagttaacgtcgacctgcagaagcttctagcta60gctagtttttat72 ( 2 ) information for seq id no : 29 :( i ) sequence characteristics :( a ) length : 73 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 29 : agctataaaaactagctagctagaagcttctgcaggctcgacgttaactacgtactagct60agctagtttttct73 ( 2 ) information for seq id no : 30 :( i ) sequence characteristics :( a ) length : 33 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 30 : gacctcgtcgacaatacgactcactatagggag33 ( 2 ) information for seq id no : 31 :( i ) sequence characteristics :( a ) length : 49 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 31 : gaagaatatgcaattccgcctaaaatagtgcattacgatacaaacttaa49 ( 2 ) information for seq id no : 32 :( i ) sequence characteristics :( a ) length : 46 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 32 : atgcactattttaggcggaattgcatattcttccttatagttattc46 ( 2 ) information for seq id no : 33 :( i ) sequence characteristics :( a ) length : 36 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 33 : atatctacgatgattttctaggttcgggacattttc36 ( 2 ) information for seq id no : 34 :( i ) sequence characteristics :( a ) length : 36 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 34 : gtcccgaacctagaaaatcatcgtagatattttctg36 ( 2 ) information for seq id no : 35 :( i ) sequence characteristics :( a ) length : 43 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 35 : cctcaggaattcgtcgactatttacacagcatcatcttctgag43 ( 2 ) information for seq id no : 36 :( i ) sequence characteristics :( a ) length : 47 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 36 : tgagatatatctaaagaagaatactttcattacgatacaaacttaac47 ( 2 ) information for seq id no : 37 :( i ) sequence characteristics :( a ) length : 18 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 37 : taatataatcttttatac18 ( 2 ) information for seq id no : 38 :( i ) sequence characteristics :( a ) length : 42 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 38 : ccgttcagcttcttcgtcaatggtacaacacggctgttagac42 ( 2 ) information for seq id no : 39 :( i ) sequence characteristics :( a ) length : 33 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 39 : gagcggtcgacaagcttataggcgggaatatgc33 ( 2 ) information for seq id no : 40 :( i ) sequence characteristics :( a ) length : 48 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 40 : tgttgtaccattgacgaagaagctgaacggtttgcatagtttgttatc48 ( 2 ) information for seq id no : 41 :( i ) sequence characteristics :( a ) length : 35 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 41 : atgaaagtattcttctttagatatatctcatccac35 ( 2 ) information for seq id no : 42 :( i ) sequence characteristics :( a ) length : 70 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 42 : aattaacccgggatccaagcttctagctagctaatttttatagcggccgctataatcgtt60aacttattag70 ( 2 ) information for seq id no : 43 :( i ) sequence characteristics :( a ) length : 67 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 43 : ctagctagaagcttggatcccgggttaattaattaataaaaagcggccgcgttaaagtag60aaaaatg67 ( 2 ) information for seq id no : 44 :( i ) sequence characteristics :( a ) length : 28 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 44 : gttacatatgtacagaatctgatcatag28 ( 2 ) information for seq id no : 45 :( i ) sequence characteristics :( a ) length : 28 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 45 : gctagaattctcttagtttttatagttg28__________________________________________________________________________