Note: Descriptions are shown in the official language in which they were submitted.
~vo 95/12682 2 1 7 ~ PCTIUS94112198
V~.AI, VEC.[~ WITH E~OV:[~ VIRAL- nTAlCT~RF:A V.Lt~Us (BVDV) Ar~L~
R~ RQuND OF THE INVENIION
Field of the Invention
This invention relates to the field of BoviDe Viral Di~rrhP~ Virus (~Vl)`V),
and v~rrina~ for t he tre-~t~np-nt thereo
Informs~tif-n Di4closllre
van ZUl, M. et a~. Live ~*~n~te~ Pseudorabies Virus E~ e_~g Envelope
Gl~ Lvl~i~ E1 of Hog Cholera Virufi ~ol~ Swine ~inct both Pseudorabies and
Hog Cholera, Journal of Virology, Vol. 65, No. 5, pp. 2761-2765 (1991). U.S. patent
4,703,011, Kit, M., and Kit, S., Thymidine Kinase Deletion Ml- I s~ ..i 4 of Bovine
He1~esv.l .18-1, issued 27 October 1987. U.S. patent 4,824,667, Kit, M., and Kit, S.
Thymidine Kinase Deletion h~ 4 of Bovine He.~esvir~ls-1, V~rr-ine~ inRt
Inf~+;oll~ Bovine Rhin~+r~rheit;R ~O 1 ~ E Same and MP+hO~1C for the Pro~ t;nn
15 and Use of Same, issued 25 April, 1989. Collett, M.S., et a~., Proteins ~-co~P~l by
Bovine Viral DiQrrheQ Virus: The Genomic O~,q..i7~ n of a r~8LVh~lB, Virology,
Vol. 165 pp. 200-208 (1988). CQ11~Ptt, M,S., et al., M*eC111~r C1nn;nE and Nll~ o+;~le
Sequence of the refflivir~ls Bovine Viral D;~ P~ virus, Virology, Vol. 166 pp. 191-
199 (1988).
20 BacL~:lou,ld
Bovine viral ~;~r~l.PQ virus (I~VL~-V) is a I~e~Liv-- u8 h-plnnEinE to the family of
1~e Flaviviridae. It causes a nllmher of .lir~.~. l con-lit;~nR in sheep, goats, and
esper;Qlly cattle. The ~y- ~t~----R ~Ppen~ upon the age, phyRiologil~l and v~rolC~i
_tate of the ~nim~l In young ~--Rc~t hlP- calves and young adults it causes a
2~ ~;Re~Re which is ch~-i~t : ed by high ~u~Ldi~y and low mortali.+y. The 15y...~-L ""R
can in~ln~P fever, de~.G ~io~, occulo-nasal liRrh~rges~ The~ and ocç5~inn~11y oral
ulcerations. Apart from t~ese ~.~y effects the virus also causes
;.. r.. o~ .l".__ion.~ )lt~h,u~ BVDVi~re~ ~.. Rarenorn~llyrelatively
mild, the virus may ~O~ Q~? or enhQn~e the pQt~ .-i ,;ly of other c~;..fe
80 mi~oo~ ,..R
In olter or 8 -Rcept;hl.~ Qnim~lQ BVDV causes similar by...l)t~-..R to those
Rm~hed above for yuu~6~ s~Rceptihle calves. In ~ --, in pregnant smim5~lR the
virus has the ability to cross the plQCPnts and il~fect the fetus. The O!~ of this
infecti~n ~ R upon the age of the fetus and whether it is at a st~ge where its
86 ;-- -------P system is fully ~ --l-etA- t The poRR;hle o k~ of inf~ nR i~ fetal
-
W 0 95112682 2 1 ~ ~ ~ 1 S PC~rrUS94/12198
reabsorption, abortion, mllmmif1c~tilm, congenital ~lPfect~, birth ~lefect~, calves born
which are persistently ;..f~cte~l with BVDV and c~mpletPJy normal calves. Calvesborn which are peraict~ntly infPct~l with BVDV, le~lese"t the most important
segm~Pnt of this BVDV pathogenesis comrl~Y PersiatPntly i~fe~t~ nim~la shed
5 large amounts of virus into their ellvilo..~-.P~nt which can i~fect susceptible ~nim~la
Furthermore, even though perfiiat~Pntly infectP(l ~nim~lc are immllnotolerant to the J-
virus which i..fe~lqd them in utero, they do develop ~ e~ce when i..f~te-l with other
closely related BVDV biotypes. These infect;~na are ch~,.c~.ized by low morbidity
(because relatively spe~king there will not be many pregnant ~nim~la i..r~ e~ at the
10 right time during pregnancy to produce BVDV perRiatently i--f~.t~l normal calves),
but high mortality. This ~liaP~ce syndrome is known as mllco~ iae~e and often
m~..ir~glY itgeLf as a y~c~le contlitir~n with calves dying of a profuse watery
h~P~ which co--~ a large ~mollnt~ of fresh blood.
The i~,uu~ l~ce of this virus and it's widespread presence m the cattle
16 population has led to the development of many vaccines in the alL~,u~ to try to
p~t~v~:~t BVDV infect;on These v~rrimpa have been built on the tr~it;on~l concepts
of inactivation or Ptt~ml~t;~n but, because of the behavior of BVDV, they have many
aiE~nific~nt dl~wbac~.
It is generally acc~ted that in~-~livaled vaccine preparations are not as0 el~iv~ as ~tt~nll~t~l live v~rrin~fi Inat ~ivaled antigen from ina~ ~iv~ted vaccine
lion undergoes ~ ge:~OU8 ~ocea~; g. Af~ter inJection into the animal the
antigen hecQmsfi part of the a~imal's soluble protein milP~n The antigen enters
antigen pL~r .~ cells through pinocytotic m~rh~niamfi and this usually produces
slnt;ho-liPa U~ tely~ hec~llfie ~nt;ho~is~ cannot gain entry`into cells, they
25 norm~lly only i~ l viral life cycles when mature virus is l~lç~nerl from the cell.
On the other hand, ~nti~Pn i~om live virus which replir~ter inside cells, undergoes
~n~ cnou8 ~)rOl~eF~ and this mP ~h~niam produces the uleft:l~ed cell m~ t,~ l
immnnP regponses. Cell mP~ t^tl immnnP rP~ponne~ can ~ 7' oell8 inf~ct
vwith viruses and have the pote-t;ql of i ~ g the virus life cycle at a much
YO earlier stage. Cell me~ te1 ~ ul ~e~ are thus tl.m~ht to be ~L~ ~ely ~o. L~
in the ;----------r~logi Pfipnae to many viral infiPcti~n~
RPc~ e of t he cell m P~ te~ 8~u~ ~e, S~ `nll~ ~ ~ live products such as
v~ inP~ ghould induce good cell mP~ çcl respQn~Pc~ With BVDV, ~ttsml~tiQn of
the virus to produce the live vaccine does not always p l~ ~_ht that vaccine virus from
35 rnn~ the ;~.~n.~.n~v~ E_ on normally ~soci~tR~ with field i~ol~te- Roth J.A.
--2-
WO 95/12682 2 1 7 2 8 1 ~i PCT/US94/12198
and Kaeberle M.L., Sll~p~es~ion of Neutrophil and Lymphocyte Fllnctio~ Induced by
a Vaccinal Strain of Bovine Viral Di~rrha~ Virus With or Without the
~lminictration of ACTH, Arnerican Journal of Veterinary Research, Vol. 44 pp. 2366-
2372 tl983). The failure of the vaccine to stop the immlln~.s~.p,ession r~Rpon~e6 creates a serious drawback to the vaccine. An animal owner may be v~crin~ting
~nims~lR to protect Ag~inRt a ~iae~Re but because of the ~rope, lies of the vaccine the
owner provides an opportunity for other liRe~ReR to afflict the ~nim~lR This forces
the owner to use inactivaled BVDV v~rr.in~R, which bec~llae of the way in which the
immllne sygtem opeL~tes, are not particularly e~. tive.
In sllmm~ry, ina~livated v~ ineR are safe but not particularly err~live while
the ~tt~n~ live vaccines are more t:~- livt: but under certain con~it;~nR may not
be very safe.
This invention comhinnR the e~tivt~less of the ~nll~tetl live v~crin~ with
the safety of the ina_liv ted v~r( inaR~ Bovine h~r~e~vil ~18 type 1(BHV-1) is another
1~ major pathogen of cattle which produces le:s~i~at~"~ ~iRe~ce. Thus, in common with
BVDV, BHV-1 also re~lic~tçR at a mllt,os~ ulr~ce. We take the gene which codes
for gp53, a major ~ ol~ of the BVDV virus and a~inat which the host
produces EnhE~t~nti~l immnn~ reRpQnRpR~ and éA~ B it in bovine herpes virus -1
(BHV-1), this reccmhin~nt virus (BHV/BVDVgp53) is used as a vaccine a~in~t
20 BVDV. Donis, R.O. and Dubovi, E.J., Gl~v~kvteills of Bovine Viral Di~rrhse~-
M11C0B~1 Disease Virus in T~f~e~ Bovine Cells, Journal of General Virology, Vol. 68,
pp. 1607-1616 (1987) and Magar, R., et al., Bovine Viral Di~rrhP~ Virus Proteins:
Helc,oge~eity of Cyl~ll.n~ . and No~ l.ngP.nic Strains and Evidence of
53K Gl~ vte~ Neutr~1;7~ -. T~ e, Veterinary Microbiology, Vol. 16, pp. 303-
25 314. Cited ~ .,ce8 are illCv~0~ led herein by ~ ce.
SUNIMARY OF T~ INVENTION.
A re~lir~t;r~ nnnr~thn~pnic virus, for lJ~cveL~ g ~ e~e caused by Bovine
Viral Di~rrhs~q Virus (~Vl)V), where said replirQt;ng .. ~ .n~e~ic virus co.. ~ s:
a gene or gene cnmhin~t~.n taken from a BVDV virus, and said rerlic~t;rlg
30 nnnr~thngenic nrus filn~;nn~lly eA~c8~e8 said gene or gene comhin~t~nnF.mhorlimP.nt~ of thig invention inr~.ln~lg the following: A virus where said rerli~ ting
.~n~ hn~e~lic virus is ~tte~ t~l~ is sel~le 1 from ~tt~ml~ Bovine Herpes Virus
type 1 (BHV~ tt~mn~ts~ adenoviruses, ~ttenll~t,ed bovine m~mmilliti~ virus,
~t~.nl~ l bovine p~rillnm~virus, or ~tt~nll~te~ pse -rl~rabies virus. A virus where
35 said repli~ting nonr~thngpni~ virus i8 sl~tP-n~ t9~l and c~ tC;~ and tAlJLa~e8 any
W09S/12682 21 72 81 5 . ~ PCT/US94/12198
comhin~tion of the following genes: the genes that code for gp48, gp25, pl4 capsid
protein, p20 N-terminal protease and pl25/p80 protein. A virus where the
~t~nn~t;on i8 created by m~kinE the thymidine kinase (tk) gene n~nfilnr~;on~
A virus where a signal peptide is inserted ~r~cee-linE the gene or gene
5 comhin~tion that codes for gp53 in said Bovine Herpes Virus type 1 (BHV-1). A
virus where said gene that codes for gp53 is inserted into the ina~ivated thymidine
kinase (tk) gene site. A virus where the filnrt;on~lly e~ as~ g gene or gene
comhin~titn, used to create the virus, comrr~RPR a recomhin~l pl~Rmi-l with intact
viral DNA, said pl~mid comprising: a) a BHV-~ genomic DNA fragmPnt cont~ining
10 the thymidine kinase (tk) gene and having a ~Pl~tio~ to the thymidine kinase (tk)
gene, b) a p~o~o~l/l.olyadenylation signal inserted in the thymidine kinase (tk)gene llelPtion, c) a signal peptide gene sequence p~ce~;. E a gp53 gene or gene
cr~mhin~t;-)n all of which is inserted b~wee~ the promoter and th~ polyadenylation
signal. A vJrus where said pl~Rmi~ is made from a pl~mi~l havin the
lB characteristics of rl~Rmi~l pHAS4. A virus where said signal peptide gene sequence
i8 taken from any well chat~c1~ ed signal peptide sequences such as any of the
thirty-nine PY~mrleR of well ch~ l n ;7erl signal peptide sequences found i~
Perlman, D., et al., J. Mol . Biol. Vol. 167 pp. 391~09 (1983), inco,~ ted by
l~fe.e,lce. A virus where said signal pept;~le gene sequence is taken from
20 Psuedorabies Virus gIII gene (PRV) and/or Bovine Growth ~ormrne (BGH).
A virus where a pl~mill is ~electecl from the following pl~smi~lR~ a)
pBHVtkex-1::BGH/p53; b) pBHVtkex-1::gIII/p53; c) pBHVt~kex-3::BGH/p53; or d)
pBHVt,kex-3::gIII/p53. A virus that produces 1~he product of a ~ t;~m~lly
e8~ g gene or gene cc~mbin~t;~n is srle~le~l from one of the following viruses,
T11-3, T11-6, or T11-8. A viruR where the r.. ~ lly e~ gene or gene
comhin~t;on, used to create the virus, c~ ;F-e~ a recnmhinPr~ Rmi-l with intact
viral DNA, said rls~Rmill C~....l.. ~in,g: a) a BHV-1 ~H~ ` DNA fragmPnt. ~...1~;"i"E
the thymidine l~inase (t k) gene and having a ~ n to the thymidine kinase (tk)
gene, b) a ~)lo"'Ot ~/~olyadenylation signal i~ldd in the thymidille kinase (tk)30 gene ~lpl~;on~ c) a gp53 gene or gene C~mhin5~ffc!n inserted ~L.v~ the ~,O~
and the polyadenylation signal. A virus where the pl~mill is pBHVtkex-3::p53. A
virus selecte l from one of ~he following viruses, T2-3#3 or T2-2#5. A vaccine for
g ~l;Res3~e caused by Bovine Viral Di~rrheA Virus tBDVD) comp~sing a
rh~rm.-r,~ lly ~:l~clive A~O ~t of the viruses l~R~ribe-l herein and a carrier.A vaccine aR described above for p,~ g rli~e~e caused by BoviDe Viral
WO 95112682 2 i 7 2 ~ 15 PCT/US94/12198
Diarrhea Viru~ (BDVD) comprising a pharm~rentic~lly e~tive amount of a virus
described above and a carrier, said carrier comprising any phy~ logic~l bu~el~ dmP~ m, i.e. about pH 7.0 to 7.4 c~ i..ing from about 2.6 to 15~o serum which does
not contsin antibodies to BHV.
S A mPthofl of ;~ i7;~g an animal against infect;ous tlice~ce caused by
Bovine Viral Diarrhea Virus (BDVD) CQmrri~inE ~3minictçring to an animal a
pharm~rent;c~lly e~ive amount of a virus or vaccine rlp~rrihe~ herein.
A ~loce88 of ~ .; ..g a virus ~l~R~ rihe~l herein comprising: a) i~ t;nn of a
ftlnrtinn~lly e~ eE~ing gene or gene comhin~t;~n that causes BVDV, b) inserting
10 said gene or gene comhin~tinn into a repli~t;ng nonr~th~genic virus, c) s~l~ct~ng a
live-virus that fimrti~n~lly e~re~les the product of said gene or gene comhin~tion
A m~otl of ~ g a virus ~e~crihetl herein where the filnrtinn~lly
e~essillg gene or gene comhin~ti~n~ used to create the virus, is produced by a
as~ comprising the recomhin~t;nn of a rl~mi~ with intact viral DNA, said
15 pl~cmi-l comprising: a) a BHV-1 genomic DNA fr~EmPnt co~t~ F the thymidine
kinase (tk) gene and having a ~ t;on to the thymidine kinase (tk) gene, b)
inserting into the thymidine kinase (tk) gene (~ ti~n of said pl5~mi~ a
promnt~r/polyadenylation signal, c) inserting a gp53 gene or gene c~mhins~t;nn
between the promoter and the polyadenylation signal, d) tr~n~fecting cells with said
20 pl~mi~ to produce a rec~ mhin~t~ virus c~ ;..;..g said fimrt;~m~l gene or gene
cQmhin~ti~ln ingerted into a live virus that does not cause ;.. - .. o~ ion in the
usual host and ~ A~ e_~g said filnrti~ n~l gene or gene c~mhins~t;~ n
A m^~o~l of ~ ; a virus described herein where the filn~i~n~lly
~Al.,e_~ gene or gene cnmhin~ti~n~ used to create the virus, is produced by a
26 ~.OCOB~ Cc~mrriRing the reCQmhin~ti~n of a rl~mi~l with intact viral DN4 said pls~mi~ l;bill~;. a) a BHV~ - --ir DNA L~ .i. CO~ g the thymidine
kinase (tk) gene and having a ~le~ n to the thymidine kinase (tk) gene, b)
inserting into the thymidine kinase (tk) gene ~ et;rm of said pl~mi~l a
prom~.t~r/polyadenylation signal, c) ins~ g a gp53 gene or gene comhin~ti-.n
30 preceded by a signal pept;~ gene sequence b~.~ the promoter and the
polyadenylation signal, d) tr~n~fec-t;ng cells with said pl~mi~ to produce a
recQmhin~ virus ~",;c;";"~ said filnrti~n~l gene or gene comhin~ti~ln inserted into
a live virus that does not cause ;~ - os--~ e~:on in the usual host and ~I:A~ illg
said filnr~ n~l gene or gene comhin-~ti~n
3~
wo9srl2682 1 7~1 5 PCT/lJS94/12198
BRIEF DESCRIPTION OF TE~ DR~AWINGS.
Figure 1. Construction of the shuttle vectors for inserting foreign genes into BHV-1.
Figure 2. Strategy for appen-ling signal peptide sequences to the BVDV gp53 gene.
Figure 3. Maps of the five shuttle plnRmitlR for inserting gp53 into BHV-1
a. EXAMPLE 1. pBHVtkex-3::p53.
b. Ti'XAlvlPLE 2: pBHVtkex-1::BGH/p53
c. EXAMPLE 3: pBHVtkex-1::gIII/p53
d. ~XAMPLE 4: pBHVtkex-3::BGHlp53
e. EXAMPLE 5:pBHVtkex-3::gIII/p53
Eigure 4. Pretlicte~l trnnRrript maps of the BHV-l/gp53 recomhinant viruses.
Figure 5. Northern blots showing transcription of gp53 meRs~Pnger RNAs in the
BHV-1 recnmhin~n~5
Figure 6. Tmmlmo~le~ it~tionR showing ~ur~-ion of gp53 protein in the BHV-1
recomhin~ntcl~
DESCRIPlION OF T~ ~ h~l) E~ODlMENTS.
All of the terIns used below will be readily unde ,,lood by one skilled in the
art. In many places the name of the manllfnt~ , of eq~ p-nt or reagents are
provided in parent~ Ri~ after the e~ ....Pnt or reagent is namatl C~ ..n..ly used
ter ns, rea~nt~ and buffers such as llplnRmitlR~Il "Klenow Fr~mPnt~," "rPli~a-~in~
20 blunt ends," "Tris," t~hela7;ng buffers such as EDTA and EGTA, and comm- nly used
chromS.~,~.d~hy c-)lllmnR are ~ere.,~ d to without Çu~ e, PYrl~n~7~on
In the de ;,uLions of the construction of the c~ u~ used in this
~vt:"liù,~, stantlard moleclllar binloE~ l te~hniques were used and are briefly named
or laR.~rihe~l here. Detailed PYpl~n~t;onR of these te^hni-lues can be found in25 ~t~n~rd la~wd~u, ~ m~nll~l~ such as ''Molp~ll~r Cloning: a La~uLa~ul~ M~nnsll"
(1989), Sambrook, et. al., Cold Spring Harbor Press, Cold Spring Harbor, New York,
or "Current nvlocols in Mrlec~ r Biology" (1991), ~l~RllhPl, F. M., et. al., eds., Wiley
..,r~ New York.
This illvt:~lion cnmhins8 t,he ~ .,ess of the 5~ live v~rrinaR with
80 the safety of the inncliv~led v~crinaR We take the gene which codes for gp53, a
major ~ v,ulvL~i~ of the BVDV v~rus and agqin~t which the ho t produces
81lhE~tqnt;q~ .a, leE~01l8e8~ and e~cB~ it in bovine herpesuirus -1 (BHV), t~is
recQmhin~nt virus (BHV/BVDVgp53) is used as a vaccine agPin~t BVDV.
Bovine h~l.esv.l .~8 (BHV) is ~nl~tll~r ~or p~ o~ll of cattle which produces
l~ rliRe~Re. Thus"n C~ Ul~ with BVDV, BHV also replir~to~R at a mncoRs31
wo 95/12682 2 1 7 2 8 1 ~ PCItUS94tl2198
~urface. With BVDV, replirsltion iB mainly at the gut mncos~l interface with less
replic~tion at the les~ to~!t interface. With BHV it is the les~ilc.lol~t interface
which ~lnmin~t~R The commnn mll~oR~l immlln~ system ensures that immlln~
responses produced at one surface will be e~-;Lve at other surfaces. Thus the
5 recnmhin~nt virus of this invention, BHV/BVDVgp53, will, when ~lmini~tAred to
cows, sheep or goats, preferably via the intranasal route, replic~t~ in the re!~,uil~tc
mns~os~p and produce an immnnP reRponRe
Prior to the t;~l las~ion of the BVDVgp53 gene in BHV, the thymidine kinase
gene was ~alPte~ from the BHV virus using a ,ulocess known to ~ttenll~tP the virus.
10 The BHV, a live ~tenll~te~l virus, will repli~te and produce a cell mptli~te~response. As part of that repli~tive pl'OCe~, the BVDV gp53 gene will be é~ulassed
and, bec~nRe the viru~ is inside the cell, the correct ,uroce~;..g for a cell me~ints~
reRponRe to the BVDV gp53 part of the recQmhin~nt vlI`U8 will alBO occur. Most
~u~ la,ltly, this reRponRe will occur without the po~RRihle side effects of
15 i~ o~u~u~e~- on~ as only part of the BVDV virus is l~la8t:llt. ThUB, the invention
comhinPR the efficacy of an ~tt~ml~te~ live virus vaccine for BVDV, with the safety
of an ina~ivated ple~ualdlion.
The PY~mrlPR in the ~locedulès section are provided for illu~L~Lve ~oses
and are in no way int~n~lpd to limit the scope of the p1ase.ll invention. A~l media
20 and buffer solnt;~nR were made up in glass AiPt;llP~ water unless otherwise
in~ ~t~
C..... l~os.~ nR and A~lmini~t~ations - A ph~rm~Pllt;rPlly ~ livè ~.. o.. t of
the vaccine of the pleE_~l, invention can be employed along with a pharm~cellt;.~lly
acco~l~ble carrier or ~ nt as a vaccine agpin~t BHV-l and BVDV in PmimP~lR, such25 a_ bovine, sheep and goat_.
~mpleR of phP~rmP c~ .1 :r~lly ac~,ul able c . . . ~ . ~ or ~ihl~nt~ useful in the
~_~t illv~ iOIl inr~ any physi~ 1 bu~ d msrlillm~ i.e., about pH 7.0 to
7.4, Go~.l s.; ..; .~g from about 2.5 to 15% serum w_ich does not c~...l ~;.. P nt;ho~iP~R to
BHV, i.e., is ~ I ve for BHV. Serum which does not ~...f ~;.. g~mm~ globulin
30 is ,ul~rt-,~d to serum which c-...~ R ~pmm~ ~lO,hlllin ~5~mple,R, of serum to be
employed in the ~ t invention in~ P fetal calf serum, lamb ~erum, horRe
serum, swine serum, and goat serum. Serum protein such as porcine P~lhnmin or
bovine serum albumin (herPin~ "BSA") in an S~ o~ l of from about 0.5 to 3.0%
can be employed as a s~ I s for the seruin. However, it is desirable to avoid the
35 use of foreign ~.ol~s in the carrier or ~ Pnt. which will induce allergic re~ponRPR
WO 95/12682 PCT/US94tl2198
~172~1S
in the animal being v~r,r,in~g~gtl
The virus may be diluted in any of the COllV~ on~l 8t5~hili7.ing golllti
contqining phosph~Q buffer, ghltDm~te, c~citon~, and sucrose or sorbose, or
c~.nt~;..i..~ phnsrh~te buffer, l~rto~e, dextran and ~ t~m~t~
It is p~ ~,ad that the vaccine viruses of the present invention be stored at a
titer of at least 105 to 106 PFU/ml at -70C to -90C or in a lyorhili7~tl state at 2C
to 7C. The lyorhili7ed virus may be reronRtit~lte-l for use with sterile ~ tillpd
water or using an aqueous diluent c~ illing yl~ ,v~lives such as ~..t~l..irin and
~mrhnl~.. c; -- B or pPnirillin and sLaptom~ ;n.
The useful dosage to be ~mini~t~vred will vary ~Pp~n~ g upon the age,
weight and species of the animal v~cr.in~t,e~l and the mode of ~-iminiRtration. A
gllit~hle dosage can be, for eY~mrle, about 104-5 to 107 PElJ/~qnim~, yl~f~ably
about 104-~ to 105-~ PPIJ.
The v~ccinP~ of the yieee.~t invention can be ~mini~tered intr~n~R~lly,
15 i~v~ginally or i~ll ~....~Rclll~rly. Intr~n~lly is the yrere~d mode of
~lminiRtration.
Utilit~r of the Invention - This invention is intQ.n~e~l to provide the user with
an e~.ilive vaccine for y'~v~lion of BVDV caused tiiRe~Re, where the vaccine canbe safely and effir~rinusly ~mini~tered illl. ~ RClll~rly~ intr~n~R~lly, or
20 i~ avaginally. Intr~n~Q~lly may be the ~l~fo,.ed route of ~-lmini~t~ation.
The v~inP~ of this i~ve~llion are ~ ed with the intpntion of treating
~iRe~Re, ~ E~ ~ably through y~ . nlion. By lJ~ ~ or p~ nlion applicant means
to keep the host ~om developing syllly~v~us of the ~liRe~Re or to mitig~e the effects
of the liRe~Re, that i8 to avert the typical ~iQp~Red state. n~ Lion imrliP~ decisive
25 action to stop, ;...l.e~iP or delay the onset of ~ e~Qe ~e~vt:~liull can in~ln~e 1~he
following cu~ el)t~ to hinder, ~ustrate, to O~hlLU~; to ,~t~_r~o~t, possibly prohibit,
imre~le or preclude. Preclude would sngEe~t the onset of the ~iiRe~Qe etate either
does not occur or the ~ e~Qe ~a~ ~LL is largely ineff~ l in c~llQ;ng the ~ e~Re
state. n~ t ûr ,u,~ lion can in~ he ~liRe~Qe state is for~t~lle~l, me~nir~g
30 that ~nt;~ action to ~evel~t or hinder the ~ Ç~Re has oc~ .uL~d but the
C n~lit;~n~ crea'dng ~he ~iiRe~e have not been ~.limin~
The ll~P*llnPQR of this invention will be illustrated by the lability of the
vaccine to provide elI~clive ~u~lk~ )n siE~inPt the spread of BVDV ~;QP~Qe in its
various m~..;r~ :---.Q. Rer~llQe the vaccine uses gp53, a major ~lyeuplol~ of
35 BVDV, and one a~in~t which the host produces a sllh~t~nt;~ ...P ~e~..Re, the
W O 95/12682 2 17 2 ~ 1 5 PCTAUS94112198
vaccine will confer enh~t~nt.i~l banafit,R upon the treated potD~nti~l host. Another
object of the invention is to provide a BVDV vaccine which can be ~mini~t~red
safely to calves and to pregnant cows in all stages of pregnancy.
~ rç~nres of ActivitY - The vaccine uses gp53, a major ~ly~iop~vteill of BVDV,
5 and one against which the host should produces a substantial immllne respon~e
Others have shown that gp53 is highly immllnogenic. Donis, RØ and Dubovi, E.J.,
Gly~;opro~il~s of Bovine Viral Diarrhoea-Mllcos~l Disease Virus in Tnfecte~ Bovine
Cells, Journal of General Virology, Vol. 68, pp. 1607-1616 (1987). It is well known
that agents that produce sllhst~nti~l immnna re~pQn~e~ can make err~ive v~c~ine~.
10 Magar, R., et al., Bovine Viral Diarrhea Virus Proteins: Hele~v~ ~eity of
Cytopathogenic and Non~l~alllogenic Strains and Evidence of 53K Gl~. vp
Neutr~li7.~t;on Epitope, Veterinary Microbiology, Vol. 16, pp. 303-314. The v~ .ina~
of this invention CC --t9;-- genes that t;~,u~e~3 large qll~ntit;eE of gp53, this iB BhOWn
in figure 5. Rec~ e of the ~:AlJr---ion of large qll~ntitia~ of gp53 the v~r.ina~ of this
15 invention will confer sllh~t~nti~l banafitc upon the l,~ated potenti~l host.
I~ef~ .l_d Co...uuullds - Any BHV-l virus ~tt~.nll~t~l with a t~ tlaht;rn and
C~l ~g the gp53 gene, the gp53 gene being preceded by a signal peptide, that
eAl,res~es ablln~l~nt A~.o!~.t~ of gp53, should be a prerelled snitqhla vaccine
c~n~ te. It ap~e~., the signal peptide ~equence may be taken from any snit~hle
20 source. We chose to e~...i..a two dirL.~t signal peptides to ensure the best
10~s~1i7.~t;~n of the gp53 ~o~ in vivo. We chose two c~n~ we call "T11-6",
~Pmho~ie~ in ~y~mrle 2, and "T11-3", amhotlip~ in ~y~mrle 3 for v~e trials. The
fonner virus was ~le~os;l ~tl to the ATCC under the lP~i~n~t;~n UC VR-58. The
latter, "T11-3" pl~mi~ was also t~ br~;le~ The virus we l~hela~ "T11-8" might
25 ~.. .l ~; .. L .. .-~ ~ e l forms of the tk L~.P .. ;1-~ and this might ~u~ Dl~ but does not
nacesQ~ - ly mean, that it would be less alL.._liv~ as a vaccine c~n~ t~ A largemlmhPr of PYiQhn~ cell lines are perRip~ntly ;..r~ with non ~L.~ BVDV
from p~RRa~c in media c~ c;--;-~g fetal bovine serum taken from i~r~lel calves. For
this illv~lltion~ it is ;"'l'~ vt: that viruses used as live, ~tt^nll~t,e l v~ n~R are free
30 of c~ t;ng BVDV.
n~..aL~lion of the Cu~uoullds
ConQl~uction of e~pr~ ~r~.n r.~ tu o for gene inOertion into
Bovine herpc..~ ~ 1;ype-1 (BHV-l).
We constructed two ~Qhnt~le v~ lwD to allow insertion of foreign genes into
35. BHV-l. Although thiB invention shows the utility of BHV-l as a vector for BVDV
W0 95/12682 217 2 ~15 ~ ; PCT/US94/12198
genes many other viruses could fill the same role. Other ~oy~mrle~ from cattle,
sheep and goats would inc]~ a cow, goat and sheep pox viruses, adenoviruses,
bovine m~mmi~iti~ virus, bovine p~pillnm~virus~ and psell-lorabies virus. A non-pathogenic virus refers to any virus which has the ability to repli~te in one of its
5 host species but does not produce any signs of ~ e~e in that species. Such non-
pathogenic viruses might arise from pathogenic parent viruses by natural mllt~ffon,
might be mutagenized by, for inrt~nce, chPmic~l~ or light to produce a non-
pathogenic virus, or could be rendered non-pathogenic through the use of
recomhin~nt DNA tqrhnl~lngi~ See, 1) Mapping Neutr~li7~t;on D!omains of Viruses,10 E.Wimmer, E.A. Emini, and D.C. Di~mnn~s~nd 2) Tmmnnogenicity of Vaccine
Products and Neutralizing Antibodies, E Norrby. Both articles are in Edited by
Notkins and Ol~tonç pllhliche-l by Springer-Verlag New York Inc. 1986.
Since we inten~scl to ~ttenl~t~ BHV-1 by ina~iv~ g the viral thymidine
kinase (tk)(M. Kit, et al., US Patent 4,703,011, (1983 )), we l~ri~l9l1 to use the BHV-
15 1 tk gene for the site of insertion. This approach not only insured the cnmrleteinactivation of the viral tk, but also allowed us to select recc-mhin~nt., t~k-negative
virus by est~hli~hs~ metbm3~ M. F. Shih, et al., Proc Natl Acad Sci USA,
81:5867-5870 (1984 ). Other m ~o~lR to ~tt~ ts BH~-1, such as dçlP~;on of other
non-e~sPnt;~l genes would also be appli~hlç to this particular invention. We started
20 with pl~mitl pHAS4 which co.~ a 2.7kb SalI subfragmPnt. of the BHV-1
T-Tin~mT A frs~mçnt cloned into pl~Qmi~l pUC18. E. relLvvLs, unpllhli~he~l dat,a. M.
Engels, et al., Vi~U8 Res, 6:57-73 (1986); J. E. Mayfield, et al., J Virol, 47:259-264
(1983); A. L. Meyer, et al., Biochim Biophys Acta, 1090:267-9 (1991). AB shown in
Fig. 1, this SalI fr~mPnt c~ 1 the entire tk gene, as well as a portion of the
2~ u~ e2~ gene h~ mnl^~ous to the HSV-1 UL24 gene, and a portion of the
~l~p.ol~ H gene. L. J. Bello, et al., Virology, 189:407-414 (1992); J. G. J~
et al., J Virol, 63:1839-1843 (1989); M.1Kit, et al., US Patent 4,703,011, (1983); A. L.
Meyer, et al., Biochim Biophys Acta, 1090:267-9 (1991).
A 424bp ~ was ~L~ duced into the tk gene by di~ pHAS4 with
80 BglII and XhoI, filling in the ends with the Klenow Fr~gm~nt of DNA polymerase I
(Klenow) and rel~t;~ the r~s~llt;ng blunt ended fr~n~nt~ ThiB m~n;P111~t;~n
.:slu.ed the BglII l~cn~ ite, but not the XhoI site (Fig. 1). The reslllffn
rls~mi~l wag named pHAS4~BX. ~is fl~J~;~n was chosen he~n8e it does not
impede on the previously i~l~nt;fed transcription init;~t;on sites for the UL24
35 h~ml~l~ which overlsps the 5' end of the tk gene. L. J. Bello, et al., Virolo~y,
-10-
WO 95/126æ PCT/US94/12198
21728~5
189:407-414 (1992); J. G. J~cobson, et al., J Virol, 63:1839-1843 (1989 ). Numerous
other ~l.qtionR within the BHV-1 tk gene would be posAible. To f~ it~te later
~loning manip~ t;~-na, we elimin~Rd the ~Tin~lTTT site in the pUC18 vector by
dige~ting pHAS4~BX with T-TintlTTT, filling in the cohesive ends with Klenow, and
5 rçli~ting the blunt ends.
We obtained a 1775bp r~Ccett~ cvllt~ ing the T-Tllm~n cytom~E~lovirus (CMV)
major imm~ t~ early promoter and the bovine growth hormone polyadenylation
sequence. R. J. Brideau, et al., J Gen Virol, 74:471-477 (1993 ). These gene
~eE -:on signals are commonly used for high levels of c"~l~ e~:on of foreign gene~
10 in a nllmher of di~c~cl.t systems, but other promoter/polyadenylation signal pairs
could also be used in this cv..~ The c~aaett~ in vector p3CLrD~ is bonn~
by unique EcoRI and BglII sites and cv..t~i..c, be~w~ the promoter and the
polyadenylation signal, unique T-Tin~TTT and SalI ~ ;nn sites for cloninE offoreign genes. The p3C~DHFR vector was digested with EcoRI, then filled in and
1~ ligated to a R~m~T linker (New h'.ngl~n-l Riol~h~ Beverly, M~aa~hllaet~c). ThiB
manipulation regenerated the EcoRI site. The construct was then digested with
R~m~T and BglII and the l~len~lc-l c~s~ett~ was ligated into the BglII site of
pHAS4~BX (Fig. 1). The lig~t;ona were transformed into E. coli strain DH5a. We
iaol~ l recomhin~nt, pl~amitla that c~ tc;~e~l the p3CL insert in both orientst;~ ~
20 relative to the BHV-1 tk gene by mapping of aBymmetric ~e~ C~ on sites. These two
constructs"la~i~n~ 1 pHAS4AR~PY-1 and pHAS4AR~Z~Y-3 (Fig. 1), c~ Pd then,
a strong promoter and polyadenylation signal bolm~P~ by the BHV-1 tk gene and
fl~nkinE~ regions to allow hnmolc!~ous recomhin~t;~n into the BHV-1 genome.
Figure 1. Construction of ~hllt~l? v~\ D for inE~ foreign genes into
25 BHV-1. PHAS4 iD a 2.7kb su~L~ ..t, from the BHV-1 ~inAm A fragmPnt, The
BglII~XhoI subfr~nPnt to be ~?I~.t~ l is shown. The l^lrt;~ n d~,;v~Liv~ of pHAS4 iD
pHAS4_BX. The ~lPl~te~l thymidine kinase (tk) gene is shown as a dark ~irrlP~l
box. The c~Q~ette C ~f ~ , the promoter and polyadenylation signal iB shown justbelow pHAS4~BX. The CMV immP~ te early promoter is shown as a light stippled
30 box, and the Bovine Growth Hormone (BGH) polyadenylation signal is shown as a~t~ ipe-l box. Finally, the inserts of the two w,l, e~:on Rhll~le pl~mi~
pHAS4AR~PY1 and pHAS4AR~ZPY~ are shown.
Addition of ~ign^l Peptide Sequences to BVDV gp53 gene.
A cDNA c~".tC".;"g the BVDV gp53 gene from strain 2724, a nu~Lu~ ic
85 strain, has been ~ u81y /lp~rihetl K~nnP~ly~ M. et al, a~ ~ of the ,~m~.ri~n
WO 95/12682 ~ 1 7 2 81 5 PCT/US94/12198
College of Veterinary Microbiologict~, 1992 workshop. Since the BVDV RNA genome
is normally tr~nRl~tRd as one long poly~ro~i~ and then post-trs~nRlntionsllly mo-lifiarl
into the various viral proteins, the gp53 portion of the BVD~T genome does not
cont~in the usual signal peptide required for tr~nRln~t;-.n of the protein to the cell
5 membrane, where the protein is normally expressed. NonPt}lelPsR, the cDNA was
sllcce~fillly ~ e~d in both cell-free systems and baculovirus, and the protein
appeared to be tr~n~loc~ o~ylated and anchored in both systems, despite thelack of a co~v~l nn~l signal peptide. We ~eci-lP~ however, to evaluate expression
of gp53 in BHV-1 both with and without various signal peptides.
In order to attach mlrlPot;~lp sequences ~nCo~lin~ signal pept;~le~ to the gp53
gene, we i,~Lo~luced a R~qmT-TT site into 5' end of the p53 gene by site directed
mutagenesis, as follows: The p53 gene was blunt-end ligated into the filled-in
R~mT-TT site of pl~Rmi~l pSP72 (Promega Corp., ~ Qn~ wiRcQncin)~ thus removing
all Rs~mT~T sites from t~e re~lllting pl~mi~ We inlloduced a single base change, a
15 C to a G, 11 bases in from the init;~tion codon used by the cDNA, using a synthetic
c.li~n~ eotide and the "Double Take" site diLe~ed mllt~eP-nPQiR kit (Stratagene, La
Jolla CA) accv~ g to ~e m~ r. t~t ~ ~ ~r s in~tr~ t;on~. This base change iL~l~vduced
a unique Rslm~TT site into the gene wililoul altering the gp53 amino acid sequence
(Fig. 2 section B). The base change was verified by nn~leot~le seql~en~ing~ and t,he
20 reRlllt~ne pl~mifl was called pP53mut. We inserted, into pP53mut seqllPnceR~
Pn~ otlinE signal peptides from the PRV gIII gene (A. K l~bhin~ et al., J Virol,58:339-347 (1986 )) and from Bovine growth hormnne R. P. Woychik, et al., Nucl
Acids Res, 10:7197-7210 (1982). (Figure 2 section A) Comrlentory oigomlcl~Qt;i~e~
4rl;"~ the two gignal peptites were srtl-P~ tl such that PnnP5~14rl oligos had
25 Sall cûhesive ends ~' and R~m~ cohesive ends 3' (E ig 2 section A). These signal
peptide Cr~~_~CF were ligated into pP53mut r~ tetl with R~m~TT and SalI, and
tr~nafolmerl into DH5a. We ~..1;- .~er7 the correct insertion of 1~he signa7 peFt;~le
~F~ D' by nnrl~ 7r seqnr~n~-;n~-
Cqmpl~nr~ntory ol~mlrl~t;~le~ ~.7;..~ any we~ ch~r -- 1~ 1 sign.,l
80 peptide can be used in 1~his iDvention. Thirty-niIle r~Y~mrle~ of we11 chals-- ~ e
signal peptide sequences found in Perlm-on~ D., et al., J. Mol . Bwl. Vol. 167 pp.
391409 (1983). Lcv.~ul6ted by .~f~ ce. These and any other well ~al~ct~.lzed
signal peptides should be ellit~ble for use as e-mhorl;..-t--.lv of this i--vt:~lion.
Figure 2. ~L~l~ for ~ >y,-rl;"~ signal pept;dç sequeIlces to the BVDV
85 gp53 gene. ~qe~;on A: Synthetic oli~Qn~lrleot;~lPy cv,,~. ~u~ tl the signal
-12- 1
WO 95/12682 2 1 7 `~ ~ 1 S PCI/US94/12198
peptide sequences of Bovine Growth Hor_one (BGH), and Pseudorabies virus gIII
(PRV gIII). ComrlçmPnt~ry oligonllrlP~otides were srtheRi7e-1 such that the
slnn~nla~ pair8 had ~lTRitAR on the 6' ends and R~mT-TT sites on the 3' ends. The
rlP~lncetl amino acid sequences of the signal peptides are also shown. In each case
5 the predicted cleavage sites for the signal peptides are just sfter the ~l~ninP (A),
three amino acids from the ends. Codons for two amino acid reRi~l~leR (F,P in BGH;
P,S in gIII) from the original native proteins were left on the signal peptide
sequences to ensure correct cleavage.
.Sect;nn B: Site di~ cted Mutagenesis of the cDNA anco~ing the BVDV gp53
10 gene. The first 60 nll- l~oti~laR of the gp53 cDNA and the col~ u~ ing amino acid
sequence are shown. A single base pair, shown by the arrow, was changed to create
a R~mT-TT le.,L,;~lion site in the sequence, shown in the box. This change does not
change the amino acid sequence. The cDNA was then digested with R~m~TT a8
shown, allowing in frame ~ n to either of the signsl peptide sequences shown in
15 section A.
Other ~ 0~3ion gene L~ t~ in ~ Qn to gp53.
E~lJres~ion of other BVDV gene or gene cnmhin~t;nn~ in a live virus vector
are also e~nho~limanta of this invention. This would inrlll~a any and all BVDV
lJlo~illB to which a v~r~in~ted animal could elicit an immllna responge. T~y~mple~
20 inrlll~, but are not limi~d to, the other two BVDV sulfL.ce ~ rol~ills, gp48 and
gp25 (Collett, M.S., et al., Virology 165:200-208 (1988)), the pl4 capsid protein
(Thiel, H.J., et al., J. Virol. 65:47054712 (1991)), and the p20 N tArmin~l ~.olease.
Wiskerchen, M., et al., J. Virol. 65:4508-4514 (1991). This group of proteins, along
with the gp53 gene, can be ~ ~e3ed t~et~ Pr from a single cDNA molPcllle~ the
25 ~ eaed poly~ v~eL itself ~uLL~_t1y into the E~ te proteins.
.~nnt~Pr BVDV ~u~Ot~ cs~n~ t e to ~~ ,.,L in a vaccine is the nonstructural
pl25/p80 ~lUk:i~l (Deregt, D., et al., Can. J. Microbiol. 37:815-122 (1991)), which
elicits a ~i~nifi~nt ~n~;hody re~pqn~e in ;..r~,le 1 cows.
Insertion of the BVDV gp58 gene into the BHV-l espression vectors.
The p53 gene, either with or ~.ilLuut added signal peptide seqllence~, was
l~tetl into the ~intlm in_~ sites of pHAS4AR~Y-1 and pHAS4AR~Y-3 by
filling in all the ~E~e Live cohe~ivt: ends of ~ _l~o and inserts followed by blunt end
li~ti~ n The lig~ n~ were ~ r.,l ..-P(l in E. coli strain DH5a. We w~ted to
ev~nt~l~lly evaluate the e,.,u~ on of gp53 in BHV-1 in ~ Jus ~1 ient~t;~n~ and
35 with at least two ~ t signal pept;~ to ensure that we achieved the most
-13-
WO 95/12682 PCT/US94/12198
217~15' '"
efflr.j~nt t~ s~,ion. The transformed coloniPc were screened by colony hybritli7~tion
using as a probe the p53 insert l~hçllP~ with Di~,o~ygt:llin-dUTP. The "Genius" DNA
hybritli7~t;~ system (Boeringer M~qnnh~im Ri~chPmir,~lc (BMB), Tn~ n~roliR, IN)
was used for this and all other DNA hybrif~ tionR ~escrihe~l in the char~rteri7~t;~
of this invention. r~,ilive reComhin~ntR were then screened by rç~t- iCtioT- analysis
for those carrying the gp53 gene in the proper oriçnt~tio~ relative to the CMV
promoter and BgH polyadenylation signal. Five pl~Rmi(lR were iRC~ , which are
grhPm~t~ic~lly ~epictç~ in Figs. 3A-E. Their des~ Lions are as follows.
~X~MPLE 1. pBHVtkex-3::p53: co..~ c the BVDV gp53 gene inserted
be~wet:-l the CMV ~l~.~ole~ and the BGH polyadenylation signal of
pHAS4ARXPY-3 with no added signal peptide. In this construct the
original gp53 gene, PRIOR to site dire_led mllt~gPns~iR~ was inserted.
See Fig. 3A. This pl~Rmill was then used to construct the virus T2-3#.
~X~MpLE 2. pBHVtkex-1::BGH/p53: c~ ;..R the mutagenized gp53 gene
16 preceded by the BGH signal peptide sequence inserted into pHAS4AR~PY-
1. See Fig. 3B. This pl~mi-l was used to create the virus T11-6. This
virus was tlPpo~;lærl
vrPTE 3. pBHVtkex-1::gIIVp~3: c~n~inR the mutagenized gp53 gene
preceded by the PRV gIII signal pepti~le sequence inserted into
pHAS4ARX~Y-1. See Fig. 3C. This pl~mi~l was used the create the virus
T11-3. This pl~mill wa8 ~ ,p~
PIE 4. pBHVtkex-3::BC~H/p53: r~ c;..~ the ~l~ui~ellized gp53 gene
preceded by the BGH signal pept;~ls sequence inserted into pHAS4AR~ZPY
3. See Eig 3D. ,l
li!~MPIE 5. pBHVtl~ex-3::gIII/p53: cr~"~ the .~ e~3 gp53 gene
~dEd by the PRV gIII signal pe~;ds sequence ~e_./Led into
pHAS4AR~PY-3. See Eig. 3E. This pl~Rmi~l was used to create the virus
T11-8. This pl~mi~l was ~l~yo~ile 1
E~gure~ 3A-E. Comrl~te maps of t~e five shuttle pl~miflq for inserting gp53
into BEV-l. The gp63 gene is shown as a solid band, the BHV-1 sequences are
shown as dark stippled bands, the CMV promoter region is ahown as a light s~ppled
band, and the BGH polyadenylation signal region is shown as a striped band. The
pl~mi~ vector, pUC18, is shown a8 a thin line. In each case the direction of
tr~n~cription of gp53 relative to the original direction of transcription of BHV-1 t~k is
35 shown. The various signal pept;~e sequenceg are in~ir~t~
WOg5/l2682 ~1 ~2~15 PCT/US94112198
a. EXAMPLE 1. pBHVtkex-3::p53.
b. ~Al\/IPLE 2. pBHVtkex-1::BGH/p53
c. EXAMPLE 3. pBHVtkex-1::gIII/p53
d. E~AMPLE 4. pBHVtkex-3::BGH/p53
e. EXAMPLE 5. pBHVtkex-3::gIII/p53
These, and all other po~Rihl~ insertions of the BVDV gp53 gene into the BHV-
1 tk gene are emho-lim~nta of this invention. These pl~Rmi~a and any ~ ami~la
~ated in this m~nn~r are known as "Principal Plasmid Vectors" and are the
pl~ami~ vtz~ used to create the virus v~inea7 of this invention.
I~l~rod~ n of the gp53 gene into BHV l 'lowa".
The five ~r.~s~iion shuttle rl~#mirl~ car~ing gp53 were lin~ri7e~ by XbaI
and cotr~n~fecte~l into Bovine Turbinate (BT) cells with unit length DNA from BHV-
1 strain Iowa (tk po~jilive) by the st~n-3~rd CaP04 m~tl~ (R. L. Grah~m, et al.,Virology, 52:456-467 (1973 )) as mo~lifi~3 by Cai (W. Cai, et al., J Virol, 61:714-721
15 (1987 )) . The cells were obtained from ATCC. The tr~nafac~;~.na were then subjected
to two rounds of sel~cti~.n either on 143tk- cella (S. K Mittal, et al., J Gen Virol,
70:(1989 )), or on Rab (BU) cells (S. Kit, et al., Virology, 130:381-389 (1983 )) in the
presence of 100ug/ml 5-Bromo-2'-DeuAyu~;dine (BDUR, Sigma ChPmi--~l Company,
St. Louis, Miaaollri) to isolate virus no longer tA~Las~ g tk. ThiB iB a st~n~l~rd
20 l~locadura described previously. M. Kit, et al., US Patent 4,703,011, (1983). Other
1 k- cell lines permissive for growth of BHV-1 can also be used. After the two rounds
of BDUR p~aaa~e~ tr~nare~ a that still showed ~l~hll.ic effect were ;..r~led onto
BT cells under comrletn media with 1% low m~ltir~ agarose to obtain single
plaques. ~nltirle single plaques were picked from each tr~nafe~;~n and the viral25 DNAs were c~ ed for the p53 gene by dot-blot DNA hybri(l; ~l :-... Alt~m~gh not
all tr~n~ a ~ iv~:d t he BDUR pas~s (particularly those on the 143 tk-
cells, as these celLa are only ~a~ ally permiaaive for BHV-1 viral growth), those
that did ~ iv~ yielded 100% rec~mhin~nt virus. Four ~ ..l, recomhin~nt~
viruses were iaol~ l and further chh~ t~
~ MPLE 1. T2-3#3 and T2-2#5 (two ide.. l :~1, but ;~ rl~ntly iR.~l~t
viral clonea): BHV-1 "Iowa" into which the insert sequences ~...l9;..9-1 in
pBHVtkex-3::p53 rec~.mhins~ Cqnt~ina the BVDV gp53 gene with no
added signal pept;~ls sequence ait l~t~ belwde~ the CMV promoter and
the BGH polyadenlyation signal, wi1~h L a~ -lional oriet41:-in the
same direction as the BHV-ltk gene.
-15~
WO 95112682 ~ 1 7 2 81 5 PCTrUS94/12198
lvlPLE 2. T11-6 (This virus was snhmitte~l to ATCC Lnder the
igns~t;on UC VR-58): BHV-1 "Iowa" into which the insert sequences
lt~ P.~l in pBHVtkex-1::BGH/p53 recomhine~ Cont~ina the BVDV
gp53 gene with the BGH signal peptide sequence ait~l~t~ bt ~ween the
CMV promoter and t-h-e BGH polyadenlyation signal, with transcriptional
orient~tion in the opposite direction relative to the BHV 1 tk gene.
li'.~AMPLE 3. T11-3: BHV-1 "Iowa" into which the insert sequences
C~llt9i ..erl in pBHVtkex-1::gIII/p53 recomhine~ Cont~ir R the BVDV gp53
gene with the PRV gIII signal peptide sequence ait~ e~ between the
CMV promoter and the BGH polyadenlyation signal, with transcriptional
nrif..ntsti~m in the opposite direction relative to the BHV-1 tk gene.
F.~AlVlPLE 5. T11-8: BHV-1 "Iowa" into which the in~ert equences
co-.ls;..P~ in pBHVtkex-3::gIIVp53 recomhinPA Cont~ir R the BVDV gp53
gene with the PRV gIII signal peptide sequence sit~l~t,e~ bel-.~ecn the
CMV promoter and the BGH polyadenlyation signal, with transcriptional
oriçnt~tion in the same direction as the BHV-1 tk gene.
A virus was not ;aQ1~t9C1 from cotr~nafect;onR with "Iowa" DNA and ~ cmi~
pBHVtl~PY3 RGH/p53~ ~AMPLE 4, but this prophetic virus, could be easily
~eated, it and any other BHV-1 viruses rv~tC;~ g the BVDVgp53 gene inserted
into thymidine kinase gene are çmho~im~nt~ of this invention. We purified DNA
from each of these viruses and cher~ for the proper insertions in the proper
ntDt;~na by Rollth~rn Hybri~i7ot;~n using bot-h- the gp~3 gene and the CMV
p~v ol~./BgH polyadenylation c~aaette as probes (data not shown~. All four of the
viruses carried the c~mplete promoL~ /~ne/polyadenylation c~et;ter in the BHV-1
2~ gene, dolete~ as pre~icte~, based on, ~ c~ " fr~EmPnt sizes. As a control, wi~h
these tr~naf~ on~, we also tr~n~f~cted the pHAS4~BX pl~mi~ with BHV-1 "Iowa"
unit length DNA and iaQl~tr~l a tk-lle~Livt: I o~.ly Cal~ ~ i~ the 424bp ~lPlpt;on in
tk (also verified by Sollt~ern Hy~ i7~t;~n). This viius is named Iowa~BX. All ofthese viruses were plaque purified twice by limit;ng ~lihlffr~n on BT cells.
A large mlmher of ~Yi~t;ng cell lines are per~i~tently ;--f~ ~e~ with non-
~p~ .ir BVDV from passage in media c~ fetal bovine serutn taken from
i~re~;t~ calves. For this invention, it is i~p~live that viIuses used as live,
~t~ml~ts-l v~rrinp~ are free of c~..tG.~ ffng BVDV. In order to ensure that the
BHV-1 viruses c~.~ the BVDV sequences were not ~ t~...;. ~te~l with non-
3~ hi~- BVD virus, we prepared DNA from each of the viruses (inrlll~lin~ the
-16-
i
Wo 9S/l2682 2 1 7 ~ 8 ~L S PCT/US941121~8
parent strain Iowa and Iowa~BX) and subjected the DNA preps to ~çnRive RNAse
treAtm~nt using a cloned RNAse (RNAse ONE, Promega CullJulation, MA~ 0n,
WiRconRin). Since BVDV has only RNA as its genetic material, this manipulation
should eliminAts any possible ~..t~ inAting BVDV sequences from the viral DNA
5 preps. We then tr~nRfecte~l these RNAsed viral DNAs into certified BVD-free
MDBK cells (ATCC) and picked virus plaques from the trAnRf~ct;-nR to use in
further manipnl~t;onR
l'ra~ucript~Q~ l aIlaly~i6 of the gp58 recQml~ -t~
We prepared RNA from each of the recomhin~nt viruses and the parent BHV-
10 1 strain Iowa and evAlllAterl transcription of gp53 by Northern hybri-li7Affon A
diagram of the poRRihl~ m~RR~ge species and the probes used is shown in Fig. 4.
Figure 4. Predicted trAnRr-~rts of the BHV-l/gp53 recomhinAnt viruses later
shown in Figure. 5. The two probes are 1) the gp53 cDNA and 2) the Sall/BglII
portion of pHAS4 (shown above the maps). The first map shows the predicted
15 tr~nR~ ~ from viruses T11-3 and T11-6, and the second map shows the predicted
trAnR~rirts from T11-8. The sites of transcript ini~i~ff~m for tk and UL24 are shown
for lcî~lc~
All of the gp53 recomhin~nt viruses made a 1.6kb m~ssa~e that hybridized
with a 32P-lAhe~ gp53 probe, the size predicted for transcription initi~t;~n at the
20 CMV promoter and termin~ n at the BgH polyadenylation site, Fig. 5, probe 1.
The T2-3#3 and T2-2#5 virus are not shown. As A~/litir~nAl major bands, T11-3 and
T11-6 made an 8.5kb trAnR~ipt and T11-8 and T2-3#3 made a 5.6kb tr~nR~irt.
These trAnRrrirts were unique to the reCQmhinAnt viruses, and were conRiPt~nt with
mPff8nePR initi~;ne at the CMV promoter, reading through the BgH poly adenylation
25 signal and t~-rminAt;r~ at the UL24 or tklgH polyadenylation ~ign~lR, ~e~E_liv~:ly.
IIyl.. ~ n with the ulJsl~ and downstream probes c~...l; r~P~ e
identity of these longer mARRAf~es. The p53 probe did not hybridize to Iowa, Ia~BX
or mock ;--fe~lecl RNAs. As a qvA~ n control we used probe p~A~6, an 867bp
salI fragmPnt that maps downstre~m of the 1~k open l~a&~ frame and iB internal to
30 1~he gH gene. A. L. Meyer, et al., Biochim Biophys Acta, 1090:267-9 (1991). All of
~he viruses made equivalent ~...o -..i~ of ~e 3.1kb gH mA~ e (data not shown).
This probe also hyl~ri~li7eri to the longer p53 m~Ass~es in T11-8 and T2-3-3, and to
the 4.3kb tk mp~a~e iIl Iowa, which iB 3' c~.l- ....i..~l with the gH transcript. L. J.
Bello, et al., Virology, 189:407-414 (1992).
To e .~ A ~he transcription ~aLl~.~s u~ ~ of the gp53 insertions, we
W O 95/12682 . , i - PCTrUS94/12198
used a probe that consisted of the pHAS4 fr~gmant from the upstream SalI site tothe BglII site in the tk gene, the beginning of the tieletion in the recomhin~ntviruses (probe 2). All of the viruses made a mPsQ7~Ee of a~r..x;...~t~ly 4.4kb which
we fletlllre~l to be UL24 (Fig. 5, probe 2). This maRsage~ however, was ,Q7m~llPr than
5 the 6.2kb UL24 mP~QQ~Ee in BHV-1 . train Cooper described by Bell~, et al (L. J.
Bello, et al., Virology, 189:407-414 (1992 )) and comigrated with the tk mee,Q,~Ee in
the wild-type ~train Iowa. Although we did not evaluate these comigrating
mPRQ7~Ee8 furt~er by using single stranded probes, we ~letecte~ a tk transcript of 4.2
kb only in the Iowa DNA with probe pHAS6 and we ~Ptects-l similarly sized
10 tr~qnRrripts in all the viral RNAs with the upstream probe, even though these other
viruses cannot be m~kinE a wild-type sized tk transcript. In T11-3 and T11-6, the
uy~7LIaall~ probe did not detect any t~-nr~t,e-1 forms of tk meRQ~aEe and hybridzed to
only the UL24 mPRR~Ee and the the 8.5kb p53 mP~R~Ee. In T11-8, on the other
hand, the probe hybridized to four ~ lit;~-n~l (minor) bands of ~. v~ tely 5.0,
15 3.7,1.8, and 1.0kb.
Figure ~i. Northern blots showing tr~nRrrirtion of gp53 me~QRPnEer RNAs in
the BHV-1 recombin~nt viruses. The first panel shows transcripts hybridizing to
probe 1, the pg53 cDNA, and the second panel shows tr~nR~irtQ hybridizing to
probe 2, the SalI/BglI subfragmant of pHAS4. KEY: M=Mock i~fa~tetl cells, I=BHV-
20 1 "Iowa" i~fe~;lptl cells, 3,6,8--T11-3, T11-6 and T11-8 ;~fe~e~l cellst RNA size
etsn~l~rds, in kilob~ReD (kB) are given to the left of each panel.
E~}~- sQn of BVDV gp68 ~_otA:n in BHV-l.
We ev~lnAt~ e. ~ioll of gp53 prot,ein in the BHV-1 rec~-mhinAntc by
immnnoy~c~ ib I :~m (IP). Detailed y~o~du~es for IPs can be found in ston~lArd
25 r~fe.~ ~ces such as "Current ~.)locols in Mol~ Ar Biology', ~ llhel, F. M., et. al.,
eds., Wiley IntersciPnre~ New York. BT cells ;~re~led with the BHV-1 r~QmhinAnt
were met~hc lirAlly lAhella-l with 35S-mPtllinninP (Amersham, Arlington ~aig~S~
noig). The viral i..fectr~ cells were lysed and soluble ~.ob~fi were .e&_~ed with
1~- .. ~.. P serum from bovine or goat ~Pin~t BVDV. VMRD, ~lllmAn,
30 Washington. ~nti~PntAnt body complPYs~ were ~ J;b~Ad staph A
(Tmmllnn~le&:pil .., Gibco/BRL, t~it~t,~ ;,, Maryland,) or protein A sepharose 4B
(Pharmacia, Uppsala, Sweden). Tmmlln~ &_Live ~obi~B were resolved by SDS-
Polyacrylamide gel electrophoresis (SDS-PAGE) and flu~o~Aphyl
Figure 6 shows that all three of the recnmhinMnt viruses c~rrying the gp53
35 gene preceded by a signal pept;~le sequence made ~ O --t~ of the lJ~o~ill.
-18-
Wo 95/12682 2 1 7 2 8 1 5 Pcr/Uss4/l2lg8
.
We did not detect any e~l les~ion of gp53 from T2-3#3, or T2-2#5 the viruses
carrying the gp53 gene, but l~rking a signal peptide, even though this virus
srt~eai~efl conRillerable amounts of gp53 mefiS~nger RNA. The clones t2-3#3 and
T2-2#B are indep.qnd~ntly ;RO1~te~ clones, which rules out the posRihility that one
5 particular virus had a defect that precl~ 3 gp53 e~e~ion (data not shown). The- po~aihlity rem~ina that gp53 ia being srth~Ri7ed from T2-3, but is rapidly
degraded, or that our ~ntiho~ly does not detect Ul.p .~cefise~ forms of the protein.
Figure 6. Tmmlln~ c. ;1~ letl proteins showing e~. as~ion of gp53 in the
BHV-1 recombinants. T ~b~ proteins were ~.~,..;~.;l~t~.l with polyclonal bovine-
10 anti-BVDV serum, this serum also had minor reactivity with BHV-1 antigens. KEY:
3,6,8=T11-3, T11-6, and T11-8 inf~cte~ cell ~ ~s, IA=BHV-1 "Iowa" inf~ctell cell
l.~ol~ins, M=Mock il~fe~Lç~l cell proteins. MW=at~ te protein moleclll~r weight
s~sn~rdR, in ~ilo~s~lton~
The gp53 protein bands in T11-3, T11-6 and T11-8 were broad, suggesting
15 1~hat the ~ ei.ls were ~r"c~rRecl, and they a~e~ed to be equivalent but not
+;~l in size to the gp53 protein in NADL (data not shown). Removal of the N-
linked sugars from the BVDV-NADL and BHV-1 ~ressed gp53 plole~s by
~lige~t;r~n with N-glyc~n~ne (Genzyme, Cambridge, M~nE~rhll~et~r) did not resolve
the size rl;lrer~ce in the proteins, but the p ~oL Lonal re~ n in size of the
20 ~Lole ll8 su~led that the native and recQmhin~nt forms of gp53 were ~-oce4~e~Eimil~rly. The slight size di~_~cl-ce belweeL~ the rec~mhin~nt and native proteins
could be due to the fact that the gp53 gene in the BHV-1 viruses can~e from a
dilr~ .~.lt BVD strain which could have a gp53 of a slightly dirr_r~llt size, or the
cDNA gp53 clone might not ~ ;-- the exact atnino acids L~socen~ from the BVDV
25 polylJio~i~ into native gp53.
The ~cst:~ invention is not to be limi~ed in scope by the cell lines ~lepo~ited
or the Pmho~limPnta ~in~lose~l herein which are intrltlP-l as single illustrations of
one aspect of the invention and any which are filn~;rn~lly equivalent are within the
scope of the invention. Tn-lPerl, various mo-l~ I ;r~ nR of the illv~Liull~ in ~lAit;~n to
30 those shown and ~es~ihe~l herein, vrill be~mP ~ 1, to those skilled in the art
from the f~eg~ .g ~eE~ m Such motlifi~t;onR are int~nded to fall within the
scope of the ~MAn~le~ claims.
It is also to be undo.~lûod that all base pair and amino acid residue n~lmherg
and sizes given for nllcleo~ e~ and peptides are ap~.u~ te and used for the
35 ~ oses of lP.e- - ;l-~ion.
-19-
W095/12682 72815 - PCTIUS94/12198
All docllmPn~c cited herein are inco~ol~ted by reference.
Deposit of Genetic Materials
One skilled in the art should be able to reconstruct all the various
embo~imentc of this invention by llt;li7.in~ only the wl;Lle~ des~iplion. However,
5 for the sake of c~mphPtAn~RR~ to ensure en~hl~mPn~, and to provide every o~l)o~ ..ity
for others to make and use this invention, certain genetic constructs of this
invention have been deposited at reco~ni~e-l ~o~; k . ;~c in accordance with theBudapest Treaty.
A viru~ was lPpositP~l with the AmPrir~n l~pe Culture CollPc.t;~ n, 12301
10 Parklawn Drive, Rockville, Maryland, zip code 20852, USA. That ~epoPit was
11PRiEn~t~ UC VR-58 by the Upjohn Co pally and given the following number by
the d~pQs;l~r.y, ATCC No. VR2436, it CO~ JU~ R to the virus described herein as
"T11-6," also known as "h'Y~mI l~ 2." This deposit was l2ceived by the American
l~pe Culture Collect;~n ~1~PQR;~ Y on 28 October 1993.
Several pl~mif~ were ~e~os;led with the Agricultural Rese~ Service
Culture Collsction (NRRL), of the U.S. D~l llent of A~ t~lre, at 1815 North
Unive~ y Street, Peoria, ~llinni~, zip code 61604, USA. One pl~mi~l was given the
Upjoh~ rleRi~n~t;~n, pUC 1564, E coli culture UC 15085, ~ to pBHVtkex-
1::gIII\p53, it culle.,~u~P to the rl~Rmil1 used to create the virus ~srrihed herein
20 as "T11-3," also known as ~Y~mrlP 3." This pl~Rmitl was given the following
mlmhf~r by the ~ n~ , NRRL B-21350. Another deposit was given the Upjohn
fl~Rign~ti~n, pUC 156~, E. coli culture UC 15086, ~f~ g to pBHVtkex-3::gIII\p53,
it corrçRpon-l~ to the rl~mi~ used to create the virus ~le~rrihed herein as, "T-11-8,"
also kllown as ''FY~mrle 5." l~ pl~Rmid was given the following mlmher by the
25 ~l~O~;lJJ~ RL B-21351. Both of the rl~mirl~ were l~;vt:d by the A~ lltnral Rese~rcl~ Service Culture Co~ n ll~oy;~ ~ on 26 October 1994.
-20- ;
W O95/12682 2 17 ~J ~ 1 ~ PCTrUS94/12198
SEQUENCE LISTING
Tt'NT'RAT, INFORNATION:
(i) APPLICANT: The Upjohn Company
v~ ORS (For U.S. Purposes only): Wardley, Richard C. and
Haanes, Elizabeth J.
(ii) TITLE OF lNV~N'l'ION: A Replicating Nonpathogenic Virus Expressing
Envelope Glycoproteins from Bovine Viral Diarrhea Virus (BVDV)
(iii) NUNBER OF ~Q~w~:S: 2
(iv) coRREspo~nT~lN~T~! ADDRESS:
(A) ADDRESSEE: Thomas A Wootton (1920-32-l), The Upjohn
Company
'B) STREET: 7000 Portage Road
C ', CITY: TCA 1 ~ - 7.oo
D STATE: Michigan
E CO~N1KY: U . S.A
~Fl ZIP: 49001-0199
(v) COMPUTER RT~'AT)~RnT~ FORM:
'A'l MEDIUM TYPE: Floppy disk
B COMPUTER: IBM PC compatible
C OPERATING SYSTEM: PC-DOS/NS-DOS
,D, SOFTWARE: PatentIn Release #l.0, Version #1.25
(vi) ~uKRhL~ APPLICATION DATA:
(A) APPLICATION NU.MBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NANE: Wootton, Thomas A.
(B) REGISTRATION NUMBER: 35,004
(C) k~:~N~/DOCRET NUMBER: 4748
(ix) TT~lnT~lcnMMTTNIcATIoN INFORMATION:
(A) TELEPHONE: 616 38 5- 7 914
(B) TELEFAX: 616 385-6897
(C) TELEX: 224 401 UPJOHN
(2) INFORMATION FOR SEQ ID NO:l:
gu~w~ CHARACTERISTICS:
~A' LENGTH: 8083 base pairs
B TYPE: nucleic acid
C STRANDEDNESS: double
~D~ TOPOLOGY: linear
(ii) NOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) O~GANT~M: Bovine viral diarrhea virus
(B) STRAIN: 2724
(C) INDIVIDUAL ISOLATE: pBHVtkex- 3:: P53
(xi) ~u~w~ DESCRIPTION: SEQ ID NO:l:
-21-
~.~1 728~S
W O95112682 PCTrUS94/12198
,; ., f, ~
GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60
CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120
CACTCATTAG GrA~CCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180
TGTGAGCGGA TAA~AATTTC ACACAGGAA~A CAGCTATGAC CATGATTACG CCAAGCTAGC 240
TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGTTGTCC 300
GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360
GTCCGCGTCC CCAACTCCGC GCGAAGACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420
GCGTA~AAAA. TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480
GCCCAGGCAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC rArAAA~AGC 540
GCC~'AAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600
CTGTTGCCCG CGTGCCTGCT GGCCGCCCAC CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660
CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCrArAGC CGCCCTTTTG CCCGTAGCCA 720
GGGGAAGCGG CTGCCCCTTC TGCCGCCGCG GCCGCGGTTG CTCGGCTTTG CGTTTGCCCC 780
GCGGCGATCG CCCCGCTCGC CGCGAACGCG CGCGCGCGAA TGGGGCGTAC TCGGCGAGCC 840
CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGCTAGC~AT CGTCTGGGCC GGCAGGCGCG 900
~0
TCACTCTGAG CACGCGCATG CCCCGCTGGG Ar.ACr-AArAr CTGrArCGGC GCTAGGACCA 960
CCGGGTCTGG GCCCGGGGGG GCGAGATCGC GrACAAC.CCG GGCCGAGTCG CGCAGCTGCC 1020
3~ GCAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGCGTGTT CAl~CG.~ r-AAAAArGGC 1080
ACGTCTTCAG CTCCACr~A~A Ar~ACAGACGG CCCGGGCGTG CC~lGC~CC GC~ACccGr~A 1140
GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC AGArArA-cGc 1200
CrArrArCTG CTTAAAAACC TCCGGGGCGC CAAACTTGCC C-AAAAGCTGG GC~AGGCGCG 1260
GGCGCAGCTT ~lGCGCGCCA Ar,CGCCGCGC GTGCGTCGCA Ar7CrAGCGCC TCGTAAAAGC 1320
45 GG~7~GGCA CCGGATCCCG GCGCGrAGGC GCGrACGTCG GTCGCGGTCG CGCGCCATGG 1380
CCGAGCCCGC GCGCG~C CGC6~C~lGC GTATCTACCT Gr-ArGGCGCG CACGGGCAGG 1440
rAAArArAAC AACGGGCCGC GCGC~CGCGG CCGCTTCCAC CGCTGGGGAG GGCGTGCTCT 1500
CCCGGA GCCGATGGCG TA~GGCGCA CGATGTTTGG TArGr-ACGCC TTAAGTGGGA 1560
~C~CGCGGC GTCTGCGCGA TGCGCCC7CAG C~CGCACGG r~Ar~cGrAcGc GCGCGGCGGG 1620
CCGGCGCACC GCGr-Ar~Ar-GC Gr-ArGCGGCG GGCCTGGTTG CGTACTArrA GGCCAGGTTC 1680
GCGGCCCCGT ACTTAATTTT GrArGCGC~ CCGCG~G CTGCGCCGCC ~GGGCCGGCG 1740
CCGGGCGGCG AGCTGGTGGA CC~C~ C~ArCGCrAC CCCG~GGCGC GC~lGCC~l 1800
GCTACCCCTT CGCCCGCTAr TGCCTCCGCG AGATCAACGC Gr.AAr.ATCCG AA~C~lCGA 1860
CCTGCAGTGA A'rAATAAAA'r ~ CCr~AAATP~CG C~ GAGAT ~ CCCG 1920
ACTAAATTCA ~CGCGC~A TA~GG~l TATCGCCrAT AGAGATGGCG ATATTGGAAA 1980
AATCGATATT Tr~AAAATA~G GCATATTGAA AATGTCGCCG ATGTGAGTTT ~GlAACT 2040
-22- ;
W O 95/12682 ~ 17 2 ~ ~ 5 PCTrUS94/12198
GATATCGCCA TTTTTCCAAA AGTTGATTTT TGGGCATACG CGATATCTGG CGATACGCTT 2100
ATAlCGl~lA CGGGGGATGG C~ATA~ACGC CTTTGGTGAC TTGGGCGATT CTGTGTGTCG 2160
6 CAAATATCGC AGTTTCGATA TAGGTGACAG A~-ATA~GAG GCTATATCGC C~A~A~-A~GC 2220
GACATCAAGC TGGCACATGG CCAATGCATA TCGATCTATA CATTGAATCA ATATTGGCCA 2280
TTAGCCATAT TATTCATTGG TTATATAGCA TAAATCAATA TTGGCTATTG GCCATTGCAT 2340
ACGTTGTATC CATATCATAA TATGTACATT TATATTGGCT CATGTCCAAC ATTACCGCCA 2400
TGTTGACATT GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT 2460
AGCCrA~A~A TGGAGTTCCG CGTTAr~TAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG 2520
CC~AAC~ACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCrA~AC.T AACGCCAATA 2580
GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA 2640
CATCAAGTGT ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC 2700
GCCTGGCATT ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC 2760
GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA 2820
TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCArCCrA TTGACGTCAA TGGGAGTTTG 2880
TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG 2940
CAAATGGGCG GTAGGCGTGT ACGGTGGGAG GTCTATA~AA GCAr.AGCTCG TTTAGTGAAC 3000
CGTCAGATCG CCTGGAGACG CCATCCACGC ~ GACC TCrATA~.AA~. ACACCGGGAC 3060
CGATCCAGCC TCCGCGGCAA GCTGATCCGT CAGGGGCCAG ATGGTACAGG GCATCCTATG 3120
GCTACTACTG ATAA~A~-GGG TACAAGGGGA CATTGACTGC AAACCTGAAC ACTrATACGC 3180
CATAGCCAGG AAT~-ATA~AA TTGGCCCATT AGGAGCTGAA GGCCTCACCA ~lG~llGGAA 3240
GGATTACTCA CATGAAATGA AGCTGGAAGA CACAATGGTC ATAGCTTGGT GrAAAGACGG 3300
TAAGTTTACA TACCTCTCAA GGTGrArAAG Ar.AAArTAr.A TATCTTGCAA TTCTGCATTC 3360
AA~.AGCCTTG rAr~A~cAGTG TGGTATTCAA AA~ACTTTTC ~AGGGGrAAA GG~AA~GGGA 3420
AACATTTGAA ATGGCTGACG ACTTTGAATT TGGACTCTGC CCATGCGATG CCAATCCCGT 3480
AG~AAr~AGGG AAGTTCAATA CAACACTGCT AAACG~ArCG GC~I~C~AGA TGGTATGCCC 3540
~ ATAG~.A~GG ArA~-~AArTG TGAGCTGTAT GT~A~CTAA~ A~G~-ACACCC TA~ArArA~c 3600
AGTAGTGCGT GTGTA~AAGA GGTCCAAACC A~CC~.lAT A~Ar.~AGGTT GTATCACCCA 3660
AA~AACTCTG GGGGAGGATC TcTA~AArTG TGATCTTGGA GGGAATTGGA ~l~lGlGAC 3720
TGGG~.ACrA~ CTArAA~ArA rAGGArGCCC TGTCGAATCT TGCAAGTGGT 6lG~l.ATAA 3780
ATTCCAAAAA AGTGAGGGGT TGCrArArTA CCCCATCGGC AAGTGTAGGT T~-AAr~AA~GA 3840
GACTGGCTAC AGATTTGTAG ArGGrAcrAr TTGCAACAr.A GAGG~ AG CCATAGTACC 3900
ArAAG~-A~TG GTAAAGTGTA A~ATAG~.Ar-A CACAATCGTA CAGGTCATAG CTCTTGACAC 3960
CAAACTTGGG CCTAlGC~ll GrAAGccATA TGAGATCATA CCAAGTGAGG GGc~lGlAGA 4020
. AAA~ArGGrA TGCACCTTCA AC~ArAC~AG GACATTA~AA AATAAA~ATT TT~-A~CCrAG 4080
-23-
WO 9S/12682 2 1 7 ~ ~ 1 5 PCTrUS94/12198
AGACAGTTAC TTCCAGCAAT ACATGCTA~A AGGAGATTAT CAATACTGGT TCIGACCTGGA 4140
GGTCACTGAC CATCATCGGG ATTACTTCGC CGAGTCCATA TTGGTGGTGG TGGTAGCTTT 4200
ACTGGGTGGA AGATACGTGC TCTGGTTACT GGTAACATAC ATGGTCCTAT ~A~.AA~AAAA 4260
GGCCTTGGGG ACCCAATATG GGGCAGGGGA AGTGGTGATG ATGGGTAACT TGCTAA~A~A 4320
TGACAGTATT GAAGTGGTGA CATATTTCTT GTTGTTATAC CTACTGCTAA GAGAGGAGGC 4380
TGTAAAr.AAC. TGGGTCTTAC TCTTATACCA CCTTGATTGA TTGAGGATCA GCTTATCCAG 4440
GGTCGACCTC AGGCATGCAA GCTCAGATCC GCTGTGCCTT CTAGTTGCCA GCCATCTGTT 4500
15 GTTTGCCCCT CCCCCGTGCC L1C~11~GACC CTGGAAGGTG CCACTCCCAC TGTCCTTTCC 4560
~AATAAAATG A~AAATTGC ATCGCATTGT CTGAGTAGGT GTCATTCTAT TCTGGGGGGT 4620
GGGGTGGGGC AGGACAGCAA GGGGGAGGAT TGG~AA~ACA ATAGCAGGCA TGCTGGGGAT 4680
GCGGTGGGCT CTATGGGTAC CCAGGTGCTG AAGAATTGAC CCG~11C~1C CTGGGCCAGA 4740
AAr.AAG~Ar.G CACATCCCCT ~ 1G1GAC ACACCCTGTC ~A~GCCCCTG G~ AGTT 4800
C~A~.CCC~AC TCATAGGACA CTCATAGCTC AGGAGGGCTC CGCTTCAATC CCACCCGCTA 4860
AAGTACTTGG AGCGGTCTCT CC~CC1CA TrA~CCrPCC AA~ACCAAACC TAGCCTCCAA 4920
GAGTGGGAAG AAATTAAAGC AA-r~ATAGGCT ATTAAGTGCA ~AGG~.A~.A~A AAATGCCTCC 4980
AACATGTGAG GAAGTAATGA TA~.AAATCAT AGAATTGAGA TCTCGAGGTG TTCGTGCTGG 5040
ACGTGTCCGC GGCGC~Ar.AC GCGTGCGCGG CCGCCGTACT GGACATGCGG CCCGC~ATGC 5100
AGGCCGCTTG CGCGGACGGG GCGGCGGGCG CGACGCTGGC GACCCTGGCG CGTCAGTTCG 5160
CGCTA~A~.AT GGCGGGGGAG GCCACGGCGG GCCCTAGGGG ACTATAAAGC T¢CCC~1GCG 5220
CTCGCTCGCT CGCTGCATTT GCGCCCC~.AT CGCCTTACGG GGACTCGGCG CTCGGCGGAT 5280
CCCCTCCCGG CCCCGCCGCG AAGCAGGCCG CCA~ArAAAA AAATGCGGCG CCCGCTCTGC 5340
GCGGCGCTAT TGGCAGCGGC 1~ C~CGCG CTCGCCGCGG GCGCCCCCGC CGCCGCCCGC 5400
GGCGGGGGCG CCqAAGC~AG GGCAGCACAG A~ACGCCC~A TAC~AAATCG A~GAGTGGGA 5460
AATGGTGGTC G~-Ar-CCGGGC CGGCC~GCA CACGTTCACC ATCCGCTGCC ~CGGGCCGCG 5520
GGGCATTGAG CGCGTGGCCC ACATTGCAAA CCTCA~CCGG CTGCTGGACG GbTA~ATAGc 5580
GGTCCACGTT GAC611GCGC GCAC~GG C~GCGG~.AC GCCATGTTTT ~C~GCCGCG 5640
CGCGGCCGTC GACTCTAGAG GATCCCCGGG TA~C~AGCTC GAATTCACTG GCCG1C~11 5700
TA~AA~GTCG TGACTGGGAA AACCCTGGCG TTACCr~A~T TAATCGCCTT G~A~ArATC 5760
CCCCTTTCGC CAGCTGGCGT ~AATA~.CGAAG AGGCCCGCAC CGATCGCCCT TCC~AA~A~,T 5820
TGCGCAGCCT GAATGGCGAA TGGCGCCTGA TGCGGTATTT ~lC~ ~ ~ACG CATCTGTGCG 5880
GTATTTCACA CCG~ATATGG TGCACTCTCA GTACAATCTG CTCTGATGCC GCATAGTTAA 5940
GCCAGCCCCG A~ArCCGC~A A~A~CCG~G ACGCGCC~1G ACGGGC11G1 ~G~,CCCGG 6000
65 CATCCGCTTA rA~A~AAGCT GTGACCGTCT CCGG~-AGCTG CA1~1G1~AG AGG1~ llCAC 6060
CGTCATCACC ~AAACGCGCG ~A~ACGAAAGG GC~1CG1~AT ACGCCTATTT TTATAGGTTA 6120
-24-
W 095/12682 ~ 1 7 ~ 8 1 ~ PCT~US94tl2198
ATGTCATGAT AATAATGGTT TCTTAGACGT CAGGTGGCAC TTTTCGGGGA AATGTGCGCG 6180
GAACCCCTAT .. ~7111ATTT TTCTAAATAC ATTCAAATAT GTATCCGCTC AT~A~ACAAT 6240
5 AACCCTGATA AATGCTTCAA TAATATTGAA AAAGGAAGAG TATGAGTATT CAACATTTCC 6300
GTGTCGCCCT TATTCCCTTT TTTGCGGCAT TTTGCCTTCC ~ GCT rACcrA~.AAA 6360
CGCTGGTGAA AGTAAAA~AT GCTGAAGATC AGTTGGGTGC ACGAGTGGGT TACATCGAAC 6420
- TGGATCTCAA CAGCGGTAAG ATCCTTGAGA GTTTTCGCCC crAA~iAAcGT TTTCCAATGA 6480
TGAGCACTTT TAAAGTTCTG CTATGTGGCG CGGTATTATC CCGTATTGAC GCCGGGCAAG 6540
AGrA~CTCGG TCGCCGCATA CACTATTCTC AGAATGACTT GGTTGAGTAC TCACCAGTCA 6600
rArAAAAGcA TcTTAcGr~AT GGCATGACAG TApr~ArAATT ATGCAGTGCT GcrATAAcr.A 6660
TGAGTGATAA CACTGCGGCC AACTTACTTC Tr~ArAArrAT CGGAGGACCG AAGGAGCTAA 6720
CCG~ l, GrArAAcA-TG GGGGATCATG TAACTCGCCT TGATCGTTGG GAACCGGAGC 6780
TGAATGAAGC r.ATAcrAAAr GACGAGCGTG ACACrACrAT GCCTGTAGCA ATGGrAA-rAA 6840
CGTTGCGCAA ACTATTAACT GGCGAACTAC TTACTCTAGC TTCCCGGCAA CAATTAATAG 6900
ACTGGATGGA GGCG~ATAAA GTTGCAGGAC CA~.~C~GCG CTCGGCCCTT CCGGCTGGCT 6960
GGTTTATTGC TrAT-AA-ATcT GGAGCCGGTG AGCGTGGGTC TCGCGGTATC ATTGCAGCAC 7020
TGGGGCCAGA TGGTAAGCCC lCCCGlATCG TAGTTATCTA rAcr~7ArGGGG AGTrA-~GrAA 7080
CTATGGATGA AC~.AAATAGA CAGATCGCTG AGATArGGTGc CTCACTGATT AAGCATTGGT 7140
AACTGTCAGA CCAAGTTTAC TCATATATAC TTTAGATTGA TTTAAAACTT CATTTTTAAT 7200
TTAA~AGGAT CTAGGTGAAG AlC~ G ATAATCTCAT r~ACr-AAAATC CCTTAACGTG 7260
A~ CGL. CCACTGAGCG TrAr-A-ccccG TA~i~AAArAT cAAAGr~ATcT TCTTGAGATC 7320
C~ l GCGCGTAATC TGCTGCTTGC AAArAAAAAA AC~-ACCGCTA CCAGCGGTGG 7380
~ 7l~LGCC GGATCAAGAG c~AcrAAcTc ~ .CCGAA GGTAACTGGC TTrAr7rArAG 7440
CG~ArATACC AAATArTGTc CTTCTAGTGT AGCCGTAGTT AGGCCACr-Ar TTrAArGAAcT 7500
cTG~Ar~rA~r-c GCCTACA~AC ~CG~I lGC TAAiCC~7~ ACCAGTGGCT GCTGCCAGTG 7560
GCr~ATAAGTC ~7~ ACC GGGTTGGACT rAAr~AcrA~A GTTAccGrA~ AAGGcGrAr~7c 7620
G~CGGGCTG AACGGGGGGT TCGTGCACAC Ar~7CCCAGCTT GrArcrAArG Acc~Ar~rcG 7680
AAcT~iAr~A~A CC~ArA,r7CGT GAGCTATGAG AAAGcGcrAr GCllCCC~iAA GGrAr~AAAr~7G 7740
55 CGGArAGGTA ~CCGGlAAGC GGCAGGGTCG r.AArAr,7r.AriA GCGCArr~Ar.7G GAGCTTCCAG 7800
GGGr~7AAA-cGc CTGGTATCTT TATAGTCCTG TCGG~llCG CCAC~ GA CTTGAGCGTC 7860
GA~ G A~GClCGlCA GGGGGGCGGA GCCTATGGAA AAACGCrAr,7C AACGCGGCCT 7920
TTTTACGGTT CCTGGCCTTT TGCTGGCCTT l~GC.~ACAT ~7~L~lC~ GCGTTATCCC 7980
CTGA~ 7l GrA~AArCGT ATTACCGCCT TTGAGTGAGC TGATAr,CGCT CGCCGrAGCC 8040
65 rAACGArCr-A GCGCAGCGAG TCAGTGAGCG AGr~AArGcGr~A AGA 8083
-25-
W O95/12682 21 7 2 81 ~ P- TrUS94/12198
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8149 base pairs
(~) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECU~E TYPE: DNA (genomic)
(iii) ~YPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISN: Bovine viral diarrhea virus
(B) STRAIN: 2724
(C) INDIVIDUAL ISOLATE: pBHVtkex-1::gBGH/p53
(xi) S~:Q~N~ DESCRIPTION: SEQ ID NO:2:
GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60
CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120
CACTCATTAG GrAcccrAGG CTTTACACTT TATGCTTCCG G~lCGlATGT TGTGTGGAAT 180
TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG CCAAGCTAGC 240
TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGllGlCC 300
GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360
86 GTCCGCGTCC CCAACTCCGC Gcr-AA~ArGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420
GCGTATAA~A TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480
GCCrAGGrAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC CAr.AAA~AGC 540
Gccr~AAATTT CGCCrAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600
Cl~llGCCCG CGTGCCTGCT GGCCGCC~r CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660
CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCrArAGC CGCC~llllG CCCGTAGCCA 720
GGGGAAGCGG C.~GCCC~lC ~C'CGCCGCG GCCGCG~-l~G ~CGG~ll~G CGTTTGCCCC 780
GcGqcrATcG CCCCG~lCGC CGCrAA~GCG CGCGCGCr~AA~ lGGGGCGlAC TCGGCrAGCC 840
CGGCTATTAT AGCCTCAAGG CGCGCCGC'GT TGC~AGCr-A~ CGTCTGGGCC GGCAGGCGCG 900
TCACTCTGAG rAr,GCGCATG CCCCG~lGGG Ar.Arr.AA~A~ CTG~A~CGGC GCTAGGACCA 960
55 CCGGGTCTGG GCCCGGGGGG GC~ArATCGC Gr-ArAAGCCG GGCCGAGTCG CGCAGCTGCC 1020
GrAGcccccc r~ArGcGcTGG TCCATCTTGC TGGGC~ l CATGTTCGTT rAAAAACGGC 1080
AcG~ AG cTcrAcrATA Ar~cArA~cGG CCCGGGCGTG CCCTGCCTCC GcrA-cccG~A 1140
GTAGGcArAc GCAATCGGGC CGCCGGClll GCAGGTTTAC CTrAAArCTC A~ArArArGC 1200
CrACrArCTG CTTAAAAACC TCCGGGGCGC CAAACTTGCC CAAAAGCTGG GCr~Ar,GCGCG 1260
GGCG~-AGCTT CTGCGCGCCA ACCGCCGCGC ~lGC~lCG~A AGCrAr-CGCC TCGTAAAAGC 1320
GGCTGTGGCA CCGGATCCCG GCGCG~AGGC GCGrACGTCG GTCGCGGTCG CGCGCCATGG 1380
-26-
W O 95/12682 ~ 17 2 ~15 PCT~US94/12198
CCGAGCCCGC GCGCGCTCTC CGCGTCGTGC GTATCTACCT GGACGGCGCG CACGGGCAGG 1440
GA~ArArAAC AACGGGCCGC GCGCTCGCGG CCGCTTCCAC CGCTGGGGAG GGCGTGCTCT 1500
TTTTCCCGGA GCCGATGGCG TACTGGCGCA CGATGTTTGG TACGGACGCC TTAAGTGGGA 1560
TCCTCGCGGC GTCTGCGCGA TGCGCCGCAG CCTCGCACGG GAGCGCACGC GCGCGGCGGG 1620
CCGGCGCACC GCGrAGACGC GGACGCGGCG GGCCTGGTTG CGTACTACCA GGCCAGGTTC 1680
GCGGCCCCGT ACTTAATTTT GCACGCGCGT GTCCGCGCTG CTGCGCCGCC TGGGCCGGCG 1740
CCGGGCGGCG AGCTGGTGGA CCCTCGTGTT CGACCGCCAC CCCGTGGCGC GCGTGCCTCT 1800
16 GCTACCCCTT CGCCCGCTAC TGCCTCCGCG AGATCAACGC GGAAGATCTC AATTCTATGA 1860
TTTCTATCAT TACTTCCTCA CATGTTGGAG GCAlll-~LC TCC~l~.G~A CTTAATAGCC 1920
TATCTTGCTT TAA111~1~C CCACTCTTGG AGGCTAGGTT 1G~111GGTG GGCTGATGAG 1980
GGAGGGAGAG ACCGCTCCAA GTACTTTAGC GGGTGGGATT GAAGCGGAGC CCTCCTGAGC 2040
TATGAGTGTC CTATGAGTGG GGCTGGAACT AAr~AA~Ar~G GGCGTGGACA GGG1~.G1CA 2100
25CAGAGAAGGG GATGTGCCTG ~C111C1G GCC~AGrAGG AACCGGGTCA A11~1~CAGC 2160
ACCTGGGTAC CrATAr~AGCC CACCGCATCC CCAGCATGCC TGCTATTGTC TTCCCAATCC 2220
TCCCCCTTGC 1~C~1GCCC rArCCCACCC CCCArAATAG AATGACACCT AcTrAr~Ar-AA 2280
TGCGATGCAA TTTCCTCATT TTATTAGGAA AG~ArAGTGG GAGTGGCACC TTCCAGGGTC 2340
AAGGAAGGCA CGGGGGAGGG GrAAA~AArA GATGGCTGGC AACTArAAGG CACAGCGGAT 2400
36 CTGAGCTTGC ATGCCTGAGG TCGACCCTGG ATAAGCTGAT CCTCAATCAA TCAAGGTGGT 2460
ATAArAGTAA rACCrACTTC TTTACAr~CCT C~ AG CAGTAGGTAT AArAACAAr~A 2520
AATATGTCAC CACTTCAATA CTGTCATGTG T~Ar-rAAGTT ACCCATCATC ACCACTTCCC 2580
CTGCCCCATA TTGGGTCCCC AAGGC~-1.. GTTCTGATAG GACCATGTAT GTTACCAGTA 2640
ACCAr~AGrAr GTA1~1~C~A CCCAGTAAAG CTArrArrAC rACrAATATG GACTCGGCGA 2700
AGTAATCCCG ATGATGGTCA GTGACCTCCA GGTCGAACCA GTATTGATAA 'l~-C~....A 2760
GCATGTATTG CTGr-AAGTAA ~.~ ~I~.GG GCTrAAAATA TTTATTTTTT AATGTCCTCG 2820
TGTAGTTGAA GGTGCATGCC ~ A ~ArGCCC~C ACTTGGTATG ATCTCATATG 2880
GCTTGCAAGG rATAGGCCrA AG1~GG~. rAAr-AGCTAT GACCTGTACG A~ lC 2940
CTATCTTACA CTTTAC~AAT C~ G~1A CTATGGCTAC ACC~1~.~lG TTGCAAGTGG 3000
56 TGCCGTCTAC AAATCTGTAG CCAGTCTCAT TCTTCAACCT ACACTTGCCG ATGGGGTAGT 3060
GTGGrAACCC CTCACTTTTT TGGAATTTAT AACrArACrA CTTGCAArAT TCGACAGGGC 3120
.C~.~.G.A TTGTAGCTGG TCCCCAGTCA CACAAGTCCA A~CC~C~A AGATCACAGT 3180
TATArA~ATC C1CCCCIA~A G1L~11~GGG T~ATACA~CC '11~ATAA GGGAATGGTT 3240
TGGACCTCTT ATArArArGC ACTACTGCTG TGTCTAGGGT ~CC~lATTA GC~AArATAr 3300
65AGCTCACAGT 1C~ AT CCTATACGGC ATArrA~CTG rAAr~GCCGGT CCG.~AGCA 3360
ATT GAA~.-CC~- CTTACTACGG GATTGGCATC GCATGGGCAG AGTCCA-AATT 3420
27-
W O 9S/12682 ~ 2 a 1 ~ PCTrUS94112198
'. .. ~ I .
CAAAGTCGTC AGCCATTTCA AATGTTTCCC CTTGCCTTTG CCCCTCGAAA AGllllllGA 3480
A~ArrArACT GGTCTGCAAG GCTCTTGAAT GCAGAATTGC AAGATATCTA ~ LLG 3540
TGCACCTTGA GAGGTATGTA AACTTACCGT CTTTGCACCA AGCTATGACC ATTGTGTCTT 3600
CCAGCTTCAT TTCATGTGAG TAATCCTTCC AAACAGTGGT GAGGCCTTCA GCTCCTAATG 3660
GGCCAATTCT ATCATTCCTG GCTATGGCGT ATGAGTGTTC AGGTTTGCAG TCAATGTCCC 3720
CTTGTACCCC TGTTATCAGT AGTAGCCATA GGATCCCTGG GAAGGCGCCC ACCACCTGAG 3780
TCCAGGGCAG GrA~AGrAC'G GC~'A~AGCCA GGAGCAGGGA GGTCCGGGGG CCTGCAGCCA 3840
TCATGTCGAA GCTTGCCGCG GAGGCTGGAT CGGTCCCGGT ~ ~LATG GAGGTCAAAA 3900
CAGCGTGGAT GGC~l~lCCA GGCGATCTGA CGGTTCACTA AACGAGCTCT GCTTA~ATAC. 3960
ACCTCCCACC GTArA-rGccT ACCGCCCATT TGCGTCAATG GGGCGGAGTT GT~AcrArA~ 4020
TTTGGAAAGT CCCGTTGATT TTGGTGCCAA AACAAArTCC CATTGACGTC AATGGGGTGG 4080
AGACTTGGAA ATCCCCGTGA GTCAAACCGC TATCCACGCC CATTGATGTA CTGCrAAAAC 4140
CGCATCACCA TGGTAATAGC GATGACTAAT ACGTAGATGT ACTGCCAAGT AGGAAAGTCC 4200
CATAAGGTCA TGTACTGGGC ATAATGCCAG GCGGGCCATT TACCGTCATT GACGTCAATA 4260
GGGGGCGTAC TTGGCATATG ATACACTTGA TGTACTGCCA AGTGGGCAGT TTACCGTAAA 4320
TACTCCACCC ATTGACGTCA ATGGAAAGTC CCTATTGGCG TTACTATGGG AArATACGTC 4380
ATTATTGACG TCAATGGGCG GGGGTCGTTG GGCGGTCAGC CAGGCGGGCC ATTTACCGTA 4440
AGTTATGTAA CGCG~AACTC rATA~A~GGG CTATGAACTA ATr-ArCCCGT AATTGATTAC 4500
TATTAATAAC TAGTCAATAA TCAATGTCAA CATGGCGGTA ATGTTGGACA TGAGCCAATA 4560
TAAATGTACA TATTATGATA TGGA~ArAAc GTATGCAATG GCrAATAGCC AATATTGATT 4620
TATGCTATAT AACrAATGAA TAATATGGCT AATGGcrAAT ATTGATTCAA TGTATA~ATC 4680
GATATGCATT GGCCATGTGC CAGCTTGATG TCGCC~AT cGGcrATA~A- GCCTCATATC 4740
45 G1C~ ACC TATATCGAAA CTGC~A~ATT TGcr~AcArAc AGAATCGCCC AAGTCACCAA 4800
AGGCGTCTAT CGCCATCCCC CGTAAA~AT ATAAGCGTAT CGC~A~ATA~ CGCGTATGCC 4860
r.AAAAA~CAA CTTTTGGAAA AATGGcr~A~A TCAGTTACAC AGAAACTCAC ATcGGc~AcA~ 4920
TTTTCAATAT GCrATATTTT CA~AA~A~CGA Lll~lCCAAT ATCGCCATCT CTATCGGCGA 4980
~AAACAcrAr TATCGCGCGA CATGAATTTA GTCGGGACAG AAATCTCAAA CGCGTATTTC 5040
Gr.ArAAAr.Ar ACATTTTATT ATTCACTGCA GGTCr~Ar,r~AA TTCGGATCTC GAG~L~l~CG 5100
TGCTGGACGT GTCCGCGGCG cr-Ar~ArGcGT GCGCGGCCGC CGTACTGGAC ATGCGGCCCG 5160
CCATGCAGGC CGCTTGCGCG GACGGGGCGG CGGGCGCGAC GCTGGCGACC CTGGCGCGTC 5220
A~llCGCGCT AGAGATGGCG GGGGAGGCCA CGGCGGGCCC TAGGGGACTA TAAAGCTGCC 5280
CCTGCGCTCG ClCG~lCGCT GCATTTGCGC CCCGATCGCC TTACGGGGAC TCGGCGCTCG 5340
GCGGATCCCC TCCCGGCCCC GCCGC~AAGC AGGCCGCrA~ A~AAAAAAAT GCGGCGCCCG 5400
CTCTGCGCGG CGCTATTGGC Ar~cGGcTGTc CTCGCGCTCG CCGCGGGCGC CCCCGCCGCC 5460
-28-
W O95/12682 2 1 7 ~ 8 1 ~ PCTrUS94/12198
GCCCGCGGCG GGGGCGCCGA AGCCAGGGCA GCACAGAGAC GCCCGATACG AAATCGAAGA 5520
GTGGGAAATG GTGGTCGGAG CCGGGCCGGC CGTGCACACG TTCACCATCC GCTGCCTCGG 5580
GCCGCGGGGC ATTGAGCGCG TGGCCCACAT TGCAAACCTC AGCCGGCTGC TGGACGGGTA 5640
CATAGCGGTC CACGTTGACG TTGCGCGCAC CTCTGGCCTG CGGGACGCCA T~lllllCCT 5700
GCCGCGCGCG GCCGTCGACT CTAGAGGATC CCCGGGTACC GAGCTCGAAT TCACTGGCCG 5760
'lc~lLllAcA ACGTCGTGAC TGGGAAAACC CTGGCGTTAC CCAACTTAAT CGCCTTGCAG 5820
CACATCCCCC TTTCGCCAGC TGGCGTAATA GCGAAGAGGC CCGCACCGAT CGCCCTTCCC 5880
15 AACAGTTGCG CAGCCTGAAT GGCGAATGGC GCCTGATGCG GTAllLl~lC CTTACGCATC 5940
TGTGCGGTAT TTCACACCGC ATATGGTGCA CTCTCAGTAC AATCTGCTCT GATGCCGCAT 6000
AGTTAAGCCA Gccccr~AcAr CCGCCAACAr CCGCTGACGC GCCCTGACGG G~ lGC 6060
TCCCGGCATC CGCTTACAGA CAAGCTGTGA CCGTCTCCGG GAGCTGCATG TGTCAGAGGT 6120
TTTCACCGTC ATrArcr-AAA CGCGCr~Ar~AC GAAAGGGCCT CGTGATACGC CTATTTTTAT 6180
AGGTTAATGT CATr~ATAA~A ATG~ lC~ L AGACGTCAGG TGGCACTTTT CGGGGA~ATG 6240
TGCGCGGAAC CCCTATTTGT TTA~lLl~ AAATArATTC AAATATGTAT CCGCTCATGA 6300
rArAATAACC cTrATAAA~G CTTCAATAAT ATTGAAAAAG GAAGAGTATG AGTATTCAAC 6360
ATTTCCGTGT CGCCCTTATT CC~llllG CGGCATTTTG CCTTCCTGTT TTTGCTCACC 6420
rAr~AAAcGcT GGTGAA~AGTA AAAGATGCTG AAGATCAGTT GGGTGCACGA GTGGGTTACA 6480
36 TCGAACTGGA TCTCAACAGC GGTAAGATCC TTGAGAGTTT TCGCCCCrAA GAACGTTTTC 6540
CAATGATGAG CACTTTTAAA GTTCTGCTAT GTGGCGCGGT ATTATCCCGT ATTGACGCCG 6600
GGrAArAGrA ACTCGGTCGC CGrA~ArArT ATTCTCAGAA TGACTTGGTT GAGTACTCAC 6660
CAGTCACAGA AAAr7rATcTT ACGGATGGCA TGACAGTAAG AGAATTATGC AGlG~GCCA 6720
~AArrA~GAG TrA~AArArT GCGGCCAACT TACTTCTGAC AACGATCGGA GrArCr.AAGG 6780
45 AGCTAACCGC ~ G~AC AACATGGGGG ATCATGTAAC lCGC~llGAT CGTTGGGAAC 6840
CGGAGCTGAA Tr.AArCrA~A CrAAArr-ArG AGCGTGACAC CACGATGCCT G~AGrAA~GG 6900
rAArAA~GTT GCGrAAACTA TTAACTGGCG AACTACTTAC TCTAGCTTCC cGGrAAcAAT 6960
~0
~ AA~Ar.ArTG GATGrAGGcG rA~AAAGTTG rAGrArrArT TCTGCGCTCG GCC~llCCGG 7020
CTGGCTGGTT TAllG~lGAT AAATCTGGAG CCGGTGAGCG TGGGTCTCGC GGTATCATTG 7080
55 CAGCACTGGG GCrAr~ATGGT AArCC~lCCC GTATCGTAGT TATCTACACG ACGGGGAGTC 7140
AGGCAACTAT GGATGAACGA AA~Ar~ArArA TCGCTGAGAT AGGTGCCTCA CTGATTAAGC 7200
ATTGGTAACT GTrAGACr,AA GTTTACTCAT ATA~ArTTTA GATTGATTTA AAACTTCATT 7260
TTTAATTTAA AAGGATCTAG GTGAAGATCC TTTTTGATAA TCTCATGACC AA~ATCCCTT 7320
AACGTGAGTT ll`'~llCCAC TGAGCGTCAG ACCCC~lAGA AAAGATCAAA GGAl~.l`ll 7380
65 GAGAlC~lll l.l~lGCGC GTAATCTGCT GCTTGCAAAC AAAAAAArrA CCGCTACCAG 7440
' CGGTGGTTTG ll~GCCGr-A~ rAAr.Ar-CTAC CAA~ TCCr-AAGGTA ACTGGCTTCA 7500
-29-
W O95/12682 2 i 7 2 ~ 1 S ` PC IrUS94112198
GCAGAGCGCA GATACCAAAT ACTGTCCTTC TAGTGTAGCC GTAGTTAGGC CACCACTTCA 7560
AGAACTCTGT AGCACCGCCT ACATACCTCG CTCTGCTAAT CCTGTTACCA GTGGCTGCTG 7620
CCAGTGGCGA TAAGTCGTGT CTTACCGGGT TGGACTCAAG ACGATAGTTA CCGGATAAGG 7680
CGCAGCGGTC GGGCTGAACG GGGGGTTCGT GCACACAGCC CAGCTTGGAG CGAACGACCT 7740
ACACCGAACT ~A~AT~CCTA CAGCGTGAGC TATGAGAAAG CGCCACGCTT CCCGAAGGGA 7800
GAAAGGCGGA CAGGTATCCG GTAAGCGGCA GGGTCGGAAC AGGAGAGCGC ACGAGGGAGC 7860
TTCCAGGGGG AAACGCCTGG TATCTTTATA GTCCTGTCGG GTTTCGCCAC CTCTGACTTG 7920
15 AGCGTCGATT TTTGTGATGC TCGTCAGGGG GGCGGAGCCT ATGr~AA.~AAr GCCAGCAACG 7980
CGGCCTTTTT ACG~lC~lG GCCTTTTGCT GGCCTTTTGC TCACATGTTC TTTCCTGCGT 8040
TATCCCCTGA ~ GGAT AACCGTATTA CCGCCTTTGA GTGAGCTGAT ACCGCTCGCC 8100
GCAGCCGAAC GACCGAGCGC AGCGAGTCAG TGAGCGAGGA AGCGGAAGA I 8149
(2) INFORMATION FOR SEQ ID NO:3:
26 (i) ~Q~r:NC~ CHARACTERISTICS:
(A) LENGTH: 8135 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA ( genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine viral diarrhea virus
(B) STRAIN: 2724
(C) INDIVIDUAL ISOLATE: pBHVtkex-l: :gIII/p53
(xi) Y7~Qu~w~ DESCRIPTION: SEQ ID NO:3:
45 GCGCCr~A~A CG~AAAccGc ~ CCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60
Cr~Ar~GGTTT CCCGACTGGA AAr-CGGGCAG T~-~GCGCAAr GCAATTAATG TGAGTTAGCT 120
CACTCATTAG GCAr,CCrAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180
TGTGAGCGGA TAArAATTTC A~r~Gr~AAA rAr,r~ATGAC CATGATTACG CCAAGCTAGC 240
TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAG~ CC 300
55 GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGC~CGCG GCr~GCTCCC GCGCGGGCGC 360
GTCCGCGTCC CCAACTCCGC GCGAAr~A~GG GC~CGlCCCA GAAGCGr-AGC GGAAAGGCCG 420
GCGTA~AAA~ l~lCG~lC~ CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480
GCCrAC-C,r-AA GCAAACTCTA AArGCCCrAG CGCCATGGCC CCGATGCCGC rACAAAr~Ar7C S40
GccG-AAATTT CGCCCAGGCA CGCCGCGCCG CCC~ACGCGT CTTTAGCG~A CCCGCCGGCG 600
~~ GCCCG CGTGCCTGCT GGCCGCCCAC CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660
. CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCr~CAGC CGCC~ G CCCGTAGCCA 720
~30~
~ W 095112682 2172$1~ PCTrUS94/12198
GGGGAAGCGG CTGCCCCTTC TGCCGCCGCG GCCGCGGTTG CTCGGCTTTG CGTTTGCCCC 780
GCGGCGATCG CCCCGCTCGC CGCGAACGCG CGCGCGCGAA TGGGGCGTAC TCGGCGAGCC 840
CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGCTAGCGAT CGTCTGGGCC GGCAGGCGCG 900
TCACTCTGAG CACGCGCATG CCCCGCTGGG AGACGAACAC CTGCACCGGC GCTAGGACCA 960
CCGGGTCTGG GCCCGGGGGG GCGAGATCGC GCACAAGCCG GGCCGAGTCG CGCAGCTGCC 1020
GCAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGCGTGTT CATGTTCGTT rAAAAACGGC 1080
ACGTCTTCAG CTCCACGATA AGACAr~ACGG CCCGGGCGTG CCCTGCCTCC GCGACCCGGA 1140
GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC ~r~A~ArAcGc 1200
CCACGACCTG CTTAAA~ACC TCCGGGGCGC CAAACTTGCC CAAAAGCTGG GCGAGGCGCG 1260
GGCGCAGCTT CTGCGCGCCA ACCGCCGCGC GTGCGTCGCA AGCCAGCGCC TCGTAAAAGC 1320
GGCTGTGGCA CCGGATCCCG GCGCGCAGGC GCGCACGTCG GTCGCGGTCG CGCGCCATGG 1380
CCGAGCCCGC GCGCGCTCTC CGCGTCGTGC GTATCTACCT GGACGGCGCG CACGGGCAGG 1440
rAAA-rArA~Ac AACGGGCCGC GCGCTCGCGG CCG~llCCAC CGCTGGGGAG GGCGTGCTCT 1500
TTTTCCCGGA GCCGATGGCG TACTGGCGCA CGATGTTTGG TACGGACGCC TTAAGTGGGA 1560
TCCTCGCGGC GTCTGCGCGA TGCGCCGCAG CCTCGCACGG GAGCGCACGC GCGCGGCGGG 1620
CCGGCGCACC GcGrArArGc GGACGCGGCG GGCCTGGTTG CGTACTACCA GGCCAGGTTC 1680
GCGGCCCCGT ACTTAATTTT GrAcGcGcGT GTCCGCGCTG CTGCGCCGCC TGGGCCGGCG 1740
CCGGGCGGCG AGCTGGTGGA CC~CG~ll c~-A-rcGcr-Ac CCCGTGGCGC GCGTGCCTCT 1800
GCTACCCCTT CGCCCGCTAC lGCClCCGCG AGATCAACGC GGAAGATCTC AATTCTATGA 1860
TTTCTATCAT TACTTCCTCA CATGTTGGAG GCAl..~lC TCCCTCTGCA CTTAATAGCC 1920
TA~ G~ll TAAll~ C CCACTCTTGG AGGCTAGGTT TGGTTTGGTG GGCTGATGAG 1980
Gr~AGGrArAr ACCGCTCCAA GTACTTTAGC GGGTGGGATT r.AAGCG~.AGC C~CC~GAGC 2040
TATGAGTGTC CTATGAGTGG GGCTGGAACT AA-r~AAcrAGG GGC~lGGACA GG~ A 2100
rArArAAGGG GA~lGC~G ~ .l`lG GccrArr~AGG ~Accr~GGTcA A~.ClLCAGC 2160
ACCTGGGTAC Cr-ATAr~A5cc rACCGrATCC CcAGrA~Gcc TGCTATTGTC TTCCCAATCC 2220
TCCCCCTTGC TGTCCTGCCC rACccrAr-cc ccr,A~AATA5 AATGACACCT ACTrA~.ArAA 2280
TGCGATGCAA .~ C~1~ATT TTATTAGGAA AG~ArA5TGG GAGTGGCACC TTC~Ar~GGTC 2340
55 AAGGA~Ar~GrA CGGGGGAGGG GrAAAcAArA GATGGCTGGC AAc~Ar~AAc~G rAr~GCGr7A~ 2400
CTGAGCTTGC ATGCCTGAGG TCGACCCTGG ATAAr.CTGAT CCTCAATCAA TCAAGGTGGT 2460
ATAA~'A~TAA ~A.~Cr-AcTTc TT~ArAr~CCT C~L ~AG CAGTAGGTAT AArAArAAr.A 2520
AATATGTCAC CACTTCAATA CTGTCATGTG TTA5rAAGTT ACCCATCATC ACCACTTCCC 2580
CTGCCCCATA TTGGGTCCCC AA~GC~ GTTCTGATAG GACCATGTAT GTTArrAc~TA 2640
ArrAC.AGrAr GTAl~l~C~A CCCAGTAAAG CTArrArrAr rACrAATATG GACTCGGCGA 2700
AGTAATCCCG ATGATGGTCA GTGACCTCCA GGTCr~AACrA GTATTGATAA l~'C~ A 2760
W O 95/12682 PC~rUS94/12198
2172~5
GCATGTATTG CTGGAAGTAA Cl~l~lClGG GCTCAAAATA TTTATTTTTT AATGTCCTCG 2820
TGTAGTTGAA GGTGCATGCC Gl~llll~iA CAGGCCCCTC ACTTGGTATG ATCTCATATG 2880
5 GCTTGCAAGG CATAGGCCCA AGTTTGGTGT CAAGAGCTAT GACCTGTACG ATTGTGTCTC 2940
CTATCTTACA CTTTAC~AAT CCTTGTGGTA CTATGGCTAC ACCCTCTCTG TTGCAAGTGG 3000
TGCCGTCTAC AAATCTGTAG CCAGTCTCAT TCTTCAACCT ACACTTGCCG ATGGGGTAGT 3060
GTGGCAACCC CTCACTTTTT TGGAATTTAT AACCACACCA CTTGCAAGAT TCGACAGGGC 3120
CTCCTGTGTA TTGTAGCTGG TCCCCAGTCA CACAAGTCCA ATTCCCTCCA AGATCACAGT 3180
15 TA~ArArATC CTCCCCCAGA ~ GGG TGA~ACAArC ll~ ATAA GGGAATGGTT 3240
TGGACCTCTT ATArACAC.GC ACTACTGCTG TGTCTAGGGT GTCCCTATTA GCTAACATAC 3300
AGCTCACAGT 'lCC~lCCAT CCTATAGGGC ATArrA~CTG GAAGGCCGGT CCGlllAGCA 3360
~ l'~l"l'~l'ATT GAACTTCCCT CTTACTACGG GATTGGCATC GCATGGGCAG AGTCCAAATT 3420
CAAAGTCGTC AGCCATTTCA AAlGlL~CCC CTTGCCTTTG CCCCTCGAAA A~l~ lGA 3480
25 ATACCACACT GGTCTGCAAG GCTCTTGAAT GCAGAATTGC AAr.ATA~CTA ~lllC~ G 3540
TGCACCTTGA GAGGTATGTA AACTTACCGT CTTTGCACCA AGCTATGACC A~ll~l~l 3600
CCAGCTTCAT TTCATGTGAG TAAlC~l~CC AAACAGTGGT GAGGCCTTCA GCTCCTAATG 3660
GGCCAATTCT ATCATTCCTG GCTATGGCGT ATGAGTGTTC AGGTTTGCAG TCAATGTCCC 3720
CTTGTACCCC TGTTATCAGT AGTAGCCATA GGATCCCCGA CGGCGCCGCG GCGATGGCCG 3780
36 CCGCGTAGAG cGcrAr~rArA- GCGAGCATCG rArGCGCr-AG CGAGGCCATG GTCGAAGCTT 3840
GCCGCGGAGG CTGGATCGGT CCCGGTGTCT TCTATGGAGG TCAAAACAGC GTGGATGGCG 3900
TCTCCAGGCG ATCTGACGGT TCACTAAACG AGCTCTGCTT A~ATAr~ArCT CCCACCGTAC 3960
ACGCCTACCG CCCATTTGCG TCAATGGGGC GGA~ A CGACATTTTG GAAAGTCCCG 4020
TTGATTTTGG TGcrAAAArA AACTCCCATT GACGTCAATG GGGTGGAGAC TTGGAAATCC 4080
45 CCGTGAGTCA AAccGcTA~c rACGCCCATT GATGTACTGC CAAAArCGrA TCACCATGGT 4140
AATAGCGATG Ar~AA~ACGT AGATGTACTG Cr-AAr-TAGr-A AAGTCCCATA AGGTCATGTA 4200
CTGGGCATAA TGCr-AGGCGG GCCATTTACC GTCATTGACG TrAA~ArGGG GCGTACTTGG 4260
CATATGATAC ACTTGATGTA CTGCCAAGTG GGCAGTTTAC CGTAAATArT C~ACCrP~TG 4320
ACGTCAATGG AAAGTCCCTA TTGGCGTTAC TATGGr~AArA TACGTCATTA TTGACGTCAA 4380
66 TGGGCGGGGG TCGTTGGGCG GTCAGCrAGG CGGGccATTT ACCGTAAGTT ATG~AACGCG 4440
GAACTCCATA TATGGGCTAT GAACTAATGA CCCC~lAATT GATTACTATT AA~AArTAGT 4500
CAA~A~cAA TGTCAACATG GCGGTAATGT TGGACATGAG CCAATATAA~A TGTACA~ATT 4560
ATGATATGGA TArAACGTAT GCAATGGCCA ATAGCrAA~A TTGATTTATG CTA~A~AArc 4620
AATr~AATAAT ATGGCTAATG GCrAA~ATTG ATTCAATGTA TAGATCGATA TGCATTGGCC 4680
ATGTGCCAGC TTGATGTCGC CTCTATCGGC r-A~ATAGCCT CATATCGTCT GTCACCTATA 4740
TCGAAACTGC GATATTTGCG ArA,rArA~AA TCGCCCAAGT CACCAA~AGGC GTCTATCGCC 4800
-32-
W O 95/12682 2 1~ 2 8 1 5 PCTrUS94/12198
ATCCCCCGTA AACGATATAA GCGTATCGCC AGATATCGCG TATGCCCAAA AATCAACTTT 4860
TG~.AAAAATG GCGATATCAG TTACACAGAA ACTCACATCG GCGACATTTT CAATATGCCA 4920
TATTTTCAAA TATCGATTTT TCCAATATCG CCATCTCTAT CGGc~ATAAA CACCACTATC 4980
GCGCGACATG AATTTAGTCG GGACAGAAAT CTCAAACGCG TATTTCGGAC AAA~A~ACAT 5040
TTTATTATTC ACTGCAGGTC GAGGAATTCG GATCTCGAGG TGTTCGTGCT GGACGTGTCC 5100
GCGGCGCCAG ACGCGTGCGC GGCCGCCGTA CTGGACATGC GGCCCGCCAT GCAGGCCGCT 5160
TGCGCGGACG GGGCGGCGGG CGCGACGCTG GCGACCCTGG CGCGTCAGTT CGCGCTAGAG 5220
ATGGCGGGGG AGGCCACGGC GGGCCCTAGG GGACTATAAA GCTGCCCCTG CGCTCGCTCG 5280
CTCGCTGCAT TTGCGCCCCG ATCGCCTTAC GGGGACTCGG CGCTCGGCGG ATCCCCTCCC 5340
GGCCCCGCCG c~A-AGrAr~Gc CGCCA~ACAA AAAAATGCGG CGCCCGCTCT GCGCGGCGCT 5400
ATTGGCAGCG GCTGTCCTCG CGCTCGCCGC GGGCGCCCCC GCCGCCGCCC GCGGCGGGGG 5460
CGCCGAAGCC AGGGCAGCAC ~r~ACGCCC GATACGAAAT C~.AAGAGTGG GAA~ATGGTGG 5520
TCGGAGCCGG GCCGGCCGTG rArACGTTCA CCATCCGCTG CCTCGGGCCG CGGGGCATTG 5580
AGCGCGTGGC CCACATTGCA AACCTCAGCC GGCTGCTGGA CGGGTACATA GCGGTCCACG 5640
TTGACGTTGC GCGCACCTCT GGCCTGCGGG ACGCCATGTT lllC~lGCCG CGCGCGGCCG 5700
~0
TCGACTCTAG AGGATCCCCG GGTAccr~AGc TCGAATTCAC TGGCC~lCGl TTTAcAAr-GT 5760
CGTGACTGGG AAAACCCTGG cGT~ArcrAA CTTAATCGCC TTGrAGrArA TCCCCCTTTC 5820
GCCAGCTGGC GTAATAGC~.A AGAGGCCCGC ACCGATCGCC ~llcc-~ArA GTTGCGCAGC 5880
CTGAATGGCG AATGGCGCCT GATGCGGTAT ~ C~LlA CGCATCTGTG CGGTATTTCA 5940
CACCGCATAT GGTGCACTCT CAGTACAATC l~l~lGATG CCGCATAGTT AAGCCAGCCC 6000
CGACACCCGC rAArArCCGC Tr~ArGcGccc TGACGGGCTT GTCTGCTCCC GGCATCCGCT 6060
TACA~ACAAG CTGTGACCGT ~CCGGr~AGC TGCATGTGTC AGAG~ C ACCGTCATCA 6120
CCr-AAArGCG c~ArAc~AAA GGGC~lC~lG A~ArGCCTAT TTTTATAGGT TAATGTCATG 6180
ATAA~AA~GG ~ AGAC GTCAGGTGGC A~ CGGG GAAATGTGCG CGr~AACCCCT 6240
AlllGlllAT ~ lAAAT ACATTCAAAT ATGTATCCGC TCATGAGACA A~AArCCTGA 6300
TAAATGCTTC AA~AATATTG AAAAAG~-AAG AGTATGAGTA TTCAACATTT CC~GlCGCC 6360
CTTAllCC~l TTTTTGCGGC ATTTTGCCTT C~l~lllllG CTrArC~A~.A AACGCTGGTG 6420
55 AAAGTAA~AG ATGCTGAAGA TCAGTTGGGT GrArr~AGTGG GTTACATCGA ACTGGATCTC 6480
AArAGCGGTA AGAlC~llGA GA~l~llCGC cCcr~AAr~AAr ~llllCCAAT GATGAGCACT 6540
TTTAAAGTTC TGCTATGTGG CGCGGTATTA lCCC~ATTG ACGCCGGGrA A~-AGr,AACTC 6600
GGTCGCCGCA TACACTATTC TCAGAATGAC ~lGGll~AGT ACTCACCAGT rArA~.AAAA~ 6660
CATCTTACGG ATGGCATGAC AGTAA~.Ar.A~A TTATGCAGTG CTGcrATAAc CATGAGTGAT 6720
AACACTGCGG CCAACTTACT TcT~ArAArG ATCGGAGGAC C~-AAG~-AGCT AACCGCTTTT 6780
TTGrArAArA TGGGGGATCA TGTAACTCGC CTTGATCGTT GGr~AArCGGA GCTGAATGAA 6840
-33-
W O95/12682 ~ 17 2 ~ 1~ PCTrUS94/12198
GCCATACCAA ACGACGAGCG TGACACCACG ATGCCTGTAG CAATGGCAAC AACGTTGCGC 6900
AAACTATTAA CTGGCGAACT ACTTACTCTA GCTTCCCGGC AACAATTAAT AGACTGGATG 6960
GAGGCGGATA AAGTTGCAGG ACCACTTCTG CGCTCGGCCC TTCCGGCTGG CTGGTTTATT 7020
GCTGATAAAT CTGGAGCCGG TGAGCGTGGG TCTCGCGGTA TCATTGCAGC ACTGGGGCCA 7080
GATGGTAAGC CCTCCCGTAT CGTAGTTATC TA~ACGArGG GGAGTCAGGC AACTATGGAT 7140
GAACGAAATA ~ ATCGC TGAGATAGGT GCCTCACTGA TTAAGCATTG GTAACTGTCA 7200
.A~AA~.TTT ACT~ATATAT ACTTTAGATT GATTTAAAAC TTCATTTTTA ATTTAAAAGG 7260
ATCTAGGTGA AGATCCTTTT TGATAATCTC ATGA~AAAA TCCCTTAACG TGAGTTTTCG 7320
TTCCACTGAG CGT~A-~A~cc CGTA~AAAA~ ATCAAAG~-AT ~llCllGAGA TC~ llll 7380
CTGCGCGTAA TCTGCTGCTT G~AAACAAAA AAAC~A~CGC TACCAGCGGT GG1~ llG 7440
CCGGATCAAG AGCTAC~AA~ l~LlLllCCG AAGGTAACTG GCTTCAGCAG AGCGCAGATA 7500
C~AAATA~TG TCCTTCTAGT GTAGCCGTAG TTAGGCCACC ACTTCAAGAA ~lcl~lAGCA 7560
CCGCCTACAT ACCTCGCTCT GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGC~.ATAA~. 7620
~lC~lGl~lLA CCGGGTTGGA CTCAAGACGA TAGTTACCGG ATAAGGCGCA GCGGTCGGGC 7680
TGAACGGGGG ~1lCGlGCAC ACAGCCCAGC TTGGAGCGAA CGACCTACAC CGAACTGAGA 7740
TACCTACAGC GTGAGCTATG A~AAA~cGcc ACG~llCCCG AAGGr~A~AAA GGCGGACAGG 7800
TAlCCG~lAA GCGGCAGGGT CGGAACAGGA GAGCGCACGA GGGAGCTTCC AGGGGGAAAC 7860
3~ GCCTGGTATC TTTATAGTCC l~CGG~lll CGCrACCTCT GACTTGAGCG TCGATTTTTG 7920
TGA~lG~lC~l CAGGGGGGCG GAGCCTATGG AAAAACGCÇA GCAACGCGGC ~l~llllACGG 7980
TTCCTGGCCT TTTGCTGGCC llllG~lCAC Al~1l~lllC CTGCGTTATC CCCTGATTCT 8040
GTG~.ATAArC GTATTACCGC CTTTGAGTGA GCT~A~ACCG ~lCGCCGCAG CC~AAC~ArC 8100
~AGCG~AGCG AGTCAGTGAG C~AG~AGCG GAAGA I 8135
4~ (2) lN~O~ATION FOR SEQ ID NO:4:
(i) S~U~N~ CHARACTERISTICS:
~A) LENGTH: 8149 base pairs
B) TYPE: nucleio acid
~0 C) STRANDEDNESS: double
,D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomlc)
(iii) ~Y~G~ CAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) OR~ANTsM: Bovine viral diarrhea virus
(B) STRAIN: 2724
(c) INDIVIDUAL ISOLATE: pBHVtkex-3::BGH/p53
(xi) ~uu~N~ D~CRTPTION: SEQ ID NO:4:
.
-34-
~ W O95/126R2 2 1 7 2 8 1 5 PCTrUS94112198
GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60
CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120
5 CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180
TGTGAGCGGA TAArAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG CCAAGCTAGC 240
TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGTTGTCC 300
GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360
GTCCGCGTCC CCAACTCCGC GCr.AArACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420
16 GCGTATAAAA TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480
GCCCAGGCAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC CACAAAGAGC 540
GCCr~AAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600
CTGTTGCCCG CGTGCCTGCT GGCCGCCr~C CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660
CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCCACAGC CGCCCTTTTG CCCGTAGCCA 720
25 GGGGAAGCGG CTGCCCCllC TGCCGCCGCG GCCGCGGTTG CTCGGCTTTG CGTTTGCCCC 780
GCGGCGATCG CCCCGCTCGC CGCGAACGCG CGCGCGCGAA TGGGGCGTAC TCGGCGAGCC 840
CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGCTAGCGAT CGTCTGGGCC GGCAGGCGCG 900
TCACTCTGAG CACGCGCATG CCCCGCTGGG ArAcrAArAr CTGCACCGGC GCTAGGACCA 960
CCGGGTCTGG GCCCGGGGGG GCGAGATCGC GrArAAr,CCG GGCCGAGTCG CGCAGCTGCC 1020
35 GCAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGC~ CAlGllC~ll r~AAAAAr,GGC 1080
ACGTCTTCAG cTcrArr~A~A ArArArAcGG CCCGGGCGTG CC~lGC~lCC Gcr~ArccGr~A 1140
GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC ArAr-ACArGC 1200
CrACrAr-CTG cT~AAAAArc TCCGGGGCGC CAAACTTGCC CAAAAGCTGG GCGAGGCGCG 1260
GGCGCAGCTT CTGCGCGCCA ACCGCCGCGC ~lGC~lCGCA AGCrAGCGCC TCGTAAAAGC 1320
45 GGCl~lGGCA CCGGATCCCG GCr,CGfAGGC GCGcArGTCG GTCGCGGTCG CGCGC~ATGG 1380
cr-r~AGcccGc GCGCG~l`.C CGC~.C~.GC GTATCTACCT Gr~AcGGcGcG rAcGGGrA~r~G 1440
rAAArArAAC AACGGGC~GC GCG~.CGCGG CCG~..CCAC CGCTGGGGAG GGCG G~l~l 1500
~ CCCGGA GCCGATGGCG TACTGGCGCA CGA~ GG ~AcGr7AcGcc TTAAGTGGGA 1560
lC~lCGCGGC GTCTGCGCr-A TGCGCCGrAG c~lcGcAr-GG rAGCGrArGC GCGCGGCGGG 1620
CCGGCGCACC GCGr-A~ArGC Gr~ACGCGGCG GGC~lG~llG CGTACTACCA GGccAGGTTc 1680
GCGGCCCCGT ACTTAATTTT GcArGcGcGT ~.CCGCG~-lG CTGCGCCGCC TGGGCCGGCG 1740
CCGGGCGGCG AGClG~lGGA CC~CG~ll C~AccGcr-Ac CCCGTGGCGC GCGTGCCTCT 1800
Gc~ArcccTT CGCCCG~lAC TGCCTCCGCG AGATCAACGC Gr'AArA~CCG AATTCCTCGA 1860
CCTGCAGTGA A~AA~AAAA~ ~ ~1~111~1 Ccr~AAA~AcG C~111~7AGAT LlCl~,lCCCG 1920
ACTAAATTCA TGTCGCGCGA TAGTGGTGTT TAlCGCC';A~ AGAGATGGCG ATATTGGAAA 1980
AATCGATATT T~AAAA~A~G GCATATTGAA AATGTCGCCG ATGTGAGTTT ~l~l~lAACT 2040
-3~-
W O 9S/12682 PCTrUS94/12198
~1~2~
GATATCGCCA TTTTTCCAAA AGTTGATTTT TGGGCATACG CGATATCTGG CGP.TACGCTT 2100
ATATCGTTTA CGGGGGATGG CGATAGACGC CTTTGGTGAC TTGGGCGATT CTCTGTGTCG 2160
rAAAT~TcGc AGTTTCGATA TAGGTGACAG ACGATATGAG GCTATATCGC Cr~TAr~AGGC 2220
GACATCAAGC TGGCACATGG CCAATGCATA TCGATCTATA CATTGAATCA ATATTGGCCA 2280
TTAC'CrA~A~ TATTCATTGG TTATATAGcA TAAATCAATA TTGGCTATTG GC¢ATTGCAT 2340
ACGTTGTATC CATATCATAA TATGTACATT TATATTGGCT CATGTCCAAC ATTACCGCCA 2400
TGTTGACATT GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT 2 460
AGCCrATATA TGGAGTTCCG CGTTArATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG 2520
CcrAAcr~Arc CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA 2580
GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AP~ACTGCCCA CTTGGCAGTA 2640
CATCAAGTGT ATCATATGCC AAG~ACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC 2700
GCCTGGCATT ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC 2 760
GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA 2820
TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG 2 880
TTTTGGCACC AAAATCAACG GGA~lllCCA AAAlGlC~lA ACAACTCCGC CCCATTGACG 2940
CAAATGGGCG GTAGGCGTGT ACGGTGGGAG GTCT~TATAA GCAGAGCTCG TTTAGTGAAC 3000
CGTCAGATCG ccTGr~ArAcG CCATCCACGC ~ ACC Tc~rATA~AAG ArArcGG~A~ 3060
CGATCCAGCC TCCGCGGCAA GCTTCGACAT GATGGCTGCA GGCCCCCGGA CCTCCCTGCT 3120
CCTGGCTTTC GCCCTGCTCT GCC,lGCC~lG GACTCAGGTG GTGGGCGCCT TCCCAGGGAT 3180
CCTATGGCTA CTACTGATAA CAGGGGTACA AGGGGACATT GACTGCAPAC CTGAACACTC 3240
A~AcGcrA~A GcrA~r~AA~G A~Ar~AATTGG CCCATTAGGA GcT~AAGGcc TCACCACTGT 3300
TTG~.AAr.rAT TACTCACATG AAATGAAGCT Gr-AArACACA ATGGTCATAG ~l~G~lGCAA 3360
AGACGGTAAG TTTArA~ACC TCTCAAGGTG rArAAr.ArAA ACTAr-A~ATC TTGCAATTCT 3420
GCATTCAAGA GC~GCAGA CCAGl~-lG~l ATTCAA~AAAA ~ CGAGG GGCAAAGGCA 3480
AGGGr.AAAC~ TTTGAAATGG CTGACGACTT TGAATTTGGA C~lGCC~AT GCGATGCCAA 3540
~0
TCCCGTAGTA AGAGGGAAGT TrAA~ArAAc ACTGCTAAAC G~ACCGGC~l TÇCAGATGGT 3600
AlGCC~A~A GGATGGACAG GAACTGTGAG CTGTATGTTA GCTAA~A~GG A~ACCCTA~-~ 3660
5~ rA~AGrA~.~A GTGC~Gl A~AA~AGGTc r.AA~CrA~TC CcTTATA~Ar AAG~ AT 3720
rAcCrAAA~A AC~C~GGGGG AGGATCTCTA TAACTGTGAT CTTG~AGGGA ATTGGACTTG 3780
TGTGACTGGG GACCAGCTAC AA~ArArAGG AGGCCClG'C GAA~CllGCA A~lGGlGlGG 3840
TTATAAATTc CAAAAAAGTG AGGGGTTGCC ACACTACCCC ATCGGCAAGT GTAGGTTGAA 3900
GAATGAGACT GGCTArA~A~ TTGTArA~GG CACCACTTGC AA~A~A~A~G GTG~AGc~A~ 3960
65 AG~ACrArAA GGATTGGTAA AGTGTAAGAT AGr.A~ArACA. ATCGTACAGG TCATAGCTCT 4020
T~ArACrAAA CTTGGGCCTA TGCCTTGCAA GCrATA~GAG ATCA~ArrAA GTGAGGGGCC 4080
-36-
_
W 095/12682 ~ ~ 7 2 ~1 5 PCT~US94/12198
TGTAGAAAAG ACGGCATGCA CCTTCAACTA CACGAGGACA TTAAAAAATA AATATTTTGA 4140
GCCCAGAGAC AGTTACTTCC AGCAATACAT GCTAAAAGGA GATTATCAAT ACTGGTTCGA 4200
CCTGGAGGTC ACTGACCATC ATCGGGATTA CTTCGCCGAG TCCATATTGG TGGTGGTGGT 4260
AGCTTTACTG GGTGGAAGAT ACGTGCTCTG GTTACTGGTA ArATACATGG TCCTATCAGA 4320
A~AAAAGGCC TTGGGGACCC AATATGGGGC AGGGGAAGTG GTGATGATGG GTAACTTGCT 4380
AACACATGAC AGTATTGAAG TGGTGACATA lll~l.~ll~ TTATACCTAC TGcTAAr~ArA 4440
GGAGGCTGTA AAGAAGTGGG TCTTACTCTT ATACr~CCTT GATTGATTGA GGATCAGCTT 4500
ATCCAGGGTC GACCTCAGGC ATGCAAGCTC AGATCCGCTG lGC~ll lAG TTGCCAGCCA 4560
lC~ lll GCCCCTCCCC CGTGCCTTCC TTGACCCTGG AAGGTGCCAC TCCCACTGTC 4620
CTTTCCTAAT AAAATGAGGA AATTGCATCG CA.l~l~GA GTAGGTGTCA TTCTATTCTG 4680
GGGGGTGGGG TGGGGCAGGA CAGCAAGGGG GAGGATTGGG AArArAA~AG CAGGCATGCT 4740
GGGGATGCGG TGGGCTCTAT GGGTACCrAr, GTGCTGAAGA ATTGACCCGG llC~lC~lGG 4800
GCrA~AAArA AGCAGGCACA TCCCCTTCTC TGT~ArArAr C~l~lCCACG CCCCTGGTTC 4860
TTAGTTCCAG CCCCACTCAT AGGACACTCA TAGCTCAGGA GGGCTCCGCT TCAATCCCAC 4920
CCGCTAAAGT ACTTGGAGCG GlCl~lCCCT CCCTCATCAG CC5ACrAAAC CAAACCTAGC 4980
CTCCAAGAGT GGGAAr~AAAT TAAAGCAAGA TAGGCTATTA AGTGCAGAGG r.Ar.Ar.AAAAT 5040
GCCTCCAACA TGTGAGGAAG TAATGATAGA AATrATAr.AA TTGAGATCTC GAG~l~llCG 5100
35 TGCTGGACGT GTCCGCGGCG C5Ar~ArGCGT GCGCGGCCGC CGTACTGGAC ATGCGGCCCG 5160
CCATGCAGGC CGCTTGCGCG GACGGGGCGG CGGGCGCGAC GClGGCGACC CTGGCGCGTC 5220
AGTTCGCGCT AGAGATGGCG GGGrAGGCCA CGGCGGGCCC TAGGGGACTA TAAAGCTGCC 5280
CCTGCGCTCG CTCGCTCGCT GCA~ GCGC CCCGATCGCC TTArGGGr~Ar TCGGCGCTCG 5340
GCGGATCCCC TCCCGGCCCC GCCGCGAAGC AGGCCGCCAG ACAAAAAAAT GCGGCGCCCG 5400
~ GCGCGG CGCTATTGGC AGCGG~lC CTCGCGCTCG CCGCGGGCGC CCCCGCCGCC 5460
GCCCGC'GGCG GGGGCGCCr~A AGCrAGGGrA GCArArAr.A5 GCCCGATACG AAATCGAAGA 5520
GTGGrAAATG ~'G~CGGAG CCGGGCCGGC CGTGrArACG TTCACCATCC G~GC~lCGG 5580
GCCGCGGGGC ATT~AGCGCG TGGCC Q rA~ TGrAAArCTC AGCCGGCTGC TGGACGGGTA 5640
CATAGCGGTC CACGTTGACG TTGCGCGrAr CTCTGGCCTG cGGr~ArGcrA 'l~lllllC~l 5700
GCCGCGCGCG GCC~lCGACT cTAr.Ar,~.ATc CCCGGGTACC GAGCTCGAAT TCA~GGCCG 5760
.C~l~llACA AC~lC~l~AC TGGr-AAAArC ~GGCGllAC CCAACTTAAT CGC~l~GCAG 5820
CACATCCCCC TTTCGCrAGC TGGCGTAATA GCr.AA~.AGGC CCGrArCr-AT CGCC~llCCC 5880
AACAGTTGCG CAGCCTGAAT GGCr.AA~GGC GCCTGATGCG GTAllll lC cT~A5GrA~c 5940
TGTGCGGTAT TTCArACCGC ATAlG~lGCA CTCTCAGTAC AA~ G~l~l GA~GCCG~i~T 6000
65 AGTTAAGcrA GCCCCr~ArA5 CCGCrAAr-AC CCG~lGACGC GCC~l~ACGG G~llGl~lGC 6060
. TCCCGGCATC CGCTTACAGA CAAGCTGTGA CCGl~lCCGG GAGCTGCATG TGTCAGAGGT 6120
-37-
W O95/12682 2 i 7 2 ~ 1 ~ PCTrUS94112198
TTTCACCGTC ATCACCGAAA CGCGCGAGAC GAAAGGGCCT CGTGATACGC CTATTTTTAT 6180
AGGTTAATGT CATGATAATA ATGGlll~ll AGACGTCAGG TGGCACTTTT CGGGGAAATG 6240
TGCGCGGAAC CCCTATTTGT TTA~ l AAATACATTC AAATATGTAT CCGCTCATGA 6300
~.ArAATAA~C CTGATAAATG CTTrAATAA~ ATTr-AAAAA~ GAAGAGTATG AGTATTCAAC 6360
ATTTCCGTGT CGCCCTTATT CC~l L 11 L LG CGGCATTTTG CCTTCCTGTT TTTGCTCACC 6420
CAGAAACGCT GGTGAAAGTA AAAGATGCTG AAGATCAGTT GGGTGCACGA GTGGGTTACA 6480
TCGAACTGGA TCTCAACAGC GGTAAGATCC TTGAGAGTTT TCGCCCCGAA GAACGTTTTC 6540
CAATGATGAG CACTTTTAAA GTTCTGCTAT GTGGCGCGGT ATTATCCCGT ATTGACGCCG 6600
GGCAAGAGCA ACTCGGTCGC CGCATACACT ATTCTCAGAA TGA~llG~.ll GAGTACTCAC 6660
CAGTCACAGA AAAGCATCTT ACGGATGGCA TGACAGTAAG AGAATTATGC AGTGCTGCCA 6720
TAACCATGAG T~A~AA~ACT GCGGC~AACT TACTTCTGAC AACGATCGGA GGACCGAAGG 6780
AGCTAACCGC ll~lGCAC AACATGGGGG ATCATGTAAC TCGCCTTGAT CGTTGGGAAC 6840
CGGAGCTGAA TGAAGCCATA C~AAPrGACG AGCGTGACAC CACGATGCCT GTAGrAATGG 6900
~AACAACGTT GCGCAAACTA TTAACTGGCG AACTACTTAC TCTAGCTTCC cGGrAA~AA~ 6960
TAATAGA~TG GATGGAGGCG GATAAAGTTG CAGGACCACT TCTGCGCTCG GCCCTTCCGG 7020
CTGGCTGGTT TATTGCTGAT AAATCTGGAG CCG~ AGCG TGGGTCTCGC GG,TATCATTG 7080
CAGCACTGGG GCCAGATGGT AAGCCCTCCC GTATCGTAGT TATCTArACG ACGGGGAGTC 7140
AGGCAACTAT GGATGAACGA AATAGACAGA TCGCTGAGAT AGGTGCCTCA CTGATTAAGC 7200
ATTGGTAACT GTrAGAcr-AA GTTTACTCAT ATATACTTTA GATTGATTTA AAACTTCATT 7260
TTTAATTTAA AAGGATCTAG GTGAAGATCC TTTTTGATAA TCTCATGACC AAAATCCCTT 7320
AACGTGAGTT ~lCG~lCCAC TGAGCGTCAG ACCCC~.~AGA AAAGATCAAA GGA'L~ L 7380
GAGATCCTTT ~ GCGC GTAATCTGCT GCTTGCAAAC AA~AAAA~rA CCGCTACCAG 7440
CGGTGGTTTG TTTGCCGGAT ~AAGAGCTAC CAACl~l~ll Tcc~AAGGTA ACTGGCTTCA 7500
GCAGA~CGrA ~.A~A~rAAA~ A~.7~ C TAGTGTAGCC GTAGTTAGGC CACCACTTCA 7560
AGAACTCTGT AGCArCGCCT ArA~ACCTCG ~l.~G~IAAT C~~ ACCA ~..GG~-G~G 7620
CCAGTGGCGA TAA~lC~ ~71 CT~A~CGGGT TGGACTCAAG A~r~ATAGTTA CÇG~-ATAAGG 7680
CGCAGCGGTC GGGCTGAACG GGGG~IC~7~ GCA~ArAGCC CAG~GGAG C~-AAC~-ACCT 7740
A~ACC~-AACT ~A~A~ArCT~ CAGCGTGAGC TATGAGAAA~- CGCCP~GCTT cçcr-AA~Gr-A 7800
GGCGGA CAGGTATCCG G~AAGCGGCA GGGTCGGAAC Ar~rA~.AGCGC A~r~Ar~Gr-Ar7c 7860
TTCCAGGGGG AAA~GccTGG TATCTTTATA ~lC~l~lCGG ~7~1 ~cGccAc CTCTGACTTG 7920
AGCGTCGATT TTTGTGATGC TCGT QGGGG GGCGr-AGCCT ATG~-AAAAAr GCrAGrAArG 7980
CGGC~ Ll ' ACGGllC~G GCC~ GCl GGCCll~lGC TCACATGTTC "llC~lGCGT 8040
65 TATCCCCTGA ~ 7~GGAT AACCGTATTA cCGC~ll GA GTGAGCTGAT ACCG~CGCC 8100
G~AGCC~AAr r-A~C~P~CGC AGCGAGTCAG TGAGCGAGGA AGCG~AA~A 8149
I
~172815
W O95/12682 ~ PCT~US94112198
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8135 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine viral diarrhea virus
(B) STRAIN: 2724
(C) INDIVIDUAL ISOLATE: pBHVtkex-3::gIII/p53
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GCGCCCAATA CGrAAAccGc CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60
CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120
CACTCATTAG GCACCCCAGG CTTTACACTT TA~ CCG GCTCGTATGT TGTGTGGAAT 180
TGTGAGCGGA ~AACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG CCAAGCTAGC 240
TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGC-,~lG CCTTCGCCAG CAG-,lL~CC 300
GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360
GTCCGCGTCC CCAACTCCGC GCr~AAr~ACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420
GCGTATAAAA TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480
GCCCAGGCAA GCAAACTCTA AArGCCCr~AG CGCCATGGCC CCGATGCCGC rArAAAr~Ar~c 540
GccrAAATTT CGCCCAGGCA CGCCGCGCCG CCCr~ArGCGT CTT~Ar,CGrA CCCGCCGGCG 600
CTGTTGCCCG CGTGCCTGCT GGCCGCCCAC CGGCGGCCGC ~CCCCGGC CTCAGCAGGG 660
CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG cGGcrA-rAGc CGCCC~ G CCCGTAGCCA 720
GGG~AAGCGG CTGCCCC~C ~GCCGCCGCG GCCGCGGTTG CTCGGCTTTG C~.l~GCCCC 780
GCGGcr.A~CG CCCCGCTCGC CGCGAACGCG CGCGCGCr-AA TGGGGCGTAC TCGGCr-AC-CC 840
CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGC~AGCGAT C~ClGGGCC GG~AGGCGCG 900
TCACTCTGAG rAr,GCGrA~G CCCCGCTGGG Ar-ACrAArAC cTGrAccGGc GCTAGGACCA 960
CCGGGTCTGG GCCCGGGGGG GCr~A~A~CGC GCArAAGCCG GGCCr~AGTCG CGCAGCTGCC 1020
GrAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGC~ CAlG~ lC~ GAAAAAcGrc 1080
AC~ CAG CTCCACGATA AGA~A~A~CGG CCCGGGCGTG CCCTGCCTCC GCr.ACCCG~A 1140
GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC Ar~AC-ArArGC 1200
crArr~ArcTG CT~AAAA~CC TCCGGGGCGC CAAACTTGCC ~AAAAr,CTGG GCGAGGCGCG 1260
GGCGCAGCTT CTGCGCGC~A ACCGCCGCGC GTGCGTCGCA Ar,CrAr,CGCC TCGTAAAAr~C 1320
GGCTGTGGCA CCGGATCCCG GCGCGCAGGC GCGCACGTCG GTCGCG~lCG CGCGCrA~GG 1380
~9
W 095/12682 ~ 17 2 8 1 5 PCTrUS~4/12198
CCGAGCCCGC GCGCGCTCTC CGCGTCGTGC GTATCTACCT GGACGGCGCG CA¢GGGCAGG 1440
~AAAf~ArAAc AACGGGCCGC GCGCTCGCGG CCGCTTCCAC CGCTGGGGAG GGCGTGCTCT 1500
5 TTTTCCCGGA GCCGATGGCG TACTGGCGCA CGATGTTTGG TACGGACGCC TTAAGTGGGA 1560
TCCTCGCGGC GTCTGCGCGA TGCGCCGCAG CCTCGCACGG GAGCGCACGC GC¢CGGCGGG 1620
CCGGCGCACC GCGCAGACGC GGACGCGGCG GGCCTGGTTG CGTACTACCA GGCCAGGTTC 1680
GCGGCCCCGT ACTTAATTTT GCACGCGCGT GTCCGCGCTG CTGCGCCGCC TGGGCCGGCG 1740
CCGGGCGGCG AGCTGGTGGA CCCTCGTGTT CGACCGCCAC CCCGTGGCGC GCGTGCCTCT 1800
GCTACCCCTT CGCCCGCTAC TGCCTCCGCG AGATCAACGC GGAAGATCCG AATTCCTCGA 1860
CCTGCAGTGA ATAATAAAAT G~ CCr.AAATA~G CGTTTGAGAT llCl~lCCCG 1920
ACTAAATTCA TGTCGCGCGA TAGTGGTGTT TATCGCCGAT AGAGATGGCG ATATTGGAAA 1980
AATCGATATT TGAAAATATG GCATATTGAA AATGTCGCCG ATGTGAGTTT ~l~l~lAACT 2040
GATATCGCCA ll~LlCCAAA AGTTGATTTT TGGGrATACG CGATATCTGG C~TACGCTT 2100
ATATCGTTTA CGGGGGATGG C~ATA~ACGC CTTTGGTGAC TTGGGCGATT ~l~lGl~lCG 2160
~AAATATCGC AGTTTCGATA TAGGTGACAG AC~A~ATGAG GCTATATCGC cr~ATAr~AGGc 2220
GACATCAAGC TGGCACATGG CCAATGCATA TCGATCTATA CATTGAATCA ATATTGGCCA 2280
TTAGCCATAT TATTCATTGG TTATATAGCA TAAATCAATA TTGGCTATTG GCCATTGCAT 2340
AC~ll~lATC CATATCATAA TATGTACATT TATATTGGCT CATGTCCAAC ATTACCGCCA 2400
3~ TGTTGACATT GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT 2460
AGCCCATATA TGGAGTTCCG CGTTArATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG 2520
CCCAACGACC CCCGCC~A~T GACGTCAATA ATGACGTATG TTccrA~A-GT AACGCCAATA 2580
GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAAClGCC~A CTTGGCAGTA 2640
CATCAAGTGT ATCATATGCC AAGTArGCCC CCTATTGACG TCAATGACGG TAAATGGCCC 2700
GCCTGGCATT ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC 2760
GTATTAGTCA TCGCTATTAC CATGGTGATG CGGllllGGC AGTArATCAA TGGGC~lGGA 2820
TAGCG~lllG ACTCACGGGG ATTTCCAAGT CTCr-ACCCr~ TTGACGTCAA TGGGAGTTTG 2880
TTTTGGCACC AAAATCAACG GGAC-llCCA AAATGTCGTA ACAACTCCGC CCCATTGACG 2940
CAAATGGGCG GTAGGC~l~. ACGGTGGGAG GTCTA~ATAA GrA~AGCTCG TTTAGTGAAC 3000
CGTCAGATCG CCTGr-Ar-ACG CCATCCACGC 'lGllL ~ GACC TCrATA~AAG ACACCGGGAC 3060
CGATCCAGCC TCCGCGGCAA GCTTCGACCA TGGC~lCG~l CGCGCGTGCG ATG~CG~C 3120
TGCTGGCGCT CTACGCGGCG GCCATCGCCG CGGCGCCGTC GGGGATCCTA TGGCTACTAC 3180
Tr~ATAA~AGG GGTAcAAGGG GACATTGACT GCAAArCTGA ACACTCATAC GCrATAr,CCA 3240
GGAATGATAG AATTGGCCCA T~AGr-AGCTG AAGGCCTCAC CACl~lllGG AAGGATTACT 3300
CACATGAAAT GAAGCTGGAA GAcAcAA-TGG TCATAGCTTG GTGCAAAr.AC GGTAAGTTTA 3360
CATACCTCTC AAGGTGCACA Ar.Ar.AAAr~A GATATCTTGC AATTCTGCAT TrAA~AGccT 3420
-40- ,
~ W 095t126æ 2~281~ PCTrUS94/12l98
TGCAGACCAG TGTGGTATTC AAAAAACTTT TCGAGGGGCA AAGGCAAGGG GAAACATTTG 3480
AAATGGCTGA CGACTTTGAA TTTGGACTCT GCCCATGCGA TGCCAATCCC GTAGTAAGAG 3540
GGAAGTTCAA TACAAr~rTG CTAAACGGAC CGGCCTTCCA GATGGTATGC CCTATAGGAT 3600
GGACAGGAAC TGTGAGCTGT ATGTTAGCTA ATAGGGACAC CCTAGACACA GCAGTAGTGC 3660
~l~lGlATAA GAGGTCCAAA CCATTCCCTT ATAGACAAGG TTGTATCACC ~AAA~-AACTC 3720
TGGGGGAGGA TCTCTATAAC TGTGATCTTG GAGGGAATTG GA~lL~,l~lG ACTGGGGACC 3780
AGCTACAATA CACAGGAGGC CCTGTCGAAT CTTGCAAGTG GTGTGGTTAT AAATTCCAAA 3840
15 AAAGTGAGGG GTTGCCACAC ~ACCCCATCG GCAAGTGTAG GTT~.AArAAT GAGACTGGCT 3900
ACAGATTTGT AGACGGCACC ACTTGCAACA GAGAGGGTGT AGCCATAGTA CCACAAGGAT 3960
TGGTAAAGTG ~AA~.A~AG~.A ~.ACArAA~CG TACAGGTCAT AGCTCTTGAC ACCAAACTTG 4020
GGCCTATGCC TTGC-AAGC~ TATGAGATCA TACCAAGTGA GGGGCCTGTA rAAAArAcGG 4080
CATGCACCTT CAACTACACG AGGACATTAA AAAATAAATA TTTTGAGCCC A~A~ACAGTT 4140
ACTTCCAGCA ATACATGCTA AAAr.rArATT ATCAATACTG GTTCGACCTG GAGGTCACTG 4200
ACCATCATCG GGATTACTTC GCCGAGTCCA TATTGGTGGT GGTGGTAGCT TTACTGGGTG 4260
rAArATAcGT GCTCTGGTTA CTGGTAACAT ACATGGTCCT ATcArAArAA AAGGCCTTGG 4320
GGACCCAATA TGGGGCAGGG GAAGTGGTGA TGATGGGTAA CTTGCTAACA CATGACAGTA 4380
TTGAAGTGGT rArATATTTC il~ llAT ACCTACTGCT AAr.ArAGrAC. GCTGTAAAGA 4440
35 AGTGGGTCTT ACTCTTATAC CACCTTGATT GATTGAGGAT CAGCTTATCC AGGGTCGACC 4500
TCAGGCATGC AAGCTCAGAT CCG~l~lGCC TTCTAGTTGC CAGCCATCTG ll~lllGCCC 4560
ClCCCCCGTG C~llC~llGA CCCTGGAAGG TGCCACTCCC ACTGTCCTTT CcTAATAAAA 4620
TGAGGAAATT GCATCGCATT GTCTGAGTAG GTGTCATTCT ATTCTGGGGG GTGGGGTGGG 4680
GrAGrArArC AAGGGGGAGG ATTGGGAAGA rAA~AGrAGG CATGCTGGGG ATGCGGTGGG 4740
45 CTCTATGGGT ArCrArGTGC T5AArAATTG ACCCGGTTCC TCCTGGGCCA r.ApAr.AAC.rA 4800
GG~Ar-ATCCC ~l-`l`l~lG A~-ArArCCTG TCrACGCCC.'C lG,~ll~llAG TTC~ArCCCC 4860
ACTCATAGGA CACTCATAGC TCAGGAGGGC lCCG~l~AA TCCr-ArCCGC TA~AGTACTT 4920
GGA~CGGTCT ~lCC~lCC~ CATrAGCC~A C~AAAC~AAA CCTAGCCTCC AAGAGTGGGA 4980
AGAPATTAAA Gr-AArATAGG CTATTAAGTG rArAGGrArA GAAAATGCCT CrAArATGTG 5040
55 Arr.AA~TAAT GA~Ar~AAA~c A~A~AA~TGA GATCTCGAGG ~llCC7-G~l GGACGTGTCC 5100
GCGGCGC~AG ACGC~,lGCGC GGCCGCC'~,lA CTGGACATGC GGCCCGCCAT G~AGGCCGCl 5160
lGCGCGr.~CG GGGCGGCGGG cGcrAcGcTG GcrArccTGG CGCGTCAGTT c~cG~l~rAr 5220
ATGGCGGGGG AGGCrACGGC GGGCCCTAGG GGACTATAPA GCTGCCCCTG CG~-CG~-CG 5280
~lCG~lGCAT lGCGCCCCG ATCGCCTTAC GGGGACTCGG CG~lCGGCGG AlCCCClCCC 5340
GGCCCCGCCG CGAAGCAGGC CGcrA~ArAA AAAAATGCGG CGCCCGCTCT GCGCGGCGCT 5400
ATTGGCAGCG G~-~,1C~lCG CG~lCGCCGC GGGCGC'CCCC.' GCCGCCGC'CC GCGGCGGGGG 5460
-41-
WO 95/12682 PC~US94/12198
2172815
, V
CGCC~.AA~CC AGGGCAGCAC AGAGACGCCC GATACGAAAT CGAAGAGTGG GAAATGGTGG 5520
TCGGAGCCGG GCCGGCCGTG CACACGTTCA CCATCCGCTG CCTCGGGCCG CGGGGCATTG 5580
5 AGCGCGTGGC CCACATTGCA AACCTCAGCC GGCTGCTGGA CGGGTACATA GCGGTCCACG 5640
TTGACGTTGC GCGCACCTCT GGCCTGCGGG ACGCCATGTT TTTCCTGCCG CGCGCGGCCG 5700
TCGACTCTAG AGGATCCCCG GGTACCGAGC TCGAATTCAC TGGCCGTCGT TTTACAACGT 5760
CGTGACTGGG AA-AACCCTGG CGTTACC~AA CTTAATCGCC TTGCAGCACA TCCCCCTTTC 5820
GCCAGCTGGC GTAATAGCGA AGAGGCCCGC ACCGATCGCC CTTCCCAACA GTTGCGCAGC 5880
CTGAATGGCG AATGGCGCCT GATGCGGTAT TTTCTCCTTA CGCATCTGTG CGGTATTTCA 5940
~ACCG~ATAT GGTGCACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT AAGC~AGCCC 6000
C~A~A~CCGC rAA~ArCCGC TGACGCGCCC TGACGGGCTT GTCTGCTCCC GGCATCCGCT 6060
TA~A~.ACAAG CTGTGACCGT CTCCGGGAGC TGCATGTGTC AGAGGTTTTC ACCGTCATCA 6120
CCGAAACGCG CGAGACGAAA GGGCC1CG1G ATACGCCTAT TTTTATAGGT TAATGTCATG 6180
25 PTAATAATGG ~ AGAC GTCAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT 6240
A111~AT 1111C1AAAT ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA 6300
TAAATGCTTC AATAATATTG AAAAAGGAAG AGTATGAGTA TTCAACATTT CCGTGTCGCC 6360
CTTATTCCCT ~ GCGGC AL~1GC~1 C~1G11~1~G CTCACCCAGA AACGCTGGTG 6420
AAAGTAA-AAG ATGCTGAAGA TCAGTTGGGT G~AC~A~TGG GTTACATCGA ACTGGATCTC 6480
35 AACAGCGGTA AGATCCTTGA GAGTTTTCGC CCCr~AA~.~AC GTTTTCCAAT GATGAGCACT 6540
TTTAAAGTTC TGCTATGTGG CGCGGTATTA TCCCGTATTG ACGCCGGGCA A~A~AACTC 6600
GGTCGCCGCA TACACTATTC TCAGAATGAC TTGGTTGAGT ACTCACCAGT CAr-ArAAAA~ 6660
CATCTTACGG ATGGCATGAC AGTAAG~r~AA TTATGCAGTG CTGC~ATAAr CATGAGTGAT 6720
AACACTGCGG CCAACTTACT TCT~-A~AArG ATCG~-A~AC C~.AA~-A~CT AACCGCTTTT 6780
TTG~A~AACA TGGGGGATCA TGTAACTCGC CTTGATCGTT GGr~AACCGr~A GCTGAATGAA 6840
GCCATACrAA AC~AC~A~CG Tr.A~ rG ATGCCTGTAG CAATGGCAAC AACGTTGCGC 6900
AAACTATTAA CTGGC~AACT ACTTACTCTA G~. lCCCGGC AACAATTAAT AGACTGGATG 6960
~ A~GCGr.ATA AAGTTGCAGG ACCACTTCTG CG~CGGCCC TTCCGGCTGG CTGGTTTATT 7020
GCTr.ATAAAT CTG~Ar~CCGG TGAGCGTGGG TCTCGCGGTA TCATTGCAGC ACTGGGGCCA 7080
~5 GATGGTAAGC CCTCCCGTAT CGTAGTTATC TA~Ar~ACGG GGAGTCAGGC AACTATGGAT 7140
~AAC~AAATA ~A~A~.ATCGC Tr.A~ATAGGT GCCTCACTGA TTAAG~ATTG GTAACTGTCA 7200
~AC~AAGTTT ACT~ATATAT ACTTTAGATT GATTTAAAAC TTCATTTTTA ATTTAAAAGG 7260
ATCTAGGTGA AGA~CC111L TGATAATCTC AT~AC~AAAA TCCCTTAACG TGA~1CG 7320
TTCCACTGAG CGTCAGACCC CGTAGAAAA~ AT~AAA~AT ~11~1~GAGA ~CC~ L ' 1 7380
65 CTGCGCGTAA TCTGCTGCTT GrAAA~AAAA AAAC~ArCGC ~Ar~A~CGGT G~1'1G111G 7440
CCGGATCAAG AGCTACrAAC ~C'-~'1CCG AAGGTAACTG GCTTCAGCAG AGCG~.A~.ATA 7500
-42-
~1~2gl5
W O95112682 PCTrUS94/12198
C~AAATACTG TCCTTCTAGT GTAGCCGTAG TTAGGCCACC ACTTCAAGAA CTCTGTAGCA 7560
CCGCCTACAT ACCTCGCTCT GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGC~ATAAC. 7620
5 TCGTGTCTTA CCGGGTTGGA CTCAAGACGA TAGTTACCGG ATAAGGCGCA GCGGTCGGGC 7680
TGAACGGGGG GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA CGACCTACAC CGAACTGAGA 7740
TACCTACAGC GTGAGCTATG AGAAAGCGCC ACGCTTCCCG AAGGGAGAAA GGCGGACAGG 7800
TATCCGGTAA GCGGCAGGGT CGGAACAGGA GAGCGCACGA GGGAGCTTCC AGGGGGAAAC 7860
GCCTGGTATC TTTATAGTCC TGTCGGGTTT CGCCACCTCT GACTTGAGCG TCGATTTTTG 7920
15 TGATGCTCGT CAGGGGGGCG GAGCCTATGG AAAAACGC~A GCAACGCGGC ~llllLACGG 7980
TTCCTGGCCT lllG~lGGCC TTTTGCTCAC A~ lC CTGCGTTATC CCCTGATTCT 8040
GTG~.ATAACC GTATTACCGC CTTTGAGTGA GCT~ATACCG CTCGCCGCAG CCGAACGACC 8100
GAGCGCAGCG AGTCAGTGAG CGAGGAAGCG GAAGA 8135
-4~-
