CA2172815A1 - Viral vector with bovine viral diarrhea virus (bvdv) antigens - Google Patents

Abstract

This invention relates to the field of Bovine Viral Diarrhea Virus (BVDV), and vaccines for the treatment thereof. This invention describes the preparation of live, attenuated Bovine Herpesvirus type I (BHV-1) as a virus, vaccine and vector for expression of BVDV antigens. A BVDV cDNA clone containing sequences corresponding to glycoprotein gp53 is inserted into an inactivated BHV-1 virus.

Description

~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.

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, 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 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 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 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.

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.

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:

~.~1 728~S

W O95112682 PCTrUS94/12198 ,; ., f, ~

CACTCATTAG GrA~CCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180 TGTGAGCGGA TAA~AATTTC ACACAGGAA~A CAGCTATGAC CATGATTACG CCAAGCTAGC 240 GCGTA~AAAA. TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480 GCCCAGGCAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC rArAAA~AGC 540 GCC~'AAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600 CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCrArAGC CGCCCTTTTG CCCGTAGCCA 720 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 ATAlCGl~lA CGGGGGATGG C~ATA~ACGC CTTTGGTGAC TTGGGCGATT CTGTGTGTCG 2160 6 CAAATATCGC AGTTTCGATA TAGGTGACAG A~-ATA~GAG GCTATATCGC C~A~A~-A~GC 2220 AGCCrA~A~A TGGAGTTCCG CGTTAr~TAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG 2520 CC~AAC~ACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCrA~AC.T AACGCCAATA 2580 TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCArCCrA TTGACGTCAA TGGGAGTTTG 2880 CAAATGGGCG GTAGGCGTGT ACGGTGGGAG GTCTATA~AA GCAr.AGCTCG TTTAGTGAAC 3000 CGTCAGATCG CCTGGAGACG CCATCCACGC ~ GACC TCrATA~.AA~. ACACCGGGAC 3060 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 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 WO 9S/12682 2 1 7 ~ ~ 1 5 PCTrUS94/12198 AGACAGTTAC TTCCAGCAAT ACATGCTA~A AGGAGATTAT CAATACTGGT TCIGACCTGGA 4140 ACTGGGTGGA AGATACGTGC TCTGGTTACT GGTAACATAC ATGGTCCTAT ~A~.AA~AAAA 4260 GGCCTTGGGG ACCCAATATG GGGCAGGGGA AGTGGTGATG ATGGGTAACT TGCTAA~A~A 4320 TGTAAAr.AAC. TGGGTCTTAC TCTTATACCA CCTTGATTGA TTGAGGATCA GCTTATCCAG 4440 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 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 W 095/12682 ~ 1 7 ~ 8 1 ~ PCT~US94tl2198 GAACCCCTAT .. ~7111ATTT TTCTAAATAC ATTCAAATAT GTATCCGCTC AT~A~ACAAT 6240 GTGTCGCCCT TATTCCCTTT TTTGCGGCAT TTTGCCTTCC ~ GCT rACcrA~.AAA 6360 CGCTGGTGAA AGTAAAA~AT GCTGAAGATC AGTTGGGTGC ACGAGTGGGT TACATCGAAC 6420 - TGGATCTCAA CAGCGGTAAG ATCCTTGAGA GTTTTCGCCC crAA~iAAcGT TTTCCAATGA 6480 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 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 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 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:

CACTCATTAG GrAcccrAGG CTTTACACTT TATGCTTCCG G~lCGlATGT TGTGTGGAAT 180 TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGllGlCC 300 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 W O 95/12682 ~ 17 2 ~15 PCT~US94/12198 GA~ArArAAC AACGGGCCGC GCGCTCGCGG CCGCTTCCAC CGCTGGGGAG GGCGTGCTCT 1500 CCGGCGCACC GCGrAGACGC GGACGCGGCG GGCCTGGTTG CGTACTACCA GGCCAGGTTC 1680 TTTCTATCAT TACTTCCTCA CATGTTGGAG GCAlll-~LC TCC~l~.G~A CTTAATAGCC 1920 TATCTTGCTT TAA111~1~C CCACTCTTGG AGGCTAGGTT 1G~111GGTG GGCTGATGAG 1980 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 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 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 W O 9S/12682 ~ 2 a 1 ~ PCTrUS94112198 '. .. ~ I .
CAAAGTCGTC AGCCATTTCA AATGTTTCCC CTTGCCTTTG CCCCTCGAAA AGllllllGA 3480 A~ArrArACT GGTCTGCAAG GCTCTTGAAT GCAGAATTGC AAGATATCTA ~ LLG 3540 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 TACTCCACCC ATTGACGTCA ATGGAAAGTC CCTATTGGCG TTACTATGGG AArATACGTC 4380 AGTTATGTAA CGCG~AACTC rATA~A~GGG CTATGAACTA ATr-ArCCCGT AATTGATTAC 4500 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 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 W O95/12682 2 1 7 ~ 8 1 ~ PCTrUS94/12198 CATAGCGGTC CACGTTGACG TTGCGCGCAC CTCTGGCCTG CGGGACGCCA T~lllllCCT 5700 'lc~lLllAcA ACGTCGTGAC TGGGAAAACC CTGGCGTTAC CCAACTTAAT CGCCTTGCAG 5820 15 AACAGTTGCG CAGCCTGAAT GGCGAATGGC GCCTGATGCG GTAllLl~lC CTTACGCATC 5940 AGTTAAGCCA Gccccr~AcAr CCGCCAACAr CCGCTGACGC GCCCTGACGG G~ lGC 6060 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 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 W O95/12682 2 i 7 2 ~ 1 S ` PC IrUS94112198 ACACCGAACT ~A~AT~CCTA CAGCGTGAGC TATGAGAAAG CGCCACGCTT CCCGAAGGGA 7800 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 (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 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 rAAA-rArA~Ac AACGGGCCGC GCGCTCGCGG CCG~llCCAC CGCTGGGGAG GGCGTGCTCT 1500 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 CTATCTTACA CTTTAC~AAT CCTTGTGGTA CTATGGCTAC ACCCTCTCTG TTGCAAGTGG 3000 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 36 CCGCGTAGAG cGcrAr~rArA- GCGAGCATCG rArGCGCr-AG CGAGGCCATG GTCGAAGCTT 3840 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 W O 95/12682 2 1~ 2 8 1 5 PCTrUS94/12198 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 GGCCCCGCCG c~A-AGrAr~Gc CGCCA~ACAA AAAAATGCGG CGCCCGCTCT GCGCGGCGCT 5400 CGCCGAAGCC AGGGCAGCAC ~r~ACGCCC GATACGAAAT C~.AAGAGTGG GAA~ATGGTGG 5520 TCGGAGCCGG GCCGGCCGTG rArACGTTCA CCATCCGCTG CCTCGGGCCG CGGGGCATTG 5580 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 W O95/12682 ~ 17 2 ~ 1~ PCTrUS94/12198 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:
.

~ W O95/126R2 2 1 7 2 8 1 5 PCTrUS94112198

5 CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180 TGTGAGCGGA TAArAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG CCAAGCTAGC 240 GTCCGCGTCC CCAACTCCGC GCr.AArACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420 GCCr~AAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600 CTGTTGCCCG CGTGCCTGCT GGCCGCCr~C CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660 25 GGGGAAGCGG CTGCCCCllC TGCCGCCGCG GCCGCGGTTG CTCGGCTTTG CGTTTGCCCC 780 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 rAAAT~TcGc AGTTTCGATA TAGGTGACAG ACGATATGAG GCTATATCGC Cr~TAr~AGGC 2220 TTAC'CrA~A~ TATTCATTGG TTATATAGcA TAAATCAATA TTGGCTATTG GC¢ATTGCAT 2340 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 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 CCTGGCTTTC GCCCTGCTCT GCC,lGCC~lG GACTCAGGTG GTGGGCGCCT TCCCAGGGAT 3180 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 _ W 095/12682 ~ ~ 7 2 ~1 5 PCT~US94/12198 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 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 ~ 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 W O95/12682 2 i 7 2 ~ 1 ~ PCTrUS94112198 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 GGCAAGAGCA ACTCGGTCGC CGCATACACT ATTCTCAGAA TGA~llG~.ll GAGTACTCAC 6660 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 ATTGGTAACT GTrAGAcr-AA GTTTACTCAT ATATACTTTA GATTGATTTA AAACTTCATT 7260 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 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 GTCCGCGTCC CCAACTCCGC GCr~AAr~ACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420 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 TCCTCGCGGC GTCTGCGCGA TGCGCCGCAG CCTCGCACGG GAGCGCACGC GC¢CGGCGGG 1620 CCTGCAGTGA ATAATAAAAT G~ CCr.AAATA~G CGTTTGAGAT llCl~lCCCG 1920 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 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 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 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 GGAAGTTCAA TACAAr~rTG CTAAACGGAC CGGCCTTCCA GATGGTATGC CCTATAGGAT 3600 ~l~lGlATAA GAGGTCCAAA CCATTCCCTT ATAGACAAGG TTGTATCACC ~AAA~-AACTC 3720 TGGGGGAGGA TCTCTATAAC TGTGATCTTG GAGGGAATTG GA~lL~,l~lG ACTGGGGACC 3780 15 AAAGTGAGGG GTTGCCACAC ~ACCCCATCG GCAAGTGTAG GTT~.AArAAT GAGACTGGCT 3900 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 rAArATAcGT GCTCTGGTTA CTGGTAACAT ACATGGTCCT ATcArAArAA AAGGCCTTGG 4320 TTGAAGTGGT rArATATTTC il~ llAT ACCTACTGCT AAr.ArAGrAC. GCTGTAAAGA 4440 TCAGGCATGC AAGCTCAGAT CCG~l~lGCC TTCTAGTTGC CAGCCATCTG ll~lllGCCC 4560 ClCCCCCGTG C~llC~llGA CCCTGGAAGG TGCCACTCCC ACTGTCCTTT CcTAATAAAA 4620 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 WO 95/12682 PC~US94/12198 , V
CGCC~.AA~CC AGGGCAGCAC AGAGACGCCC GATACGAAAT CGAAGAGTGG GAAATGGTGG 5520 CGTGACTGGG AA-AACCCTGG CGTTACC~AA CTTAATCGCC TTGCAGCACA TCCCCCTTTC 5820 ~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 25 PTAATAATGG ~ AGAC GTCAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT 6240 A111~AT 1111C1AAAT ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA 6300 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 ~1~2gl5 W O95112682 PCTrUS94/12198 C~AAATACTG TCCTTCTAGT GTAGCCGTAG TTAGGCCACC ACTTCAAGAA CTCTGTAGCA 7560 CCGCCTACAT ACCTCGCTCT GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGC~ATAAC. 7620 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 -4~-

Claims

441. An attenuated replicating non-pathogenic virus, for preventing disease caused by Bovine Viral Diarrhoea Virus (BVDV), which comprises a gene or gene combination from a BVDV virus, which functionally expresses the BVDV
glycoprotein gp53, gp48 or gp25, or any combination thereof.
2. A virus of claim 1, which is selected from attenuated Bovine Herpes Virus type 1 (BHV-1), attenuated adenoviruses, attenuated bovine mammillitis virus, attenuated bovine papillomavirus or attenuated pseudorabies virus.
3. A virus of claim 1 or claim 2, which is attenuated because the thymidine kinase (tk) gene is non-functional.
4. A virus of claim 3, which is attenuated BHV-1.
5. A virus of claim 4, which contains and expresses the gene that codes for BVDV gp53.
6. A virus of claim 5, where a signal peptide is inserted preceding the gene or gene combination that codes for gp53 in BHV-1.
7. A virus of claim 5 or claim 6, where the gene that codes for gp53 is inserted into the inactivated tk gene site.
8. A virus of claim 7, where the gene or gene combination comprises a recombined plasmid with intact viral DNA, said plasmid comprising:
a) a BHV-1 genomic DNA fragment containing the tk gene and having a deletion to the tk gene;
b) a promoter/polyadenylation signal inserted in the tk gene deletion; and c) a signal peptide gene sequence preceding a gp53 gene or gene combination, all of which is inserted between the promoter and the polyadenylation signal.
9. A virus of claim 8, where the signal peptide gene sequence is any of the thirty-nine examples of well-characterised signal peptide sequences found in Perlman et al, J. Mol. Biol., Vol. 167:391-409 (1983).

10. A virus of claim 9, where the signal peptide gene sequence is taken from Pseudorabies Virus gIII gene and/or Bovine Growth Hormone.
11. A virus of claim 10, where the plasmid is selected from a) pBHVtkex-a::BGH/p53; b) pBHVtkex-1::gIII/p53; c) pBHVtlex-3::BGH/p53; and d) pBHVtkex-3::gIII/p53.
12. A virus of claim 11, selected from T11-3, T11-6 and T11-8, e.g. T11-6.
13. A virus of claim 7, where the gene or gene combination comprises a recombined plasmid with intact viral DNA, said plasmid comprising:
a) a BHV-1 genomic DNA fragment as defined in claim 8;
b) a promoter/polyadenylation signal as defined in claim 8; and c) a gp53 gene or gene combination inserted between the promoter and the polyadenylation signal.
14. A virus of claim 13, where the plasmid is made from a plasmid having the characteristics of plasmid pHAS4, e.g.
pBHVtkex-3::p53.
15. A virus of claim 14, where the virus is T2-3#3 or T2-2#5.
16. A vaccine for preventing disease caused by BVDV, comprising a virus of claim 1 and a carrier.
17. A vaccine as claimed in claim 16, where the carrier comprises a physiological buffered medium, pH 7.0 to 7.4, containing 2.5 to 15% serum which does not contain antibodies to BHV.
18. A process of preparing a virus of claim 1, comprising:
a) isolation of a functionally expressing gene or gene combination that causes BVDV;
b) inserting the gene or gene combination into a replicating non-pathogenic virus; and c) selecting a live virus that functionally expresses the product of said gene or gene combination.
19. A method of preparing a virus of claim 8, comprising transfecting cells with the plasmid to produce a recombinant virus containing the functional gene or gene combination inserted into a live virus that does not cause immunosuppression in the usual host and expresses the functional gene or gene combination.
20. Use of a virus of any of claims 1 to 15, for the manufacture of a medicament for use in immunisation against a disease caused by BVDV.
21. A method of immunising an animal against infectious disease caused by BVDV comprising administering to the animal a pharmaceutically-effective amount of a virus of

claim 1.