Selection of a Less Pathogenic BVDV Strain for the Construction of Avirulent Chimeric Pestivirus

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1 Journl of Bcteriology nd Virology Vol. 40, No. 1 p DOI /jbv Originl Article Selection of Less Pthogenic BVDV Strin for the Construction of Avirulent Chimeric Pestivirus Jejo Kim, Seong-In Lim, Dong-Seob Trk, Je-Young Song nd Byounghn Kim * Ntionl Veterinry Reserch nd Qurntine Service, Anyng, Kore To select less pthogenic bovine virl dirrhe virus (BVDV) strin for the construction of chimeric pestivirus hrboring clssicl swine fever virus (CSFV) E2 gene, five Koren BVDV isoltes (four type 1 isoltes nd type 2 isolte) were evluted for their pthologicl nd biologicl properties with respect to porcine infection. Ech of five groups of 100-dy-old pigs ws inoculted intrnslly with one of the five BVDV isoltes. No clinicl sign or leukopeni ws observed in ny pig throughout the durtion of the experiment, but viruses were detected in blood, nsl dischrges nd postmortem smples using RT-PCR. These results indicted tht lthough the five BVD viruses could infect pigs, they did not cuse cliniclly pprent symptoms. Becuse of its proper infection dynmics shown in this preliminry niml study nd its fst growth rte nd quick CPE in cell culture, one isolte (KD26-1) ws chosen mong the five isoltes to test its virulence nd immunogenic properties in 40-dy-old piglets. Neither clinicl sign nor pthologicl lesion ws observed in 40-dy-old piglets during the course of infection of isolte KD26-1. The first neutrlizing ntibodies were detectble 14 dys post-inocultion (PI) nd incresed to 1:128~1: dys PI. A BVDV specific gene ws detectble by RT-PCR in tonsil, spleen, inguinl lymph node nd brin until 14 dys PI. According to this study, it cn be concluded tht isolte KD26-1 hs little pthologicl effect in pigs nd is cndidte for construction of chimeric pestivirus hrboring CSFV E2 gene. Key Words: Bovine virl dirrhe virus, Clssicl swine fever virus, Animl study, Pigs, Pthologicl effect INTRODUCTION Along with clssicl swine fever virus (CSFV) nd border disese virus (BDV), bovine virl dirrhe virus (BVDV) belongs to the genus Pestivirus in the fmily Flviviride (1). BVDV is the custive gent of bovine virl dirrhe tht is minly found in cttle, but it cn lso present itself in pigs while CSFV infects strictly pigs (2~5). Phylogenic nlysis hs reveled two genotypes of Received: August 25, 2009/ Revised: December 2, 2009 Accepted: December 7, 2009 * Corresponding uthor: Byoung-Hn Kim. Ntionl Veterinry Reserch nd Qurntine Service, 480, Anyng 6 dong, Mnn-gu, Anyng City, Gyeonggi-do, , Kore. Phone: , Fx: e-mil: kimbh@nvrqs.go.kr BVDV, type 1 nd type 2 (6). Regrdless of genotype, the virus cn lso be divided into two biotypes, cytopthogenic (CP) or non-cytopthogenic (NCP), depending on the cellulr outcome of infection (1, 7~9). Becuse BVDV nd CSFV re of the sme genus, their serology is littered with cross-rections. As result, pigs infected with BVDV hve often misled in-field serosurveillnce of CSFV infection (2, 10~12). It hs been previously demonstrted tht experimentl inocultion of BVDV in pigs cn interfere with, or even prevent, infection of virulent CSFV (13). Interestingly, the clinicl symptoms of pigs infected with BVDV vried from bortion to inpprent infection (3~5, 14, 15). Recently, chimeric pestiviruses hve been constructed using reverse genetics s cndidtes for vccines ginst clssicl swine fever (CSF), nd these chimeric viruses lso re puttive mrker vccines for the DIVA (differentiting 39

2 40 J Kim, et l. Tble 1. The properties of five BVDV isoltes used in niml experiments BVD isolte Origin CPE (dpi ) Genotype b KD26-1 Dirrhe 3~ Persistent infection 4~ Respirtory infection 6~ Dirrhe 4~ Dirrhe 4~5 1 dpi: the number of dys fter which more thn 80% cell deth hd occurred fter inocultion with 0.1 MOI of ech virus b Genotype determined by RT-PCR (19) infected from vccinted individuls) strtegy (16~18). To develop virulent chimeric pestivirus for the CSFV mrker vccine using BVDV bckbone, it is prerequisite to test sfety of the cndidte BVDV in pigs. Therefore, we decided to evlute virulence nd immunogenicity of the BVDV Koren isoltes in pigs nd to select one isolte s cndidte for the construction of mrker vccine ginst CSF. In the present study, we demonstrted tht the five Koren BVDV isoltes exhibited wek or no pthogenicity to pigs. We lso selected BVDV isolte to be used for the construction of chimeric pestivirus. MATERIALS AND METHODS Cells nd viruses Mdin-Drby bovine kidney (MDBK) cells were propgted in lph miniml essentil medium (lph MEM; Gibco-BRL, Githersburg, MD, USA) supplemented with sodium pyruvte, non-essentil mino cids, 5% inctivted horse serum (HS) nd ntibiotics. Cells were mintined t 37 with 5% CO 2. Four isoltes (KD26-1, 32669, 95589, 32527) of BVDV type 1 nd one isolte (95002) of BVDV type 2 were prepred for inocultion of pigs. Isoltes of BVDV were recovered from cttle with dirrhe or respirtory symptoms in Kore between 1994 nd Detils of ech isolte re presented in Tble 1. For infection of MDBK cells, virus, diluted in lph MEM, ws dsorbed for 1 h t 37, nd then the inoculum ws removed nd replced with fresh lph MEM-HS. Cultures were incubted t 37 for 3 to 6 dys until cytopthic effect (CPE) ws observed. Virus stocks were prepred by three freeze-thw cycles of infected cells in culture medium nd clrified by centrifugtion t 1000 g for 10 min. Animl experiments Two sets of experiments in swine were performed in order to nlyze the five isoltes' chrcteristics of infection. In the first experiment, virl pthogenicity ws exmined using dy-old pigs. Pigs were seprted into five groups corresponding to the five different virus isoltes; ech group consisted of six inoculted pigs nd two sentinels. Four dys before inocultion, blood ws collected to confirm tht every pig ws negtive for BVDV specific ntigen nd ntibody. The titers of KD26-1, 32669, 95002, 95589, nd inoculnts were , , , , nd TCID 50 /ml, respectively. Pigs were inoculted intrnslly with 1 ml of inoculnts. Heprinized blood smples nd nsl swbs were collected t 0, 1, 2, 3, 4, 5, 7, 9 nd 11 dys post-inocultion (PI) to check for BVDV ntigen by RT-PCR. Additionlly, white blood cell count ws performed. Rndomly selected pigs were euthnized t 7, 9 nd 10 dys PI for necropsy to check for gross lesions while tissues from tonsil, bronchil lymph node, lung, spleen, mesenteric lymph node, nd ileum were collected for histopthology nd to probe for the presence of BVDV ntigen by RT-PCR. In the second experiment, fifteen 40-dy-old pigs were used to re-exmine the virulence of the KD26-1 isolte. Pigs were seprted into two groups. Ech of 10 pigs ws intrmusculrly inoculted with TCID 50 of KD26-1 while five pigs s sentinels were housed together with inoculted pigs. Blood smples nd nsl swbs were collected t 0, 3, 7, 14, 21 nd 28 dys PI. Two inoculted pigs nd one sentinel were rndomly selected nd euthnized for necropsy t 3, 7, 14, 21 nd 28 dys PI. After necropsy, tissues from tonsil, lung, hert, spleen, liver, kidney, mesenteric lymph node, ileum, cecum, inguinl lymph node, nd brin were collected nd exmined for BVDV ntigen by RT-PCR nd for ny gross nd histopthologicl lesions tht might hve resulted from BVDV infection.

3 Selection of Less Pthogenic BVDV 41 RNA extrction nd RT-PCR RNA ws extrcted from 300 μl of virus suspension, heprinized blood nd nsl swbs using RNesy Miniprep kit (Qigen, Hilden, Germny) ccording to the mnufcturer's instructions. For RNA extrction from tissues, tissue smples were wshed three times with utoclved phosphte-buffered sline (PBS) nd 1 g of tissue ws removed nd ground with se-snd in mortr. Ground tissue ws mixed with 9 ml of lph-mem contining ntibiotics. After centrifugtion t 1,000 g for 10 min, 300 μl liquots of superntnt were used for RNA extrction. RNA ws eluted in 50 μl of RNse-free wter nd stored t -70 until use. Estblished methods described by Gilbert et l. (19) were slightly modified to crry out cdna synthesis nd RT-PCR for moleculr detection nd genotyping. Briefly, first-strnd cdna ws synthesized using the One Step RT-PCR kit (Qigen) in volume of 25 μl contining 5 μl of templte RNA, 5 μl of 5 RT-PCR buffer, 1 μl of dntps (10 mm ech), 1 μl of enzyme mix, 0.5 μl of forwrd nd reverse primers (5 pmol ech) nd 12 μl of RNse-free wter. The rection mixture ws incubted t 42 for 30 min nd then subjected to the following cycles: 94 for 15 min followed by 25 cycles t 94 for 20 s, 50 for 30 s nd 72 for 30 s. A single 5 min extension step t 72 cpped the mplifiction process. For nested PCR, Qigen Tq PCR Mster Mix Kit ws used ccording to the mnufcturer's instructions. The PCR ws crried out in 25 μl volumes contining 1 μl of cdna, 12.5 μl of Tq PCR mster mix, 1 μl of nested forwrd nd reverse primers (5 pmol ech), nd 10 μl of RNse-free wter, nd the rection mixture ws subjected to the following cycles: 97 for 15 min followed by 30 cycles t 94 for 20 s, 50 for 30 s nd 72 for 30 s. A single 5 min extension step t 72 ws gin used to cp the mplifiction process. The PCR products were nlyzed by electrophoresis on 1% grose gel. Virus neutrliztion test To detect BVDV-specific neutrlizing ntibodies, virus neutrliztion test (VNT) ws crried out ccording to the stndrd mnul of the OIE (20) with flt-bottomed 96-well microtiter pltes nd MDBK cells. The ser were inctivted for 30 min t 56. Briefly, 50 μl of porcine serum ws serilly diluted two-fold in 50 μl of lph MEM. A cytopthogenic BVDV (KD26-1 isolte) suspension (200 TCID 50 /50 μl) ws dded to the wells nd mixed on microplte shker for 20 s. The pltes were then incubted t 37 for 1 h nd 50 μl of growth medium contining cells/ml ws dded to ech well. The pltes were then incubted for 3 to 4 dys in n incubtor contining 5% CO 2. The cells were exmined for CPE under low-mgnifiction microscopy nd trnslted to virus neutrliztion (VN) titer. RESULTS Virus chrcteristics Five BVDV isoltes were ctegorized into two genetic types using the PCR method suggested by Gilbert et l. (19). Among the five, only isolte ws confirmed by PCR to be type 2. Genetic differences observed for 32669, nd KD26-1 re in greement with prior sequencing dt (21). BVDV type 1 isoltes, which were of the CP type, cused more thn 80% cell deth in MDBK cultures within 4 dys fter cultivtion. The CP biotype of ws not confirmed until 4 dys fter cultivtion. The titer of ech isolte did not rise with seril pssges, but rther fluctuted up nd down. Pthogenicity of five BVDV isoltes in 100-dy-old pigs No significnt difference between inoculted pigs nd sentinels ws found with respect to the leukocyte count fter virus inocultion. Most counts fell within norml rnges (Fig. 1). No leukopeni occurred in ny of the inoculted or contct pigs, nor were ny clinicl signs of pthogenic pestivirus observed in this niml experiment. And ny gross pthologicl chnges nd histopthologicl findings were not found in euthnized pigs. Initil virus detection in nsl swbs vried from one

4 42 J Kim, et l. dy to three dys PI (Tble 2) while virl ntigens were detected in RNA purified from blood smples s of three dys PI (Tble 3). In the inoculted pigs, ntigen ws detectble in nsl swbs nd blood smples up to seven dys PI. In the sentinel pigs, virl ntigen ws detected initilly from nsl swbs t two dys PI. Even though BVD ntigens were detected in nsl swbs of inoculted pigs nd sentinels in the groups inoculted with isolte KD26-1, 95589, nd 32527, the positive ptterns of blood Leukocyte Leukocyte Counts counts (10 3 cells/mm 3 cells/mm 3 ) 3 ) Dys Dys Post post inocultion Inocultion KD26-1, 32669, 95589, 32527, 95002, contct pigs Figure 1. Leukocyte counts (10 3 cells/mm 3 ) during the course of intrnsl infection in the first niml study. Men leukocyte counts in pigs inoculted with isoltes KD26-1, 32669, 95589, nd nd those of contct pigs re given. Stndrd devitions re shown s error brs. smples were not consistent with those of nsl swbs. In the group inoculted with KD26-1, BVDV ntigens were detected in inoculted pigs nd sentinel. But in the group inoculted with isolte 95589, positive results were shown only in inoculted pigs, nd in the group inoculted with isolte 32527, those were shown only in sentinel. Though no symptom or clinicl sign ws pprent in ny niml, including sentinels, BVDV ntigens were detected in the orgns of ll inoculted groups except those inoculted with isolte (Tble 4). In the cse of the group inoculted with isolte 32669, BVDV ntigens were detected in orgns of four inoculted pigs nd sentinel. In the group inoculted with KD26-1, virl ntigens were detected from tonsil, bronchil lymph node, nd lung of inoculted pigs t nine nd eleven dys PI. And in the group inoculted with isolte nd 32527, virl ntigen were detected from tonsil of inoculted pigs. However, spleens, mesenteric lymph nodes, nd ileums collected from ll groups were negtive by RT-PCR. In summry, pigs inoculted with BVDV type 1 were 42%, 38%, nd 33% virl ntigen positive in nsl, blood nd tissue smples, respectively, nd pigs inoculted with BVDV type 2 exhibited 67%, 17%, nd 0% virl ntigen positive rtes in nsl, blood nd tissues, respectively (Tble 5). Tble 2. RT-PCR results for detection of virl ntigen from nsl swbs of five pig groups during the course of intrnsl infection Dys post inocultion Group Inoculted virus I KD26-1 0/6 0/6 0/6 2/6 0/6 1/6 0/6 2/6 0/4 0/2 Contct 0/2 0/2 0/2 0/2 0/2 0/2 1/2 1/2 0/1 1/1 II /6 0/6 1/6 1/6 0/6 0/6 0/6 0/6 0/4 0/2 Contct 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/1 0/1 III /6 0/6 2/6 0/6 0/6 1/6 1/6 2/6 0/4 0/2 Contct 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/1 0/1 IV /6 0/6 0/6 0/6 1/6 0/6 1/6 1/6 0/4 0/2 Contct 0/2 0/2 0/2 1/2 1/2 0/2 1/2 0/2 0/1 0/1 V /6 0/6 0/6 0/6 2/6 1/6 1/6 0/6 0/4 0/2 Contct 0/2 0/2 0/2 0/2 0/2 0/2 1/2 1/2 1/1 0/1 Number of positive pigs / Totl number of pigs

5 Selection of Less Pthogenic BVDV 43 Tble 3. RT-PCR results for detection of virl ntigen from blood smples of five pig groups during the course of intrnsl infection Dys post inocultion Group Inoculted virus I KD26-1 0/6 0/6 0/6 0/6 1/6 1/6 1/6 0/6 0/4 0/2 Contct 0/2 0/2 0/2 0/2 0/2 0/2 1/2 0/2 0/1 0/1 II /6 0/6 0/6 0/6 0/6 1/6 1/6 1/6 0/4 0/2 Contct 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/1 0/1 III /6 0/6 0/6 0/6 0/6 0/6 1/6 0/6 0/4 0/2 Contct 0/2 0/2 0/2 0/2 0/2 1/2 0/2 0/2 0/1 0/1 IV /6 0/6 0/6 0/6 0/6 2/6 2/6 0/6 0/4 0/2 Contct 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/1 0/1 V /6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/4 0/2 Contct 0/2 0/2 0/2 0/2 0/2 0/2 0/2 1/2 0/1 0/1 Number of positive pigs / Totl number of pigs Tble 4. RT-PCR detection of virl ntigen from tissues smples of five pig groups t 7, 9, nd 11 dys PI Group I II III IV V Inoculted virus 7 dpi 9 dpi 11 dpi KD b T c, B d, L e T, B, L Contct - NP f T, 2B g, L L L Contct T NP Contct - NP T - Contct - NP T Contct - NP - dpi: dys post inocultion, b -: negtive in tissues, c T: positive in tonsil, d B: positive in bronchil lymph node, e L: positive in lung, f NP: not performed, g 2B: Two pigs were positive in bronchil lymph node. Animl experiment with KD26-1 isolte in 40-dy-old piglets The second niml experiment with KD26-1 lso filed to revel ny indiction of leukopeni. In the course of infection, leukocyte counts were scttered within norml rnges with one exception; the pig mong contct pigs presented leukocyte concentrtion of cells Tble 5. Summry of virl ntigen positive pigs (%) in thirty 100- dy-old pigs inoculted intrnslly with BVDV type 1 nd 2 Virus type Antigen positive smples Nsl swbs Blood Tissues /mm 3 t 14 dys PI nd ws dignosed s Glässer's disese infection by clinicl symptoms (Fig. 2). No clinicl signs ssocited with CSFV infection were observed during the course of infection. According to serologicl dt for the second experiment (Fig. 3), the BVDV specific ntibody ws not detected until seven dys PI. The VN titer rnged from 1:4 to 1:16 t fourteen dys PI. By twenty-eight dys PI, it hd reched to 1:128 nd 1:256. Virl ntigen ws initilly detected in orgns t three dys PI (Tble 6). The orgns tht were positive for BVDV by RT-PCR were tonsil, spleen, inguinl lymph node nd brin. The ntigen ws detected up to fourteen dys PI. DISCUSSION No. of pigs tested Type 1 10 (42) 9 (38) 8 (33) 24 (100) Type 2 4 (67) 1 (17) 0 ( 0) 6 (100) This study ws conducted in order to select BVDV

6 44 J Kim, et l. KD26-1 inoculted pigs, Contct pigs Figure 2. Leukocyte counts (10 3 /mm 3 ) of pigs fter intrmusculr inocultion with KD26-1 ( TCID 50 /hed). Men leukocyte counts in pigs inoculted with isoltes KD26-1 nd those of contct pigs re given. Stndrd devitions re shown s error brs. KD26-1 inoculted pigs, Contct pigs Figure 3. Virus neutrliztion titers of ser from pigs fter intrmusculr inocultion with KD26-1 ( TCID 50 /hed). Men virus neutrliztion titers in pigs inoculted with isoltes KD26-1 nd those of contct pigs re given. Stndrd devitions re shown s error brs. Tble 6. RT-PCR results for detection of virl ntigen from orgns of euthnized pigs fter intrmusculr inocultion with KD26-1 ( TCID 50 /hed) Orgn KD26-1 inoculted pigs Contct pigs 3 d 7 d 14 d 21 d 28 d 3 d 7 d 14 d 21 d 28 d Tonsil 1/2 b 1/2 1/2 0/2 0/2 0/1 0/1 0/1 0/1 0/1 Lung 0/2 0/2 0/2 0/2 0/2 0/1 0/1 0/1 0/1 0/1 Hert 0/2 0/2 0/2 0/2 0/2 0/1 0/1 0/1 0/1 0/1 Spleen 0/2 1/2 0/2 0/2 0/2 0/1 0/1 0/1 0/1 0/1 Liver 0/2 0/2 0/2 0/2 0/2 0/1 0/1 0/1 0/1 0/1 Kidney 0/2 0/2 0/2 0/2 0/2 0/1 0/1 0/1 0/1 0/1 Mesenteric LN 0/2 0/2 0/2 0/2 0/2 0/1 0/1 0/1 0/1 0/1 Ileum 0/2 0/2 0/2 0/2 0/2 0/1 0/1 0/1 0/1 0/1 Cecum 0/2 0/2 0/2 0/2 0/2 0/1 0/1 0/1 0/1 0/1 Inguinl LN 0/2 0/2 1/2 0/2 0/2 0/1 0/1 0/1 0/1 0/1 Brin 1/2 0/2 0/2 0/2 0/2 0/1 0/1 0/1 0/1 0/1 d: dys post-inocultion, b Number of pigs positive to virl ntigen / Number of pigs tested strin tht could be used for the construction of recombinnt pestiviruses. At first, it ws essentil to remove possibly pthogenic strins mong our five isoltes. Clinicl symptoms nd gross nd histopthologicl chnges were probed becuse CSF-like pthogenic BVDV isoltes hve been reported (4, 5, 15), lthough BVDV normlly cuses symptomtic infection in pigs (2, 3, 13, 14). Fortuntely, no pprent clinicl sign of BVDV infection ws presented during the course of infection, nor ws ny histopthologicl chnge detected. Next, we exmined the infection dynmics of BVDV virl vector cndidte in pigs. It ws greed tht suitble virl vector for construction of BVDV/CSFV chimeric vccine should infect swine but dispper from tissues s soon s possible, nd it should lck the bility to trnsmit the virus between pigs. According to the ntigen positive

7 Selection of Less Pthogenic BVDV 45 rte of nsl swbs, blood smples, nd tissues in the first intrnsl pig inocultion experiment, it seemed tht five Koren BVDV isoltes did not infect pigs effectively. However, the intrnsl route of infection to pigs might cuse loss of significnt mount of inoculnts. And, due to the host specificity nd the low titer of inoculnts, BVDV in pigs might not lst long enough to propgte over the detection limit of RT-PCR. In the cse of infection with isolte KD26-1, ll inoculted pigs in the first niml experiment were not positive by RT-PCR, but VN results in the second niml experiment indicted tht serologicl response to BVDV rouse effectively in pigs. Therefore, the RT-PCR results of the first niml experiment my men tht most of the BVDV isoltes were wekly infectious to pigs by intrnsl inocultion. Most isoltes exhibited similr dynmics during the course of infection except for the nd isoltes. We considered the isolte to be lest desirble mong the five isoltes since it might be more infectious in pigs thn the others, bsed on ntigen detection by the RT-PCR from tissues. In contrst, the isolte, which is BVDV type 2, seemed to be too non-infectious to be vector. Furthermore, due to its slow nd mbiguous CPE in cell culture nd the derth of prevlence dt regrding BVDV type 2 infection in Kore, it ws inpproprite to develop into virl vector for CSF control. Among the remining three isoltes, KD26-1 ws chosen s cndidte virl vector becuse of its fst growth rte nd quick CPE in cell culture. To ese ny concern of pthogenicity in younger pigs, nd to obtin serologicl dt fter inocultion, we conducted the second inocultion experiment with KD26-1 in piglets. Chrcteriztion of BVDV nturlly isolted from pigs nd experimentl BVDV infection of pigs hd been conducted for decdes (2~5, 11, 13~15, 22). Persistent virus shedding ws demonstrted in congenitlly infected pigs tht were experimentlly induced with ruminnt pestiviruses (22). But presently it is ccepted tht BVDV infection in pig is self-limiting (13). In our first niml experiment, the five BVDV isoltes seemed to be ctively trnsmitted between pigs. However, the trnsmission between pigs could be the result of contmintion from remining inoculnt, which hd been dministered intrnslly. This scenrio is supported by the fct tht ntigen ws detected one dy PI in nsl swbs of inoculted pigs from groups nd nd two dys PI in the KD26-1 group, while nsl swb from contct pigs in the group were positive two dys PI. Yet, the second niml experiment, in which inoculnt ws dministered intrmusculrly, filed to demonstrte ny cross infection of KD26-1 between pigs despite inoculting with 500-fold higher titer of KD26-1 thn in the first experiment. Therefore, limited trnsmission of BVDV ws demonstrted mong pigs with respect to infection with the KD26-1 isolte. However, when pigs re infected with immunosuppressive diseses such s porcine reproductive nd respirtory syndrome (PRRS) or post-wening multisystemic wsting syndrome (PMWS), the possibility of its trnsmission between pigs remins question for further study. Interestingly, the route of infection could explin the vrible RT-PCR results observed for tissue smples in both experiments. We ttempted to use RT-PCR to detect virl ntigen from tonsil, bronchil lymph node, lung, spleen, mesenteric lymph node nd ileum of ll euthnized pigs in the first experiment; however, virl ntigen ws only detected in tonsil, bronchil lymph node nd lung, even though lung tissue from the second experiment filed to revel virl ntigen. As BVDV is very closely relted to CSFV in serology, fers of cross rection between these often led to multiple differentil dignoses in order to void errors (1, 2, 10, 11, 13). In the second experiment, VN ntibodies to BVDV t 28 dys PI rnged from 1:128 to 1:256, which might explin elimintion of the virus from piglets fter only 14 dys PI; the VN titer to CSFV ws negtive during the course of the second experiment. Thus, we cn stte tht serologicl cross rection between BVDV nd CSFV ws not induced by KD26-1 up to 28 dys PI. In the present study, we inoculted pigs with BVDV in order to select virl vector for development of live mrker vccine. Though BVDV could be cndidte vccine to protect pigs from CSFV infection, the min concern of BVDV infection in swine is interference with serologicl surveillnce nd the msking of virulent CSFV

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