Detection of Bovine Viral Diarrhea Virus Genome in Leukocytes from Persistently Infected Cattle by RNA-cDNA Hybridization J. Jensen, J. Aiken and R.D. Schultz
ABSTRACT A bovine viral diarrhea virus (BVDV) cDNA library was constructed. One cloned complementary DNA sequence was used as a probe to detect BVDV RNA by hybridization in infected cell cultures and in mononuclear leukocytes from persistently infected cattle by dot blot and in situ hybridization. The cDNA probe hybridized with all cytopathic and noncytopathic BVDV isolates tested. The hybridization results were consistent with results obtained using conventional subculturing and immunofluorescent staining methods and by inoculation of seronegative test cattle.
RESUME Cette experience visait a etablir une banque d'ADN complementaires specifiques au virus de la diarrhee virale bovine (BVD-MD). Une sequence d'ADN complementaire clonee fut utilisee comme sonde pour la detection de l'ARN genomique du virus du BVD-MD dans les cultures cellulaires infectees experimentalement et les leucocytes mononuclees de bovins porteurs asymptomatique de la maladie. Les techniques dhybridation sur filtres de nitrocellulose ou d'hybridation in situ ont ete evaluees. Des reactions dhybridation positives ont ete obtenues avec toutes les souches cytopathogenes et non-cytopathogenes du virus. Les resultats obtenus par la technique dhybridation etaient
compatibles a ceux d'immunofluorescence indirecte et, des essais d'inoculation experimentale de bovins seronegatifs.
cattle and to develop a rapid method for screening large numbers of blood samples for BVDV. MATERIALS AND METHODS
INTRODUCTION Bovine viral diarrhea virus (BVDV) is ubiquitous among the world's cattle population (1,2). Infection with BVDV has different clinical manifestations depending on the age and immune status of the animal at the time of infection and the biotype of the infecting BVDV (1-4). Infection of the bovine fetus in utero with noncytopathic (NCP) BVDV can result in persistent infection (1,2,4). Persistently infected (PI) cattle are the major disseminators of BVDV in the cattle population and are the animals which succumb to mucosal disease (MD) (3,4). Most often, PI cattle are clinically normal and are identified by isolation of NCP BVDV from the animal's serum or peripheral blood mononuclear leukocytes (PBML) after amplification of the virus by one or more passages in cell culture. While technologies such as nucleic acid hybridization have been used for rapid detection of other cattle viruses directly in infected tissues (5,6), there is little published information regarding methods for direct detection of BVDV in PBML from PI cattle. This study was designed to determine if RNA-cDNA hybridization could be used for direct detection of BVDV genome in PBML from PI
VIRUSES AND CELL CULTURES
Bovine viral diarrhea virus cytopathic (CP) strains NADL and Singer and bovine herpesvirus-I (BHV-1) were obtained from the stock virus collection at the James A. Baker Institute for Animal Health, Cornell University. Oregon C24V CP and NYI NCP were obtained from Dr. S.R. Bolin, National Animal Disease Center, Ames, Iowa. Bluetongue virus-17 (BTV-17) was obtained from Dr. A. Luedke, Arthopod-borne Animal Disease Research Laboratory, USDA-ARS, Laramie, Wyoming. Primary bovine testicle cells, obtained from calves less than two months old at a local abattoir, were used to propagate BVDV and for virus isolation and serum neutralization (SN) tests. Cells were grown in minimal essential media (MEM) with 10% fetal bovine serum (FBS) (Sigma, St. Louis, Missouri) and 1% penicillinstreptomycin and were determined negative for NCP BVDV by immunofluorescent staining. Fetal bovine serum was determined negative for NCP BVDV by inoculation of a third trimester bovine fetus as previously published (7). MONOCLONAL ANTIBODIES
Monoclonal antibodies (MAb) CA1 and CA-80 were obtained from Dr.
Department of Pathobiological Sciences (Jensen, Schultz) School of Veterinary Medicine, and Department of Veterinary Science (Aiken), University of Wisconsin-Madison, Madison, Wisconsin 53706. This work was supported in part by grants from Eastern Artificial Insemination Cooperative, Ithaca, New York and the Large Animal Research Fund, School of Veterinary Medicine, University of Wisconsin. Submitted August 14, 1989.
256
Can J Vet Res 1990; 54: 256-259
S.R. Bolin. These MAb have been and first strand cDNA synthesized demonstrated to react with serologi- using M-MLV reverse transcriptase cally diverse BVDV variants (8). (Bethesda Research Labs (BRL), Gaithersburg, Maryland). Second EXPERIMENTAL ANIMALS strand synthesis was performed using Care and housing of cattle was in Escherichia coli DNA polymerase I accordance with National Institute of and E. coli RNase H (BRL) according Health Guidelines (9). Six cattle to the manufacturer's instructions. persistently infected with BVDV and Double-stranded cDNA was filled in 46 non-PI cattle were used. Cattle using T4 polymerase and blunt ligated confirmed as PI were viremic for into the Sma I site of the plasmid BVDV for a minimum of a two-month pGEMT"-blue (Promega Biotec Madiperiod as determined by isolation of son, Wisconsin). Ligated plasmid NCP BVDV from PBML and by the DNA was then transformed into E. animal inoculation test (7). For coli strain TB1 in the presence of Xisolation of NCP BVDV, PBML were Gal. Twelve individual clones were harvested after centrifugation over randomly chosen from the library, Ficoll-hypaque (p = 1.080)(10) and plasmid DNA extracted by the boiling passaged in bovine testicle cells (three method (12), inserts removed by passages at 3-5 day intervals). Isola- digestion with Eco RI-Bam HI (BRL) tion of NCP BVDV was confirmed by and insert size estimated by gel indirect fluorescent antibody (IFA) electrophoresis in 1.0% agarose. staining of infected cell cultures using Complementary DNA inserts in these MAb CA-1 or CA-80 tissue culture clones ranged in size from 300-2000 fluids (diluted 1:2) as primary antib- base pairs. ody and rabbit antimouse IgG(l:10) (ICN, Lisle, Illinois) as a second RNA-cDNA HYBRIDIZATION antibody (8). For animal inoculation, Bovine viral diarrhea virus-infected 10 PBML, isolated as described and uninfected cell monolayers were above, were inoculated into seronega- rinsed with cold buffer (0.01 M Tristive test cattle. Serum neutralizing HCI, pH 8.0-2 mM MgCl2-.0I M antibody titers to BVDV were deter- NaCl) the cells resuspended in cold mined four weeks postinoculation by buffer by scraping, the mixture the varying serum-constant antigen adjusted to 0.5% Nonidet P-40 method using 100 TCID50 BVDV (Sigma, St. Louis, Missouri) -1% (NADL). deoxycholate (Fisher Scientific, Fair Lawn, New Jersey) and cells lysed on CONSTRUCTION OF BVDV cDNA ice for 5 min. The nuclear pellet was LIBRARY removed by microcentrifugation Bovine viral diarrhea virus (NADL) (12,500 revolutions per min for 5 min). was cultivated in bovine testicle cell Cytoplasmic RNA was extracted from monolayers to 4+ cytopathic effect the supernatant with an equal volume (CPE), the cells freeze-thawed and the of phenol, followed by extraction with supernatant clarified by centrifuga- an equal volume of phenoltion at 8000 x g for 10 min at 4°C. chloroform (1:1) and then with an Polyethylene glycol (PEG) 6000 was equal volume of chloroform, ethanoladded to 7% final concentration and precipitated and redissolved in water. virus precipitated for 6 h at 4°C with Mononuclear leukocytes were isolated stirring. The virus precipitate was by centrifugation over Ficoll hypaque, collected by centrifugation at 14,000 x resuspended in cold lysis buffer and g for 30 min at 4°C, resuspended in cytoplasmic RNA extracted as desTNE (10 mM Tris-HCl(pH 8.0)-100 cribed above for monolayer cells. mM NaCI-I mM EDTA(pH 8.0) and Typically, 1.0 mL of bovine blood layered over a linear 45%-30% potas- yielded 106 mononuclear leukocytes. sium tartrate:glycerol gradient (11). Ribonucleic acid yields were deterFollowing centrifugation at 200,000 x mined spectrophotometrically. g for 3 h at 40C, the viral band was Ribonucleic acid was diluted in 0.44 M harvested and genomic RNA phenol- formaldehyde-20X standard saline extracted. The RNA genome was citrate (SSC) buffer, heated to 60°C randomly primed using an N6 primer for 15 min, blotted onto nitrocellulose (Boehinger Mannheim, W. Germany) presoaked in 20X SSC using a slot blot
apparatus (BRL) and affixed by heating at 800 C for 2 h in vacuo. Following prehybridization at 420C for 2-4 h, heat-denatured nicktranslated 32p labelled cDNA probe was added to the hybridization mixture at 0.1 ,ug/mL (10 cpm/,ug) and hybridization continued at 420C
for 18 h. The prehybridization mixture consisted of 50% deionized formamide, 5X Denhardt solution, 5X SSC and 350 ,ug/ mL denatured salmon sperm DNA. The hybridization mixture in addition contained 10% dextran sulfate. Following hybridization, nitrocellulose was washed in 3X SSC at room temperature (three washes, 15 min per wash) followed by two 30 min washes in 0.1X SSC at 500 C. Autoradiography was done for 4-18 h using Kodak XAR film at -70° C. The modified in situ hybridization was done as described by Paeratakul et al (13). Briefly, 105 cells were blotted onto nitrocellulose using a dot blot apparatus (BRL), fixed in 10% glutaraldehyde (Sigma) for 60 min at 40C and digested with 20 Ag/ mL proteinase K(Boehinger-Mannheim) Prehybridization and hybridization were done as described above for RNA extracts except that dextran sulfate was omitted from the hybridization mixture. Supernatants from infected and uninfected cells were treated with proteinase K (250 ,ug/ mL)-1% sodium dodecyl sulfate (Sigma) for 30 min at 370C (12), the RNA phenol-extracted and hybridization done as described above for extracts from cell monolayers. RESULTS
Approximately 1500 ampicillinresistant, lac Z- transformants were obtained. Recombinant plasmids from 8 of 52 (15%) randomly selected transformants hybridized with RNA extracted from BVDV(NADL)infected cells but not with RNA extracted from uninfected cells. One of these cloned cDNA sequences (clone 3), a cDNA segment 1 kb in length, was chosen for evaluation as a diagnostic probe for BVDV. The results of RNA-cDNA hybridization using clone 3 with RNA extracts from cells infected with serologically divergent (14, 15) CP and 257
NCP BVDV strains are shown in Fig. 1. Clone 3 hybridized with cytopathic strains NADL, Singer and C24V, with NY-1 NCP and with all six NCP BVDV field isolates from our six PI cattle, but not with RNA from uninfected bovine testicle cells or with DNA from cells infected with BHV-1 or RNA from BTV-17 infected cells. The detection limit of hybridization with RNA extracted from BVDV (NADL)-infected cells was about 200 pg; no hybridization signal was found using up to 800 ng of RNA from uninfected cells (data not shown). Using supernatants from cell cultures infected with BVDV NADL and BVDV NY-1 NCP, hybridization signals were obtained at dilutions of 1 x 10-7 and 1 x 10-6 as compared with detection by cell culture at dilutions of l x 10-6 and 2 x 10-5 for BVDV NADL and BVDV NY-1 NCP, respectively. No hybridization signals were obtained using supernatants from uninfected cell cultures. The feasibility of using RNA-cDNA hybridization to detect BVDV genome
Fig. 1. Slot blot hybridization of RNA extracted from cells infected with BVDV NADL (A-1); BVDV C24V CP (A-2); NY-I NCP (A-3); BVDV Singer (A-4); NCP BVDV isolates from PI cattle I through 6 (A5 - A-10); uninfected cell cultures (B-i); BTV-17 (B-2); and DNA extracted from BHV-I infected cells (B-3). Nucleic acid was blotted at 20 ng and hybridized with clone 3.
258
in RNA extracted directly from PBML of PI cattle without prior amplification in cell culture was examined (Fig. 2). RNA was extracted from 107 PBML and blotted at 20 ng. Typically, 107 PBML yielded 0.5 to 1 jig of RNA. Bovine viral diarrhea virus clone 3 hybridized with RNA extracted from the six PI cattle; no hybridization signal was obtained with PBML RNA extracts from control animals. The comparison between RNAcDNA hybridization and conventional methods for detecting persistent BVDV infection in cattle is shown in Table I for the six PI and 46 non-PI cattle from our research herd. Viral RNA was detected by RNA-cDNA hybridization in PBML from all six of the cattle identified as PI by conventional subculturing and/ or animal inoculation. Of the 46 non-PI cattle, 45 were found negative for BVDV by hybridization, immunofluorescent staining of infected cell cultures and the animal inoculation test. One animal was positive for BVDV by hybridization and animal inoculation but negative by immunofluorescent staining. Later specimens taken from this animal were negative for BVDV by hybridization and thus this animal may have been acutely infected with BVDV at the time of the initial sampling.
Fig. 2. Slot blot hybridization of RNA extracted from mononuclear leukocytes of PI cattle 1 through 6 (Ai-6) and six non-PI, nonviremic control cattle (Bi-6) Nucleic acid was blotted at 20 ng (equivalent to RNA from 2-4 x 105 cells) and hybridized with clone 3.
To facilitate screening large numbers of blood specimens for BVDV, we adopted a modified in situ hybridization method (13). Figure 3 shows the results obtained with PBML from PI and control cattle. Bovine viral diarrhea virus RNA was detectable with as few as 105 PBML from PI cattle with no detectable hybridization with PBML from control cattle.
DISCUSSION The development of procedures for the rapid detection of viral infections is an active area of diagnostic research. This study was designed to test the feasibility of detecting persistent BVDV infection in cattle by RNAcDNA hybridization. A BVDV (NADL) cDNA library was constructed and one of the cDNA clones, which hybridized with serologically diverse strains of BVDV in cell culture, was used to detect BVDV RNA in PBML from PI cattle. Bovine viral diarrhea virus RNA could be detected using 105 cells from PI cattle by an in situ hybridization method and with RNA extracted from 2-5 x 105 PBML. No hybridization was detectable with PBML from 45 non-PI, nonviremic control cattle. The ability to detect BVDV RNA in PBML from PI cattle probably reflects not only the sensitivity of RNA-cDNA hybridization but also the percentage of BVDV-positive PBML in PI cattle. Other investigators have reported that 0.5-30% of PBML in PI cattle are BVDV antigenpositive as determined by isolation of infectious virus or immunolabelling with polyclonal antiserum to BVDV (16,17). Detection of BVDV antigen in PBML of acutely infected cattle by immunofluorescent staining has also been reported (18). While the percentage of BVDV RNA-positive PBML for the six PI cattle used in this study was not determined, IFA staining of PBML from these cattle using MAb CA-1 and CA-80 showed that 0.5-12% were positive for BVDV antigen (19). While other investigators have reported construction of BVDV cDNA libraries (20,21), the application of RNA-cDNA hybridization to detect persistent infection with BVDV has not been previously reported.
TABLE 1. Identification of persistently infected cattle by conventional subculturing, animal inoculation and RNA-cDNA hybridization Animal
Cell culturea
Animal inoculationa
Hybridizationa
I
+
2 3 4 5 6 7-12 13-51 52
+ + + +
+ +
+ +
+
+
+
+ +
+
ND ND ND
+
+
+
aAnimals were scored positive or negative for BVDV infection by subculturing mononuclear leukocytes in cell culture followed by IFA staining of infected cells or by inoculation of mononuclear leukocytes into seronegative test cattle. Mononuclear leukocytes were examined for BVDV genome by RNA-cDNA hybridization with clone 3 ND = not done
Use of an in situ hybridization method to probe cells fixed on nitrocellulose for BVDV RNA obviates the need for amplification of NCP-BVDV in cell culture and allows large numbers of samples to be examined at one time.
The feasibility of using RNA-cDNA hybridization to detect BVDV in serum or whole blood specimens from PI cattle is currently under investigation in our laboratory.
REFERENCES
Fig. 3 In situ hybridization of mononuclear leukocytes from PI animal I (A-1); PI animal 2 (A-2); and 28 non-PI, nonviremic control cattle (A-3 through C-10). Mononuclear leukocytes (I x 105) were blotted and hybridized with clone 3.
1. RADOSTITS OM, LITTLEJOHNS IR. New concepts in the pathogenesis, diagnosis and control of diseases caused by the bovine viral diarrhea virus. Can Vet J 1988; 29: 513528. 2. PERDRIZET JA, REBHUN WC, DUBOVI EJ, DONIS RO. Bovine virus diarrhea-clinical syndromes in dairy herds. Cornell Vet 1987; 77: 46-74. 3. MALMQUIST WA. Bovine viral diarrheamucosal disease: etiology, pathogenesis and applied immunity. J Am Vet Med Assoc 1968; 152: 763-768. 4. LITTLEJOHNS IR, WALKER KH. Aetiology and pathogenesis of mucosal disease of cattle; current concepts, observation and speculation. Aust Vet J 1985; 62: 101-103. 5. ROY P, RITTER GD, AKASHI H, COLLISSON E, INABA Y. A genetic probe for identifying bluetongue virus infections in vivo and in vitro. J Gen Virol 1985; 66: 1613-1619. 6. DORMAN M, BLAIR CD, COLLINS JK, BEATY BJ. Detection of bovine herpesvirus 1 DNA immobilized on nitrocellulose by hybridization with biotinylated DNA probes. J Clin Microbiol 1985; 22: 990-995. 7. SCHLAFER DH, SCHULTZ RD, SCOTT FW, DUNCAN JR. Bovine fetal inoculations with calf rotavirus. Can J Comp Med 1979; 43: 405-414. 8. BOLIN S, MOENNIG V, GOURLEY NE, RIDPATH J. Monoclonal antibodies with neutralizing activity segregate isolates of bovine viral diarrhea virus into groups. Arch Virol 1988; 99: 117-123.
9. UNITED STATES DEPARTMENT OF HEALTH AND HUMAN SERVICES. Guide for the care and use of laboratory animals, 1985. National Institute of Health Publication No. 85-23. 10. SCHULTZ RD. Assays of cellular immunity. J Am Vet Med Assoc 1982; 181: 11691176. 11. TRENT DW, GRANT JA, ROSEN L, MONATH TP. Genetic variation among dengue-2 viruses of different geographic origin. Virology 1983; 128: 271-284. 12. MANIATIS T, FRITSCH EF, SAMBROOK J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, 1982. 13. PAERATAKUL U, DE STASIO PR, TAYLOR MW. A fast and sensitive method for detecting specific viral RNA in mammalian cells. J Virol 1988; 62: 11321135. 14. CORAPI WV, DONIS RO, DUBOVI EJ. Monoclonal antibody analysis of cytopathic and noncytopathic viruses from fatal bovine viral diarrhea virus infections. J Virol 1988; 62: 2813-2817. 15. MAGER R, MINOCHA HC, MONTPETIT C, CARMAN PS, LECOMTE J. Typing of cytopathic and noncytopathic bovine viral diarrhea virus reference and Canadian field strains using a neutralizing monoclonal antibody. Can J Vet Res 1988; 52: 4245. 16. BOLIN SR, SACKS JM, CROWDER SV. Frequency of association of noncytopathic bovine viral diarrhea virus with mononuclear leukocytes from persistently infected cattle. Am J Vet Res 1987; 48: 1441-1445. 17. BIELEFELDT OHMANN H, RONSHOLT L, BLOCH B. Demonstration of bovine viral diarrhoea virus in peripheral blood mononuclear cells of persistently infected, clinically normal cattle. J Gen Virol 1987; 68: 1971-1982. 18. BEZEK DM, BAKER JC, KANEENE JB. Immunofluorescence of bovine virus diarrhea viral antigen in white blood cells from experimentally infected immunocompetent calves. Can J Vet Res 1988; 52: 288290. 19. JENSEN J, SCHULTZ RD, BOLIN SR. Detection of persistent infection with bovine viral diarrhea virus (BVDV) by immunofluorescent staining using monoclonal antibodies. Presented at American Society for Virology, Annual Meeting, University of Texas, Austin, 1988. 20. COLLET MS, LARSON R, GOLD C, STRICK D, ANDERSON DK, PURCHIO AF. Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus. Virology 1988; 165: 191199. 21. BROCK KV, BRIAN DA, ROUSE BT, POTGEITER LND. Molecular cloning of complementary DNA from a pneumopathic strain of bovine viral diarrhea virus and its diagnostic application. Can J Vet Res 1988; 52: 451457.
259
ABSTRACT A bovine viral diarrhea virus (BVDV) cDNA library was constructed. One cloned complementary DNA sequence was used as a probe to detect BVDV RNA by hybridization in infected cell cultures and in mononuclear leukocytes from persistently infected cattle by dot blot and in situ hybridization. The cDNA probe hybridized with all cytopathic and noncytopathic BVDV isolates tested. The hybridization results were consistent with results obtained using conventional subculturing and immunofluorescent staining methods and by inoculation of seronegative test cattle.
RESUME Cette experience visait a etablir une banque d'ADN complementaires specifiques au virus de la diarrhee virale bovine (BVD-MD). Une sequence d'ADN complementaire clonee fut utilisee comme sonde pour la detection de l'ARN genomique du virus du BVD-MD dans les cultures cellulaires infectees experimentalement et les leucocytes mononuclees de bovins porteurs asymptomatique de la maladie. Les techniques dhybridation sur filtres de nitrocellulose ou d'hybridation in situ ont ete evaluees. Des reactions dhybridation positives ont ete obtenues avec toutes les souches cytopathogenes et non-cytopathogenes du virus. Les resultats obtenus par la technique dhybridation etaient
compatibles a ceux d'immunofluorescence indirecte et, des essais d'inoculation experimentale de bovins seronegatifs.
cattle and to develop a rapid method for screening large numbers of blood samples for BVDV. MATERIALS AND METHODS
INTRODUCTION Bovine viral diarrhea virus (BVDV) is ubiquitous among the world's cattle population (1,2). Infection with BVDV has different clinical manifestations depending on the age and immune status of the animal at the time of infection and the biotype of the infecting BVDV (1-4). Infection of the bovine fetus in utero with noncytopathic (NCP) BVDV can result in persistent infection (1,2,4). Persistently infected (PI) cattle are the major disseminators of BVDV in the cattle population and are the animals which succumb to mucosal disease (MD) (3,4). Most often, PI cattle are clinically normal and are identified by isolation of NCP BVDV from the animal's serum or peripheral blood mononuclear leukocytes (PBML) after amplification of the virus by one or more passages in cell culture. While technologies such as nucleic acid hybridization have been used for rapid detection of other cattle viruses directly in infected tissues (5,6), there is little published information regarding methods for direct detection of BVDV in PBML from PI cattle. This study was designed to determine if RNA-cDNA hybridization could be used for direct detection of BVDV genome in PBML from PI
VIRUSES AND CELL CULTURES
Bovine viral diarrhea virus cytopathic (CP) strains NADL and Singer and bovine herpesvirus-I (BHV-1) were obtained from the stock virus collection at the James A. Baker Institute for Animal Health, Cornell University. Oregon C24V CP and NYI NCP were obtained from Dr. S.R. Bolin, National Animal Disease Center, Ames, Iowa. Bluetongue virus-17 (BTV-17) was obtained from Dr. A. Luedke, Arthopod-borne Animal Disease Research Laboratory, USDA-ARS, Laramie, Wyoming. Primary bovine testicle cells, obtained from calves less than two months old at a local abattoir, were used to propagate BVDV and for virus isolation and serum neutralization (SN) tests. Cells were grown in minimal essential media (MEM) with 10% fetal bovine serum (FBS) (Sigma, St. Louis, Missouri) and 1% penicillinstreptomycin and were determined negative for NCP BVDV by immunofluorescent staining. Fetal bovine serum was determined negative for NCP BVDV by inoculation of a third trimester bovine fetus as previously published (7). MONOCLONAL ANTIBODIES
Monoclonal antibodies (MAb) CA1 and CA-80 were obtained from Dr.
Department of Pathobiological Sciences (Jensen, Schultz) School of Veterinary Medicine, and Department of Veterinary Science (Aiken), University of Wisconsin-Madison, Madison, Wisconsin 53706. This work was supported in part by grants from Eastern Artificial Insemination Cooperative, Ithaca, New York and the Large Animal Research Fund, School of Veterinary Medicine, University of Wisconsin. Submitted August 14, 1989.
256
Can J Vet Res 1990; 54: 256-259
S.R. Bolin. These MAb have been and first strand cDNA synthesized demonstrated to react with serologi- using M-MLV reverse transcriptase cally diverse BVDV variants (8). (Bethesda Research Labs (BRL), Gaithersburg, Maryland). Second EXPERIMENTAL ANIMALS strand synthesis was performed using Care and housing of cattle was in Escherichia coli DNA polymerase I accordance with National Institute of and E. coli RNase H (BRL) according Health Guidelines (9). Six cattle to the manufacturer's instructions. persistently infected with BVDV and Double-stranded cDNA was filled in 46 non-PI cattle were used. Cattle using T4 polymerase and blunt ligated confirmed as PI were viremic for into the Sma I site of the plasmid BVDV for a minimum of a two-month pGEMT"-blue (Promega Biotec Madiperiod as determined by isolation of son, Wisconsin). Ligated plasmid NCP BVDV from PBML and by the DNA was then transformed into E. animal inoculation test (7). For coli strain TB1 in the presence of Xisolation of NCP BVDV, PBML were Gal. Twelve individual clones were harvested after centrifugation over randomly chosen from the library, Ficoll-hypaque (p = 1.080)(10) and plasmid DNA extracted by the boiling passaged in bovine testicle cells (three method (12), inserts removed by passages at 3-5 day intervals). Isola- digestion with Eco RI-Bam HI (BRL) tion of NCP BVDV was confirmed by and insert size estimated by gel indirect fluorescent antibody (IFA) electrophoresis in 1.0% agarose. staining of infected cell cultures using Complementary DNA inserts in these MAb CA-1 or CA-80 tissue culture clones ranged in size from 300-2000 fluids (diluted 1:2) as primary antib- base pairs. ody and rabbit antimouse IgG(l:10) (ICN, Lisle, Illinois) as a second RNA-cDNA HYBRIDIZATION antibody (8). For animal inoculation, Bovine viral diarrhea virus-infected 10 PBML, isolated as described and uninfected cell monolayers were above, were inoculated into seronega- rinsed with cold buffer (0.01 M Tristive test cattle. Serum neutralizing HCI, pH 8.0-2 mM MgCl2-.0I M antibody titers to BVDV were deter- NaCl) the cells resuspended in cold mined four weeks postinoculation by buffer by scraping, the mixture the varying serum-constant antigen adjusted to 0.5% Nonidet P-40 method using 100 TCID50 BVDV (Sigma, St. Louis, Missouri) -1% (NADL). deoxycholate (Fisher Scientific, Fair Lawn, New Jersey) and cells lysed on CONSTRUCTION OF BVDV cDNA ice for 5 min. The nuclear pellet was LIBRARY removed by microcentrifugation Bovine viral diarrhea virus (NADL) (12,500 revolutions per min for 5 min). was cultivated in bovine testicle cell Cytoplasmic RNA was extracted from monolayers to 4+ cytopathic effect the supernatant with an equal volume (CPE), the cells freeze-thawed and the of phenol, followed by extraction with supernatant clarified by centrifuga- an equal volume of phenoltion at 8000 x g for 10 min at 4°C. chloroform (1:1) and then with an Polyethylene glycol (PEG) 6000 was equal volume of chloroform, ethanoladded to 7% final concentration and precipitated and redissolved in water. virus precipitated for 6 h at 4°C with Mononuclear leukocytes were isolated stirring. The virus precipitate was by centrifugation over Ficoll hypaque, collected by centrifugation at 14,000 x resuspended in cold lysis buffer and g for 30 min at 4°C, resuspended in cytoplasmic RNA extracted as desTNE (10 mM Tris-HCl(pH 8.0)-100 cribed above for monolayer cells. mM NaCI-I mM EDTA(pH 8.0) and Typically, 1.0 mL of bovine blood layered over a linear 45%-30% potas- yielded 106 mononuclear leukocytes. sium tartrate:glycerol gradient (11). Ribonucleic acid yields were deterFollowing centrifugation at 200,000 x mined spectrophotometrically. g for 3 h at 40C, the viral band was Ribonucleic acid was diluted in 0.44 M harvested and genomic RNA phenol- formaldehyde-20X standard saline extracted. The RNA genome was citrate (SSC) buffer, heated to 60°C randomly primed using an N6 primer for 15 min, blotted onto nitrocellulose (Boehinger Mannheim, W. Germany) presoaked in 20X SSC using a slot blot
apparatus (BRL) and affixed by heating at 800 C for 2 h in vacuo. Following prehybridization at 420C for 2-4 h, heat-denatured nicktranslated 32p labelled cDNA probe was added to the hybridization mixture at 0.1 ,ug/mL (10 cpm/,ug) and hybridization continued at 420C
for 18 h. The prehybridization mixture consisted of 50% deionized formamide, 5X Denhardt solution, 5X SSC and 350 ,ug/ mL denatured salmon sperm DNA. The hybridization mixture in addition contained 10% dextran sulfate. Following hybridization, nitrocellulose was washed in 3X SSC at room temperature (three washes, 15 min per wash) followed by two 30 min washes in 0.1X SSC at 500 C. Autoradiography was done for 4-18 h using Kodak XAR film at -70° C. The modified in situ hybridization was done as described by Paeratakul et al (13). Briefly, 105 cells were blotted onto nitrocellulose using a dot blot apparatus (BRL), fixed in 10% glutaraldehyde (Sigma) for 60 min at 40C and digested with 20 Ag/ mL proteinase K(Boehinger-Mannheim) Prehybridization and hybridization were done as described above for RNA extracts except that dextran sulfate was omitted from the hybridization mixture. Supernatants from infected and uninfected cells were treated with proteinase K (250 ,ug/ mL)-1% sodium dodecyl sulfate (Sigma) for 30 min at 370C (12), the RNA phenol-extracted and hybridization done as described above for extracts from cell monolayers. RESULTS
Approximately 1500 ampicillinresistant, lac Z- transformants were obtained. Recombinant plasmids from 8 of 52 (15%) randomly selected transformants hybridized with RNA extracted from BVDV(NADL)infected cells but not with RNA extracted from uninfected cells. One of these cloned cDNA sequences (clone 3), a cDNA segment 1 kb in length, was chosen for evaluation as a diagnostic probe for BVDV. The results of RNA-cDNA hybridization using clone 3 with RNA extracts from cells infected with serologically divergent (14, 15) CP and 257
NCP BVDV strains are shown in Fig. 1. Clone 3 hybridized with cytopathic strains NADL, Singer and C24V, with NY-1 NCP and with all six NCP BVDV field isolates from our six PI cattle, but not with RNA from uninfected bovine testicle cells or with DNA from cells infected with BHV-1 or RNA from BTV-17 infected cells. The detection limit of hybridization with RNA extracted from BVDV (NADL)-infected cells was about 200 pg; no hybridization signal was found using up to 800 ng of RNA from uninfected cells (data not shown). Using supernatants from cell cultures infected with BVDV NADL and BVDV NY-1 NCP, hybridization signals were obtained at dilutions of 1 x 10-7 and 1 x 10-6 as compared with detection by cell culture at dilutions of l x 10-6 and 2 x 10-5 for BVDV NADL and BVDV NY-1 NCP, respectively. No hybridization signals were obtained using supernatants from uninfected cell cultures. The feasibility of using RNA-cDNA hybridization to detect BVDV genome
Fig. 1. Slot blot hybridization of RNA extracted from cells infected with BVDV NADL (A-1); BVDV C24V CP (A-2); NY-I NCP (A-3); BVDV Singer (A-4); NCP BVDV isolates from PI cattle I through 6 (A5 - A-10); uninfected cell cultures (B-i); BTV-17 (B-2); and DNA extracted from BHV-I infected cells (B-3). Nucleic acid was blotted at 20 ng and hybridized with clone 3.
258
in RNA extracted directly from PBML of PI cattle without prior amplification in cell culture was examined (Fig. 2). RNA was extracted from 107 PBML and blotted at 20 ng. Typically, 107 PBML yielded 0.5 to 1 jig of RNA. Bovine viral diarrhea virus clone 3 hybridized with RNA extracted from the six PI cattle; no hybridization signal was obtained with PBML RNA extracts from control animals. The comparison between RNAcDNA hybridization and conventional methods for detecting persistent BVDV infection in cattle is shown in Table I for the six PI and 46 non-PI cattle from our research herd. Viral RNA was detected by RNA-cDNA hybridization in PBML from all six of the cattle identified as PI by conventional subculturing and/ or animal inoculation. Of the 46 non-PI cattle, 45 were found negative for BVDV by hybridization, immunofluorescent staining of infected cell cultures and the animal inoculation test. One animal was positive for BVDV by hybridization and animal inoculation but negative by immunofluorescent staining. Later specimens taken from this animal were negative for BVDV by hybridization and thus this animal may have been acutely infected with BVDV at the time of the initial sampling.
Fig. 2. Slot blot hybridization of RNA extracted from mononuclear leukocytes of PI cattle 1 through 6 (Ai-6) and six non-PI, nonviremic control cattle (Bi-6) Nucleic acid was blotted at 20 ng (equivalent to RNA from 2-4 x 105 cells) and hybridized with clone 3.
To facilitate screening large numbers of blood specimens for BVDV, we adopted a modified in situ hybridization method (13). Figure 3 shows the results obtained with PBML from PI and control cattle. Bovine viral diarrhea virus RNA was detectable with as few as 105 PBML from PI cattle with no detectable hybridization with PBML from control cattle.
DISCUSSION The development of procedures for the rapid detection of viral infections is an active area of diagnostic research. This study was designed to test the feasibility of detecting persistent BVDV infection in cattle by RNAcDNA hybridization. A BVDV (NADL) cDNA library was constructed and one of the cDNA clones, which hybridized with serologically diverse strains of BVDV in cell culture, was used to detect BVDV RNA in PBML from PI cattle. Bovine viral diarrhea virus RNA could be detected using 105 cells from PI cattle by an in situ hybridization method and with RNA extracted from 2-5 x 105 PBML. No hybridization was detectable with PBML from 45 non-PI, nonviremic control cattle. The ability to detect BVDV RNA in PBML from PI cattle probably reflects not only the sensitivity of RNA-cDNA hybridization but also the percentage of BVDV-positive PBML in PI cattle. Other investigators have reported that 0.5-30% of PBML in PI cattle are BVDV antigenpositive as determined by isolation of infectious virus or immunolabelling with polyclonal antiserum to BVDV (16,17). Detection of BVDV antigen in PBML of acutely infected cattle by immunofluorescent staining has also been reported (18). While the percentage of BVDV RNA-positive PBML for the six PI cattle used in this study was not determined, IFA staining of PBML from these cattle using MAb CA-1 and CA-80 showed that 0.5-12% were positive for BVDV antigen (19). While other investigators have reported construction of BVDV cDNA libraries (20,21), the application of RNA-cDNA hybridization to detect persistent infection with BVDV has not been previously reported.
TABLE 1. Identification of persistently infected cattle by conventional subculturing, animal inoculation and RNA-cDNA hybridization Animal
Cell culturea
Animal inoculationa
Hybridizationa
I
+
2 3 4 5 6 7-12 13-51 52
+ + + +
+ +
+ +
+
+
+
+ +
+
ND ND ND
+
+
+
aAnimals were scored positive or negative for BVDV infection by subculturing mononuclear leukocytes in cell culture followed by IFA staining of infected cells or by inoculation of mononuclear leukocytes into seronegative test cattle. Mononuclear leukocytes were examined for BVDV genome by RNA-cDNA hybridization with clone 3 ND = not done
Use of an in situ hybridization method to probe cells fixed on nitrocellulose for BVDV RNA obviates the need for amplification of NCP-BVDV in cell culture and allows large numbers of samples to be examined at one time.
The feasibility of using RNA-cDNA hybridization to detect BVDV in serum or whole blood specimens from PI cattle is currently under investigation in our laboratory.
REFERENCES
Fig. 3 In situ hybridization of mononuclear leukocytes from PI animal I (A-1); PI animal 2 (A-2); and 28 non-PI, nonviremic control cattle (A-3 through C-10). Mononuclear leukocytes (I x 105) were blotted and hybridized with clone 3.
1. RADOSTITS OM, LITTLEJOHNS IR. New concepts in the pathogenesis, diagnosis and control of diseases caused by the bovine viral diarrhea virus. Can Vet J 1988; 29: 513528. 2. PERDRIZET JA, REBHUN WC, DUBOVI EJ, DONIS RO. Bovine virus diarrhea-clinical syndromes in dairy herds. Cornell Vet 1987; 77: 46-74. 3. MALMQUIST WA. Bovine viral diarrheamucosal disease: etiology, pathogenesis and applied immunity. J Am Vet Med Assoc 1968; 152: 763-768. 4. LITTLEJOHNS IR, WALKER KH. Aetiology and pathogenesis of mucosal disease of cattle; current concepts, observation and speculation. Aust Vet J 1985; 62: 101-103. 5. ROY P, RITTER GD, AKASHI H, COLLISSON E, INABA Y. A genetic probe for identifying bluetongue virus infections in vivo and in vitro. J Gen Virol 1985; 66: 1613-1619. 6. DORMAN M, BLAIR CD, COLLINS JK, BEATY BJ. Detection of bovine herpesvirus 1 DNA immobilized on nitrocellulose by hybridization with biotinylated DNA probes. J Clin Microbiol 1985; 22: 990-995. 7. SCHLAFER DH, SCHULTZ RD, SCOTT FW, DUNCAN JR. Bovine fetal inoculations with calf rotavirus. Can J Comp Med 1979; 43: 405-414. 8. BOLIN S, MOENNIG V, GOURLEY NE, RIDPATH J. Monoclonal antibodies with neutralizing activity segregate isolates of bovine viral diarrhea virus into groups. Arch Virol 1988; 99: 117-123.
9. UNITED STATES DEPARTMENT OF HEALTH AND HUMAN SERVICES. Guide for the care and use of laboratory animals, 1985. National Institute of Health Publication No. 85-23. 10. SCHULTZ RD. Assays of cellular immunity. J Am Vet Med Assoc 1982; 181: 11691176. 11. TRENT DW, GRANT JA, ROSEN L, MONATH TP. Genetic variation among dengue-2 viruses of different geographic origin. Virology 1983; 128: 271-284. 12. MANIATIS T, FRITSCH EF, SAMBROOK J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, 1982. 13. PAERATAKUL U, DE STASIO PR, TAYLOR MW. A fast and sensitive method for detecting specific viral RNA in mammalian cells. J Virol 1988; 62: 11321135. 14. CORAPI WV, DONIS RO, DUBOVI EJ. Monoclonal antibody analysis of cytopathic and noncytopathic viruses from fatal bovine viral diarrhea virus infections. J Virol 1988; 62: 2813-2817. 15. MAGER R, MINOCHA HC, MONTPETIT C, CARMAN PS, LECOMTE J. Typing of cytopathic and noncytopathic bovine viral diarrhea virus reference and Canadian field strains using a neutralizing monoclonal antibody. Can J Vet Res 1988; 52: 4245. 16. BOLIN SR, SACKS JM, CROWDER SV. Frequency of association of noncytopathic bovine viral diarrhea virus with mononuclear leukocytes from persistently infected cattle. Am J Vet Res 1987; 48: 1441-1445. 17. BIELEFELDT OHMANN H, RONSHOLT L, BLOCH B. Demonstration of bovine viral diarrhoea virus in peripheral blood mononuclear cells of persistently infected, clinically normal cattle. J Gen Virol 1987; 68: 1971-1982. 18. BEZEK DM, BAKER JC, KANEENE JB. Immunofluorescence of bovine virus diarrhea viral antigen in white blood cells from experimentally infected immunocompetent calves. Can J Vet Res 1988; 52: 288290. 19. JENSEN J, SCHULTZ RD, BOLIN SR. Detection of persistent infection with bovine viral diarrhea virus (BVDV) by immunofluorescent staining using monoclonal antibodies. Presented at American Society for Virology, Annual Meeting, University of Texas, Austin, 1988. 20. COLLET MS, LARSON R, GOLD C, STRICK D, ANDERSON DK, PURCHIO AF. Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus. Virology 1988; 165: 191199. 21. BROCK KV, BRIAN DA, ROUSE BT, POTGEITER LND. Molecular cloning of complementary DNA from a pneumopathic strain of bovine viral diarrhea virus and its diagnostic application. Can J Vet Res 1988; 52: 451457.
259
