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Tissue homogenates from 60 specimens submitted to the Veterinary Diagnostic Center were evaluated by polymerase chain reaction (PCR) for detection of bovine viral diarrhea virus (BVDV)..[r]

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Journal of Veterinary Diagnostic

http://vdi.sagepub.com/content/6/1/44 The online version of this article can be found at:

DOI: 10.1177/104063879400600109 1994 6: 44

J VET Diagn Invest

Beverly J Schmitt, Osvaldo J Lopez, Julia F Ridpath, Judith Galeota-Wheeler and Fernando A OsorioEvaluation of PCR for Diagnosis of Bovine Viral Diarrhea Virus in Tissue Homogenates

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- Jan 1, 1994 Version of Record

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Evaluation of PCR for diagnosis of bovine viral diarrhea virus in tissue homogenates

Beverly J Schmitt, Osvaldo J Lopez, Julia F Ridpath, Judith Galeota-Wheeler, Fernando A Osorio

Abstract Tissue homogenates from 60 specimens submitted to the Veterinary Diagnostic Center were evaluated by polymerase chain reaction (PCR) for detection of bovine viral diarrhea virus (BVDV) Conventional virus isolation procedures showed the specimens contained BVDV The BVDV RNA was extracted from the homogenates and subjected to a reverse transcription reaction followed by PCR amplification The PCR product was blotted onto a nylon membrane and hybridized with a 30-base pair oligonucleotide probe labeled with 32P One set of PCR primers detected BVDV in 46/60 (77%) of the tissue homogenates An additional set of primers was used to detect 10/11 samples that had escaped detection with the first set of primers The results indicate that BVDV can be detected by PCR directly out of tissue homogenates generated in a diagnostic setting

Bovine viral diarrhea virus (BVDV) is an important using the polymerase chain reaction (PCR) tech-pathogen of cattle causing a variety of diseases that nique.1,2,5,6,10,11,15,23,24,26

range in severity from mild inapparent infections to Application of PCR in a diagnostic setting requires fatal mucosal disease.20,21 BVDV is classified within adaptation for use on clinical samples Previous reports the family Flaviviridae and is a member of the genus on the use of PCR for detection of BVDV have not Pestivirus The viral genome consists of a single-strand- addressed the problems associated with clinical spec-ed nonpolyadenylatspec-ed RNA about 12.5 kb in length.8 imens The purpose of this study was to evaluate PCR Isolates of BVDV are divided into biotypes, cyto- detection of BVDV in field specimens using primers15,22 that amplify sequences near opposite ends of the BVDV genome.

pathic and noncytopathic, based on effects in cell cul-ture.25 Infections of cattle with noncytopathic BVDV during early gestation may cause abortion or birth of calves immunologically tolerant to the infecting vi-rus 12,17 These calves are persistent carriers of BVDV and can be a source of infection to the herd.4 Super-infection of persistently infected cattle with a cyto-pathic BVDV may induce mucosal disease.4,7 Detec-tion of persistently infected cattle is important for control of BVD In addition, contamination of fetal bovine serum and other bovine products used in cell culture work with BVDV presents a problem in pre-paring veterinary biologics.3,19 Traditionally, virus iso-lation with confirmation by immunofluorescence has been the method used to detect BVDV in clinical samples and in contaminated cell cultures and biolog-ical products These techniques are time consum-ing and require the use of cell culture Several reports have described rapid diagnostic tests for BVDV

From the Department of Veterinary Science, University of Ne-braska, Lincoln, NE 68583 (Schmitt, Lopez, Galeota-Wheeler, Oso- rio), and the Virology Cattle Research Unit, National Animal Dis-ease Center, Agricultural Research Service, USDA, Ames, IA 50010 (Ridpath) Current address (Schmitt): Diagnostic Virology Labora-tory, National Veterinary Services Laboratories, Veterinary Services, Animal and Plant Health Inspection Service, USDA, Ames, IA

Received for publication March 26, 1993

Materials and Methods

Preparation of clinical samples. Tissue samples from cat-tle with clinical signs of BVD were submitted to the Uni-versity of Nebraska Veterinary Diagnostic Center virology laboratory All samples came from the state of Nebraska between 1988 and 1990 The samples were prepared as fol-lows Minimum essential medium (MEM) with 2% horse serum was added to tissue at a ratio of 3:l and then ho-mogenized in a stomacher for 2-4 The homogenate was centrifuged at 2,000 rpm for 30 at C, after which the supernatant was poured into 15-ml snap cap tubes After initial detection of BVDV by virus isolation, homogenates were preserved by freezing at -70 C

Cell cultures Tissue homogenates were inoculated onto primary bovine lung cells and bovine turbinate cellsa that were free of BVDV Cultures were maintained in MEM sup-plemented with 10% horse serum in Leighton tubes Cell monolayers were stained for BVDV antigen using fluores-cein-labeled antibodya and examined by fluorescent micros-copy Tissue homogenates that were positive for BVDV were used for further analysis by PCR

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Use of PCR in diagnosis of BVDV infections 45

strain) End point was determined by observing inhibition of cytopathic effect?

Primers for PCR. The primers used in this study are listed in Table Primer set 115 amplifies a 205-base segment that contains a BstIII site The primers were synthesizedb and purified by either polyacrylamide gel electrophoresis (PAGE) or high-performance liquid chromatography (HPLC) A sec-ond set of primers was used on samples that were not am-plified by primer set with the exception of samples that lacked sufficient quantity for testing These primers are lo-cated at the 5' end immediately upstream of the open reading frame 22

Extraction of BVDV RNA from tissue homogenates. A 100-µl volume of tissue homogenate was added to 450 µ1 of STE buffer (100 mM NaCl, 50 mM Tris, mM ethylene-diaminetetraacetic acid [EDTA]; pH 8) in a microcentrifuge tube Proteinase K was added at a concentration of 20 µg/ ml, the solution was incubated at room temperature for 10 min, and µl of 10% sodium dodecyl sulfate (SDS, 0.2% wt/ vol) was added After incubation at 56 C for 20 min, phenol/ chloroform extractions were done, and the nucleic acid was precipitated with 60 µl of M sodium acetate (pH 5.2) plus

1 ml cold (-20 C) 100% ethyl alcohol The precipitated RNA was pelleted by centrifugation for 15 at 12,000 x g at C, washed with cold 70% ethanol, and dried in a vacuum desiccator

Reverse transcription (R T). After resuspension of the RNA pellet in µl of sterile distilled water, µl (40 µmol) of each primer was added and the sample was incubated for at 70 C The sample was chilled on ice for and µl of deoxynucleoside triphosphatesc (7.5 µM), µl Schimke solution (10 mM dithiothreitol, 10 mM MgCl,, 70 mM KCl, 80 mM Tris; pH 8), µl of reverse transcriptased (19.6 u/ml), and µl of RNase inhibitore (28 u/µl) were added The re-sulting RT reaction mixture was incubated at 42 C for hr Reaction mixtures were then chilled on ice for immediate testing or stored at -20 C

PCR Viral strains NADL and Singer were used as pos-itive controls for PCR Negative controls consisted of MEM with 1) 10% horse serum and gentamicin and 2) tissue ho-mogenate from a gnotobiotic calf A mixture of PCR reagents without template was used as control for detection of con-tamination with extraneous DNA

A master mix was made of the following reagents: distilled water, 58.5 µl; 10x buffer, 9.5 µl (15 mM MgCl2, 400 mM

Tris-HCl, 500 mM KCl, 1% gelatin); dNTPS, 16 µl of equi-molar mixture of dNTPs (5 mM); µl (40 µmol) of each primer A 5-µl aliquot of RT reaction mixture was added; the resulting mixture was heated to 95 C for and chilled on ice Then 2.5 units of Taq polymerasee in µl of x buffer (10 x Promega buffer diluted 1: 10) were added for PCR am-plification A layer of 60 µl of Nujol mineral oilf was added

The PCR program consisted of at 95 C (denaturation), at 55 C (primer annealing) and at 72 C (primer extension) for 40 cycles Primer set was used at a concen-tration of 15 µmol per reaction during both RT and PCR All other parameters remained as previously described

Nucleic acid hybridization. PCR products using primer set were analyzed by electrophoresis in 2% agaroseg in TBE buffer (0.045 M Tris borate, 0.001 M EDTA) Gels were blotted onto a nylon membrane, denatured, and cross-linked by UV light The hybridization reaction used a 30-base probe with the following sequence: ACCTAAACCGAAGCAGGT-TACCAAGGAAGC The probe was end labeled with 32P using T4 polynucleotide kinase.h Unincorporated label was removed by chromatography using a Sephadex G-50 spin column Prehybridization was done in x SSPE-1 x BP (2% bovine serum albumin, 2% polyvinylpyrrolidone)-1% sodi-um dodecyl sulfate (SDS) for 30 at 55 C The hybrid-ization was performed using the same buffer with addition of at least x 106 cpm probe/ml for 0.5 hr at 55 C The nylon filter was washed in 1% SSPE-1% SDS times for at room temperature and time for at 55 C The filter was dried, exposed to radiographic film, and developed Analysis of the amplified product from primer set was performed using a probe made from a 365-base pair segment of the BVDV-Singer genome’ and cloned into the pBluescript II KS +/- vectorj The probe aligned with bases 182-545 of the BVDV NADL genome Phage T3 polymerase with

32

P-UTP was used to radiolabel the probe

Prehybridization was for at least hr at 55 C in hybrid-ization buffer consisting of x standard saline citrate (SSC), x Denhardt’s reagent, 0.1% SDS, and 100 u/ml denatured salmon sperm Hybridization with the same buffer was done at 55 C overnight The blots were washed twice for at room temperature in x SSC and 0.1% SDS followed by washes for 30 in 0.2 x SSC and 0.1% SDS at 55 C Autoradiography was performed by exposing the blot to ra-diographic film

Results

Titration of BVDV infectivity in clinical samples.

Fifty-five of 60 tissue homogenates had sufficient quan-tity to be tested for virus concentration In 23 ho-mogenates, the concentration of BVDV (either cyto-pathic or noncytocyto-pathic) was 101.7-103.3CCID,,/ml. The concentration of BVDV in 32 homogenates was

< 101.7 CCID50/ml

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of 77% (46/60) Primer set allowed detection of BVDV in 10/11 homogenates that were negative using primer set These complementary primers boosted the de-tection level to 93% (56/60).

Discussion

Practical application of PCR for diagnosis of BVDV requires that it be useful for field samples such as tissue homogenates Consideration should be given to the sensitivity of the test and ability of the primers to identify most, if not all, BVDV strains In this study, PCR was evaluated for direct detection of BVDV in tissue homogenates from field cases of BVD Using 2 sets of primers, 55/60 tissue homogenates positive for BVDV by virus isolation were positive by PCR The PCR procedure used here required a single amplifi-cation cycle and used primers derived from either the 5' noncoding region or the 3' coding region of the viral genome.

In an earlier study, a PCR technique was evaluated on tissue samples without cell culture passage.2 These authors tested their set of nested primers targeting the genomic region of gp48 on organ suspensions posi-tive for BVDV by virus isolation They were able to detect BVDV in the suspensions only after second stage amplification by PCR.

In the present study, primer set detected samples that had detectable virus titer but were negative with primer set Primer set may amplify a more con-served area of the BVDV genome The genomic di-versity of BVDV is well documented and presents a challenge to researchers looking for a nucleic acid-based test that will detect all isolates 13,14,22

Further work in the development of a diagnostic PCR procedure for detection of BVDV should focus on the selection of a single universal set or mixture of primers that can detect all strains of BVDV Also,

re-sitivity of PCR as a diagnostic test. Routine use of PCR in a diagnostic setting will require the use of non-radioactively labeled oligonucleotide probes in con-firmatory hybridizations of amplified products.

Acknowledgements

We thank Dr E Berry (North Dakota State University, Fargo, ND) for sequence information and M Palmer and D Reichenbach for their excellent technical assistance The use of a particular manufacturer’s product does not constitute an endorsement on behalf of the USDA

1

verse transcriptases that act as DNA polymerases during both reverse transcription and PCR can streamline the procedure by combining the reactions in tube.18 10. Preventing the RNA destroying activity of RNases by addition of RNAase inhibitor in sample preparation 11. could also be an important step in increasing the

sen-Sources and manufacturers National Veterinary Services Laboratories, Ames, IA Genosys, The Woodlands, TX

Pharmacia, Alameda, CA Life Sciences, St Petersburg, FL Promega, Madison, WI Perkin-Elmer, Norwalk, CT FMC, Rockland, ME Biolabs, Beverly, MA

Dr E Berry, North Dakota State University, Fargo, ND Stratagene, La Jolla, CA

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