Molecular and Cellular Probes (1991) 5, 291-298
Hybridization analysis of genomic variability among isolates of bovine viral diarrhoea virus using cDNA probes Julia F. Ridpath* and Steven R . Bolin Virology Cattle Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, lA 50010, USA (Received 27 December 1990, Accepted 15 February 1991)
Genomic variability among 28 cytopathic and 37 non-cytopathic isolates of BVDV was evaluated using hybridization analysis . Total RNA, isolated from cell cultures infected with BVDV, was probed with each of three plasmids containing cDNA derived from the cytopathic BVDV-NADL isolate . The three cDNA plasmid probes represented about 40% of the BVDV-NADL genome . The probes originated from the 5' end of the viral genome, the region of the genome coding for the p80 polypeptide and the 3' end of the genome . Three temperatures, 63 ° C, 53 ° C and 43 ° C, were used for hybridization and washing and allowed approximately 29%, 36%, and 43% mismatch, respectively . Fifteen of the BVD viral isolates differed by less than 29% from BVDV-NADL in all three of the regions probed . Seventeen of the BVD viral isolates differed between 36% and 43% from BVDV-NADL in all three of the regions probed . Ten viral isolates differed by more than 43% from BVDV-NADL in all the regions probed . The remaining viral isolates exhibited different levels of similarity to BVDV-NADL in the three areas probed .
KEYWORDS : BVDV, genome, homology, RNA, hybridization .
INTRODUCTION Bovine viral diarrhoea (BVD) virus, an ubiquitous and economically important pathogen of cattle, is the prototype member of the pestivirus genus . Although currently classified as non-arthropod-borne members of the Togaviridae family' ,' the replication and expression strategies of pestiviruses more closely resemble those of the Flaviviridae family .' Two biotypes of BVD virus, non-cytopathic and cytopathic, are differentiated by their effect on cultured cells . ^ While only one serotype of BVD virus is recognized, there appears to be antigenic diversity among BVD viruses .'-" The BVD virus has a single positive strand RNA genome . Generally, high mutation frequencies are associated with genomes of this type ."' Thus, a degree of genomic heterogeneity would be expected among BVD viral genomes . Genomes of two cytopathic BVD viruses, BVDV-NADL and BVDV-Osloss, have been cloned and sequenced ."" These ' Author to whom correspondence should be addressed . 0890-8508/91/040291 + 08 $03 .00/0
genomes have an aligned sequence identity of approximately 74% . 2' The extent of heterogeneity among BVD viral isolates becomes important for development of diagnostic tests based on recognition of genomic sequences and for estimation of BVD viral mutation rates . While direct sequence comparison is the most precise measure of genomic heterogeneity, it is not practical for comparison of large numbers of viral isolates . Hybridization analysis is better suited for large scale comparisons and has been used to study genomic relationships among viruses .21,2' The purpose of this study was to evaluate genomic similarity among BVD viral isolates by hybridization analysis . To this end, total RNA was isolated from cell cultures infected with 65 isolates of BVD virus . The RNAs were blotted and probed with cloned sequences representing approximately 40% of the genome of the BVD-NADL virus . The genomic sequences used as
291
292
J . F . Ridpath and S . R. Bolin by three successive plaque purifications . The noncytopathic viral isolates were biologically cloned by serial passage at limiting dilution . Stock viruses were propagated in bovine turbinate (BT) cells, harvested
probes were selected because of their relatively high conservation between BVD-NADL and BVDV-Osloss viruses .
after one freeze-thaw cycle, and stored at -90 ° C . The BT cells were grown in minimal essential media
MATERIALS AND METHODS
(F15 Eagle's medium, GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum . Fetal bovine serum was tested and found free of adventitious BVD viruses, by virus isolation procedures, and free of viruses, by indirect antibodies to BVD
BVD viral isolates The BVD viruses (n=65) used consisted of 28 cytopathic (Cl through C28) and 37 non-cytopathic (NC1 through NC37) viral isolates. All but two of the viruses were isolated from animals or cell cultures originating in the United States . Viral isolates C3 and NC35 were isolated from fetal calf serum obtained from New Zealand . As denoted in Tables 1 and 2, 52 viral
immunoperoxidase staining, viral neutralization, and radioimmunoprecipitation (RIP) procedures . The BT cells were free of adventitious BVD viruses as determined by indirect immunoperoxidase staining .
isolates were isolated from bovine tissue, eight from fetal calf sera, two from porcine cell lines, and one each from a rabbit cell line, a bovine cell line, and deer tissue . Cytopathic viral isolates were biologically cloned
Table 1 .
RNA slot-blots To generate RNA blots, confluent monolayers of BT cells were grown in 25 cm 2 flasks and inoculated with
Hybridization of cytopathic BVD viral RNA pBV -18
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26 C27 C28 Positive % Positive
pBV - p80
pBV - KPB
Source
63C
53C
43C
63C
53C
43C
63C
53C
43C
a a b a a a b a a a a a a a a a b a a a a a a a a a a b
+ + + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + + + + +
+
+ + + + + + + + + + + + + + + + + + + -
14 50
16 57
22 79
17 61
23 82
15 54
16 57
23 82
a : cattle tissue; b: fetal calf serum .
-
15 54
-
BVDV genomic homology Table 2 .
293
Hybridization of non - cytopathic BVD viral RNA pBV - 18
NC1 NC2 NC3 NC4 NC5 NC6 NC7 NC8 NC9 NC10 NC11 NC12 NC13 NC14 NC15 NC16 NC17 NC18 NC19 NC20 NC21 NC22 NC23 NC24 NC25 NC26 NC27 NC28 NC29 NC30 NC31 NC32 NC33 NC34 NC35 NC36 NC37
Positive Positive
pBV4- p80
pBV - KPB
Source
63C
53C
43C
63C
53C
43C
a a a a b a a c a a a a b a
+ -
+ + -
+ + + + + + + + + + + + + +
+ + + -
+ + + -
+ + + +
+ + + + + + -
+ + + + + + +
d
-
-
+
a a a a a
-
-
+ + + + +
e
-
-
+
a a c a a a a f a a a a a b b a
-
-
+ + + + + -
63C
+ +
53C
43C
+ + + + + -
+ + + + + +
+
+ + +
1 3
2 5
26 70
3 8
9 24
31 84
3 8
6 16
29 78
a : cattle tissue; b : fetal calf serum ; c : porcine cell line; d : rabbit cell line; e: deer tissue ; f: bovine cell line .
BVD virus at a multiplicity of infection (moi) of 10. Cell cultures infected with cytopathic viruses were harvested when 90% of cells exhibited cytopathic effect, usually 24 to 30 h after inoculation . Cell cultures infected with non-cytopathic viruses were harvested 48 h post-inoculation . Cell cultures were harvested by one freeze-thaw cycle . Cell debris and culture fluids were collected and a 500 gl aliquot was removed for titration of the virus . End points of virus concentration were determined in microtitration plates seeded with BT cells using cytopathic effect for cytopathic viruses and indirect immunoperoxidase staining for non-cytopathic viruses ." Only harvested
material that had at least 10 6 CCID ml -1 of BVDV were used . RNA was prepared for blotting by adding 10 ml PK buffer (0 . 2m Tris-Cl, pH 8 . 0, 25 mm EDTA, 0.3 M NaCl, 2% SDS, and 200 tg ml -1 proteinase K) to 10 ml of harvested material (equivalent to approximately 1 x 10' cells) . This mixture was incubated at 37° C for 30 min, extracted once with an equal volume of a 1 :1 mixture of phenol and chloroform, and once with an equal volume of chloroform . After extraction, 20 ml of formaldehyde [6 . 15 M in 20 X SSC (3-00m NaCl, 0 . 34m sodium citrate, pH 7 . 0)1 was added followed by incubation at 65 ° C for 15 min . A volume of 0. 5 ml of this solution (equivalent to ap-
294
J . F . Ridpath and S . R. Bolin reagent, 0-1% SDS, and 100 µg ml - ' of denatured salmon sperm DNA) . After prehybridization blots were hybridized for 16 h at the aforementioned temperatures . The hybridization buffer was the same as that used for prehybridization with the addition of 0. 1 .tg of denatured cDNA probe (specific activity at
proximately 1 . 25 x 10 5) was blotted onto nitrocellu-
lose. Nucleic acid from cells infected with BVDVNADL virus and uninfected cells was blotted as positive and negative controls, respectively . After blotting, nitrocellulose membranes were rinsed in 2
X
SSC and baked for 2 h at 80 ° C .
least 2 x 108 cpm
lag -1 ) .
Hybridized filters were
washed for 20 min at room temperature in 1 x SSC, 0. 1% SDS, followed by two washes of 1 h each in 0. 2 x SSC, 0. 1 % SDS at the temperature used for hybridization ." After washing, blots were exposed to
Preparation of probes and hybridization Three plasmids, pBV-18, pBV4-p80, and pBV-KPB, derived from BVDV-NADL (courtesy of M . Collett, Molecular Vaccines Inc ., Gaithersburg, MD) were
X-Omat AR film (Eastman Kodak Company, Oak Brook, IL) . The RNA from cultures infected with certain viral isolates did not hybridize to any of the probes. To determine if sufficient viral RNA was blotted onto the
used as probes for evaluation of genomic similarity (Fig . 1). The plasmids contained inserts encompassing nucleotides 24 to 1308 (pBV-18), nucleotides 5644 to 7949 (pBV4-p80), and nucleotides 9857 to 12020 (pBV-KPB) . The probes were selected on the basis of conservation between published nucleotide sequences of BVD viral genomes as determined using
membrane for detection, these samples were probed with a 30-base oligomer. The 30-base oligomer (5' act cca tgt gcc atg tac agc aga gat ttt 3') was complementary to a sequence conserved in both the BVDVOsloss and BVDV-NADL genomes . In previous stud-
the Align sequence alignment program (Scientific and Educational Software, Silver Spring, MD) . The overall aligned sequence identity for the genomes of BVDNADL and BVDV-Osloss (minus the extreme 5' and 3'
ies, oligomers containing the same sequence hybridized with 86% of the BVD viruses tested ." The oligomer was end labelled using T4 polynucleotide
ends) is approximately 74% .2' The aligned sequence identities for the portions of the genome contained in plasmids pBV-18, pBV4-p80, and pBV-KPB are 84%, 82%, and 77%, respectively . Radiolabelled doublestranded probes were prepared from plasmids by nick translation following the method of Koch et al." as modified by Sambrook et al." Spin columns (5 Prime-3 Prime, Paoli, PA) were used to remove unin-
kinase. 26 Unincorporated label was removed by pre-
corporated label . Hybridization reactions, using the above plasmids as probes, were performed as follows. Blots were prehybridized for at least 6 h at either 63 ° C, 53 ° C, or
tions were carried out at 50° C for 2 h in 6 x NET, 0. 1 SDS, 5 x Denhardt's and 5-10 ng ml - ' of end labelled oligomer probe (specific activity at least 5 . 0 x 10 7 cpm gg -1 ) . After hybridization, the blots were
43 ° C, in hybridization buffer (6 x SSC, 2 x Denhardt's
washed three times, for 5 min each, in 6 x SSC, 0 . 1
pBV-18
p20
ization reactions with the oligomer were performed as described by Lewis et al." In short, blots probed with oligomers were prehybridized at 65 ° C for 4-5 h in 6 x NET [0 . 15 M NaCl, 15 mm Tris-HCI (pH 8 . 3), 1 mm EDTA], 0-1% SDS, 5 x Denhardt's, and 100 lag ml - ' of
denatured salmon sperm DNA . Hybridization reac-
pBV-KpB
pBV4-p80
gp25 gp48
cipitation with cetylpyridinium bromide ." Hybrid-
p125 gp53
p58
p75
p80
Location of BVDV-NADL cDNA probes in open reading frame . The location of the cDNA plasmids (∎) in relationship to the BVDV-NADL open reading frame (∎) and sequences coding for BVD viral polypeptides'-20 is shown. Fig. 1 .
295
BVDV genomic homology SDS at room temperature . The blots were then washed twice for 30 min each in 3 x SSC, 0 . 1 % SDS at 50° C, and twice for 20 min in 1 x SSC, 0 . 1 % SDS at 50 ° C . After washing, blots were exposed to X-Omat AR or XAR-5 film (Eastman Kodak Company, Oak
averaged about 600 by in length as determined by gel electrophoresis followed by autoradiography . Formamide was not present in the hybridization buffer used for the study . Using this formula, the T m for the cDNA probe and the bound viral RNA is 107 ° C . The T m of a nucleic acid hybrid is depressed by base mismatching, with 1 % mismatch reducing the T m between 0 . 5 ° C and 1 .4°C .30-34 The exact value of this ratio depends upon fraction of guanine and cytosine
Brook, IL) for 48 h .
Calculation of melting temperature
(G+C) residues and the distribution of mismatched bases . While the G + C value can be estimated, the distribution of mismatches within the duplex cannot be determined by the methods used . Using a value of 0 . 5 ° C of Tm lowering per each percent mismatch, 34
The melting temperature (T m) for DNA probes hybridized to immobilized RNA has been defined by the following formula:28,29 = 79 .8 + 18 . 51ogM + 58 . 4(mole fraction GC) + 11 . 8(mole fraction GC)2 -820/L-0 . 5 (% formamide)
Tm
failure of probes to hybridize at 63 ° C, indicates a mismatch of at least 29% between cDNA probe and viral RNA target . Similarly, lack of hybridization at 53 ° C and 43 ° C indicates a minimum percentage mismatch of 36% and 43%, respectively .
Where M is the concentration of monovalent cation in moles per litre . For the hybridization conditions used in this study M equaled 0 . 99 . Mole fraction GC is the base composition expressed as mole fraction of guanine and cytosine residues . The mole fraction GC for BVDV-NADL is 0 .46 .21 L is the length of the
RESULTS
hybridized duplex and is determined by the length of the probe . Nick translated probes used in this study
63C
Hybridization results are summarized in Tables 1 and 2 and an example is shown in Fig . 2a . Genomic
pBV4-p80 63C 53C 43C
pBV-18 53C 43C
63C
pBV-KPB 53C 43C
- C9 -C13 -NCI - NC5 -C15
(b) 63C
43C -No dilution -1 :2
00
%40
-1 :10 -1 :100
Fig . 2 . Hybridization of cDNA plasmids to cytopathic and non-cytopathic BVD viral RNA . Examples of hybridization of the three cDNA probes at the three stringencies used to cytopathic and non-cytopathic BVD viral RNA is shown in (a) . A control comparing the sensitivity of the hybridization reaction at different stringencies is shown in (b) . Shown are dilutions (no dilution, 1 :2, 1 :10, 1 :100) of RNA isolated from BVDV-NADL infected cells probed with pBV-18 at 63 ° C and 43 ° C. All hybridizations shown were performed as described in Materials and Methods .
J. F. Ridpath and S. R. Bolin
2%
variation was evaluated based on the hybridization of probes, corresponding to the 5' end of the BVDVNADL genome, the region of the BVDV-NADL genome coding for the p80 polypeptide and the 3' end of the BVDV-NADL genome, at different stringencies. Fifteen of the BVD viral isolates differed by less than 29% from BVDV-NADL in all three of the regions probed . Seventeen of the BVD viral isolates differed between 36% and 43% from BVDV-NADL in all three of the regions probed . Ten viral isolates differed by more than 43% from BVDV-NADL in all the regions probed . The remaining viral isolates exhibited different levels of similarity to BVDV-NADL in the three areas probed (Tables 1 and 2) . To if the difference seen in hybridization at stringencies was due to the amount of BVD, present on the blot dilutions of total RNA
determine different viral RNA from BVD-
NADL infected cells were hybridized to the probes at 63 ° C, 53° C, and 43 ° C. An example of the results from this test is shown in Fig . 2b . No difference was seen in hybridization results performed at 63 °C, 53 ° C, and 43 ° C . The highest dilution detected at all three stringencies was 1 :10 . Total cell RNAs, isolated from cell cultures infected with the 10 isolates which did not bind to any of the plasmids, were blotted and probed with a 30-base oligomer to determine if viral RNA was present . An example of the hybridization reactions is shown in Fig . 3 . Nine of the 10 isolates which did not hybridize
A
to the plasmid probes were detected with the oligomer probe, indicating that viral RNA was present on the blot . One isolate, C28, did not hybridize to the oligomer probe . The failure to hybridize may have been due to low homology between the probe and the viral genome or insufficient viral RNA present for detection . All viral preparations used had approximately the same infective titre . However, infective titre does not take into account the presence of unpackaged viral RNA . Thus it is possible that a BVD isolate which packaged the majority of the RNA present would go undetected while a virus which was less efficient at packaging progeny RNA would be detected even though both viruses were grown to the same titre . As noted above, similarity to BVDV-NADL in the areas probed was not uniform with all viral isolates (Tables 1 and 2) . For example, eight viral isolates were more similar to BVDV-NADL in the p80 coding region than in the other two regions probed and five viral isolates were more similar in the 3' end region . A difference in hybridization was observed between non-cytopathic and cytopathic BVD viral isolates . At 43 ° C the percentage of cytopathic and non-cytopathic BVDV isolates recognized by at least one of the three probes were similar (86% versus 84%) . However, the percentage of cytopathic BVDV isolates bound by at least one of the plasmids at 63 ° C (58%) and 53° C (61 %) was greater than the percent-
B
C
D
NADL
Cell control -
C25-
C26-
C27-
C28 -
NC32--
Fig. 3 . Detection of BVD viral RNA with an oligonucleotide probe . To determine if failure of cDNA plasmids to hybridize with RNA from some BVDV isolates was due to insufficient RNA present for detection of low similarity, viral RNA was also probed with a 30-base oligomer complementary to BVDV-NADL . Lane A, B, C, and D represent blotted viral RNAs probed with plasmids pBV-18, pBV4-p80, and pBV-KPB, and the 30-base oligomer, respectively . Hybridizations with plasmids were performed at 43 ° C. Hybridizations with the oligomer were performed as described in the Materials and Methods section .
BVDV genomic homology
297
age of non-cytopathic BVDV isolates (11% and 32%, respectively) bound at these stringencies .
hybridized to at least one of the probes while only 4 out of 37 (11%) non-cytopathic BVD viruses hybridized to at least one of the probes . Under the lower stringency of 43 ° C, the percentage of cytopathic and
DISCUSSION
non-cytopathic viral RNAs that hybridized to at least one of the probes was similar (89% and 84%, respectively) .
In this study, we examined the nucleotide similarity among RNA from isolates of BVDV to plasmid probes derived from BVDV-NADL . The extent of genomic variability observed the BVDV-NADL probes and 50 of the BVD viral isolates evaluated was higher than the 74% reported between BVDV-NADL and BVDVOsloss . 21 Similarity to portions of the nucleotide sequence of BVDV-NADL was 57% or more for 55 viral isolates . Of these 55, 29 were 64% or more similar and 15 were 71% or more similar . Ten BVD viral isolates failed to hybridize to the BVDV-NADL cDNA probes under conditions which theoretically allowed 43% mismatch . No attempt was made in this study to determine the correspondence between similarity estimations based on hybridization analysis and similarity based on aligned sequence identity . However, it is intriguing to note that the Alfort strain of hog cholera virus, another member of the pestivirus family, has an overall aligned sequence identity to BVDV-NADL of 68% (32% mismatch) . 35 Because the BVD viral genome is a single-stranded RNA, a degree of genomic heterogeneity would be expected among BVD viral genomes . The recognition of only one serotype of BVD virus, however, suggests conservation of at least portions of the BVDV genome . Little information is available regarding genomic variability among pestiviruses . A number of studies have examined genomic variability among flaviviruses, which have similar replication and expression strategies to those of the pestiviruses . 3 Two strains of Dengue 2 virus belonging to the same serotype have sequence similarity of 91 % . 36 The similarity between Dengue viruses of two different serotypes are in the range of 64% to 66% . 37 The similarity among published sequences of yellow fever virus, 38 West Nile virus, 39 Kunjin virus,40 and Dengue 2 virus, Jamaica genotype37 are in the 45% to 55% range as determined using the Fastdb software program (Intelligenetics, Inc ., Mt . View, CA) . As a group genomic variability among the BVDV viruses observed in this study is similar to that observed among the flaviviruses as a group . However, when compared to the genomic similarity observed between flaviviruses belonging to the same serotype it is higher. A marked difference was observed between the hybridization of cytopathic and non-cytopathic BVD viral isolates to the plasmid probes at 63 ° C . At this stringency 16 out of 28 (57%) cytopathic viruses
The possibility of greater than 40% variation in BVD viral genomes increases the degree of difficulty in devising a reliable diagnostic probe based on genomic sequences . However, genomic variation in BVD viral isolates may be exploited to the researcher's benefit in epidemiology and pathogenesis studies .
ACKNOWLEDGEMENTS The authors would like to thank Dr Marc Collett for his generosity in supplying the cloned cDNA plasmids . In addition, the authors would like to thank Rebecca Zaworski, Shari Steadham, and Sharon Stark for technical assistance, and Sandy Johnson, Gene Hedberg, and Tom Glasson for manuscript preparation .
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10 . Liess, B ., Frey, H : R ., Orban, S . & Hafez, S. M . (1983) . Bovine virus diarrhea (BVD)-mucosal disease : persistent BVD field virus infections in serologically selected cattle . Deutsche Tieraerztliche Wochenschrift 990, 261-6. 11 . Itoh, 0 ., Sasaki, H . & Hanaki, T. (1984) . A study of serologic relationships among non-cytopathogenic strains of bovine viral diarrhea-mucosal disease virus by reverse plaque technique . Japanese Journal of Veterinary Science 46, 669-75 . 12 . Howard, C. J ., Brownlie, J . & Clarke, M . C . (1987) . Comparison by the neutralization assay of pairs of noncytopathogenic and cytopathogenic strains of bovine virus diarrhea virus from cases of mucosal disease . Veterinary Microbiology 13, 361-9 . 13 . Bolin, S . R ., Moennig, V ., Kelso Gourley, N . E . & Ridpath, J . F . (1988) . Monoclonal antibodies with neutralizing activity segregate isolates of bovine viral diarrhea virus into groups . Archives of Virology 99, 117-24 . 14. Corapi, W . V ., Donis, R. O . & Dubovi, E . F . (1988) . Monoclonal antibody analyses of cytopathic and noncytopathic viruses from fatal bovine viral diarrhea virus infections . Journal of Virology 62, 2823-7 . 15. Domingo, E. (1989) . RNA virus evolution and the control of viral disease . Progress in Drug Research 33, 93133 . 16. Domingo, E ., Martinez-Salas, E ., Sobrino, R . et al. (1985) . The quasispecies (extremely heterogeneous) nature of viral RNA genome populations : biological relevancea review. Gene 4, 1-8. 17 . Holland, J . J ., Spindler, K ., Horodyski, F., Grabau, E ., Nichol, S . & VandePol, S . (1982) . Rapid evolution of RNA genomes. Science 215, 1577-85 . 18 . Renard, A ., Guiot, C ., Schmetz, D et al . (1985) . Molecular cloning of bovine viral diarrhea virus sequences . DNA 4, 429-38. 19 . Renard, A ., Dino, D . & Martial, J . (1987) . Vaccines and diagnostics derived from bovine diarrhea virus. European Patent Number 0208672 . 20 . Collett, M . S ., Larson, R ., Gold, C ., Strick, C ., Anderson, D. K . & Purchio, A . F . (1988b) . Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus . Virology 165, 191-9 . 21 . Collett, M . S ., Moennig, V . & Horzinek, M . C. (1989) . Recent advances in pestivirus research . Journal of General Virology 70, 253-66 . 22 . Howley, P. M., Israel, M . A ., Law, M .-F . & Martin, M. A . (1979). A rapid method for detecting and mapping homology between heterologous DNAs. Journal of Biological Chemistry 254, 4876-83 . 23 . Bornkamm, G . W ., Desgranges, C . & Gissmann, L . (1983). Nucleic acid hybridization for the detection of viral genomes. Current Topics in Immunology and Microbiology 104, 287-98 . 24 . Ridpath, J . F ., Lewis, T . L., Bolin, S . R . & Berry, E . S . (1991) . Antigenic and genomic comparison between noncytopathic and cytopathic bovine viral diarrhea viruses isolated from cattle that had spontaneous mucosal disease . Journal of General Virology 72, 725-9 . 25 . Koch, J ., Kovraa, S. & Bolund, L . (1986) . An improved method for labelling of DNA probes by nicktranslation . Nucleic Acids Research 14, 7132 . 26. Sambrook, J ., Fritsch, E. F . & Maniatis, T . (1989). Molecu-
lar Cloning, a Laboratory Manual. New York: Cold Spring Harbor Laboratory . 27 . Lewis, T . L ., Ridpath, J . F ., Bolin, S . R . & Berry, E . S . (1991) . Detection of BVD viruses using synthetic oligonucleotides . Archives of Virology (in press) . 28 . Casey, J ., Davidson, N . (1977) . Rates of formation and thermal stabilities of RNA :DNA and DNA :DNA duplexes at high concentrations of formamide . Nucleic Acids Research 4, 1539-52 . 29 . Wahl, G . M., Berger, S . L . & Kimmel, A . R . (1987) . Molecular hybridization of immobilized nucleid acids : Theoretical concepts and practical considerations . In Guide to Molecular Cloning Techniques, Methods in Enzymology (Berger, S . L . & Kimmel, A . R ., eds), Vol .152 . pp . 399-407 . New York : Academic Press . 30 . Marmur, J . & Doty, P . (1962) . Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature . Journal of Molecular Biology 5, 109-18 . 31 . Bonner, T . I ., Brenner, D . J ., Neufeld, B . R . & Britten, R . J . (1973) . Reduction in the rate of DNA reassociation by sequence divergence . Journal of Molecular Biology 81, 123-35 . 32 . Hyman, R . W ., Brunovskis, I . & Summers, W. C . (1973) . DNA base sequence homology between coliphages T7 and Oil and between T3 and Oil as determined by heteroduplex mapping in the electron microscope . Journal of Molecular Biology 77, 189-96 . 33 . Britten, R . J., Graham, D . E . & Neufield, B . R . (1974). Analysis of repeating DNA sequences by reassociation . Methods in Enzymology 29, 363-405 . 34. Yang, R . C. A ., Young, A. & Wu, R . (1980) . Bk virus DNA sequence coding for the t and T antigens and evaluation of methods for determining sequence homology. Journal of Virology 34, 416-30 . 35. Meyers, G., Rumenapf, T. & Theil, H .-J . (1989). Molecular cloning and nucleotide sequence of the genome of hog cholera virus . Virology 171, 555-67 . 36 . Hahn, Y . S ., Caller, R ., Hunkapiller, T ., Dalrymple, J . M., Strauss, J . H . & Strauss, E . G. (1988) . Nucleotide sequence of Dengue 2 RNA and comparison of the encoded proteins with those of other Flaviviruses . Virology 162, 167-80 . 37 . Deuble, V ., Kinney, R . M . & Trent, D . W . (1988) . Nucleotide sequence and deduced amino acid sequence of the nonstructural proteins of Dengue type 2 virus, Jamaica genotype : comparative analysis of the fulllength genome . Virology 165, 234-44 . 38 . Rice, C . M ., Lencher, E . M., Eddy, S . R., Shin, S . J ., Sheets, R . L . & Strauss, J . H . (1985). Nucleotide sequence of yellow fever virus : implications for flavivirus gene expression and evolution . Science 229, 726-33 . 39 . Castle, E ., Nowak, T ., Leidner, U ., Wengler, G . & Wengler, G . (1985) . Sequence analysis of the viral core protein and the membrane-associated proteins V1 and NV2 of the flavivirus West Nile virus and of the genome sequence for these proteins . Virology 147, 227-36 . 40 . Coia, G ., Parker, M . D ., Speight, G ., Byrne, M . E . & Westaway, E . G . (1988). Nucleotide and complete amino acid sequences of Kunjin virus : definitive gene order and characteristics of the virus-specific proteins . Journal of General Virology 69, 1-21 .
Hybridization analysis of genomic variability among isolates of bovine viral diarrhoea virus using cDNA probes Julia F. Ridpath* and Steven R . Bolin Virology Cattle Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, lA 50010, USA (Received 27 December 1990, Accepted 15 February 1991)
Genomic variability among 28 cytopathic and 37 non-cytopathic isolates of BVDV was evaluated using hybridization analysis . Total RNA, isolated from cell cultures infected with BVDV, was probed with each of three plasmids containing cDNA derived from the cytopathic BVDV-NADL isolate . The three cDNA plasmid probes represented about 40% of the BVDV-NADL genome . The probes originated from the 5' end of the viral genome, the region of the genome coding for the p80 polypeptide and the 3' end of the genome . Three temperatures, 63 ° C, 53 ° C and 43 ° C, were used for hybridization and washing and allowed approximately 29%, 36%, and 43% mismatch, respectively . Fifteen of the BVD viral isolates differed by less than 29% from BVDV-NADL in all three of the regions probed . Seventeen of the BVD viral isolates differed between 36% and 43% from BVDV-NADL in all three of the regions probed . Ten viral isolates differed by more than 43% from BVDV-NADL in all the regions probed . The remaining viral isolates exhibited different levels of similarity to BVDV-NADL in the three areas probed .
KEYWORDS : BVDV, genome, homology, RNA, hybridization .
INTRODUCTION Bovine viral diarrhoea (BVD) virus, an ubiquitous and economically important pathogen of cattle, is the prototype member of the pestivirus genus . Although currently classified as non-arthropod-borne members of the Togaviridae family' ,' the replication and expression strategies of pestiviruses more closely resemble those of the Flaviviridae family .' Two biotypes of BVD virus, non-cytopathic and cytopathic, are differentiated by their effect on cultured cells . ^ While only one serotype of BVD virus is recognized, there appears to be antigenic diversity among BVD viruses .'-" The BVD virus has a single positive strand RNA genome . Generally, high mutation frequencies are associated with genomes of this type ."' Thus, a degree of genomic heterogeneity would be expected among BVD viral genomes . Genomes of two cytopathic BVD viruses, BVDV-NADL and BVDV-Osloss, have been cloned and sequenced ."" These ' Author to whom correspondence should be addressed . 0890-8508/91/040291 + 08 $03 .00/0
genomes have an aligned sequence identity of approximately 74% . 2' The extent of heterogeneity among BVD viral isolates becomes important for development of diagnostic tests based on recognition of genomic sequences and for estimation of BVD viral mutation rates . While direct sequence comparison is the most precise measure of genomic heterogeneity, it is not practical for comparison of large numbers of viral isolates . Hybridization analysis is better suited for large scale comparisons and has been used to study genomic relationships among viruses .21,2' The purpose of this study was to evaluate genomic similarity among BVD viral isolates by hybridization analysis . To this end, total RNA was isolated from cell cultures infected with 65 isolates of BVD virus . The RNAs were blotted and probed with cloned sequences representing approximately 40% of the genome of the BVD-NADL virus . The genomic sequences used as
291
292
J . F . Ridpath and S . R. Bolin by three successive plaque purifications . The noncytopathic viral isolates were biologically cloned by serial passage at limiting dilution . Stock viruses were propagated in bovine turbinate (BT) cells, harvested
probes were selected because of their relatively high conservation between BVD-NADL and BVDV-Osloss viruses .
after one freeze-thaw cycle, and stored at -90 ° C . The BT cells were grown in minimal essential media
MATERIALS AND METHODS
(F15 Eagle's medium, GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum . Fetal bovine serum was tested and found free of adventitious BVD viruses, by virus isolation procedures, and free of viruses, by indirect antibodies to BVD
BVD viral isolates The BVD viruses (n=65) used consisted of 28 cytopathic (Cl through C28) and 37 non-cytopathic (NC1 through NC37) viral isolates. All but two of the viruses were isolated from animals or cell cultures originating in the United States . Viral isolates C3 and NC35 were isolated from fetal calf serum obtained from New Zealand . As denoted in Tables 1 and 2, 52 viral
immunoperoxidase staining, viral neutralization, and radioimmunoprecipitation (RIP) procedures . The BT cells were free of adventitious BVD viruses as determined by indirect immunoperoxidase staining .
isolates were isolated from bovine tissue, eight from fetal calf sera, two from porcine cell lines, and one each from a rabbit cell line, a bovine cell line, and deer tissue . Cytopathic viral isolates were biologically cloned
Table 1 .
RNA slot-blots To generate RNA blots, confluent monolayers of BT cells were grown in 25 cm 2 flasks and inoculated with
Hybridization of cytopathic BVD viral RNA pBV -18
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26 C27 C28 Positive % Positive
pBV - p80
pBV - KPB
Source
63C
53C
43C
63C
53C
43C
63C
53C
43C
a a b a a a b a a a a a a a a a b a a a a a a a a a a b
+ + + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + + + + +
+
+ + + + + + + + + + + + + + + + + + + -
14 50
16 57
22 79
17 61
23 82
15 54
16 57
23 82
a : cattle tissue; b: fetal calf serum .
-
15 54
-
BVDV genomic homology Table 2 .
293
Hybridization of non - cytopathic BVD viral RNA pBV - 18
NC1 NC2 NC3 NC4 NC5 NC6 NC7 NC8 NC9 NC10 NC11 NC12 NC13 NC14 NC15 NC16 NC17 NC18 NC19 NC20 NC21 NC22 NC23 NC24 NC25 NC26 NC27 NC28 NC29 NC30 NC31 NC32 NC33 NC34 NC35 NC36 NC37
Positive Positive
pBV4- p80
pBV - KPB
Source
63C
53C
43C
63C
53C
43C
a a a a b a a c a a a a b a
+ -
+ + -
+ + + + + + + + + + + + + +
+ + + -
+ + + -
+ + + +
+ + + + + + -
+ + + + + + +
d
-
-
+
a a a a a
-
-
+ + + + +
e
-
-
+
a a c a a a a f a a a a a b b a
-
-
+ + + + + -
63C
+ +
53C
43C
+ + + + + -
+ + + + + +
+
+ + +
1 3
2 5
26 70
3 8
9 24
31 84
3 8
6 16
29 78
a : cattle tissue; b : fetal calf serum ; c : porcine cell line; d : rabbit cell line; e: deer tissue ; f: bovine cell line .
BVD virus at a multiplicity of infection (moi) of 10. Cell cultures infected with cytopathic viruses were harvested when 90% of cells exhibited cytopathic effect, usually 24 to 30 h after inoculation . Cell cultures infected with non-cytopathic viruses were harvested 48 h post-inoculation . Cell cultures were harvested by one freeze-thaw cycle . Cell debris and culture fluids were collected and a 500 gl aliquot was removed for titration of the virus . End points of virus concentration were determined in microtitration plates seeded with BT cells using cytopathic effect for cytopathic viruses and indirect immunoperoxidase staining for non-cytopathic viruses ." Only harvested
material that had at least 10 6 CCID ml -1 of BVDV were used . RNA was prepared for blotting by adding 10 ml PK buffer (0 . 2m Tris-Cl, pH 8 . 0, 25 mm EDTA, 0.3 M NaCl, 2% SDS, and 200 tg ml -1 proteinase K) to 10 ml of harvested material (equivalent to approximately 1 x 10' cells) . This mixture was incubated at 37° C for 30 min, extracted once with an equal volume of a 1 :1 mixture of phenol and chloroform, and once with an equal volume of chloroform . After extraction, 20 ml of formaldehyde [6 . 15 M in 20 X SSC (3-00m NaCl, 0 . 34m sodium citrate, pH 7 . 0)1 was added followed by incubation at 65 ° C for 15 min . A volume of 0. 5 ml of this solution (equivalent to ap-
294
J . F . Ridpath and S . R. Bolin reagent, 0-1% SDS, and 100 µg ml - ' of denatured salmon sperm DNA) . After prehybridization blots were hybridized for 16 h at the aforementioned temperatures . The hybridization buffer was the same as that used for prehybridization with the addition of 0. 1 .tg of denatured cDNA probe (specific activity at
proximately 1 . 25 x 10 5) was blotted onto nitrocellu-
lose. Nucleic acid from cells infected with BVDVNADL virus and uninfected cells was blotted as positive and negative controls, respectively . After blotting, nitrocellulose membranes were rinsed in 2
X
SSC and baked for 2 h at 80 ° C .
least 2 x 108 cpm
lag -1 ) .
Hybridized filters were
washed for 20 min at room temperature in 1 x SSC, 0. 1% SDS, followed by two washes of 1 h each in 0. 2 x SSC, 0. 1 % SDS at the temperature used for hybridization ." After washing, blots were exposed to
Preparation of probes and hybridization Three plasmids, pBV-18, pBV4-p80, and pBV-KPB, derived from BVDV-NADL (courtesy of M . Collett, Molecular Vaccines Inc ., Gaithersburg, MD) were
X-Omat AR film (Eastman Kodak Company, Oak Brook, IL) . The RNA from cultures infected with certain viral isolates did not hybridize to any of the probes. To determine if sufficient viral RNA was blotted onto the
used as probes for evaluation of genomic similarity (Fig . 1). The plasmids contained inserts encompassing nucleotides 24 to 1308 (pBV-18), nucleotides 5644 to 7949 (pBV4-p80), and nucleotides 9857 to 12020 (pBV-KPB) . The probes were selected on the basis of conservation between published nucleotide sequences of BVD viral genomes as determined using
membrane for detection, these samples were probed with a 30-base oligomer. The 30-base oligomer (5' act cca tgt gcc atg tac agc aga gat ttt 3') was complementary to a sequence conserved in both the BVDVOsloss and BVDV-NADL genomes . In previous stud-
the Align sequence alignment program (Scientific and Educational Software, Silver Spring, MD) . The overall aligned sequence identity for the genomes of BVDNADL and BVDV-Osloss (minus the extreme 5' and 3'
ies, oligomers containing the same sequence hybridized with 86% of the BVD viruses tested ." The oligomer was end labelled using T4 polynucleotide
ends) is approximately 74% .2' The aligned sequence identities for the portions of the genome contained in plasmids pBV-18, pBV4-p80, and pBV-KPB are 84%, 82%, and 77%, respectively . Radiolabelled doublestranded probes were prepared from plasmids by nick translation following the method of Koch et al." as modified by Sambrook et al." Spin columns (5 Prime-3 Prime, Paoli, PA) were used to remove unin-
kinase. 26 Unincorporated label was removed by pre-
corporated label . Hybridization reactions, using the above plasmids as probes, were performed as follows. Blots were prehybridized for at least 6 h at either 63 ° C, 53 ° C, or
tions were carried out at 50° C for 2 h in 6 x NET, 0. 1 SDS, 5 x Denhardt's and 5-10 ng ml - ' of end labelled oligomer probe (specific activity at least 5 . 0 x 10 7 cpm gg -1 ) . After hybridization, the blots were
43 ° C, in hybridization buffer (6 x SSC, 2 x Denhardt's
washed three times, for 5 min each, in 6 x SSC, 0 . 1
pBV-18
p20
ization reactions with the oligomer were performed as described by Lewis et al." In short, blots probed with oligomers were prehybridized at 65 ° C for 4-5 h in 6 x NET [0 . 15 M NaCl, 15 mm Tris-HCI (pH 8 . 3), 1 mm EDTA], 0-1% SDS, 5 x Denhardt's, and 100 lag ml - ' of
denatured salmon sperm DNA . Hybridization reac-
pBV-KpB
pBV4-p80
gp25 gp48
cipitation with cetylpyridinium bromide ." Hybrid-
p125 gp53
p58
p75
p80
Location of BVDV-NADL cDNA probes in open reading frame . The location of the cDNA plasmids (∎) in relationship to the BVDV-NADL open reading frame (∎) and sequences coding for BVD viral polypeptides'-20 is shown. Fig. 1 .
295
BVDV genomic homology SDS at room temperature . The blots were then washed twice for 30 min each in 3 x SSC, 0 . 1 % SDS at 50° C, and twice for 20 min in 1 x SSC, 0 . 1 % SDS at 50 ° C . After washing, blots were exposed to X-Omat AR or XAR-5 film (Eastman Kodak Company, Oak
averaged about 600 by in length as determined by gel electrophoresis followed by autoradiography . Formamide was not present in the hybridization buffer used for the study . Using this formula, the T m for the cDNA probe and the bound viral RNA is 107 ° C . The T m of a nucleic acid hybrid is depressed by base mismatching, with 1 % mismatch reducing the T m between 0 . 5 ° C and 1 .4°C .30-34 The exact value of this ratio depends upon fraction of guanine and cytosine
Brook, IL) for 48 h .
Calculation of melting temperature
(G+C) residues and the distribution of mismatched bases . While the G + C value can be estimated, the distribution of mismatches within the duplex cannot be determined by the methods used . Using a value of 0 . 5 ° C of Tm lowering per each percent mismatch, 34
The melting temperature (T m) for DNA probes hybridized to immobilized RNA has been defined by the following formula:28,29 = 79 .8 + 18 . 51ogM + 58 . 4(mole fraction GC) + 11 . 8(mole fraction GC)2 -820/L-0 . 5 (% formamide)
Tm
failure of probes to hybridize at 63 ° C, indicates a mismatch of at least 29% between cDNA probe and viral RNA target . Similarly, lack of hybridization at 53 ° C and 43 ° C indicates a minimum percentage mismatch of 36% and 43%, respectively .
Where M is the concentration of monovalent cation in moles per litre . For the hybridization conditions used in this study M equaled 0 . 99 . Mole fraction GC is the base composition expressed as mole fraction of guanine and cytosine residues . The mole fraction GC for BVDV-NADL is 0 .46 .21 L is the length of the
RESULTS
hybridized duplex and is determined by the length of the probe . Nick translated probes used in this study
63C
Hybridization results are summarized in Tables 1 and 2 and an example is shown in Fig . 2a . Genomic
pBV4-p80 63C 53C 43C
pBV-18 53C 43C
63C
pBV-KPB 53C 43C
- C9 -C13 -NCI - NC5 -C15
(b) 63C
43C -No dilution -1 :2
00
%40
-1 :10 -1 :100
Fig . 2 . Hybridization of cDNA plasmids to cytopathic and non-cytopathic BVD viral RNA . Examples of hybridization of the three cDNA probes at the three stringencies used to cytopathic and non-cytopathic BVD viral RNA is shown in (a) . A control comparing the sensitivity of the hybridization reaction at different stringencies is shown in (b) . Shown are dilutions (no dilution, 1 :2, 1 :10, 1 :100) of RNA isolated from BVDV-NADL infected cells probed with pBV-18 at 63 ° C and 43 ° C. All hybridizations shown were performed as described in Materials and Methods .
J. F. Ridpath and S. R. Bolin
2%
variation was evaluated based on the hybridization of probes, corresponding to the 5' end of the BVDVNADL genome, the region of the BVDV-NADL genome coding for the p80 polypeptide and the 3' end of the BVDV-NADL genome, at different stringencies. Fifteen of the BVD viral isolates differed by less than 29% from BVDV-NADL in all three of the regions probed . Seventeen of the BVD viral isolates differed between 36% and 43% from BVDV-NADL in all three of the regions probed . Ten viral isolates differed by more than 43% from BVDV-NADL in all the regions probed . The remaining viral isolates exhibited different levels of similarity to BVDV-NADL in the three areas probed (Tables 1 and 2) . To if the difference seen in hybridization at stringencies was due to the amount of BVD, present on the blot dilutions of total RNA
determine different viral RNA from BVD-
NADL infected cells were hybridized to the probes at 63 ° C, 53° C, and 43 ° C. An example of the results from this test is shown in Fig . 2b . No difference was seen in hybridization results performed at 63 °C, 53 ° C, and 43 ° C . The highest dilution detected at all three stringencies was 1 :10 . Total cell RNAs, isolated from cell cultures infected with the 10 isolates which did not bind to any of the plasmids, were blotted and probed with a 30-base oligomer to determine if viral RNA was present . An example of the hybridization reactions is shown in Fig . 3 . Nine of the 10 isolates which did not hybridize
A
to the plasmid probes were detected with the oligomer probe, indicating that viral RNA was present on the blot . One isolate, C28, did not hybridize to the oligomer probe . The failure to hybridize may have been due to low homology between the probe and the viral genome or insufficient viral RNA present for detection . All viral preparations used had approximately the same infective titre . However, infective titre does not take into account the presence of unpackaged viral RNA . Thus it is possible that a BVD isolate which packaged the majority of the RNA present would go undetected while a virus which was less efficient at packaging progeny RNA would be detected even though both viruses were grown to the same titre . As noted above, similarity to BVDV-NADL in the areas probed was not uniform with all viral isolates (Tables 1 and 2) . For example, eight viral isolates were more similar to BVDV-NADL in the p80 coding region than in the other two regions probed and five viral isolates were more similar in the 3' end region . A difference in hybridization was observed between non-cytopathic and cytopathic BVD viral isolates . At 43 ° C the percentage of cytopathic and non-cytopathic BVDV isolates recognized by at least one of the three probes were similar (86% versus 84%) . However, the percentage of cytopathic BVDV isolates bound by at least one of the plasmids at 63 ° C (58%) and 53° C (61 %) was greater than the percent-
B
C
D
NADL
Cell control -
C25-
C26-
C27-
C28 -
NC32--
Fig. 3 . Detection of BVD viral RNA with an oligonucleotide probe . To determine if failure of cDNA plasmids to hybridize with RNA from some BVDV isolates was due to insufficient RNA present for detection of low similarity, viral RNA was also probed with a 30-base oligomer complementary to BVDV-NADL . Lane A, B, C, and D represent blotted viral RNAs probed with plasmids pBV-18, pBV4-p80, and pBV-KPB, and the 30-base oligomer, respectively . Hybridizations with plasmids were performed at 43 ° C. Hybridizations with the oligomer were performed as described in the Materials and Methods section .
BVDV genomic homology
297
age of non-cytopathic BVDV isolates (11% and 32%, respectively) bound at these stringencies .
hybridized to at least one of the probes while only 4 out of 37 (11%) non-cytopathic BVD viruses hybridized to at least one of the probes . Under the lower stringency of 43 ° C, the percentage of cytopathic and
DISCUSSION
non-cytopathic viral RNAs that hybridized to at least one of the probes was similar (89% and 84%, respectively) .
In this study, we examined the nucleotide similarity among RNA from isolates of BVDV to plasmid probes derived from BVDV-NADL . The extent of genomic variability observed the BVDV-NADL probes and 50 of the BVD viral isolates evaluated was higher than the 74% reported between BVDV-NADL and BVDVOsloss . 21 Similarity to portions of the nucleotide sequence of BVDV-NADL was 57% or more for 55 viral isolates . Of these 55, 29 were 64% or more similar and 15 were 71% or more similar . Ten BVD viral isolates failed to hybridize to the BVDV-NADL cDNA probes under conditions which theoretically allowed 43% mismatch . No attempt was made in this study to determine the correspondence between similarity estimations based on hybridization analysis and similarity based on aligned sequence identity . However, it is intriguing to note that the Alfort strain of hog cholera virus, another member of the pestivirus family, has an overall aligned sequence identity to BVDV-NADL of 68% (32% mismatch) . 35 Because the BVD viral genome is a single-stranded RNA, a degree of genomic heterogeneity would be expected among BVD viral genomes . The recognition of only one serotype of BVD virus, however, suggests conservation of at least portions of the BVDV genome . Little information is available regarding genomic variability among pestiviruses . A number of studies have examined genomic variability among flaviviruses, which have similar replication and expression strategies to those of the pestiviruses . 3 Two strains of Dengue 2 virus belonging to the same serotype have sequence similarity of 91 % . 36 The similarity between Dengue viruses of two different serotypes are in the range of 64% to 66% . 37 The similarity among published sequences of yellow fever virus, 38 West Nile virus, 39 Kunjin virus,40 and Dengue 2 virus, Jamaica genotype37 are in the 45% to 55% range as determined using the Fastdb software program (Intelligenetics, Inc ., Mt . View, CA) . As a group genomic variability among the BVDV viruses observed in this study is similar to that observed among the flaviviruses as a group . However, when compared to the genomic similarity observed between flaviviruses belonging to the same serotype it is higher. A marked difference was observed between the hybridization of cytopathic and non-cytopathic BVD viral isolates to the plasmid probes at 63 ° C . At this stringency 16 out of 28 (57%) cytopathic viruses
The possibility of greater than 40% variation in BVD viral genomes increases the degree of difficulty in devising a reliable diagnostic probe based on genomic sequences . However, genomic variation in BVD viral isolates may be exploited to the researcher's benefit in epidemiology and pathogenesis studies .
ACKNOWLEDGEMENTS The authors would like to thank Dr Marc Collett for his generosity in supplying the cloned cDNA plasmids . In addition, the authors would like to thank Rebecca Zaworski, Shari Steadham, and Sharon Stark for technical assistance, and Sandy Johnson, Gene Hedberg, and Tom Glasson for manuscript preparation .
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