Restriction Endonuclease Analysis of Bovine Herpesvirus 1 DNA and Nucleic Acid Homology between Isolates
By B R U C E S. SEAL, 1 S T E P H E N C. ST. J E O R 1. AND R O B E R T E. L E E T A Y L O R 2
1Departments of Microbiology and Biochemistry, University of Nevada School of Medicine, Reno, Nevada 89557 and 2Agriculture Experiment Station, University of Nevada School of Veterinary
Medicine, Reno, Nevada 89502, U.S.A.
(Accepted 20 August 1985)
SUMMARY
Isolates of bovine herpesvirus 1 (BHV-1) are associated with a variety of clinical manifestations. To determine if a single form of BHV-I was responsible for the different virus-associated diseases or whether subpopulations of various isolates produced different clinical symptoms, studies were initiated to examine the DNA restriction enzyme patterns and nucleic acid homology between virus isolates from respiratory infections and other clinical syndromes. Differences between the genomes of several virus isolates were detected using DNA restriction enzyme analyses. However, nucleic acid hybridization studies of the virus DNAs using filter and liquid hybridization indicated at least a 95~ genetic homology between the virus isolates from different types of infections. Additionally, these studies demonstrated that the DNA of BHV-1 had an average molecular weight of 84 x 106.
Bovine herpesvirus 1 (BHV-1) has been associated with a variety of clinical syndromes. These are primarily respiratory, but may also inclfide genital infections, encephalitis, ocular carcinoma and abortion. The virus associated with respiratory diseases has been referred to as infectious bovine rhinotracheitis (IBR) virus, whereas the virus isolate associated with genital infections has been termed infectious pustular vulvovaginitis (IPV) virus (Kahrs, 1977; Pastoret et al., 1982). Misra et al. (1983) reported that isolates of BHV-I could be divided into three distinct groups based upon the virus DNA restriction enzyme patterns. As in the situation of herpes simplex virus types 1 and 2 in humans, the possibility exists that respiratory and genital infections may be caused by partially related viruses that appear serologically similar, but by analysis of the virus DNA represent different virus types. Consequently, the investigations reported here were initiated to determine the amount of genetic homology between a respiratory isolate of BHV-1 and other clinical isolates using restriction enzyme analysis and nucleic acid hybridization of virus DNA.
Since reports indicated that DNA from BHV-1 isolates associated with respiratory infections (IBR virus) had different restriction patterns from the DNA isolated from viruses associated with genital infections (IPV virus) (Engels et al., 1981), we initially differentiated virus isolates using restriction enzyme analysis and nucleic acid hybridization. The BHV-1 isolates used in these studies were IBR virus Los Angeles (LA), a respiratory disease isolate, obtained from the American Type Culture Collection and an IPV virus isolate obtained from the Department of Microbiology, School of Veterinary Medicine, University of California, Davis. A variety of isolates from various clinical disease states were also obtained from the School of Veterinary Medicine at Washington State University, Pullman, Washington and from the Agriculture Experiment Station at the University of Nevada. Bovine embryonic lung cells were used for all experiments and cultured in Dulbecco's MEM with 5 ~ foetal calf serum. Virus DNA for use in restriction enzyme analysis was isolated and purified essentially as described by Huang et al.
(1973). For hybridization analyses virus D N A was isolated from caesium chloride density gradient fractions corresponding to a density of 1-730 g/ml (Graham et al., 1972).
Various isolates of BHV-1 representing a variety of clinical syndromes were compared by restriction enzyme analysis and Southern blot hybridization as illustrated in Fig. 1 (a, b). Even though there were differences in virus D N A restriction enzyme patterns there appeared to be no correlation between the type of pattern obtained and the corresponding clinical manifestation. The D N A restriction enzyme patterns depicted in Fig. 1 lanes 3, 9, 10, 11 and 13 are from 'respiratory' isolates and conform to patterns obtained from vaginal isolates (lanes 2, 4, 5 and 6), an isolate from an aborted foetus (lane 8) or from bovine ocular carcinoma (lane 7). It is of interest to note that the IPV virus isolate illustrated (lane 2) has a pattern exactly like that of a respiratory isolate (lane 13). Additionally, genital isolates (lanes 5 and 6) may have identical patterns to IBR virus LA isolate (lane 3). Another difference is that of a restriction enzyme pattern obtained from the D N A of an encephalitis isolate of BHV-1 (Fig. 1, lane 12). Although very faint in the illustration of the gel it demonstrated a pattern conforming to that of the 'Cooper ' isolate of BHV-1 (Mayfield et al., 1983).
To initially determine the nucleic acid homology between isolates, the BHV-1 D N A fragments in Fig. 1 (a) were transferred to nitrocellulose paper (Southern, 1975) and hybridized (Maniatis et al., 1982) with a nick-translated (Rigby et al., 1977) IBR virus LA isolate 32p_ labelled D N A probe (Fig. 1 b). In every case the probe D N A hybridized to all the fragments present for each BHV-1 isolate while not hybridizing to the lambda phage D N A used as a control. This demonstrates the extensive homology between D N A sequences present in all the BHV-1 isolates examined. When the IPV virus isolate D N A (lane 2) was used as a probe of an identical gel blotted and hybridized, duplicate results were obtained (data not shown).
It was apparent from the initial restriction enzyme analysis and hybridization data (Fig. 1 a, b) that a high percentage of homology existed between isolates of BHV-1 regardless of the clinical manifestation. Therefore, more extensive studies were conducted using the LA isolate (Fig. 1, lane 3) and an IPV isolate (Fig. 1, lane 2), two isolates from different clinical syndromes which show the greatest variation in restriction enzyme pattern. One ~tg of D N A isolated from the IBR LA and IPV virus was digested with 5 units of the restriction enzymes HindlII , EcoRI or B a m H I (Bethesda Research Laboratories), end-labelled with [~-32p]dATP, using the Klenow fragment of D N A polymerase I in the presence of unlabelled dGTP, dCTP and dTTP (Maniatis et al., 1982) and electrophoresed (Fig. 2). Molecular weights of the resultant fragments were determined by comparison with phage lambda DN A standards (Bearden, 1979). Molar amounts of the virus D N A restriction fragments were calculated from D N A obtained from an in vitro infection using 32p-labelled H3PO 4 to label the virus D N A uniformly. Following electrophoresis and extraction of the virus D N A fragments from agarose gel slices, molarities were determined as described by Wharton et al. (1981) and the data are presented in Table 1. The sum of the molecular weights of individual restriction fragments indicated average molecular weights for IBR virus and IPV virus D N A to be 84 × 106. Genome molecular weights for both BHV-1 isolates obtained by summation of the molecular weights of virus D N A fragments were lower than that reported by Engels et al. (1981), but concurred with that of Mayfield et al. (1983) and Misra et al. (1983).
Although the results of blot hybridizations indicated homology between all D N A fragments
Fig. 1. Comparison of HindIII restriction enzyme cleavage patterns and Southern blot hybridization of clinical isolates of BHV-I. Following digestion with HindIII 10 ~tl of tracking dye (50~ Ficoll, 0.75~ bromophenol blue, 0.1 ~ EDTA) was added to the reaction mixture and electrophoresed in a horizontal 0-7~ agarose slab gel for 14 h at 75 mA in Tris-acetate buffer (40 mM-Tris, 20 mM-sodium acetate, 18 mM-NaCI, 2 mM-EDTA, pH 8.4). The gel was stained with ethidium bromide and photographed with Polaroid type 667 film during u.v. transillumination (a). The DNA fragments were transferred to nitrocellulose and hybridized with a nick-translated 32pqabelled IBR LA virus DNA probe (b). Lane 1, lambda phage DNA restricted with HindllI; lane 2, IPV; lane 3, IBR LA isolate; lane 4, vaginal isolate following vaccination; lanes 5 and 6, vaginal isolates from genital infections; lane 7, isolate from bovine ocular carcinoma; lane 8, isolate from an aborted foetus; lane 9, Colorado isolate obtained from U.S.D.A., National Veterinary Service, Ames, Iowa; lanes 10 and 11, respiratory disease isolates; lane 12, isolate from an encephalitis infection; lane 13, respiratory disease isolate.
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Fig. 2. Comparative restriction endonuclease cleavage patterns of IBR virus and IPV virus DNA. IBR virus D N A (lanes 1, 3 and 5) and IPV virus D N A (lanes, 2, 4 and 6) were digested with HindllI (lanes 1 and 2), EcoRl (lanes 3 and 4) and BamHI (lanes 5 and 6). Following digestion, fragments were end- labelled and then electrophoresed as described in Fig. 1. The gel was dried on to Wha tman 3MM filter paper with a Bio-Rad slab gel dryer and exposed to Kodak XAR-5 autoradiography film. Lambda phage DNA digested with H/ndll l and EcoRI was co-electrophoresed with restricted BHV-1 virus D N A and used as a reference to determine fragment sizes by regression analysis (Bearden, 1979).
T a b l e l . Sizes* oJ" bovine herpesvirus 1 virus isolate DNA fragments produced by restriction endonuclease cleavage
HindI I I Eco R I Bam H I A . k ~,
IBR IPV IBR IPV IBR IPV
A 14.3 A 14-3 A 28.4 A 28-4 A 18-8 A 18,8 B 12.9 B 12,9 B 15.0 B 15.0 B 14-3 B 14-3 C 11.37 C 11.37 C 12.4 C 12.4 C 12,4 C 12.4 D 10.4? D 10.4? D 10-9 D 11.8 D 11.8 D 10.9 E,F 8.6+ + E,F 8.65 E 8.9 E 8.9 E 10.9 E 8.3 G,H 8.0:~ G,H 8.0:~ F 5.8 F 5.8 F,G 8-35 F 8.2 I 6,1 I 6.8 G 2.1 G 1.0 H 1.0 G,H 3.7§ J 5.8 J 6.1 H 1.2 I 1.0 K 5-5 K 5.5 L 2.8 L 4.6 M 1.8 M 2.8
N 1.7 O 1.1 P 1.0
* Molecular weights ( x 10 -6) were determined by regression analysis of fragments electrophoresed with bacteriophage | ambda D N A fragments.
? 0.5 M amount approximately. All other values are approximately 1-0 M. The molar abundance of each fragment was determined by the formula M = (Fragment c.p.m./total c .p.m.)/(Fragment tool. wt./mol, wt. of intact molecule).
++ Present in 1.5 M amount. § Present in 2.0 M amount.
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Fig. 3. Comparison of IBR and IPV virus DNA by DNA DNA reassociation. (a) Hybridization of32P - labelled IBR virus DNA in the presence of unlabelled DNA from IBR virus (O), IPV virus (O) and lambda phage ( . ) . (b) Hybridization of 32p-labelled IPV virus DNA in the presence of unlabelled DNA from IPV virus (O), IBR virus ((3) and tambda phage ( I ) . Following nick translation (Rigby et al., 1977) and heat denaturation, single-stranded probe DNA was collected by hydroxylapatite column chromatography (Martinson, 1973). The labelled probe, 4 ~tg unlabelled virus DNA and 20 btg salmon sperm DNA were sonically disrupted to approximately 5S in size (Cedar, 1976), heat-denatured at 100 °C for 15 min and then brought to 1 ml in 1 x PIPES buffer (0.72 M-NaCI, 0.01 M-PIPES, 0.001 M- EDTA, pH 7.0). The DNA was allowed to reassociate at 61 °C (T m - 25 °C) and following initiation of the reaction, samples were taken at 0.5, 1, 2, 5, 8, 16 and 24 h. The fraction of reassociated 32P-labelled DNA was analysed by $1 enzyme differential digestion (Vogt, 1973). Co t (tool. s/1 deoxynucleotide) was calculated on the basis of the total amount of virus DNA present in each assay. Background values were subtracted and the data normalized following five separate hybridizations with three samples taken per time point.
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of the isolates examined, partial homology between a specific fragment and the probe D N A could produce a similar pattern by autoradiography, as would complete hybridization to the same fragment. To quantify differences in genetic composition between the isolates of BHV-1, which exhibited the greatest variation in restriction pattern, liquid hybridization between the DNA of the IBR LA and IPV virus isolates was conducted and the results plotted as the percent DNA reassociated versus Cot and incubation time (Britten & Kohne, 1968). In both sets of hybridizations, the 32p-labelled virus probes did not reassociate significantly in the presence of lambda phage DNA used as a control. As illustrated in Fig. 3 (a) there were no differences in the rates of reassociation of 32p_labelled IBR LA virus DNA with unlabelled IBR LA virus D N A or with unlabelled IPV virus DNA. Conversely, 32p-labelled IPV virus D N A reannealed at the same rate with unlabelled IPV virus DNA as it did with unlabelled IBR LA virus D N A (Fig. 3b). The C0tl/2 value of 4 x 10 -2 was the same in both sets of reactions. Consequently, there is very little detectable quantitative difference in sequence homology between these isolates of BHV-1.
The results of the liquid hybridization studies indicate a high degree of homology between the DNA of IBR and IPV virus isolates examined. Although a 5 ~ mismatch during D N A hybridizations may occur (Sugino & Kingsbury, 1976), the conditions used for both the Southern blot hybridizations and the liquid hybridizations were relatively stringent. Southern blots were hybridized at 15 °C below the Tm of BHV-1 DN A in 5 0 ~ formamide followed by extensive washing at 60 °C in 0-1 x SSC, while the liquid hybridizations were conducted at 25 °C below the T~ of BHV-1 DNA in 0.72 M-NaCl.
The results presented indicate that, although the D N A from the IBR LA virus isolate, IPV and other BHV-1 virus isolates may vary in their restriction pattern, the D N A sequences of these isolates are at least 95 ~ homologous. Differences in restriction enzyme patterns are probably due to specific point mutations resulting in the loss or gain of certain restriction endonuclease sites. Considering the high degree of D N A homology between various isolates, the reason for differences in clinical syndromes is still not well understood. In agreement with Misra et al. (1983), it may be that routes of infection and/or other environmental factors determine whether a particular isolate causes a typical respiratory tract infection or some other type of disease such as an encephalitis or genital infection. Also, the relationship between
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2792 Short communication
BHV-1 isolates does no t appea r analogous to tha t o f h u m a n herpes s implex virus types 1 and 2 where there is a 5 0 ~ nucleic acid homology di f ference (Kie f f et al., 1972; Ludwig et al., 1972).
The authors thank James Evermann, D.V.M. (W.S.U.) and D. G. McKercher, D.V.M. (U.C.D.) for clinical isolates of bovine herpesvirus 1 and Jan Amesbury for typing the manuscript. Appreciation is extended to Mark R. Hall, Jesse Martinez, Berch E. Henry and Elizabeth Griffith for their critical review of the manuscript. These investigations were partially supported by Public Health service grant R01 CA28029, the Research Advisory Board of the University of Nevada, Reno (S.S.J.) and the Agriculture Experiment Station, University of Nevada, Reno (R.E.L.T.). In partial fulfilment for the Ph.D. in biochemistry (B.S.S.).
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