Journal of Medical Virology 38:1621 (1992)
Genomic Variation Among Herpes Simplex Virus Type 1 Strains: Virus DNA Analysis of Isolates From Saudi Patients Mohammed N. Al-Ahdal, George Kessie, Mohamed A. Taha, Fahad J. Al-Shammary, and Mohamed Ettayebi Departments of Biological and Medical Research (M.N.A.-A., G.K., M.A.T.) and Pathology and Laboratory Medicine (M.N.A.-A.), King Faisal Specialist Hospital and Research Centre, and College of Applied Medical Sciences, King Saud University (F.J.A.-S.), Riyadh, Saudi Arabia; Biotechnology Laboratory, College of Sciences, Sidi Mohamed Bin Abdallah Uniuersity, Fes, Morocco (M.E.I Fifty-two clinical isolates of herpes simplex virus type 1 (HSV-1) from Saudi Arabian patients were analysed by restriction endonuclease digestion of the virus DNA using the enzymes Hindlll and BarnHI, followed by hybridization with 32P labelled DNA of laboratory strain F. Of the isolates, 17 were resolved into four distinct cleavage patterns with Hindlll restriction enzyme. The remaining 35 strains had the same cleavage pattern as the standard HSV-1-F. Further investigation of the 52 isolates with BarnHI, which is a multicut enzyme and therefore capable of higher resolution, differentiated 47 of the 52 isolates and were assigned into nine cleavage groups. Comparing our findings with similar studies reported elsewhere suggest geographic clustering of HSV-1 strains. Fragments giving rise t o the observed DNA polymorphism were mapped t o the unique region of the long and short segments of the genome. GI1992 Wiley-Liss, Inc.
KEY WORDS: HSV-1 DNA analysis, HSV-1 DNA cleavage pattern
INTRODUCTION Herpes simplex virus type 1(HSV-1)is a n important human pathogen, which after primary infection can remain latent in the relevant sensory ganglia [Brown e t al., 1978; Lonsdale et al., 19801. Periodic reactivation of the virus serves as a source of infection of individuals in close contact with the carrier. The spread of HSV-1 is, however, restricted within a community or a group of individuals, such as family members. Previous attempts to classify HSV-1 strains and to establish interrelationships among geographic origins of clinical isolates were carried out by virion polypeptide analysis [Pereira et al., 19761. Recent molecular techniques have refined the methods of determining intratypic variations. Viral genome polymorphism can be visual0 1992 WILEY-LISS, INC.
ized by treating the DNA with restriction endonucleases (RE). Such treatment produces strain specific cleavage patterns, thus allowing unambiguous classification of the strains. This technique has been used to trace nosocomial transmission of HSV-1 [Buchman et al., 1978; Linneman eta]., 19781. In addition, i t was used to differentiate between strains from different individuals [Ueno et al., 1982; Whitley et al., 19821, different sites of infection in the same individual [Alam et al., 19891, and geographic origin ISakaoka et al., 1985, 19861, a s well a s in family clusters [Sakaoka e t al., 19841. The basis of such studies is that the cleavage patterns of two unrelated strains of HSV-1 are not identical [Roizman and Buchman, 1979; Roizman, 1980a, b]. The variation in the fragments generated can be distinguished by simply referring to the published cleavage maps of the relevant restriction enzyme [Wilkie, 1976; Locker and Frenkel, 19791. Whereas numerous studies have been performed on DNA polymorphism among HSV-1 isolates from different parts of the world [Sakaoka et al., 1985, 1986; Alam e t al., 19891, we are unaware of any investigation carried out in the Middle East. King Faisal Specialist Hospital and Research Centre is a 530-bed, tertiary care hospital in Riyadh, Saudi Arabia. Since it is a referral medical centre, the hospital receives a large number of patients from various parts of the kingdom. We therefore used this opportunity to study DNA polymorphism among 52 clinical isolates of HSV-1 obtained from Saudi patients. The extent of genomic variation among these isolates was determined, based solely on the gain andlor loss of restriction enzyme cleavage sites.
Accepted for publication February 6, 1992. Address reprint requests to Dr. Mohammed N. Al-Ahdal, Biological and Medical Research Department (MBC-03),King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia.
HSV-1 DNA Polymorphism in Saudi Arabia
B H ~ ~ I I I n
17
B=D+G
10 I
I
I
J
I
A
1
K
/
L
I
D
M l N l G
. I
C=D+M E-HIG
F-H+M
Fig. 1. A Structure of HSV-1 genome. Unique sequences U, and Us are bound by terminal reiterated sequences ITR,, TR,], which are also present in inverted forms [IR,, IR,] at the junction of the L and S components. B: HindIII and BarnHI cleavage maps of strain F [Locker and Frenkel, 1979; Roizman and Buchman, 1979; Post et al., 1980; Poffenberger et al., 19831. Only the prototype arrangements are shown. The three other isomeric forms can be generated by inverting L, S, or both components. Arrows under each map identify the positions of variable cleavage sites found in the HSV-1 isolates.
MATERIALS AND METHODS Viruses, Cells, and Isolates Strain F of HSV-1 (HSV-1-F, ATCC VR-733) was used a s a standard laboratory strain and purchased from ATCC (Rockville, MD). Clinical isolates were initially isolated and identified as HSV-1 by monoclonal antibody immunofluorescence a t the Diagnostic Virology Laboratory. Baby hamster kidney cells (BHK-BlI C13, Cat.# 03-420, Flow Laboratories, Irvine, Scotland) were used for propagation of the virus. The cells were maintained in Eagle's minimal essential medium (EMEM) containing 10% fetal calf serum (FCS), 50 Uiml penicillin and 50 pg/ml streptomycin. Growth of Virus Confluent monolayers of BHK-21iC13 cells in roller bottles were infected with 5 PFUiml of HSV-1-F or stock virus of HSV-1 clinical isolates. At 1hour postinfection, 25 ml of EMEM containing 1%FCS were added to each culture bottle and incubated a t 37°C for 36 hours or until maximum cytopathic effect was evident. The medium containing virus particles was collected and centrifuged a t low speed to remove cellular debris, then a t 30,000 g for 1hour a t 4°C. The pellet was resuspended in TNE buffer (0.1 M Tris-HC1, 0.01 M NaC1, 0.01 M EDTA, pH 7.4), and stored a t -20°C. Preparation of Virus DNA Virus pellets were layered on a linear 20-60% (w/v) sucrose gradient in TNE buffer and centrifuged at 50,000 g for 2 hours a t 4°C. The band containing purified virus was harvested and dialysed against TNE buffer. The dialysate was centrifuged at 30,000 g for 1 hour at 4°C. The pelleted purified virus was disrupted with 1% SDS, 1 mM EDTA, and 250 pg/ml proteinase
TABLE I. Frequency of Variable Restriction Endonuclease Cleavage Sites of HSV-1 Clinical Isolates Among Saudi Patients Hind111 pattern (1) Fusion fragment 1-0 11/52a (2) Fusion fragment K-L 4/52 (3) Fusion fragment M-N gain of cleavage 1/52 site in 0 (4) Two fusion fragments 1-0 & K-L 1/52 BarnHI pattern (1) Fusion fragment A-A' 14/52 (2) Fusion fragment D-H 11/52 (3) Two fusion fragments A-A' & D-H 13/52 (4) Two fusion fragments D-H & W - K 3/52 (5) Three fusion fragments A-A', D-H & B-L 2/52 (6) Gain of cleavage site in A 1/52 (7) Two fusion fragments A-A' & C'-K' 1/52 (8) Fusion fragment W-K' 1/52 (9) Three fusion fragments A-A', D-H & C'-K' 1/52 'Frequency is expressed as t h e number of isolates with the indicated fragment(s)/total number of isolates.
K. The mixture was incubated a t 37°C for 2 hours. Viral DNA was extracted three times with a mixture of phenol/chloroform (1:l)and subsequently twice with a mixture of chloroform/Isoamyl alcohol (24:l).The DNA was then precipitated with 2.5 volumes of ethanol and kept at -20°C overnight. DNA was dissolved in distilled water to final concentration of 1 mg/ml for RE analysis.
Restriction Enzyme Digestion, Agarose Gel Electrophoresis, and Southern Blot HindIII and BarnHI were purchased from BRL (Bethesda, MD). Purified viral DNA (3-5 pg) in a reaction volume of 25 pl was digested with 5 units of RE/pg of DNA at 37°C for a minimum of 8 hours. Appropriate reaction buffer recommended by the manufacturer was used for each enzyme. The reaction was stopped by the addition of 5 pl of 0.1 M EDTA followed by the addition of 2.5 pl of 01% bromophenol blue. The limit digests were then loaded onto 0.5% agarose slab gels (0.5 >( 20 x 20 cm) and electrophoresed overnight in running buffer (90 mM Tris, 90 mM boric acid, 2 mM EDTA, pH 8.3) at 60 volts. The gels were stained with ethidium bromide (1 pg/ml) and photographed under UV light with a Polaroid MP4 camera using Type 665 film. The gels were rinsed briefly in sterile distilled water and the DNA fragments transferred onto nitrocellulose paper (Bio-Rad Laboratories, Richmond, CA) a s described by 3outhern [19751 and fixed by baking for 2 hours at 80°C. Hybridization HSV-1-F DNA was used as a probe and labelled by nick translation [Rigby et al., 19771 with [ c L - ~ dCTP ~P] (3,000 Ci/mmol, Amersham, Buckinghamshire, England). Hybridization was carried out in 25 ml of 6X SSC (1X SSC is 0.15 M sodium chloride and 0.015 M sodium citrate), 5X Denhardt's solution ( 1X Denhardt's solution is 0.02% bovine serum albumin, 0.02% polyvi-
Al-Ahdal et al.
18
Fig. 2. Hind111 digestion profiles of DNA from HSV-1-F and representative clinical isolates. Panel I shows the electrophoretic mobility of the fragments. Panel I1 shows the corresponding autoradiograms of the same samples after Southern blot and hybridization. Lanes 3 and 5 illustrate pattern 1. Lane 4 is characteristic of pattern 2. Lane 6 depicts pattern 3. Lanes 1 and 2 show same pattern as strain F.
nylpyrrolidone, and 0.02%,Ficoll 400), 0.5% SDS, 50% formamide and 100 pg/ml heat denatured salmon sperm DNA a t 42°C for 16 hours. The membrane was then washed thrice in 100 ml of 2X SSC containing 0.1% SDS a t room temperature and thrice in 0.1X SSC containing 0.1% SDS a t 50°C. The membrane was air dried and exposed to a Kodak X-ray film (XK-6) with .intensifying screens at -70°C for 16 hours.
RESULTS Viral DNA Fragment Patterns Obtained With Hind111 The DNA from 52 clinical isolates of HSV-1 were digested with HzndIII and BamHI restriction enzymes. The fragments were separated on 0.5% agarose gels, transferred onto nitrocellulose membranes, and hybridized to "P-labelled DNA of standard laboratory HSV-1-F strain. Differences in the HSV-1 strains as compared to HSV-I-F were reflected a s differences in cleavage patterns and were identified based on the gain or loss of restriction enzyme cleavage sites by referring to the published cleavage maps of strain F for these endonucleases (Fig. 1B). Table I summarizes the frequency of variable cleavage sites obtained with HindIII and BamHI restriction endonucleases in the HSV-1
clinical isolates. Analysis with HzndIII resolved 17 of the clinical HSV-1 isolates and these were classified into four distinct cleavage patterns. Fig. 2 shows a representative endonuclease profile of three of the four patterns obtained with HindIII digestion a s well as the corresponding autoradiograms. The most common variation found was the presence of a fusion fragment 1 - 0 due to a loss of cleavage site between I and 0. This occurred in 11 of 52 isolates (Fig. 2, pattern 1).Four of the isolates exhibited the presence of fusion fragment K-L caused by the loss of a site between K and L (Fig. 2, pattern 2). One of the isolates showed a loss of a site between M and N, resulting in a fusion fragment M-N. In addition, there was a gain in a cleavage site in fragment 0,yielding two smaller fragments, m, m2 (Fig. 2, pattern 3). A single isolate showed two fusion fragments in the same isolate, namely 1 - 0 and K-L (Table I, pattern 4). The remaining 35 isolates could not be distinguished from strain F using HindIII, a s represented in Figure 2.
Viral DNA Fragment Patterns Obtained With BamHI The genomic variation in the isolates was investigated further with BamHI, which cleaves the HSV-1
19
HSV-1 DNA Polymorphism in Saudi Arabia
Fig. 3. BarnHI digestion profiles of DNA from HSV-1-F and representative clinical isolates. Panel I shows the electrophoretic mobility of the fragments. Panel I1 shows the corresponding autoradiograms of the same samples after Southern blot and hybridization. Darker autoradiogram is due to longer exposure to achieve sufficient clarity ofsmaller fragments. Lanes 3,4, and 5 illustrate pattern 1.Lanes 6,8,9,and 10 are characteristic of pattern 2. Lanes 7,1,2,and 11 depict patterns 3 , 4 , 5 , and 7, respectively.
genome more frequently than HindIII, thus providing a higher resolution of the fragments. Typical restriction endonuclease profiles of the DNAs from the HSV-1 isolates cleaved with BarnHI with the corresponding autoradiograms are shown in Figure 3. Of the 52 clinical isolates, 47 were distinguished with BamHI and were classified into nine cleavage patterns. Fourteen of the isolates exhibited the presence of a fusion fragment A-A' due to the loss of a restriction site between A and A' (pattern 1). Eleven of the isolates showed the presence of a fusion fragment D-H caused by a loss of a cleavage site between D and H (pattern 2 ) . Pattern 3, which occurred in 13 of the isolates, showed the presence of two fusion fragments in each isolate namely A-A' and D-H. The three patterns mentioned above were predominant in the HSV-1 isolates cleaved with BarnHI. Three of the isolates showed the presence of two fusion fragments D-H and W-K' (pattern 4). In pattern 5, which occurred in two of the isolates, each
had three fusion fragments, namely, A-A', D-H, and B-L. The four remaining patterns obtained by BarnHI are indicated in Table I. Five of the isolates could not be differentiated from HSV-1-F with BarnHI.
DISCUSSION The purpose of this study was to determine genomic variation of HSV-1 clinical strains isolated from Saudi patients. We used purified HSV-1 particles for virus DNA extraction to minimize contamination with cellular DNA. Pilot studies with several enzymes led us to select HindIII and BarnHI as the most useful enzymes for our work since they provided a clear and unambiguous identification of the restriction fragments. The criteria used for the classification was based on the gain or loss of RE cleavage site(s), which has been found to be more stable and thus serve as a useful marker for epi-
Al-Ahdal et al.
20
demiological and genetic studies. However, a closer examination of the results revealed that there was considerable variation in mobility of fragments derived from the terminal region of the genome (e.g., HindIII-M and BamHI-S), the reiterated sequences of the L and S segments of the genome (e.g., BamHI K and El, and the junction spanning the unique and reiterated sequences of the L and S components (e.g.,BamHI X and Z). Such variation has been observed in plaque purified stocks of a single virus strain [Wagner and Summers, 1978; Lonsdale et al., 19801, in recurrent isolates from a single individual [Ueno et al., 19821,and in paired isolates from siblings [Sakaoka et al., 19841. Variation due to mobility of restriction fragments was not included in the classification of our HSV-1 strains. The most essential feature of the cleavage patterns obtained with HindIII was the predominance of the fusion fragment 1-0,which occurred in 11 of the isolates (Fig. 2, pattern 1). Another essential feature was noted with BamHI where three predominant cleavage patterns (Fig. 3, patterns 1,2, and 3) were recognized but occurred with almost equal frequency (Table I). Results with HindIII also demonstrated two novel cleavage patterns, one of which is Figure 2, pattern 3, and the other is pattern 4, Table I. Pattern 3 is characterized by the presence of fusion fragment M-N as well as a gain of cleavage site in 0 yielding two smaller fragments m, and m2. In pattern 4 there were two fusion fragments in the same isolate, namely, 1-0and K-L. Although patterns 3 and 4 occurred at low frequency in the isolates (1 isolate each), they were highly reproducible and could not be an artifact. Using the published cleavage maps for these endonucleases (Fig. 1B) as a reference, it is obvious that with exception of HindIII M and BamHI B, fragments giving rise to the observed polymorphism were derived from the unique region of the long and short components of the genome. The HSV-1 strains were classified into 13 patterns when HindIII and BamHI were used, but there is the possibility of existence of other variant forms, which could be differentiated using other enzymes [Umene and Sakaoka, 19911. Sakaoka et al. 119861 studied HSV-1 strains isolated from Kenya and Japan and found significant differences in frequency of cleavage patterns between the two countries. For example, HindIII 1-0 fusion fragments, which were predominant in Kenyan isolates, were totally absent in Japanese isolates. Furthermore, two BamHI fusion fragments A-A’ and D-H, which were predominant in Saudi isolates, were absent in the Kenyan and Japanese isolates. It has been postulated that mutations of HSV-1 have occurred by repeated person-to-person transmission throughout the millennia, giving rise to an unlimited number of variants in the population [Buchman et al., 1980; Roizman, 1980a,b]. Similarities in variation among strains from each country might have originated from a common ancestor [Umene et al., 19841. This study, therefore, provides further evidence that there is a geographic clustering of HSV-1 strains.
ACKNOWLEDGMENTS The authors thank Ofelia Frias for excellent secretarial assistance and the Medical Photography Department for illustrations. This work was supported by funds from King Faisal Specialist Hospital and Research Centre (Project Number 87-0012).
REFERENCES Alam TM, Joncas JH, Ozanne G (1989): DNA polymorphism among isolates from multiple sites of a patient with chronic Herpes Simplex Virus type 1 infection. Journal of Medical Virology 29:186191. Brown SM, Subak-SharpeJH, Warren KA, Wroblewska Z, Kaprowski H (1978): Detection by Complementation of defective or uninducible (herpes simplex type 1)Virus genomes latent in human ganglia. Proceedings of the National Academy of Sciences of the USA 76:2364-2368. Buchman TG, Roizman B, Adams G, Stover BH (1978): Restriction endonucleases fingerprinting of Herpes Simplex Virus DNA. A novel epidemiological tool applied to a nosocomial outbreak. Journal of Infectious Diseases 138:488-498. Buchman TG, Simpson T, Nosal C, Roizman B, Nahmias AJ (1980): The structure of Herpes Simplex Virus DNA and its application to molecular epidemiology. Annals of New York Academy of Sciences 354:279-290. Linneman CC, Buchman TA, Light IJ, Ballard J L , Roizman B (1978): Transmission of herpes simplex virus type 1 in a nursery for the newborn identification of viral isolates by DNA “fingerprinting”. Lancet 13964-966. Locker H, Frenkel N (1979):BarnHI, Kpnl and Sall restriction enzyme maps of the DNAs of herpes simplex virus strains Justin and F: Occurrence of heterogeneities in defined regions of the viral DNA. Journal of Virology 32:429-441. Lonsdale DM, Brown SM, Lang J, Subak-Sharpe JH, Kaprowski H, Warren KA (1980): Variations in herpes simplex virus isolated from human ganglia and a study of clonal variation in HSV-1. Annals of New York Academy of Sciences 354:291-308. Pereira L, Casai E, Honess RW, Roizman B, Terni M, Nahmias A (1976):Variability in the structural polypeptides of herpes simplex virus 1 strains: Potential application in molecular epidemiology. Infection and Immunity 13:211-220. Poffenberger KL, Tabares E, Roizman B (1983):Characterization of a viable, non-inverting herpes simplex virus 1 genome derived by insertion and deletion of seauences a t the iunction of comoonents L and S. Proceedings of the National AcadeAy of Sciences &the USA 80:2690-2694. Post LE, Conley AJ, Mocurski ES, Roizman B (1980):Cloning of reiterated and non-reiterated herpes simplex virus 1 sequences as BamHI fragments. Proceedings of the National Academy of Sciences of the USA 77:42014205. Rigby PWJ, Dieckmann M, Rhodes C, Berg P (1977):Labelling DNA to high specific activity in vitro by nick translation with DNA polymerase 1.Journal of Molecular Biology 113:237-251. Roizman B (1980a):The structure and isomerization of herpes simplex virus genomes. Cell 16:481-494. Roizman B (1980b): Genome variation and evolution among herpes simplex virus. Annals of New York Academy of Sciences 354:472483. Roizman B, Buchman T (19791: The molecular epidemiology of herpes simplex viruses. Hospital Practice 14:95-104. Sakaoka H, Aomori T, Ozaki I, Ishida S, Fujinaga K (1984):Restriction endonuclease cleavage analysis of herpes simplex virus isolates obtained from three pairs of siblings. Infection and Immunity 43:771-774. Sakaoka H, Aomori T, Honda 0, Saheki U, Ishida S, Yamanishi S, Fujinaga K (1985): Sub-types of herpes simplex virus type 1 in Japan: Classification by restriction endonucleases and analysis of distribution. Journal of Infectious Diseases 150:190-197. Sakaoka H, Aomori T, Saito H, Sat0 S, Kawana R, Hazelett DT, Fujinaga K (1986): A comparative analysis by restriction endonucleases of herpes simplex virus type 1 isolated in Japan and Kenya. Journal of Infectious Diseases 153:612-616.
HSV-1 DNA Polymorphism in Saudi Arabia Southern EM (19751:Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98:503-517. Ueno T, Suzuki N, Sakaoka H, Fujinaga K (1982): A simple and practical method for typing and strain differentiation of herpes simplex virus using infected cell DNAs. Microbiology and Immunology 26:1159-1170. Umene K, Sakaoka H (1991):Homogeneity and diversity of genome polymorphism in a set of herpes simplex virus type 1strains classified as the same genotypic group. Archives of Virology 119:53-65. Umene K, Eto T, Mori R, Takagi U, Enquist WL (1984):Herpes Simplex Virus Type 1 restriction fragment polymorphism determined using southern hybridization. Archives of Virology 80:275-290.
21 Wagner MJ, Summers WC (1978):Structure of the joint regions and termini of the DNA of herpes simplex virus type 1. Journal of Virology 27:374-387. Whitley R, Lakeman A, Nahmias A, Roizman B (1982):DNA restriction enzyme analysis of herpes simplex virus isolates obtained from patients with encephalitis. New England Journal of Medicine 307:1060-1062. Wilkie NM (1976):Physical maps for herpes simplex virus type 1DNA for restriction endonucleases HindIII, Hpal and Xbal. Journal of Virology 20:222-233.
Genomic Variation Among Herpes Simplex Virus Type 1 Strains: Virus DNA Analysis of Isolates From Saudi Patients Mohammed N. Al-Ahdal, George Kessie, Mohamed A. Taha, Fahad J. Al-Shammary, and Mohamed Ettayebi Departments of Biological and Medical Research (M.N.A.-A., G.K., M.A.T.) and Pathology and Laboratory Medicine (M.N.A.-A.), King Faisal Specialist Hospital and Research Centre, and College of Applied Medical Sciences, King Saud University (F.J.A.-S.), Riyadh, Saudi Arabia; Biotechnology Laboratory, College of Sciences, Sidi Mohamed Bin Abdallah Uniuersity, Fes, Morocco (M.E.I Fifty-two clinical isolates of herpes simplex virus type 1 (HSV-1) from Saudi Arabian patients were analysed by restriction endonuclease digestion of the virus DNA using the enzymes Hindlll and BarnHI, followed by hybridization with 32P labelled DNA of laboratory strain F. Of the isolates, 17 were resolved into four distinct cleavage patterns with Hindlll restriction enzyme. The remaining 35 strains had the same cleavage pattern as the standard HSV-1-F. Further investigation of the 52 isolates with BarnHI, which is a multicut enzyme and therefore capable of higher resolution, differentiated 47 of the 52 isolates and were assigned into nine cleavage groups. Comparing our findings with similar studies reported elsewhere suggest geographic clustering of HSV-1 strains. Fragments giving rise t o the observed DNA polymorphism were mapped t o the unique region of the long and short segments of the genome. GI1992 Wiley-Liss, Inc.
KEY WORDS: HSV-1 DNA analysis, HSV-1 DNA cleavage pattern
INTRODUCTION Herpes simplex virus type 1(HSV-1)is a n important human pathogen, which after primary infection can remain latent in the relevant sensory ganglia [Brown e t al., 1978; Lonsdale et al., 19801. Periodic reactivation of the virus serves as a source of infection of individuals in close contact with the carrier. The spread of HSV-1 is, however, restricted within a community or a group of individuals, such as family members. Previous attempts to classify HSV-1 strains and to establish interrelationships among geographic origins of clinical isolates were carried out by virion polypeptide analysis [Pereira et al., 19761. Recent molecular techniques have refined the methods of determining intratypic variations. Viral genome polymorphism can be visual0 1992 WILEY-LISS, INC.
ized by treating the DNA with restriction endonucleases (RE). Such treatment produces strain specific cleavage patterns, thus allowing unambiguous classification of the strains. This technique has been used to trace nosocomial transmission of HSV-1 [Buchman et al., 1978; Linneman eta]., 19781. In addition, i t was used to differentiate between strains from different individuals [Ueno et al., 1982; Whitley et al., 19821, different sites of infection in the same individual [Alam et al., 19891, and geographic origin ISakaoka et al., 1985, 19861, a s well a s in family clusters [Sakaoka e t al., 19841. The basis of such studies is that the cleavage patterns of two unrelated strains of HSV-1 are not identical [Roizman and Buchman, 1979; Roizman, 1980a, b]. The variation in the fragments generated can be distinguished by simply referring to the published cleavage maps of the relevant restriction enzyme [Wilkie, 1976; Locker and Frenkel, 19791. Whereas numerous studies have been performed on DNA polymorphism among HSV-1 isolates from different parts of the world [Sakaoka et al., 1985, 1986; Alam e t al., 19891, we are unaware of any investigation carried out in the Middle East. King Faisal Specialist Hospital and Research Centre is a 530-bed, tertiary care hospital in Riyadh, Saudi Arabia. Since it is a referral medical centre, the hospital receives a large number of patients from various parts of the kingdom. We therefore used this opportunity to study DNA polymorphism among 52 clinical isolates of HSV-1 obtained from Saudi patients. The extent of genomic variation among these isolates was determined, based solely on the gain andlor loss of restriction enzyme cleavage sites.
Accepted for publication February 6, 1992. Address reprint requests to Dr. Mohammed N. Al-Ahdal, Biological and Medical Research Department (MBC-03),King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia.
HSV-1 DNA Polymorphism in Saudi Arabia
B H ~ ~ I I I n
17
B=D+G
10 I
I
I
J
I
A
1
K
/
L
I
D
M l N l G
. I
C=D+M E-HIG
F-H+M
Fig. 1. A Structure of HSV-1 genome. Unique sequences U, and Us are bound by terminal reiterated sequences ITR,, TR,], which are also present in inverted forms [IR,, IR,] at the junction of the L and S components. B: HindIII and BarnHI cleavage maps of strain F [Locker and Frenkel, 1979; Roizman and Buchman, 1979; Post et al., 1980; Poffenberger et al., 19831. Only the prototype arrangements are shown. The three other isomeric forms can be generated by inverting L, S, or both components. Arrows under each map identify the positions of variable cleavage sites found in the HSV-1 isolates.
MATERIALS AND METHODS Viruses, Cells, and Isolates Strain F of HSV-1 (HSV-1-F, ATCC VR-733) was used a s a standard laboratory strain and purchased from ATCC (Rockville, MD). Clinical isolates were initially isolated and identified as HSV-1 by monoclonal antibody immunofluorescence a t the Diagnostic Virology Laboratory. Baby hamster kidney cells (BHK-BlI C13, Cat.# 03-420, Flow Laboratories, Irvine, Scotland) were used for propagation of the virus. The cells were maintained in Eagle's minimal essential medium (EMEM) containing 10% fetal calf serum (FCS), 50 Uiml penicillin and 50 pg/ml streptomycin. Growth of Virus Confluent monolayers of BHK-21iC13 cells in roller bottles were infected with 5 PFUiml of HSV-1-F or stock virus of HSV-1 clinical isolates. At 1hour postinfection, 25 ml of EMEM containing 1%FCS were added to each culture bottle and incubated a t 37°C for 36 hours or until maximum cytopathic effect was evident. The medium containing virus particles was collected and centrifuged a t low speed to remove cellular debris, then a t 30,000 g for 1hour a t 4°C. The pellet was resuspended in TNE buffer (0.1 M Tris-HC1, 0.01 M NaC1, 0.01 M EDTA, pH 7.4), and stored a t -20°C. Preparation of Virus DNA Virus pellets were layered on a linear 20-60% (w/v) sucrose gradient in TNE buffer and centrifuged at 50,000 g for 2 hours a t 4°C. The band containing purified virus was harvested and dialysed against TNE buffer. The dialysate was centrifuged at 30,000 g for 1 hour at 4°C. The pelleted purified virus was disrupted with 1% SDS, 1 mM EDTA, and 250 pg/ml proteinase
TABLE I. Frequency of Variable Restriction Endonuclease Cleavage Sites of HSV-1 Clinical Isolates Among Saudi Patients Hind111 pattern (1) Fusion fragment 1-0 11/52a (2) Fusion fragment K-L 4/52 (3) Fusion fragment M-N gain of cleavage 1/52 site in 0 (4) Two fusion fragments 1-0 & K-L 1/52 BarnHI pattern (1) Fusion fragment A-A' 14/52 (2) Fusion fragment D-H 11/52 (3) Two fusion fragments A-A' & D-H 13/52 (4) Two fusion fragments D-H & W - K 3/52 (5) Three fusion fragments A-A', D-H & B-L 2/52 (6) Gain of cleavage site in A 1/52 (7) Two fusion fragments A-A' & C'-K' 1/52 (8) Fusion fragment W-K' 1/52 (9) Three fusion fragments A-A', D-H & C'-K' 1/52 'Frequency is expressed as t h e number of isolates with the indicated fragment(s)/total number of isolates.
K. The mixture was incubated a t 37°C for 2 hours. Viral DNA was extracted three times with a mixture of phenol/chloroform (1:l)and subsequently twice with a mixture of chloroform/Isoamyl alcohol (24:l).The DNA was then precipitated with 2.5 volumes of ethanol and kept at -20°C overnight. DNA was dissolved in distilled water to final concentration of 1 mg/ml for RE analysis.
Restriction Enzyme Digestion, Agarose Gel Electrophoresis, and Southern Blot HindIII and BarnHI were purchased from BRL (Bethesda, MD). Purified viral DNA (3-5 pg) in a reaction volume of 25 pl was digested with 5 units of RE/pg of DNA at 37°C for a minimum of 8 hours. Appropriate reaction buffer recommended by the manufacturer was used for each enzyme. The reaction was stopped by the addition of 5 pl of 0.1 M EDTA followed by the addition of 2.5 pl of 01% bromophenol blue. The limit digests were then loaded onto 0.5% agarose slab gels (0.5 >( 20 x 20 cm) and electrophoresed overnight in running buffer (90 mM Tris, 90 mM boric acid, 2 mM EDTA, pH 8.3) at 60 volts. The gels were stained with ethidium bromide (1 pg/ml) and photographed under UV light with a Polaroid MP4 camera using Type 665 film. The gels were rinsed briefly in sterile distilled water and the DNA fragments transferred onto nitrocellulose paper (Bio-Rad Laboratories, Richmond, CA) a s described by 3outhern [19751 and fixed by baking for 2 hours at 80°C. Hybridization HSV-1-F DNA was used as a probe and labelled by nick translation [Rigby et al., 19771 with [ c L - ~ dCTP ~P] (3,000 Ci/mmol, Amersham, Buckinghamshire, England). Hybridization was carried out in 25 ml of 6X SSC (1X SSC is 0.15 M sodium chloride and 0.015 M sodium citrate), 5X Denhardt's solution ( 1X Denhardt's solution is 0.02% bovine serum albumin, 0.02% polyvi-
Al-Ahdal et al.
18
Fig. 2. Hind111 digestion profiles of DNA from HSV-1-F and representative clinical isolates. Panel I shows the electrophoretic mobility of the fragments. Panel I1 shows the corresponding autoradiograms of the same samples after Southern blot and hybridization. Lanes 3 and 5 illustrate pattern 1. Lane 4 is characteristic of pattern 2. Lane 6 depicts pattern 3. Lanes 1 and 2 show same pattern as strain F.
nylpyrrolidone, and 0.02%,Ficoll 400), 0.5% SDS, 50% formamide and 100 pg/ml heat denatured salmon sperm DNA a t 42°C for 16 hours. The membrane was then washed thrice in 100 ml of 2X SSC containing 0.1% SDS a t room temperature and thrice in 0.1X SSC containing 0.1% SDS a t 50°C. The membrane was air dried and exposed to a Kodak X-ray film (XK-6) with .intensifying screens at -70°C for 16 hours.
RESULTS Viral DNA Fragment Patterns Obtained With Hind111 The DNA from 52 clinical isolates of HSV-1 were digested with HzndIII and BamHI restriction enzymes. The fragments were separated on 0.5% agarose gels, transferred onto nitrocellulose membranes, and hybridized to "P-labelled DNA of standard laboratory HSV-1-F strain. Differences in the HSV-1 strains as compared to HSV-I-F were reflected a s differences in cleavage patterns and were identified based on the gain or loss of restriction enzyme cleavage sites by referring to the published cleavage maps of strain F for these endonucleases (Fig. 1B). Table I summarizes the frequency of variable cleavage sites obtained with HindIII and BamHI restriction endonucleases in the HSV-1
clinical isolates. Analysis with HzndIII resolved 17 of the clinical HSV-1 isolates and these were classified into four distinct cleavage patterns. Fig. 2 shows a representative endonuclease profile of three of the four patterns obtained with HindIII digestion a s well as the corresponding autoradiograms. The most common variation found was the presence of a fusion fragment 1 - 0 due to a loss of cleavage site between I and 0. This occurred in 11 of 52 isolates (Fig. 2, pattern 1).Four of the isolates exhibited the presence of fusion fragment K-L caused by the loss of a site between K and L (Fig. 2, pattern 2). One of the isolates showed a loss of a site between M and N, resulting in a fusion fragment M-N. In addition, there was a gain in a cleavage site in fragment 0,yielding two smaller fragments, m, m2 (Fig. 2, pattern 3). A single isolate showed two fusion fragments in the same isolate, namely 1 - 0 and K-L (Table I, pattern 4). The remaining 35 isolates could not be distinguished from strain F using HindIII, a s represented in Figure 2.
Viral DNA Fragment Patterns Obtained With BamHI The genomic variation in the isolates was investigated further with BamHI, which cleaves the HSV-1
19
HSV-1 DNA Polymorphism in Saudi Arabia
Fig. 3. BarnHI digestion profiles of DNA from HSV-1-F and representative clinical isolates. Panel I shows the electrophoretic mobility of the fragments. Panel I1 shows the corresponding autoradiograms of the same samples after Southern blot and hybridization. Darker autoradiogram is due to longer exposure to achieve sufficient clarity ofsmaller fragments. Lanes 3,4, and 5 illustrate pattern 1.Lanes 6,8,9,and 10 are characteristic of pattern 2. Lanes 7,1,2,and 11 depict patterns 3 , 4 , 5 , and 7, respectively.
genome more frequently than HindIII, thus providing a higher resolution of the fragments. Typical restriction endonuclease profiles of the DNAs from the HSV-1 isolates cleaved with BarnHI with the corresponding autoradiograms are shown in Figure 3. Of the 52 clinical isolates, 47 were distinguished with BamHI and were classified into nine cleavage patterns. Fourteen of the isolates exhibited the presence of a fusion fragment A-A' due to the loss of a restriction site between A and A' (pattern 1). Eleven of the isolates showed the presence of a fusion fragment D-H caused by a loss of a cleavage site between D and H (pattern 2 ) . Pattern 3, which occurred in 13 of the isolates, showed the presence of two fusion fragments in each isolate namely A-A' and D-H. The three patterns mentioned above were predominant in the HSV-1 isolates cleaved with BarnHI. Three of the isolates showed the presence of two fusion fragments D-H and W-K' (pattern 4). In pattern 5, which occurred in two of the isolates, each
had three fusion fragments, namely, A-A', D-H, and B-L. The four remaining patterns obtained by BarnHI are indicated in Table I. Five of the isolates could not be differentiated from HSV-1-F with BarnHI.
DISCUSSION The purpose of this study was to determine genomic variation of HSV-1 clinical strains isolated from Saudi patients. We used purified HSV-1 particles for virus DNA extraction to minimize contamination with cellular DNA. Pilot studies with several enzymes led us to select HindIII and BarnHI as the most useful enzymes for our work since they provided a clear and unambiguous identification of the restriction fragments. The criteria used for the classification was based on the gain or loss of RE cleavage site(s), which has been found to be more stable and thus serve as a useful marker for epi-
Al-Ahdal et al.
20
demiological and genetic studies. However, a closer examination of the results revealed that there was considerable variation in mobility of fragments derived from the terminal region of the genome (e.g., HindIII-M and BamHI-S), the reiterated sequences of the L and S segments of the genome (e.g., BamHI K and El, and the junction spanning the unique and reiterated sequences of the L and S components (e.g.,BamHI X and Z). Such variation has been observed in plaque purified stocks of a single virus strain [Wagner and Summers, 1978; Lonsdale et al., 19801, in recurrent isolates from a single individual [Ueno et al., 19821,and in paired isolates from siblings [Sakaoka et al., 19841. Variation due to mobility of restriction fragments was not included in the classification of our HSV-1 strains. The most essential feature of the cleavage patterns obtained with HindIII was the predominance of the fusion fragment 1-0,which occurred in 11 of the isolates (Fig. 2, pattern 1). Another essential feature was noted with BamHI where three predominant cleavage patterns (Fig. 3, patterns 1,2, and 3) were recognized but occurred with almost equal frequency (Table I). Results with HindIII also demonstrated two novel cleavage patterns, one of which is Figure 2, pattern 3, and the other is pattern 4, Table I. Pattern 3 is characterized by the presence of fusion fragment M-N as well as a gain of cleavage site in 0 yielding two smaller fragments m, and m2. In pattern 4 there were two fusion fragments in the same isolate, namely, 1-0and K-L. Although patterns 3 and 4 occurred at low frequency in the isolates (1 isolate each), they were highly reproducible and could not be an artifact. Using the published cleavage maps for these endonucleases (Fig. 1B) as a reference, it is obvious that with exception of HindIII M and BamHI B, fragments giving rise to the observed polymorphism were derived from the unique region of the long and short components of the genome. The HSV-1 strains were classified into 13 patterns when HindIII and BamHI were used, but there is the possibility of existence of other variant forms, which could be differentiated using other enzymes [Umene and Sakaoka, 19911. Sakaoka et al. 119861 studied HSV-1 strains isolated from Kenya and Japan and found significant differences in frequency of cleavage patterns between the two countries. For example, HindIII 1-0 fusion fragments, which were predominant in Kenyan isolates, were totally absent in Japanese isolates. Furthermore, two BamHI fusion fragments A-A’ and D-H, which were predominant in Saudi isolates, were absent in the Kenyan and Japanese isolates. It has been postulated that mutations of HSV-1 have occurred by repeated person-to-person transmission throughout the millennia, giving rise to an unlimited number of variants in the population [Buchman et al., 1980; Roizman, 1980a,b]. Similarities in variation among strains from each country might have originated from a common ancestor [Umene et al., 19841. This study, therefore, provides further evidence that there is a geographic clustering of HSV-1 strains.
ACKNOWLEDGMENTS The authors thank Ofelia Frias for excellent secretarial assistance and the Medical Photography Department for illustrations. This work was supported by funds from King Faisal Specialist Hospital and Research Centre (Project Number 87-0012).
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