Oncology 36: 245-247 (1979)
Cytomegalovirus Strain Differentiation by DNA Restriction Analysis1 H. W. Doerr, A. Kiinzler and H. Schmitz Institut für Medizinische Virologie der Universität Heidelberg, Heidelberg, and Institut für Virologie, Zentrum für Hygiene der Universität Freiburg. Freiburg
Key Words. CMV strain differentiation • DNA cleavage patterns • Clinical aspects
Introduction Differences in the antigenic composition of cyto megalovirus isolates have been reported by several authors [1, 2]. Strain-specific reactions were demon strated by cross-neutralization and more recently by complement fixation tests [3, 4], blit they have not been confirmed by other investigators [5-7], The problem of CMV subtyping is important for the ques tion of exogenous or endogenous reinfections and for the efficiency of vaccination. Further, the evaluation of the oncogenic potential of CMV [8, 9] may be type specific, as has been supposed from herpes simplex virus from clinical data [10]. Small distinctions were detected in the polypeptide patterns of different CMV isolates [11]. DNA fragments produced by restriction enzymes were presented and could possibly be a power ful technique to examine the homology of CMV strains [ 12]. Methods and Materials Beside the standard strains AD 169 |14| and Davis |1 | the following CMV isolates were investigated: RAU-75 (kindly provided 1 This work was supported by the 'Deutsche Forschungsgemein schaft'(Do 157/2).
by H. zur Hausen), FEI-75 (kindly provided by G. F.nders), KUL-73, KUR-74 (own isolates). All these strains were isolated from patients’ urine specimens. Additionally, we investigated the vaccination strain Towne 125 [15) (kindly provided by M. Just (16)). The strains w'ere propagated in human embryonic lung fibroblasts, which were maintained with Eagle's MEM + 5% FCS. Cells were infected with a multiplicity of 0.5 PFU/ cell. Virus was harvested, after a maximal cytopathic effect had de veloped, usually 8-10 days after infection. The infected cells of 3-5 roller bottles (surface 1,500 cm2) were shaken into the nutrient medium and sedimented (4,000 g, 10 min). The cell sediment was homogenized in 5 ml destilled water with a Douncc glass homogenizer, and centrifuged again (4,000 g, 10 min). The supernatant was pooled with the virus, which had been sedimented from the nutrient medium by ultracentrifugation (40,000 g, 90 min). The virus was further purified and DNA extractions were performed, as described in detail by lleckenstein et at. [17). The DNA fragmentation using the restric tion enzyme Eco RI was carried out in the following assay: 330 /d CMV DNA (= 0.5-2 ,«g) was incubated for 3 h at 37 °C with 10//I Eco RI (= 10 U/min; Miles Co., Ind.) + 4 0 /d 10 mM Tris pH 9.0 containing 5 mM MgCl2, 7 mAi /1-mcrcaptoethanol, 15 mM KC1. After addition of 30 //I 0.5 M EDTA, 70 ftl 3 M sodium acetate, 500 «1 H,(). the DNA fragments were further purified by chloroform-isoamylalcohol extraction and pelleted after ethanol precipita tion (-20 °C) at 135,000 g for 30 min. The pellet was resuspended in 30 /(I H20 and 10 «1 bromphenol blue sucrose (5 mg bromphenol blue + 11 g sucrose in 9 ml H20 ) and electrophoresed on a 3 mm thick 0.7% agarose gel (Sigma, St. Louis, Mo.) at 25 V for 16 h. The DNA fragments were stained by adding 0.5 /d/ml ethidium bromide to the electrophoresis buffer (40 mM Tris pH 7.8, 10 mM sodium acetate, 1 mM EDTA). The stained bands were visualized by UV light and photographed. In the same way CMV DNA fragmenta-
Downloaded by: Lund University Libraries 130.235.66.10 - 11/23/2018 6:29:03 PM
Abstract. The heterogeneity of CMV DNA obtained from standard strains and new isolates, including a vac cination strain (Towne 125), was investigated. The cleavage patterns produced by the restriction endonucleases Eco RI and Bam 1 revealed stable strain specificities of CMV. On the other hand, a remarkable homology of se quence-specific CMV DNA fragmentation was demonstrated. A CMV subtyping relevant to clinical questions seems to be improbable.
246
Doerr/Künzler/Schmitz
lion by the restriction enzyme Bam 1 was performed (Biolabs, Be verly, Mass.; 10 p\ Bam + 330 p\ CMV DNA + 40 «1 10 mM Tris pH 7.5, 10 mM MgCl2 + 20/tl H20 ).
15 10 8
6
l -• 3
Results Figure 1 shows the results of DNA fragmentation by Eco RI restriction endonuclease. Although distinct dif ferences could be detected, most of the fragments comigrated throughout the different CMV strains in vestigated. Among the new isolates KUL-73 displayed a good agreement to AD 169, while RAU-75 is more similar to Davis and FE1-75 to Towne 125. The pat terns for the DNA’ of the standard strains Davis and AD 169 are obviously identical to those found by Kilpatrick et at. [12], This is especially remarkable, since the strains were obtained from different sources and the AD 169 had been propagated for at least 6 years in our laboratory. Thus, the virus strains seem to be relatively stable over a great number of cell pas sages. The vaccination strain Towne 125 also showed no significant difference to the original isolate [12]. Using Bam 1 restriction enzyme differences of nu cleotide sequence between AD 169 and KUL-73 could be detected extending the result seen with Eco RI
- -
2
-■
1
Fig. 2. DNA cleavage pattern of six CMV strains produced by the restriction enzyme Bam 1; from left to right: RAU-75, KUL-73, Towne 125, Davis, KUR-74, AD 169, Eco RI cleavage pattern of KUL-73 with the molecular weight scale (x 106 daltons).
(fig. 2): it produced different bands in the MW region of about 2.0 x 106 daltons. In general, however, Bam 1 digestion of CMV DNA rendered similar results to those obtained with Eco RI: most of the fragments of the different CMV isolates comigrated, while some bands seem to be strain specific. CMV DNA electro phoresis without previous enzyme digestion revealed no fragmentation in all strains investigated.
Fig. 1. DNA cleavage pattern of six CMV strains produced by the restriction enzyme Eco RI (from left to right: Towne 125, Davis. R A U -75. FE I-75. AD 169, K U L-73). The num bers represent the
molecular weights (x 106 daltons); from Kilpatrick et al. [12],
The investigations described here have demonstrated that it is possible to identify different CMV isolates exactly and reproducibly by analyzing DNA base se quences. The findings are in agreement with previous results [4, 12]. They revealed stable strain specificities which may encourage studies about a molecular CMV epidemiology. On the other hand, the small number of CMV strains investigated up to date does not permit a classification into types, as has been done for herpes simplex virus (HSV). On the basis of studies with HSV types 1 and 2, which considerably differ in their DNA sequences, but display only minor antigenic differences [13], we suppose that CMV strains with relatively similar DNA base sequences could not be expected to show much serological variance of a seroepidemiological significance analogous to HSV antibody typing. We were unable, to find statistically significant differences in a great series of patients’ serum samples using
Downloaded by: Lund University Libraries 130.235.66.10 - 11/23/2018 6:29:03 PM
Discussion
247
Cytomegalovirus Strain Differentiation by DNA Restriction Analysis
7
8
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10
Acknowledgement We thank IS. Fleckenstein for kind methodical advice, and we greatly appreciate the helpful technical assistance of R. Lepacli and S. Schnierda.
11
12
13
References 1 Weller, T. H.; 1ianshaw, J. B., and Scott, D, E.: Serologic differen tiation of viruses responsible for cytomegalic inclusion disease. Virology 12: 130-132(1960). 2 Birnbaum, G.; Lynch, J.I.; Margileth, A.M.; Longergan, W.M., and Sever, J.L.: Cytomegalovirus infections in newborn infants. J. Pediat. 75: 789-795 (1969). 3 Huang, Y.T.: Huang, E.S., and Pagano, J.S.: Antisera to human cytomegaloviruses prepared in the guinea pigs: specific immuno fluorescence and complement-fixation tests. J. Immun. 112: 528-532 (1974). 4 Huang, E.S.; Kilpatrick, B. A.; Huang, Y.T., and Pagano, J.S.: Detection of human cytomegalovirus and analysis of strain varia tion. Yale J. Biol. Med. 49: 29-44 (1975). 5 Stern. H. and Elek. S.D.: The incidence of infection with cyto megalovirus in a normal population. A serological study in Greater London. J. Hyg., Camb. 63: 79-87 (1965). 6 Andersen. H.K.: Complement-fixing and virus-neutralizing anti bodies in cytomegalovirus infections as measured against homol
14
15
16
17
18
ogous and heterologous antigens. Acta path, microbiol. scand. 78: 504-508 (1970). Haines, H.G.; v. Essen, R., and Benyesh-Melnick, M.: Prepara tion of specific antisera to cytomegaloviruses in goats. Proc. Soc. exp. Biol. Med. 138: 846-849 (1971). Albrecht, T. and Rapp, F.: Malignant transformation of hamster embryo fibroblasts following exposure to ultraviolet-irradiated human cytomegalovirus. Virology 55: 56-61 (1973). Rapp, F.; Geder, L.; Murasko, D.; Lausch, R.; Laddka, R.; Huang, E.S., and Webber, M.M.: Long-term persistence of CMV genome in cultivated human cells of prostatic origin. J. Virol. 16: 982-990(1975). Nahmias, A.J. and Roizman, B.: Infection with herpes-simplex viruses 1 and 2. il. New Engl J. Med. 289: 719-725 (1973). Gupta, P.; St. Jeor, S., and Rapp, F.: Comparison of the poly peptides of several strains of human cytomegalovirus. J. gen. Virol. 34: 447-454 (1977). Kilpatrick, B.A.; Huang, E.S., and Pagano, J.S.: Analysis of cytomegalovirus genomes with restriction endonucleases HinD 111 and Eco R-l. J. Virol. 18: 1095-1105 (1976). Sugino, W.M. and Kingsbury, D.T.: DNA homologies between strains of herpes simplex virus. Virology 71: 605-608 (1976). Rowe, W.P.; Harley, J.W.; Waterman, S.; Turner, A.C., and Huebner, R.J.: Cytopathogenic agent resembling human salivary gland virus recovered from tissue cultures of human adenoids. Proc. Soc. exp. Biol. Med. 92: 418-424 (1956). Plotkin, S.; Furukawa, T.; Zygraich, N.; Vamos, E.. and Haygden, C.: Candidate cytomegalovirus strain for vaccination. Infect. Im munity 12: 521-527 (1975). Just, M.; Buergin-Wolff, A.; Emoedi, G., and Hernandez, R.: Immunisation trials with live attenuated cytomegalovirus Towne 125. Infection 3: 111-114 (1975). Fleckenstein, B.; Bornkamm. G.W.. and Ludwig, H.: Repetitive sequences in complete and defective genomes of herpes virus saimiri. J. Virol. 15: 398-406 (1975). Schmitz, 1L; Docrr, H.W., and Obrig, M.: Envelope and nucleo capsid antigens of cytomegalovirus (CMV). Med. Microbiol. Immunol. 161: 155-162 (1975). Dr. H.W. Docrr, Institut für Medizinische Virologie der Universität Heidelberg, im Neuenheimer Feld 324, 6900 Heidelberg (FRG)
Downloaded by: Lund University Libraries 130.235.66.10 - 11/23/2018 6:29:03 PM
neutralization or complement fixation tests with puri fied viral envelopes as antigen prepared from Davis and AD 169 [18]. This is in agreement with the results of Gupta et al. [11], who found a remarkable similarity in the polypeptide composition of four dif ferent CMV strains. If crude antigens are used in the complement fixation test [3], a different distribution of antibodies to both envelope and nucleocapsid anti bodies may be responsible for apparent differences in some strains.
Cytomegalovirus Strain Differentiation by DNA Restriction Analysis1 H. W. Doerr, A. Kiinzler and H. Schmitz Institut für Medizinische Virologie der Universität Heidelberg, Heidelberg, and Institut für Virologie, Zentrum für Hygiene der Universität Freiburg. Freiburg
Key Words. CMV strain differentiation • DNA cleavage patterns • Clinical aspects
Introduction Differences in the antigenic composition of cyto megalovirus isolates have been reported by several authors [1, 2]. Strain-specific reactions were demon strated by cross-neutralization and more recently by complement fixation tests [3, 4], blit they have not been confirmed by other investigators [5-7], The problem of CMV subtyping is important for the ques tion of exogenous or endogenous reinfections and for the efficiency of vaccination. Further, the evaluation of the oncogenic potential of CMV [8, 9] may be type specific, as has been supposed from herpes simplex virus from clinical data [10]. Small distinctions were detected in the polypeptide patterns of different CMV isolates [11]. DNA fragments produced by restriction enzymes were presented and could possibly be a power ful technique to examine the homology of CMV strains [ 12]. Methods and Materials Beside the standard strains AD 169 |14| and Davis |1 | the following CMV isolates were investigated: RAU-75 (kindly provided 1 This work was supported by the 'Deutsche Forschungsgemein schaft'(Do 157/2).
by H. zur Hausen), FEI-75 (kindly provided by G. F.nders), KUL-73, KUR-74 (own isolates). All these strains were isolated from patients’ urine specimens. Additionally, we investigated the vaccination strain Towne 125 [15) (kindly provided by M. Just (16)). The strains w'ere propagated in human embryonic lung fibroblasts, which were maintained with Eagle's MEM + 5% FCS. Cells were infected with a multiplicity of 0.5 PFU/ cell. Virus was harvested, after a maximal cytopathic effect had de veloped, usually 8-10 days after infection. The infected cells of 3-5 roller bottles (surface 1,500 cm2) were shaken into the nutrient medium and sedimented (4,000 g, 10 min). The cell sediment was homogenized in 5 ml destilled water with a Douncc glass homogenizer, and centrifuged again (4,000 g, 10 min). The supernatant was pooled with the virus, which had been sedimented from the nutrient medium by ultracentrifugation (40,000 g, 90 min). The virus was further purified and DNA extractions were performed, as described in detail by lleckenstein et at. [17). The DNA fragmentation using the restric tion enzyme Eco RI was carried out in the following assay: 330 /d CMV DNA (= 0.5-2 ,«g) was incubated for 3 h at 37 °C with 10//I Eco RI (= 10 U/min; Miles Co., Ind.) + 4 0 /d 10 mM Tris pH 9.0 containing 5 mM MgCl2, 7 mAi /1-mcrcaptoethanol, 15 mM KC1. After addition of 30 //I 0.5 M EDTA, 70 ftl 3 M sodium acetate, 500 «1 H,(). the DNA fragments were further purified by chloroform-isoamylalcohol extraction and pelleted after ethanol precipita tion (-20 °C) at 135,000 g for 30 min. The pellet was resuspended in 30 /(I H20 and 10 «1 bromphenol blue sucrose (5 mg bromphenol blue + 11 g sucrose in 9 ml H20 ) and electrophoresed on a 3 mm thick 0.7% agarose gel (Sigma, St. Louis, Mo.) at 25 V for 16 h. The DNA fragments were stained by adding 0.5 /d/ml ethidium bromide to the electrophoresis buffer (40 mM Tris pH 7.8, 10 mM sodium acetate, 1 mM EDTA). The stained bands were visualized by UV light and photographed. In the same way CMV DNA fragmenta-
Downloaded by: Lund University Libraries 130.235.66.10 - 11/23/2018 6:29:03 PM
Abstract. The heterogeneity of CMV DNA obtained from standard strains and new isolates, including a vac cination strain (Towne 125), was investigated. The cleavage patterns produced by the restriction endonucleases Eco RI and Bam 1 revealed stable strain specificities of CMV. On the other hand, a remarkable homology of se quence-specific CMV DNA fragmentation was demonstrated. A CMV subtyping relevant to clinical questions seems to be improbable.
246
Doerr/Künzler/Schmitz
lion by the restriction enzyme Bam 1 was performed (Biolabs, Be verly, Mass.; 10 p\ Bam + 330 p\ CMV DNA + 40 «1 10 mM Tris pH 7.5, 10 mM MgCl2 + 20/tl H20 ).
15 10 8
6
l -• 3
Results Figure 1 shows the results of DNA fragmentation by Eco RI restriction endonuclease. Although distinct dif ferences could be detected, most of the fragments comigrated throughout the different CMV strains in vestigated. Among the new isolates KUL-73 displayed a good agreement to AD 169, while RAU-75 is more similar to Davis and FE1-75 to Towne 125. The pat terns for the DNA’ of the standard strains Davis and AD 169 are obviously identical to those found by Kilpatrick et at. [12], This is especially remarkable, since the strains were obtained from different sources and the AD 169 had been propagated for at least 6 years in our laboratory. Thus, the virus strains seem to be relatively stable over a great number of cell pas sages. The vaccination strain Towne 125 also showed no significant difference to the original isolate [12]. Using Bam 1 restriction enzyme differences of nu cleotide sequence between AD 169 and KUL-73 could be detected extending the result seen with Eco RI
- -
2
-■
1
Fig. 2. DNA cleavage pattern of six CMV strains produced by the restriction enzyme Bam 1; from left to right: RAU-75, KUL-73, Towne 125, Davis, KUR-74, AD 169, Eco RI cleavage pattern of KUL-73 with the molecular weight scale (x 106 daltons).
(fig. 2): it produced different bands in the MW region of about 2.0 x 106 daltons. In general, however, Bam 1 digestion of CMV DNA rendered similar results to those obtained with Eco RI: most of the fragments of the different CMV isolates comigrated, while some bands seem to be strain specific. CMV DNA electro phoresis without previous enzyme digestion revealed no fragmentation in all strains investigated.
Fig. 1. DNA cleavage pattern of six CMV strains produced by the restriction enzyme Eco RI (from left to right: Towne 125, Davis. R A U -75. FE I-75. AD 169, K U L-73). The num bers represent the
molecular weights (x 106 daltons); from Kilpatrick et al. [12],
The investigations described here have demonstrated that it is possible to identify different CMV isolates exactly and reproducibly by analyzing DNA base se quences. The findings are in agreement with previous results [4, 12]. They revealed stable strain specificities which may encourage studies about a molecular CMV epidemiology. On the other hand, the small number of CMV strains investigated up to date does not permit a classification into types, as has been done for herpes simplex virus (HSV). On the basis of studies with HSV types 1 and 2, which considerably differ in their DNA sequences, but display only minor antigenic differences [13], we suppose that CMV strains with relatively similar DNA base sequences could not be expected to show much serological variance of a seroepidemiological significance analogous to HSV antibody typing. We were unable, to find statistically significant differences in a great series of patients’ serum samples using
Downloaded by: Lund University Libraries 130.235.66.10 - 11/23/2018 6:29:03 PM
Discussion
247
Cytomegalovirus Strain Differentiation by DNA Restriction Analysis
7
8
9
10
Acknowledgement We thank IS. Fleckenstein for kind methodical advice, and we greatly appreciate the helpful technical assistance of R. Lepacli and S. Schnierda.
11
12
13
References 1 Weller, T. H.; 1ianshaw, J. B., and Scott, D, E.: Serologic differen tiation of viruses responsible for cytomegalic inclusion disease. Virology 12: 130-132(1960). 2 Birnbaum, G.; Lynch, J.I.; Margileth, A.M.; Longergan, W.M., and Sever, J.L.: Cytomegalovirus infections in newborn infants. J. Pediat. 75: 789-795 (1969). 3 Huang, Y.T.: Huang, E.S., and Pagano, J.S.: Antisera to human cytomegaloviruses prepared in the guinea pigs: specific immuno fluorescence and complement-fixation tests. J. Immun. 112: 528-532 (1974). 4 Huang, E.S.; Kilpatrick, B. A.; Huang, Y.T., and Pagano, J.S.: Detection of human cytomegalovirus and analysis of strain varia tion. Yale J. Biol. Med. 49: 29-44 (1975). 5 Stern. H. and Elek. S.D.: The incidence of infection with cyto megalovirus in a normal population. A serological study in Greater London. J. Hyg., Camb. 63: 79-87 (1965). 6 Andersen. H.K.: Complement-fixing and virus-neutralizing anti bodies in cytomegalovirus infections as measured against homol
14
15
16
17
18
ogous and heterologous antigens. Acta path, microbiol. scand. 78: 504-508 (1970). Haines, H.G.; v. Essen, R., and Benyesh-Melnick, M.: Prepara tion of specific antisera to cytomegaloviruses in goats. Proc. Soc. exp. Biol. Med. 138: 846-849 (1971). Albrecht, T. and Rapp, F.: Malignant transformation of hamster embryo fibroblasts following exposure to ultraviolet-irradiated human cytomegalovirus. Virology 55: 56-61 (1973). Rapp, F.; Geder, L.; Murasko, D.; Lausch, R.; Laddka, R.; Huang, E.S., and Webber, M.M.: Long-term persistence of CMV genome in cultivated human cells of prostatic origin. J. Virol. 16: 982-990(1975). Nahmias, A.J. and Roizman, B.: Infection with herpes-simplex viruses 1 and 2. il. New Engl J. Med. 289: 719-725 (1973). Gupta, P.; St. Jeor, S., and Rapp, F.: Comparison of the poly peptides of several strains of human cytomegalovirus. J. gen. Virol. 34: 447-454 (1977). Kilpatrick, B.A.; Huang, E.S., and Pagano, J.S.: Analysis of cytomegalovirus genomes with restriction endonucleases HinD 111 and Eco R-l. J. Virol. 18: 1095-1105 (1976). Sugino, W.M. and Kingsbury, D.T.: DNA homologies between strains of herpes simplex virus. Virology 71: 605-608 (1976). Rowe, W.P.; Harley, J.W.; Waterman, S.; Turner, A.C., and Huebner, R.J.: Cytopathogenic agent resembling human salivary gland virus recovered from tissue cultures of human adenoids. Proc. Soc. exp. Biol. Med. 92: 418-424 (1956). Plotkin, S.; Furukawa, T.; Zygraich, N.; Vamos, E.. and Haygden, C.: Candidate cytomegalovirus strain for vaccination. Infect. Im munity 12: 521-527 (1975). Just, M.; Buergin-Wolff, A.; Emoedi, G., and Hernandez, R.: Immunisation trials with live attenuated cytomegalovirus Towne 125. Infection 3: 111-114 (1975). Fleckenstein, B.; Bornkamm. G.W.. and Ludwig, H.: Repetitive sequences in complete and defective genomes of herpes virus saimiri. J. Virol. 15: 398-406 (1975). Schmitz, 1L; Docrr, H.W., and Obrig, M.: Envelope and nucleo capsid antigens of cytomegalovirus (CMV). Med. Microbiol. Immunol. 161: 155-162 (1975). Dr. H.W. Docrr, Institut für Medizinische Virologie der Universität Heidelberg, im Neuenheimer Feld 324, 6900 Heidelberg (FRG)
Downloaded by: Lund University Libraries 130.235.66.10 - 11/23/2018 6:29:03 PM
neutralization or complement fixation tests with puri fied viral envelopes as antigen prepared from Davis and AD 169 [18]. This is in agreement with the results of Gupta et al. [11], who found a remarkable similarity in the polypeptide composition of four dif ferent CMV strains. If crude antigens are used in the complement fixation test [3], a different distribution of antibodies to both envelope and nucleocapsid anti bodies may be responsible for apparent differences in some strains.
