119
~~u~n~~ of I/iro/qqicui ~~1~~)~s. 40 (I 992) 119 126 .Cj 1992 Elsevier Science Publishers B.V. i All rights reserved / 0166-0934/92~$05.00 VIRMET 01402
Rapid DNA diagnosis of herpes simplex virus serotypes Toshiya
Matsumoto~, Toshio
“Ikpurtment
of‘ Pathology.
iJupun) , ‘Shimane
Pr&wd
Institute
Heulth,
,fbr Public
Osamu Yamadaa, Asao Itagakib, Shigeru Kamahora” and Takashi Kurimura” Rcwarch
Institulr
Institute,fir Tottori
(Jupunj
,fbr Microbial
Public
Heulth.
and “Totrori )
Discuses, Mutsw
Universit.v
Osaka
(Jupanj School
Ishida”,
University,
( ‘ Tottori
Suitu
Prqfktural
of’ Medicine,
Yonugo
(Jupun (Accepted
IO June 1992)
Summary The presence of nucleotide sequences specific for each of herpes simplex virus (HSV) serotypes was demonstrated. These sequences were applied for dot DNA-DNA hybridization and for PCR for rapid DNA diagnosis of HSV infections. These sequences were found by molecular cloning of HSV-DNA fragments after digestion of DNA by KpnI enzyme. The type l-specific sequence was found around the 5’ end of BumHI B-fragment in the L region of type 1 DNA (corresponds to CIgene 27, promotor-regulatory region) and the type 2-specific sequence was around the junction region of the L and S of type 2 DNA (corresponds to a’ sequence). Both simple dot blot hybridization and PCR of HSV DNA’s, employing these type-specific nucieotide sequences, were proven to be much more useful than immunofluorescence in terms of typespecific diagnosis of HSV infections. HSV; PCR;
Dot blot; DNA
diagnosis
Introduction Molecular biological studies on herpes simplex virus (HSV) have been extensively performed by many researchers, but no one has so far reported on T. Kurimura, Dept. of Pathology, Research Institute for Microbial Diseases, Osaka University. Suita 565, Japan.
e~~rrr.~pun{i~nc~ to:
120
type-specific DNA diagnosis of HSV infections. For the typing of HSV. neutralisation tests, immunofluorescence, biological characteristics of isolated virus and the restriction pattern of viral DNA have been employed frequently (Balachandran et al., 1982; Pereira et al., 1982; Peterson et al., 1983: Sakaoka et al., 1985). It is well-known that intratypic antigenic variations are frequently observed (Pereira et al., 1982) so it is very important to locate conserved regions in HSV DNA sequences for DNA diagnosis. especially when the polymerase chain reaction (PCR) is used. Dot blot DNA-DNA hybridization and PCR (Saiki et al., 1985; Saiki et al., 1988; Ehrlich et al., 1991) have been introduced widely into laboratory diagnosis of viral infections, but this report is the first to introduce type-specific DNA diagnosis of HSV infections.
Materials and Methods
HSV type 1 (strain WT-51) (Hirano et al., 1979) and type 2 (strain UW 26X) were used as the reference strains. Thirty-five HSV-1 fresh isolates and 29 HSV2 fresh isolates were used, as well as reference strains. Typing of these virus isolates was performed by analysing restriction pattern profiles. These viruses were propagated in CV- 1, an African green monkey kidney cell line, which was grown at 37‘C in Eagle’s minimal essential medium supplemented with 2 mM glutamine, 2.5 mM sodium bicarbonate and 4% newborn calf serum.
Clinical specimens (swabs) from herpetic lesions were placed in 2 ml of sterile phosphate-buffered saline (pH 7.2) (PBS). Fifty-p1 aliquots of these diluted specimens were used for dot-blot DNA-DNA hybridization assay and for combination of PCR and dot-blot hybridization. Viral DNA CV-1 cells were infected with virus at an MO1 of 1 plaque-forming unit~cell and, after an appropriate incubation period at 37”C, viral DNA was prepared by the method of Pignatti et al. (1979). For PCR, DNA from clinical specimens in PBS were extracted as shown below. Fifty ,LII of 6 M guanidine isothiocyanate and 10 ~1 of glass powder suspension (Yamada et al., 1990) were mixed with 50 ~1 of a specimen in a 0.5 ml tube. The tube was left at the room temperature for 10 min and spun for 2 min at I5 000 rpm in a microcentrifuge. The precipitate was washed twice with 300 ~1 of ethanol wash solution (50% ethanol. 10 mM Tris (pH 7.4), 1 mM EDTA, 50 mM NaCl). The precipitate was suspended in 50 p-11 of distilled water and incubated at 55 ‘C for 15 min. After centrifugation at 15 000 rpm for 2 min. the supernatant was
121
collected
and used for PCR.
Dot-Blot method Type-specific DNA probes were hybridized directly with clinical specimens treated with NaOH on nylon membrane (Gene Screen Plus, DuPont, MA, USA). DNA probes were labelled with [a-‘2P]dCTP (ICN, Irvine) using Sweden) or non-radioisotopic labelling was oligolabelling kits (Pharmacia, performed using the method of Jablonski et al. (1986). HSV-1 or 2 type-specific DNA sequences were cloned as shown in Results. Type-.specijic PC R A primer pair TM 1 l/12 was selected from a gene, a promotor-regulatory region, of HSV- 1. This primer pair is expected to amplify the region between 3 1 bases upstream and 212 bases downstream of BamHI B fragment (5’ end) of the HSV-1 sequence described by Roizman et al. (1982). Another primer pair, TM21/22, was selected from the a’ sequence of HSV-2. This primer pair is expected to amplify the region between five bases downstream and 221 bases downstream of the a’ sequence (5’ end) of HSV-2 sequence described by Wilkie et al. (1981). Oligonucleotide probe TM I3 spans the region between 50 bases downstream and 89 bases downstream of the BarnHI B fragment (5’ end) of HIV-l (Table 1). Another probe, specific for HSV-2, TM23, spans the region between 148 bases downstream and 187 bases downstream of the a’ sequence (5’ end) (Table 1). Fifty ~1 of DNA extract for PCR was transferred to a 0.5 ml tube containing 50 ~1 of reaction buffer (20 mM Tris (pH 8.8), 50 mM KCl, 1.5 mM MgC12, 0.01% gelatin, 125 PM dATP, 125pM dCTP, 125 PM dTTP, 3 1.25 PM dGTP, 93.75 PM 7-deaza-2’-deoxyguanosine triphosphate, 10% dimethyl sulfoxide, 2.5 units of Taq polymerase and 1 PM each of primers) and 100 ~1 of mineral oil. HSV-1 PCR (92°C for 1 min, 60°C for 1 min, 72°C for 2 min: 30 cycles) and HSV-2 PCR (93°C for 1 min, 65°C for 1 min, 73°C for 1 min, 30 cycles) were TABLE
I
Primers and probes selected for type-specific dot blot hybridization Designation
Nucleotide sequence
HSV-I TMII TM12 TM13
T-CACGGGTATAAGGACATCCA-3’ 5’-GGGTCCTCGTCCAGATCGCT-3’ T-CCCCGATTCGGGCCCGGTCGCTCGCTACCGGTGCGCCACC-3’
HSV-2 TM21 TM22 TM23
T-GCCTCTTTTCCCCCGGGGAG-3’ T-GGGAAAAAAGCCGCGCGGGG-3’ T-CCCCGCGGGCGCCGCCCCTCCCCCCGCGCGCCGCGGGCTG-3’
122
carried out with a programmable Dri-Block PHC-1 (Techne, Cambridge, UK). The experimental conditions were selected according to the GC-content in the nucleotide sequences to be amplified, respectively, for HSV-I and HSV-2. The resultant product of PCR was examined for the presence of type-specific amplified nucleotide sequence by dot blot hybridization on nylon membrane (Gene Screen Plus, DuPont, MA, USA) employing TM13 and TM23 as the probes. The specificity of the reaction was confirmed by Southern blot hybridization (Southern, 1975).
Smears of swabs from herpetic lesions were fixed on glass slides and tested by Micro Track Herpes (Syva, CA, USA).
Results Selection of’ type-specifics HS V DNA ,fi+ugments KpnI digests of HSV type I (strain WT-51) or type 2 (strain UW268) DNA were randomly cloned in pUC 19 DNA and propagated in E. cofi (MVll90). After colony hybridization with strain WT-51 or UW268 DNA, candidates of type-specific DNA clones were checked for their sizes by Southern blot hybridization. Out of 63 clones of type 1 DNA fragments and 43 clones of type 2 DNA fragments, 10 clones and five clones, respectively, for types 1 and 2 were chosen as the candidates and DNA clones of different sizes were crosshybridized within each type to check for homology. Finally, one clone each from type 1 and type 2 was selected and tested for typing of 35 HSV-I isolates and 29 HSV-2 isolates. The selected type I DNA clone (clone 56) hybridized with all of DNA from 35 HSV-1 isolates and with none of 29 HSV-2 isolates. On the other hand, the selected type 2 DNA clone (clone 5) hybridized with all of 29 type 2 DNA samples and with 2 of 35 type 1 DNA samples, maybe because of partial homology of large-sized DNA clone 5 with DNA of some of HSV-1 isolates. DNA sequencing of clone 56 and clone 5 was done partially (about 400 nucleotides for both clones) and their loci on the viral genome were presumed by referring to published nucleotide sequences of HSV- 1 (Roizman et al., 1982) and HSV-2 (Wilkie et al., 1981). After trial and error, TM 11,12,13 and TM21,22,23 were selected as type-specific primers and probes (see Materials and Methods).
Vesicular fluid specimens of 18 patients with herpes simplex were tested for the presence of HSV by dot-blot hybridization and by immunofluorescence (Table 2). The results clearly show that, even in the absence of cells infected
123
TABLE 2 Correlation of the results of type-specific fluid was used as the specimen
dot blot method and immunofluorescence
when vesicular
Result of typing
Method
Dot blot Immunofluorescence
HSV-1
HSV-2
indeterminate
ND=
Total
12 7
6
0 2b
0 8”
18 18
I
-I_
“Not done. ‘Positive for both types I and 2. “Lack of cells in the specimens.
with HSV, dot-blot hybridization can be carried out satisfactorily, while immuno~uorescence cannot be done without cells and fails to identify serotypes in some cases. The usefulness of PCR for HSV typing 58 swab specimens from patients with oral or genital herpes were tested for virus isolation and typing by restriction pattern of isolated virus, dot blot hybridization of the swabs and PCR (Table 3). The virus isolation rate was relatively low (46/58), possibly because of virus inactivation during storage of the specimens. The PCR assay was sensitive and detected virus DNA in 51 out of 58 specimens. Because of the inhibitory effect of saliva, the dot-blot assay often failed to detect HSV DNA in throat swabs particularly when the amount of DNA was small. To demonstrate the accuracy of typing of HSV serotypes by PCR-dot blot assay, 28 clinical specimens were tested by the procedure shown in Fig. 1. Other specimens were also distinguished clearly by the procedure. Type-specific PCR was proven to be able to differentiate serotypes of these specimens without difficulty.
TABLE 3 Correlation
of the results of dot blot method and PCR assay
Method
Virus isolation Dot blot PCR
Typing HSV-1
HSV-2
Indeterminate
34(O) LO(O) 32(o)
12(12) 14(14) 19(19)
1O(O)
O(0) O(0)
Negative
Total
12(10) 24(g) 7(3)
58(22) 58(22) 58(22)
58 Clinical specimens (throat swab. 36; genital swab, 22) were tested. Numbers in parentheses vesicular fluid specimens from patients with genital herpes.
are
124
12
34
5
67
8
910
Fig. 1. Dot-blot hybridization of type-spccitic PCR products. DNA probes were labelled with alkaline phosphatase. Lanes A to C, HSV-1 type-specific PCR: Lanes D to F. HSV-2 type-specific PCR: 0. F9, HSV-I reference strain (KOS): ClO, Fl0. HSV-2 reference strain (r!WhS); Al X8. Dl FX. clinical specimens.
Discussion HSV 1 and HSV 2 have closely related nucleotide sequences (Ludwig et al., 1972). The existence of highly type-specific nucleotide sequences in HSV DNA was confirmed by DNA-DNA hybridization. These DNA sequences can be used for detection and typing of HSV in clinical specimens both by dot blot hybridization and PCR, combined with dot blot hybridization. The procedure requires only a very small amount of specimen and does not require cells infected with the virus or handling of infectious virus. The type-specific sequences do not cross-react with other human herpes viruses or DNA from human cells (data not shown). Practically, DNA diagnostic procedures are more sensitive and specific than immunofluorescence. In the case of immunofluorescence, specimens from the uterine cervix are often difficult and cells infected with the virus are required. The contamination of saliva in the specimen often interferes with dot blot hybridization, especially when the amount of viral DNA is small. This problem may be overcome by treatment of the specimens with guanidine isothiocyanate before hybridization. Employing our primers and probes, it will be possible to identify the specific serotype of HSV in clinical specimens with high sensitivity.
125
References Balachandran, N., Frame, B., Chernesky, M., Kraiseburd, E., Kouri, Y., Garcia, D., Lavery, C. and Rawis, W.E. (1982) Identification of herpes simplex viruses with monoclonal antibodies. J. Clin. Microbial. 16, 2055208. Davis, L.G., Dibner, M.D. and Battey, J.F. (1986) Basic Methods in Molecular Cloning. Elsevier, Amsterdam, pp. 147 149. Erlich, H.A., Gelfand, D. and Sninsky, J.J. (1991) Recent advances in the polymerase chain reaction. Science 252, 1643-1651. Hirano. A., Yumura, K., Kurimura, T., Katumoto, T. Moriyama, H. and Manabe, H. (1979) Analysis of herpes simplex virus isolated from patients with recurrent herpes keratitis exhibiting ‘treatment-resistance’ to 5’.iodo-2’-deoxyuridine. Acta Virol. 23, 226-230. Jablonski, E., Moomaw. E.W., Tullis, R.H. and Ruth, J.L. (1986) Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes. Nucleic Acids Res. 14, 6115 6128. Ludwig, H.O., Biswal, N. and Benyesh-Melnick, M. (1972) Studies on the relatedness of herpes viruses through DNA-DNA hybridization. Virology 49, 95 -101. Pereira, L.D., Dondero, D.V., Devlin, V. and Woodie, J.D. (1982) Serological analysis of herpes simplex virus types 1 and 2 with monoclonal antibodies. Infect. Immun. 35, 3655367. Peterson, E., Schmidt, O.W., Goldstein, L.C., Nowinsky, R.C. and Corey, L. (1983) Typing of clinical herpes simplex virus isolation with mouse monoclonal antibodies to herpes simplex virus type I and 2: comparison with type-specific rabbit antisera and restriction endonuclease analysis of viral DNA. J. Clin. Microbial. 17, 92-96. Pignatti. P.F., Cassai, E., Meneguzzi, G., Chenciner, N. and Milanesi, G. (1979) Herpes simplex virus DNA isolation from infected cells with a novel procedure. Virology 93, 260-264. Roizman, B. and Mackem, S. (I 982) Structural features of the herpes simplex virus GIgene 4,0, and 27 promoter-regulatory sequences which confer 2 regulation on chimeric thymidine kinase genes. J. Virol. 44, 939 949. Saiki. R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, D.T. and Erlic, H.A. (1985) Enzymatic amplification of /%globulin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350-1354. Saiki, R.K., Gelfland, D.H., Stoffel, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487~491. Sakaoka. H., Aomori, T., Honda, O., Saheki, Y., Ishida, S., Yamanishi, Y. and Fujinaga. K. (1985) Subtypes of herpes simplex virus type I in Japan: classification by restriction endonucleases and analysis of distribution. J. Infect. Dis. 152, 190- 197. Southern. E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 5033517. Wilkie, N.M. and Davison, A.J. (1975) Nucleotide sequences of the joint between the L and S segments of herpes simplex virus type I and 2. J. Cert. Virol. 55, 3 l5- 33 I. Yamada, O., Matsumoto, T., Nakashima, M., Hagari. S., Kamahora, T., Ueyama, H., Kishi, Y., Uemura, H. and Kurimura, T. (1990) A new method for extracting DNA or RNA for polymerase chain reaction. J. Virol. Methods 27, 203-~210.
~~u~n~~ of I/iro/qqicui ~~1~~)~s. 40 (I 992) 119 126 .Cj 1992 Elsevier Science Publishers B.V. i All rights reserved / 0166-0934/92~$05.00 VIRMET 01402
Rapid DNA diagnosis of herpes simplex virus serotypes Toshiya
Matsumoto~, Toshio
“Ikpurtment
of‘ Pathology.
iJupun) , ‘Shimane
Pr&wd
Institute
Heulth,
,fbr Public
Osamu Yamadaa, Asao Itagakib, Shigeru Kamahora” and Takashi Kurimura” Rcwarch
Institulr
Institute,fir Tottori
(Jupunj
,fbr Microbial
Public
Heulth.
and “Totrori )
Discuses, Mutsw
Universit.v
Osaka
(Jupanj School
Ishida”,
University,
( ‘ Tottori
Suitu
Prqfktural
of’ Medicine,
Yonugo
(Jupun (Accepted
IO June 1992)
Summary The presence of nucleotide sequences specific for each of herpes simplex virus (HSV) serotypes was demonstrated. These sequences were applied for dot DNA-DNA hybridization and for PCR for rapid DNA diagnosis of HSV infections. These sequences were found by molecular cloning of HSV-DNA fragments after digestion of DNA by KpnI enzyme. The type l-specific sequence was found around the 5’ end of BumHI B-fragment in the L region of type 1 DNA (corresponds to CIgene 27, promotor-regulatory region) and the type 2-specific sequence was around the junction region of the L and S of type 2 DNA (corresponds to a’ sequence). Both simple dot blot hybridization and PCR of HSV DNA’s, employing these type-specific nucieotide sequences, were proven to be much more useful than immunofluorescence in terms of typespecific diagnosis of HSV infections. HSV; PCR;
Dot blot; DNA
diagnosis
Introduction Molecular biological studies on herpes simplex virus (HSV) have been extensively performed by many researchers, but no one has so far reported on T. Kurimura, Dept. of Pathology, Research Institute for Microbial Diseases, Osaka University. Suita 565, Japan.
e~~rrr.~pun{i~nc~ to:
120
type-specific DNA diagnosis of HSV infections. For the typing of HSV. neutralisation tests, immunofluorescence, biological characteristics of isolated virus and the restriction pattern of viral DNA have been employed frequently (Balachandran et al., 1982; Pereira et al., 1982; Peterson et al., 1983: Sakaoka et al., 1985). It is well-known that intratypic antigenic variations are frequently observed (Pereira et al., 1982) so it is very important to locate conserved regions in HSV DNA sequences for DNA diagnosis. especially when the polymerase chain reaction (PCR) is used. Dot blot DNA-DNA hybridization and PCR (Saiki et al., 1985; Saiki et al., 1988; Ehrlich et al., 1991) have been introduced widely into laboratory diagnosis of viral infections, but this report is the first to introduce type-specific DNA diagnosis of HSV infections.
Materials and Methods
HSV type 1 (strain WT-51) (Hirano et al., 1979) and type 2 (strain UW 26X) were used as the reference strains. Thirty-five HSV-1 fresh isolates and 29 HSV2 fresh isolates were used, as well as reference strains. Typing of these virus isolates was performed by analysing restriction pattern profiles. These viruses were propagated in CV- 1, an African green monkey kidney cell line, which was grown at 37‘C in Eagle’s minimal essential medium supplemented with 2 mM glutamine, 2.5 mM sodium bicarbonate and 4% newborn calf serum.
Clinical specimens (swabs) from herpetic lesions were placed in 2 ml of sterile phosphate-buffered saline (pH 7.2) (PBS). Fifty-p1 aliquots of these diluted specimens were used for dot-blot DNA-DNA hybridization assay and for combination of PCR and dot-blot hybridization. Viral DNA CV-1 cells were infected with virus at an MO1 of 1 plaque-forming unit~cell and, after an appropriate incubation period at 37”C, viral DNA was prepared by the method of Pignatti et al. (1979). For PCR, DNA from clinical specimens in PBS were extracted as shown below. Fifty ,LII of 6 M guanidine isothiocyanate and 10 ~1 of glass powder suspension (Yamada et al., 1990) were mixed with 50 ~1 of a specimen in a 0.5 ml tube. The tube was left at the room temperature for 10 min and spun for 2 min at I5 000 rpm in a microcentrifuge. The precipitate was washed twice with 300 ~1 of ethanol wash solution (50% ethanol. 10 mM Tris (pH 7.4), 1 mM EDTA, 50 mM NaCl). The precipitate was suspended in 50 p-11 of distilled water and incubated at 55 ‘C for 15 min. After centrifugation at 15 000 rpm for 2 min. the supernatant was
121
collected
and used for PCR.
Dot-Blot method Type-specific DNA probes were hybridized directly with clinical specimens treated with NaOH on nylon membrane (Gene Screen Plus, DuPont, MA, USA). DNA probes were labelled with [a-‘2P]dCTP (ICN, Irvine) using Sweden) or non-radioisotopic labelling was oligolabelling kits (Pharmacia, performed using the method of Jablonski et al. (1986). HSV-1 or 2 type-specific DNA sequences were cloned as shown in Results. Type-.specijic PC R A primer pair TM 1 l/12 was selected from a gene, a promotor-regulatory region, of HSV- 1. This primer pair is expected to amplify the region between 3 1 bases upstream and 212 bases downstream of BamHI B fragment (5’ end) of the HSV-1 sequence described by Roizman et al. (1982). Another primer pair, TM21/22, was selected from the a’ sequence of HSV-2. This primer pair is expected to amplify the region between five bases downstream and 221 bases downstream of the a’ sequence (5’ end) of HSV-2 sequence described by Wilkie et al. (1981). Oligonucleotide probe TM I3 spans the region between 50 bases downstream and 89 bases downstream of the BarnHI B fragment (5’ end) of HIV-l (Table 1). Another probe, specific for HSV-2, TM23, spans the region between 148 bases downstream and 187 bases downstream of the a’ sequence (5’ end) (Table 1). Fifty ~1 of DNA extract for PCR was transferred to a 0.5 ml tube containing 50 ~1 of reaction buffer (20 mM Tris (pH 8.8), 50 mM KCl, 1.5 mM MgC12, 0.01% gelatin, 125 PM dATP, 125pM dCTP, 125 PM dTTP, 3 1.25 PM dGTP, 93.75 PM 7-deaza-2’-deoxyguanosine triphosphate, 10% dimethyl sulfoxide, 2.5 units of Taq polymerase and 1 PM each of primers) and 100 ~1 of mineral oil. HSV-1 PCR (92°C for 1 min, 60°C for 1 min, 72°C for 2 min: 30 cycles) and HSV-2 PCR (93°C for 1 min, 65°C for 1 min, 73°C for 1 min, 30 cycles) were TABLE
I
Primers and probes selected for type-specific dot blot hybridization Designation
Nucleotide sequence
HSV-I TMII TM12 TM13
T-CACGGGTATAAGGACATCCA-3’ 5’-GGGTCCTCGTCCAGATCGCT-3’ T-CCCCGATTCGGGCCCGGTCGCTCGCTACCGGTGCGCCACC-3’
HSV-2 TM21 TM22 TM23
T-GCCTCTTTTCCCCCGGGGAG-3’ T-GGGAAAAAAGCCGCGCGGGG-3’ T-CCCCGCGGGCGCCGCCCCTCCCCCCGCGCGCCGCGGGCTG-3’
122
carried out with a programmable Dri-Block PHC-1 (Techne, Cambridge, UK). The experimental conditions were selected according to the GC-content in the nucleotide sequences to be amplified, respectively, for HSV-I and HSV-2. The resultant product of PCR was examined for the presence of type-specific amplified nucleotide sequence by dot blot hybridization on nylon membrane (Gene Screen Plus, DuPont, MA, USA) employing TM13 and TM23 as the probes. The specificity of the reaction was confirmed by Southern blot hybridization (Southern, 1975).
Smears of swabs from herpetic lesions were fixed on glass slides and tested by Micro Track Herpes (Syva, CA, USA).
Results Selection of’ type-specifics HS V DNA ,fi+ugments KpnI digests of HSV type I (strain WT-51) or type 2 (strain UW268) DNA were randomly cloned in pUC 19 DNA and propagated in E. cofi (MVll90). After colony hybridization with strain WT-51 or UW268 DNA, candidates of type-specific DNA clones were checked for their sizes by Southern blot hybridization. Out of 63 clones of type 1 DNA fragments and 43 clones of type 2 DNA fragments, 10 clones and five clones, respectively, for types 1 and 2 were chosen as the candidates and DNA clones of different sizes were crosshybridized within each type to check for homology. Finally, one clone each from type 1 and type 2 was selected and tested for typing of 35 HSV-I isolates and 29 HSV-2 isolates. The selected type I DNA clone (clone 56) hybridized with all of DNA from 35 HSV-1 isolates and with none of 29 HSV-2 isolates. On the other hand, the selected type 2 DNA clone (clone 5) hybridized with all of 29 type 2 DNA samples and with 2 of 35 type 1 DNA samples, maybe because of partial homology of large-sized DNA clone 5 with DNA of some of HSV-1 isolates. DNA sequencing of clone 56 and clone 5 was done partially (about 400 nucleotides for both clones) and their loci on the viral genome were presumed by referring to published nucleotide sequences of HSV- 1 (Roizman et al., 1982) and HSV-2 (Wilkie et al., 1981). After trial and error, TM 11,12,13 and TM21,22,23 were selected as type-specific primers and probes (see Materials and Methods).
Vesicular fluid specimens of 18 patients with herpes simplex were tested for the presence of HSV by dot-blot hybridization and by immunofluorescence (Table 2). The results clearly show that, even in the absence of cells infected
123
TABLE 2 Correlation of the results of type-specific fluid was used as the specimen
dot blot method and immunofluorescence
when vesicular
Result of typing
Method
Dot blot Immunofluorescence
HSV-1
HSV-2
indeterminate
ND=
Total
12 7
6
0 2b
0 8”
18 18
I
-I_
“Not done. ‘Positive for both types I and 2. “Lack of cells in the specimens.
with HSV, dot-blot hybridization can be carried out satisfactorily, while immuno~uorescence cannot be done without cells and fails to identify serotypes in some cases. The usefulness of PCR for HSV typing 58 swab specimens from patients with oral or genital herpes were tested for virus isolation and typing by restriction pattern of isolated virus, dot blot hybridization of the swabs and PCR (Table 3). The virus isolation rate was relatively low (46/58), possibly because of virus inactivation during storage of the specimens. The PCR assay was sensitive and detected virus DNA in 51 out of 58 specimens. Because of the inhibitory effect of saliva, the dot-blot assay often failed to detect HSV DNA in throat swabs particularly when the amount of DNA was small. To demonstrate the accuracy of typing of HSV serotypes by PCR-dot blot assay, 28 clinical specimens were tested by the procedure shown in Fig. 1. Other specimens were also distinguished clearly by the procedure. Type-specific PCR was proven to be able to differentiate serotypes of these specimens without difficulty.
TABLE 3 Correlation
of the results of dot blot method and PCR assay
Method
Virus isolation Dot blot PCR
Typing HSV-1
HSV-2
Indeterminate
34(O) LO(O) 32(o)
12(12) 14(14) 19(19)
1O(O)
O(0) O(0)
Negative
Total
12(10) 24(g) 7(3)
58(22) 58(22) 58(22)
58 Clinical specimens (throat swab. 36; genital swab, 22) were tested. Numbers in parentheses vesicular fluid specimens from patients with genital herpes.
are
124
12
34
5
67
8
910
Fig. 1. Dot-blot hybridization of type-spccitic PCR products. DNA probes were labelled with alkaline phosphatase. Lanes A to C, HSV-1 type-specific PCR: Lanes D to F. HSV-2 type-specific PCR: 0. F9, HSV-I reference strain (KOS): ClO, Fl0. HSV-2 reference strain (r!WhS); Al X8. Dl FX. clinical specimens.
Discussion HSV 1 and HSV 2 have closely related nucleotide sequences (Ludwig et al., 1972). The existence of highly type-specific nucleotide sequences in HSV DNA was confirmed by DNA-DNA hybridization. These DNA sequences can be used for detection and typing of HSV in clinical specimens both by dot blot hybridization and PCR, combined with dot blot hybridization. The procedure requires only a very small amount of specimen and does not require cells infected with the virus or handling of infectious virus. The type-specific sequences do not cross-react with other human herpes viruses or DNA from human cells (data not shown). Practically, DNA diagnostic procedures are more sensitive and specific than immunofluorescence. In the case of immunofluorescence, specimens from the uterine cervix are often difficult and cells infected with the virus are required. The contamination of saliva in the specimen often interferes with dot blot hybridization, especially when the amount of viral DNA is small. This problem may be overcome by treatment of the specimens with guanidine isothiocyanate before hybridization. Employing our primers and probes, it will be possible to identify the specific serotype of HSV in clinical specimens with high sensitivity.
125
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