Original Paper
Intervirology 1992;33:1-5
Department o f Pediatrics, Hokkaido University School of Medicine, Sapporo; Pediatric Clinic, Kitami Red Cross Hospital, Kitami; Pediatric Clinic, Sapporo Kohsei Hospital, Sapporo, Japan
Key Words Kawasaki disease Epstein-Barr virus Polymerase chain reaction
Detection of Epstein-Barr Virus Sequences in Patients with Kawasaki Disease by Means of the Polymerase Chain Reaction
Summary We used a selective DNA amplification technique to detect Epstein-Barr virus (EBV) DNA in peripheral blood mononu clear cells from patients with Kawasaki disease (KD). By means of the polymerase chain reaction EBV sequences were indentified directly in 21 (60%) of 35 KD patients within 2 weeks after the onset of KD. Furthermore, EBV sequences were detected in all of 6 repeatedly tested patients with KD within 3 months after disease onset. In contrast, only 2 (12%) of 17 control DNA samples were polymerase chain reaction positive. These results indicate that an unusual EBV-cell interaction may exist in KD.
Introduction Kawasaki disease (KD), an acute vasculitis of infants and young children, is characterized by fever, rash, mucositis, lymphadenopathy, and coronary artery damage [1], Epidemiologic data on KD strongly suggest that an infectious agent is involved. However, the etiology is still un known. We have reported some relationship be tween KDand Epstein-Barrvirus(EBV)[2-6],
Received: November 13,1990 Accepted : January 7,1991
The nucleic acid amplification procedure, termed the polymerase chain reaction (PCR) has been successfully used in recent years in the clinical diagnosis of genetic and infectious diseases [7-9], The PCR enables the use of very small amounts of tissue for DNA analysis and provides results within a few days. The objective of this study was to directly deter mine the presence of genetic EBV information in the DNA of patients with KD.
Dr. Hideaki Kikuta Department of Pediatrics Hokkaido University School of Medicine North 15, West 7, Kita-ku, Sapporo 060 (Japan)
© 1992 S. Karger AG, Basel 0300-5526/92/ 0331 -0001 S2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/13/2018 8:09:48 PM
Hideaki Kikuta a Masonori Nakanishia Nobuyoshi Ishikawa b Mutsuko Konnoc Shuzo Matsumotoa
Clinical Samples In this study we used peripheral blood mononuclear cell (PBMC) DNA samples from 35 patients with KD and from 17 age-matched patients with acute febrile illnesses. The blood samples were collected from 35 patients within 2 weeks after the onset of KD. The mean age of the KD patients was 20 months (range from 2 months to 5 years). Multiple blood samples were collect ed at irregular intervals during several months in 6 of the 35 KD patients. The mean age of the controls was 22 months (range from 4 months to 5 years). PBMCs were obtained by centrifugation of heparinized peripheral blood on a Ficoll-Conray gradient. Human DNA sam ples from Raji cells and Molt-4 cells were used as positive and negative controls, respectively. Human em bryonic fibroblast cells infected with HSV-1 (Seibert strain), VZV (wild type), and human CMV (wild type) and HHV-6-(OK strain) [10] infected cord blood mono nuclear cells were used for these studies. Amplification o f EBV DNA Sequences The PCR was performed using a protocol by Cetus Corp. The PCR reaction mixture consisted of 200 prnol of each deoxyribonucleotide, 2.5 U of Taq (Thermits aquaticus) DNA polymerase, 50 nmol/l of potassium chloride, 10 mmol/l of Tris-HCI (pH 8.3), 1.5 mmol/1 of magnesium chloride, 0.01% (v/w) of gelatin, 20 pmol of each oligonucleotide primer, and I pg of DNA in a volume of 100 pi. The mixtures were overlaid with 50 pi of mineral oil. Before thermal cycling, the mixture was incubated at 95° for 5 min to denature the genomic DNA. Samples were then subjected to 35 cycles of PCR, each consisting of 1 min of denaturation at 94°, 2 min of annealing at 55°, and 3 min of polymerization at 72° by a DNA thermal cycler (Perkin-Elmer/Cetus). After the last cycle, all samples were incubated for an additional 7 min at 72° to ensure that the final extension step was complete. The oligonucleotide primers used to amplify a 410 base pair (bp) sequence in the region within the EBV BamHI-W fragment were 5-GTGACTTCACCAAAGGTCAG-3' and 5'-TTAAAGTCCACTTACCTCTG-3' [II]. Analysis o f Amplified Samples Ten microliters of the PCR product was subjected to electrophoresis on a 1.5% NuSeive gel (FMC BioProducts, Denmark) and run at 45 mA for I h. The gel was stained with 1pg/m l of ethidium bromide for 5 min, and the DNA bands were visualized under ultraviolet light. The PCR product was transferred onto nitrocellu
2
lose membrane filters. Each filter was hybridized with the -,:P-Labeled cloned fragment BomHI-W of the EBV genome for 48 h at 41° in 1x standard saline citrate (0.15 M sodium chloride and 0.015 M sodium citrate), 50% formamide, 0.5% SDS, and heat-denatured salmon sperm DNA (100 pg/ml). After hybridization, the filters were washed three times at room temperature in 0.1 x standard saline citrate with 0.1% SDS, then incu bated three times for I h at 50°. The filters were then dried and exposed to Sakura X-ray film (Tokyo) at -80°.
Results Specificity o f PCR The PCR-amplified genomic Raji cell DNA was confirmed on ethidium bromide stained gel and by Southern blotting with a radiola beled EBV ZtowHI-W fragment. Furthermore, the PCR product was identified from the Pst\ cleavage pattern, because it cleaves inside the 410-bp region (fig. 1). When DNA samples from cells infected with HSV-I, VZV, human CMV, and HHV-6 were used as templates in the PCR reaction, no amplification was noted by direct gel analysis or Southern blot hybridization (fig. 2). Sensitivity o f PCR To assess the sensitivity of the technique, 1 pg of Raji cell DNA was serially diluted to 104. A positive signal after PCR was visible on ethidium bromide stained gel and Southern blot analyses for dilutions from 1in 10° to 1in 103 (fig. 1,2). Detection o f EBV DNA in PBMCs o f Patients with KD EBV sequences, as indicated by the 410-bp DNA band identical to that of the positive controls, were detected in 21 (60%) of 35 KD patients within 2 weeks after the onset of KD. EBV sequences were detected within 3 months after the onset of KD in the DNA samples repeatedly collected from 6 individual patients
Kikuta/Nakanishi/lshikawa/Konno/ Matsumoto
KBV Sequences and Kawasaki Disease
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/13/2018 8:09:48 PM
Material and Methods
(fig. 3). In contrast, only 2 (12%) of the 17 control DNA samples were PCR positive.
Discussion
Fig. 1. Electrophoretic analysis of the PCR pro ducts. The DNA used for enzymatic amplification was as follows: 10»(lane 1), 10 '(la n e2), 10 : (lane3), 10 -'(lane 4), and 10 4 (lane 5) pg of Raji cell DNA. R E = Rr/I-digested PCR product: MW = molecular weight marker:
Intervirology 1992;33:1-5
Department o f Pediatrics, Hokkaido University School of Medicine, Sapporo; Pediatric Clinic, Kitami Red Cross Hospital, Kitami; Pediatric Clinic, Sapporo Kohsei Hospital, Sapporo, Japan
Key Words Kawasaki disease Epstein-Barr virus Polymerase chain reaction
Detection of Epstein-Barr Virus Sequences in Patients with Kawasaki Disease by Means of the Polymerase Chain Reaction
Summary We used a selective DNA amplification technique to detect Epstein-Barr virus (EBV) DNA in peripheral blood mononu clear cells from patients with Kawasaki disease (KD). By means of the polymerase chain reaction EBV sequences were indentified directly in 21 (60%) of 35 KD patients within 2 weeks after the onset of KD. Furthermore, EBV sequences were detected in all of 6 repeatedly tested patients with KD within 3 months after disease onset. In contrast, only 2 (12%) of 17 control DNA samples were polymerase chain reaction positive. These results indicate that an unusual EBV-cell interaction may exist in KD.
Introduction Kawasaki disease (KD), an acute vasculitis of infants and young children, is characterized by fever, rash, mucositis, lymphadenopathy, and coronary artery damage [1], Epidemiologic data on KD strongly suggest that an infectious agent is involved. However, the etiology is still un known. We have reported some relationship be tween KDand Epstein-Barrvirus(EBV)[2-6],
Received: November 13,1990 Accepted : January 7,1991
The nucleic acid amplification procedure, termed the polymerase chain reaction (PCR) has been successfully used in recent years in the clinical diagnosis of genetic and infectious diseases [7-9], The PCR enables the use of very small amounts of tissue for DNA analysis and provides results within a few days. The objective of this study was to directly deter mine the presence of genetic EBV information in the DNA of patients with KD.
Dr. Hideaki Kikuta Department of Pediatrics Hokkaido University School of Medicine North 15, West 7, Kita-ku, Sapporo 060 (Japan)
© 1992 S. Karger AG, Basel 0300-5526/92/ 0331 -0001 S2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/13/2018 8:09:48 PM
Hideaki Kikuta a Masonori Nakanishia Nobuyoshi Ishikawa b Mutsuko Konnoc Shuzo Matsumotoa
Clinical Samples In this study we used peripheral blood mononuclear cell (PBMC) DNA samples from 35 patients with KD and from 17 age-matched patients with acute febrile illnesses. The blood samples were collected from 35 patients within 2 weeks after the onset of KD. The mean age of the KD patients was 20 months (range from 2 months to 5 years). Multiple blood samples were collect ed at irregular intervals during several months in 6 of the 35 KD patients. The mean age of the controls was 22 months (range from 4 months to 5 years). PBMCs were obtained by centrifugation of heparinized peripheral blood on a Ficoll-Conray gradient. Human DNA sam ples from Raji cells and Molt-4 cells were used as positive and negative controls, respectively. Human em bryonic fibroblast cells infected with HSV-1 (Seibert strain), VZV (wild type), and human CMV (wild type) and HHV-6-(OK strain) [10] infected cord blood mono nuclear cells were used for these studies. Amplification o f EBV DNA Sequences The PCR was performed using a protocol by Cetus Corp. The PCR reaction mixture consisted of 200 prnol of each deoxyribonucleotide, 2.5 U of Taq (Thermits aquaticus) DNA polymerase, 50 nmol/l of potassium chloride, 10 mmol/l of Tris-HCI (pH 8.3), 1.5 mmol/1 of magnesium chloride, 0.01% (v/w) of gelatin, 20 pmol of each oligonucleotide primer, and I pg of DNA in a volume of 100 pi. The mixtures were overlaid with 50 pi of mineral oil. Before thermal cycling, the mixture was incubated at 95° for 5 min to denature the genomic DNA. Samples were then subjected to 35 cycles of PCR, each consisting of 1 min of denaturation at 94°, 2 min of annealing at 55°, and 3 min of polymerization at 72° by a DNA thermal cycler (Perkin-Elmer/Cetus). After the last cycle, all samples were incubated for an additional 7 min at 72° to ensure that the final extension step was complete. The oligonucleotide primers used to amplify a 410 base pair (bp) sequence in the region within the EBV BamHI-W fragment were 5-GTGACTTCACCAAAGGTCAG-3' and 5'-TTAAAGTCCACTTACCTCTG-3' [II]. Analysis o f Amplified Samples Ten microliters of the PCR product was subjected to electrophoresis on a 1.5% NuSeive gel (FMC BioProducts, Denmark) and run at 45 mA for I h. The gel was stained with 1pg/m l of ethidium bromide for 5 min, and the DNA bands were visualized under ultraviolet light. The PCR product was transferred onto nitrocellu
2
lose membrane filters. Each filter was hybridized with the -,:P-Labeled cloned fragment BomHI-W of the EBV genome for 48 h at 41° in 1x standard saline citrate (0.15 M sodium chloride and 0.015 M sodium citrate), 50% formamide, 0.5% SDS, and heat-denatured salmon sperm DNA (100 pg/ml). After hybridization, the filters were washed three times at room temperature in 0.1 x standard saline citrate with 0.1% SDS, then incu bated three times for I h at 50°. The filters were then dried and exposed to Sakura X-ray film (Tokyo) at -80°.
Results Specificity o f PCR The PCR-amplified genomic Raji cell DNA was confirmed on ethidium bromide stained gel and by Southern blotting with a radiola beled EBV ZtowHI-W fragment. Furthermore, the PCR product was identified from the Pst\ cleavage pattern, because it cleaves inside the 410-bp region (fig. 1). When DNA samples from cells infected with HSV-I, VZV, human CMV, and HHV-6 were used as templates in the PCR reaction, no amplification was noted by direct gel analysis or Southern blot hybridization (fig. 2). Sensitivity o f PCR To assess the sensitivity of the technique, 1 pg of Raji cell DNA was serially diluted to 104. A positive signal after PCR was visible on ethidium bromide stained gel and Southern blot analyses for dilutions from 1in 10° to 1in 103 (fig. 1,2). Detection o f EBV DNA in PBMCs o f Patients with KD EBV sequences, as indicated by the 410-bp DNA band identical to that of the positive controls, were detected in 21 (60%) of 35 KD patients within 2 weeks after the onset of KD. EBV sequences were detected within 3 months after the onset of KD in the DNA samples repeatedly collected from 6 individual patients
Kikuta/Nakanishi/lshikawa/Konno/ Matsumoto
KBV Sequences and Kawasaki Disease
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/13/2018 8:09:48 PM
Material and Methods
(fig. 3). In contrast, only 2 (12%) of the 17 control DNA samples were PCR positive.
Discussion
Fig. 1. Electrophoretic analysis of the PCR pro ducts. The DNA used for enzymatic amplification was as follows: 10»(lane 1), 10 '(la n e2), 10 : (lane3), 10 -'(lane 4), and 10 4 (lane 5) pg of Raji cell DNA. R E = Rr/I-digested PCR product: MW = molecular weight marker:
