G Model PEDOT 7131 No. of Pages 6
International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Coexistence of Epstein–Barr virus and Parvovirus B19 in tonsillar tissue samples: Quantitative measurement by real-time PCR lu c,2 , Fatih Şahiner a, *, Ramazan Gümral b,1, Üzeyir Yildizog d,3 e,4 it , Abdullah Durmaz , Nuri Yig it f,5 , Mehmet Ali Saraçli g,6 Mustafa Alparslan Babayig , Ayhan Kubar h,7 a
Division of Medical Virology, Department of Medical Microbiology, Gulhane Military Medical Academy, Ankara, Turkey Department of Medical Microbiology, Gulhane Military Medical Academy, Ankara, Turkey Department of Otorhinolaryngology, Gulhane Military Medical Academy, Ankara, Turkey d Department of Public Health, Gulhane Military Medical Academy, Ankara, Turkey e Department of Otorhinolaryngology, Gulhane Military Medical Academy, Ankara, Turkey f Department of Medical Pathology, Gulhane Military Medical Academy, Ankara, Turkey g Department of Medical Microbiology, Gulhane Military Medical Academy, Ankara, Turkey h Division of Medical Virology, Department of Medical Microbiology, Gulhane Military Medical Academy, Ankara, Turkey b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 March 2014 Received in revised form 3 May 2014 Accepted 6 May 2014 Available online xxx
Objective: In this study, we aimed to investigate the presence and copy number of six different viruses in tonsillar tissue samples removed surgically because of chronic recurrent tonsillitis or chronic obstructive tonsillar hypertrophy. Methods: In total, 56 tissue samples (tonsillar core) collected from 44 children and 12 adults were included in this study. The presence of viruses was investigated using a new TaqMan-based quantitative real-time PCR assay. Results: Of the 56 tissue samples, 67.9% (38/56) were positive for at least one of the six viruses. Epstein– Barr virus was the most frequently detected virus, being found in 53.6% (30/56), followed by human Parvovirus B19 21.4% (12/56), human adenovirus 12.5% (7/56), human Cytomegalovirus 5.4% (3/56), BK polyomavirus 1.8% (1/56), and Herpes simplex virus 1.8% (1/56). Precancerous or cancerous changes were not detected in the tonsillar tissue samples by pathologic examination, whereas lymphoid hyperplasia was observed in 24 patients. In contrast to other viruses, B19 virus was present in high copy number in tonsillar tissues. The rates of EBV and B19 virus with high copy number (>500.000 copies/ml) were higher in children than in adults, and a positive relationship was also found between the presence of EBV and the presence of B19 virus with high copy number (P = 0.037). Conclusions: It is previously reported that some viral agents are associated with different chronic tonsillar pathologies. In the present study, the presence of B19 virus in tonsillar core samples was investigated quantitatively for the first time, and our data suggests that EBV infections could be associated with B19 virus infections or could facilitate B19 virus replication. However, further detailed studies are needed to clarify this observation. ã 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Adenovirus Parvovirus B19 EBV CMV Tonsillitis
* Corresponding author at: Gulhane Military Medical Academy, Department of Medical Microbiology, Asagieglence Mah. Mercimek Sk. 37/20, Etlik, Kecioren, Ankara, Turkey. Tel.: +90 312 304 3481; fax: +90 312 304 3402. lu), [email protected] (M.A. Babayig it), E-mail addresses: [email protected] (F. Şahiner), [email protected] (R. Gümral), [email protected] (Ü. Yildizog it), [email protected] (M.A. Saraçli), [email protected] (A. Kubar). [email protected] (A. Durmaz), [email protected] (N. Yig 1 Tel.: +90 312 304 3433. 2 Tel.: +90 312 304 5702. 3 Tel.: +90 312 304 4672. 4 Tel.: +90 312 304 5713. 5 Tel.: +90 312 304 3734. 6 Tel.: +90 312 304 3402. 7 Tel.: +90 312 304 3414. http://dx.doi.org/10.1016/j.ijporl.2014.05.012 0165-5876/ ã 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: F. Şahiner, et al., Coexistence of Epstein–Barr virus and Parvovirus B19 in tonsillar tissue samples: Quantitative measurement by real-time PCR, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.05.012
G Model PEDOT 7131 No. of Pages 6
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2
1. Introduction Chronic infections in the palatine tonsils and adenoids (also known as pharyngeal or nasopharyngeal tonsils) produce immunological reactions which cause hypertrophic and hyperplasic changes [1,2]. These tissues are immunologically more active in childhood, and may be infected acutely or chronically with a variety of microorganisms, most commonly bacterial pathogens [1,3,4]. In some situations, hypertrophy with upper respiratory airway obstruction and/or recurrent infections can adversely affect the quality of life [2]. Nowadays, the first step in managing recurrent tonsillitis is medical treatment, and surgical treatment typically can be also performed in some cases where medical therapy has failed [4]. However, tonsillectomy has been the most commonly performed major surgical procedure in otorhinolaryngology, mainly in the pediatric population [5,6]. Chronic and/or recurrent tonsillitis and chronic obstructive tonsillar hypertrophy are the two most common indications for tonsillectomy. However, the underlying mechanisms of these pathologies have not yet been fully clarified [1,5]. Misuse of antibiotic therapy in acute tonsillitis, changes in the tonsillar microflora, structural changes in tonsillar crypt epithelium, and viral infections have been recognized as the main causes of recurrent tonsillitis [1]. Numerous studies have been conducted to investigate the relationship between different viral infections and tonsillar hypertrophy and recurrent tonsillitis. The Epstein–Barr virus (EBV) is one of the most commonly-detected infectious agents in recurrent tonsillitis attacks and is associated with tonsillar hypertrophy [2,7]. It has been reported previously that some human adenovirus types are associated with upper and lower respiratory tract infections, and these viruses cause latent infections in the lymphoid tissue of tonsils [8,9]. Human Parvovirus B19 (B19 virus) is another virus associated with latency in tonsillar tissue. After primary infection, B19 virus establishes a persistent latent infection by its retention in bone marrow, skin, synovium, tonsil, liver, and myocardial tissues [10–12]. BK polyomavirus (BK virus) that has been claimed to be associated with recurrent upper respiratory tract infections can also remain latent in various tissues, especially kidneys, after the primary infection, and this virus can be reactivated in case of immunodeficiency [13,14]. In addition to those mentioned above, human herpesvirus 7 (HHV-7),
HHV-6, Enteroviruses, rhinoviruses, Herpes simplex virus type 1 (HSV-1), and human Cytomegalovirus (CMV) have all been detected in adenoid and tonsil tissues [3,15]. EBV, CMV, HSV, B19 virus, BK virus and adenoviruses that were investigated in tonsil tissues in this study have some important common features. These viruses are most frequently transmitted to humans through the oral-respiratory secretions, and enter the body via the oropharyngeal region [1,2,9,14,16–20]. Infections caused by these viruses usually occur in early childhood and adolescence, and are often asymptomatic [2,9,14,16–20]. After primary infection, these viruses have the ability to establish latent infections in a variety of solid tissues including tonsils, and may be responsible for recurrent infections associated with different organs and systems [2,3,14–21]. In this study, we aimed to investigate the presence of some common viral agents in tonsillar tissue samples using a new quantitative real-time PCR assay. 2. Materials and methods 2.1. Study group and sample collection This study was conducted after the approval from the local ethical committee (Gulhane Military Medical Academy, Ankara, Turkey; Decision number: 13/1648.4–2145), and informed consent was obtained from the study subjects who underwent surgical removal of their tonsils because of chronic recurrent tonsillitis (for at least 2 years with 5 or more acute attacks) or obstructive tonsillar hypertrophy (patients with clinical manifestation of tonsillar obstruction, such as snoring, mouth open sleep, restless sleep, sleep apnea in the period of infectiousness). In total, 56 tissue samples taken from the core of tonsils that were collected from 22 children (12 female and 10 male) with recurrent tonsillitis, 20 children (8 female and 12 male) with tonsillar hypertrophy, 2 children (2 male) with both indications, 2 adults (2 male) with recurrent tonsillitis, and 10 adults (4 female and 6 male) with tonsillar hypertrophy were included in this study. 2.2. Histopathological examination Tonsillectomy materials were passed into sterilized containers, which included formaldehyde (10.0%), and transported to the
Table 1 Primers and probes used in real-time PCR assays. Virus/internal control
Target gene
Primer/prob sequences
UL20 type 1 membrane protein gene F: 50 -ggaagtagcgtcggtgttttatg-30 R: 50 -gccacaacggcatctacgatc-30 P: 50 -FAM-cagcgtcgtcgtcactcgtggc-BHQ-30 HSV (common primers/probe for HSV-1 and HSV-2) Thymidine kinase UL23 gene F: 50 -gcataaggcrtgcycattgtta-30 R: 50 -cgcgcgacratatcgtctac-30 P (antisense): 50 -FAM-ccgagccgatgacttactggcrggt-BHQ-30 Adenovirus Hexon gene F: 50 -ccwtcgatgmtgccscartg-30 R: 50 -csgtgggrttyctraacttgt-30 P: 50 -FAM-tacatgcacatcgcsggncagga-BHQ-30 BK virus Major capsid protein VP1 gene F: 50 -gagtgtccaggggcagctc-30 R: 50 -gcattctacctctgtwatagcatc-30 P: 50 -FAM-aggaacccgtgcaagtgccaaaactac-BHQ-30 EBV Major tegument protein, F: 50 -gaacctggtcatcctttgcca-30 BNRF1/p140 gene region R: 50 -ccagtgcttcgttatagccgta-30 P: 50 -FAM-agtacgagtgcctgcgaccagatc-BHQ-30 B19 virus Minor capsid protein VP1 gene F: 50 -tacacaagcctgggcawgtt-30 R: 50 -cagcactgtcaacagcactt-30 P: 50 -FAM-actacccggtactaactatgttggg-BHQ-3' GAPDH Human GAPDH gene F: 5'-tcctgcaccaccaactgcttag-30 R: 50 -catcacrccacagyttyccagag-30 P: 50 -FAM-aggtcatccatgacaactttggyatcg-BHQ-30
CMV
Amplicon size 118 bp
178 bp
139 bp
126 bp
102 bp
105 bp
145 bp
Abbreviations: F: forward primer, R: reverse primer, and P: probe. *M, N, R, S, W, and Y codes indicate degenerate bases; M = (A/C), N = (A/C/G/T), R = (A/G), S = (G/C), W = (A/T), and Y = (C/T).
Please cite this article in press as: F. Şahiner, et al., Coexistence of Epstein–Barr virus and Parvovirus B19 in tonsillar tissue samples: Quantitative measurement by real-time PCR, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.05.012
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Table 2 Viral DNA quantification of all positive samples.
female male male female female male male male male male male male male female male male male female male male female female male female female female female male female male female male male male male female male male
3 3 3 4 4 4 4 4 4 4 5 5 5 6 6 6 6 7 7 7 8 8 8 9 10 10 11 11 20 20 23 24 24 25 26 29 30 31
EBV
B19 virus
ADV
CMV
HSV
BK virus
4.0 103 2.1 103 5.2 103 9.4 104 8.6 105 8.4 104 9.2 104 1.0 106 5.2 104 1.0 103 8.3 103 8.9 106 6.0 105 2.0 103 9.3 104 8.9 103 1.2 106 9.0 104 7.5 104 1.5 105 9.6 103 1.0 103 7.6 104 8.6 104 1.1 103 2.0 104 8.6 104 9.7 104 6.5 104 1.6 104
1.3 108 9.6 105 6.3 109 3.2 108 4.0 105 1.1 104 8.8 104 2.6 107 2.2 103 2.0 104 5.8 104 1.6 104
9.5 104 2.3 104 2.0 104 5.5 104 6.7 103 8.6 103 3.5 106
2.5 104 1.6 104 1.0 103
6.5 104
4.3 102
Abbreviations: ; negative, +; present. The positive results with high copy number (>500.000 copies/ml) are written in bold.
pathology laboratory. All samples were embedded in paraffin. The paraffin embedded tissue sections were stained using hematoxylin and eosin, and examined for pathological abnormalities using a light-microscope. 2.3. DNA extraction Template DNA was extracted from excision materials using the standard phenol–chloroform–isoamyl alcohol method [22]. Briefly, approximately 40 mg of tissue samples were minced and suspended in 500 ml of TE buffer (10 mM Trishydrochloride, 1 mM EDTA, pH 8), and homogenized by vigorous mixing on a vortex. A 10 ml aliquot of protease solution (65 mg/ml) (Sigma–Aldrich Corp., St. Louis, MO, USA) and 250 ml of K buffer were added to the 250 ml of mixed specimen and incubated for 60 min at 45 C. Following centrifugation at 10,000 g for 10 min at 12 C, DNA was extracted from the supernatant using a mixture of 250 ml alkali phenol and 250 ml chloroform–isoamyl alcohol (24:1), and then precipitated using 500 ml isopropyl alcohol. The DNA was washed in 75% ethyl alcohol, centrifuged at 10,000 g for 5 min at 4 C, airdried at 37 C and dissolved in 100 ml distilled water. 2.4. Real time PCR analyses TaqMan-based real-time PCR assays were used for the detection and quantification of six different viruses (EBV, CMV, BK virus, B19 virus, adenovirus, and HSV) in tonsillar tissue samples. Real-time PCR reactions and quantitative analyses were performed primarily
based on the previously described methods [23,24]. The primers and probes used in this study are shown in Table 1. The reaction mixture was prepared for all real-time PCR assays as follows: 1.25 U Hot Start Taq DNA polymerase (Bioron, Germany), 10 pmol of each primer, 2.5 pmol TaqMan probe, 0.2 mM dNTP mix, and 2.5 mM MgCl2. PCR amplifications were carried out in a final volume of 25 ml of the PCR reaction mixture, after the addition of 5 ml of the sample containing template DNA. The amplification conditions were as follows: initial denaturation for 10 min at 95 C, followed by 40 amplification cycles of 15 s each at 95 C and 1 min at 60 C (annealing-extension step) [23]. The primers and probes were designed by using the OligoYap 4.0 software program [25], and confirmed by using a new software program (OligoBee 1.0) that was developed in our laboratory for molecular biology research. All primer and probe sequences were analyzed with the GenBank BLAST database for specificity, and were synthesized by MWGBiotech (Ebersberg, Germany). The TaqMan probes were labeled with a fluorescent reporter dye (FAM: 6-carboxy fluorescein) at the 50 end, and with black hole quencher (BHQ) as the non-fluorescent quencher at the 30 end. The human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control [26], and the PCR mixture without the template DNA was used as a negative control. Viral isolates used as a positive control were originated from the strains of our laboratory. The virus-specific amplicons and the GAPDH amplicon were cloned into plasmid vectors using a TOPO TA cloning system (Invitrogen, USA). Serial plasmid dilutions (108–101 copies/ml) were prepared for the determination of the detection sensitivities of the PCR assays
Please cite this article in press as: F. Şahiner, et al., Coexistence of Epstein–Barr virus and Parvovirus B19 in tonsillar tissue samples: Quantitative measurement by real-time PCR, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.05.012
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4
3. Results
Fig. 1. The positive cases of B19 virus and EBV with high copy number in adults and children. Samples containing high-copy-number of viral-DNA are shown as sphere (not circle), in accordance with the number of copies. *!; indicated cases with low copy number (
International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Coexistence of Epstein–Barr virus and Parvovirus B19 in tonsillar tissue samples: Quantitative measurement by real-time PCR lu c,2 , Fatih Şahiner a, *, Ramazan Gümral b,1, Üzeyir Yildizog d,3 e,4 it , Abdullah Durmaz , Nuri Yig it f,5 , Mehmet Ali Saraçli g,6 Mustafa Alparslan Babayig , Ayhan Kubar h,7 a
Division of Medical Virology, Department of Medical Microbiology, Gulhane Military Medical Academy, Ankara, Turkey Department of Medical Microbiology, Gulhane Military Medical Academy, Ankara, Turkey Department of Otorhinolaryngology, Gulhane Military Medical Academy, Ankara, Turkey d Department of Public Health, Gulhane Military Medical Academy, Ankara, Turkey e Department of Otorhinolaryngology, Gulhane Military Medical Academy, Ankara, Turkey f Department of Medical Pathology, Gulhane Military Medical Academy, Ankara, Turkey g Department of Medical Microbiology, Gulhane Military Medical Academy, Ankara, Turkey h Division of Medical Virology, Department of Medical Microbiology, Gulhane Military Medical Academy, Ankara, Turkey b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 March 2014 Received in revised form 3 May 2014 Accepted 6 May 2014 Available online xxx
Objective: In this study, we aimed to investigate the presence and copy number of six different viruses in tonsillar tissue samples removed surgically because of chronic recurrent tonsillitis or chronic obstructive tonsillar hypertrophy. Methods: In total, 56 tissue samples (tonsillar core) collected from 44 children and 12 adults were included in this study. The presence of viruses was investigated using a new TaqMan-based quantitative real-time PCR assay. Results: Of the 56 tissue samples, 67.9% (38/56) were positive for at least one of the six viruses. Epstein– Barr virus was the most frequently detected virus, being found in 53.6% (30/56), followed by human Parvovirus B19 21.4% (12/56), human adenovirus 12.5% (7/56), human Cytomegalovirus 5.4% (3/56), BK polyomavirus 1.8% (1/56), and Herpes simplex virus 1.8% (1/56). Precancerous or cancerous changes were not detected in the tonsillar tissue samples by pathologic examination, whereas lymphoid hyperplasia was observed in 24 patients. In contrast to other viruses, B19 virus was present in high copy number in tonsillar tissues. The rates of EBV and B19 virus with high copy number (>500.000 copies/ml) were higher in children than in adults, and a positive relationship was also found between the presence of EBV and the presence of B19 virus with high copy number (P = 0.037). Conclusions: It is previously reported that some viral agents are associated with different chronic tonsillar pathologies. In the present study, the presence of B19 virus in tonsillar core samples was investigated quantitatively for the first time, and our data suggests that EBV infections could be associated with B19 virus infections or could facilitate B19 virus replication. However, further detailed studies are needed to clarify this observation. ã 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Adenovirus Parvovirus B19 EBV CMV Tonsillitis
* Corresponding author at: Gulhane Military Medical Academy, Department of Medical Microbiology, Asagieglence Mah. Mercimek Sk. 37/20, Etlik, Kecioren, Ankara, Turkey. Tel.: +90 312 304 3481; fax: +90 312 304 3402. lu), [email protected] (M.A. Babayig it), E-mail addresses: [email protected] (F. Şahiner), [email protected] (R. Gümral), [email protected] (Ü. Yildizog it), [email protected] (M.A. Saraçli), [email protected] (A. Kubar). [email protected] (A. Durmaz), [email protected] (N. Yig 1 Tel.: +90 312 304 3433. 2 Tel.: +90 312 304 5702. 3 Tel.: +90 312 304 4672. 4 Tel.: +90 312 304 5713. 5 Tel.: +90 312 304 3734. 6 Tel.: +90 312 304 3402. 7 Tel.: +90 312 304 3414. http://dx.doi.org/10.1016/j.ijporl.2014.05.012 0165-5876/ ã 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: F. Şahiner, et al., Coexistence of Epstein–Barr virus and Parvovirus B19 in tonsillar tissue samples: Quantitative measurement by real-time PCR, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.05.012
G Model PEDOT 7131 No. of Pages 6
F. Şahiner et al. / International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx
2
1. Introduction Chronic infections in the palatine tonsils and adenoids (also known as pharyngeal or nasopharyngeal tonsils) produce immunological reactions which cause hypertrophic and hyperplasic changes [1,2]. These tissues are immunologically more active in childhood, and may be infected acutely or chronically with a variety of microorganisms, most commonly bacterial pathogens [1,3,4]. In some situations, hypertrophy with upper respiratory airway obstruction and/or recurrent infections can adversely affect the quality of life [2]. Nowadays, the first step in managing recurrent tonsillitis is medical treatment, and surgical treatment typically can be also performed in some cases where medical therapy has failed [4]. However, tonsillectomy has been the most commonly performed major surgical procedure in otorhinolaryngology, mainly in the pediatric population [5,6]. Chronic and/or recurrent tonsillitis and chronic obstructive tonsillar hypertrophy are the two most common indications for tonsillectomy. However, the underlying mechanisms of these pathologies have not yet been fully clarified [1,5]. Misuse of antibiotic therapy in acute tonsillitis, changes in the tonsillar microflora, structural changes in tonsillar crypt epithelium, and viral infections have been recognized as the main causes of recurrent tonsillitis [1]. Numerous studies have been conducted to investigate the relationship between different viral infections and tonsillar hypertrophy and recurrent tonsillitis. The Epstein–Barr virus (EBV) is one of the most commonly-detected infectious agents in recurrent tonsillitis attacks and is associated with tonsillar hypertrophy [2,7]. It has been reported previously that some human adenovirus types are associated with upper and lower respiratory tract infections, and these viruses cause latent infections in the lymphoid tissue of tonsils [8,9]. Human Parvovirus B19 (B19 virus) is another virus associated with latency in tonsillar tissue. After primary infection, B19 virus establishes a persistent latent infection by its retention in bone marrow, skin, synovium, tonsil, liver, and myocardial tissues [10–12]. BK polyomavirus (BK virus) that has been claimed to be associated with recurrent upper respiratory tract infections can also remain latent in various tissues, especially kidneys, after the primary infection, and this virus can be reactivated in case of immunodeficiency [13,14]. In addition to those mentioned above, human herpesvirus 7 (HHV-7),
HHV-6, Enteroviruses, rhinoviruses, Herpes simplex virus type 1 (HSV-1), and human Cytomegalovirus (CMV) have all been detected in adenoid and tonsil tissues [3,15]. EBV, CMV, HSV, B19 virus, BK virus and adenoviruses that were investigated in tonsil tissues in this study have some important common features. These viruses are most frequently transmitted to humans through the oral-respiratory secretions, and enter the body via the oropharyngeal region [1,2,9,14,16–20]. Infections caused by these viruses usually occur in early childhood and adolescence, and are often asymptomatic [2,9,14,16–20]. After primary infection, these viruses have the ability to establish latent infections in a variety of solid tissues including tonsils, and may be responsible for recurrent infections associated with different organs and systems [2,3,14–21]. In this study, we aimed to investigate the presence of some common viral agents in tonsillar tissue samples using a new quantitative real-time PCR assay. 2. Materials and methods 2.1. Study group and sample collection This study was conducted after the approval from the local ethical committee (Gulhane Military Medical Academy, Ankara, Turkey; Decision number: 13/1648.4–2145), and informed consent was obtained from the study subjects who underwent surgical removal of their tonsils because of chronic recurrent tonsillitis (for at least 2 years with 5 or more acute attacks) or obstructive tonsillar hypertrophy (patients with clinical manifestation of tonsillar obstruction, such as snoring, mouth open sleep, restless sleep, sleep apnea in the period of infectiousness). In total, 56 tissue samples taken from the core of tonsils that were collected from 22 children (12 female and 10 male) with recurrent tonsillitis, 20 children (8 female and 12 male) with tonsillar hypertrophy, 2 children (2 male) with both indications, 2 adults (2 male) with recurrent tonsillitis, and 10 adults (4 female and 6 male) with tonsillar hypertrophy were included in this study. 2.2. Histopathological examination Tonsillectomy materials were passed into sterilized containers, which included formaldehyde (10.0%), and transported to the
Table 1 Primers and probes used in real-time PCR assays. Virus/internal control
Target gene
Primer/prob sequences
UL20 type 1 membrane protein gene F: 50 -ggaagtagcgtcggtgttttatg-30 R: 50 -gccacaacggcatctacgatc-30 P: 50 -FAM-cagcgtcgtcgtcactcgtggc-BHQ-30 HSV (common primers/probe for HSV-1 and HSV-2) Thymidine kinase UL23 gene F: 50 -gcataaggcrtgcycattgtta-30 R: 50 -cgcgcgacratatcgtctac-30 P (antisense): 50 -FAM-ccgagccgatgacttactggcrggt-BHQ-30 Adenovirus Hexon gene F: 50 -ccwtcgatgmtgccscartg-30 R: 50 -csgtgggrttyctraacttgt-30 P: 50 -FAM-tacatgcacatcgcsggncagga-BHQ-30 BK virus Major capsid protein VP1 gene F: 50 -gagtgtccaggggcagctc-30 R: 50 -gcattctacctctgtwatagcatc-30 P: 50 -FAM-aggaacccgtgcaagtgccaaaactac-BHQ-30 EBV Major tegument protein, F: 50 -gaacctggtcatcctttgcca-30 BNRF1/p140 gene region R: 50 -ccagtgcttcgttatagccgta-30 P: 50 -FAM-agtacgagtgcctgcgaccagatc-BHQ-30 B19 virus Minor capsid protein VP1 gene F: 50 -tacacaagcctgggcawgtt-30 R: 50 -cagcactgtcaacagcactt-30 P: 50 -FAM-actacccggtactaactatgttggg-BHQ-3' GAPDH Human GAPDH gene F: 5'-tcctgcaccaccaactgcttag-30 R: 50 -catcacrccacagyttyccagag-30 P: 50 -FAM-aggtcatccatgacaactttggyatcg-BHQ-30
CMV
Amplicon size 118 bp
178 bp
139 bp
126 bp
102 bp
105 bp
145 bp
Abbreviations: F: forward primer, R: reverse primer, and P: probe. *M, N, R, S, W, and Y codes indicate degenerate bases; M = (A/C), N = (A/C/G/T), R = (A/G), S = (G/C), W = (A/T), and Y = (C/T).
Please cite this article in press as: F. Şahiner, et al., Coexistence of Epstein–Barr virus and Parvovirus B19 in tonsillar tissue samples: Quantitative measurement by real-time PCR, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.05.012
G Model PEDOT 7131 No. of Pages 6
F. Şahiner et al. / International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx
3
Table 2 Viral DNA quantification of all positive samples.
female male male female female male male male male male male male male female male male male female male male female female male female female female female male female male female male male male male female male male
3 3 3 4 4 4 4 4 4 4 5 5 5 6 6 6 6 7 7 7 8 8 8 9 10 10 11 11 20 20 23 24 24 25 26 29 30 31
EBV
B19 virus
ADV
CMV
HSV
BK virus
4.0 103 2.1 103 5.2 103 9.4 104 8.6 105 8.4 104 9.2 104 1.0 106 5.2 104 1.0 103 8.3 103 8.9 106 6.0 105 2.0 103 9.3 104 8.9 103 1.2 106 9.0 104 7.5 104 1.5 105 9.6 103 1.0 103 7.6 104 8.6 104 1.1 103 2.0 104 8.6 104 9.7 104 6.5 104 1.6 104
1.3 108 9.6 105 6.3 109 3.2 108 4.0 105 1.1 104 8.8 104 2.6 107 2.2 103 2.0 104 5.8 104 1.6 104
9.5 104 2.3 104 2.0 104 5.5 104 6.7 103 8.6 103 3.5 106
2.5 104 1.6 104 1.0 103
6.5 104
4.3 102
Abbreviations: ; negative, +; present. The positive results with high copy number (>500.000 copies/ml) are written in bold.
pathology laboratory. All samples were embedded in paraffin. The paraffin embedded tissue sections were stained using hematoxylin and eosin, and examined for pathological abnormalities using a light-microscope. 2.3. DNA extraction Template DNA was extracted from excision materials using the standard phenol–chloroform–isoamyl alcohol method [22]. Briefly, approximately 40 mg of tissue samples were minced and suspended in 500 ml of TE buffer (10 mM Trishydrochloride, 1 mM EDTA, pH 8), and homogenized by vigorous mixing on a vortex. A 10 ml aliquot of protease solution (65 mg/ml) (Sigma–Aldrich Corp., St. Louis, MO, USA) and 250 ml of K buffer were added to the 250 ml of mixed specimen and incubated for 60 min at 45 C. Following centrifugation at 10,000 g for 10 min at 12 C, DNA was extracted from the supernatant using a mixture of 250 ml alkali phenol and 250 ml chloroform–isoamyl alcohol (24:1), and then precipitated using 500 ml isopropyl alcohol. The DNA was washed in 75% ethyl alcohol, centrifuged at 10,000 g for 5 min at 4 C, airdried at 37 C and dissolved in 100 ml distilled water. 2.4. Real time PCR analyses TaqMan-based real-time PCR assays were used for the detection and quantification of six different viruses (EBV, CMV, BK virus, B19 virus, adenovirus, and HSV) in tonsillar tissue samples. Real-time PCR reactions and quantitative analyses were performed primarily
based on the previously described methods [23,24]. The primers and probes used in this study are shown in Table 1. The reaction mixture was prepared for all real-time PCR assays as follows: 1.25 U Hot Start Taq DNA polymerase (Bioron, Germany), 10 pmol of each primer, 2.5 pmol TaqMan probe, 0.2 mM dNTP mix, and 2.5 mM MgCl2. PCR amplifications were carried out in a final volume of 25 ml of the PCR reaction mixture, after the addition of 5 ml of the sample containing template DNA. The amplification conditions were as follows: initial denaturation for 10 min at 95 C, followed by 40 amplification cycles of 15 s each at 95 C and 1 min at 60 C (annealing-extension step) [23]. The primers and probes were designed by using the OligoYap 4.0 software program [25], and confirmed by using a new software program (OligoBee 1.0) that was developed in our laboratory for molecular biology research. All primer and probe sequences were analyzed with the GenBank BLAST database for specificity, and were synthesized by MWGBiotech (Ebersberg, Germany). The TaqMan probes were labeled with a fluorescent reporter dye (FAM: 6-carboxy fluorescein) at the 50 end, and with black hole quencher (BHQ) as the non-fluorescent quencher at the 30 end. The human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control [26], and the PCR mixture without the template DNA was used as a negative control. Viral isolates used as a positive control were originated from the strains of our laboratory. The virus-specific amplicons and the GAPDH amplicon were cloned into plasmid vectors using a TOPO TA cloning system (Invitrogen, USA). Serial plasmid dilutions (108–101 copies/ml) were prepared for the determination of the detection sensitivities of the PCR assays
Please cite this article in press as: F. Şahiner, et al., Coexistence of Epstein–Barr virus and Parvovirus B19 in tonsillar tissue samples: Quantitative measurement by real-time PCR, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.05.012
G Model PEDOT 7131 No. of Pages 6
F. Şahiner et al. / International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx
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3. Results
Fig. 1. The positive cases of B19 virus and EBV with high copy number in adults and children. Samples containing high-copy-number of viral-DNA are shown as sphere (not circle), in accordance with the number of copies. *!; indicated cases with low copy number (
