FRET-based detection and genotyping of HPV-6 and HPV-11 causing recurrent respiratory papillomatosis

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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet

FRET-based detection and genotyping of HPV-6 and HPV-11 causing recurrent respiratory papillomatosis

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Catharina E. Combrinck a , Riaz Y. Seedat b , Felicity J. Burt a,c,∗

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a

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b

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c

Department of Medical Microbiology and Virology, University of the Free State, Bloemfontein, Free State, South Africa Department of Otorhinolaryngology, University of the Free State and Universitas Academic Hospital, Bloemfontein, Free State, South Africa Department of Medical Microbiology and Virology, National Health Laboratory Service, Bloemfontein, Free State, South Africa

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a b s t r a c t 8

Article history: Received 13 November 2012 Received in revised form 24 January 2013 Accepted 30 January 2013 Available online xxx

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Recurrent respiratory papillomatosis (RRP) is a potentially life-threatening disease caused by human papillomavirus (HPV), usually HPV types 6 and 11. The conventional method used for detection and typing the RRP isolates in our laboratory is the polymerase chain reaction (PCR) and DNA sequencing method. A real-time PCR assay based on fluorescence resonance energy transfer (FRET) probe technology was developed for the detection and rapid genotyping of HPV-6 and-11 isolates from biopsy material. The primers and probes were designed using multiple alignments of HPV-6 and HPV-11 partial E6 and E7 sequences that included prototypic and non-prototypic variants. Real-time PCR followed by probe-specific meltingcurve analysis allowed differentiation of HPV-6 and HPV-11. HPV-6 and HPV-11 amplicons were used to determine detection limits and inter- and intra-assay variability. The detection limit of the assay was 12.8 DNA copies for HPV-6 and 22.5 DNA copies for HPV-11. A total of 60 isolates were genotyped using the FRET real-time PCR assay and a 100% concordance was obtained when results were compared with genotyping based on conventional DNA sequencing. The real-time PCR assay based on FRET technology was able to detect and rapidly genotype HPV from tissue biopsy obtained from patients with RRP. The assay reduces the time required for genotyping from three working days to less than a day. © 2013 Elsevier B.V. All rights reserved.

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Keywords: HPV genotyping HPV-6 HPV-11 FRET Real-time PCR Recurrent respiratory papillomatosis (RRP)

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1. Introduction

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Human papillomaviruses (HPV) are a group of viruses which may cause warts on the cutaneous epithelium of the skin or cause cancer and warts in the mucosal epithelium of the anogenital and oral region of both men and women (de Villiers et al., 2004; Maver et al., 2010; Molijn et al., 2005). To date, more than 150 HPV types have been identified and fully characterized (Burk et al., 2011). These HPV types have been divided into low risk and high risk types according to their ability to induce malignancy (Bonagura et al., 2010; Draganov et al., 2006; Molijn et al., 2005; Seaman et al., 2010). Low risk HPV types include HPV-6 and HPV-11, which are primarily associated with genital warts and recurrent respiratory papillomatosis (RRP) (Bonagura et al., 2010; Burk et al., 2011; Garland et al., 2007; Kocjan et al., 2011; Maver et al., 2011). Both HPV-6 and HPV-11 belong to the genus Alphapapillomavirus – species 10, of the Papillomaviridae family (Bernard et al., 2010; Burk et al., 2011; de Villiers et al., 2004). Based on the genetic

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∗ Corresponding author at: Department of Medical Microbiology and Virology (G23), P.O. Box 339, Faculty of Health Sciences, University of the Free State, Bloemfontein 9300, South Africa. Tel.: +27 51 405 3348. E-mail addresses: [email protected], [email protected] (F.J. Burt).

relationship and the percentage differences of the nucleotide sequence of aligned complete genomes, Burk et al. (2011) classified HPV-6 into two lineages – lineage A and lineage B, and three sublineages – B1, B2 and B3. Lineage A contains the prototype HPV6b, while the non-prototypic variants HPV-6a and HPV-6vc belong to sublineages B3 and B1, respectively. HPV-11 has been classified into two sublineages – A1 and A2, with sublineage A1 containing the HPV-11 prototype (Burk et al., 2011). RRP, predominantly caused by HPV-6 and HPV-11 (Derkay and Wiatrak, 2008; Goon et al., 2008; Larson and Derkay, 2010; Seedat et al., 2010) is a chronic disease characterized by benign exophytic, verrucous proliferation within the respiratory tract (Derkay and Wiatrak, 2008; Larson and Derkay, 2010; Wiatrak et al., 2004). RRP usually involves the larynx of the patient, but may also extend to the esophagus, trachea, nasal cavity and even the lungs (Derkay and Wiatrak, 2008; Draganov et al., 2006; Wiatrak et al., 2004). RRP has the potential to cause significant morbidity and episodic mortality due to complete airway obstruction or malignant transformation (Bonagura et al., 2010; Goon et al., 2008; Larson and Derkay, 2010; Wiatrak et al., 2004). The course of the disease is unpredictable. Patients may recover completely after the first presentation of the disease, while others may present with aggressive disease requiring multiple surgical debridement of the papillomas (Bonagura et al., 2010; Larson and Derkay, 2010; Wiatrak et al., 2004).

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Please cite this article in press as: Combrinck, C.E., et al., FRET-based detection and genotyping of HPV-6 and HPV-11 causing recurrent respiratory papillomatosis. J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.01.025

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Various molecular assays have been developed for the detection of different HPV types. Several universal primer sets have been designed in order to identify a number of HPV types in a single PCR run; the most frequently used include MY09/MY11 (Draganov et al., 2006; Gravitt et al., 2000; International Agency for Research on Cancer, 2007; Seaman et al., 2010; Seedat et al., 2010). Little is known about the immune correlates of protection and why certain patients present with a severe disease. Previous studies have suggested that HPV-11 infection tends to be more aggressive with patients having an earlier onset and requiring more frequent surgical intervention, although severity of disease is likely dependent on various factors including age, immune status and undetermined host factors (Bonagura et al., 2010; Buchinsky et al., 2008; Donne et al., 2010; Gillison et al., 2012; Larson and Derkay, 2010; Seedat et al., 2010; Wiatrak et al., 2004). The conventional method used for genotyping an HPV isolate from an RRP patient in our laboratory is by PCR, using the MY09/MY11 primer set followed by DNA sequencing. This process takes about three working days, excluding the extraction process, in order to obtain the result. Genotyping will provide epidemiological data on HPV strains currently circulating and may provide useful data to consider the impact of prophylactic vaccination of mothers on the occurrence of RRP in children. The aim of this study was to design a realtime fluorescence resonance energy transfer (FRET) based assay for detection of HPV-6 and -11 isolates circulating in our region from tissue biopsy specimens of patients with RRP that would have application in diagnosis and genotyping.

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2. Materials and methods

62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88

Table 1 Isolates used in the multiple alignment for design of primers and probes. Sequence data from GenBank represents all known mutations from 77 HPV-6 and 63 HPV-11 isolates (Kocjan et al., 2009; Maver et al., 2011; Combrinck et al., 2012). Isolatea

GenBank acc. no.

HPV type

Country

HPV-6b prototype HPV-6b prototype HPV-6a HPV-6vc VBD09/09 VBD77/09 VBD04/09 VBD07/09 VBD22/10 VBD44/08 VBD19/10 VBD12/09 VBD80/09 VBD61/08 VBD02/10 VBD46/08 LP23 HPV-11 prototype VBD21/10 VBD34/08 VBD26/10 CAC321 LP27 LP19 LP16 LP14 LP13 LP12 LP6 LP1

NC 001355 X00203 L41216 AF092932 JN573165 JN573169 JN573168 JN573171 JN573173 JN573163 JN573172 JN573166 JN573170 JN573174 JN573167 JN573164 FM897022 M14119 N/Sb N/S N/S FN870454 FN870448 FN870447 FN870446 FN870445 FN870444 FN870443 FN870442 FN870441

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 11 11 11 11 11 11 11 11 11 11 11 11 11

Unknown Unknown Unknown Unknown South Africa South Africa South Africa South Africa South Africa South Africa South Africa South Africa South Africa South Africa South Africa South Africa Slovenia Unknown South Africa South Africa South Africa Slovenia Slovenia Slovenia Slovenia Slovenia Slovenia Slovenia Slovenia Slovenia

a b

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2.1. Samples and DNA extraction

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Genotyping was performed on 60 HPV isolates obtained from tissue biopsy collected from patients who were treated for RRP at the Universitas Academic hospital in Bloemfontein from 2008 until 2011. Of the cohort, 19 were reported previously and 41 were determined subsequently on submission to the laboratory based on sequence data from the L segment (Seedat et al., 2010). Ten isolates had previously been genotyped as HPV-6a and two isolates as HPV-6vc based on E6 sequence data (Combrinck et al., 2012). The results obtained were compared with the genotype determined based on a real-time FRET assay. Briefly, tissue samples were obtained from the first endoscopic procedure after enrolment and submitted for HPV typing. Laryngeal biopsies were submitted to the laboratory and processed immediately or frozen at −20 ◦ C until the DNA was extracted. Total DNA was extracted from biopsy material using the QIAamp DNA Mini Kit according to manufacturer’s instructions (QIAGEN, Valencia, CA, USA).

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2.2. Genotyping HPV based on L1 segment

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Consensus primers, previously identified for a region of the genome that is well conserved for most HPV types, were used to amplify a region of the major viral capsid L1 gene, using a standard PCR technique (Seedat et al., 2010). The primer pair (designated MY09 and MY11) target and amplify a 469 base pair (bp) region between positions 6722 and 7190 of the HPV-6 genome (Ting and Manos, 1990). The nucleotide sequences of the amplicons were determined using Big DyeTM Terminator Sequencing Ready Reaction kits with AmpliTaq DNA polymerase FS (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions and after sequence editing the genotypes of HPV were determined by BLAST analysis.

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Laboratory numbers assigned to isolates where available. N/S: unpublished laboratory data not submitted to GenBank.

2.3. Primer and probe design for FRET assay

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Sequence data, obtained from a previous study in the Department of Medical Microbiology and Virology, University of the Free State, for the E6 region of twelve HPV-6 isolates and three HPV-11 isolates were aligned with sequence data retrieved from GenBank for five HPV-6, ten HPV-11 isolates (prototypic and non-prototypic HPV-6, and prototypic HPV-11 variants included) and the closely related HPV types, HPV-44 (GenBank acc. no. U31788) and HPV74 (GenBank acc. no. AF436130) using Clustal X version 2.1 (Larkin et al., 2007) (Table 1). The sequence data for the HPV-6 and HPV-11 isolates obtained from GenBank represented all the known mutations in this region from analysis of a total of 77 HPV-6 isolates and 63 HPV-11 isolates. The alignment included specifically an HPV-6 variant, GenBank acc. no. FM897022 (Kocjan et al., 2009), and an HPV-11 variant, GenBank acc. no. FN870454 (Maver et al., 2011). These two variants contain a specific mutation near the 3 -end of the E6 genomic region. The donor FRET probe, designated probe 1 and the anchor probe, designated probe 2, were designed using sequence data from a conserved region at the 3 -end of the E6. The probes were designed to have 100% base pairing with HPV6 and two base changes in the donor probe relative to HPV-11, to allow differentiation in the melt curve analysis (Table 2). The anchor probe differed by one base change from HPV-11. Degenerated bases at this point mutation were not included to avoid reducing specificity. A primer pair, designated HPV Geno F and HPV Geno R, were designed to amplify a 232 base pair fragment including the probe site. The forward primer was designed based on the alignment of the isolates from Table 1. The reverse primer is the combined and modified version of the reverse primers designed by Kocjan et al. (2009) for HPV-6 and Maver et al. (2011) for HPV-11 in order to identify variants. This reverse primer was designed within

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Please cite this article in press as: Combrinck, C.E., et al., FRET-based detection and genotyping of HPV-6 and HPV-11 causing recurrent respiratory papillomatosis. J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.01.025

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Table 2 Sequences of the primers and probes designed for genotyping an HPV isolate. Primer

5 –3 Sequence

Genome position (bp)a

Tm

HPV Geno F HPV Geno R

TGT GTC ACA A(A/G)C CG(C/T) TGT G CAC (C/T)T(T/C) GTC CAC CTC ATC T

418–436 649–631

56.1 ◦ C 54.1 ◦ C

Probes

5 –3 Sequence

Genome position (bp)

Probe 1 Probe 2

GGA AGG GTC GCT GCC TAC AC GCT GGA CAA CAT GCA TGG AAG ACA TGT TAC CC

499–518 520–551

a

153 154 155 156

Genomic positions are relative to the HPV-6b prototype (GenBank acc. no. X00302).

the E7 genomic region using multiple alignments of the prototypic and non-prototypic HPV-6 variants (Genbank acc. no. NC 001355, X00203, L41216, and AF092932, respectively), and prototypic HPV11 variant (GenBank acc. no. M14119).

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2.4. Controls

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Positive controls for the optimization of the FRET real-time PCR were prepared using a representative HPV-6 isolate (designated VBD12/09) and HPV-11 isolate (designated VBD21/10) which has three mismatches relative to the hybridization probes. In order to evaluate the sensitivity and reproducibility of the assay a 232 bp amplicon was generated from each representative isolate using the primer pair HPV Geno F and HPV Geno R. PCR amplification was performed using Phusion® HotStart II High-Fidelity DNA Polymerase according to the manufacturer’s instructions (Finnzymes, Espoo, Finland). Briefly, the reaction mix comprised 10 ␮l of 10× Phusion HF Buffer, 0.2 mM dNTPs, 0.5 ␮M of each primer, 2% DMSO, 0.5 ␮l (0.02 U/␮l) of HotStart DNA polymerase, 10–100 ng of DNA template and water up to 50 ␮l. The reaction mixtures were cycled on a 9700 Gene Amp® PCR System. Cycling conditions involved an initial denaturation step at 98 ◦ C for 30 s, followed by 98 ◦ C for 10 s, 47 ◦ C for 30 s, and 72 ◦ C for 15 s for a total of 30 cycles. A final elongation step was carried out at 72 ◦ C for 10 min, followed by a 4 ◦ C hold. PCR amplicons were purified using the SV Gel PCR purification kit according to the manufacturer’s instructions (Promega, Fitchburg, WI, USA). The number of copies of the DNA template was calculated by measuring the DNA concentration with a Nanodrop. The calculation was based on the number of copies = (amount of dsDNA(ng) × 6.022 × 1023 )/(length of dsDNA(bp) × 109 × 650).

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2.5. FRET real-time PCR

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The FRET real-time PCR assay was performed on the LightCycler 2.0 Real-Time PCR system (Roche Diagnostics, Mannheim, Germany) using a LightCycler® Faststart DNA Master HybProbe kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s protocol. Briefly, the optimized reaction mixture consisted of 2 mM MgCl2 , 0.5 ␮M of each primer, 0.2 ␮M of probe 1, 0.4 ␮M of probe 2, 2 ␮l of LightCycler DNA Master Enzyme, 1 ␮l of DNA template, and water up to a total volume of 20 ␮l. A negative control sample that contained nuclease-free water as template, was included in each run. Cycling commenced at 95 ◦ C for 10 min, followed by amplification of the DNA for 45 cycles at 95 ◦ C for 10 s, 45 ◦ C for 20 s and 72 ◦ C for 9 s. Single acquisition of the fluorescence signal was performed at the end of the annealing step of each cycle. Melting curve analysis post amplification was performed at a denaturation temperature of 95 ◦ C for 0 s, followed by annealing of the probes at 37 ◦ C for 1 min and then 95 ◦ C for 0 s at a transition rate of 0.1 ◦ C/s, with continuous monitoring of the fluorescence. A final step consisted of cooling at 40 ◦ C for 30 s. Real-time fluorescence was monitored at channel 640. A PCR product of estimate 232 bp in size could be visualized by electrophoresis on a 1% agarose gel. For determining the sensitivity, a FRET assay was performed using

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10-fold dilutions of each control, 104 –1011 , and the results used to calculate the detection limit of the FRET real-time PCR.

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2.6. Intravariability and intervariability of the real-time FRET assay

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The intra- and inter-assay reproducibility were evaluated using triplicates of 104 dilution of each control in a single and two independent runs, respectively. The inter-assay reproducibility was expressed as a coefficient of variation based on mean cycle threshold, (CV%) where the standard deviation was divided by the mean cycle threshold (Ct) times 100.

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3. Results

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3.1. Sensitivity

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The aim was to identify probes in a highly conserved region that would readily distinguish between HPV-6 and HPV-11 in our cohort of patients and would exclude other genotypes. Hence, FRET hybridization probes, probe 1 and probe 2, were designed with 100% base pair homology with HPV-6a, -6b and -6vc. There was one base change between the probes and a previously identified novel variant of HPV-6 (Kocjan et al., 2009). In addition, the probes were identified to have three base changes compared to the HPV11 prototype and two base changes compared to a known HPV-11 variant (Maver et al., 2011). HPV-44 and HPV-74 are members of the alpha-10 species group and are the most closely related HPV types to HPV-6 and HPV-11 (Burk et al., 2011; Kocjan et al., 2008). At the 3 -end of the E6 gene, encompassing the region where the probes were designed to bind, HPV-44 has eleven base differences and HPV-74 has nine base differences when compared with HPV-6 (Fig. 1) and they are unlikely to be detected with this assay. FRET real-time PCR amplification using the selected HPV-6 and HPV-11 controls, yielded two distinguishable melting curve peaks at 66.33 ◦ C and 50.68 ◦ C, respectively. The DNA concentrations of the HPV-6 and HPV-11 controls were 1.28 × 1011 DNA copies/␮l and 2.25 × 1011 DNA copies/␮l, respectively. The performance and sensitivity of the assay was evaluated using 10-fold dilution replicates of the HPV-6 and HPV-11 controls representing an input equivalent to 1.28 × 100 to 1.28 × 1011 DNA copies and 2.25 × 10◦ to 2.25 × 1011 DNA copies per reaction, respectively. The real-time PCR was able to detect at least 12.8 and 22.5 DNA copies per assay for HPV-6 and HPV-11, respectively (Fig. 2). To evaluate the intraassay reproducibility, three 104 dilution samples of each control were run in duplicate corresponding to an input equivalent to 1.28 × 107 and 2.25 × 107 DNA copies per ␮l per assay for HPV-6 and HPV-11, respectively. In the first run, all six samples had average Ct values of 17 and in the following run, the Ct ranged from 15 to 16. The inter-assay reproducibility was expressed as a percentage CV. The mean CV (%) obtained for the inter-assay variability was 1.43% and 1.13% for the first and second run, respectively.

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(A) 418

Forward primer HPV-6bprototype HPV-6a HPV-6vc HPV-6variant HPV-11prototype HPV-11variant

(B) FRET probes HPV-6bprototype HPV-6a HPV-6vc HPV-6variant HPV-11prototype HPV-11variant HPV-13 HPV-44 HPV-74

a

436

649

TGT GTC ACA ARC CGY TGT G

… … … .A. ..C … . … … … .A. ..C … . … … … .A. ..C … . … … … .A. ..C … . … … … .G. ..T … . … … … .G. ..T … . Probe 1 499

631

CAC YTY GTC CAC CTC ATC T

Reverse primer

… T.C … … … … . … T.C … … … … . … T.C … … … … .

HPV-6bprototype HPV-6a HPV-6vc

… C.T … … … … .

Probe 2

HPV-11prototype

551

GGA AGG GTC GCT GCC TAC AC – GCT GGA CAA CAT GCA TGG AAG ACA TGT TAC CC

... ... ... ... ... ... .. – ... ... ... ... ... ... ... ... ... ... .. ... ... ... ... ... ... .. – ... ... ... ... ... ... ... ... ... ... .. ... ... ... ... ... ... .. – ... ... ... ... ... ... ... ... ... ... .. ... ... .C. ... ... ... .. – ... ... ... ... ... ... ... ... ... ... .. ... ... ... .T. ..T ... .. – ... ... ... ... ... ... ... ..T ... ... .. ... ... ... .T. ... ... .. – ... ... ... ... ... ... ... ..T ... ... .. ... .A. .C. ... .TT .T. .T – ... ..T ..T ... ... ... ..A .T. .CC ... .T ... ... ... ... ..T .C. .T – .T. ... ..T ... ... ... ..A CT. .AC ... .T ... .A. .G. ... ..T ... .T – ... ... ... ... ... ... ..A .T. .CC ... .T

Fig. 1. Schematic representation of the FRET primers and probes and corresponding binding sites on HPV-6 and HPV-11, prototypes, non-prototypes and variants illustrating conserved sites and mismatches. (A) Forward and reverse primers, base position relative to HPV-6 prototype. (B) Probes 1 and 2 base position relative to HPV-6 prototype. Sequence data for HPV-13, HPV-44 and HPV-74 are included to illustrate number of mismatches in the probe region likely excluding detection of these types in the assay. GenBank accession numbers for sequence data: HPV-6b prototype, NC 001355; HPV-6a, L41216; HPV-6vc, AF092932; HPV-6variant, FM897022; HPV-11prototype, M14119; HPV-11variant, FN870454; HPV-13, X62843; HPV-44, U31788; HPV-74, AF436130. All sequence data are shown in 5 –3 direction. a Base positions relative to the prototype HPV-6b.

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3.2. Genotyping HPV-6 and HPV-11 clinical samples

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DNA extracted from tissue biopsies of laryngeal papillomas from 60 patients in our cohort was available for the study. Based on L segment sequence data a total of 32 HPV-6 types were identified and 28 HPV-11 types. The primer pair designed for the real-time PCR adequately amplified HPV DNA from all the isolates using the amplification conditions described. Amplicons from the real-time assay were separated on agarose gels which confirmed the presence of 232 base pair products from all the isolates (data not shown). The genotyping confirmed 32 HPV-6 and 28 HPV-11 isolates in concordance with the genotype identified using MY09 and MY11 DNA sequencing. The melting peaks for the HPV-6 isolates ranged between 65.66 ◦ C and 67.36 ◦ C, consistent with the HPV-6 control. Melting peaks for samples that had previously been identified as HPV-6a or HPV-6vc ranged from 65.95 ◦ C to 67.36 ◦ C. Melting peaks for HPV-11 isolates ranged between 49.64 ◦ C and 50.95 ◦ C, which were consistent with the HPV-11 control included in the assay.

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4. Discussion

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Rapid real-time PCR assays have previously been described for detection and genotyping of genomic variants of medically significant pathogens. Rapid methods for detection are frequently useful for implementing appropriate treatment regimens and genotype determination can be a useful prognostic indicator or important for identifying source of infection and outbreaks. For HPV infections

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causing RRP, genotyping is useful for monitoring strains circulating. There are various techniques available for real-time PCR assays and detection of amplified products including FRET probes, hydrolysis probes, molecular beacons, Simple-Probes and short probes designed using locked nucleic acids. The choice of probe is dependent on a range of factors including required outcome and application and genomic variation in the gene of interest that will influence design of probes and probe length depending on heterogeneity. For our application, the aim was to design an assay that would rapidly detect HPV infection in biopsy material and simultaneously genotype the sample. The patients that are treated at the Universitas Hospital in Bloemfontein and included in our cohort reside in rural areas and often need to travel great distances for management. Fatalities have been recorded in our cohort of patients who could not receive urgent surgical intervention. Hence having a result from a potential prognostic indicator before a patient is discharged would be a useful tool in planning the further management of the patient. In a previous study, in which HPV isolates from patients with RRP disease were genotyped within our laboratory using the L1 gene, patients infected with HPV-11 required more frequent surgical intervention to remove papillomas, suggesting a more aggressive form of the disease (Seedat et al., 2010). As HPV-11 tends to be a more aggressive disease than HPV6 disease, a rapid genotyping assay could be used as a prognostic indicator for the course of RRP disease. Hence a rapid detection and genotyping assay would have application in diagnosis and prognosis.

Please cite this article in press as: Combrinck, C.E., et al., FRET-based detection and genotyping of HPV-6 and HPV-11 causing recurrent respiratory papillomatosis. J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.01.025

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Fig. 2. Amplification plots of serially diluted HPV-6 and HPV-11 DNA controls to determine the sensitivity of the FRET assay. (A) 104 –107 dilution of HPV-6 and (B) 108 –1011 dilution of HPV-6 and (C) 104 –1011 dilution of HPV-11.

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A real-time FRET-based PCR assay was developed in which real-time technology provides a rapid detection method and the FRET probes allow differentiation of genotypes based on sequence variation between probe and gene. The melting temperature of the probe and template is dependent on the number of sequence mismatches allowing a melting-curve analysis to

generate specific melting peaks dependent on the sequence composition of the gene in the probe binding region. Similarly, Kocjan et al. developed a FRET-based real-time PCR based on the E2 genomic region of HPV (Kocjan et al., 2008). Sequence data for the E2 region was not available for our cohort of isolates from South African patients and hence the E6 gene,

Please cite this article in press as: Combrinck, C.E., et al., FRET-based detection and genotyping of HPV-6 and HPV-11 causing recurrent respiratory papillomatosis. J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.01.025

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for which sequence data had previously been established, was selected (Combrinck et al., 2012). Within each type, variants are known to occur. However a region was selected that was sufficiently conserved to include all known variants but sufficiently diverse between different types. This assay was shown to have 100% concordance with the genotyping performed using conventional PCR and partial sequence determination of the L1 gene. The assay has been designed to detect specifically and differentiate between HPV-6 and HPV-11. Previous studies have indicated that patients with HPV-11 have a more severe disease (Bonagura et al., 2010; Larson and Derkay, 2010; Seedat et al., 2010; Wiatrak et al., 2004). However in a recent study in which age of diagnosis was taken into account, the clinical course was shown to be more closely associated with age at diagnosis (<3 or 4 years) than HPV type, and that HPV-11 is more closely associated with early diagnosis than HPV-6 hence giving the impression that HPV-11 is the cause of more aggressive disease (Buchinsky et al., 2008; Gillison et al., 2012). Although early onset of disease could also be an indicator of the virulence of HPV-11 there are likely multiple factors associated with disease severity. Donne et al., addressed the role of HPV type in disease severity, concluding that the role in the pathogenesis of RRP of specific variants of HPV-6 and HPV-11 and non-HPV infections remains undetermined. In contrast to other studies, age of diagnosis of the patients in our cohort is higher (for HPV-11 mean age at diagnosis was 5.4, range 1.4–9.5) which is likely a consequence of inadequate healthcare facilities in rural areas and regardless of age at diagnosis, patients with HPV-11 as the dominant infection in our cohort have required more frequent surgical intervention (Seedat et al., 2010). Nevertheless the pathogenesis of RRP is likely influenced by a variety of factors including age of the patient, specific host immune responses and genetic factors and the role of type is more frequently considered less significant. The assay was designed to target specifically HPV-6 and HPV-11. HPV-44 and HPV-74 are the most closely related types. However, the additional mismatches between the probes and the target region of HPV-44 and HPV-74 would likely exclude the detection of these types. A limitation of the FRET assay described in this study is that co-infections of HPV-6 and HPV-11 are unlikely to be detected. The main advantage of the assay is that it can replace the current method used based on PCR amplification and sequencing of region of the L gene. To conclude, this study has demonstrated the application of a rapid real-time based assay for detection and genotyping of HPV using DNA extracted from clinical samples. Implementing this technique in our laboratory would reduce the time required for genotyping from three working days to several hours.

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Please cite this article in press as: Combrinck, C.E., et al., FRET-based detection and genotyping of HPV-6 and HPV-11 causing recurrent respiratory papillomatosis. J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.01.025

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