Resequencing Microarray Technology for Genotyping Human Papillomavirus in Cervical Smears Nicolas Berthet 1,2 * ¤ , Michael Falguie ` res 3 , Claudia Filippone 1,2 , Chloe ´ Bertolus 4,5 , Christine Bole-Feysot 6 , Sylvain Brisse 7 , Antoine Gessain 1,2 , Isabelle Heard 3,8 , Michel Favre 3,9 1 Institut Pasteur, Epide ´miologie et Physiopathologie des Virus Oncoge `nes, 75724 Paris Cedex 15, France, 2 Centre National de la Recherche Scientifique, UMR 3569, 75724 Paris Cedex 15, France, 3 Institut Pasteur, Centre National de Re ´fe ´rence des Papillomavirus, 75724 Paris Cedex 15, France, 4 AP-HP, Ho ˆ pital Pitie ´ -Salpe ˆtrie `re, Service de Chirurgie Maxillo-Faciale et Stomatologie, 75013 Paris, France, 5 UPMC, Universite ´ Paris 06, CIMI-Paris, UMRS CR7, INSERM U1135, 75005, Paris, France, 6 Imagine, Institut des maladies ge ´ne ´tiques - Plateforme Ge ´nomique, Ho ˆ pital Necker - Enfants Malades, 75743 Paris cedex 15, France, 7 Institut Pasteur, Plate-forme Ge ´ notypage des Pathoge `nes et Sante ´ Publique, 75724 Paris Cedex 15, France, 8 UPMC Universite ´ Paris 06, Groupe hospitalier Pitie ´ -Salpe ˆtrie `re, Paris Cedex 13, France, 9 Institut Pasteur, Unite ´ de Ge ´ne ´tique, Papillomavirus et Cancer humain, 75724 Paris Cedex 15, France Abstract There are more than 40 human papillomaviruses (HPVs) belonging to the alpha genus that cause sexually transmitted infections; these infections are among the most frequent and can lead to condylomas and anogenital intra-epithelial neoplasia. At least 18 of these viruses are causative agents of anogenital carcinomas. We evaluated the performance of a resequencing microarray for the detection and genotyping of alpha HPV of clinical significance using cloned HPV DNA. To reduce the number of HPV genotypes tiled on microarray, we used reconstructed ancestral sequences (RASs) as they are more closely related to the various genotypes than the current genotypes are among themselves. The performance of this approach was tested by genotyping with a set of 40 cervical smears already genotyped using the commercial PapilloCheck kit. The results of the two tests were concordant for 70% (28/40) of the samples and compatible for 30% (12/40). Our findings indicate that RASs were able to detect and identify one or several HPV in clinical samples. Associating RASs with homonym sequences improved the genotyping of HPV present in cases of multiple infection. In conclusion, we demonstrate the diagnostic potential of resequencing technology for genotyping of HPV, and illustrate its value both for epidemiological studies and for monitoring the distribution of HPV in the post-vaccination era. Citation: Berthet N, Falguie ` res M, Filippone C, Bertolus C, Bole-Feysot C, et al. (2014) Resequencing Microarray Technology for Genotyping Human Papillomavirus in Cervical Smears. PLoS ONE 9(11): e109301. doi:10.1371/journal.pone.0109301 Editor: Richard C. Willson, University of Houston, United States of America Received November 21, 2013; Accepted September 10, 2014; Published November 10, 2014 Copyright: ß 2014 Berthet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This program was supported by the Programme Transversal de Recherche DEVA No. 246 financed by the Institut Pasteur (Paris, France), sponsored by the Fondation Total-Institut Pasteur and the Elisabeth Taub award from the Acade ´mie Nationale de Me ´de ´cine. This study was funded by the French Institut National du Cancer (Canceropole 2011-1VADS 03-IP1). The funders had no role in study design, data analysis or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: nicolas.berthet@ird.fr ¤ Current address: CIRMF, De ´partement Zoonose et Maladies Emergentes, BP769 Franceville, Gabon Introduction More than 170 human papillomavirus (HPVs) have been characterized and they are classified into the alpha, beta, gamma, mu, and nu genera [1]. Some alpha papillomaviruses presenting a mucous tropism cause some of the most frequent sexually transmitted infections: around 300 million women worldwide carry a HPV infection of the uterine cervix. About 40 HPV genotypes able to infect genital mucous membranes have been identified. Low-risk mucosal HPVs (LR HPVs) are responsible for condylomas found in 1–2% of the sexually active population. High-risk HPVs (HR HPVs) are causative agents of more than 80% of high-grade cervical intra-epithelial neoplasia [2]. Persistent lesions associated with HR HPVs may evolve towards invasive cervical carcinomas. HPV DNA sequences are detected in the vast majority of adenocarcinomas, adenosquamous carcinomas and squamous cell carcinomas of the cervix, all of which are preceded by premalignant lesions [3]. HR HPVs also contributes to the pathogenesis of other anogenital and aero-digestive tract cancers [4]. The need for effective screening for cervical cancer is now recognized worldwide and testing for HPVs is a potentially powerful approach to the detection of cervical or anogenital lesions. Other than the hybrid capture II and the Cervista detection tests [5], most of the available tests for HPVs are based on PCR methods using degenerate or consensus primers. All these methods allow the detection, and some allow genotyping, of a wide spectrum of HPVs. In addition to their clinical value, robust methods of detection are required for epidemiological studies of HPV infections. In particular, they could be used to study the prevalence and distribution of circulating HPV genotypes in vaccinated and non-vaccinated populations. Several low or high-density microarrays have been developed and successfully used for the detection of several viral families such as Papillomaviridae. Most of them are based on long oligonucle- otide probes and virus identification is based only on hybridization patterns. This approach leads to a lack of information for pathogen identification at the single-pair resolution level [6,7]. However, this limitation has since been overcome with the use of the resequencing microarrays (RMAs) method. Resequencing PLOS ONE | www.plosone.org 1 November 2014 | Volume 9 | Issue 11 | e109301