Rosario Nicola Brancaccio

Nouvelles stratégies pour l'identification et la caractérisation génomique complète de types inconnus d'HPV à partir d'échantillons d'ADN humain: Les papillomavirus humains (HPV) sont de petits virus icosaédriques non-enveloppés à ADN circulaire double brin. À ce jour, plus de 200 HPV ont été identifiés. La classification des Papillomaviridae est basée sur les identités de séquence nucléotidique du gène L1. La découverte de nouveaux HPV est d'une importance cruciale pour élargir nos connaissances sur ces virus. Alors qu'un sous-groupe HPV du genre alpha communément appelé "à haut risque", a été clairement associé aux cancers anogénitaux, et à certains cancers de la tête et du cou, il existe peu d’informations sur le rôle des HPV des genres bêta et gamma-HPVs dans les cancers humains. Des études récentes montrent que des bêta-HPVs pourraient jouer un rôle dans les cancers de la peau de type non-mélanome (NMSC), en association avec le rayonnement ultraviolet (UV). Toutefois, des études supplémentaires sont nécessaires pour confirmer cette hypothèse chez l’homme. Jusqu’à présent, les méthodes de choix utilisées pour l'identification de nouveaux HPVs dans des échantillons humains reposaient sur l’utilisation de la PCR et le séquençage de Sanger. Plusieurs amorces spécifiques ou à large spectre ciblant le gène L1, ont été développées pour l'amplification de séquences de HPV. De nos jours, avec l'avènement des méthodes de séquençage à haut débit (NGS), la possibilité d’élargir ces familles virales s’est accrue. Cependant, de grandes quantités de données sont générées par ces nouvelles méthodes de séquençage nécessitant le développement de méthodes bioinformatiques d'analyse spécifiques pour une identification et une reconstruction efficaces des séquences virales. Le but de ce travail de thèse a été de développer une stratégie pour l'identification des HPV, ainsi que la caractérisation du génome complet de nouveaux types, dans des échantillons humains. Au cours de ce travail de thèse, l’utilisation d’amorces à large spectre connues et validées, mais aussi l’utilisation d’amorces améliorées par notre laboratoire, toutes ciblant une portion du gène L1, ont été utilisées pour la recherche de séquences HPV dans des échantillons cutanés et oraux. L'analyse des données NGS a permis l'identification de 105 possible nouvelles séquences de PVs, ainsi que l’identification de 296 HPV connus. Une méthode d’analyse bioinformatique a été développée pour l’analyse des données de séquençage. La deuxième partie de ma thèse a consisté, à partir des données NGS précédemment obtenues, d’obtenir le génome entier de plusieurs HPVs. Ainsi, le génome entier de HPV227, un nouveau bêta-HPV, a été obtenu, en combinant plusieurs méthodes c.à.d. l'amplification par cercle roulant (ou rolling circle amplification - RCA), PCR pour amplification de longs fragments (ou “Long Range PCR”), le séquençage de Sanger par “primer walking”, et le clonage. Enfin, la technologie de séquençage par nanopores (MinION, Oxford Nanopore Technologies) a été utilisée afin d’obtenir le génome entier du HPV227, directement à partir de l'échantillon cutané d'origine et ainsi permettre un gain de temps important dans la caractérisation du génome complet des HPV. Pour ce faire, une méthode d’analyse bioinformatique des données de séquençage MinION pour la reconstruction des génomes de HPV a été développée. L'ensemble du génome du HPV227 a été reconstruit, confirmant l'efficacité de cette stratégie. Dans l'ensemble, cette thèse décrit et valide plusieurs approches pour l'identification et la caractérisation complète de nouveaux génomes de HPV. De plus, la découverte de 105 possible nouveaux PVs élargit nos connaissances sur cette famille de virus, bien que pour des analyses supplémentaires soient nécessaires pour la caractérisation complète de ces nouveaux virus

The nasopharyngeal tract contains a complex microbial community essential to maintaining host homeostasis. Recent studies have shown that SARS-CoV-2 infection changes the microbial composition of the nasopharynx. Still, little is known about how it affects the fungal microbiome, which could provide valuable insights into disease pathogenesis. Nasopharyngeal swabs were collected from 55 patients, during three distinct COVID-19 waves that occurred in the Campania Region (southern Italy). An RNA-seq-based analysis was performed to evaluate changes in mycobiota diversity, showing variations depending on the disease’s severity and the sample collection wave. The phyla Basidiomycota and Ascomycota were shown to have higher abundance in patients with severe symptoms. Furthermore, the diversity of the fungal population was greater in the second wave. Conclusion: According to our research, COVID-19 induces significant dysbiosis of the fungal microbiome, which may contribute to disease pathogenesis, and understanding its underlying mechanisms could contribute to developing effective treatments.

To study the interaction between HIV and other carcinogenic infections in conjunctival squamous cell carcinoma (SCC), we evaluated the presence of a broad spectrum of human viruses in conjunctiva specimens. Beta Human papillomavirus (HPV; n = 46), gamma HPV (n = 52), polyomaviruses (n = 12) and herpes viruses (n = 3) was determined in DNA extracted from 67 neoplastic and 55 non-neoplastic conjunctival tissues of HIV-positive and HIV negative subjects by Luminex-based assays. Next-generation sequencing (NGS) was also used to further characterize the presence of cutaneous HPVs. Detection of beta-2 HPV infections was associated with the risk of neoplasia (adjusted odds ratio [aOR] 3.0; 95% confidence interval [CI] 1.3-6.8), regardless of HIV status (HIV positive, aOR 2.6, 95% CI 0.9-7.7; HIV negative, aOR 3.5, 95% CI 0.9-14.4). EBV was strongly associated with the risk of neoplasia (aOR 12.0, 95% CI 4.3-33.5;P < .01) mainly in HIV individuals (HIV positive, aOR 57.5; 95% CI: 10.1-327.1; HIV negative aOR 2.6; 95% CI: 0.2-34.7). NGS allowed to identify 13 putative novel HPVs in cases and controls. Our findings suggest a role of beta HPV types and EBV, in conjunctival SCC. However, additional studies of viral expression in tumor tissue are required to confirm the causal association.

Both mucosal and cutaneous Human Papillomaviruses (HPVs) can be detected in the oral cavity, but investigations regarding the epidemiology of cutaneous HPVs at this site are scarce. We assessed mucosal (alpha) and cutaneous (beta and gamma) HPV infection in oral samples of HIV-infected and uninfected men who have sex with men (MSM). Oral rinse-and-gargles were collected from 310 MSM. Alpha HPVs were detected using the Linear Array, whereas beta and gamma HPVs were detected using multiplex PCR and Luminex technology. An amplicon-based next-generation sequencing (NGS) protocol was applied to a subset of samples collected from 30 HIV-uninfected and 30 HIV-infected MSM. Beta HPVs were significantly more common than alpha types (53.8% vs. 23.9% for HIV-infected subjects, p < 0.0001; 50.3% vs. 17.1% for HIV-uninfected subjects, p < 0.0001). Gamma HPVs were also frequently detected (30.8% and 25.9% in HIV-infected and uninfected MSM, respectively). NGS produced 2,620,725 reads representative of 146 known HPVs (16 alpha-PVs, 53 beta-PVs, 76 gamma-PVs, one unclassified) and eight putative new HPVs, taxonomically assigned to the beta genus. The oral cavity contains a wide spectrum of HPVs, with beta types representing the predominant genus. The prevalence of beta and gamma HPVs is high even in immunorestored HIV-infected individuals. NGS confirmed the abundance of cutaneous HPVs and identified some putative novel beta HPVs. This study confirms that cutaneous HPVs are frequently present at mucosal sites and highlights that their pathological role deserves further investigation since it may not be limited to skin lesions.

Background: The detection of known human papillomaviruses (PVs) from targeted wet-lab approaches has traditionally used PCR-based methods coupled with Sanger sequencing. With the introduction of next-generation sequencing (NGS), these approaches can be revisited to integrate the sequencing power of NGS. Although computational tools have been developed for metagenomic approaches to search for known or novel viruses in NGS data, no appropriate tool is available for the classification and identification of novel viral sequences from data produced by amplicon-based methods. Results: We have developed PVAmpliconFinder, a data analysis workflow designed to rapidly identify and classify known and potentially new Papillomaviridae sequences from NGS amplicon sequencing with degenerate PV primers. Here, we describe the features of PVAmpliconFinder and its implementation using biological data obtained from amplicon sequencing of human skin swab specimens and oral rinses from healthy individuals. Conclusions: PVAmpliconFinder identified putative new HPV sequences, including one that was validated by wet-lab experiments. PVAmpliconFinder can be easily modified and applied to other viral families. PVAmpliconFinder addresses a gap by providing a solution for the analysis of NGS amplicon sequencing, increasingly used in clinical research. The PVAmpliconFinder workflow, along with its source code, is freely available on the GitHub platform: https://github.com/IARCbioinfo/PVAmpliconFinder.

Ethiopia lies in the high-risk corridor of esophageal squamous cell carcinoma in East Africa, where individuals with this malignancy often do not report established risk factors, suggesting unidentified etiologies. Here, we report the prevalence of mucosal human papillomavirus (HPV) and of Helicobacter pylori (H. pylori) detection in endoscopy-obtained esophageal and gastroesophageal junction biopsies and in oral cell specimens taken at the time of esophageal cancer diagnosis in a case–control study in Addis Ababa, Ethiopia. DNA extraction was performed from fresh frozen tissue and oral cell pellets obtained with saline solution gargling subsequently fixed with ethanol. Mucosal HPV and H. pylori DNA was detected using highly sensitive assays that combine multiplex polymerase chain reaction and bead-based Luminex technology. The proportions of specimens testing positive were expressed as percentages, with binomial 95% confidence intervals. Agreement of results between tissue biopsy and oral cell specimens was estimated using the kappa statistic. Comparison of study participants’ characteristics by test results was done using the Pearson chi-square test. HPV DNA was detected in 1 of 62 tumor specimens (2, 95% confidence interval (CI): 0–9%), corresponding to HPV16 type. HPV DNA was detected in the oral cavity of 7 cases (11, 95% CI: 5–22%) and 4 of 56 matched healthy controls (7, 95% CI: 2–17%), with multiple HPV types detected. Detection of H. pylori DNA was 55% (95% CI: 42–68%), and 20 of 34 H. pylori-positive specimens (59, 95% CI: 41–75%) were positive for the cagA gene. Agreement of detection rates between tissue and oral cells in cases was poor for HPV and for H. pylori. The prevalence of mucosal-type HPV was very low, whereas H. pylori was more commonly detected, with a high proportion testing positive for the pro-inflammatory gene cagA. These novel findings remain to be replicated in larger studies and with the addition of serological determinations to better understand their biological significance in the context of esophageal and gastroesophageal junction cancers.

We report the complete genome characterization of a novel human papillomavirus (HPV) (ICB2) isolated from a skin swab. The L1 region of HPV ICB2 shares 87.9% nucleotide similarity with its closest relative, HPV37, and thus constitutes a novel human betapapillomavirus.