RESUMO
BACKGROUND: Human papillomaviruses (HPVs) have been divided into mucosal and cutaneous types according to their primary epithelial tissue tropism. However, recent studies showed the presence of several cutaneous types in mucosal lesions and healthy mucosa from different anatomical sites. METHODS: Here, the HPV prevalence and type-specific distribution were assessed in a variety of mucosal samples from 435 individuals using a combination of two established broad-spectrum primer systems: Gamma-PV PCR and CUT PCR. RESULTS: Overall HPV prevalence in anal canal swabs, cervical cancer biopsies, genital warts and oral swabs was 85, 47, 62 and 4%, respectively. In anal canal swabs, Alpha-PVs were most frequently found (59%), followed by Gamma- (37%) and Beta-PVs (4%). The prevalence and persistence of HPV infection in the anal canal of 226 individuals were further explored. Overall HPV, Gamma-PVs and multiple HPV infections were significantly higher in men vs. women (p = 0.034, p = 0.027 and p = 0.003, respectively); multiple HPV infections were more common in individuals ≤40 years (p = 0.05), and significantly higher prevalence of Gamma-PVs and multiple HPV infections was observed in HIV-1-positive vs. HIV-1-negative individuals (p = 0.003 and p = 0.04, respectively). Out of 21 patients with follow-up anal swabs, only one persistent infection with the same type (HPV58) was detected. CONCLUSIONS: Our findings suggest that Gamma-PVs (except species Gamma-6) are ubiquitous viruses with dual muco-cutaneous tissue tropism. Anal canal Gamma-PV infections may be associated with sexual behavior and the host immune status. This study expands the knowledge on Gamma-PVs' tissue tropism, providing valuable data on the characteristics of HPV infection in the anal canal.
Assuntos
Doenças do Ânus/complicações , Gammapapillomavirus/genética , Soropositividade para HIV/complicações , HIV-1/imunologia , Mucosa Bucal/virologia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/epidemiologia , Reação em Cadeia da Polimerase/métodos , Adolescente , Adulto , Idoso , Doenças do Ânus/virologia , Sequência de Bases/genética , Condiloma Acuminado/virologia , Epitélio/virologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Papillomavirus/virologia , Prevalência , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Adulto JovemRESUMO
Genus Gammapapillomavirus (Gamma-PV) is the most diverse and largest clade within the Papillomaviridae family. A novel set of degenerate primers targeting the E1 gene was designed and further used in combination with the well-known CUT PCR assay to assess HPV prevalence and genus distribution in a variety of cutaneous samples from 448 immunocompetent individuals. General HPV, Gamma-PV and mixed infections prevalence were significantly higher in actinic keratosis with respect to benign and malignant neoplasms, respectively (pâ¯=â¯0.0047, pâ¯=â¯0.0172, pâ¯=â¯0.00001). Gamma-PVs were significantly more common in actinic keratosis biopsies than Beta- and Alpha-PVs (pâ¯=â¯0.002). The full-length genome sequence of a novel putative Gamma-PV type was amplified by 'hanging droplet' long-range PCR and cloned. The novel virus, designated HPV210, clustered within species Gamma-12. This study provides an additional tool enabling detection of HPV infections in skin and adds new insights about possible early roles of Gamma-PVs in the development of cutaneous malignant lesions.
Assuntos
Gammapapillomavirus/genética , Gammapapillomavirus/isolamento & purificação , Ceratose Actínica/virologia , Reação em Cadeia da Polimerase/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Gammapapillomavirus/classificação , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Papillomavirus/virologia , Adulto JovemRESUMO
A modified pan-PV consensus-degenerate hybrid oligonucleotide primer (CODEHOP) PCR was developed for generic and sensitive detection of a broad-spectrum of human papillomaviruses (HPVs) infecting the cutaneous epithelium. To test the analytical sensitivity of the assay we examined 149 eyebrow hair follicle specimens from immunocompetent male patients. HPV DNA was detected in 60â% (89/149) of analysed eyebrow samples with a total of 48 different HPV sequences, representing 21 previously described HPVs and 27 putative novel HPV types. Evidence for ten novel HPV subtypes and seven viral variants, clustering to three out of five genera containing cutaneous HPVs, was also obtained. Thus, we have shown that the modified pan-PV CODEHOP PCR assay is able to identify multiple HPV types, even from different genera, in the same clinical sample. Overall, these results demonstrate that the pan-PV CODEHOP PCR is an excellent tool for screening and identification of novel cutaneous HPVs, even in samples with low viral loads.
Assuntos
Betapapillomavirus/isolamento & purificação , Primers do DNA/química , DNA Viral/genética , Gammapapillomavirus/isolamento & purificação , Genótipo , Infecções por Papillomavirus/diagnóstico , Reação em Cadeia da Polimerase/métodos , Adulto , Sequência de Bases , Betapapillomavirus/classificação , Betapapillomavirus/genética , Primers do DNA/metabolismo , Sobrancelhas/virologia , Gammapapillomavirus/classificação , Gammapapillomavirus/genética , Folículo Piloso/virologia , Humanos , Masculino , Tipagem Molecular/métodos , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/virologia , Filogenia , Prevalência , Sensibilidade e Especificidade , Eslovênia/epidemiologiaRESUMO
More than 170 human papillomavirus (HPV) types have been completely sequenced, curated and divided into five genera: Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Mupapillomavirus and Nupapillomavirus. With the application of PCR methods, hundreds of putative novel HPV types have been identified as PCR amplicons in mucosa and skin. However, at present there are no studies reporting a systematic search of the currently known L1 amplicons and their phylogenetic relationships. This survey revealed the existence of at least 202 different putative HPV types that are pending for full-genome characterization: five alphapapillomaviruses, 37 betapapillomaviruses, 159 gammapapillomaviruses and one mupapillomavirus. All potential viruses of the genera Alphapapillomavirus and Betapapillomavirus were grouped in the defined species, while 59 putative gammapapillomaviruses types were segregated in 21 unidentified putative species. These data highlight the need for progress in the identification of additional taxa of the family Papillomaviridae in order to elucidate the diversity, evolution and medical implications of these viruses.
Assuntos
Variação Genética , Papillomaviridae/classificação , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Filogenia , Alphapapillomavirus/classificação , Alphapapillomavirus/genética , Betapapillomavirus/classificação , Betapapillomavirus/genética , Proteínas do Capsídeo/genética , Primers do DNA , DNA Viral/análise , DNA Viral/genética , Gammapapillomavirus/classificação , Gammapapillomavirus/genética , Genoma Viral , Humanos , Mupapillomavirus/classificação , Mupapillomavirus/genética , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/epidemiologia , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
This study developed a hanging-droplet long PCR, a generic and highly sensitive strategy to facilitate the identification of new human papillomavirus (HPV) genomes. This novel procedure used for the first time the hanging-droplet PCR technique for the amplification of long DNA fragments with generic primers targeting the L1 and E1 regions. It was first applied to the amplification of types belonging to the highly divergent genus Gammapapillovirus (γ-PV). The hanging-droplet long PCR was 100-fold more sensitive than a simple long PCR procedure, detecting as few as ten copies of HPV-4. Nineteen skin samples, potentially containing putative HPV types from the γ-PV genus, were also screened. The method identified four γ-PV genomic halves from new and previously described putative types, and made the full characterization of HPV-156 possible. This novel virus meets the criteria for a new species within the γ-PV genus, with nucleotide identities in the L1 ORF ranging from 58.3 to 67.3â% compared with representative types of the current γ-PV species. HPV-156 showed the highest identity to HPV-60 (67.3â%) from species γ-4, and was consistently closely related to it in both late- and early-gene-derived phylogenies. In conclusion, this report provides a versatile and highly sensitive approach that allowed identification of the prototype of a new species within the γ-PV genus. Its application with primers targeting the different genera in which both human and non-human PVs are distributed may facilitate characterization of the missing members of the family Papillomaviridae.
Assuntos
Alphapapillomavirus/classificação , DNA Viral/genética , Gammapapillomavirus/classificação , Gammapapillomavirus/genética , Reação em Cadeia da Polimerase/métodos , Alphapapillomavirus/genética , Especiação Genética , Humanos , Dados de Sequência Molecular , FilogeniaRESUMO
RNAi (RNA interference) is a natural process by which eukaryotic cells silence gene expression through small interference RNAs (siRNA) which are complementary to messenger RNA (mRNA). In this process, the siRNA that are 21-25 nucleotides long and are known as microRNA (miRNA), either associate with the RNA-induced silencing complex (RISC), which targets and cleaves the complementary mRNAs by the endonucleolytic pathway, or repress the translation. It is also possible to silence exogenous gene expression during viral infections by using DNA templates to transcribe siRNA with properties that are identical to those of bioactive microRNA. Persistent human papillomavirus (HPV) infection is the main etiological agent during cervical cancer development and the HPV E6 and E7 oncogenes, which induce cellular transformation and immortalization, represent strategic targets to be silenced with siRNA. In several in vitro and in vivo studies, it has been demonstrated that the introduction of siRNA directed against the E6 and E7 oncogenes in human tumoral cervical cells transformed by HPV, leads to the efficient silencing of HPV E6 and E7 oncogene expression, which induces the accumulation of the products of the p53 and pRb tumor suppressor genes and activates the mechanism of programmed cell death by apoptosis; thus, the progression of the tumoral growth process may be prevented. The goal of this review is to analyze the microRNA biogenesis process in the silencing of gene expression and to discuss the different protocols for the use of siRNA as a potential gene therapy strategy for the treatment of cervical cancer.