Prueba de concepto de una pcr multiplex de primera generación (cualitativa) para detección del oncogén e7 de vphs de alto riesgo

Introducción: Infección persistente con el virus de papiloma humano de alto riesgo (VPH-AR) causa cáncer de cuello uterino (CCU). Existen ensayos moleculares para la detección y la genotipificación del gen L1 de VPH, sin embargo, L1 puede perderse durante la integración viral. La expresión e integración del oncogén E7 es fundamental para el desarrollo de CCU. Objetivo: Estandarizar una PCR multiplex (mPCR) del oncogén E7 (E7-mPCR) para genotipificación de los VPH-AR de mayor frecuencia en CCU (VPH-16, -18, -31, -33, -45 y -52). Métodos: Se obtuvieron cepillados cervicales de voluntarias y se analizaron amplificando por PCR el gen L1 con subsecuente hibridación reversa. Posteriormente, se escogieron 59 muestras positivas para VPH-AR y se analizaron por E7-mPCR. Resultados: Se evidenció una elevada concordancia entre los resultados del ensayo E7- mPCR y los de la PCR de L1 (concordancia observada de 95,1%, Kappa de Cohen = 0,88), encontrándose mayor número de infecciones por VPH- AR en el 15,8% con E7-mPCR. Conclusión: E7-mPCR es una herramienta diagnóstica con alta concordancia y económica que puede adaptarse a una plataforma de mayor complejidad para procesar y detectar mayor cantidad de muestras y genotipos de VPH-AR.

Introduction: The persistent infection of the high-risk Human Papiloma Virus (VPH-AR in Spanish) causes uterine cervix cancer (CCU in Spanish). There are molecular essays for detection and genotyping of gen L1 of VPH. However, L1 may get lost during the viral integration. The expression and integration of oncogene E7 is fundamental for the development of CCU. Objective: To standardize a multiplex PCR (mPCR) of oncogene E7 (E7-mPCR) for genotyping the VPH- AR of highest frequency in CCU (VPH-16, -18, -31, -33, -45 y -52). Method: We obtained cervix brushing simples from volunteers and we analyzed them by amplifying the L1 gene through PCR with a subsequent reverse hybridization. After that, we chose 59 positive VPH- AR samples and we analyzed them for E7-mPCR. Results: We found out a high concordance between the results of the essay E7-mPCR and those of L1 PC (Observed concordance was of 95.1%, Cohen's Kappa = 0.88), and we revealed a higher number of infections for VPH-AR in a 15.8% with E7-mPCR. Conclusion: E7-mPCR is an economic diagnostic tool with high concordance which can be adapted to a platform with more complexity to process and detect a higher number of samples and VPH-AR genotypes.

Introdução: a infecção persistente com o virus de papiloma humano de alto risco (HPV-AR) causa cáncer de colo do útero (CCU). Existem ensaios moleculares para detecção e para a genotipificação do gene L1 de HPV; contudo, L1 pode ser perdido durante a integrado viral. A expressão e integração do oncogênese E7 é fundamental para o desenvolvimento do CCU. Objetivo: padronizar uma PCR multiplex (mPCR) do oncogênese E7 (E7-mPCR) para genotipificação dos HPV-AR de maior frequência no CCU (HPV-16, -18, -31, -33, -45 e -52). Métodos: foram realizadas raspagens com escova cervical rodada em voluntárias e foram analisadas a partir da amplificação do gene L1 por PCR com subsequente hibridação inversa. Em seguida, foram escolhidas 59 amostras positivas para HPV-AR, as quais foram analisadas por E7-mPCR. Resultados: foi evidenciada elevada concordância entre os resultados do ensaio E7-mPCR e os da PCR de L1 (concordância observada de 95,1%, Kappa de Cohen = 0,88), encontrando-se maior número de infecções por HPV-AR em 15,8% com E7-mPCR. Conclusão: E7-mPCR é uma ferramenta diagnóstica com alta concordância e económica que pode ser adaptada a uma plataforma de maior complexidade para processar e detectar maior quantidade de amostras e genótipos de HPV-AR.

Identification of canine papillomavirus type 1 (CPV1) DNA in dogs with cutaneous papillomatosis / Identificação do DNA de Papillomavirus canino tipo 1 em cães com papilomas cutâneos no Brasil

Canine oral papillomavirus (COPV), also known as Canine Papillomavirus type 1 (CPV1), induces papillomas at the mucous membranes of the oral cavity and at the haired skin of dogs. The classification of Papillomavirus (PV) types is based on the L1 capsid protein and nucleotide sequence; so far, 14 CPV types have been described in several countries, but the molecular characterization of CPV in Brazil is lacking. This study investigated the presence of the PV in seven papillomas from four mixed breed dogs from Londrina/PR, Southern Brazil, by partial sequencing of the L1 gene. Seven exophytic cutaneous lesions were surgically removed and processed for histopathological and molecular characterization. Histopathology confirmed the lesions as viral papillomas due to typical histological features. Polymerase Chain Reaction (PCR) assay using the FAP59 and FAP64 primers targeted the L1 gene followed by sequence analysis of the amplicons identified CPV1 in all evaluated papilloma samples. This study represents the first description of CPV1 DNA associated with canine papillomatosis in Brazil.

O papilomavírus oral canino (COPV), também denominado Papillomavirus canino tipo 1 (CPV1), tem a capacidade de induzir papilomas na mucosa da cavidade oral e também em pele de cães. A classificação dos tipos de papilomavírus (PV) é baseada na proteína L1 do capsídeo e na sequência de nucleotídeos que a codifica. Atualmente são descritos 14 tipos de CPV, no entanto, ainda faltam estudos moleculares relacionados à identificação dos tipos de CPV no Brasil. O objetivo deste estudo foi investigar a presença de PV em fragmentos de papilomas obtidos de quatro cães sem raça definida, provenientes de Londrina/PR, região sul do Brasil, e definir o tipo viral por meio da análise da sequência parcial de nucleotídeos do gene L1. Sete lesões cutâneas foram cirurgicamente removidas e processadas ​​para a caracterização histopatológica e molecular. O exame histopatológico confirmou as lesões como papilomas. Foi realizada reação em cadeia de polimerase (PCR), utilizando os primers FAP59 FAP64 para a amplificação parcial do gene L1, seguida por análise das sequências dos produtos amplificados, que confirmou a presença do CPV1 em todas as amostras avaliadas. Este estudo representa a primeira identificação do DNA de CPV1 associado com papilomatose canina no Brasil.

The role of hypermethylation in promoter region of ubiquitin carboxyl-terminal hydrolase L1 in human esophageal cancer / 中华内科杂志

Objective To study the association of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) promoter region methylation with human esophageal cancer. Methods Promoter region methylation of UCHL1 was dctcctcd by rnethylation specific PCR (MSP) in esophageal cancer cell lines and tissue samples.The expression of UCHL1 was detected by semi-quantitative RT-PCR and Western blot in esophagcal cancer cell lines.5-Aza-2'-deoxycytidine (5-Aza) was applied to reactivate methylated cell lines.ResultsComplete methylation of UCHL1 promoter region was detected in 8 cell lincs (KYSE30,KYSE150,KYSE140,KYSE450,KYSE510,TE3,TE7,TE10).Loss of UCHL1 expression was found in7 cell lines ( KYSE30,KYSE150,KYSE140,KYSE450,KYSE510,TE3,TE7).Reduced expression was found in TE10 cell line. Promoter region hypermethylation was correlated with UCHL1 expression in esophageal cancer cell lines.Re-expression of UCHL1 was induced by 5-Aza treatment in KYSE150 and TE3 cell lines.UCHL1 was frequently methylated in human primary esophageal cancer (74.51％,38/51 ),while no methylation was detected in normal esophageal mucosa (0/10). No association was found between promoter region methylation and age,gender,tumor location,tumor stage or lymph node metastasis.Conclusions UCHL1is silenced by promoter region hypermethylation in human esophageal cancer.Methylation of UCHL1 is frequently happened to primary esophageal cancer and may play an important role in the tumorigenesis.

Characterization of canine oral papillomavirus by histopathological and genetic analysis in Korea

In August 2008, forty dogs out of 400 developed oral warts in a breeding farm in Korea. Canine oral papilloma infection is a common disease in dogs. However, there has been no report of an outbreak of canine oral papillomavirus (COPV) in a group of dogs or in dog breeding farms in Korea, and the genetic analysis of COPV in Korea has yet to be performed. This study diagnosed canine oral papilloma from the oral samples of these dogs based on histopathological examination and immunohistochemistry. Polymerase chain reaction was applied to amplify the corresponding products using pre-existing primer sets for COPV and a universal human papillomavirus targeting L1 gene. Further genetic analysis of the major viral capsid gene L1 confirms the sequences of Korean COPV, which shows a close relationship to previously reported COPV. This study describes the histopathological and immunohistochemical characteristics of canine oral papilloma in a group of breeding dogs in Korea and discloses the complete L1 gene sequences of Korean COPV.

Phylogenetic position of an uncharacterized Brazilian strain of bovine papillomavirus in the genus Xipapillomavirus based on sequencing of the L1 open reading frame

The use of PCR assays with degenerate primers has suggested the existence of numerous as yet uncharacterized bovine papillomaviruses (BPV). Despite the endemic nature of BPV infections, the identification of BPV types in Brazilian cattle is still only sporadic. However, in a recent analysis of a partial segment of the L1 gene, we observed notable diversity among the BPV types detected. The aim of this study was to determine the phylogenetic position of the previously identified wild strain BPV/BR-UEL2 detected in the state of Paraná in Brazil. Since previous analysis of the partial L1 sequence had shown that this strain was most closely related to BPV type 4, genus-specific primers were designed. Phylogenetic analysis using complete L1 ORF sequences revealed that BPV/BR-UEL2 was related to BPV types classified in the genus Xipapillomavirus and shared the highest L1 nucleotide sequence similarity with BPV type 4 (78 percent). This finding suggests that BPV/BR-UEL2 should be classified as a potential new type of BPV in the genus Xipapillomavirus.

Comparison between L and E gene amplification analytical methods for human papillomavirus typing / 부인종양

OBJECTIVE: L and E6/E7 gene amplification analyses were compared to identify human papillomavirus (HPV) infection and verify the HPV type, with the intent to minimize HPV typing errors. METHODS: L1 gene verified HPV typing was accomplished via polymerase chain reaction (PCR) and membrane assays. Verification of HPV typing via E6/E7 genes was accomplished through nested multiplexed PCR. The results from 104 samples were compared. RESULTS: The rates of accordance and difference were 35% and 65%, respectively. For 29% of the analyses, nested multiplexed PCR was more diversified than the membrane assay. CONCLUSION: HPV can be classified into low-risk HPV and high-risk HPV groups. In parallel amplifications of the L and E genes is more efficient for accurate diagnosis in light of the different symptoms and attendant precautions of the risk groups.

Comparison between L and E gene amplification analytical methods for human papillomavirus typing / 부인종양

OBJECTIVE: L and E6/E7 gene amplification analyses were compared to identify human papillomavirus (HPV) infection and verify the HPV type, with the intent to minimize HPV typing errors. METHODS: L1 gene verified HPV typing was accomplished via polymerase chain reaction (PCR) and membrane assays. Verification of HPV typing via E6/E7 genes was accomplished through nested multiplexed PCR. The results from 104 samples were compared. RESULTS: The rates of accordance and difference were 35% and 65%, respectively. For 29% of the analyses, nested multiplexed PCR was more diversified than the membrane assay. CONCLUSION: HPV can be classified into low-risk HPV and high-risk HPV groups. In parallel amplifications of the L and E genes is more efficient for accurate diagnosis in light of the different symptoms and attendant precautions of the risk groups.