Complete genome sequence of a novel human gammapapillomavirus isolated from a cervical swab in Luxembourg

A novel human papillomavirus genotype was detected in a cervical swab specimen by next-generation sequencing after rolling circular amplification. It was fully cloned and characterized. The L1 open reading frame showed 77% nucleotide similarity with the closest genotype, HPV101, belonging to the gamma-6 species

Deregulation of LIMD1-VHL-HIF-1α-VEGF pathway is associated with different stages of cervical cancer.

To understand the mechanism of cellular stress in basal-parabasal layers of normal cervical epithelium and during different stages of cervical carcinoma, we analyzed the alterations (expression/methylation/copy number variation/mutation) of HIF-1α and its associated genes LIMD1, VHL and VEGF in disease-free normal cervix (n = 9), adjacent normal cervix of tumors (n = 70), cervical intraepithelial neoplasia (CIN; n = 32), cancer of uterine cervix (CACX; n = 174) samples and two CACX cell lines. In basal-parabasal layers of normal cervical epithelium, LIMD1 showed high protein expression, while low protein expression of VHL was concordant with high expression of HIF-1α and VEGF irrespective of HPV-16 (human papillomavirus 16) infection. This was in concordance with the low promoter methylation of LIMD1 and high in VHL in the basal-parabasal layers of normal cervix. LIMD1 expression was significantly reduced while VHL expression was unchanged during different stages of cervical carcinoma. This was in concordance with their frequent methylation during different stages of this tumor. In different stages of cervical carcinoma, the expression pattern of HIF-1α and VEGF was high as seen in basal-parabasal layers and inversely correlated with the expression of LIMD1 and VHL. This was validated by demethylation experiments using 5-aza-2'-deoxycytidine in CACX cell lines. Additional deletion of LIMD1 and VHL in CIN/CACX provided an additional growth advantage during cervical carcinogenesis through reduced expression of genes and associated with poor prognosis of patients. Our data showed that overexpression of HIF-1α and its target gene VEGF in the basal-parabasal layers of normal cervix was due to frequent inactivation of VHL by its promoter methylation. This profile was maintained during different stages of cervical carcinoma with additional methylation/deletion of VHL and LIMD1.This work was supported by CSIR (Council of Scientific and Industrial Research, Government of India)-JRF/NET grant [File No.09/030(0059)/2010-EMR-I] to Mr. C.Chakraborty, grant [Sr. No. 2121130723] from UGC (University Grants Commission, Government of India) to Mr. Sudip Samadder, grant [SR/SO/HS-116/2007] from DST (Department of Science and Technology, Government of India) to Dr. C. K. Panda and grant [ No. 60(0111)/14/EMR-II of dt.03/11/2014] from CSIR (Council of Scientific and Industrial Research, Government of India) to Dr. C. K. Pand

MinION nanopore sequencing and assembly of a complete human papillomavirus genome

BACKGROUND: The MinION sequencer belongs to the third generation of sequencing technology that allows for the generation of ultra-long reads, representing a potentially more effective approach to characterize entire viral genome sequences than other time-consuming and low-throughput methodologies. METHODS: We report the use of the MinION nanopore sequencer to sequence the full-length genome of human papillomavirus (HPV)-ICB2 (7441 bp), which was previously characterized in our laboratory. Three independent MinION libraries were prepared and sequenced using either three consecutive 12 -h runs (Protocol A) or a single run of 48 h starting from a pool of three barcoded DNA libraries (Protocol B). A fully automated bioinformatics pipeline was developed for the reconstruction of the viral genome. RESULTS: Protocols A and B generated 9,354,933 and 3,255,879 reads, respectively. Read length N50 values ranged between 6976 and 7360 nucleotides over the four sequencing runs. Bioinformatics analysis showed that both protocols allowed for the reconstruction of the whole viral genome, with pairwise percentages of identity to HPV-ICB2 of 100 % for protocol A and 99.98 % for protocol B. CONCLUSION: Our results show that the use of the MinION nanopore sequencer represents an effective strategy for whole-genome sequencing of HPVs with a minimal error rate