Characterization of High Risk Human Papillomavirus (HPV) by Molecular and Phylogenetic Analysis

Abstract

Human papillomavirus (HPV) is recognized as a major causative agent for cervical carcinomas. Cervical cancer is the second leading cause of deaths due to cancer in females worldwide. HPV belongs to family Papillomaviridae and is velY diverse group of viruses as so for one hundred and fifty distinct subtypes have been reported. In Pakistan HPV prevalence and its con-elation with cervical cancer is unknown. The cun-ent investigations aimed at genetic characterization of high risk HPV types from Pakistan and understanding of their phylogenetic relationships with other already reported isolates from the world. To achieve this, universal primers designed against high risk types of HPV. HRTI primer set designed for HPV types 16, 18,31,33,45,52,56,58,59,68 and 69. While HRT2 designed for HPV types 18, 45, 56, 59, 68, and 69. These primers were designed on the conserved region of HPV El gene. Samples were collected from females reported with gynecological problems at Benazir Bhutto Shaheed Hospital Rawalpindi, Nuclear Medicine, Oncology and Radiotherapy Institute (NORl) Hospital Islamabad and Pakistan Institute of Medical Sciences (PIMS) Hospital Islamabad Pakistan. A primer set designed against human actin gene from nuclear genome was also used as control for establishing success of DNA isolation. The initial screening with this assay resulted in positive results from two patients out of fifty, indicating a prevalence rate of four percent in this small data set. PCR amplified product ofHPV DNA was ligated in plasmid pTZ57R1T and cloned in E.coli DH5a strain by electroporation technique. Out of two HPV positive samples one was successfully cloned and sequenced. Neighbor Joining (NJ) trees were constructed by using HPVPAKI sequence, selected sequences of all other types of HPV and all sequences of HPV 16 available in NCBI data base respectively. The sequence analysis of HPVPAKI indicated 92% bootstraps supports with the HPV 16 Asian American variant in the NCB I data base. Phylogenetic analysis confirmed that HPVP AKI belonged to high risk HPV 16 type. Nucleotide identities and homology were calculated by using MetGat. HPVPAKI showed 99.4% nucleotide identity with HPV 16. Our study showed for the first time that HPV high risk type 16 is found in Pakistan and playing role in cervical cancer among women.