Sequence variants of human papillomavirus type 16 in clinical samples permit verification and extension of epidemiological studies and construction of a phylogenetic tree

Abstract

Genomic variability between different viral isolates provides a powerful epidemiological tool for verifying ultrasensitive diagnostic procedures, understanding infectious pathways in individuals and human populations, and studying viral evolution. The potential of this approach has not yet been exploited for the diagnosis of human papillomaviruses (HPVs) like HPV type 16 (HPV-16), which are involved in genital cancer. Toward this end, we amplified by polymerase chain reaction, cloned, and sequenced a 364-bp noncoding segment of the HPV-16 genome from cell lines, cervical biopsy specimens, and cervical smears. The HPV-16 genomes in the cell lines SiHa and CaSki showed an identical point mutation, and in the SiHa cell line it had an additional 38-bp deletion. Only 4 of 22 cervical lesions biopsied from patients at several hospitals in Singapore contained HPV-16 DNA with the prototype sequence, while the DNAs of the other 18 cervical lesions differed by 1 to 10 mutations. This excludes contaminations with cloned HPV-16 DNA as the source of this DNA. To test whether this diversity was a geographic idiosyncrasy, we analyzed 25 cervical biopsy specimens from Brazil. Eight of these contained the prototype sequence, while 17 were mutated. Altogether, 11 genomic variants were found in the Singaporean samples and 12 genomic variants were found in the Brazilian samples, and only 5 of these occurred identically in both cohorts. All variants could be connected to form a phylogenetic tree, with some branches being specific for each cohort. This suggests that the variants did not originate over a short period in the individual patient but, rather, evolved consecutively while spreading throughout humankind. Repeated amplification with the low-error Vent polymerase suggested that at least five patients each had two variants in the same biopsy specimen, while another patient exhibited different variants in two separate biopsy specimens. This documents the possibility of multiple infections with variants of the same papillomavirus type. We do not believe that the observed variants have greater carcinogenicity, since similar variants were found in cervical smears from asymptomatic women. In chloramphenicol acetyltransferase assays, enhancer segments of some selected samples that were analyzed did not show altered transcriptional activity. This was expected since most of the mutations did not coincide with known cis-responsive elements. The observed sequence variability can be used as a natural tag of the viral genome to unambiguously assess a variety of epidemiological questions.

Association of human papillomavirus type 16 and its genetic variants with cervical lesion in Korea

“…HPV16 E6 polymorphisms have been shown, although inconsistently, to be associated with CIN or cervical cancer (6). The D25E and L83V variants are both associated with an elevated risk of cervical carcinoma and the risk that they confer appears to vary geographically based on genetic differences between populations (14,29,(33)(34)(35)(36)(37)(38). In Europe, L83V was reported to be associated with an increased risk of persistent infection and higher grades of cervical lesions (33,39).…”

Human papillomavirus genotypes and HPV16 variants in penile carcinoma

“…It is well known that HPV16 present well conserved genomic variants distinctive of geographical origin/population ethnicity, 29 which have been clustered by phylogenetic analysis into five distinct groups: European (E), Asian (As), Asian-American (AA), African 1 (Af-1) and African 2 (Af-2). [30][31][32][33][34][35][36][37][38][39] Several epidemiological studies, focused on the association of HPV16 variants and risk progression of cervical carcinoma, have observed an increased risk of disease associated with either European variants 34,40 or with non-European variants of HPV16. 37,38,[41][42][43] Much less is known about the involvement of HPV variants in the development of HPVrelated tumors other than cervical neoplasia.…”

Sites of disruption within E1 and E2 genes of HPV16 and association with cervical dysplasia

“…It is the most frequently identified HPV type, with a prevalence of 50% in cervical cancer cases [Ferlay et al, 2010;De Sanjose et al, 2010;Li et al, 2011]. HPV16 is classified in five major intratypic variant lineages known as HPV16 European, European Asian, African type I, African type II, and Asian American [Ho et al, 1991, ;1993;Chen et al, 2005]. The variant lineages have been associated with the geographic origin of the populations in which they are prevalent [Chan et al, 1992;Yamada et al, 1995].…”