L. L. Villa and others
HPVs have co-evolved with their natural hosts over a periodof several million years. Data obtained from 25 diﬀerentgeographical regions in the world indicate that HPV-16 hasevolved along ﬁve major branches, two being present mainlyin Africa, two in Asia, and one in Europe and India (L. Ho
., 1993). These studies revealed that coloniz-ation of the New World by Europeans and Africans is reﬂectedin the distribution of HPV-16 variants in the Americancontinent. It also became clear that these variants representgenuine HPV genomes since they have been found in diﬀerentindividuals from diﬀerent regions around the world. Fur-thermore, variation studies performed on the long controlregion (LCR), E6, L1 and L2 genes of HPV-16 indicate thatrecombination between variants is either rare or non-existent(L. Ho
., 1993; Yamada
., 1995, 1997).Few nucleotide diﬀerences found in HPV variants cor-respond to changes in amino acids. Nevertheless, it is of greatinterest to deﬁne alterations that may interfere with thefunctional or antigenic properties of speciﬁc viral proteins. Forinstance, variant 114K of HPV-16 assembles into virus-likeparticles (VLP) in a heterologous expression system, whereasthe reference (prototype) clone of HPV-16 does not have thesame property (Kirnbauer
., 1992, 1993). This diﬀerencehas been attributed to a single amino acid change at residue202 (Asp to His) of the L1 gene. Other changes in the L1
L2regions of the HPV genomes may be important for dis-criminating between the infectious potential of diﬀerentvariants as well as in deﬁning epitopes relevant to vaccinedesign. It has also been reported that a speciﬁc variation in theHPV-16 E6 protein, isolated from cervical cancers of HLA-B7individuals, interferes with the T-cell cytotoxic immuneresponse (Ellis
., 1995).In studies examining the course of HPV infection and therole of the virus in the genesis of cervical cancer, issues such aspolyclonality of lesions and frequency of particular HPVvariantsmaybecriticaltoourunderstandingofvirusinfectivityand pathogenicity (Zehbe & Tommasino, 1999). Nucleotidesequence variation has been used as an important tool forepidemiological studies of virus transmission and persistence(Franco
., 1994). The utility of this approach was indicatedin a study of the sexual transmission of HPV-16 (G. Ho
.(1995)found16 diﬀerent HPV-16 variants, one of which persisted overtime, while the other variants were transiently detected. Inaddition, intratypic variation of HPV-16 has also been shownto be an important predictor of progression to clinicallyrelevant cervical lesions (Xi
., 1997).In this report, we present results from an ongoing study of the natural course of HPV infection and cervical neoplasia inBrazil showing the association between variants as deﬁned bygeographical relatedness and characteristics of the infectionand lesion development. Unlike most previous studies, wehave analysed variants of both HPV-16 and -18 and assessedtheir correlation with virus persistence and virus burden aswell as with risk of prevalent and incident pre-invasivelesions.
Details of the Ludwig–McGill cohort study havebeen presented elsewhere (Franco
). Since 1993, womenenrolled in a cancer screening program catering for low-income familiesin Sa
o Paulo, Brazil have been followed-up in scheduled returns every 4months in the ﬁrst year post-enrolment (0, 4, 8 and 12 months) and onceevery 6 months thereafter. In each of these visits, a questionnaire-basedinterview was carried out. Subjects also had a cervical specimen taken forPap cytology and HPV testing and a blood sample was drawn forserologic testing for HPV antibodies. A cervicography was performedonce in the ﬁrst year and every 2 years thereafter. This investigation wasapproved by ethical review boards of the authors’ institutions.
Cervical cell specimens.
An Accelon biosampler (Medscand) wasused to collect a sample of ectocervical and endocervical cells at each of the visits. After the smear was prepared on a glass slide and ﬁxed in 95%ethanol, the exfoliated cells remaining in the sampler were immersed in atube containing Tris–EDTA buﬀer pH 7
4, kept at 4
C at the clinic forat most 5 days, and then frozen until testing. Cervical smears were sentto Montreal and were evaluated by the Bethesda system. Cytologyreadings were done blindly (A.F.).
HPV DNA detection and typing.
Cervical specimen DNA wasextracted and puriﬁed following standard techniques. In brief, cells weredigested with 100
ml proteinase K for 3–18 h at 55
C and the DNAwas puriﬁed by spin-column chromatography. Specimens were tested forthe presence of HPV DNA by a previously described PCR protocolamplifying a highly conserved 450 bp segment in the L1 viral gene(ﬂanked by primers MY09
11). Typing of the ampliﬁed products wasperformed by hybridization with individual oligonucleotide probesspeciﬁc for 27 HPV genital types (Bauer
., 1991; Hildesheim
.,1994). Ampliﬁed products hybridizing to the generic probe but not toany of the type-speciﬁc probes were further tested by restrictionfragment length polymorphism analysis (Bernard
). Thisextends the range of identiﬁable HPVs to over 40 genital types. In orderto check the integrity of the host DNA material extracted from thespecimens, assays also included an additional set of primers (GH20 andPC04), which amplify a 268 bp region of the human
-globin gene (Saiki
., 1988). In most analyses, infections with HPV-16 and -18 wereconsidered together and the remaining HPV types were groupedaccording to their oncogenic potential. We followed the classiﬁcationscheme of Liaw
. (1999), which grouped HPV-31, -33, -35, -39, -45,-51, -52, -56, -58, -59 and -68 as oncogenic types and considered all otherHPV types (except HPV-16 and -18) as non-oncogenic. Unknown typeswere included in the latter group.
Determination of virus load.
We measured virus load in thecervical specimens by a quantitative PCR protocol as previouslydescribed (Caballero
., 1995). This test consists of employing lowstringency conditions to allow co-ampliﬁcation of the speciﬁc HPV DNAfragment along with certain DNA sequences from the human genomepresent in the starting mixture. After ampliﬁcation, the ratio of thespeciﬁcHPVbandtothatofthehostgenomeinternalbandwasmeasuredby densitometry and followed by quantiﬁcation via interpolation from astandard curve; the standard curve was prepared with pre-selectedamounts of reference HPV plasmid added to a constant background of normal human DNA (Caballero
., 1995; Villa