www.elsevier.com/locate/yviro

Virology 340 (20

Worldwide genomic diversity of the human papillomaviruses-53, 56, and

66, a group of high-risk HPVs unrelated to HPV-16 and HPV-18

Jose C. Pradoa, Itzel E. Calleja-Maciasb, Hans-Ulrich Bernardb,*, Mina Kalantarib,

Sylvia A. Macayb, Bruce Allanc, Anna-Lise Williamsonc,d, Lap-Ping Chunge,

Robert J. Collinse, Rosemary E. Zunaf, S. Terence Dunnf, Rocio Ortiz-Lopezg,

Hugo A. Barrera-Saldanag, Heather A. Cubieh, Kate Cuschierih,

Magnus von Knebel-Doeberitz i, Gloria I. Sanchezj,

F. Xavier Boschk, Luisa L. Villaa

aLudwig Institute for Cancer Research, Sao Paulo, BrazilbDepartment of Molecular Biology and Biochemistry, University of California Irvine, Irvine, CA 92697, USA

cInstitute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, South AfricadNational Health Laboratory Service, University of Cape Town, South Africa

eQueen Mary Hospital and the University of Hong Kong, Hong KongfDepartment of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA

gDepartamento de Bioquimica, Facultad de Medicina, Universidad Autonoma de Nueva Leon, Monterrey, MexicohRoyal Infirmary of Edinburgh, Edinburgh, Scotland

iInstitute for Molecular Pathology, University of Heidelberg, Heidelberg, GermanyjHPV Laboratory, IDIBELL, Institut Catala d’Oncologia, Barcelona, Spain

kEpidemiology and Cancer Registration Unit, IDIBELL, Institut Catala d’Oncologia, Barcelona, Spain

Received 8 April 2005; returned to author for revision 14 June 2005; accepted 15 June 2005

Abstract

Among more than 200 human papillomavirus (HPV) types presumed to exist, 18 ‘‘high-risk’’ HPV types are frequently found in

anogenital cancer. The best studied types are HPV-16 and 18, which are only distantly related to one another and form two separate

phylogenetic branches, each including six closely related types. HPV-30, 53, 56, and 66 form a third phylogenetic branch unrelated to HPV-

16 and 18. Worldwide comparison of HPV-16 and 18 isolates revealed a distribution of variant genomes that correlated with the geographic

origin and the ethnicity of the infected cohort and led to the concept of unique African, European, Asian, and Native American HPV-16 and

18 variants. Here, we address the question whether similar phylogenies are found for HPV-53, 56, and 66 by determining the sequence of the

long control regions (LCR) of these HPVs in samples from Europe, Asia, and Africa, and from immigrant societies in North and South

America. Phylogenetic trees calculated from point mutations and a few insertions/deletions affecting 2–4.2% of the nucleotide sequences

were distinct for each of the three HPVs and divergent from HPV-16 and 18. In contrast to the ‘‘star-phylogenies’’ formed by HPV-16 and 18

variants, 44 HPV-53 isolates represented nine variants, which formed two deep dichotomic branches reminiscent of the beginning split into

two new taxa, as recently observed for subtypes of HPV-44 and 68. A total of 66 HPV-56 isolates represented 17 variants, which formed

three branches preferentially containing European, Asian, and African variants. Variants of a fourth branch, deeply separated from the other

three, were characterized by a 25 bp insertion and created a dichotomy rather than star-like phylogeny. As it contained isolates from cohorts

in all continents, it may have evolved before the spread of humans into all continents. 18 of 31 HPV-66 isolates represented the prototype

clone, which was found in all parts of the world, while the remaining 13 clones formed 11 branches without any geographic association. Our

findings confirm the notion of a quantitatively limited genomic diversity of each HPV type with some correlation to the geographic origin of

0042-6822/$ - s

doi:10.1016/j.vi

* Correspondi

E-mail addr

05) 95 – 104

ee front matter D 2005 Elsevier Inc. All rights reserved.

rol.2005.06.024

ng author. Fax: +1 949 824 8551.

ess: hbernard@uci.edu (H.-U. Bernard).

J.C. Prado et al. / Virology 340 (2005) 95–10496

the sample. In addition, we observed in some variants of these three HPV types mutations that affect the amino acid sequence of the E6

oncoproteins and the L1 capsid protein, supporting the possibility of immunogenic and oncogenic diversity between variants of any HPV

type.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Genomic diversity; HPV-53; HPV-56; HPV-66

Introduction

The taxonomy of papillomaviruses (PVs) based on

phylogeny is now a mature field of research. PVs form a

separate family and are further classified into genera and

species as recognized by the International Committee on

Taxonomy of Viruses (de Villiers et al., 2004). On lower

taxonomic levels, PVs are classified as types, subtypes, and

variants, and molecular, clinical, and epidemiological

research normally addresses PVs on these latter three levels

of taxonomy. Among more than 200 different human

papillomavirus (HPV) types, close to one hundred being

formally described (Antonsson et al., 2000; Chan et al.,

1995; de Villiers, 1989; de Villiers et al., 2004), 18 HPV

types are of particular interest as they are more likely to be

associated with anogenital cancer and therefore classified as

‘‘high-risk’’ HPV types (Munoz et al., 2003). Paradigms for

the high-risk HPVs are HPV-16 and 18, which are only

remotely related to one another. Each of HPV-16 and 18

form, together with six other HPV types, two separate

phylogenetic branches, which are now called ‘‘species’’ (de

Villiers et al., 2004). A third branch (species) of high-risk

HPV types is formed by HPV-30, 53, 56, and 66.

An HPV type is defined as a separate taxon, when the

nucleotide sequence of its L1 gene differs from that of any

other HPV type by at least 10%. The additional term

‘‘subtype’’ was defined to identify HPV genomes whose L1

nucleotide sequence is 2–10% different from that of the

closest type (de Villiers et al., 2004). Presently, we know

only of four HPV subtypes, and recent analyses of two of

them have shown that these evolved dichotomically into

independent taxa, i.e., into future HPV types (Calleja-

Macias et al., in press).

In contrast to the scarce occurrence of subtypes, each

HPV type can be re-isolated in the form of genomic

variants, whose number is small in comparison to the quasi-

species formed by rapidly evolving RNA viruses. Variants

of HPV types differ by less than 2% of their L1 nucleotide

sequence, but slightly more in the long control region

(LCR), which does not encode genes and is therefore less

restricted to give rise to viable mutations (Stewart et al.,

1996; Yamada et al., 1997). Previous studies have shown

that specific genomic variants have increased prevalence in

some parts of the world and sometimes correlate with the

predominant ethnic group of that geographic region (Deau

et al., 1993; Ho et al., 1993; Ong et al., 1993; Heinzel et al.,

1995; Stewart et al., 1996; Chan et al., 1997; Yamada et al.,

1997). Most notably, variants of HPV-16 and HPV-18 form

phylogenetic trees with multiple short branches (‘‘star-

phylogenies’’) with high prevalence of variants of each

branch either in Africa, or Europe, or East Asia. One branch

of each type extends from East Asia into the Americas.

Thus, these trees are reminiscent of the evolution and

worldwide spread of the human host of the virus (Bernard,

1994) and suggested that certain variants diverged approx-

imately at the same time when the major human ethnic

groups formed.

Here, we report a study of the genomic diversity of

variants of the closely related high-risk types HPV-53, 56,

and 66. The three phylogenetic trees show similarities to

but also differences from the patterns observed for HPV-16

and 18. Several geographic clusters of HPV-53 and 56

variants established similarities, while discrepancies include

the deep dichotomic branching of the HPV-53 and 56 trees,

possibly the early stage of a split into two taxa and a lack

of clear geographic association of HPV-66 variants. In

contrast to the HPV-16 and 18 trees, these phylogenies

indicate that intratype diversification may have occurred

well before and after the evolution of human ethnic groups

and not necessarily in linkage to the evolution of the human

host. The low prevalence of HPV-53, 56, and 66 may

indicate a slower evolution and slower spread of these

viruses than that observed in the more common HPV types.

Our data bases confirm that each HPV type shows only

modest diversification of its genome sequence and the

encoded proteins, confirming that HPVs evolve slowly and

could be much more accessible targets of vaccination and

pharmaceutical approaches than many rapidly evolving

RNA viruses. Our study also reports diversity between the

E6 oncoproteins and the L1 capsid proteins of variants of

each type, alerting us to the need to consider variant

diversity in epidemiological, etiological, and vaccination

research.

Results

Genomic diversity of HPV-53 isolates

HPV-53 was originally isolated from a cervical swab of a

pregnant woman without cytological or clinical abnormality

(Gallahan et al., 1989). Subsequently, HPV-53 been detected

in smears graded as normal (Wheeler et al., 1993; Lazcano-

Ponce et al., 2001) as well as dysplastic lesions (Isacson et

al., 1996; Meyer et al., 2001) and is considered a

‘‘probable’’ high risk HPV type (Munoz et al., 2003). While

J.C. Prado et al. / Virology 340 (2005) 95–104 97

it is not among the eight most common HPV types in

cervical cancer (Munoz et al., 2003), epidemiological

studies have reported prevalence up to 6.3% (Lazcano-

Ponce et al., 2001).

Among 44 HPV-53 variants, we observed mutations in

21 positions, namely 20 single nucleotide exchanges and

one 4 bp (GGGA) insertion. In the 501 bp segment that was

phylogenetically evaluated, maximal distances between any

two variants were 21 mutations (4.2%). A phylogenetic tree

calculated from these variant sequences shows deeply

dichotomical branching (Fig. 1). Such dichotomy has been

recently observed in the trees of the HPV subtype pairs

HPV-44/55 and 68a/b (Calleja-Macias et al., 2005), but it

has not been described in a tree of HPV variants. One

branch contains the HPV-53 reference clone and 15 isolates

representing four variants. Most isolates of this branch

originated from Africa or potential African immigrant

populations. The second branch contained 28 isolates

representing five variant genomes. One variant was repre-

sented in four out of five isolates from Asia (Hong Kong)

but included an equal number of isolates from Scotland.

Therefore, we cannot identify a phylogenetic cluster likely

predominating in Asia.

Genomic diversity of HPV-56 isolates

HPV-56 DNA was originally identified by low-strin-

gency hybridization with an HPV-31 probe, in a specimen

(GU56) of cervical intraepithelial neoplasia (CIN) obtained

from a woman in the Washington D.C. metropolitan area

(Lorincz et al., 1989). HPV-56 has also been found in

cervical cancer and all grades of precursor lesions. Some

authors believe that this type is under-represented in

invasive cancer compared to preinvasive cervical lesions

(Clifford et al., 2003). It is generally considered to be a

high-risk type (Munoz et al., 2003), and while not being

among the eight most common HPV types in cervical

cancer, prevalence of this type is still generally observed

between 1.1–10.9% (Franceschi et al., 2005).

The 59 HPV-56 isolates represented 17 different genomic

variants based on point mutations in 25 positions and a 41

bp deletion. Maximal distances in the phylogenetically

evaluated 549 bp segment (counting the deletion as a single

mutation) between any two variants were 11 mutations

(2%). Although we counted the 41 bp deletion only as a

single mutational event, presence or absence of this segment

divided the phylogenetic tree into two deep dichotomically

separated branches. One branch was formed by six variants,

which contained the 41 bp sequence, and included the

reference clone, four closely related variants, and the

remotely related variant BR4114. The other branch con-

tained eleven variants: three variants of this branch had a

high prevalence in the South African and Hong Kong cohort

as well as in cohorts from Europe or presumed European

immigrants and might have a specificity for geographic

region or ethnicity (Fig. 2).

Genomic diversity of HPV-66 isolates

HPV-66 was originally detected in a biopsy of a 38-year-

old patient with a stage I invasive squamous-cell carcinoma

of the uterine cervix (Tawheed et al., 1991) and is

considered a ‘‘probable’’ high-risk type (Munoz et al.,

2003). HPV-66 has been reported with a prevalence up to

14.5% in CIN II/III (De Vuyst et al., 2003) but was not

found among the eight most common types in cervical

cancer (Munoz et al., 2003).

Among the HPV-66 variants, there were seventeen

nucleotide exchanges and one 2 bp insertion, with a total

of 12 different variants and maximal distances of 16

mutations in the 621 bp (2.6%) of the phylogenetically

evaluated segment. 18 of the 31 isolates contained the HPV-

66 reference clone, and the other eleven variants did not

show any geographic clustering. The most distantly related

variant came from a patient from Morocco, a country whose

intratype HPV diversity has never been studied before

(Fig. 3).

Diversity in the E6 oncogene and a conserved part of the L1

gene

In order to evaluate the potential for functional changes

of HPV proteins, whose genes are linked to LCR variation,

we compared HPV-53, 56, and 66 variants selected from

remote branches of the phylogenetic tree and determined

the sequence of their E6 genes and the corresponding E6

oncoproteins as well as the sequence of that part of the L1

gene that is bracketed by the MY09/11 amplification

primers, encoding about a third of the major capsid protein.

These sequence data are summarized in Fig. 4.

Surprisingly, the E6 genes of HPV-53 variants showed

significant distances between any two variants and between

the variants and the reference clone, namely up to 16

nucleotides (3.6% of the nucleotide sequence), approaching

the diversity of the LCR. In the case of one variant

(ED1817), this affected eleven amino acid residues or

7.3% of the E6 oncoprotein. Diversity was much lower in

the case of HPV-56 and 66, with maximal distances among

any two variants of seven and six nucleotides (1.5 and

1.3%). In many cases, these changes did not affect the

amino acid sequence, while one, two, and three amino acids

were exchanged in others. Seven, six, and six, respectively,

nucleotides were affected in the L1 gene segment, amount-

ing to diversities of 1.6 and 1.3% and affecting normally

none of the amino acids, but leading in some variants to one

and two amino acid exchanges.

Discussion

The high-risk HPV types involved in anogenital carcino-

genesis form three phylogenetic groups (now called

‘‘species’’), two of which are affiliated with HPV-16 and

J.C. Prado et al. / Virology 340 (2005) 95–10498

J.C. Prado et al. / Virology 340 (2005) 95–104 99

HPV-18 (HPV species 9 and 7, respectively), and the third

consisting of the little studied HPV types 30, 53, 56, and 66

(HPV species 6) (de Villiers et al., 2004). It was the

objective of our research to analyze this latter group, on the

one hand, to investigate whether past models of intratype

diversification of HPV types, built largely on the paradigms

of HPV-16 and 18, but possibly supported by HPV-31 and

35 (Calleja-Macias et al., 2004), would apply to all genital

and cancer associated HPVs, and on the other hand, to begin

the establishment of a data base for future epidemiological,

etiological, pharmaceutical, and vaccination studies should

the genomic diversity affect the virally encoded proteins.

The most important outcome of this study is the low level

of genomic distances (2–4.2%) that were observed for the

various geographic isolates of HPV-53, 56, and 66. This is a

remarkable observation when one considers that the LCR is

under less mutations restrictions and therefore more diverse

in sequence than coding regions of the HPV genome. This

corresponded to a much lower diversity of the E6 and L1

genes for the most distant variants of each type. This finding

may imply that normally only moderate functional differ-

ences exist between variants within each type, which is of

relevance for epidemiological, etiological, pharmaceutical,

and vaccination research. These small intragenic mutations

may, however, lead to functional change, which are

particularly likely to be expected from the highly diverse

HPV-53 E6 variants, one showing eleven amino acid

exchanges. It should be noted that transcriptional as well

as epidemiological studies have identified quite significant

functional differences between variants of HPV-16, which

seem to stem from diversity in the LCR as well as in genes

(Stewart et al., 1996; Yamada et al., 1997; Xi et al., 1997,

1998; Kammer et al., 2000, 2002; Villa et al., 2000; Sichero

et al., 2005).

Phylogenetically, it is remarkable that most HPV types

investigated so far show a similar and very limited diversity

of genomes. This also appears to apply to HPV-53, 56, and

66. Each isolate in the current study was typed in the

respective laboratory of origin by amplification with

consensus primers and hybridization to type-specific probes.

The two laboratories at the Ludwig Institute in Sao Paulo

and at the University of California Irvine, which centrally

evaluated all samples, were able to amplify with separate

type-specific primers almost 100% of the samples. There-

fore, we are confident that no hypothetical highly diverse

variants were lost due to primer design problems.

To date, only four HPV types had given rise to subtypes,

namely HPV-20, 34, 44, and 68 as well as two ape PV (Van

Ranst et al., 1992; de Villiers et al., 2004). We recently

Fig. 1. The intratype diversity of HPV-53. The phylogenetic trees represent the re

control region (LCR). The large tree is based on the UPGMA, the small tree on the

(BR), Hong Kong (HK), Monterrey in Mexico (MX), Edinburgh in Scotland (ED),

(ML). The natural root of this UPGMA tree and those shown in Figs. 2 and 3 are n

the deepest branches.

investigated two of these, HPV-44 and 68, and found

distances of up to 10% of the LCR sequences between the

prototypes and the subtypes (Calleja-Macias et al., 2005).

Each of the subtypes has evolved by a dichotomic split,

giving rise to clusters of closely related variants, each cluster

representing one of the two subtypes. All other HPV types

studied, namely HPV-2, 5, 6, 11, 16, 18, 27, and 57 (Deau et

al., 1993; Ho et al., 1993; Ong et al., 1993; Heinzel et al.,

1995; Stewart et al., 1996; Chan et al., 1997; Yamada et al.,

1997), gave rise to multiple short branches, which we refer

to as ‘‘star-phylogenies’’. Interestingly, our study of HPV-53

and 56 showed a dichotomic branching pattern similar to

that observed for subtypes pointing to evolutionary pro-

cesses that may lead to new HPV subtypes and eventually

types.

The process that gives rise to phylogenetic branches, be it

the short branches typical of many HPV variants, or the

dichotomic branches of subtypes, or the branching events that

lead to new HPV types, are not well understood. Processes

such as simple genetic drift and some positive selection

would possibly suffice to explain these patterns since variants

with favorable properties (such as faster replication, more

efficient infection) would over time predominate in a

population, and less fit variants would arithmetically dis-

appear, without hypothetical competition or selection. The

quantitative limitation to diversification of each HPV type

may be best explained by a genetic bottleneck. For example,

the human population at the origin of the human host species

may have been a very small number of individuals, limiting

the number and therefore also diversity of HPV genomes that

entered the subsequently expanding population.

Lastly, we did observe a limited amount of specificity of

variants for geographic and ethnic origin, but not as strictly

correlative as in the case of HPV-16 and HPV-18 (Ho et

al., 1993; Ong et al., 1993, Stewart et al., 1996; Yamada et

al., 1997). It may be noteworthy that this poorer correlation

was observed with HPV types that are rare in comparison

to HPV-16 and HPV-18. It is not understood why different

HPV types have quite divergent prevalence, but intuitively

one may propose that abundant HPV types may multiply

and spread faster than rare types. This could imply that the

diversification of rare HPV types took place on a different

time scale to that of HPV-16 and 18 that is not consistent

with the spread of human ethnic groups around the world.

For instance, the observation that the HPV-66 prototype

was found abundantly in patients from South Africa and

Scotland could be explained if the HPV-66 genome as it

exists today was already extant more than 100,000 years

ago.

lationship between HPV-53 variants based on a 501 bp segment of the long

neighbor joining algorithm. Samples were obtained from Sao Paulo in Brazil

Cape Town in South Africa (SA), Oklahoma, United States (OK), and Mali

ot known but generated by the computer program at the mid-point between

Fig. 2. The intratype diversity of HPV-56. The phylogenetic trees represent the relationship between HPV-56 variants based on a 549 bp segment of the long

control region (LCR). The large tree is based on the UPGMA, the small tree on the neighbor joining algorithm. Origin of samples as in Fig. 1, three samples

were from Heidelberg, Germany (HE).

J.C. Prado et al. / Virology 340 (2005) 95–104100

Fig. 3. The intratype diversity of HPV-66. The phylogenetic trees represent the relationship between HPV-66 variants based on a 621 bp segment of the long

control region (LCR). The large tree is based on the UPGMA, the small tree on the neighbor joining algorithm. Origin of samples as in Fig. 1, and one sample

was from Morocco (MR).

J.C. Prado et al. / Virology 340 (2005) 95–104 101

Fig. 4. Diversity of the E6 genes (three panels on the right side of the figure) and part of the L1 genes (left side of the figure) in distantly related variants of HPV-53, 56, and 66. Within each panel, the first column

lists the variants, whose relative phylogenetic position can be found in Figs. 1, 2, and 3. The central part of the figure identifies nucleotide exchanges (letters) or maintenance of the sequence of the reference

genome (gray squares). The box on the right side of each panel informs whether the amino acid sequence of the reference clone has been maintained (‘‘prototype’’) or whether and what kind of amino acid

exchanges had occurred.

J.C.Pradoet

al./Viro

logy340(2005)95–104

102

J.C. Prado et al. / Virology 340 (2005) 95–104 103

Materials and methods

Sample collection

In order to explore the intratype phylogeny of HPV-53,

56, and 66, we examined the sequence diversity within the

long control region (LCR) of isolates from six geographi-

cally and ethnically remote cohorts from Brazil (BR), Hong

Kong (HK), Mexico (MX), Scotland (ED, Edinburgh),

South Africa (SA), Oklahoma, and United States (OK) with

inclusion of some samples from Germany (HE, Heidelberg),

Mali (ML), and Morocco (MR). All patients from Hong

Kong were ethnic Chinese, all samples from South Africa

were obtained from Cape Town patients with an exclusive

or predominant African genetic background. All samples

had been collected during ongoing diagnostic and epide-

miological research of cervical smears in studies that are

unrelated to the objectives of our project. No patient was

sampled solely for the purpose of our research.

HPV-53

Genomic segments of HPV-53 were amplified by

polymerase chain reaction (PCR) using the primers HPV-

53-F, 5V-TTTGCATGTTGTTAATAAATAT-3V, and HPV-

53-R, 5V-AGTAGGATTGTTACTTTC-3V, which generated

a 602 bp segment, between the genomic positions 7270 and

15. The amplicons were directly sequenced with the primers

HPV-53-Fa, 5V-TGGTGTCCCTAGGCAGTTGG-3V, and

HPV-53-R. The genomic sequence was established for 501

bp of the 602 bp fragment. The sequences of all 44 isolates

are published with the GenBank access codes AY949045–

AY949087. In order to exclude possible PCR artifacts, all

samples were amplified twice, and both strands were

sequenced twice. The same precaution applied to the treat-

ment of the HPV-56 and HPV-66 samples.

HPV-56

Genomic segments of HPV-56 were amplified by PCR

with the primers 56-F (5V-TGTGTCATTATTGTGG-

CTTTTGTTTTGT-3V) and 56-R (5V-AGCTGCCTTTTA-TATGTACCGTTTTC-3V), which generated a 603 bp

segment between the genomic positions 7323 and 81. The

genomic sequence was established for 549 bp of the 603 bp

fragment. The sequences of all 59 isolates are published

with the GenBank access codes AY949088–AY949146.

HPV-66

Genomic segments of HPV-66 were amplified by PCR

with the 66-F (5V-AACGATAGTTGTGTGTTGTG-3V) and66-R (5V-ACCTGTTTTAGCACGACC-3V), resulting in a

659 bp fragment between genomic positions 7151 and

7810. The genomic sequence was established for 621 of

the 659 bp fragment. The sequences of all 31 isolates are

deposited in GenBank under the access codes AY949147–

AY949176.

Amplification with E6 and L1 consensus primers

The HPV-53, 56, and 66 E6 genes were amplified with

the consensus primers LCR-S (5V-AAGGGAGTAACC-GAAAACGGT-3V) and E7-AS (5V-TCATCCTCCTCCTCT-GAG-3V) (Noda et al., 1998), which generated a 650 bp

segment. The genomic sequence was established for 464 bp

(HPV-53 and 56) and 467 bp (HPV-66), respectively, of

these 650 bp. 450 bp fragments of the L1 genes were

amplified with the consensus primers MY09/MY11 (Bauer

et al., 1991; Bernard et al., 1994), and the genomic sequence

was established for 390 bp of these 450 bp. The sequences

of all isolates are published with the GenBank access codes

DQ007159–DQ007188.

PCR amplification, sequence analysis, and phylogenetic

evaluations

The PCR amplicons were electrophoretically separated on

2% agarose gel, purified with ExoI and alkaline phosphatase

(USB, Cleveland OH), and applied to enzymatic extension

reactions for DNA sequencing using the ABI PRISM

BigDye Cycle Sequencing Kit (AB Applied Biosystems).

Both strands were sequenced with the same forward and

reverse primers as those used for PCR amplification of the

LCR unless stated differently. The sequencing reactions were

purified by ethanol/sodium acetate precipitation and then run

on an ABI Prism 3100 sequencer. The mutations were

analyzed and determined by the ALIGN program at the

GENESTREAM network server (Pearson et al., 1997, http://

www2.igh.curs.fr/bin/align-guess.cgi). The Neighbor-join-

ing and unweighted pair-group method with arithmetic

average (UPGMA) trees were constructed using Mega

version 2.1.

Acknowledgments

This research was supported by funds of the Ludwig

Institute for Cancer Research to L.L.V., by NIH grant ROI

CA-91964, and by funds from the Chao Family Compre-

hensive Cancer Center of the University of California Irvine

to H.U.B., as well as by a Collaborative Research Grant from

UCMEXUS-CONACYT to I.E.C.M., H.A.B.S., and H.U.B.

References

Antonsson, A., Forslund, O., Ekberg, H., Sterner, G., Hansson, B.G.,

2000. The ubiquity and impressive genomic diversity of human skin

papillomaviruses suggest a commensalic nature of these viruses.

J. Virol. 74, 11636–11641.

Bauer, H.M., Ting, Y., Greer, C.E., Chambers, J.C., Tashiro, C.J., Chimera,

J., Reingold, A., Manos, M.M., 1991. Genital human papillomavirus

J.C. Prado et al. / Virology 340 (2005) 95–104104

infection in female university students as determined by a PCR-based

method. JAMA 265, 472–477.

Bernard, H.U., 1994. Coevolution of papillomaviruses and human

populations. Trends Microbiol. 2, 140–143.

Bernard, H.U., Chan, S.Y., Manos, M.M., Ong, C.K., Villa, L.L., Delius,

H., Bauer, H.M., Peyton, C., Wheeler, C.M., 1994. Assessment of

known and novel human papillomaviruses by polymerase chain

reaction, restriction digest, nucleotide sequence, and phylogenetic

algorithms. J. Infect. Dis. 170, 1077–1085.

Calleja-Macias, I.E., Kalantari, M., Huh, J., Ortiz-Lopez, R., Rojas-

Martines, A., Gonzales-Guerrero, J.F., Williamson, A.L., Hagmar, B.,

Wiley, D.J., Villarreal, L., Bernard, H.U., Barrera-Saldana, H.A., 2004.

High prevalence of specific variants of human papillomavirus-16, 18,

31, and 35 in a Mexican population. Virology 319, 315–323.

Calleja-Macias, I.E., Kalantari, M., Allan, B., Williamson, A.L., Chung,

L.P., Collins, R.J., Zuna, R.E., Dunn, S.T., Ortiz-Lopez, R., Barrera-

Saldana, H.A., Cubie, H.A., Villa, L.L., Bernard, H.U., 2005.

Papillomavirus subtypes are natural and old taxa: phylogeny of the

Human papillomavirus (HPV) types 44/55 and 68a/b. J. Virol. 79,

6565–6569.

Chan, S.Y., Delius, H., Halpern, A.L., Bernard, H.U., 1995. Analysis of

genomic sequences of 95 papillomavirus types: Uniting typing,

phylogeny, and taxonomy. J. Virol. 69, 3074–3083.

Chan, S.Y., Chew, S.H., Egawa, K., Grussendorf-Conen, E.I., Honda,

Y., Ruebben, A., Tan, K.C., Bernard, H.U., 1997. Phylogenetic

analysis of the Human papillomavirus type 2 (HPV-2), HPV-27, and

HPV-57 group, which is associated with common warts. Virology

239, 296–302.

Clifford, G.M., Smith, J.S., Plummer, M., Munoz, N., Franceschi, S., 2003.

Human papillomavirus types in invasive cervical cancer worldwide: a

meta-analysis. Br. J. Cancer 8, 63–73.

Deau, M.C., Favre, M., Jablonska, S., Rueda, L.A., Orth, G., 1993. Genetic

heterogeneity of oncogenic human papillomavirus type 5 (HPV5) and

phylogeny of HPV5 variants associated with epidermodysplasia

verruciformis. J. Clin. Microbiol. 31, 2918–2926.

de Villiers, E.M., 1989. Heterogeneity of the human papillomavirus group.

J. Virol. 63, 4893–4898.

de Villiers, E.M., Fauquet, C., Broker, T.R., Bernard, H.U., zur Hausen, H.,

2004. Classification of papillomaviruses. Virology 324, 17–27.

De Vuyst, H., Steyaert, S., Van Renterghem, L., Claeys, P., Muchiri, L.,

Sitati, S., Vansteelandt, S., Quint, W., Kleter, B., Van Marck, E.,

Temmerman, M., 2003. Distribution of human papillomavirus in a

family planning population in Nairobi, Kenya. Sex. Transm. Dis. 30,

137–142.

Franceschi, S., Rajkumar, R., Snijders, P.J., Arslan, A., Mahe, C., Plummer,

M., Sankaranarayanan, R., Cherian, J., Meijer, C.J., Weiderpass, E.,

2005. Papillomavirus infection in rural women in southern India. Br. J.

Cancer 92, 601–606.

Gallahan, D., Muller, M., Schneider, A., Delius, H., Kahn, T., de Villiers,

E.M., Gissmann, L., 1989. Human papillomavirus type 53. J. Virol. 63,

4911–4922.

Heinzel, P.A., Chan, S.Y., Ho, L., O’Connor, M., Balaram, P., Campo,

M.S., Fujinaga, K., Kiviat, N., Kuypers, J., Pfister, H., Steinberg, B.M.,

Tay, S.K., Villa, L.L., Bernard, H.U., 1995. Variation of human

papillomavirus type 6 (HPV-6) and HPV-11 genomes sampled

throughout the world. J. Clin. Microbiol. 33, 1746–1754.

Ho, L., Chan, S.Y., Burk, R.D., Das, B.C., Fujinaga, K., Icenogle, J.P.,

Kahn, T., Kiviat, N., Lancaster, W., Mavromara, P., Labropoulou, V.,

Mitrani-Rosenbaum, S., Norrild, B., Pillai, M.R., Stoerker, J., Syrjaenen,

K., Syrjaenen, S., Tay, S.K., Villa, L.L., Wheeler, C.M., Williamson,

A.L., Bernard, H.U., 1993. The genetic drift of human papillomavirus

type 16 is a means of reconstructing prehistoric viral spread and

movement of ancient human populations. J. Virol. 67, 6413–6414.

Isacson, C., Kessis, T.D., Hedrick, L., Cho, K.R., 1996. Both cell

proliferation and apoptosis increase with lesion grade in cervical

neoplasia but do not correlate with human papillomavirus type. Cancer

Res. 56, 669–674.

Kammer, C., Warthorst, U., Torrez-Martinez, N., Wheeler, C.M., Pfister, H.,

2000. Sequence analysis of the long control region of human

papillomavirus type 16 variants and functional consequences for P97

promoter activity. J. Gen. Virol. 81, 1975–1981.

Kammer, C., Tommasino, M., Syrjanen, S., Delius, H., Hebling, U.,

Warthorst, U., Pfister, H., Zehbe, I., 2002. Variants of the long control

region and the E6 oncogene in European human papillomavirus type 16

isolates: implications for cervical disease. Br. J. Cancer 86, 269–273.

Lazcano-Ponce, E., Herrero, R., Munoz, N., Cruz, A., Shah, K.V., Alonso,

P., Hernandez, P., Salmeron, J., Hernandez, M., 2001. Epidemiology of

HPV infection among Mexican women with normal cervical cytology.

Int. J. Cancer 91, 412–420.

Lorincz, A.T., Quinn, A.P., Goldsborough, M.D., McAllister, P., Temple,

G.F., 1989. Human papillomavirus type 56: a new virus detected in

cervical cancers. J. Gen. Virol. 70, 3099–3104.

Meyer, T., Arndt, R., Beckmann, E.R., Padberg, B., Christophers, E.,

Stockfleth, E., 2001. Distribution of HPV 53, HPV 73 and CP8304 in

genital epithelial lesions with different grades of dysplasia. Int. J.

Gynecol. Cancer 11, 198–204.

Munoz, N., Bosch, F.X., de Sanjose, S., Herrero, R., Castellsague, X., Shah,

K.V., Snijders, P.J.F., Meijer, C.J.L.M., 2003. Epidemiological classi-

fication of human papillomavirus types associated with cervical cancer.

N. Engl. J. Med. 348, 518–527.

Noda, T., Sasagawa, T., Dong, Y., Fuse, H., Namiki, M., Inoue, M., 1998.

Detection of human papillomavirus (HPV) DNA in archival specimens

of benign prostatic hyperplasia and prostatic cancer using a highly

sensitive nested PCR method. Urol. Res. 26, 165–169.

Ong, C.K., Chan, S.Y., Campo, M.S., Fujinaga, K., Mavromara, P.,

Labropoulou, V., Pfister, H., Tay, S.K., ter Meulen, J., Villa, L.L.,

Bernard, H.U., 1993. Evolution of human papillomavirus type 18: an

ancient phylogenetic root in Africa and intratype diversity reflect

coevolution with human ethnic groups. J. Virol. 67, 6424–6431.

Pearson, W.R., Wood, T., Zhang, Z., Miller, W., 1997. Comparison of DNA

sequences with protein sequences. Genomics 46, 24–36.

Sichero, L., Franco, E.L., Villa, L.L., 2005. Different P105 promoter

activities among natural variants of human papillomavirus type 18.

J. Infect. Dis. 191, 739–742.

Stewart, A.C., Eriksson, A.M., Manos, M.M., Munoz, N., Bosch, F.X., Peto,

J., Wheeler, C.M., 1996. Intratype variation in 12 human papillomavirus

types: a worldwide perspective. J. Virol. 70, 3127–3136.

Tawheed, A.R., Beaudenon, S., Favre, M., Orth, G., 1991. Characterization

of human papillomavirus type 66 from an invasive carcinoma of the

uterine cervix. J. Clin. Microbiol. 29, 2656–2660.

Van Ranst, M., Fuse, A., Fiten, P., Beuken, E., Pfister, H., Burk, R.D., g.

Opdenakker, G., 1992. Human papillomavirus type 13 and pigmy

chimpanzee papillomavirus type 1: comparison of the genome

organization. Virology 190, 587–596.

Villa, L.L., Sichero, L., Rahal, P., Caballero, O., Ferenczy, A., Rohan, T.,

Franco, E.L., 2000. Molecular variants of human papillomavirus types

16 and 18 preferentially associated with cervical neoplasia. J. Gen.

Virol. 81, 2959–2968.

Wheeler, C.M., Parmenter, C.A., Hunt, W.C., 1993. Determinants of genital

human papillomavirus infection among cytologically normal women

attending the University of New Mexico student health center. Sex.

Transm. Dis. 20, 286–289.

Xi, L.F., Koutsky, L.A., Galloway, D.A., Kuypers, J., Hughes, J.P.,

Wheeler, C.M., Holmes, K.K., Kiviat, N.B., 1997. Genomic variation

of human papillomavirus type 16 and risk for high grade cervical

intraepithelial neoplasia. J. Natl. Cancer Inst. 89, 796–802.

Xi, L.F., Critchlow, C.W., Wheeler, C.M., Koutsky, L.A., Galloway, D.A.,

Kuypers, J., Hughes, J.P., Hawes, S.E., Surawicz, C., Goldbaum, G.,

Holmes, K.K., Kiviat, N.B., 1998. Risk of anal carcinoma in situ in

relation to human papillomavirus type 16 variants. Cancer Res. 58,

3839–3844.

Yamada, T., Manos, M.M., Peto, J., Greer, C.E., Munoz, N., Bosch, F.X.,

Wheeler, C.M., 1997. Human papillomavirus type 16 sequence variation

in cervical cancers: a worldwide perspective. J. Virol. 71, 2463–2472.