Proc. Nati. Acad. Sci.
USA
Vol. 75, No. 3, pp. 11537-1541, March 1978
Medical Sciences
Characterization of two types of human papillomaviruses in lesions
of epidermodysplasia verruciformis
(malignant transformation/molecular hybridization/antigenic properties/ skin/wart viruses)
GERARD ORTH*, STEFANIA JABLONSKAt, MICHEL FAVRE*, ODILE CROISSANT*,
MARIA JARZABEK-CHORZELSKAt, AND GENOWEFA RZESAt
* Unit6 de Recherches sur l'Etiologie Virale des Cancers humains, LA 147 du Centre National de la Recherche Scientifique, U 140 de l'Institut National de la
Sante et de la Recherche MWdicale, Institut Gustave-Roussy, 94800, Villejuif, France; t Department of Dermatology, Warsaw School of Medicine, Warsaw,
Poland; and * Unit6 d'Oncologie Virale, DMpartement de Virologie, Institut Pasteur, 75015, Paris, France
Communicated by Franqois Jacob, November 17,1977
ABSTRACT Human papillomaviruses (HPVs) found in le-
sions of 11 patients suffering from epidermodysplasia verruci-
formis were compared to HPV type 1 (HPV-1) and HPV type 2
(HPV-2) previously characterized in plantar and common warts,
respctively. Complementary RNAs (cRNAs) to HPV-1, HPV-2,
and viruses obtained from two patients with epidermodysplasia
verruciformis (J.D. HPV and J.K HPV) were used in cRNAkDNA
filter hybridization experiments. No sequence homology was
detected between HPV-1 or HPV-2 DNAs and DNAs obtained
from the 11 epidermodysplasia verruciformis HPV isolates.
Furthermore, with J.D. and J.K. HPV cRNAs, epidermodys-
plasia verruciformis HPV DNAs fell into two groups showing
little, if any, sequence homology. A lower extent of annealing
was observed for the DNAs of some isolates showing a genetic
heterogeneity within each of the two groups. Almost no anti-
genic crossreaction was detected by immunodiffusion and in-
direct immunofluorescence tests, either between epidermo-
dysplasia verruciformis HPVs and HPV-1 or HPV-2 or between
J.D. and J.K. HPVs. Viruses belonging to the same group have
common antigenic properties, but antigenic differences were
observed when two of the viruses sharing only partial DNA se-
quence homology were compared. Viruses related to J.D. HPV
were preferentially associated with flat wart-like lesions of
epidermodysplasia verruciformis and were further found in the
lesions of five patients bearing multiple flat warts. Viruses re-
lated to JK HPV were found in morphologically distinct lesions
(red spots) present in some patients with epidermodysplasia
verruciformis. Thus, we propose to distinguish two other types
of HPVs designated provisionally as HPV type 3 (HPV-3) and
HPV type 4 (HPV-4), with J.D. and J.K. HPVs as prototypes,
respectively. Malignant conversion of some epidermodysplasia
verruciformis lesions is more frequently associated with HPV-4
than with HPV-3 infection.
Epidermodysplasia verruciformis (EV) is a rare disease with
a frequent familial occurrence, characterized by a lifelong
generalized eruption of skin lesions which usually resemble flat
warts; a malignant transformation of some of the lesions is ob-
served in about 25% of the patients with EV, generally on areas
exposed to the sun (1-3). The wart-like lesions are transmittable
(2), and intranuclear papillomavirus particles are regularly
observed in the benign lesions (3-8) but are no longer detected
in the carcinomas (2, 6, 8-11). Genetic (8), immunological (12),
and extrinsic (2, 13) factors play an important role in the
pathogenesis of EV. However, the role of the virus, especially
in the malignant conversion, remains unclear. It was long held
that all human lesions associated with a papillomavirus were
due to the same virus (14). However, the existence of two dis-
tinct types of human papillomavirus (HPV) that showed little,
if any, DNA sequence homology and no antigenic crossreaction,
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1537
has been reported recently (15-17). One of these viruses, HPV
type 1 (HPV-1), was characterized in deep, painful plantar
warts (18, 19), while the other, HPV type 2 (HPV-2), was found
preferentially associated with common warts (16, 17). Studies
on the viruses found in the lesions of 11 patients with EV re-
ported in this paper have led to the characterization of at least
two other types of human papillomaviruses, HPV type 3 and
HPV type 4. This may indicate that some HPVs possess a higher
oncogenic potential than others.
MATERIALS AND METHODS
Virus Purification. Scrapings of the lesions of six patients
with EV (J.D., D.D., E.D., J.G., J.K., and S.M.) were collected
repeatedly and kept at -20° in tissue culture medium. Virions
were extracted from pooled samples of each patient by ho-
mogenization in 50 mM sodium phosphate/0.15 M NaCl, at pH
7.2. After a 10-min centrifugation at 7000 X g at 40 and reex-
traction of the pellets, pooled supernatants were centrifuged
at 100,000 X g. Pelleted viral particles were fractionated by
equilibrium centrifugation in a CsCl gradient and/or by sedi-
mentation in a sucrose gradient (17). HPV-1 and HPV-2 were
purified from plantar warts and hand common warts, respec-
tively, as reported (17). Viral suspensions were kept at -20°,
in 0.1 M NaCl/1 mM EDTA/50 mM sodium phosphate, pH
7.3.
Viral DNA Preparation. DNAs were extracted from virions
by adding a 'ho volume of a 5% Sarkosyl solution to viral sus-
pensions. After a 10-min incubation at 250, the mixtures were
extracted twice with phenol saturated with 0.1 M Tris, pH 8;
DNAs were precipitated with ethanol, then dissolved in 200 Al
of 10 mM Tris/1 mM EDTA, pH 7.9. Form I DNA molecules
of HPV-1 and HPV-2 were prepared as described (17). For
most of the patients studied, viral DNAs were selectively ex-
tracted from the lesions (17). Viral DNA concentration was
determined as described by Raleigh and Davis (20), with the
replicative form (RF) of 4X174 phage DNA as a standard.
HPV-1 DNA (50,ug/ml), HPV-2 DNA (5 ,g/ml), and EV HPV
DNAs (0.2-4 ,g/ml) were kept at -200 in Tris/EDTA.
Electron Microscopy. Viral particle and DNA molecule
preparations (17) were observed with a Siemens Elmiskop 101
electron microscope. Contour lengths of DNA molecules were
measured on electron micrographs, with 4X174 RF DNA
molecules as the length standard (17).
Hybridization Experiments. HPV-1 DNA, HPV-2 DNA,
and DNAs extracted from J.D., J.K., J.G. and S.M. HPVs were
transcribed in vitro into complementary RNAs (cRNAs) with
Escherichia coil RNA polymerase containing sigma factor (a
Abbreviations: HPV, human papillomavirus; cRNA, complementary
RNA; EV, epidermodysplasia verruciformis; RF, replicative form.
1538 Medical Sciences: Orth et al. Proc. Natl. Acad. Sci. USA 75 (1978)
Table 1. Summarized EV case reports
Age, Age at Lesions Malignant HPV
Patienta yrs onset, yrs Typeb Spreadc Activitycd conversions CMIf Remarks types
J.K., 2 37 5 A+B ++++ ++++ +++ (17) - Sister of H.D.; predominance of type B lesions 4
H.D., 9 45 11 A +++ - - - Mother of E.D., D.D., and W.D. 3
E.D., 4 21 7 A ++ ± - - 3
D.D., 9 20 7 A+B +++ +++ + (15) - Predominance of type A lesions 3,4
W.D., d 7 5 A + - - + Warts disappeared after biopsies 3
A.P., 9 24 10 A + + - + Father with abortive EV lesions since early 3
childhood
E.I., 2 17 Early A + ++ - - Father with abortive EV lesions 3
infancy
J.D., d 42 Early A+C ++++ +++ +++ (37) - Nonfamilial case 3
infancy
J.G., d 23 7 A + + - - Nonfamilial case; some hand common warts due 3
to HPV-2g
R.M., d 35 2 A+B +++ +++ + (35) + Nonfamilial case; predominance of type B lesions 4
S.M.9d 21 8 A+B +++ +++ ++ (20) - Nonfamilial case; predominance of type B lesions 4
a Detailed case reports corresponding to the first five patients have already been published (2, 4, 9, 12).
b Type A: flat wart type, usually slightly elevated when localized on hands, dorsum of the feet, and legs. Type B: red spots of irregular shape,
sometimes depigmented in sun-exposed areas. Type C: hyperpigmented, partially confluent, large plaques of irregular shape.
c + Hands and face; ++, hands, face, and some at other locations; +++, widespread; ++++, generalized.
d-, No new lesions for many years; + to ++++, from a few new lesions to extensive spreading at the time of studies.
e +, Premalignant lesions of Bowen's type; ++, Bowen's carcinomas; +++, carcinomas at various sites, sometimes invasive and destructive.
Numbers in parentheses, age at first signs of lesion conversion.
f Depressed (-), lowered (+), or preserved (+) cell-mediated immunity (CMI), as checked by in vitro methods and by cutaneous tests (12).
g Virus type, as deduced from data in this paper.

gift of S. Saragosti, Institut Pasteur, Paris) (17). cRNA-DNA
filter hybridizations under paraffin (21) were performed as
described (17). When cRNAs transcribed from EV HPV DNAs
were used as probes, heat-denatured fetal human thymus DNA
was added to the hybridization mixture (0.5 Ag per assay) to
eliminate any hybridization due to cellular DNA contamination
of viral DNA preparations.
Serological Studies. Guinea pigs were immunized by four
intramuscular inoculations of J.D. or E.D. full particles and J.K.
or S.M. empty particles in complete Freund's adjuvant, at
weekly intervals (about 10-20 ,g of viral protein per inocula-
tion). Animals were bled 2 weeks after the last injection. HPV-1
and HPV-2 guinea pig antisera were those used in previous
studies (17). Antiviral antibodies were assayed by indirect im-
munofluorescence test and immunodiffusion in agarose gels
as described (17).
RESULTS
Patients Studied. This study involved 11 patients with EV
attending the Department of Dermatology in Warsaw School
of Medicine. They were considered as having EV on the basis
of the duration and familial occurrence of the disease, the
morphology and spread or the lesions, the malignant transfor-
mation of some lesions, mainly on the forehead, and a depressed
cell-mediated immune response (Table 1). Five additional
patients bearing multiple flat warts on face and hands for 1-11
years were included in this study; Detailed case reports have
already been published (2, 4, 9, 12), or will be reported else-
where.
Electron Microscopy of EV Virions and Viral DNA Mol-
ecules. In negatively stained preparations, particles purified
from lesions of six EV patients appear as isolated naked particles
showing the characteristic morphology and size of a papillo-
mavirus (diameter ranging from 54 to 58 nm) (17). Circular
supercoiled (form I) or relaxed (form II) DNA molecules with
a length of about 2.5 ,um, typical of HPV DNAs (18), were ob-
served in all viral DNA preparations either extracted from EV
virions or selectively extracted from EV lesions or flat warts.
A similar homogeneous length distribution was observed for
the DNAs from J.D., J.G., J.K., and S.M. HPVs. When expressed
in 4X174 DNA units (5375 base pairs) (22), the lengths of these
DNAs as determined on 200 molecules, were estimated to be
1.442 + 0.027, 1.479 ± 0.021, 1.410 ± 0.031, and 1.474 + 0.025,
corresponding to molecular weights of 5.12 X 106, 5.25 X 106,
5.02 X 106, and 5.22 X 106, respectively. These values are close
to those of HPV-1 DNA (5.23 X 106) or HPV-2 DNA (5.26 X
106) (17).
Sequence Homology between HPV DNAs. Data reported
in Table 2 show that little, if any, annealing was observed after
hybridization of EV or flat wart HPV DNAs with HPV-1 or
HPV-2 cRNAs, except for J.G.2 HPV DNA obtained from
typical common warts; no annealing was observed either be-
tween HPV-1 or HPV-2 DNA and four EV HPV cRNAs. This
demonstrates clearly that HPV DNAs isolated from EV lesions
or flat warts are distinct from HPV-1 and HPV-2 DNAs.
Taking the first characterized EV viruses, J.D. HPV and J.K.
HPV, as prototypical viruses, we may arrange EV HPV DNAs
into two groups showing little, if any, sequence homology be-
tween them (Table 2). The only exception, namely, the results
obtained with D.D. HPV DNA preparations, is discussed below.
J.D. HPV cRNA annealed to comparable extents with J.D.,
H.D., E.D., and W.D. HPV DNAs and to lower extents with
J.G.1, A.P., and E.I. HPV DNAs, all obtained from EV patients
bearing flat wart-like (type A) lesions (Table 1). In addition, J.D.
HPV cRNA annealed to variable extents with the five flat wart
HPV DNAs. No annealing was detected with J.K., S.M., and
R.M. HPV DNAs. Conversely, J. K. HPV cRNA annealed only
with J.K. HPV DNA and, to a lower extent, with S.M. and R.M.
HPV DNAs, i.e., DNAs obtained from patients bearing mainly
irregularly shaped reddish macules (type B lesions) (Table
1).
Data obtained with J.G.1 and S.M. HPV cRNAs (Table 2)
confirm the existence of two groups of EV HPV DNAs. These
cRNAs were transcribed from DNAs that annealed to a lesser
 Medical Sciences: Orth et al. Proc. Natl. Acad. Sci. USA 75 (1978) 1539

Table 2. Extent of sequence homology between the DNAs of Table 3. Characterization of HPVs present in different types of
HPV-1, HPV-2, and HPVs present in EV lesions or flat warts, as EV lesions by cRNA.DNA hybridizations
detected by cRNA-DNA hybridizationa % radioactivity bound on filters after
% radioactivity bound on filters after hybridization DNA on hybridization with different [3HlcRNAs
with [3H]RNAs complementary to different DNAsC filtersb J.D. cRNA J.K. cRNA S.M. cRNA
DNA on J.D. J.K. J.G.1 S.M. 0.3 0.2
filtersb HPV-1 HPV-2 HPV HPV HPV HPV D.D. (leg; A) 54.5
D.D. (neck; A + B) 17.8 16.3 21.4
HPV-1 62.3 0.7 0.1 0.3 0.1 0.4 J.K. (arm; A) -0.6 42.6 NDc
HPV-2 0.3 51.7 1.1 0.1 0.2 0.8 J.K. (forearm; A) 1.8 41.7 ND
EV HPVs S.M. (hand; A) 1.0 ND 46.6
J.D. -0.1 0.3 49.2 0.2 13.3 0.4 S.M. (forearm; A) 4.5 ND 54.1
H.D. 0.1 3.2 66.6 0.8 8.1 0.7 S.M. (back; B) 0.7 ND 52.6
E.D. 0.3 0.2 61.7 0.1 13.2 0.1 J.D. (forearm; A) 44.3 0.8 -0.2
D.D., 1.5 0.2 52.8 0.3 27.2 0.8 J.D. (leg; C) 42.6 1.0 0.1
D.D.2 0.6 0.5 40.9 10.2 NDd 13.7 Human thymus 1.1 0.8 0.2
W.D. 0.2 3.0 71.5 0.3 9.3 0.8 a cRNA-DNA filter hybridization experiments were performed as
J.G., 0.1 0.4 14.2 0.1 58.8 0.1
described in the legend to Table 2, with the same cRNAs.
J.G.2 -0.1 37.6 2.0 0.2 0.5 0.8 b Viral DNAs were selectively extracted from scrapings or biopsies
A.P. 0.1 -0.1 15.9 0.1 10.4 0.5 of lesions collected from patients D.D., J.K., S.M., and J.D. Ana-
E.I. 0.1 0.1 8.1 0.3 10.3 0.3 tomical sites and types of lesions are indicated in parentheses.
J.K. 0.3 -0.2 0.5 52.5 0.5 8.4 c ND, not determined.
S.M. -0.4 0.4 0.8 14.7 0.2 51.9
R.M. 0.5 -0.1 1.0 15.3 0.6 6.1 in Table 3 show that the DNA obtained from type A lesions
Flat wart HPVs annealed only with J.D. HPV cRNA, while DNA extracted
J.W. 0.1 0.3 18.2 0.2 7.9 0.7 from scrapings from an area bearing both type A and B lesions
K.M. 0.1 0.4 68.7 0.6 14.1 -0.5 annealed to comparable extents with the three EV HPV cRNAs.
G.M. 0.2 0.1 65.9 0.1 14.7 -0.3
W.M. 0.1 0.5 28.9 0.4 ND 0.3
These data show that patient D.D. is infected with two viruses;
K.Z. 0.2 0.3 52.8 0.9 ND 0.5 one is of J.D. HPV type and the other one may be either of J.K.
Human 0.1 0.5 0.5 0.1 0.6 0.4 HPV type or a virus related to both types.
thymus The three patients bearing mainly type B lesions also had
type A lesions on their hands and upper limbs, although these
lesions were flatter than -those observed in patient J.D. Data
aFilter hybridization in paraffin (21) was performed at 370, for 17 hr, reported in Table 3 show that type A lesions of patients J.K. and
in 0.30 M NaCl/0.030 M sodium citrate/50% formamide, with about S.M. are not due to a virus belonging to the J.D. HPV group,
50 ng of DNA and 3000 cpm per filter of [3HlcRNA (specific activity, although the low extent of hybridization between J.D. HPV
3-4 X 107 cpm/,gg) (17).
b Viral DNAs were obtained from pooled plantar warts (HPV-1), hand cRNA and one S.M. HPV DNA preparation does not exclude
common warts of a single patient (HPV-2), EV lesions, and flat infection by two viruses. Finally, no difference can be detected
warts. DNAs from D.D. EV HPV were obtained from two samples between the viral DNAs obtained from the type A lesions and
of pooled scrapings of lesions (D.D.1 and D.D.2). DNA from J.G. the hyperpigmented, confluent, large plaques of irregular shape
HPV was obtained from flat wart-like lesions (J.G.1) or common (type C lesions) found on the legs of patient J.D. (Table 1).
warts (J.G.2) of patient J.G. Viral DNAs were either extracted from Antigenic Relationships of HPVs. Antisera raised against
virions (HPV-1, HPV-2, J.D., E.D., D.D., J.G., J.K., and S.M.) or
selectively extracted from lesions (17). HPV-1, HPV-2, J.D. and E.D. HPV full particles, or J.K. and
c [3H]cRNAs were obtained by in vitro transcription with E. coli RNA S.M. HPV empty particles showed high antibody titers against
polymerase of purified form I HPV-1 or HPV-2 DNA molecules and homologous antigens by indirect immunofluorescence (17),
of J.D., J.K., J.G.1, and S.M. HPV DNA molecules extracted from which allows the detection of EV HPV antigens in the nuclei
virions.
d ND, not determined.
of upper Malpighian cells and in keratinized cells (Fig. 1 a and
b), and by an immunodiffusion test (Fig. 2). Hardly any
crossreaction was detected by both techniques (Table 4 and Fig.
extent with the prototypical J.D. and J.K. HPV cRNAs, re- 2), either between HPV-1 or HPV-2 and EV HPVs or between
spectively, as compared to homologous DNAs. Reciprocally, the two prototypical J.D. and J.K. HPVs. One-way crossreac-
the extent of annealing of J.G. and S.M. HPV cRNAs with tions were observed by the immunodiffusion test between
prototypical DNAs and other related DNAs was less than that HPV-1 and HPV-2 (Fig. 2b) and between HPV-2 and J.D. HPV
observed with homologous DNAs. These results demonstrate or the related E.D. and D.D. (type A lesions) HPVs (Fig. 2c)
a genetic heterogeneity within the two groups of EV HPV after increased times of diffusion and with undiluted antisera.
DNAs. When used undiluted, J.K. HPV antiserum gave a precipitin
For almost all EV patients, at least two DNA preparations line with J.D. HPV and the related viruses that seems com-
were obtained from scrapings of lesions collected at different pletely distinct from the line found with J.D. HPV antiserum
times and yielded similar results. However, conflicting results (Fig. 2c).
were observed with the DNA preparations obtained from J.D. HPV antiserum gave a positive stain with lesion sections
scrapings of lesions of patient D.D. (Table 2). Several prepa- from seven patients infected with a virus related to J.D. HPV
rations, including D.D.1, annealed only with J.D. and J.G. HPV (shown by cRNA-DNA hybridization experiments). In contrast,
cRNAs, while one preparation (D.D.2) annealed both with J.D. no reaction was observed with lesion sections (including D.D.
HPV cRNA and, to a lesser extent, with J.K. and S.M. HPV type B lesions) from the three patients infected with a virus
cRNAs. Typical type A lesions were predominant in this patient, related to J.K. HPV. Opposite results were obtained with J.K.
but type B lesions were also observed on the neck. Data reported HPV antiserum and sections of the same lesions. Antisera raised
1540 Medical Sciences: Orth et al. Proc. Natl. Acad. Sci. USA 75 (1978)

FIG. 1. Detection of viral antigens in the superficial layers of EV lesions by indirect immunofluorescence. Frozen sections of (a) J.D. and (b)
S.M. lesions were incubated either with (a) antiserum against J.D. HPV or (b) antiserum against S.M. HPV at a %/s60 dilution and, after washing,
with fluorescein-labeled anti-guinea pig IgG rabbit globulins. (X 160.)

against J.D. and E.D. HPVs, whose DNAs were closely related
(Table 2) exhibited the same reactivity (Table 4); in particular,
they had similar titers by immunodiffusion and immunofluo-
rescence with J.D., E.D., and D.D. (type A lesions) HPV anti-
gens. However, antigenic differences were detected between
J.K. and S.M. HPVs, which have only partial DNA sequence
homology. J.K. and S.M. HPV antisera gave lower antibody
titers when tested on heterologous lesion sections by immu-
nofluorescence (Table 4), and a dramatic difference (320
compared to 1) when assayed by immunodiffusion with J.K.
HPV as antigen, as shown in Fig. 2d, where the precipitin band
given by undiluted S.M. HPV antiserum is hardly detectable.
Finally, the reaction of S.M. HPV antiserum with a section from
a J.D. lesion, in contrast to immunodiffusion and reciprocal
immunofluorescence experiments (Table 4), may indicate ei-
ther that patient S.M. is infected by two viruses or that J.D. and
S.M. HPVs have a common antigenic determinant.

DISCUSSION
cRNA-DNA hybridization experiments and serological studies
demonstrate that HPVs found in the lesions of 11 patients with
EV are distinct from the HPVs previously characterized in deep
plantar warts (HPV-1) and in common warts (HPV-2) (17). This
is in agreement with the serological data of Pass et al. (23).
Furthermore, the data reported in this paper permit the ar-
rangement of EV HPVs into two groups whose prototypical
viruses, J.D. HPV and J.K. HPV, are as distinct from one an-
other as they are from HPV-1 or HPV-2. Thus, J.D. HPV and
J.K. HPV define two new types of HPVs, HPV type 3 (HPV-3)
and HPV type 4 (HPV-4).
HPVs related to J.D. HPV were found in eight patients with

Table 4. Serological comparison of HPVs of different origin by

indirect immunofluorescencea
Antibody titers with sections of
Antiserab Plantar Common J.D. J.K. S.M.
against wart wart lesion lesion lesion
HPV-1 2560 <20' <20 <20 <20
FIG. 2. Double-immunodiffusion patterns of different HPVs HPV-2 <20 2560 <20 <20 <20
against specific guinea pig antisera. Antisera against HPV-1, HPV-2, J.D. HPV <20 <20 640 <20 <20
J.D. HPV, J.K. HPV, and S.M. HPV were those used for immu- E.D. HPV <20 <20 640 <20 <20
nofluorescence experiments (Table 4). The first four antisera gave J.K. HPV <20 <20 <20 640 160
a precipitin line with homologous antigens until the 1/320, 1/640,
1heo,
S.M.HPV <20 <20 40 80 640
and 1/320 dilution, respectively. No S.M. HPV antigen was available
for immunodiffusion experiments. The central wells contained pu- a Frozen tissue sections were successively incubated for 30 min at 370,
rified (a) HPV-1, (b) HPV-2, (c) D.D. (type A lesions) HPV, and (d) in the presence of serial dilutions of the sera and, after washing, in
J.K. HPV viral suspensions. (a-c) Peripheral wells contained sera the presence of fluorescein-labeled anti-guinea pig IgG rabbit
against (1) HPV-1, (2), J.K. HPV, (3) J.D. HPV, (4) S.M. HPV, (5) globulins (17). Titers were expressed as the reciprocal of the highest
HPV-2, and (6) phosphate-buffered saline. Sera were used undiluted, dilution of the sera giving a medium intensity staining of the viral
except for antisera against (a) HPV-1, (b) HPV-2, and (c) J.D. HPV, capsid antigens.
used at a 1/80 dilution. (d) Peripheral wells contained antisera at two b Antisera were prepared by immunization of guinea pigs with HPV-1
different dilutions: antiserum against J.D. HPV, (1) undiluted and obtained from a single plantar wart (G 260), HPV-2 obtained from
(2) 1/5 dilution; antiserum against J.K. HPV, (3) 1/80 and (4) 1/20 dilu- pooled common warts of a single patient (G 206), and viral particles
tion; antiserum against S.M. HPV, (5) undiluted and (6) 1/1o dilution. obtained from EV lesions of patients J.D. (G 280), E.D. (G 281), J.K.
Diffusion was allowed to proceed for 2 or (b) 5 days. (G 251), and S.M. (G 264).
 Medical Sciences: Orth et al.
EV and in five patients bearing multiple flat warts. However,
cRNA-DNA hybridization experiments reveal only partial se-
quence homology for three of these EV viruses and two of the
flat wart viruses. Four patients with EV were found to be in-
fected by viruses related to J.K. HPV, but molecular hybrid-
ization and immunological data indicate a substantial genetic
heterogeneity among the isolates. The genetic heterogeneity
inside each group may result from a genetic variability of the
virus favored by multiple reinfections occurring during this
lifelong disease. This hypothesis is supported by the hetero-
geneity found for an HPV-3 isolate obtained from an additional
EV patient, as shown by restriction enzyme analysis of the viral
DNA (0. Croissant and F. Pass, unpublished results), and by
the existence of up to 8% of sequence heterogeneity in a HPV-2
DNA preparation obtained from a single patient (17). However,
further restriction enzyme analysis, molecular hybridization
experiments, and serological studies are required to exclude the
existence of additional HPV types.
At the present time, viruses related to J.D. HPV and J.K. HPV
may be considered as belonging to type 3 and type 4, respec-
tively. This is further warranted since different biological
properties seem to be associated with the two groups of viruses
(Table 1). HPV-3 was always found in lesions of patients bearing
disseminated flat warts of long duration and of EV patients
bearing flat wart-like lesions, as well as in the brown, confluent,
large plaques (type C lesions) of patient J.D. HPV-4 was always
found in irregularly shaped, reddish macules (type B lesions)
and also in the flat wart-like lesions observed in these patients.
Furthermore, while a malignant conversion was observed in
all patients infected with HPV-4, malignant conversion was
observed only in two of the eight patients with EV infected with
HPV-3, one of them (D.D.) being infected also by HPV-4 and
the other one showing type C lesions.
Data reported here suggest that the role of the virus in the
pathogenesis of EV may have to be reconsidered. Genetic
factors have been thought so far to play the major role in this
disease. The genetic constitution of the host may result in (i) a
higher susceptibility of keratinocytes to HPV-3 or HPV-4; (ii)
some deficiency in the immune functions (Table 1), resulting
in a generalized and lifelong infection (12); or (iii) a decreased
capacity of the host cell to repair UV-damaged DNA, a possible
factor in malignant conversion (13). However, the role of the
virus may well be causative, since, as we show here, EV can be
associated with particular types of HPVs and the clinical aspect
of the lesions and the probability of a malignant conversion
seem to depend on the virus type. In addition, the genetic
variability detected within each group of virus should favor the
formation of variants responsible for the polymorphism of the
lesions or of variants with increased oncogenic potential. A role
of the virus in the production of EV carcinomas would strongly
be suggested by the detection of viral genomes in these tu-
mors.
In any case, our data indicate that at least one HPV may have
oncogenic potential, as do rabbit Shope papilloma virus and
bovine alimentary tract papilloma virus (W. F. H. Jarrett, et
al., unpublished data), and may play an initiating or promoting
role in the origin of some human cancers (16). In this respect,
EV, involving as it does both specific viruses and genetic, im-
munological and extrinsic factors, constitutes an outstanding
model system.
Note Added in Proof. Recent restriction enzyme analysis with Hae
III endonuclease confirms that HPV-1, HPV-2, and J.D. and J.K. HPVs
Proc. Nati. Acad. Sci. USA 75 (1978) 1541
are distinct viruses. It further shows that, in contrast to J.D. HPV DNA,
J.K. HPV DNA behaves as a highly heterogeneous DNA molecule
population. Finally, it clearly shows that HPV-2, J.D. HPV, and J.K.
HPV are distinct from a virus recently characterized and designated
HPV-4 by Gissmann et al. (15), while HPV-1 corresponds to their
closely related HPV-1, HPV-2, and HPV-3 (unpublished).
We are especially grateful to Dr. S. Obalek for his most efficient
collaboration in collection of lesion samples. We thank Drs. P. Shel-
drick, M. Yaniv, and F. Breitburd for critical reading of the manuscript.
We also thank C. Dauguet for technical collaboration, N. Jibard and
D. Fortin for excellent assistance, and D. Cany for preparation of il-
lustrations. This investigation was supported by Grants A.T.P. 28.76.60.
and C.R.L. 76.4.100.1 from the Institut National de la Sante et de la
Recherche Medicale and A.T.P. A 651-1921 from the Centre National
de la Recherche Scientifique, and by Grant PR6. 26/76 from the Polish
Government for Cancer Research.
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