MICROBIOLOGICAL REVIEWS, June 1982, p. 191-207 Vol. 46, No.

2
0146-0749/8V020191-17$02.W0O

Animal Papillomaviruses
WAYNE D. LANCASTER'* AND CARL OLSON2
Departments of Obstetrics and Gynecology and Pathology, Georgetown University Medical Center,
Washington, D.C. 20007,1 and Department of Veterinary Science, University of Wisconsin, Madison,
Wisconsin 537062
INTRODUCTION ....................................... 191
CLASSIFICATION ....................................... 192
PHYSICOCHEMICAL PROPERTIES ........................................ 192
PAPILLOMA TYPES ....................................... 193
PATHOGENESIS ....................................... 193
Mkroscopic Changes ...................................... 193
Natural and Experinentad Infections ........................................ 193
Cottontail rabbit (Shope) papi viornavus (CRPV) ................... ............. 194
Bovine papilomavirus (BPV) .......................................1 94
(i) BPV types 1 and 2 ........... ............................ 194
(i) BPV type 3 ............................................ 195
(iii) BPV type 4 ............ ................................ 195
(iv) BPV type 5 ....................................... 196
Other papillomaviruses ....................................... 1%
Infectivity in Cel Culture ........... ............................ 196
Human papiMlomavirus .......... ............................. 196
BPV ............................................ 197
CRPV ....................................... 197
IMMUNITY ....................................... 198
CRPV ....................................... 198
BPV ....................................... 199
Human PapiDlomavirus ....................................... 200
EVOLUTIONARY RELATEDNESS . ....................................... 200
Antigenic Cross-Reactivity ........... ............................ 200
Conserved Polynuckotide Sequences ........................................ 201
MOLECULAR BIOLOGY ....................................... 202
Lack of Integratfon ....................................... 202
Cloning Vector ....................................... 202
Transcription ....................................... 203
CONCLUDING REMARKS ........... ............................ 203
LITERATURE CITED ....................................... 204

INTRODUCTION transmitted in 1898 (105) and equine skin warts

Infectious papillomatosis (warts) are conta-
gious in the animals in which they naturally
occur. These lesions may be regarded as either
hyperplasia or benign neoplasms since they do
not metastasize and kill the host. However, in at
least three species of lower animals, papillomas
may undergo malignant transformation to carci-
noma. In some instances virus can produce
tumors in alien hosts both experimentally and by
natural infection. This oncogenic potential
makes the papillomaviruses an important group
of tumor viruses, but surprisingly little is known
about their biology.
Warts (or verrucae) have been recognized in
animals for centuries. For example, equine pap-
illomas were described in the 9th century A.D.
by a stablemaster of the Caliph of Bagdad (38).
Experimental transmission of warts did not oc-
cur until the end of the 19th and beginning of the
20th century, with canine oral papillomas being
191
in 1901 (18). The viral nature of warts was
inferred by transmission experiments of sterile
filtrates of human warts in 1907 (26) and was
subsequently demonstrated for a number of ani-
mal papillomaviruses. Although the cottontail
rabbit papilloma was the first to be examined in
depth (147), the transformation of papilloma to
squamous carcinoma, which was noted over 45
years ago (144), is still not completely under-
stood.
Until recently, the papillomavirus literature
dealt mainly with transmission experiments, vi-
rus ultrastructure and chemical composition,
and pathological description of lesions. This is
understandable since the main impediment to
analysis of these viruses has been the lack of a
reproducible cell culture system permissive for
their replication. However, the papillomavirus
field has enjoyed renewed interest due to recent
advances in the techniques of molecular biology,
especially molecular cloning, which have made
192 LANCASTER AND OLSON
these viruses more amenable to experimenta-
tion. Although the molecular aspects of papillo-
mavirus research is in its infancy, much of the
information thus far obtained is intriguing, and
some of the wart viruses recently have been
shown to possess unusual biological properties
which would appear to make them unique in the
field of tumor virology. The purpose of this
review is to summarize these findings and to
reconsider the older literature.
CLASSIFICATION
The papillomaviruses represent genus A of the
family Papovaviridae; the polyomaviruses con-
stitute genus B (111). These genera differ in size,
with the papillomaviruses having a larger capsid
and genome. The genera also differ in their
biological properties. In general, the papilloma-
viruses are relatively inactive in vitro and pro-
duce benign cutaneous and mucosal tumors in
their hosts as a result of natural infection. The
polyomaviruses, on the other hand, are not
tumorigenic under conditions of natural infec-
tion and are capable of producing a wide variety
of effects in cultured cells.
Recently, papillomavirus nomenclature has
been revised following the discovery of multiple,
minimally related viruses infecting a given spe-
cies. Most notable are the human papillomavi-
ruses (HPVs) for which eight types have been
identified (for review see 58). Although these
individual viruses will not be described in detail,
the group will be discussed with respect to
infectivity in cell culture and host immunity. The
proposed nomenclature changes provide for
classification of viruses into types or subtypes
based on polynucleotide sequence homology.
To be classified as a new virus type, a maximum
of 50%o polynucleotide sequence homology with
other classified viruses should exist in conjunc-
tion with significant serological deviations in
reciprocal assays. Those viruses with >50% but
MICROBIOL. REV.
<100% deoxyribonucleic acid (DNA) sequence
homology are subtypes (27).
Table 1 lists a number of characterized animal
papillomavirus and one avian virus infecting the
chaffinch (Fringilla), which is the only nonmam-
malian papillomavirus thus far described. Virus-
es have been detected in papillomas from a
variety of other mammalian species including
cutaneous lesions of the opossum, dog, and pig,
oral lesions in the coyote and rabbit, and papillo-
mas on the udders of goats (see reference 4).
These viruses have not been well characterized
and will not be considered further.
A virus isolated from hamsters has also been
classified a member of the papillomavirus genus
(111). However, this virus, although it matures
in epithelial cells like papillomaviruses, has a
smaller icosahedral capsid (45 nm) and a smaller
double-stranded, covalently closed circular
DNA genome (3 x 106 daltons) (13) and should
not be classified a papillomavirus but rather a
member of the polyomavirus genus.
PHYSICOCHEMICAL PROPERTIES
Papillomaviruses are naked icosahedral cap-
sids 50 to 55 nm in diameter. Human, cottontail
rabbit, and the chaffinch papillomaviruses have
72 capsomers, with the rabbit having a left-hand
surface lattice and the human and chaffinch
viruses having a right-hand lattice (45, 76, 130).
Both "full" and "empty" particles can be isolat-
ed from papillomas, with full particles having
sedimentation coefficients of 2% to 300S and
empty particles having sedimentation coeffi-
cients of 168 to 172S (30). Full particles have a
buoyant density in CsCl of 1.34 g/ml, and empty
particles have a buoyant density of 1.29 g/ml
(30). Tubular variants of papillomavirus have
been observed in some preparations (24).
The papillomavirus genome is a double-
stranded, covalently closed circular DNA mole-
cule (16, 29) with molecular weights ranging

TABLE 1. Animal papillomaviruses

Virus Host Site Histology Reference
Bovine papillomavirus
Type 1 Cattle Cutaneous Fibropapilloma (90)
Type 2 Cattle Cutaneous Fibropapilloma (90)
Type 3 Cattle Cutaneous Papilloma (136)
Type 4 Cattle Alimentary tract Papilloma (19)
Type 5 Cattle Teat Papilloma (20)
Cottontail rabbit (Shope) papillomavirus Cottontail rabbit Cutaneous Papilloma (147)
Equine papillomavirus Horse Cutaneous Papilloma (49)
Canine oral papillomavirus Dog Oral mucosa Papilloma (21, 137)
Sheep papillomavirus Sheep Cutaneous Fibropapilloma (52)
European elk papiliomavirus Elk Cutaneous Fibropapilloma (113)
Deer fibromavirus Deer Cutaneous Fibroma (149, 153)
Mastomys natalensis papiliomavirus Multimammate mouse Cutaneous Papilloma (116)
Chaffinch papillomavirus Bird Cutaneous Papillomas (103, 130)
VOL. 46, 1982
from 4.5 x 106 for bovine types 3 and 4 to 5.5 x
106 for the canine oral virus (19, 136, 137). The
viral genome is sufficient to code for about
300,000 daltons of protein. The guanine-plus-
cytosine content of papillomavirus DNA ranges
from 41 mol% for HPV to 50 mol% for Mas-
tomys natalensis virus, and nearest-neighbor
analysis indicates a similarity to host species
DNA (30, 115, 116). HPV type 1 (HPV-1) DNA
has adenine-plus-thymine-rich areas (46) as well
as two regions of inverted repeats each repre-
senting about 2% of the viral genome (53).
Restriction endonuclease analysis of HPV-1
DNA from virions indicates partial methylation
at the HpaII recognition sequence (33).
Virion protein represents 88% of the mass of
the particle (29, 74), and up to 10 polypeptides
have been resolved by dodecyl sulfate poly-
acrylamide gel electrophoresis (43, 44, 90, 150).
The major structural polypeptides have molecu-
lar weights in the range of 50,000 to 60,000.
Minor high-molecular-weight polypeptides have
been observed in some preparations, but their
detection has been variable and may represent
purification artifacts or aggregates. Polypeptides
in the molecular weight range of 30,000 to 40,000
are consistently resolved in many virus prepara-
tions. Four low-molecular-weight polypeptides
closely associated with viral DNA have been
resolved and are similar to cellular histones. No
serological cross-reactivity among major capsid
proteins was detected in papillomaviruses of
different species (78, 98), and no DNA sequence
homology was detected between selected mem-
bers of this group (29, 90) when standard assays
were employed.
PAPILLOMA TYPES
Based on host response, papillomaviruses can
be subdivided further as to tissue tropism and
the histology of the lesions. One group, com-
posed of equine, chaffinch, bovine types 3 and 5,
and the cottontail rabbit papillomavirus, induces
neoplasia of cutaneous stratified epithelium,
whereas the canine oral and bovine papilloma-
virus type 4 constitutes a second group that
primarily induces hyperplasia of either normal
nonstratified squamous epithelium or metaplas-
tic squamous epithelium. The third group in-
cludes bovine types 1 and 2, sheep, and Europe-
an elk papillomavirus, which induce not only a
cutaneous papilloma but, in addition, an under-
lying fibroma of connective tissue (fibropapil-
loma). The deer fibromavirus represents the
fourth group, with the production of fibromas
with a minimally hyperplastic cutaneous epithe-
lium. In all histological types, including deer
fibromas, intact virus can be demonstrated only
in the outer layers of keratinizing cells of the
epithelium (23, 49, 153).
ANIMAL PAPILLOMAVIRUSES 193
PATHOGENESIS
Microscopic Changes
Productive papillomavirus infection of epithe-
lial cells results in hyperplasia of cells in the
spinous layer (acanthosis). These cells show an
increase in the size and number of desmosomes
and tonofibrils, whereas other epithelial cells
show degenerative changes with loss of tonofi-
brils, detachment of desmosomes, focal nuclear
atypia, and cytoplasmic vacuolization. Towards
the upper layers of the epithelium, these changes
are more pronounced. In the granular layer,
nuclear degeneration is evident, with margin-
ation and condensation of chromatin. Electron
microscopic analysis reveal virions in crystalline
array in nuclei of degenerated cels in the kera-
tinizing layer. In contrast are the fibromas in-
duced by the deer fibromavirus occurring on the
white-tailed deer (149). These fibromas show
extensive fibroblastic proliferation, with the
cells arranged in whorls and irregular patterns.
The overlying epithelium exhibits mild acantho-
sis.
Fibropapillomas exhibit a combination of
these effects. From infectivity studies, the first
reaction of the skin is fibroblastic stimulation in
the dermis accompanied by an inflammatory
response with congestion, edema, and leukocyte
infiltration. About 1 week after infection, the
inflammatory reaction subsides but the fibro-
blastic stimulation continues followed by inva-
sion of the papillary layer of the dermis by
fibroblasts. The epithelium overlying the area of
dermal hyperplasia begins to proliferate and
shows acanthosis and hyperkeratosis.
Natural and Experimental Infections
Natural infection by papillomavirus is general-
ly limited to the skin or mucous membranes of
the host species depending on the tissue tropism
of the virus. However, canine oral papilloma-
virus infection is not limited to the oral cavity
and has been identified in lesions of conjunctival
epithelium, eyelid, and skin around the nose and
mouth (21). Fibromas in the lungs of deer have
been reported in animals with extensive cutane-
ous lesions; however, the presence of virus in
the lung lesions was not verified (77).
In squamous papillomas of viral origin, virus
generally is detectable using immunological
methods or electron microscopy. In contrast,
when the papilloma has undergone malignant
transformation to carcinoma, the structural in-
tegrity of the virus is lost and virus or viral
structural antigen is not present. Similarly, tu-
mors induced in unnatural hosts as well as cell
lines established from these tumors contain no
detectable mature virus. This phenomenon of
virus masking is intriguing and may be related to
194 LANCASTER AND OLSON
the process of differentiation of epithelial cells
since virions are almost always confined to
mature cells in the outer keratinizing epithelium
of a papilloma.
Cottontail rabbit (Shope) papillomavirus
(CRPV). Cottontail rabbit (Shope) papilloma-
virus (CRPV) produces tumors which either
regress, persist, or undergo transformation to
carcinomas. The tumors occur in nature as a
spontaneous enzootic disease of wild cottontail
rabbits (Sylvilagus floridanus) predominately in
the high plains of the midwestern United States
even though the susceptible host is widespread
throughout the country. However, the disease
does occur in Kansas cottontail rabbits imported
to Whelbley Island in Puget Sound, Washington
(39). Factors responsible for this curious distri-
bution are unknown but could be related to
vector range, host susceptibility, environmental
influences, or to factors which modify the im-
mune status of the host.
Experimentally, CRPV produces cutaneous
papillomas in cottontail rabbits, jackrabbits,
snowshoe rabbits, and domestic rabbits (Orycto-
lagus cuniculus) (7). Snowshoe rabbits and jack-
rabbits produce infectious virus, whereas papil-
lomas induced in domestic rabbits rarely contain
virus (148). In some instances, virus structural
antigens have been detected in domestic rabbit
papillomas, but not consistently (110, 118, 129,
151). This lack of infectious virus production
appears to be mediated by the host genotype.
The importance of genetic factors in malignant
transformation is also reflected by the threefold
higher frequency of squamous carcinoma in vi-
rus-induced papillomas of domestic rabbits as
compared to cottontail rabbits (144, 152). The
complex interaction between virus production,
genetic background, and malignant transforma-
tion is a fascinating problem which undoubtedly
will receive much attention.
No cell culture system is available for CRPV
productive infection or transformation. Since
the disease is sporadic in the wild, only limited
supplies of CRPV are available for study. This
problem has now been overcome as a result of
successful cloning of the viral genome (155),
which should allow for further experimentation
on the system since viral DNA is infectious for
the domestic rabbit (63-65).
Bovine papillomavirus (BPV). Bovine papillo-
mavirus (BPV) comprises at least five distinct
virus types, each producing a specific type of
lesion. In addition to their lack of immunological
cross-reactivity, these viruses are readily distin-
guished by characteristic restriction endonucle-
ase cleavage patterns of their genomes and de-
gree of polynucleotide sequence homology
(Table 2).
(i) BPV types 1 and 2. BPV types 1 and 2
MICROBIOL. REV.
(BPV-1, -2) both hemagglutinate mouse erythro-
cytes but not erythrocytes from humans, oxen,
rabbits, rats, and a number of other species, and
the agglutination is not inhibited by pretreatment
of cells with neuraminidase (42). Although these
viruses are closely related (50%o DNA sequence
homology), they differ in their efficiency of
hemagglutination, with BPV-1 requiring about
fivefold fewer virions to agglutinate a standard
suspension of erythrocytes (90). No major dif-
ferences were noted in the molecular weights of
their structural polypeptides in sodium dodecyl
sulfate-polyacrylamide gels, and the restriction
endonuclease cleavage maps of their genomes
are colinear with respect to a unique cleavage
site present on both DNAs (86).
BPV-1 and -2 have a wide tissue range in
cattle. Experimental inoculation of calves with
BPV-1 and -2 results in polyploid tumors of the
urinary bladder and genital mucosa (15, 121,
123). The viruses also can induce meningiomas
of the brain after intracranial inoculation irre-
spective of the immune status of the animal to
the virus (54). Meninges in the terminal spinal
chord and cauda equine, peripheral nerves,
tongue, abomasum, rumen, small intestine, tes-
ticle, and kidney all show moderate fibroblastic
stimulation in response to experimental infec-
tion, whereas muscle, marrow, cornea, lymph
node, and oral mucosa are refractory (54).
BPV-1 and -2 have the broadest experimental
host range exhibited by the papillomavirus ge-
nus. Fibroblastic tumors can be induced in the
hamster which show no age dependence of the
animal (23). Within 9 months o1 inoculation,
fibromas and/or fibrosarcomas can develop in
the subcutis, meningiomas can develop in the
brain, and chondromas can develop in the ear
(141, 142). Tumors of the subcutis can metasta-
size to internal organs, especially the lungs. One
strain of inbred mouse (C3H/eB) (11), rabbits
(14), and pikas (138) develop fibromas at the
site of inoculation. When assayed by molecular
hybridization, hamster fibromas and calf menin-
giomas have been shown to contain large quanti-
ties of viral DNA sequences (100 to 700 copies
per cell) (92).
TABLE 2. Sensitivity of different BPV DNAs to
restriction endonucleases
Viral No. of cleavage sites Mol wt Ref-
DNA BamHl EcoRI Hindll HindIII (x 10') er-
BPV-1 1 1 3 1 5.0 (86)
BPV-2 2 0 4 1 5.0 (86)
BPV-3 3 1 4 4 4.5 (136)
BPV-4 1 2 3 3 4.5 (19)
BPV-5 1 0 4 4 5.1 (20)
VOL. 46, 1982
The horse is also susceptible to experimental
infection by BPV-1 and -2 (119). The tumor
presents a fibrosarcoma-like histology and has a
tendency to regress. A common naturally occur-
ring horse tumor, termed sarcoid (67), is similar
in histology to experimentally induced tumors.
The natural disease is characterized by a gener-
ally lifelong course and slow growth, with the
animal being susceptible to infection by BPV
(140). Although the response of the host to
experimental infection shows major differences
from the natural disease (tumor regression, im-
munity to BPV, rapid growth), viral sequences
have been detected in high copy number by
molecular hybridization in both experimental
and natural cases of the disease (93, 94). These
results, along with the occurrence of the disease
in epizootics (139), strongly indicates a BPV
etiology. The differences between experimental
and naturally acquired sarcoids could be due to
the amount of virus to which the animal is
exposed or the mode of transmission.
Jarrett et al. (70) indicated that BPV-1 and -2
are predominately associated with paragenital
lesions and cutaneous fibropapillomas, respec-
tively. However, in a survey of 10 individual
fibropapillomas occurring on the head, neck,
and flank, four were identified as BPV-2 and the
remainder were identified as BPV-1 (90). These
lesions were collected over a period of years
from various geographic locations. In another
study, Pfister (133) obtained similar results and,
in addition, found both BPV-1 and -2 in
fibropapillomas of the udder.
(il) BPV type 3. BPV-3 was isolated from
cutaneous papillomas lacking a fibrous compo-
nent (136). BPV-3-containing papillomas were
collected in Australia, but Pfister et al. (136),
although surveying only a limited number of
animals with warts, were unable to detect BPV-3
in papillomas collected in southern Germany.
The virus is transmissible to the skin of cattle
but not to conjunctiva or other sites. A papillo-
mavirus has been demonstrated in similar le-
sions occurring in the United States (6). This
virus, which is immunologically distinct from
BPV-1 and -2, may be similar to BPV-3. Infec-
tion can occur concurrently with fibropapillo-
mas. The papillomas had a tendency to persist,
and in some animals whose papillomas re-
gressed, histologically similar papillomas later
developed. No antibodies to the virus could be
detected by immunodiffusion while substantial
titers were present against virus from fibropapil-
lomas in sera of animals with both lesions.
Vaccination against the disease with papilloma
homogenates had no influence on the course or
frequency of the disease. This is in sharp con-
trast to fibropapilloma-derived vaccines which
are effective prophylactically (124). Transmis-
ANIMAL PAPILLOMAVIRUSES 195
sion experiments in other species known to be
susceptible to BPV-1 and -2 were unsuccessful.
Especially notable was the inability to transmit
the disease to calves.
(iii) BPV type 4. Alimentary tract papillomato-
sis of cattle is a widespread disease in the United
Kingdom, with an incidence of about 19% (71).
The disease occurs as true epithelial papillomas
and has been shown to be caused by a virus
classified as BPV-4 (19). A striking feature of the
disease is the presence of papillomas at the same
sites as squamous carcinomas. Although alimen-
tary tract papillomatosis is widespread, the oc-
currence of alimentary tract carcinoma is sharp-
ly confined to the upland areas of Scotland and
northern England (69). Necropsy studies of cat-
tle in low-incidence areas indicate that the pres-
ence of three or more papillomas in a given
animal is rare; however, 80%o of the animals in
high cancer areas have papillomas and 55% of
those have multiple papillomas. Furthermore,
90o of the cattle with squamous carcinoma of
the alimentary tract have multiple papillomas
(69).
Experimentally the soft palate of calves is
uniformly susceptible to BPV-4 infection but
the skin is refactory (19). Viral DNA recovered
from experimentally induced lesions has restric-
tion enzyme cleavage patterns identical to the
inoculated virus (70). Evidence that BPV-4 is
associated with alimentary tract carcinomas is
the complete concordance of the sites of papillo-
mas and carcinoma in which all stages of trans-
formation of papilloma to carcinoma can be
found (68). However, in some instances, carci-
noma can be found without histological evidence
of papilloma. More convincingly, Campo et al.
(19) have identified BPV-4 DNA sequences by
molecular hybridization in malignancies in
which no intact virus was detected.
The area of high cancer incidence corresponds
with grazing areas in which there is severe
infestation by bracken fern (69). Bracken is
tumorigenic in laboratory animals and has been
associated with acute bracken poisoning in high-
incidence areas. Although the carcinogenic com-
pound has not been identified, its effects appear
to be cumulative with chronic feeding.
Another interesting feature of the findings of
Jarrett et al. (69) is the presence of urinary
bladder tumors in about 30% of the animals in
high cancer areas. Similar bladder tumors have
been associated with chronic enzootic hematu-
ria, a disease localized in certain areas of the
world, which is characterized by progressive
neoplasia of epithelial and mesenchymal ele-
ments (120). These tumors have glandular and
squamous metaplasia of the epithelium, as well
as hemangiomatous areas and fibromatous
changes in the submucosa. In some cases there
1% LANCASTER AND OLSON
appears to be transitional cell carcinoma. A
major contributing factor to the disease appears
to be the ingestion of bracken fern (131). Inter-
estingly, BPV occasionally has been detected in
some of these lesions since bladder tumor ex-
tracts have induced cutaneous fibropapillomas
in calves (122). One isolate has been identified as
BPV-2 (our unpublished observation).
The production of tumors by the combined
effects of bracken fern and papillomavirus is an
attractive model for the study of multifactorial
carcinogenesis. Although removal of bracken
fern from high-incidence farms eliminates the
development of enzootic hematuria, evidence
for such an association is circumstantial. The
putative carcinogen of bracken apparently de-
velops only with certain soil conditions. For
example, bracken fern grown in Wisconsin does
not seem to produce bladder lesions in cattle
whether ingested in the field or when experimen-
tally fed (34). Enzootic hematuria has been
reported from rather restricted areas of the
world and has ceased to occur where improved
pastures provide sufficient forage and cattle no
longer feed on bracken (34).
(iv) BPV type 5. Meischke (108) surveyed
papillomas of the teats of cattle and was able to
distinguish three papillomavirus-containing his-
tological types: (i) papilloma, (ii) "rice grain"
lesions consisting of a white elongated protuber-
ance, and (iii) fibropapilloma. Animals with rice
grain or papillary lesions were susceptible to
infection with virus from fibropapillomas. How-
ever, the presence of rice grain lesions or papil-
lomas conferred immunity to challenge with the
reciprocal virus. These results suggest that mul-
tiple immunologically distinct viruses infect the
teats of cattle. The virus from fibropapilloma
teat lesions induces typical fibropapillomas on
the skin and probably represents either BPV-1
or -2. Virus from the other histological types
were transmitted to the integument and pro-
duced the same lesions from which they were
derived.
Recently, the virus in rice grain lesions of the
teat has been characterized as a new BPV type
(BPV-5) (20). DNA from this virus has unique
restriction endonuclease cleavage patterns dis-
tinct from those BPVs previously described and
has about 5% DNA sequence homology with
BPV-2 and no sequence homology with BPV-4;
its relation to BPV-1 and -3 is unknown.
The presence of teat papillomas is relatively
common, occurring in about 48% of cows. It was
noted by Jarrett et al. (71) that alimentary tract
and teat papillomatosis had a similar range. It is
possible that the BPV-4 may be derived from a
teat papilloma by transmission to nursing calves.
Artificially suckled calves may also be exposed
to infectious virus since 2 of 10 samples of milk
MICROBIOL. REV.
from dairies examined by electron microscopy
showed the presence of virions indistinguishable
from BPV (107).
Other papillomaviruses. The deer fibroma-
virus and sheep papillomavirus can also induce
tumors in the hamster (52, 78). It is of interest to
note that those viruses which have been suc-
cessfully transmitted experimentally to alien
hosts all have a dermal involvement in the
natural host. The somewhat broader tissue tro-
pism for these viruses may reflect their wider
experimental host range. From these observa-
tions it would be of interest to determine if the
recently described papillomavirus from the Eu-
ropean elk, which was isolated from fibropapil-
lomas, is transmissible to laboratory animals.
In addition to CRPV and BPV4, the papillo-
mavirus of an inbred strain of M. natalensis
(GRA Geissen) exhibits the transformation of
papilloma to carcinoma (116). About 3% of adult
animals have epithelial proliferations. These le-
sions can be classified as papillomas, keratiniz-
ing squamous cell carcinomas, and keratoac-
anthomas. Information available on this virus is
limited, but it should provide an additional ani-
mal model for the study of papillomavirus onco-
genesis.
Infectivity in Cell Culture
Human papillomavirus. Many attempts have
been made to demonstrate infectivity of HPV in
vitro, and early observations with the virus have
been reviewed by Rowson and Mahy (145). In an
extensive study, Butel (17) was unable to detect
HPV activity in cultured cells while employing
virus isolated from various types of human pap-
illomas under a variety of culture conditions,
including coinfection with other viruses. One
observation of interest, however, was a tran-
sient stimulation of fetal rabbit kidney cell DNA
synthesis about 24 h after infection with virus
isolated from plantar warts. Although no cyto-
pathic effect was observed, infected cultures
could be passaged more times than the uninfect-
ed controls. In a similar experiment (89), fetal
human lung fibroblast DNA synthesis was stim-
ulated, again transiently, after infection with
virus purified from plantar warts. No cytopathic
effect was observed, but virus DNA sequences
were detectable by molecular hybridization in
cells after 15 doublings and cells pooled from 30
and 40 doublings at about 0.2 viral genomes per
cell. However, these cells had the same life span
as the uninfected controls. Eisinger et al. (37)
reported cytopathic effects and incorporation of
radiolabeled thymidine into a particle banding at
1.34 g/ml in CsCl, identical in morphology to
human wart virus, and which cross-reacted with
anti-plantar wart virus serum. Unfortunately,
VOL. 46, 1982
the cell line which was used in the study was lost
(M. Eisinger, personal communication).
Cubie (31) observed "cytotoxic" effects (cell
rounding, medium pH changes, multinucleation)
in human skin fibroblasts infected only at very
high multiplicities of infection with HPV purified
from plantar warts. These effects could not be
inhibited by specific immune serum; however,
control cultures infected with extracts of plantar
callus did not show any effect on the cells.
Infection of cells with HPV DNA did produce an
interesting effect, however. Infected cells were
maintained at 33°C, and 6 weeks after inocula-
tion a bright perinuclear fluorescence was ob-
served by indirect immunofluorescence using
serum from a patient with recurrent plantar
warts. The fluorescence did not increase signifi-
cantly over the next 2 weeks; however, the cells
were not passaged.
Obtaining a reproducible culture system per-
missive for HPV replication or cell transforma-
tion has been frustrating. Since skin is a complex
organ, certain necessary cell interactions which
would allow for the replication of the virus may
not occur in culture. Attempts to circumvent
this problem have been made by grafting human
skin infected with HPV to nude or antithymo-
cyte serum-treated mice (32, 132). Although the
grafts were successful, there was no evidence of
the development of warts for observation peri-
ods of up to 14 weeks. However, at the time
these studies were undertaken, it was presumed
that regardless of the histology of the papilloma,
human wart virus represented a single agent.
Since it is now known that a number of different
viruses exist, these studies should perhaps be
reevaluated using different HPV types.
BPV. The only papillomaviruses shown to
have a reproducible effect in tissue culture are
bovine viruses which induce cutaneous fibro-
papillomas (types 1 and 2). In early studies, the
virus type used was unknown, but it is presumed
that they represented either BPV-1 or -2 since
these viruses are present in the most obvious of
the cutaneous lesions. Black et al. (10) observed
cytopathic changes in cultures of bovine con-
junctival cells after infection with bovine papil-
loma extracts. These changes included altered
cell morphology, piling-up, and an increase in
the acidity of the medium. Subsequently, cell-
free extracts of bovine papillomas were shown
to induce morphological transformation of pri-
mary embryonic bovine skin cells (107), mouse
embryo cell cultures (11, 154), and hamster
embryo cells (50). Meischke (107) indicated that
only virus obtained from fibropapillomas (cuta-
neous and teat) was effective in transformation
of fetal bovine cells, whereas virus from lesions
lacking a fibrous component were inactive.
Recently, an in vitro focus assay has been
ANIMAL PAPILLOMAVIRUSES 197
developed for the quantitation of BPV-1 and -2
(36). Discrete foci of transformants develop in
infected cultures of NIH3T3 mouse cells and
C127 cells derived from RIII mouse breast tis-
sue. Secondary cultures of NIH and BALB/c
embryo cells respond to BPV infection with the
production of indistinct areas of heaped-up cells.
Focus formation followed single-hit kinetics,
indicating that a single particle was responsible
for the induction of a focus. These cells contain
multiple copies of the BPV genome based on
reassociation kinetics and are tumorigenic for
the nude mouse. A number of human cell lines in
this study were infected with BPV with no
indication of morphological transformation.
Similar to other DNA viruses, DNA from
BPV-1 and -2 is infectious in vitro. Boiron et al.
(12) first reported transformation of fetal bovine
skin cells with DNA obtained from virus isolated
from a pool of bovine papillomas. The trans-
forming activity of the DNA was abolished by
digestion with deoxyribonuclease but not by
ribonuclease or pretreatment with anti-BPV se-
rum. The DNA preparation was also infectious
for hamsters, and inoculation resulted in fibro-
mas and fibrosarcomas identical to those pro-
duced by intact virus.
Recently, the genomes of BPV-1 and -2 have
been molecularly cloned (59). Linearized BPV
DNAs released from flanking vector sequences
transform mouse C127 cells with approximately
the same efficiency as DNA purified from viri-
ons. Furthermore, a cloned fragment of BPV-1
DNA representing 69o of the viral genome will
also transform mouse cells but with a somewhat
lower efficiency than unit length BPV-1 DNA
(59, 104). Further cleavage of this subgenomic
fragment at either end interrupts transforming
activity. The biological activity of this fragment
and cloned genomes in hosts susceptible to BPV
infection has not been determined.
CRPV. In vitro studies with other members of
the papillomavirus genus are very limited. Char-
donnet et al. (22) were unsuccessful in obtaining
CRPV replication in or transformation of human
cells or cells derived from rabbit epidermis.
They did note, however, some effects on CRPV-
infected cells superinfected with other DNA
viruses. Adenovirus type 5 reportedly replicated
more efficiently in human cells infected 24 h
previously with CRPV than in untreated control
cultures. Herpes simplex virus replication, on
the other hand, was inhibited in cells derived
from either CRPV-induced papillomas or carci-
nomas, whereas virus replication was efficient in
cells derived from normal rabbit epidermis. The
polyomavirus simian virus 40 had no effect on
normal rabbit epidermal lines but, curiously,
infected cells derived from papillomas or cells
previously infected with CRPV expressed simi-
198 LANCASTER AND OLSON MICROBIOL. REV.

an virus 40 T antigen. These cells maintained the leading to regression or persistence and progres-
same morphology as uninfected controls, and the sion to carcinoma of CRPV-induced papillomas
production of T antigen could be inhibited by have not been determined. However, several
pretreatment of CRPV with specific immune serum. lines of evidence suggest regression is mediated
Attempts to obtain papillomavirus replication by an immunological reaction directed against
in cells derived from papillomas have been nega- the tumor cells of the papilloma. First, once
tive. It has been assumed that cells of the regression begins, all papillomas regress simul-
germinal epithelial layer of a papilloma contain taneously. This indicates that the rejection
wart virus sequences since cells in the keratiniz- mechanism is systemic (40). Second, vaccina-
ing layer which contain virus would be derived tion with homologous or autochthonous tumor
from the basal layer. Keratinocytes derived preparations has been reported to induce an
from the basal germinal layer of human warts increase in the regression frequency (40). Third,
replicate in culture and appear to undergo some autologous rabbit skin infected in vitro will
stages of differentiation (117). However, these develop warts in those animals in which papillo-
cells did not contain intact HPV demonstrable mas persist but not in animals whose warts are
by electron microscopy nor did they contain rejected (79). Fourth, suppression of the im-
viral structural antigens. In situ hybridization mune system inhibits regression (106). Fifth,
experiments on cottontail rabbit papillomas whereas rabbits with regressing or persistent
failed to demonstrate the presence of viral se- papillomas have antiviral antibodies, papillomas
quences in the cells of the germinal layer al- can be induced by inoculation of CRPV DNA
though viral DNA was readily detectable in large only in rabbits with persisting papillomas (39).
amounts in the outer layer of keratinizing cells This suggests that those animals whose warts
(127). The inability to detect viral sequences in have regressed have immunity against papilloma
the basal layer may be the result of the lack of tumor cells.
sensitivity of the probe. However, viral DNA As with papillomas from other systems, re-
can be detected by in situ hybridization in virtu- gressing rabbit papillomas contain a mononucle-
ally every cell of CRPV-induced carcinomas and ar infiltrate whose distribution is often uneven
BPV-induced connective tissue tumors (108, (80). This could represent a specific response of
151). It would have been of interest to determine the host to the tumor, but the superficial location
by sensitive molecular hybridization techniques of the lesions exposes them to trauma and
whether proliferating keratinocytes derived infection which also induce focal leukocytic
from warts contain viral sequences. infiltration. Kreider (80) indicated that there was
Some stages of differentiation required for a significant increase in the number of leuko-
expression of virus structural antigens apparent- cytes in the germinal and spinous layers and
ly occur in the domestic rabbit CRPV-induced immediately below the basement membrane in
Vx7 and Vx2 transplantable carcinomas (75, regressing papillomas as compared with persis-
126, 143). CRPV structural antigens have been tent lesions. However, the infiltrates were not
regularly detected in these tumors (110, 128). coincidental with those areas of the tumor in
The percentage of cells expressing antigen can which tumor cell proliferation was inhibited,
be dramatically increased by treatment with thus suggesting that direct cell contact may not
iododeoxyuridine (60). Since papillomavirus be necessary and that tumor regression could be
only matures in the outer layers of keratinizing the result of soluble factors.
cells of the epithelium, it appears that some step Lymph node cells from rabbits whose papillo-
in the process of differentiation is required for mas regressed or persisted reduced the plating
virus maturation. Indeed, in those areas of Vx7 efficiency of tumor cells from papillomas or
carcinomas in which structural antigen can be carcinomas but not normal skin fibroblasts taken
detected, there is cytological evidence for cell from the same rabbits as the tumor cells (57).
maturation (110). These results would suggest the presence of cell-
mediated immunity to a specific tumor-associat-
E4MUNITY ed antigen(s). It is unknown if this antigen is
Host immune responses to papillomavirus in- virus coded or virus induced, but tumor-associ-
fection are not well understood. In general, the ated antigens have been demonstrated by immu-
infection is acquired by the young, and warts nofluorescence on papilloma and carcinoma
persist for variable periods after which the tu- cells (61, 62). The plating efficiency of tumor
mors regress. The host is left immune to reinfec- cells pretreated With sera from rabbits bearing
tion with the same virus. persistent papillomas or carcinomas was not
inhibited by lymph node cells previously shown
CRPV to be active against the tumor cells. Serum from
Virus-host interactions in the rabbit papillo- rabbits with regressing papillomas was not in-
mavirus system are most intriguing. The factors hibitory (57). These results would suggest that
VOL. 46, 1982
the immune rejection phenomenon may be mod-
ulated by soluble factors. However, serum mod-
ulation of immune reactions may be limited to in
vitro systems since passive transfer of sera from
rabbits with persisting warts to CRPV-infected
rabbits had no effect on the percentage of rabbits
whose warts subsequently regressed (41).
BPV
Information available on host immune re-
sponses to BPV infection is generally limited to
virus from fibropapillomas (BPV-1 and -2). In-
oculation of BPV by scarification induces an
initial dermal fibromatosis evident at about 3
weeks followed by epidermal papillomatosis at
about 2 to 3 months after infection; however,
virus usually will induce only a fibroma after
intradermal inoculation. There is a wide range of
susceptibility of calves to primary BPV infec-
tion, and not all calves develop mature fibropa-
pillomas. Tumors may regress spontaneously
during any stage of development (25, 48, 101).
Calves respond to BPV infection by generating
precipitating antibodies of the 19S class within
the 1st week. These antibodies persist for the
first 8 weeks and are undetectable after 16
weeks. Precipitating antibodies of the 7S class
can be detected at about 6 weeks and persist for
at least 26 weeks after infection (102). Of interest
is that the precipitin response does not correlate
with either growth or regression of fibropapillo-
mas (2).
Multiple bovine warts usually regress on an
individual animal simultaneously, but this oc-
curs later than the development of resistance to
reinfection with BPV (100). Regressing fibromas
are infiltrated with mononuclear leukocytes,
mainly lymphocytes; this occurs generally in
perivascular areas but also as a diffuse scattering
throughout the tumor. The intensity of infiltra-
tion is proportional to the rate of fibroma regres-
sion. Unusual is the finding that in animals with
both fibromas and fibropapillomas, the fibromas
will regress whereas the fibropapillomas will
continue to grow and not regress until much
later. This is correlated with a lack of leukocyte
infiltration in the persisting fibropapillomas. The
presence of precipitating antibodies does not
protect against reinfection unless the warts have
undergone resolution. However, a small per-
centage of animals whose fibromas have re-
gressed are still susceptible to reinfection, but
the resultant lesions undergo early regression.
The ability of the immune system to reject a
fibroma without the concurrent rejection of fi-
bropapilloma presents a paradox since autolo-
gous fibropapilloma cells cannot be transplanted
successfully once the immune response to fibro-
mas has been mounted (100).
ANIMAL PAPILLOMAVIRUSES 199
Treatment of bovine papillomatosis with wart
vaccines is difficult to evaluate since the disease
is self-limiting and its duration varies in individ-
ual animals. In a study designed to control some
of the variables involved (such as time of infec-
tion), vaccination had no effect on the regression
rates of warts. A significant proportion of ani-
mals failed to reject their warts despite vaccina-
tion with autologous tumor preparations (120).
In rather rare instances of naturally acquired
papillomatosis, the tumors can become quite
extensive. These lesions persist and show no
evidence of regression. In one such case in
which immune parameters were partially deter-
mined, a calf with extensive warts of long dura-
tion was unable to respond to tuberculin skin
tests after adjuvant vaccination. Its humoral
response was normal against a selected immuno-
gen, and antibody titers against BPV rose after
vaccination with wart vaccine. Prevaccination
serum inhibited phytohemagglutinin stimulation
of normal bovine lymphocytes, thus suggesting
the presence of a humoral immunosuppressive
factor(s) (35). However, there was no mention of
control reactions. These observations suggest
that cell-mediated immunity plays a role in the
rejection of viral warts and that the virus or
virus-induced tumor factors may suppress cellu-
lar immunity in certain susceptible animals.
Meischke (109) reported that calves inoculat-
ed with BPV-transformed fetal bovine skin or
meningeal cells were resistant to challenge by
BPV, whereas untransformed counterparts were
unable to confer resistance. The virus-trans-
formed cells did not contain intact virus as
judged by electron microscopy. However, virus
structural proteins could have been present in
the cells. This seems unlikely, however, since a
number of BPV-transformed cells have been
assayed for virus structural antigens with high
titer antisera by immunofluorescence and have
been uniformly negative (3, 5). Meischke (107)
also observed that adsorption of serum from an
infected cow with these virus-transformed cells
significantly decreased its neutralizing titer
against BPV. This effect was also seen with
fragments of tissue derived from fibropapilloma
and fibroma but not from virus-containing papil-
lomas of non-BPV-1 or -2 origin. Similarly,
CsCl-banded virus from cutaneous fibropapillo-
mas also decreased the neutralizing titer of the
serum. It is uncertain what factor(s) is being
adsorbed from the serum, but it would have
been of interest to determine if the effect was
additive using a combination of virus-trans-
formed cells and purified virus as adsorbants.
Barthold and Olson (3, 5) detected tumor-
specific antigens by immunofluorescence on the
surface membranes of cells explanted from
BPV-induced cattle and horse fibromas using
200 LANCASTER AND OLSON
sera from tumor-bearing cattle and horses. The
sera were cross-reactive and did not totally
inhibit the reaction in serum-blocking studies,
suggesting the presence of inter- and intraspe-
cies antigens. Hamster cells derived from BPV-
induced tumors were nonreactive with the horse
and ox sera and sera from tumorous hamsters
failed to react with horse and ox fibroma cells.
However, Geraldes (51) was able to detect new
antigens in hamster embryo and mouse cells
transformed in vitro by BPV. The test serum
was obtained from tumor-bearing hamsters and
reacted in the cytoplasm of the cells rather than
on the surface membrane. It is unknown if these
antigens are induced or encoded by BPV.
Human Papillomavirus
Regression of human flat warts also appears to
be associated with some form of cell-mediated
immune response. Histologically, regressing flat
warts show perivascular infiltration of mononu-
clear leukocytes in the upper dermis with epider-
mal invasion, with the infiltrate being confined
sharply to the papilloma (9). These observations
have been on lesions with which HPV-3 fre-
quently is associated. As with CRPV and BPV,
once warts begin to regress, distant lesions also
will regress simultaneously. However, patients
with regressing flat warts will maintain plantar
or palmer warts (8). This is consistent with a
different HPV type etiology for these lesions
(see reference 58). A similar differential regres-
sion has been observed in cattle infected with
various types of BPV (6).
Immune responses to HPV infection may be
difficult to assess due to the plurality of immuno-
logically distinct viruses infecting humans.
However, in many of the studies, wart antigen
prepared either from a pool of hand and plantar
warts or from an individual wart was tested
against the patient from whom the wart was
obtained.
The antibody response to HPV infection is
characterized by the appearance of immuno-
globulin M (IgM) before the onset of regression;
after regression, both IgG and IgM antibody
classes are present, and long after regression
only IgG remains (S. Pyrhonen, Ph.D. thesis,
University of Helsinki, Finland, 1978). This pro-
gression from IgM to IgG is similar to that found
in cattle responding to BPV infection (102). The
incidence of antibody to HPV extracted from
plantar warts (HPV-1) in an unselected sample
population is about 35% (135); however, the
incidence to other HPV types is unknown.
An in vitro cell-mediated immune response
can be detected towards HPV infection. Lee and
Eisinger (99) showed that, in general, patients
with warts of <1 year duration were responsive
to wart virus antigen as assayed by lymphocyte
MICROBIOL. REV.
transformation and lymphocyte migration inhibi-
tion. However, lymphocytes from patients with
warts of long duration were unresponsive to the
antigen even though their cells responded nor-
mally to phytohemagglutinin stimulation. Curi-
ously, the cells of a high percentage of individ-
uals who had no recollection of warts responded
to HPV. In a similar study, Ivanyi and Morison
(66) observed that only 28% of the control
subjects were responsive. However, both stud-
ies did concur with the increased responsiveness
of lymphocytes to viral antigen in individuals
with a past history of warts or regressing warts.
Cells from patients with regressing warts re-
sponded to a wart tissue extract, whereas those
with active disease also responded but to a
lesser extent. However, the lymphocyte re-
sponse was increased in those patients whose
warts later regressed (114). These results would
suggest an altered immune response during re-
gression. It is unknown if this response is
mounted as a result of regression or its cause.
However, an intriguing study of a single patient
with warts of 20 years' duration suggested the
presence of a soluble factor in the wart itself
which inhibited leukocyte responsiveness in
lymphocyte migration inhibition (47). This indi-
vidual had normal lymphocyte responsiveness
to phytohemagglutinin and produced normal de-
layed hypersensitivity reaction to purified pro-
tein derivative. Although leukocytes were re-
sponsive to purified protein derivative in the
lymphocyte migration inhibition reaction, they
did not respond to a saline extract of the pa-
tient's warts. When purified protein derivative
and wart extract were used together, the lym-
phocyte migration inhibition index indicated
blockage of migration inhibition.
It is difficult to comment on this work since no
control reactions with extracts of normal tissue
from the patient were evaluated. However, if a
factor were elicited by recalcitrant warts which
inhibited a cellular immune response in vitro, it
would provide evidence for immune modulation
by papillomaviruses in certain individuals.
An obvious need exists for more detailed and
comprehensive studies on the cellular host im-
mune responses to papillomavirus tumor induc-
tion. This information may provide an explana-
tion as to why some individuals are able to reject
their tumors whereas others appear to be immu-
nologically paralyzed. These differences in host
immune response may involve histocompatibil-
ity antigens or immune response genes.
EVOLUTIONARY RELATEDNESS
Antigenic Cross-Reactivity
No immunological cross-reactivity has been
detected between main members of the papillo-
mavirus genus using hyperimmune sera or sera
VOL. 46, 1982
from animals with warts. However, Orth et al.
(126) observed immunological cross-reactivity
between HPV-1 and CRPV with sera obtained
from rabbits bearing the transplantable, CRPV-
induced, Vx7 carcinoma. Antisera prepared
against intact CRPV or HPV-1 only reacted with
the homologous virus; however, when alkali-
disrupted or purified HPV-1 structural polypep-
tides were used as immunogen, the resultant
sera were reactive with CRPV. Immunoptecipi-
tation studies indicated that the main common
antigenic determinant(s) resided on the major
structural polypeptide. Cross-reactivity of anti-
sera directed against disrupted HPV-1 was not
affected by absorption with intact HPV-1, but
was eliminated when disrupted virus was used.
These results would indicate an evolutionary
relatedness among the papillomaviruses and that
the common antigenic determinant(s) is not nor-
mally exposed to the immune system of the
infected host. The antigen being developed by
the Vx7 carcinoma may be from synthesis of
virus structural polypeptides which are not as-
sembled into virions since virus structural anti-
gens have routinely been detected in this tumor
but not infectious virus.
Jenson et al. (73) demonstrated cross-reactiv-
ity between diverse members of the papilloma-
virus genus while employing an antiserum di-
rected against detergent-disrupted HPV-1. The
antiserum reacted by indirect immunofluores-
cence on frozen sections of fibropapillomas from
cattle containing BPV-1 or -2, canine oral papil-
lomas, and human warts containing either HPV-
1 or -2. Type-specific sera were not cross-
reactive. The cross-reaction was localized to the
nuclei of cells of the granular layer and was
identical to the pattern observed with homolo-
gous antiserum. An antiserum against detergent-
disrupted simian virus 40 was cross-reactive
with other members of the polyomavirus genus
but not with papillomavirus, thus indicating no
relation between the two genera of the papovavi-
rus family.
It was also noted that papillomavirus structur-
al antigens could be detected in Formalin-fixed
paraffin-embedded tissues using the peroxidase-
antiperoxidase test. Studies on Formalin-fixed
juvenile laryngeal papillomas indicate that
roughly 50%o of the lesions contain papilloma-
virus structural antigens using antisera against
detergent-disrupted HPV-1 or BPV-1 (28, 84,
85). Although only a few cells in the outer
keratinizing layer contain antigen, the presence
of virions was verified by electron microscopy in
positive areas excised from the tissue block (84).
Similarly, papillomavirus antigens have been
detected in -50%o of human oral cavity verru-
cae, condlyomas, multiple papillomas (72), and
vulvar condylomas and dysplasias of the uterine
ANIMAL PAPILLOMAVIRUSES 201
cervix (83); the latter is considered a premalig-
nant condition. A large number of Formalin-
fixed human plantar and common warts as well
as animal papillomas have been examined for
virus, and in all instances about 50% of the
lesions contain demonstrable antigen (A. B.
Jenson, personal communication). The reason
that only half of all warts are positive for virus is
unknown but may be related to the immune
status of the host, age of the lesion, sampling
error, or natural cycling of antigen synthesis and
virus maturation.
Papillomas of undetermined etiologies occur
in almost all organs such as breast, salivary
gland, urinary bladder, and brain that have an
epithelial component. The use of a broadly
cross-reactive antiserum would be useful for
screening tissues for papillomavirus antigens,
especially those tissues previously characterized
and stored in paraffin blocks.
Conserved Polynucleotide Sequences
The immunological relatedness is paralleled
by the occurrence of conserved DNA sequences
among the genomes of papillomaviruses. Under
standard conditions of DNA-DNA hybridization
(25°C below the melting temperature of the DNA
[Tm - 25°C]), no sequence homology can be
detected between main members of the papillo-
mavirus genus. However, Law et al. (96), by
lowering the stringency of hybridization, were
able to detect regions of sequence homology
between the genomes of BPV-1 and -2 and HPV-
1 and CRPV. The conditions of hybridization
were equivalent to Tm- 43°C which represents
about 25 to 35% base mismatch. The regions of
homology were limited to two of the four HinclI
cleavage fragments of BPV-2 DNA which are
noncontiguous on the BPV-2 genome. These
regions may represent two genes since one re-
gion of homology is contained within the 69%o
transforming fragment of BPV DNA and thus
may code for transforming proteins, and the
other region may encode information for struc-
tural polypeptides. They were unable to detect
stable heteroduplexes between papillomavirus
DNA and DNA from members of the polyoma-
virus genus, suggesting these two virus genera
were derived from different ancestors.
The thermal stabilities of heteroduplexes
formed between BPV, HPV-1, and CRPV DNAs
were similar, thus indicating the same degree of
relatedness among these viruses. Interestingly,
an analysis of HPV-1, -2, and -4 relatedness
suggested that all three viruses were equally
related based on thermal stability of heterodu-
plexes (56). Furthermore, heteroduplexes
formed with BPV indicated that the human
viruses were as related to each other as they
were to BPV. These results would suggest that
202 LANCASTER AND OLSON MICROBIOL. REV.
the bovine and human viruses were derived from DNA which have lost the unique restriction site
a common ancestor very early in their evolution. used to excise the sequences from plasmid. Cells
Since the pipillomaviruses appear to have transformed by the 69% subgenomic fiagment of
similar degrees of relatedness regardless of the BPV-1 DNA contain only extrachromosomal
species of origin, it should be possible to assay sequences present as supercoiled, linear, and
for the presence of papillomavirus DNA se- nicked circular molecules. Since this DNA has
quences in tumors by using any papillomavirus noncohesive termini (BamHI and HindlIl ends),
DNA as a probe in molecular hybridization it may not be able to recircularize, which could
experiments, providing the conditions are re- explain the reduced efficiency of cell transfor-
laxed. In recent studies papillomavirus genus- mation as compared to unit length viral DNA
specific DNA sequences have been detected in with homologous cohesive termini. Analysis of
human juvenile laryngeal papillomas (88) and these viral DNA sequences in transformed cells
cervical dysplasias (82) using a BPV-1 DNA indicates either molecular rearrangement and
probe, and in anogenital warts with HPV-1 or -2 duplication of viral DNA sequences or addition
probes (81) under nonstringent hybridization of host or carrier DNA sequences during recir-
conditions. cularization. Such rearrangements may occur in
nature since a naturally occurring equine tumor
MOLECULAR BIOLOGY has been shown to contain BPV sequences in
which 9% of the viral genome has been deleted
Lack of Integration (1).
In general, cellular transformation by DNA A similar lack of viral DNA integration has
and RNA viruses is accompanied by integration been observed in carcinomas induced by CRPV
of the viral sequences required for maintenance in domestic rabbits (155). However, the predom-
of transformation. Although extrachromosomal inant form of viral DNA is in high-molecular-
forms of DNA have been described in cells weight complexes with only trace amounts of
transformed by DNA viruses, it is always in as- supercoiled DNA. These complexes are con-
sociation with integrated viral DNA. However, verted to unit length linear molecules when
BPV-1 and -2, and perhaps other papillomavi- digested with a single-cut restriction enzyme.
ruses, may be exceptions since there is no The majority of high-molecular-weight complex-
evidence that BPV DNA is covalently linked to es appear to represent concatenated viral DNA
host cell sequences in virus-transformed cells. rather than catenated structures since limited
Lack of integration of BPV-1 and -2 genomes digestion with a single-cut restriction enzyme
has been observed in naturally occurring and yields viral DNA migrating as linear monomers,
experimental BPV-induced equine tumors (1, dimers, trimers, and tetramers as well as higher-
87, 91), cell lines established from equine and molecular-weight multimeric forms (157). In ad-
bovine tumors (87, 91), BPV-induced hamster dition to unit-length viral DNA, sequences with
tumor cells (112, 134), and BPV-transformed higher and lower molecular weights were also
mouse cells (87, 97). Viral DNA sequences exist observed in one of four carcinomas. The viral
predominantly as supercoiled molecules, unit sequences had the properties of viral-cellular
length linear, and open circular forms. In addi- junction pieces based on analysis of limited
tion, undigested DNA preparations contain digestion with single-cut restriction enzymes
high-molecular-weight viral sequences which (157). These results would suggest that CRPV
convert to unit-length linear molecules when DNA sequences can integrate into cellular DNA
digested with a single-cut restriction enzyme. in at least some of the tumors induced by the
These slowly migrating viral sequences may virus.
represent intertwined catenated circular mole- Cloning Vector
cules since limited nuclease S1 digestion or mild
shearing results in conversion to monomeric The property of the 69%o transforming frag-
linear and circular forms (97). The viral se- ment of the BPV-1 genome to remain extrachro-
quences exist exclusively extrachromosomally mosomal as a supercoiled molecule at high copy
since reconstruction experiments designed to number and to carry additional DNA sequences
detect 0.1 to 0.2 viral genomes per cell fail to would make this viral DNA an attractive eucary-
resolve viral sequences migrating other than otic cloning vector. Recently, Sarver et al. (146)
unit-length linear molecules in agarose gels after constructed a 69%o BPV-1 transforming frag-
digestion of the DNA with a single-cut restric- ment/rat preproinsulin recombinant. This DNA
tion enzyme (97). was able to transform mouse cells at high effi-
Modification of the viral genome can occur in ciency. Analysis of selected transformed clones
cells transfected with linearized BPV DNA. indicated that the recombinant molecule was
Analysis of virus sequences in mouse cells indi- intact in high copy number and was present as
cates that a percentage of clones contain viral supercoiled DNA. The transformants contained
VOL. 46, 1982
correctly spliced transcripts of the preproinsulin
gene as well as protein immunoreactive with
anti-insulin serum and the protein identified as
rat proinsulin.
BPV-1 DNA has also been used to introduce a
procaryotic gene into mouse cells. Law et al.
(95) constructed hybrid DNAs consisting of the
69%o BPV-1 DNA-transforming region and the
xanthine-guanine phosphoribosyltransferase
gene of Escherichia coli contained in a modified
simian virus 40 early region transcriptional unit.
Both morphologically transformed cells and
cells able to grow under selective pressure for
phenotypic expression of the xanthine-guanine
phosphoribosyltransferase gene were detected
regardless of the orientation of the xanthine-
guanine phosphoribosyltransferase gene. In all
lines, multiple copies of sequences could be
detected which hybridized to both BPV-1 DNA
and the xanthine-guanine phosphoribosyltrans-
ferase gene. In many instances the hybrid mole-
cules were extrachromosomal but often highly
rearranged from the input DNA. About 17% of
the selected lines had lost one of the phenotypic
markers, and in all cases the input DNA had
been rearranged.
The ability of a BPV-1 DNA fragment to act as
a eucaryotic vector is a unique property and may
provide alternatives to procaryotic vector sys-
tems. The usefulness of BPV DNA as a vector is
manifold: (i) BPV DNA and a defined subgeno-
mic fragment are efficient in transforming sus-
ceptible mouse cells; (ii) BPV-transformed cells
contain multiple copies which exist exclusively
as extrachromosomal sequences; (iii) since inte-
gration of the viral DNA does not occur, the
integrity of the "'passenger" DNA should re-
main intact; (iv) selectable markers other than
cell transformation may be employed which
could expand the host range of the BPV plasmid
vector system.
Transcription
Analysis of BPV-1-transformed mouse cells
has indicated the presence of virus-specific
RNA transcripts (55). The transcripts were se-
lected for polyadenylated termini and consisted
of five distinct size classes ranging from 1,050 to
4,050 bases. These RNAs are all transcribed
from the same DNA strand, all map within the
699o transforming region of BPV-1 DNA, and
all share a common 3' terminus. Estimates of the
copy number per cell of the virus-specific RNA
species indicate that they represent 0.006 to
0.01% of the total polyadenylated RNA pool in
the transformed cells. However, in another
transformed mouse cell clone in which the BPV-
1 genome copy number was about fourfold high-
er, the relative abundance of virus-specific RNA
was increased about twofold. In addition, cells
ANIMAL PAPILLOMAVIRUSES 203
transformed by a hybrid molecule consisting of
the 69%o transforming fragment of BVP-1 DNA
and the rat preproinsulin gene was found to
contain an 8- to 10-fold increase in the amount of
virus-specific RNA. Analysis of the most abun-
dant RNA species indicated a lack of internal
splicing; however, the presence of a remote 5'
leader sequence could not be ruled out.
Wettstein and Stevens (156) have assayed
total cellular RNA in CRPV-containing cotton-
tail rabbit papillomas and virus-induced carcino-
mas in the domestic rabbit for virus-specific
RNA transcripts. Virus-containing papillomas
had RNA sequences complementary to between
9 and 25% of the viral genome, whereas primary
and metastatic carcinomas contained RNA se-
quences which were homologous to between 6
and 12% of CRPV DNA. This fluctuation in the
percent homology is probably a reflection of the
lack of abundance of virus-specific RNA in
these tumors.
These analyses of virus-specific transcripts in
the bovine and rabbit systems indicate that there
is limited viral RNA synthesis in cells malignant-
ly transformed by papillomavirus and that the
bulk of the RNA is complementary to only a
small proportion of the viral genome. It is un-
known if these viral RNAs are translated in the
malignant cells. New polypeptides have not
been detected in extracts of tumor cells by
radioimmunoprecipitation or by immunohisto-
chemical staining of tumors using sera from
tumor-bearing animals (14, 156).
CONCLUDING REMARKS
Significant progress in papillomavirus re-
search has been made in recent years. However,
numerous aspects of the biology of this unique
group of tumor viruses remain to be elucidated.
Although the genomes of many of the papilloma-
viruses have been molecularly cloned and de-
tailed physical maps of the viral DNA have been
constructed, a major impediment for analysis of
this virus genus is the lack of a culture system
permissive for their replication. Transcription of
portions of the viral genome in tumor and trans-
formed cells indicates the potential for the syn-
thesis of virus-specific proteins. However, rea-
sons for the inability to detect unique proteins in
these cells remain obscure. The rejection of
papillomas clearly is associated with a cell-
mediated phenomenon directed against the tu-
mor cell. The cellular determinant that is recog-
nized by the immune system is unknown. The
finding of multiple, minimally related types with-
in a single species would suggest that the papillo-
maviruses have evolved in response to pressure
of the host immune system since regions of the
capsid which would not normally be exposed to
the immune system have remained conserved
 204 LANCASTER AND OLSON
throughout their evolution. The papillomavi-
ruses may offer an interesting system for the
analysis of evolution at the polynucleotide level,
since a large number of viruses have been identi-
fied from diverse species and all show conserved
regions of DNA sequence homology. Informa-
tion as to why some animals can reject papillo-
mas while lesions persist (and progress to carci-
noma in some instances) in other animals could
provide insights into host immune defense
against neoplasia. The transformation of papillo-
mas to carcinoma appears to involve not only
virus but other extrinsic factors such as genetic
background or environmental factors. Multifac-
torial carcinogenesis is thought to be involved in
the generation of many forms of cancer. Lack of
integration of papillomavirus genomes in trans-
formed cells would suggest that these viruses
may have evolved an alternative mechanism for
maintenance of the transformed state.
ACKNOWLEDGMENTS
We thank A. Bennett Jenson and Robert J. Kurman for
critical review of the manuscript.
This work was supported by Public Health Service grants
CA32603 and CA32638 from the National Cancer Institute.
LITERATURE CITED
1. Amtnann, E., H. Muller, and G. Sauer. 1980. Equine
connective tissue tumors contain unintegrated bovine
papilloma virus DNA. J. Virol. 35:962-964.
2. Barthold, S. W., and C. Olsm. 1974. Fibroma regression
in relation to antibody and challenge immunity to bovine
papilloma virus. Cancer Res. 34:2436-2431.
3. Barthold, S. W., and C. Olson. 1974. Membrane antigen
of bovine papilloma virus-induced fibroma cells. J. Natl.
Cancer Inst. 52:737-741.
4. Barthold, S. W., and C. Olson. 1974. Papovavirus-in-
duced neoplasia 70-75. In E. C. Melby, Jr., and N. H.
Altman (ed.), Handbook of laboratory animal science.
CRC Press, Cleveland.
5. Barthold, S. W., and C. Olson. 1978. Common membrane
neoantigen on bovine papilloma virus-induced fibroma
cells from cattle and horses. Am. J. Vet. Res. 39:1643-
1645.
6. Barthold, S. W., L. D. Koller, C. Olson, E. Studer, and A.
Holtan. 1974. Atypical warts in cattle. J. Am. Vet. Med.
Assoc. 165:276-280.
7. Beard, J. W., and P. Rous. 1935. Effectiveness of Shope
papilloma virus in various American rabbits. Proc. Soc.
Exp. Biol. Med. 33:191-193.
8. Berman, A., and J. E. Berman. 1978. Efflorescence of
new warts: a sign of onset of involution in flat warts. Br.
J. Dermatol. 99:179-182.
9. Berman, A., and R. K. Wlnkelmna. 1977. Flat warts
undergoing involution: histopathological findings. Arch.
Dermatol. 113:1219-1221.
10. Black, P. H., J. W. Hartley, W. P. Rowe, and R. J.
Huebner. 1963. Transformation of bovine tissue culture
cells by bovine papilloma virus. Nature (London)
199:1016-1018.
11. Bodron, M., J. P. Levy, M. Tboma, J. C. Fredman, and
J. Bernard. 1964. Some properties of bovine papilloma
virus. Nature (London) 201:423.
12. Boiron, M., M. Thms, and P. H. CheulaDle. 1965. A
biological property of deoxyribonucleic acid extracted
from bovine papilloma virus. Virology 52:150-153.
13. Bottger, M., D. Blerwolf, V. Wunderlich, and A. Grffi.
1971. New calibration correlations for molecular weights
of the DNA of an oncogenic papovavirus of the Syrian
MICROBIOL. REV.
hamster. Biochim. Biophys. Acta 232:21-31.
14. Brdtburd, F., M. Favre, R. Zoorob, D. Fortin, and G.
Orth. 1981. Detection and characterization of viral ge-
nomes and search for tumoral antigens in two hamster
cell lines derived from tumors induced by bovine papillo-
mavirus type 1. Int. J. Cancer 27:693-702.
15. Brobst, D. F., and C. Olson. 1965. Histopathology of
urinary bladder tumors induced by bovine cutaneous
papilloma agent. Cancer Res. 25:12-19.
16. Bujard, H. 1967. Studies on circular deoxyribonucleic
acid. I. Isolation of bovine papilloma virus and charac-
terization of its deoxyribonucleic acid. J. Virol. 1:1135-
1138.
17. Butel, J. S. 1972. Studies with human papilloma virus
modeled after known papovavirus systems. J. Natl.
Cancer Inst. 48:285-299.
18. Cadeac, M. 1901. Sur la transmission experimentale des
papillomes des diverses especes. Bull. Soc. Sci. Vet.
Med. Comp. Lyon 4:280-286.
19. Campo, M. S., M. H. Moar, W. F. H. Jarrett, and H. M.
Laird. 1980. A new papillomavirus associated with ali-
mentary tract cancer in cattle. Nature (London) 286:180-
182.
20. Campo, M. S., M. H. Moar, H. M. Laird, and W. F. H.
Jarrett. 1981. Molecular heterogeneity and lesion site
specificity of cutaneous bovine papillomaviruses. Virolo-
gy 113:323-335.
21. Chambers, V. C., and C. A. Evans. 1959. Canine oral
papillomatosis. I. Virus assay and observations of the
various stages of the experimental infection. Cancer Res.
19:1188-1195.
22. Chardonnet, Y., T. Greenland, M. Lyon, and R. Sohier.
1978. Interactions of Shope papilloma virus with some
other DNA viruses. Bull. Cancer 65:195-206.
23. Chevlle, N. F. 1966. Studies on connective tissue tumors
in the hamster produced by bovine papilloma virus.
Cancer Res. 26:2334-2339.
24. Cheville, N. F., and C. Olson. 1964. Cytology of the
canine oral papilloma. Am. J. Pathol. 45:849-872.
25. Chevie, N. F., and C. Olson. 1964. Epithelial and
fibroblastic proliferation in bovine cutaneous papilloma-
tosis. Pathol. Vet. 1:248-257.
26. Cluffo, G. 1907. Innesto positivo con ifitrato di verrucae
volgare. Giorlp. Ital. Mal. Venereol. 48:12-17.
27. Coggin, J. R., Jr., and H. zur Hane. 1979. Workshop
on papillomaviruses and cancer. Cancer Res. 39:545-
546.
28. Costa, J., P. M. Howley, M. C. Bowlig, R. Howard, and
W. C. Bauer. 1981. Presence of human papilloma viral
antigens in juvenile multiple laryngeal papilloma. Am. J.
Clin. Pathol. 75:194-197.
29. Crawford, L. V. 1969. Nucleic acids of tumor viruses.
Adv. Virus Res. 14:89-152.
30. Crawford, L. V., and E. M. Crawford. 1963. A compara-
tive study of polyoma and papilloma viruses. Virology
21:258-263.
31. Cuble, H. A. 1974. Experiments with human papilloma
virus in cell culture. Br. J. Dermatol. 91:569-571.
32. Cuble, H. A. 1976. Failure to produce warts on human
skin grafts on 'nude' mice. Br. J. Dermatol. 94:659-665.
33. Danos, O., M. Katinka, and M. Yaniv. 1980. Molecular
cloning, refined physical map and heterogeneity of meth-
ylation sites of papilloma virus type la DNA. Eur. J.
Biochem. 109:457-461.
34. Doberener, J., C. Olon, R. R. Brown, J. M. Prie, and
N. Yess. 1966. Metabolites in urine of cattle with experi-
mental bladder lesions and fed bracken fern. Presq.
Agropec. Bras. 1:189-199.
35. Duncan, J. R., L. B. Corbeil, D. H. Davies, R. D. Shultz,
and R. H. Whiock. 1975. Persistent papillomatosis
associated with immunodeficiency. Cornell Vet. 65:202-
211.
36. Dvoretzky, I., R. Shober, S. K. Chattopadhy, and D. R.
Lowy. 1980. A quantitative in vitro focus fonning assay
for bovine papilloma virus. Virology 103:369-375.
VOL. 46, 1982
37. Elshlger, M., 0. Kucarova, N. H. Sarkar, and R. A.
Good. 1975. Propagation of human wart virus in tissue
culture. Nature (London) 256:432-434.
38. Erk, N. 1976. A study of Kitab al-Hail wal-Baitara by
Muhammed Ibu ahi Hazam. Historia Medicinae Veterin-
ariae. 1:101-104.
39. Evans, C. A., and Y. Ito. 1966. Antitumor immunity in
the Shope papilloma-carcinoma complex of rabbits. III.
Response to reinfection with viral nucleic acid. J. Natl.
Cancer Inst. 36:1161-1166.
40. Evans, C. A., L. R. Gorman, Y. Ito, and R. S. Weiser.
1962. Antitumor immunity in the Shope papilloma-carci-
noma complex of rabbits. I. Papilloma regression in-
duced by homologous and autologous tissue vaccines. J.
Natl. Cancer Inst. 29:277-285.
41. Evans, C. A., R. S. Weiser, and Y. Ito. 1963. Antiviral
and antitumor immunologic mechanisms operative in the
Shope papilloma-carcinoma system. Cold Spring Harbor
Symp. Quant. Biol. 27:453-462.
42. Favre, M. F. Breitburd, 0. Croissant, and G. Orth. 1974.
Hemmagglutinating activity of bovine papilloma virus.
Virology 60:572-578.
43. Favre, M., F. Breltburd, 0. CroIssat, and G. Orth. 1975.
Structural polypeptides of rabbit, bovine, and human
papilloma viruses. J. Virol. 15:1239-1247.
44. Favre, M., F. Bredtburd, 0. Crosant, and G. Orth. 1977.
Chromatin-like structures obtained after aLkaline disrup-
tion of bovine and human papiilomaviruses. J. Virol.
21:1205-1209.
45. FInch, J. T., and A. Klog. 1965. The structure of viruses
of the papilloma-polyoma type Ill. Structure of rabbit
papilloma virus, with an appendix on the topography of
contrast in negative staining for electron microscopy. J.
Mol. Biol. 13:1-12.
46. Follet, E. A. C., and L. V. Crawford. 1967. Electron
microscopic study of denaturation of human papilloma
virus DNA II. The specific location of denatured regions.
J. Mol. Biol. 28:461-467.
47. Freed, D. L. J., and K. E. Evans. 1979. Persistent warts
protected from immune attack by a blocking factor. Br.
J. Dermatol. 100:731-733.
48. Fujimoto, Y., and C. Olson. 1966. The fine structure of
the bovine wart. Pathol. Vet. 3:659-684.
49. Fulton, R. E., F. W. Doane, and L. W. Macpherson. 1970.
The fine structure of equine papilloma and the equine
papilloma virus. J. Ultrastruct. Res. 30:328-343.
50. Geraldes, A. 1969. Malignant transformation of hamster
cells by cell-free extracts of bovine papillomas (in vitro).
Nature (London) 222:1283-1284.
51. Geraldes, A. 1970. New antigens in hamster embryo cells
transformed in vitro by bovine papilloma extracts. Na-
ture (London) 226:81-82.
52. Gibbs, E. P. J., C. J. Smale, and M. J. P. Lauman. 1975.
Warts in sheep. J. Comp. Pathol. 85:327-334.
53. G_ssnn, L., and H. zor Hansen. 1977. Inverted repeti-
tive sequences in human papilloma virus 1 (HPV-1)
DNA. Virology 83:271-276.
54. Grdon, D. E., and C. Obon. 1968. Meningiomas and
fibroblastic neoplasia in calves induced with the bovine
papilloma virus. Cancer Res. 28:2423-2431.
55. Heglman, C. A., L. Engel, D. R. Lowry, and P. M.
Howley. 1982. Virus-specific transcription in bovine
papillomavirus transformed mouse cells. Virology
119:22-34.
56. HeUlman, C. A., M.-F. Law, M. A. Isael, and P. M.
Howley. 1981. Cloning of human papilloma virus genomic
DNAs and analysis of homologous polynucleotide se-
quences. J. Virol. 36:395-407.
57. Hdbromn, I., C. A. Evans, and K. E. Helstrom. 1969.
Cellular immunity and its serum-mediated inhibition in
Shope-virus-induced rabbit papillomatosis. Int. J. Can-
cer 4:601-607.
58. Howley, P. M. Papoviruses-search for evidence of
possible association with human cancer. In L. Phillips
(ed.), Viruses associated with human cancer. Marcel
ANIMAL PAPILLOMAVIRUSES 205
Dekker, New York, in press.
59. Howley, P. M., M.-F. Law, C. Heil m, L. Engel, M. C.
Alonso, M. A. Israel, D. R. Lowy, and W. D. Lancaster.
1980. Molecular characterization of papillomavirus ge-
nomes. Cold Spring Harbor Conf. Cell Prolif. 7:233-247.
60. Inokuchi, T., S. Ikejivi, F. Mlnuzo, and T. Osato. 1975.
Activation by 5-iododeoxyuridine of Shope papilloma
viral genome in cultured Vx2 and Vx7 carcinomas. Arch.
Virol. 48:275-277.
61. Ishinoto, A., and Y. Ito. 1969. Specific surface antigens
in Shope papilloma cells. Virology 39:595-597.
62. Ishmoto, A., and Y. Ito. 1971. Further studies on surface
antigen of Shope papilloma cells: trypsin sensitivity. J.
Natl. Cancer Inst. 46:353-357.
63. Ito, Y. 1960. A tumor-producing, factor extracted by
phenol from papillomatous tissue (Shope) of cottontail
rabbits. Virology 12:596-601.
64. Ito, Y. 1963. Relationship of components of papilloma
virus to papilloma and carcinoma cells. Cold Spring
Harbor Symp. Quant. Biol. 27:387-394.
65. Ito, Y., and C. A. Evans. 1961. Induction of tumors in
domestic rabbits with nucleic acid preparations from
partially purified Shope papilloma virus and from ex-
tracts of papillomas of domestic and cottontail rabbits. J.
Exp. Med. 114:485-500.
66. Ivanyl, L., and W. L. Morison. 1976. In vitro lymphocyte
stimulation by wart antigen in man. Br. J. Dermatol.
94:523-527.
67. Jackson, C. 1936. The incidence and pathology of tumors
of domesticated animals in South Africa. Onderstepoort
J. Vet. Sci. Anim. Ind. 6:378-385.
68. Jarrett, W. F. H. 1978. Transformation of warts to
malignancy in alimentary carcinomas in cattle. Bull.
Cancer 65:191-194.
69. Jarrett, W. F. H., P. E. McNeil, W. T. R. Grmshaw, I. E.
Sehlan, and W. I. M. McIntyre. 1978. High incidence
area of cattle cancer with a possible interaction between
an environmental carcinogen and a papilloma virus.
Nature (London) 274:215-217.
70. Jarrett, W. F. H., P. E. McNeil, H. M. Laird, B. W.
O'Nefl, J. Murphy, M. S. Campo, and M. H. Moar. 1980.
Papilloma viruses in benign and malignant tumors of
cattle. Cold Spring Harbor Conf. Cell Prolif. 7:215-222.
71. Jarrett, W. F. H., J. Murphy, B. W. O'Neil, and H. M.
Laird. 1978. Virus-induced papillomas of the alimentary
tract of cattle. Int. J. Cancer 22:323-328.
72. Jenson, A. B., W. D. Lancaster, D.-P. Hartmann, and
E. L. Shafer. 1982. Frequency and distribution of papillo-
mavirus structural antigens in verrucae, multiple papillo-
mas and condylomata of the oral cavity. Am. J. Pathol.
107:212-218.
73. Jenson, A. B., J. R. Rosenthal, C. Obon, F. Pass, W. D.
Lancaste, and K. Shah. 1980. Immunological related-
ness of papillomaviruses from different species. J. Natl.
Cancer Inst. 64:495-500.
74. Kass, S. J., and C. A. Knight. 1965. Purification and
chemical analysis of Shope papilloma virus. Virology
27:273-281.
75. Kidd, J. G., and P. Rous. 1940. A transplantable rabbit
carcinoma originating in a virus-induced papilloma and
containing the virus in a masked or altered state. J. Exp.
Med. 71:813-838.
76. Khng, A., and J. T. FInch. 1968. Structure of viruses of
the papilloma-polyoma type IV. Analysis of tilting ex-
periments in the electron microscope. J. Mol. Biol. 31:1-
12.
77. KoUler, L. D., and C. Olson. 1971. Pulmonary fibroblasto-
mas in a deer with cutaneous fibromatosis. Cancer Res.
31:1373-1375.
78. Koler, L. D., and C. Olson. 1972. Attempted transmis-
sion of warts from man, cattle, and horses and of deer
fibroma, to selected hosts. J. Invest. Dermatol. 58:366-
368.
79. Kreider, J. W. 1963. Studies on the mechanism responsi-
ble for the spontaneous regression of the Shope rabbit
206 LANCASTER AND OLSON
papilloma. Cancer Res. 23:1593-1599.
80. Krader, J. W. 1960. Neoplastic progression ofthe Shope
rabbit papilloma. Cold Sping Harbor Conf. Cell Prolif.
7:283-300.
81. Kryzyk, R. A., S. L. Watb, D. L. Andeo, A. J. Far,
and F. Pas 1980. Anogenital warts contain several
distinct species of human papillomavirus. J. Virol.
36:236-244.
82. Kurma, R. J., L. E. Saz, A. B. Jesuos, S. Perry, and
W. D. Lscatr. 1982. Papilomavirus infection of the
cervix I. Conelation of histology with viral structural
antigens and DNA sequences. Int. J. Gynecol. Pathol.
1:17-28.
83. Kmnan, R. J., K. H. Shah, W. D. LAncaser, ad A. B.
Jeman. 1981. Immunoperoxidase localization of papillo-
mavirus antigens in cervical dysplasia and vulvar condy-
lomas. Am. J. Obstet. Gynecd. 140931-935.
84. La*c, E. E., A. B. Jesu, H. G. Smit, G. B. Healy, F.
Pas, G. F. Vawter. 1980. Immunoperoxidase localiza-
tion of human papillomavirus in laryngeal papillomas.
Intervirol. 14:148-154.
85. Lack, L E., G. F. Vawte, H. G. Smit, G. B. Healy,
W. D. Lanate, and A. B. Jen. 1980. Immunohisto-
chemincl localisation of human papillomavirus in squa-
mous papillomas of the larynx. Lancet 1:592.
86. Lancae, W. D. 1979. Physical maps of bovine papillo-
mavirus type 1 and type 2 gepomes. J. Virol. 32:684-67.
87. Lancaer, W. D. 1981. Apparent lack of integrtion of
bovine papiliomavirus DNA in virus-induced equine and
bovine tumors and vimas-trnsformed mouse cells. Virol-
ogy 16:251-255.
88. Laater, W. D., ad A. B. Jee.. 1981. Evidence for
papillomavirus antigens and DNA sequences in laryngeal
papiliona. Intervirology 15:204-212.
89; Lancater, W. D., and W. Mdeue. 1975. Persisten c of
viral DNA in human cel cultures infected with human
papilioma virus. Natue (London) 256434-436.
90. Lancaste W. D., ad C. Olson. 1978. Demonstration of
two distinct classes of bovine papilloma virus. Virology
89:371-379.
91. La r, W. D.* an C. Olo. 1980. State of bovine
papilloma virus DNA in, connective tissue tumors. Cold
Spring Hwbor Conf. Cel Proif. 7:223-232.
92. L , W. D, C. Olon, and W. Mdako. 1976.
Quantitation of bovine papilloma viral DNA in viral-
induced tumors. J. Virol. 17:824-831.
93. L1-anaw, W. D)., C. Ohmo, ad W. Mdelke 1977.
Bovine ppiloma virus: presence of virus-specific DNA
sequences in natually occurring equine tumors. Proc.
Natl. Acad. Sci. U.S.A. 74:524-528.
94. La_ncase, W. D., G. H. Tfalen, ad C. Olsn. 1979.
Hybridization of bovine papilloma virus type 1 and type
2 DNA to DNA from virus-induced hamster tumors and
naturally occurring equine connective tissue tumors.
Intervirology 11:227-233.
95. Law, M.-F., B. Howard, N. Sane, and P. M. Bowley.
1982. Expression of selective traits in mouse cells trans-
formed with a BPV DNA derived hybrid molecule con-
taining E. coil p, p. 79-85. In Y. Gluzman (ed.), Viral
vectors. Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York.
96. Law, M.-F., W. D. Lancaer, nd P. M. How0ey. 1979.
Conserved sequences amg the genomes of papilloma-
viruses. J. Virol. 32:199-07.
97. Law, M.-F., D. R. Lowy, . D r , and P. M. Hewley.
1981. Mouse cells transformed by bovine papillomavirus
contain only extrachromosomal viral DNA sequences.
Proc. Natl. Acad. Sci. U.S.A. 7&82727-2731.
98. Le Boevier, G. L., M. Sasmu, ad L. V. Crawford.
1966. Antigenic diversity of mammalian papilloma virus-
es. J. Gen. Microbiol. 45:497-501.
99. Le, A. L. Y., and M. Eliugr. 1976. Cell-mediated
immunity (CMI) to human wart virus and wart associated
tissue antigens. Clin. Exp. Immunol. 26:419-424.
100. Lee, K. P., ad C. Obo. 1968. Response of calves to
MICROB3IOL. REV.
intravenous and repeated inlradermal inoculation of bo-
vine papilloma virus. Am. J. Vet. Res. 16:517-520.
101. Loe, K. P., and C. Olo. 1969. Histochenical study of
experimntally produced bovine fibropapillomas. J. In-
vest. Dermatol. 52:454-464.
102. Lee, K. P., and C. Olso. 1969. Precipitin response of
cattle to bovine papilloma virus. Cancer Res. 29:1393-
1397.
103. Lina, P. H. C., M. J. van Noord, ad F. G. de Groot.
1973. Detection of virus in sqous p s of the
wild bird species Fringella coelebs. J. NaIl. Cancer Inst.
5k;567-571.
104. Lw y, D. R., DVWty, Shober, M.-F. Law, L.
aged, sd P. M. Howley. 1980. In vitro turmorigenic
transformation by a defined subgenomic ragment of
bovine papilloma virus DNA. Nature (London) 217:72-
74.
105. MeFdyes, J., ad F. Hodbqy. 1896. Note on the
experimental transmission of warts in the dog. J. Comp.
Pathol. Ther. 11:341-344.
106. McMlc l, H. 1967. Inhibition by methylprednisolone
of regression of the Shope rabbit papiloma. J. Natl.
Cancer Inst. 39:55-65.
107. M E,H R. C. 1979. In vitro transformation by
bovine papilloma virus. J. Gen. Virol. 43:471-487.
108. MN eko, H. R. C. 1979. A survey of bovine teat
ppilomatosis. Vet. Rec. 104:28-31.
109. Mdschk, H. R. C. 1979. Experimental transmission of
bovine papilloma virus (BPV) extracted from morpho-
locally distinct teat and cutaneous lesions and the
effects of inoculation of BPV transformed fetal bovine
cells. Vet. Rec. 1044:360-366.
110. Mlsr, R. C. 1960. Tumor cell localization of the
antigens of the Shope papilloma virus and Rous sarcoma
virs. Cancer Res. 2t.774-753.
111. Melnlck, J. L., A. C. Alb=on, J. S. B.., W. Eckhat,
B. E. Eddy, S. Kit, A. J. Leaee,J. A. R. Me, J. S.
Paa, L. Sachs, and V. Vonka. 1974. Papovaviridae.
Intervirology 3:106-12W.
112. Mo_, M. H., M. S. Cmpo, H. M. Lard, an W. F. H.
Jarrett. 1981. Unintegrated virl DNA sequences in
hamster tumor induced by bovine papilloma virus. J.
Virol. 39:945-49.
113. MormLapes, J., U. Potteress., Z. Dter, aW L.
Phls_. 1981. Characterization of a papi-losa virus
from the European elk (EEPV). Viroly 11258 95.
114. Moth. W. L. 1975. In vitro assay of immunity to
human wart antigen. Br. J. Dermatol. 93:545-552.
115. Morrios, J. M., H. M. Ker, H. Si Shar, 1nd L. V.
Crawford. 1967. Nearest neighbour base sequn analy-
sis of the dexoxyribonucleic acid of a futher three
mammali viruses: simian virus 40, human papilloma
virus and adenovirus type 2. J. Gen. Virol. 1:101-108.
116. Muier, H., sm L. Gsma. 1978. Masonnys natalensis
papilloma virus (MnPV), the causative aget of epithelial
proliferations: characterization of the virus particle. J.
Gen. Virol. 41:315-323.
117. NilpMr, M., Fi P_,IL Weoley, nd C. A. Soutwr. 1975.
Primary tissue culture of human wart-derived epidermal
cells (keratinocytes). J. Natl. Cancer Int. 5:563-5466.
118. Noys, W. F., ad R. C. Mdlwrs. 1957. Fluorescent
antibody detection of the antigens of the Sbope papillo-
ma virus in p mas of the wild and domestic rabbit. J.
Exp. Med. 106:555-562.
119. Olo, C., Jr., and R. H. Cook. 1951. Cutaneous sarco-
ma-like lesions of the horse caused by the agont of
bovine papilloma. Proc. Soc. Exp. Biol. Med. 77:281-
284.
120. Oson, C., D. E. Godo, M. G. RebM, and K. P. Lao.
1969. Oncogenicity of bovine pillom virus. Arch.
Environ. Hialth 19.827-837.
121. Oblo, C., A. J. Lsedke, ad D. F. Broh1t. 1962. Induced
immunity of skin, vagina, and urinary bladder to bovine
papillomatosis. Cancer Res. 22:463-468.
122. Olson, C., A. M. PamIckl, and D. F. Brebst. 1965.
VOL. 46, 1982
Papilloma-like virus from bovine urinary bladder tumors.
Cancer Res. 25:840-849.
123. Ohs, C., A. M. Pamucka, D. F. Brobst, T. Kowakzyk,
E. J. Slatter, and J. M. Prie. 1959. A urinary bladder
tumor induced by a bovine cutaneous papilloma agent.
Cancer Res. 19:779-782.
124. Olon, C., M. G. RobI, and L. L. Larso. 1968. Cutane-
ous and penile bovine fibropapillomatosis. J. Am. Vet.
Med. Assoc. 153:1189-1194.
125. Obon, C., and L. V. Skkdnore. 1959. Therapy of experi-
mentally produced bovine cutaneous papillomatosis with
vaccines and excision. J. Am. Vet. Med. Assoc.
135:339-343.
126. Orth, G., F. Breitbrd, and M. Favre. 1978. Evidence for
antigenic determinants shared by the structural polypep-
tides of (Shope) rabbit papillomavirus and human papil-
lomavirus type 1. Virology 91:243-255.
127. Orth, G., P. Jeanteur, and 0. Cro_ssat. 1971. Evidence
for and localization of vegetative viral DNA replication
by autographic detection of RNA-DNA hybrids in sec-
tions of tumors induced by Shope papilloma virus. Proc.
Natl. Acad. Sci. U.S.A. 68:1876-1880.
128. Osato, T., and Y. Ito. 1967. In vitro cultivation and
immunofluorescence studies of transplantable carcino-
mas Vx2 and Vx7. Persistence of a Shope virus-related
antigenic substance in the cells of both tumors. J. Exp.
Med. 126:8814886.
129. Osato, T., and Y. Ito. 1968. Immunofluorescence studies
of Shope papilloma virus in cottontail rabbit kidney
tissue cultures. Proc. Soc. Exp. Biol. Med. 128:1205-
1209.
130. Osterhaus, A. D. M. E., D. J. Eles , and M. C. Horzinek.
1977. Identification and characterization of a papilloma-
virus from birds (Frigihlidae). Intervirology 8:351-359.
131. Pamwcku, A. M., S. K. Goksoy, and J. M. Price. 1967.
Urinary bladder neoplasms induced by feeding bracken
fern (Pteris aquilina) to cows. Cancer Res. 27:917-924.
132. Pa, F., M. Niunmra, and J. W. Kreler. 1973. Pro-
longed survival of human skin xenografts on antithymo-
cyte serum-treated mice: failure to produce vurrucae by
inoculation with extracts of human warts. J. Invest.
Dermatol. 61:371-374.
133. Pister, H. 1980. Comparative aspects of papillomatosis,
p. 93-106. In P. A. Bachmann (ed.), Munich symposium
on microbiology. Taylor and Francis, London.
134. POster, H., B. Fink, and C. Thomas. 1981. Extrachromo-
somal bovine papillomavirus type 1 DNA in hamster
fibromas and fibrosarcomas. Virology 115:414-418.
135. Plster, H., L. Glssmann, H. zur Hausen, ad G. Grow.
1980. Characterization of human and bovine papilloma
viruses and of the humoral immune response to papillo-
ma virus infection. 1980. Cold Spring Harbor Conf. Cell
Prolif. 7:249-258.
136. Pllster, H., U. LInz, L. Gs.an, B. Huchthausen, D.
Homnana, and H. zur Hamsen. 1979. Partial characteriza-
tion of a new type of bovine papilloma virus. Virology
96:1-8.
137. Pister, H., and J. Mesaz . 1980. Partial characteriza-
tion of a canine oral papillomavirus. Virology 104:243-
246.
138. Paget, A., M. Favre, and G. Orth. 1975. Induction de
tumeurs fibroblastiques cutanees ob sous-cutanees chez
l'Ochotone afghan (Ochotono rufescens rufescens) par
inoculation du virus du papillome bovin. C. R. Acad.
Sci. Ser. D 280:2813-2816.
ANIMAL PAPILLOMAVIRUSES 207
139. Rnglad, W. L., G. H. Koewn, and J. R. Gorbam. 1966.
An epizootic of equine sarcoid. Nature (London)
210:1399.
140. Ragland, W. L., and G. R. Spencer. 1968. Attempts to
relate bovine papilloma virus to the cause of equine
sarcoid: immmunity to bovine papilloma virus. Am. J. Vet.
Res. 29:1363-1366.
141. Robi, M. G., D. E. Gordon, K. P. Lee, and C. Olson.
1972. Intracranial fibroblastic neoplasms in the hamster
from bovine papilloma viws. Cancer Res. 32:2221-2225.
142. Robl, M. G., and C. Olson. 1968. Oncogenic action of
bovine papilloma virus in hamsters. Cancer Res.
28:1596-1604.
143. Rogers, S. D., J. G. Kidd, and P. Rous. 1960. Relation-
ships of the Shope papilloma virus to cancers it deter-
mines in domestic rabbits. Acta Unio Int. Contra Can-
crum 16:129-130.
144. Ros, P., and J. W. Beard. 1935. The progression to
carcinoma of viws-induced rabbit papilloma (Shope). J.
Exp. Med. 62:523-548.
145. Rowson, K. E. K., and B. W. J. Mahy. 1967. Human
papova (wart) virus. Bacteriol. Rev. 31:110-131.
146. Server, N., P. Gnus, M.-F. Law, G. Khoury, and P. M.
Howley. 1981. Bovine papilloma virus deoxyribonucleic
acid: novel eukaryotic cloning vector. Mol. Cell. Biol.
1:486-496.
147. Shope, R. E. 1933. Infectious papillomatosis of rabbits;
with a note on the histopathology. J. Exp. Med. 58:607-
624.
148. Shope, R. E. 1935. Serial transmission of virus of infec-
tious papillomatosis in domestic rabbits. Proc. Soc. Exp.
Biol. Med. 32:830-832.
149. Shope, R. E., R. Mangold, L. G. McNamara, and K. R.
Dumbell. 1958. An infectious cutaneous fibroma of the
Virginia white-tailed deer (Odocoileus virginianus). J.
Exp. Med. 108:797-802.
150. Spira, G., M. K. Estes, G. R. Dreesnan, J. S. Butel, and
W. E. Rawls. 1974. Papovavirus structural polypeptides:
comparison of human and rabbit papilloma viruses with
simian virus 40. Intervirology 3:220-231.
151. Stevens, J. G., and F. 0. Wettstein. 1979. Multiple copies
of Shope virus DNA are present in cells of benign and
malignant non-virus-producing neoplasms. J. Virol.
30:891-898.
152. Syverton, J. T. 1952. The pathogenesis of the rabbit
papilloma-to-carcinoma sequence. Ann. N. Y. Acad.
Sci. 54:1126-1140.
153. Tajnima, M., D. E. Gordon, and C. Obon. 1968. Electron
microscopy of bovine papilloma and deer fibroma virus-
es. Am. J. Vet. Res. 29:1185-1194.
154. Tboma, M., M. Borlon, J. Taner, J. P. Lev, and J.
Benard. 1964. In vitro transformation of mice cells by
bovine papilloma virus. Nature (London) 202:709-710.
155. Wettsteln, F. O., and J. G. Stevens. 1980. Distribution
and state of viral nucleic acid in tumors induced by
Shope papilloma virus. Cold Spring Harbor Conf. Cell
Prolif. 7:301-307.
156. Wettsteln, F. O., and J. G. Stevens. 1981. Transcription
of the viral genome in papillomas and carcinomas in-
duced by the Shope virus. Virology 109:448-451.
157. Wettteln, F. O., and J. G. Stevens. 1982. Variable-sized
free episomes of Shope papilloma virus DNA are present
in all non-virus-producing neoplasms and integrated epi-
somes are detected in some. Proc. Natl. Acad. Sci.
U.S.A. 79:790-794.