213Fenner’s Veterinary Virology. DOI:© 2011 Elsevier Inc. All rights reserved.

10.1016/B978-0-12-375158-4.00011-0

Papillomaviridae and Polyomaviridae

Chapter ContentsMembers of the Family Papillomaviridae 213Properties of Papillomaviruses 213

Classification 213Virion Properties 215Virus Replication 215

Bovine Papillomavirus 218Clinical Features and Epidemiology 218Pathogenesis and Pathology 219Diagnosis 219Immunity, Prevention, and Control 219

Equine Papillomavirus 219Equine Sarcoid 219Canine Papillomavirus 220Feline Papillomavirus 221Papillomaviruses of Other Mammalian Species 221Papillomaviruses of Birds 221Members of the Family Polyomaviridae 221

Avian Polyomaviruses, Including Budgerigar Fledgling Disease Polyomavirus 222

Polyomaviruses of Primates and Laboratory Animals 223Bovine Polyomavirus Infection 223

Viruses in the families Papillomaviridae and Polyomaviridae are taxonomically and biologically distinct, but they share striking similarities in their genome organization, virion structure, and mechanisms of replication, cell cycle regula-tion, and tumor induction.

Papillomaviruses are the cause of papillomas (warts), which have been recognized in animals for centuries: a stable master for the Caliph of Baghdad described equine “warts” in the 9th century. That papillomas have a viral etiology was recognized as long ago as 1907, and in the 1970s it was determined that papillomas in cattle and in other species are caused by several different viruses. In 1935, Peyton Rous observed that benign rabbit papillomas occasionally progressed to carcinomas; this was one of the earliest associations of viruses with cancer. Cutaneous pap-illomas are common in cattle, and less so in other domestic species. Papillomas occur with some frequency in cer-vids (deer, moose, etc.). Young animals preferentially are affected, and in both dogs and goats there is breed predis-position to development of cutaneous papillomas.

Papillomaviruses cannot yet be grown in conventional cell cultures, but their genomes readily can be sequenced with molecular methods, without need for culture. The discovery in the 1980s that specific papillomaviruses are a primary cause of cervical and certain other carcinomas in humans prompted much research on the nature and mechanisms of papilloma-virus-induced oncogenesis, which in turn has advanced under-standing of papillomavirus infections in animals.

Polyomaviruses are ubiquitous, but individual viruses are highly host-species specific. Cell lines contaminated with simian polyomavirus [simian virus 40 (SV40)] were

used to propagate some batches of the original human polio vaccine, which was disconcerting at the time because of the proven oncogenic potential of the SV40 virus in laboratory animals. However, it is now clear that polyoma-viruses are ubiquitous in humans and animals but generally do not cause significant disease even though they often persist in a life-long subclinical state. They may cause rare neurological (progressive multifocal leukoencephalopathy) and renal diseases (polyomavirus nephropathy) in immu-nocompromised hosts, including humans and non-human primates, and may be associated with various types of neoplasia. Pathogenic polyomavirus infections occur with some frequency in psittacine birds.

MeMbers of the faMily PaPillomaviridae

ProPerties oF PaPilloMaviruses

Classification

The family Papillomaviridae, like the family Polyomaviridae, includes viruses with circular double-stranded DNA genomes. The Papillomaviridae currently are divided into some 16 genera (Alpha-, Beta-, Gamma-, Delta-, Epsilon-, Zeta-, Eta-, Theta-, Iota-, Kappa-, Lambda-, Mupa-, Nupa-, Xipa-, Omikron- and Pipapapillomavirus), the member viruses of each being distinguished on the basis of host range, DNA sequence relatedness and genome organization, and their biological properties, including the disease that they cause. The distinction of individual virus types (species)

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is difficult and confusing, which has led to frequent misclas-sification. For example, current taxonomic criteria identify some 100 human papillomavirus types and 10 bovine pap-illomavirus types in at least three different genera, with additional types proposed. There also are several papillo-maviruses of dogs (canine oral papillomavirus and several novel cutaneous canine papillomaviruses), cats, horses, rabbits (cottontail rabbit and rabbit oral papillomaviruses),

birds (chaffinch and psittacine papillomaviruses), and pap-illomaviruses have been described in cetaceans (Phocoena spinipinnis papillomavirus), deer (deer fibroma virus), elk and reindeer, non-human primates, elephant, opossum, and rodents (Mastomys natalensis papillomavirus), although many of these viruses have been only partially characterized (Table 11.1). There is little sequence homology between the genomes of papillomaviruses from different species.

FiGure 11.1 (Left) Atomic rendering of a papillomavirus capsid with combined image reconstructions from electron cryomicroscopy of bovine papillomavirus (BPV) at 9 A resolution with coordinates from the crystal structure of small virus-like particles of the human papillomavirus 16 (HPV-16) L1 protein (Modis, et al., 2002). (Center) Schematic diagram representing the 72 capsomers in a T 7 arrangement of a papillomavirus capsid (the icosahedral structure includes 360 VP1 subunits arranged in 12 pentavalent and 60 hexavalent capsomers). (Right) Negative contrast elec-tron micrograph of human papillomavirus 1 (HPV-1) virions. The bar represents 100 nm.) [From Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses (C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger, L. A. Ball, eds.), p. 239. Copyright © Elsevier (2005), with permission.]

table 11.1 Diseases Caused by Papillomaviruses

Virus Principal Species Affected

Disease

Bovine papillomaviruses 1–10 Cattle Cutaneous fibropapilloma and papilloma, teat papilloma, and intestinal papilloma (type 4)

Horses Sarcoid (bovine papillomaviruses 1, 2)

Feline papillomavirus(es) Cat Cutaneous fibropapilloma (feline sarcoid), plaques and papillomas, squamous cell carcinoma

Ovine papillomavirus Sheep Cutaneous fibropapilloma

Equine papillomaviruses 1, 2 Horses Cutaneous papilloma, aural plaques

Porcine genital papillomavirus Swine Cutaneous papilloma

Canine papillomaviruses 1–4 Dogs Oral papilloma (type 1), endophytic papilloma (type 2), pigmented cutaneous plaques (types 3 and 4)

Deer papillomavirus Deer Fibropapilloma, papilloma, fibroma

Cottontail rabbit papillomavirusa and rabbit papillomavirus

Rabbits Cutaneous papilloma (may become malignant)

Fringilla (finch) papillomavirus Finches Papilloma

Avian papillomavirus Parrots Papilloma

aAlso called Shope papillomavirus.

Chapter | 11 Papillomaviridae and Polyomaviridae 215

The human papillomaviruses exhibit a remarkable genetic diversity. Of the animal papillomaviruses, bovine papilloma-viruses are especially genetically divergent. Like the human papillomaviruses, the properties of individual strains of bovine papillomavirus differ, with some clearly being more capable of inducing neoplasms than others. In addition to cutaneous papillomas and fibropapillomas, bovine papillomaviruses also can induce papillomas of the mucosal lining of the upper gas-trointestinal tract and, in concert with the chemical, quercetin, that is contained in bracken fern, these viruses can induce car-cinomas in both the gastrointestinal and urinary tracts of cat-tle. Papillomaviruses also cause transmissible fibropapillomas of the genitalia of young cattle, with venereal spread analo-gous to that of human genital warts. In dogs, papillomaviruses are the cause of epithelial plaques and papillomas of the skin and mucosal lining of the oral cavity, conjunctiva, and exter-nal genitalia. In horses, they are the cause of papillomas, aural plaques, and sarcoids. Papillomaviruses are the cause of a similar spectrum of lesions in other animal species, although there is considerable variation in the lesions that are most common in each species.

Papillomaviruses can also be categorized according to their tissue tropism and the histologic character of the lesions they cause: for example, some bovine papilloma-viruses (types 3, 4, 6, 9, and 10) and cottontail rabbit papil-lomavirus infect solely keratinocytes and induce squamous papillomas, whereas other bovine papillomaviruses (types 1, 2, and 5) infect both keratinocytes and underlying der-mal fibroblasts to produce fibropapillomas, which consist of proliferative masses of both epithelium and fibrovascu-lar (mesenchymal) tissue. Bovine papillomavirus type 4 can infect the epithelium of the mucosal lining of the upper gastrointestinal tract to produce purely epithelial papillo-mas. In contrast, other papillomaviruses such as deer pap-illomavirus infect mesenchymal cell to induce primarily fibromas, with minimal infection or proliferation of the overlying epidermis. Nevertheless, productive virus repli-cation is restricted to the epithelial component of fibropap-illomas and fibromas.

virion Properties

Papillomavirus virions are non-enveloped, spherical, 55 nm in diameter, with icosahedral symmetry. Virions are composed of 72 hexavalent (six-sided) capsomers arranged in pentameric (five-sided) arrays (Figure 11.1). Both “empty” and “full” virus particles are seen by electron microscopy. The genome consists of a single molecule of circular double-stranded DNA, 6.8–8.4 kb in size. The DNA circle is covalently closed, supercoiled, associated with histones, and is infectious. The genome encodes some 8–10 proteins, two of which (L1 and L2) form the capsid (Figure 11.2). The remainder are non-structural proteins (designated E1–E8, depending on the individual virus) that exert important regulatory and replicative

functions. The genome organization differs between the indi-vidual genera of papillomaviruses, precise details of which are beyond the scope of this text (Figure 11.3).

Papillomaviruses are resistant to diverse environmental insults: infectivity survives lipid solvents and detergents, low pH, and high temperatures.

virus replication

Replication of papillomaviruses is linked intimately to the growth and differentiation of cells in stratified squamous epithelium of the skin and some mucous membranes, from their origin in basal layers to their shedding at the surface. Actively dividing basal cells in the stratum germinativum are infected initially, and maintain the virus in a proviral, possibly latent, state throughout cellular differentiation. Virus-induced hyperplasia, induced by early viral gene products produced during replication, leads to increased basal cell division and delayed maturation of cells in the stratum spinosum and stratum granulosum. These cells become massed into nascent papillomas. Late viral genes encoding capsid proteins are expressed first in cells of the stratum spinosum, and virions appear at this stage of cel-lular differentiation. The accumulation of large numbers of virions and associated cytopathology are most pronounced in the stratum granulosum. Virions are shed with exfoliated cells of the stratum corneum (keratinized layer) of the skin or non-keratinized cells of mucosal surfaces (Figure 11.4).

Virions attach to cellular receptors, enter via receptor- mediated endocytosis, and are transported to various loca-tions, including the endoplasmic reticulum where they are

FiGure 11.2 Diagram of the genome of the bovine papillomavirus (BPV 1). The viral dsDNA (size in bp—7946; origin of replication—ori). Inner arrows indicated the viral proteins encoded by each open reading frame, as well as the direction of transcription (L1, L2—capsid proteins; E1–E8—non-strctural proteins). [From Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses (C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger, L. A. Ball, eds.), p. 240. Copyright © Elsevier (2005), with permission.]

Part | ii Veterinary and Zoonotic Viruses216

completely or partially disassembled. The genome and some viral proteins enter the nucleus, where they are rep-licated. The heparan sulfate proteoglycan, syndecan-3, can serve as a human papillomavirus receptor on dendritic cells, but receptors for other papillomaviruses remain to be thoroughly characterized. During productive infection, transcription of the viral genome is divided into early and late stages. Transcription of early and late coding regions is controlled by separate promoters and occurs on the same DNA strand. First, the half of the genome that contains the early genes is transcribed, forming messenger RNAs (mRNAs) that direct the synthesis of enzymes involved in virus replication and cell regulation. Late mRNAs that direct the synthesis of the structural proteins (L1, L2) that are involved in capsid assembly are transcribed from the

other half of the viral genome after DNA synthesis has begun. The regulated expression of the late (L1, L2) pro-teins of the papillomaviruses occurs only in differentiated epithelial cells, or in differentiating keratinocytes. Progeny DNA molecules serve as additional template, greatly amplifying the production of structural proteins. Several different translational strategies are utilized to enhance the limited coding capacity of the viral genomes.

DNA replication begins at a single unique origin of rep-lication (ori) and proceeds bidirectionally, terminating about 180° away on the circular DNA (Figure 11.2). An initiation complex binds to the origin and unwinds a region (the rep-lication bubble and fork); nascent DNA chains are formed, one strand being synthesized continuously in the direction of unwinding, the other synthesized discontinuously in the

FiGure 11.3 Comparison of the genome organization of the viruses corresponding to the type species of each genus in the family Papillomaviridae. The circular viral dsDNA genomes have been flattened for convenience and the origin of replication (ori) has been taken as the opening site. Similar open reading frames are indicated in similar colors for genes encoding structural (L1, L2) and non-structural proteins (E1–E9), and less well- characterized proteins described for individual viruses (V, W, X, Y). HPV, human papillomavirus; EEPV, European elk papillomavirus; BPV, bovine pap-illomavirus; EcPV, equine (Equus caballus) papillomavirus; FcPV, Fringilla coelebs papillomavirus; PePV, Psittacus erithacus timneh papillomavirus, MnPV, mastomys natalensis papillomavirus, CRPV, cottontail rabbit papillomavirus; COPC, canine oral papillomavirus; PsPV, Phocoena spinipinnis papillomavirus; HaOPV, hamster oral papillomavirus. [From Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses (C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger, L. A. Ball, eds.), p. 241. Copyright © Elsevier (2005), with permission.]

Chapter | 11 Papillomaviridae and Polyomaviridae 217

opposite direction. As replication proceeds, the torsional strain created by the unwinding of the parental strands of DNA is released by the action of a specific viral helicase. Bidirectional replication proceeds around the full genomic DNA circle, at which point the progeny DNA circles separate.

Virions are assembled in the nucleus and are released on cell death, often simply as a consequence of cellu-lar replacement in epithelia through obsolescence. Some cells exhibit a characteristic cytopathic effect, marked by cytoplasmic vacuolization. An infected cell may produce 10,000 to 100,000 virions.

Some papillomaviruses are able to transform cells through the activities of one or more of their non-structural proteins that corrupt normal cellular control signals. Specific strains of papillomavirus express proteins with particular affinity for cellular proteins such as Rb and p53, which are involved in the regulation of the cell cycle and/or apopto-sis. For example, the E6 protein is a key component of the

transforming ability of some bovine papillomaviruses, whereas in other bovine papillomaviruses the E6 protein is absent but its oncogenic functions are expressed by other non-structural viral proteins. The E6 protein is a transcriptional activator, and interacts with and inhibits or degrades a variety of cellular proteins, including the transcription coactivator, CBP/p300, and p53 tumor suppressor. It also interacts with activating protein 1 in trans-Golgi processes, and it blocks the activity of the cellular molecule, paxillin, that contributes to focal adhesions between cells. Papillomavirus proteins also reduce the expression of major histocompatibility complex class I protein through a number of processes, compromising recognition of viral antigens and elimination of virus-infected cells by the immune system.

The host immune response to papillomavirus infection is directed against the virus, affording protective immunity to subsequent infection, and against the virus-induced tumor, resulting in regression of the papilloma or fibropapilloma. This dichotomous response involves separate antigenic

(A)

(B)

(1) (2) (3)

FiGure 11.4 (A) Schematic representation of the events in papillomavirus infection of keratinocytes. (1) The primary infection occurs in a cell of the striatum germinativum, with the virus gaining entry via an abrasion, etc. (2) This results in a proliferating clone of infected cells that spreads laterally in association with virus-induced delay in the maturation of infected cells. (3) Cellular differentiation occurs eventually and large numbers of virions are produced in association with the formation of a papilloma. This is most pronounced in the stratum granulosum. Virions are shed with exfoliated cells of the stratum corneum. (B) Immunoperoxidase staining of papillomavirus antigen in a squamous plaque from a dog. Note intense, focally extensive nuclear staining (brown) of cells in a discrete region of the stratum spinosum (the granular cytoplasmic pigment in the other cell layers is melanin). (A: Courtesy of H. zur Hausen. B: Courtesy of J. Luff, University of California, Davis.)

Part | ii Veterinary and Zoonotic Viruses218

targets, and was initially described by Richard Shope in early studies with rabbit papillomavirus-induced tumors, leading to the first descriptions of “T” (tumor) antigen and tumor-specific transplantation antigens. Antiviral immunity could be induced by vaccination with inactivated virus, but could be circumvented by inoculation of rabbits with infectious DNA (which lacks the viral antigen). However, once rabbits became immune to the tumor, neither virus nor DNA could induce papillomas. The tumor-directed immune response is cell mediated, and results in regression of tumors. Because of this dichotomous response, hosts may be immune to induction of new papillomas, but existing papillomas may continue to flourish, or regress, independent of immunity. These principles pertain to both papillomas and fibropapillomas of several spe-cies, in which regressing tumors typically contain infiltrates of T cells. Once an animal is immune to virus or has undergone tumor regression, it is strongly resistant to reinfection, but that immunity is virus-strain specific. Clinical approaches to papil-loma therapy often continue erroneously to immunize against virus in an effort to induce tumor regression.

Bovine PaPilloMavirus

Papillomas or warts occur more commonly in cattle than in any other domestic animal. All ages can be affected, but the incidence is highest in calves and yearlings. There currently are at least 10 recognized types of bovine pap-illomavirus, and additional types have been proposed. Bovine papillomavirus types 1 and 2 belong to the genus Deltapapillomavirus and exhibit a somewhat broader host range and tissue tropism than other types; they cause fibro-papillomas in cattle and sarcoids in horses. Bovine pap-illomavirus types 3, 4, 6, 9, and 10 belong to the genus Xipapillomavirus; they are restricted to cattle and infect only epithelial cells to induce true papillomas. Bovine papillomavirus types 5 and 8 are members of the genus Epsilonpapillomavirus and appear to cause both fibropap-illomas and true papillomas. Bovine papillomavirus type 7 is currently unclassified. The biological properties of many bovine papillomaviruses remain to be adequately defined, including those of many of the proposed additional types.

Clinical Features and epidemiology

Bovine papillomavirus are probably transmitted between animals by fomites, including contaminated milking equipment, halters, nose leads, grooming and earmarking equipment, rubbing posts and wire fences, and other arti-cles contaminated by contact with affected cattle. Sexual transmission of venereal warts in cattle is likely, as such lesions are rare in animals that are artificially inseminated. The disease is more common in housed cattle than in cat-tle on pasture. Natural bovine papillomavirus infection of horses may occur after exposure of the horses to cattle or to fomites contaminated from exposure to infected cattle.

Surveys for papillomavirus DNA in normal bovine skin and dermal tissues show that the viruses are widespread, most often without any obvious lesions. This apparently latent DNA may be reactivated at sites of injury, leading to papilloma formation. Where disease occurs, the various pap-illomaviruses in cattle are associated with distinct lesions, some in different anatomic locations. Many of the viruses are associated with teat and udder lesions, probably as a result of transmission during milking. Bovine papillomavirus types 1, 2, and 5 infect both mesenchymal and epithelial cells to cause “teat frond” warts, common cutaneous warts, and “rice grain” fibropapillomas. Papillomas have a fibrous core covered to a variable depth with stratified squamous epithelium, the outer layers of which are hyperkeratinized. The lesions vary from small firm nodules to large cauliflower-like growths; they are grayish to black in color and rough and spiny to the touch (Figure 11.5). Large masses are subject to abrasion and may bleed. Fibropapillomas are common on the udder and teats and on the head, neck, and shoulders; they may also occur in the omasum, vagina, vulva, penis, and anus.

In contrast, bovine papillomaviruses 3, 4, 6, 9, and 10 induce epithelial and cutaneous lesions without fibroblast proliferation. Lesions caused by bovine papillomavirus 3 have a tendency to persist, and are usually flat with a broad base, unlike the more usual fibropapillomas that protrude and are often pedunculated. In upland areas of Scotland and northern England, and in other parts of the world where bracken fern (Pteridium aquilinum) or closely related ferns are common, progressive papillomas caused by bovine pap-illomavirus 4 may occur in the alimentary tract, which can progress to squamous cells carcinomas. Although bovine papillomavirus 4 can cause transient papillomas in the ali-mentary tract, ingestion of the bracken fern is a major con-tributing factor, because the carcinogens, mutagens, and immunosuppressive chemicals that it contains are essential for the transition to invasive carcinoma of the alimentary tract. In cattle that eat bracken fern, papillomavirus types 1 and 2 may also contribute to the syndrome of “enzootic hematuria” that is characterized by hematuria and/or

FiGure 11.5 Cattle with papillomas.

Chapter | 11 Papillomaviridae and Polyomaviridae 219

urinary bladder cancer; over half the bladder tumors include both (mixed) mesenchymal and epithelial compo-nents, and the remainder are exclusively of one or the other tissue type. Like fibromas, these tumors exhibit cellular transformation, but do not manifest virus replication.

Pathogenesis and Pathology

Papillomas develop after the introduction of virus through abrasions of the skin, or by activation of viruses already present. Infection of epithelial cells results in hyperplasia, with subsequent degeneration and hyperkeratinization. These changes begin usually 4–6 weeks after exposure. In gen-eral, papillomas persist for 1–6 months before spontaneous (immune-mediated) regression; multiple warts usually regress simultaneously. There is a sequential pattern of papilloma development: stage 1 papillomas appear as slightly raised plaques, starting—at about 4 weeks after exposure—with fibroplasia of the underlying dermis and early epithelial pro-liferation in association with nascent fibroma. Stage 2 fibro-papillomas are characterized by virus-induced cytopathology, virus replication, and crystalline aggregates of virions in the keratinizing epithelium of lesions, starting at about 8 weeks. During this stage, the proliferating epidermis extends inva-sively into the underlying fibroma. Stage 3 fibropapillomas are characterized by fibrotic, pedunculated bases and rough, lobate, or fungiform surfaces, starting after about 12 weeks.

Diagnosis

The clinical appearance of papillomas is characteristic, and laboratory diagnosis is seldom necessary. Virions can be seen by electron microscopic examination but are restricted to the keratinizing epithelium. Polymerase chain reaction (PCR) tests can be readily used to detect papillo-mavirus DNA, and for typing the virus involved, but the common presence of papillomavirus DNA in samples from many clinically unaffected animals makes it important to interpret any positive results with care, and to associate the virus with the clinical disease. Immunohistochemical staining with appropriate antisera is very useful to confirm the presence of papillomavirus antigen within proliferative (“wart-like”) lesions in the skin and mucosal surfaces.

immunity, Prevention, and Control

Prevention and treatment of papillomas are difficult to evaluate, because the disease is self-limiting and its dura-tion varies. Bovine interferon- and photodynamic ther-apy have been used to treat affected cattle, but not widely. Inoculation with homogenized, autologous wart tissue, treated with formalin, has been used for many years, but its efficacy has always been evaluated anecdotally. Vaccination with viral capsid proteins produced by recombinant DNA technology has been encouraging for protection against viral infection, but vaccines must contain several virus

types, because there is no cross protection between types. “Debulking” the overall tumor mass on an affected animal may stimulate general fibropapilloma regression, and the same appears to be true for papillomas in other species.

equine PaPilloMavirus

Equine papillomaviruses are the cause of aural plaques and cutaneous papillomas. Papillomas are usually small, elevated, keratinized lesions that are most common around the lips and noses of young horses, but that also can occur on the ears, eyelids, genitalia, and limbs. They generally regress after 1–9 months. Warts that interfere with the bit, bridle, or other tack can be removed surgically. Congenital equine papillomas have been described.

Aural plaques are discrete, raised, smooth or hyper-keratotic depigmented plaques or nodules on the inner sur-face of the pinnae of the ear. They are neither pruritic nor painful and, unlike papillomas, they do not spontaneously regress. The presence of papillomavirus in these lesions can be confirmed by immunohistochemical staining, PCR assays, or by electron microscopy.

equine sarCoiD

Sarcoid is the most common skin tumor of horses, mules, and donkeys. Sarcoids are most common in horses less than 4 years of age, and may occur singly or in groups, with a predilection for the head, ventral abdomen, and limbs (Figure 11.6A). Although sarcoids are locally aggressive and frequently recur after surgical excision, they are not malignant, and they do not metastasize despite their histo-logical resemblance to that of locally invasive fibrosarco-mas. Specifically, the majority of the tumor consists of an irregular mass of proliferating, haphazardly arranged fibrob-lasts, accompanied by characteristic proliferation of the overlying epidermis with extensions into the dermal mass (Figure 11.6B). Superficial ulceration and secondary trauma are common. On the basis of their appearance, sarcoids have been classified into several types, including a verrucous (wart-like) type, a fibroblastic type, a mixed type, and a flat type.

It has been repeatedly confirmed that distinct variants of bovine papillomavirus types 1 or 2 are present in equine sarcoids, along with their E5 transforming protein that can modulate cellular regulatory signals. Interestingly, bovine papillomavirus DNA also can be detected in the unaffected skin of horses with sarcoids, and sometimes even in the skin of completely unaffected horses. However, transmission tri-als with sarcoid material are usually unsuccessful, and there is no strong evidence that productive replication of bovine papillomaviruses with production of infectious virions occurs in horses. Viral DNA persists episomally (not inte-grated into cellular genome) within the nucleus of infected fibroblasts and is not present within epithelial cells.

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Horses are susceptible to experimental infection with bovine papillomaviruses 1 and 2, and the tumors produced are morphologically similar to sarcoids. Bovine papillomavi-rus DNA sequences have been detected in high copy number by hybridization or quantitative PCR in both experimen-tal and natural lesions. Also, bovine papillomaviruses have been shown to transform equine fibroblasts in vitro. These data, together with the observation that sarcoids can occur in epizootic form, indicate that certain bovine papillomaviruses cause equine sarcoids. However, unlike their natural counter-parts, the induced tumors regress spontaneously, and horses infected experimentally develop antibodies against bovine papillomaviruses, which are absent in horses with naturally occurring sarcoids. Thus much remains to be determined regarding the precise mechanisms whereby specific strains of papillomavirus naturally induce equine sarcoids.

The variable success and hazards of attempted therapies (surgery, laser surgery, radiation, topical drugs) have led to an interest in immunotherapy. Stimulation of cell-mediated responses by the injection of immunopotentiators has met with limited success. Ocular sarcoids have regressed after the injection of viable bacille Calmette–Guérin (BCG) mycobacteria. Experimental vaccines containing mixtures of bovine papillomavirus type 1 virus-like particles and the E7 protein were associated with increased regression of sarcoids, suggesting that therapeutic or protective vaccines may be possible in the future.

Canine PaPilloMavirus

Several genetically distinct canine papillomaviruses have been identified and sequenced, each associated with a spe-cific clinical manifestation—specifically, canine oral pap-illomavirus from oral papillomas, canine papillomavirus 2 from endophytic papillomas, and canine papillomaviruses 3 and 4 associated with pigmented cutaneous plaques.

The most commonly encountered infection is canine oral papillomavirus (genus Lamdapapillomavirus), which is associated with, often multiple, exophytic lesions within the oral mucosa of young dogs. The warts usually begin on the lips, and can spread to the buccal mucosa, tongue, palate, and pharynx (Figure 11.7). They are characterized histologically by epithelial hyperplasia and cytoplasmic vacuolization (koilocytosis, which is a feature of all papil-lomas, regardless of species) with occasional intranuclear viral inclusion bodies. There is a 4–8-week incubation period, and lesions typically regress spontaneously after a further 4–8 weeks. The lesions occasionally become exten-sive and/or may not regress spontaneously, and progression to squamous cell carcinoma occurs very rarely.

Canine cutaneous exophytic and endophytic papillo-mas usually occur as single masses in older, often immu-nosuppressed, dogs. Exophytic papillomas are proliferative

FiGure 11.7 Oral papillomas in a dog. (Courtesy of R. A. Rosychuk, Colorado State University, and S. White, University of California, Davis.)

(A) (B)

FiGure 11.6 Equine sarcoid. (A) Sarcoid on the face of a horse. (B) Histological appearance, with proliferation of dermal fibroblasts and hyperplastic overlying epithelium. (A: Courtesy of H. Hilton, University of California, Davis. B: Courtesy of V. Affolter, University of California, Davis.)

Chapter | 11 Papillomaviridae and Polyomaviridae 221

masses that exhibit acanthosis, koilocytosis, and occa-sional intranuclear viral inclusions, whereas endophytic papillomas are cup-shaped and occur below the level of surrounding normal skin. Progression of these lesions to highly invasive, metastatic squamous cell carcinoma has been described in a research colony of dogs with canine X-linked severe combined immunodeficiency.

Pigmented plaques are heavily pigmented cutaneous lesions that occur most frequently in pug dogs. The lesion is characterized by locally extensive epithelial hyperplasia often lacking distinct viral inclusions, with rare progres-sion to squamous cell carcinoma.

Feline PaPilloMavirus

Papillomavirus sequences can be amplified (using PCR) from the skin of apparently normal, healthy cats. However, viral sequences have been identified in characteristic prolifera-tive cutaneous lesions in both domestic cats and various wild felids, including leopards, bobcats, and panthers. Some of these viral sequences have been designated as Felis domes-ticus papillomavirus type 1 (genus Lambdapapillomavirus), and these viruses are associated with dermal plaques and with squamous cell carcinomas. Viral sequences from other felid species are divergent, and suggest that these viruses may have co-evolved over time with their respective hosts, typically causing little or no disease.

In cats, individual papillomaviruses are variously associ-ated with feline cutaneous fibropapillomas (feline sarcoids), viral plaques in immunosuppressed animals, cutaneous pap-illomas, and, recently, in-situ (early) squamous cell carci-nomas. Feline cutaneous fibropapillomas are characterized by dermal fibroblastic proliferation and occur most often in young cats, on the head, neck, and digits. Partial sequence analysis of a papillomavirus present in these lesions sug-gests that it is a potentially novel virus most closely related to bovine papillomavirus 1. Feline hyperkeratotic viral plaques associated with feline papillomavirus 1 infection are most common in old, immunosuppressed cats. Feline in-situ carcinoma, also referred to as multicentric squamous cell carcinoma in situ, appears as multiple cutaneous, often pigmented, plaques that rarely invade the underlying dermis; genetically distinct strains of papillomavirus have been iden-tified in these lesions. Recently, a portion of a human papillo-mavirus 9 was amplified from a feline cutaneous papilloma.

PaPilloMaviruses oF other MaMMalian sPeCies

Classic studies on viral oncogenesis were carried out in the late 1930s with the Shope rabbit papillomavirus. Papillomas caused by this virus often progress to carcinomas both in their natural host, the cottontail rabbit (Sylvilagus spp.), and in laboratory rabbits infected experimentally. Virus replica-tion, however, only occurs in the natural host.

Oral papillomatosis occurs naturally in domestic rab-bits (Oryctolagus cuniculus); the tumors are small, gray-white, filiform or pedunculated nodules (5 mm in diameter) and are localized mostly on the underside of the tongue. The causative papillomavirus is distinct from the Shope rabbit papillomavirus (cottontail rabbit papillomavirus). Experimentally, oral papillomatosis has been reproduced in various rabbit species, and in nature it is widespread among domestic rabbits, particularly young animals. Virus spread in animal rooms seems not to occur, but transmis-sion from the mother to offspring during the suckling period is common. Oral papillomas of rabbits show no ten-dency to malignancy, and may persist for many months.

Papillomaviruses are remarkably absent in rodents, despite concerted efforts to find murine variants to serve as models in the laboratory. Exceptions are the papilloma-viruses in the “multimammate rat” or African soft-furred rat (Mastomys natalensis) and the European harvest mouse (Micromys minutus). Because of the strong species spe-cificity of papillomaviruses, these cannot be transmitted to laboratory mice or rats.

PaPilloMaviruses oF BirDs

The Fringilla (finch) papillomavirus causes papillomas in wild common chaffinch (Fringilla coelebs), brambling (Fringilla montifringilla) and Eurasian bullfinch (Pyrrhula pyrrhula). Infection with a second papillomavirus-like virus has been described in African grey parrot (Psittacus erithacus). Both viruses have been demonstrated in papil-lomatous lesions in their respective host species. In finches, papillomas occur exclusively on the toes and distal legs, and show stages of development from a slight node on a digit to heavy involvement of the foot and adjacent regions, with obscuring of the individual digits and resulting overgrowth and distortion of the claws. In severe cases the tumor may account for up to 5% of the bird’s total body weight, but affected birds seem to remain in good condition otherwise. Cutaneous papillomas have been described in various psit-tacine birds, but many of these lesions are associated with herpesvirus infections and not papillomaviruses.

MeMbers of the faMily PolyomaviridaePolyomaviruses have highly restricted host ranges, and these viruses typically cause life-long, inapparent infec-tions in their respective hosts. The oncogenic potential of polyomaviruses is only evident when they are inocu-lated experimentally into heterologous hosts or their cells in culture. The family Polyomaviridae contains a single genus, Polyomavirus, which includes viruses identified in: humans; non-human primates (African green monkey, baboon, stump-tail and rhesus macaque); rodents, includ-ing mice (polyomavirus and K virus), hamsters (hamster

Part | ii Veterinary and Zoonotic Viruses222

polyomavirus), and rats (rat polyomavirus); rabbits (rabbit kidney vacuolating agent); birds (avian polyomaviruses, including budgerigar fledgling disease polyomavirus); cat-tle (bovine polyomavirus); equine (equine polyomavirus). The avian polyomaviruses segregate independently of the mammalian ones on the basis of genome sequence analy-ses, indicating long-standing evolutionary divergence that may result in their being included in different genera in the future. Polyomaviruses of veterinary importance occur in laboratory animals and birds.

The genome organization, virion structure, and replica-tion strategy of polyomaviruses is generally similar to that of papillomaviruses (Table 11.2). Virions (40–45 nm) and the genome (approximately 5 kb) of polyomaviruses are slightly smaller than those of papillomaviruses, and their replication is analogous to that of papillomaviruses, except that transcription of coding regions occurs on opposite DNA strands in the case of polyomaviruses and on the same strand with papillomaviruses. Like the papillomaviruses, polyoma-viruses regulate the cell cycle and can transform infected cells through the activities of one or more of their non-structural proteins (so-called T proteins). Transformation of infected cells occurs in some instances by specific inactiva-tion of the cellular p53 tumor suppressor gene that normally limits infection by inducing apoptosis of virus-infected cells.

Reactivation of persistent, latent polyomavirus infec-tions can occur as a consequence of immunosuppression, regardless of the cause, potentially as a result of mutations in the transcriptional control region of the viral genome. Reactivation of virus in the kidney leads to shedding of virus in the urine of infected humans.

avian PolyoMaviruses, inCluDinG BuDGeriGar FleDGlinG Disease PolyoMavirus

Polyomaviruses have been identified in numerous species of birds, although most infections are not associated with clinical disease. The avian polyomaviruses are all related, but there is considerable genetic diversity amongst them. Disease syndromes associated with polyomavirus infec-tions in birds include increased mortality in a variety of young captive psittacine birds [lovebirds (Agapornis spp.), macaws (Ara spp.), conures (Aratinga and Pyrrhula spp.), ring-necked parakeets (Psittacula krameri), caiques (Pionites spp.), Eclectus parrots (Eclectus roratus), occa-sionally nestlings of Amazon parrots (Amazona spp.) and cockatoos (Cacatua spp.)], and an acute generalized dis-ease in fledgling budgerigars (Melopsittacus undulatus). So-called budgerigar fledgling disease polyomavirus may also be responsible for “French molt,” a milder disease of budgerigars that results in chronic disorders of feather formation. Subclinical polyomavirus infection has been described in European raptors, zebra finches (Poephila gut-tata), Ross’s turaco (Musophaga rossae) and a kookaburra (Dacelo novaeguineae). The only species shown to be infected in its natural setting is the sulfur-crested cockatoo (Cacatua gallerita) in Australia. A polyomavirus has been described in 3–16-week-old domestic geese with bloody diarrhea, subcutaneous hemorrhage, tremors, ataxia, and variable mortality. An avian polyomavirus of unknown significance has been isolated from wild buzzards (Buteo buteo) and Eurasian kestrel (Falco tinnunculus).

Avian polyomavirus-associated diseases have repeat-edly been reported from captive populations. Infection and disease are widespread in budgerigars and lovebirds, with mortality rates ranging between 30 and 80% of infected nestlings. The virus can be shed in the feces for up to 6 months. Affected birds typically die suddenly with mini-mal clinical warning, but they briefly manifest weakness, pallor, subcutaneous hemorrhages, anorexia, dehydration, inappetence, and crop stasis. Two additional, but less fre-quent presentations are described. The first is an acute form of disease that results in birds that present with gener-alized edema and ascites, full crops, reddened skin, feather dystrophy, and acute death. Gross lesions in affected birds include hydropericardium, cardiomegaly (enlarged heart), hepatomegaly (enlarged liver), and scattered cutaneous hemorrhages. Histologic lesions include focal necrosis in several organs, including the liver, with characteristic clear-to-light basophilic intranuclear inclusions in cells adjacent to necrotic foci. Polyomavirus virions can be visualized by electron microscopy in the nuclei of epithelial cells—for example, in renal tubules. The second atypical form occurs mostly in cockatoo, which have reduced weight gains, and develop interstitial pneumonia and pulmonary edema.

table 11.2 Properties of Papillomaviridae and Polyomaviridae

Virions are non-enveloped, spherical in outline, with icosahedral symmetry. Virions are 55 nm (Papillomaviridae) or 45 nm (Polyomaviridae) in diameter

The genome consists of a single molecule of circular double-stranded DNA, 6.8–8.4 kbp (Papillomaviridae) or 5 kbp (Polyomaviridae) in size. The DNA has covalently closed ends, is circular and supercoiled, and is infectious

Members of both families replicate in nucleus; members of the Polyomaviridae grow in cultured cells; most members of the Papillomaviridae have not been grown in conventional cultured cells, but will transform cultured cells; infectious virions produced only in terminally differentiated epithelial cells

During replication, Polyomaviridae DNA is transcribed from both strands, whereas Papillomaviridae DNA is transcribed from one strand

Integrated (Polyomaviridae) or episomal (Papillomaviridae) DNA may be oncogenic

Chapter | 11 Papillomaviridae and Polyomaviridae 223

PolyoMaviruses oF PriMates anD laBoratory aniMals

Progressive multifocal leukoencephalopathy caused by sim-ian polyomaviruses (SV40) occurs in immunosuppressed non-human primates, typically rhesus macaques (Macaca mulatta) infected with simian immunodeficiency virus. The same disease occurs in some humans with acquired immu-nodeficiency syndrome, but is generally associated with a distinct human polyomavirus, JC virus.

Polyomavirus infections involve a number of common laboratory animal species, including mice, rats, hamsters, and rabbits. Polyomavirus of mice is the namesake virus for this family, but its previous status as the prototype virus for the family has been usurped by SV40 virus. Components of the T antigen genes of polyomavirus are often used in transgenic constructs in order to induce transformation. Polyomavirus biology is well understood in the mouse. Young mice are globally immunodeficient at birth and, when experimentally inoculated with polyomavirus as neonates, they develop multisystemic infections with cytolytic rep-lication of the virus in several organs. If the mice survive, foci of transformed cells arise, resulting in the evolution of many (poly) types of tumor (oma), including tumors of mes-enchyme as well as epithelium. Tumors of skin resemble papillomas, with virus replication in the keratinizing epithe-lium, but, otherwise, tumors do not contain replicating virus. Under natural conditions, neonatal mice in enzootically infected populations are protected from infection by mater-nal antibody, and acquire infection when maternal antibody is waning. Under these circumstances, infection is subclini-cal, without tumor induction, and may be persistent, with chronic shedding of virus in the urine. This feature of sub-clinical persistent urinary shedding is common among many species of polyomaviruses.

The mouse is host to another polyomavirus, known as K virus, or Kilham’s virus, and more recently erroneously named as “murine pneumotropic virus.” K virus is not truly pneumotropic, but rather may induce pulmonary edema and hemorrhage as a result of its tropism for, and cytolytic replication in, vascular epithelium. This only occurs when mice develop disseminated infections as immunodeficient neonates. Like polyomavirus of mice, K virus may be chron-ically shed in the urine, but, unlike polyomavirus of mice, it has no known oncogenic activity. Both polyomavirus and K virus are essentially non-existent as naturally occur-ring infections in contemporary mouse colonies. However, because polyomavirus continues to be studied experimen-tally, it has occasionally been the cause of iatrogenic con-tamination of laboratory mice. This is most significant in athymic nude (T-cell-deficient) mice, which have been found to develop neoplasia following natural exposure.

Both laboratory and pet Syrian hamsters (Mesocricetus auratus) and European hamsters (Cricetus cricetus) have

been found to be infected with a hamster polyomavirus of unknown origin, which probably arose in Eastern Europe when wild European hamsters were mixed with laboratory Syrian hamsters. Hamster polyomavirus is biologically similar to most other polyomaviruses, with initial multisys-temic cytolytic infection, tumor induction in immunodefi-cient animals, and chronic infection with shedding of virus in the urine. This cycle is enhanced in the hamster, because Syrian hamsters are highly inbred, with an undefined cel-lular immune deficiency which renders them susceptible to the virus and to tumor induction at all ages. When initially introduced to a naïve population of hamsters, hamster polyomavirus will induce massive epizootics of transmis-sible lymphomas among young hamsters, generally arising initially in the mesenteric lymph nodes, but involving sev-eral organs. These epizootics can be devastating, and have resulted in the loss of several valuable inbred lines of labo-ratory hamsters. When infection becomes enzootic within the population, the prevalence of lymphomas decreases, and animals often manifest multiple cutaneous epithelio-mas. As in polyomavirus of mice, lymphomas represent transformed cells without virus replication, whereas virus replication occurs in the keratinizing epithelium of epithe-liomas. This feature initially resulted in the erroneous clas-sification of the hamster agent as a papillomavirus.

Other common laboratory animals may also incur polyomavirus infections. One of the earliest polyoma- viruses to be discovered was the rabbit kidney vacuolating agent, which induced cytopathic change in cottontail rabbit kidney cultures. Thus the virus is indigenous to Sylvilagus spp., but polyomavirus-like intranuclear inclusion bodies may also be found in renal tubules of Oryctolagus spp. rab-bits, suggesting the presence of a similar virus in this species. Polyomaviruses are clinically inconsequential in rabbits.

Finally, laboratory rats may be infected with an as yet to be characterized polyomavirus, which has been manifest clinically on several occasions in athymic nude rats with respiratory disease. These animals have robust intranuclear inclusion bodies in the respiratory epithelium and lungs, as well as salivary glands, but not involving the kidneys. This observation parallels the recent discovery of human polyo-maviruses in children with respiratory disease (KI and WU viruses), and in adults with Merkel cell carcinomas (MC virus).

Bovine PolyoMavirus inFeCtion

Bovine polyomavirus is frequently present in bovine sera, especially fetal and neonatal calf sera. Furthermore, cross-reacting antibodies have been detected in the serum of a high proportion of veterinarians in some regions. However, despite the ubiquitous presence of this virus in cattle, no disease has been associated with bovine polyomavirus infection, and its significance remains uncertain.