DNA VIRAL INFECTIONS

Adenovirus Infection

Adenoviral enteritis has been documented in commer­cial rabbits in Hungary. Peak losses occurred at 6-8 weeks of age. Profuse diarrhea was observed in severely affected animals, with low mortality.

Herpesvirus Infections

There are 4 known herpesviruses of rabbits, known as leporid herpesvirus (LHV) -1, -2, -3, and -4. LHV-1, -2, and -3 belong to the subfamily Gammaherpesvirinae, whereas LHV-4 belongs to the genus Simplex of the subfamily Alphaherpesvirinae. In addition, rabbits are naturally susceptible to Human herpesvirus 1 (Herpes sim­plex 1). Among the 4 rabbit herpesviruses, LHV-1 (cot­tontail herpesvirus) and LHV-3 (Herpesvirus sylvilagus) are indigenous to Sylvilagus rabbits and do not infect Oryctolagus rabbits. They are covered in this chapter because they are traditionally listed among rabbit viruses. LHV-2 and LHV-4 naturally infect domestic rabbits, but only LHV-4 has clinical significance.

Leporid Herpesvirus 1 and 3 Infection

Both LHV-1 and LHV-3 were isolated from primary kidney cell cultures of weanling cottontail (Sylvilagus) rabbits. LHV-3 enjoyed a brief period of interest as a model of human Epstein-Barr virus infection.

Leporid Herpesvirus 2 Infection

LHV-2 has also been termed “Virus III" and Herpesvirus cuniculi. The virus was originally discovered during serial testicular transfer following inoculation of blood from a human varicella patient, and subsequently isolated from rabbit cell kidney cultures. It appears to be a subclinical infection among seropositive rabbits under natural con­ditions, but experimental intracerebral inoculation results in nonsuppurative encephalitis with intranuclear inclusion bodies.

Leporid Herpesvirus 4 Infection

Systemic infections with a LHV-4 have been observed in several commercial rabbitries in Canada. Outbreaks have mysteriously occurred among isolated rabbit popula­tions, as well as following introduction of outside rabbits to rabbitries. The disease, which affects animals of vari­ous ages, is characterized by sudden onset with mortality up to 30%, with highest mortality among young rabbits.

Animals are frequently found dead, with no previous evidence of illness. Clinical signs include anorexia, con­junctivitis, periocular and facial swelling, subcutaneous swellings, respiratory distress, diarrhea, and abortion.

FIG. 6.6. Integument from a rabbit with leporid herpesvirus 4 infection, depicting necrosis of the epidermis and papillary dermis with underlying dermal hemorrhage. (Source: Brash et al. 2010. Reproduced with permission from Canadian Veterinary Medical Association.)

FIG. 6.7. Leporid herpesvirus 4 infection of the skin from a rabbit, depicting a multinucleate syncytium and intranuclear inclusion bodies (arrows) in the follicular epithelium.

Herpes Simplex Virus Infection

For decades, domestic rabbits have served as an animal model for experimental Human herpesvirus-1 (Herpes sim­plex-1 virus) encephalitis. Sporadic naturally occurring cases of fatal encephalitis due to H. simplex virus infec­tion have been observed in pet rabbits. Affected rabbits may present with conjunctivitis (Fig. 6.8) and neurologic signs. Necropsy reveals nonsuppurative meningo­encephalitis with neuronal necrosis and prominent amphophilic intranuclear inclusion bodies in neurons and astroglial cells (Fig. 6.9). Lesions in other organs have not been described. Typical herpesvirus particles have been observed in affected cells by electron micros­copy, and H. simplex-1 has been confirmed as the

FIG. 6.8. Acute conjunctivitis in a rabbit naturally infected with herpes simplex virus infection following transmission from an infected human. (Source: Muller et al. 2009. Reproduced with permission from the American Veterinary Medical Association.)

FIG. 6.9. Cerebrum from a rabbit with naturally acquired herpes simplex virus encephalitis. Note the prominent intranuclear inclusion bodies in neurons (arrows) and astroglia.

causative agent. Contact with humans infected with H. simplex virus has been documented in most reported cases in rabbits.

Lapine Parvovirus Infection

A parvovirus has been isolated from clinically normal rabbits in Japan. In a serological survey of commercial rabbits, approximately 60% of animals evaluated had antibody to lapine parvovirus. Oral or intravenous inoc­ulation of 1 month old rabbits with lapine parvovirus resulted in transient depression and anorexia, with no mortality. The virus could be isolated from a variety of organs for up to 2 weeks postinoculation and from the small intestine on day 30 postinoculation.

Necrotic hepatitis associated with a parvovirus was reported in Mexico in 1989. Rabbits also had splenic necrosis, and microinfarctions in the myocardium, kid­ney, and lung believed to be associated with intra­vascular coagulation. These lesions were reminiscent of rabbit hemorrhagic disease (RHD), but affected rabbits also had severe segmental necrosis of the small intestinal crypts and villi, which is not a feature of RHD. Further­more, liver lesions contained intranuclear paracrystal­line arrays of parvoviral-like virions by electron microscopy, and intranuclear staining with monoclonal antibodies against porcine parvovirus and murine par­vovirus (MVM) was present. No other reports of this syndrome have been published, and this case may have been due to activation of parvovirus during the course of an RHD epizootic.

Papillomavirus Infections

Papilloma viruses were once classified as members of the family Papoviridae that included 2 genera, Papillomavi­ruses and Polyomaviruses, which differ in size, biology, and DNA sequence. That family has been eliminated and papilloma viruses are now classified within the family Papillomaviridae. Rabbits can be infected with 2 papil­loma viruses, cottontail rabbit papillomavirus (CRPV) and rabbit oral papilloma virus (ROPV), both of which belong to the genus Kappapapillomavirus. Both of these viruses are used as experimental models of papilloma virus pathogenesis and immunity in the laboratory rabbit.

Cottontail Rabbit Papillomavirus (CRPV) Infection

CRPV, which is also known as Shope papilloma virus, is native to New World Sylvilagus spp.

Epizootiology and Pathogenesis

Primarily a benign disease of cottontail rabbits, papillo­matosis due to CRPV has occurred in spontaneous out­breaks among domestic rabbits. Insect vectors are the usual means of mechanical spread of the virus from cottontail rabbits to domestic rabbits, and ticks are important for infection among Sylvilagus rabbits. When the CRPV is inoculated into Oryctolagus rabbits, it produces papillomas with a high incidence (75%) of progression to squamous cell carcinomas. Transforma­tion to carcinomas also occurs in Sylvilagus, but to a lesser degree. Papillomas induced by this virus in Oryc- tolagus rabbits produce minimal or no infectious virus, in contrast to papillomas induced in the natural Sylvilagus host or in hares (Lepus spp.). Papillomas typically undergo immune-mediated resolution if they do not progress to carcinomas. Host immunity has been shown to have 2 distinct targets: virus structural antigens, invoking protection against virus reinfection, and tumor antigens, invoking papilloma regression. If rabbits are immunized against the virus, viral immunity does not affect papilloma status, and rabbits remain susceptible to induction of papillomas with infectious DNA. Once tumors have undergone immune-mediated regression, rabbits resist both virus and DNA challenge. Virus DNA can remain latent, with no histologic changes in the epidermis, until nonspecific irritants activate the forma­tion of papillomas.

Pathology

Naturally occurring papillomas on Orycolagus rabbits occur most frequently on the eyelids and ears, which are sites most often accessed by insects. Papillomas

FIG. 6.10. Multiple cutaneous papillomas (horns) projecting from

the lips of a Sylvilagus rabbit naturally infected with cottontail rabbit papilloma virus (CRPV). (Source: Flicker © C Forry wd45/364229280.)

consist of a pedunculated, cornified surface overlying a fleshy central core. Papillomas in Sylvilagus rabbits vary in size and number, up to numerous large cutaneous horns that debilitate the host (Fig. 6.10). Papillomas due to CRPV, which arise in naturally infected Oryctolagus rabbits, tend to be small and few in number (Fig. 6.11). Histologic findings are consistent with squamous papil­loma. In those tumors that progress to malignancy, squamous cell carcinomas are locally invasive as well as metastatic to regional lymph nodes and lung. Differ­ential diagnosis must discriminate from spontaneous nonviral papillomas, which tend to arise in haired skin.

Rabbit Oral Papillomavirus (ROPV) Infection

ROPV is distinct from CRPV. The natural host of ROPV is the Oryctolagus rabbit. Like CRPV, ROPV is also used as an experimental model for papilloma virus pathogenesis

FIG. 6.11. Solitary papilloma on the ear of a laboratory rabbit naturally infected with cottontail rabbit papilloma virus (CRPV). Since CRPV papillomas arise in an abnormal host (domestic rabbit), these tumors do not support virus replication and are prone to malignant transformation.

FIG. 6.12. Oral papillomas involving the tongue and gingiva of a

juvenile New Zealand White rabbit naturally infected with rabbit oral papilloma virus. These lesions undergo regression with no tendency toward malignancy.

and immunity. Natural infection is common among domestic rabbits, and infection is spread by direct con­tact, including from does to their kits.

Lesions most frequently occur in rabbits between 2 and 18 months of age. Pedunculated and sessile lesions are most commonly found along the ventral aspect of the tongue (Fig. 6.12), but may also involve other areas of oral and labial mucosa. These papillomas usually regress spontaneously within a few weeks. They are typical squamous papillomas on microscopic examina­tion. Persistent papillomas of the conjunctiva have also been attributed to ROPV. Basophilic intranuclear inclu­sions and viral antigen may be present in the stratum spinosum. There are no reports of malignant transfor­mation of oral papillomas in rabbits. Viral DNA can be detected in oral swabs in rabbits without lesions, latent virus remains in epithelium following papilloma regres­sion, and latent virus can be activated by nonspecific injury.

Polyomavirus Infection

Cottontail rabbits are frequently subclinically infected with rabbit kidney vacuolating virus, a nononcogenic member of the Polyomaviridae family. The virus was isolated from CRPV papillomas inoculated into primary kidney cultures derived from Sylvilagus rabbits. This virus has no known pathogenic effect in cottontail or domes­tic rabbits but can be a contaminant of papilloma virus stocks. Seroconversion to the virus has been found in Sylvilagus rabbits, but antibodies have not been detected against the virus in domestic rabbits. Intranuclear inclu­sions in renal tubular epithelium, consistent with

FIG. 6.13. Intranuclear inclusion body (arrow), found as an incidental finding in the renal tubular epithelium of an adult New Zealand White rabbit. These inclusions are suspected to be due to an uncharacterized polyomavirus.

polyomavirus-like inclusions, may be seen as an inci­dental finding in domestic rabbits (Fig. 6.13), but their etiology has not been defined.

Poxvirus Infections

Rabbits are host to several poxviruses with considerable clinical significance. The family Poxviridae is divided into 2 subfamilies, Entomopoxvirinae (which infect insects) and Chordopoxvirinae (which infect mam­mals). The latter contains the Leporipoxvirus genus, which includes the lagomorph pathogens known as myxoma, rabbit fibroma, and hare fibroma viruses. These viruses, particularly myxoma and rabbit fibroma viruses, are closely related and are represented by a number of strains with an overlapping spectrum of virulence. Another Leporipoxvirus is squirrel fibroma virus, which is antigenically and genetically closely related to, but distinct from myxoma and rabbit fibroma viruses. Finally, rabbitpox virus is closely related to vaccinia virus in the Orthopox genus. A comprehensive review of poxviral infections of rabbits is available in Brabb and DiGiacomo (2012).

Myxoma Virus Infection: Myxomatosis

Myxomatosis was first recognized in laboratory Orycto- lagus rabbits acquired for experiments in a South Ameri­can laboratory in the late 19th century. The name “infectious myxomatosis” was used to denote the myx­oid appearance of the subcutaneous masses associated with the disease. In the original cases described, the virus was believed to have been transmitted by insect vectors from the relatively resistant tropical forest rabbit (S. braziliensis). Myxomatosis was also recognized in North America in 1930, where outbreaks of the disease occurred in rabbitries in southern California. The brush rabbit (S. bachmani) has been implicated as the reservoir host in that area. Myxomatosis is enzootic in the western

United States, with sporadic cases spreading into domes­tic rabbits, and is also endemic in wild Sylvilagus and feral Oryctolagus rabbits in South and Central America. Around 1950, a highly virulent South American strain of myxoma virus was introduced into Australia in an effort to reduce (or eliminate) the overpopulation of feral Oryctolagus cuniculus, which had become a major eco­nomic problem in that country. Mortality rates of up to 99% subsequently dropped to around 25% within a few years. The dramatic reduction in mortality was related to natural selection for genetically resistant rabbits and the emergence of attenuated strains of the virus. In 1953, myxoma virus was released into France by a citizen who was disenchanted with the wild rabbit problem. The virus subsequently spread to other countries in western Europe, including England, where it is now a well-established enzootic among wild Oryctolagus rabbits. Transmission of the virus is usually mechanical and by arthropod vectors, primarily mosquitoes in the Americas and Australia and fleas in Europe, but also by direct contact and fomites. There is considerable variation in virulence among myxoma virus strains that have evolved from these events. Attenuated strains of myxoma virus and rabbit fibroma virus are used as live vaccines in Europe to protect commercially raised rabbits.

Pathology

Disease severity is highly variable, depending upon virus strain and host species. Sylvilagus rabbits (natural hosts) tend to be resistant, and develop localized cutaneous lesions similar to those induced by fibroma viruses. Following inoculation of Oryctolagus rabbits by an arthropod vector, viral replication results in the devel­opment of a primary subcutaneous myxoid mass, usu­ally within 3-4 days. Within 6-8 days, mucopurulent conjunctivitis, facial edema, and multiple cutaneous skin masses (myxomas) are usually observed (Fig. 6.14), often involving the base of the ears. Swelling of the anogenital region is also common, with scrotal edema. In rabbits that die with a peracute form of the disease, the animal may be found dead, and other than redness of the conjunctiva, there may be no other

FIG. 6.14. Mucopurulent conjunctivitis, periocular swelling, and facial edema in a rabbit with myxomatosis.

FIG. 6.15. Skin of a rabbit with myxomatosis. Note the blue­staining mucoid material in the superficial dermis.

evidence of disease. Microscopically, there is dermal and subcutaneous proliferation of large, stellate mesen­chymal cells (myxoma cells) interspersed within a homogeneous mucinoid matrix (Fig. 6.15), with a sprin­kling of inflammatory cells. Hypertrophy and prolifera­tion of endothelial cells occur, and changes in the epithelium overlying the lesions may vary from hyper­plasia to degeneration. Intracytoplasmic inclusions are often present in the affected epidermis and in epithelial cells of the conjunctiva (Fig. 6.16), and in some cases may be found in respiratory epithelium. Remarkably, systemic changes are minimal, except lymphoid tissue. These include proliferation of alveolar epithelium, pul­monary hemorrhage, and orchitis in bucks. Myxoma virus is T lymphocytotropic, and viremic dissemination involves lymphocytes and monocytes, as well as rapid local dissemination to draining lymph nodes. Lymph nodes are initially enlarged with formation of syncytia

FIG. 6.16. Intracytoplasmic inclusion bodies (arrows) in the conjunctival epithelium of a rabbit with myxomatosis.

FIG. 6.17. Lymph node of a rabbit with myxomatosis, depicting hypertrophy and hyperplasia of stellate myxoma cells and severe lymphocytic depletion.

among apoptotic lymphoid cells, followed by marked lymphocytic depletion, particularly the T-cell zones. Affected lymph nodes, and to a lesser extent spleen, have hypertrophy and hyperplasia of stellate myxoma cells (Fig. 6.17), as well as focal necrosis, hemorrhage, and proliferative vasculitis. The cause of death in myxomato­sis has never been fully defined, and suggested to be related to cytokine-mediated shock. Limited analysis of natural cases suggests that acute sepsis may contribute to mortality due to massive immunodeficiency. Mild disease may occur in rabbits inoculated with live attenuated vaccine strains, or partially immune-vaccinated rabbits exposed to myxoma virus. Under these circumstances, rabbits may develop nodular fibromatous masses of the conjunctiva.

"Amyxomatous" myxomatosis is also recognized, in which rabbits have minimal or small skin nodules, and present with respiratory signs. Rabbits develop pneumo­nia, and pox inclusions may be visualized in respiratory epithelium (Fig. 6.18). Presumably, this form of myxo­matosis is spread through respiratory contact.

FIG. 6.18. Intracytoplasmic inclusion bodies (arrows) in the respiratory epithelium of a rabbit with “amyxomatous” myxomatosis. (Source: S. Diab, University of California, Davis, CA. Reproduced with permission from S. Diab.)

reservoir host, with spread by insect vectors. In naturally infected cottontail and European rabbits, firm, flattened tumors occur on the legs and feet (Fig. 6.19), sometimes with involvement of the muzzle, periorbital, and peri­neal regions. These cutaneous tumors may be up to 7 cm in diameter, are usually freely movable, and may persist for several months, and then undergo spontaneous res­olution. In young rabbits, fibromatous lesions may arise in abdominal viscera and bone marrow. There is local­ized fibroblast proliferation, with mononuclear and polymorphonuclear cell infiltration. Affected fibroblasts are characteristically fusiform to polygonal. In European domestic rabbits, subcutaneous masses may vary from myxoid in type to typical fibromas. Large, intracytoplas- mic, eosinophilic inclusion bodies may be present in fibromatous cells (Fig. 6.20) and in epidermal cells over­lying the tumors. The typical gross and histological appearance of the circumscribed masses should

Rabbit Fibroma Virus Infection

Rabbit fibroma virus, also known as Shope fibroma virus, is closely related antigenically to myxoma virus and to the hare and squirrel fibroma viruses. Shope fibroma virus was first isolated from a cottontail rabbit (S. flori- danus) in the United States in 1932. The virus is trans­missible to Oryctolagus rabbits, producing localized fibromas. Shope fibroma virus infections are relatively widespread in wild cottontail rabbits in the eastern United States and Canada. Infection is usually a benign, self-limiting disease in the wildlife population. The virus may persist for several months within lesions, and mechanical transmission by arthropod vectors appears to be the primary means of spread. In rare occasions, fibromatosis has been diagnosed in commercial rabbit- ries. Wild cottontail rabbits in the area are the likely

FIG. 6.19. Nodular fibroma on the forelimb of a Sylvilagus rabbit

naturally infected with Shope rabbit fibroma virus.

FIG. 6.20. Skin of a Sylvilagus rabbit infected with Shope fibroma virus. There is a dense network of fusiform to polyhedral fibroblasts in the dermis, many of which contain prominent intracytoplasmic inclusion bodies (arrows).

facilitate the differentiation of rabbit fibromatosis from myxomatosis and from the raised, horny, epidermal growths seen in papillomatosis. However, the myxoid forms sometimes seen histologically can be confused with myxomatosis. Considerable variation in micro­scopic appearance may occur.

Hare Fibroma Virus Infection

Hare fibroma virus is closely related to rabbit fibroma virus and myxoma virus and is an infection of European hares. Hare fibroma virus has been documented in Euro­pean hares prior to the introduction of myxoma virus to Europe, and outbreaks have been documented in cap­tive-bred European hares. Fibromatous lesions, 1-3 cm in diameter, typically arise on the ears and legs of affected hares, and spontaneously regress in adult hares. Lesions consist of large spindle and stellate cells with prominent intracytoplasmic inclusions. Virus is trans­mitted by insects and contact. Myxoma virus can also infect hares, resulting in formation of firm nodular cutaneous masses of the head, back, and limbs, which resemble hare fibroma virus-induced lesions.

Rabbitpox (Vaccinia) Virus Infection

Rabbit pox is relatively rare and has generally been associated with inadvertent exposure to vaccinia virus. Recent sequence analysis of rabbitpox virus has con­firmed its close relationship to vaccinia virus. During outbreaks of rabbit pox, virus is highly contagious. It is readily transmitted by aerosol, with the respiratory tract being the primary site of replication, followed by viremic dissemination. Papular lesions may occur in the oro­pharynx, respiratory tract, spleen, and liver. In the "pockless" form, a few pocks were present in the oral cavity, and focal hepatic necrosis, pleuritis, and spleno­megaly were observed. Histologic changes include dis­seminated focal necrosis with leukocytic infiltration in the skin and affected viscera, as well as necrosis of lymphoid tissue. Because of the similarity to smallpox, rabbitpox virus has been used as a model of variola virus infection, thereby emphasizing the possibility of future outbreaks due to the marked ability of natural spread among susceptible rabbits.