237Fenner’s Veterinary Virology. DOI:© Elsevier Inc. All rights reserved.2011

10.1016/B978-0-12-375158-4.00013-4

Circoviridae

Chapter ContentsProperties of Circoviruses 237

Classification 237Virion Properties 237Virus Replication 239

Beak and Feather Disease Virus 239Clinical Features and Epidemiology 239Pathogenesis and Pathology 239Diagnosis 240Immunity, Prevention, and Control 240

Other Avian Circoviruses 240

Porcine Circoviruses 240Clinical Features and Epidemiology 240Pathogenesis and Pathology 241Diagnosis 241Immunity, Prevention, and Control 241

Chicken Anemia Virus 242Clinical Features and Epidemiology 242Pathogenesis and Pathology 242Diagnosis 242Immunity, Prevention, and Control 242

The family Circoviridae includes viruses with circular sin-gle-stranded DNA genomes, and which share common phys-icochemical and genomic properties. Together with members of the family Parvoviridae, these are the smallest known DNA viruses of vertebrates, and have similarities to single-stranded DNA viruses of plants. The family Circoviridae includes important pathogens of birds and swine. Torque teno viruses are genetically heterogeneous single-stranded DNA viruses that are morphologically similar to circo-viruses, but which are classified in a free-standing genus, Anellovirus, because of other distinctive characteristics (see Chapter 32).

ProPerties of CirCoviruses

Classification

The member viruses of the family Circoviridae have some-what similar virion and genome properties, but are eco-logically, biologically, and antigenically quite distinct. The family currently contains two genera (Circovirus, Gyrovirus). Porcine circovirus 1 is the type species of the genus Circovirus, members of which use an ambisense genome strategy, with viral genes in different orientations. The genus Circovirus includes beak and feather disease virus, canary circovirus, goose circovirus, pigeon circovi-rus, and porcine circoviruses 1 and 2. Chicken anemia virus is the type (and only) member of the genus Gyrovirus, in which the viral genes are all in the same orientation.

virion Properties

The properties of circovirus virions are summarized in Table 13.1. They are small (approximately 20–25 nm in diameter), non-enveloped, spherical in outline, with T 1 icosahedral symmetry. Virions are made up of 60 capsid sub-units that package the viral circular single-stranded DNA. Virions of individual circoviruses differ in surface structure, with chicken anemia virus having 12 trumpet-like struc-tures that are less obvious in the other circoviruses (Figure 13.1). Mature virions often appear in infected cells and free

Chapter 13

Table 13.1 Properties of Circoviruses

Virions are small (20–25 nm), non-enveloped, spherical in outline, with icosahedral symmetry

Mature virions can be seen in infected cells as well as in linear arrays in cell-free diagnostic specimens

The genome consists of a single molecule of circular (covalently closed ends) single-stranded ambisense (genus Circovirus) or positive-sense (genus Gyrovirus) DNA, 1.7–2.3 kb in size

Chicken anemia virus encodes a protein (VP3) that induces apoptosis in chicken lymphocytes (apoptin)

Replication takes place in the nucleus of cycling cells, producing large intranuclear inclusion bodies

Virions are very stable, resisting 60°C for 30 minutes and pH 3 to pH 9

PArt | ii Veterinary and Zoonotic Viruses238

in diagnostic specimens and in linear “strings of pearls” in cell-free diagnostic specimens. The genome consists of a single molecule of circular (covalently closed ends) single-stranded ambisense (genus Circovirus) or negative sense (genus Gyrovirus) DNA, approximately 1.7–2.3 kb in size.

Beak and feather disease virus, porcine circoviruses 1 and 2, and the other members of the genus Circovirus utilize an ambisense transcription strategy—that is, some genes are encoded in the viral sense DNA and others in the complementary strand (Figure 13.2). Beak and feather disease virus has three open reading frames and porcine circovirus has four; in each case there is one major capsid

protein. In contrast, the genes of chicken anemia virus, genus Gyrovirus, are all encoded in the complementary positive-sense DNA strand that is transcribed to give a single polycistronic transcript (Figure 13.3); however, the existence of minor spliced transcripts was also recently described. Chicken anemia virus has three open reading frames, one of which encodes the major capsid protein (VP1) that is present in virions. Another virus-encoded protein (VP3), termed apoptin, induces apoptosis of

figure 13.1 (Left upper) Cryo-electron microscopy image of a particle of an isolate of chicken anemia virus. A structural model comprising 60 subunits (T1) arranged in 12 protruding pentagonal trumpet-shaped units pentameric rings has been proposed. (Left lower) Cryo-electron microscopy image of a particle of an isolate of porcine circovirus 2. A structural model comprising 60 subunits (T1) arranged in 12 flat pentameric morpho-logical units has been proposed. (Right) Negative contrast electron microscopy of particles of an isolate of chicken anemia virus (black arrow) and beak and feather disease virus (BFDV) (white arrow), stained with uranyl acetate. The bar represents 20 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. 327. Copyright © Elsevier (2005), with permission.]

figure 13.2 Genome organization of an isolate of porcine circovirus 1 (PCV-1). The origin of replication is located between the start sites of the two major, divergently-arranged ORFs, cap and rep (thick arrows). The cap gene, encoding the capsid protein (CP), is expressed from a spliced transcript, and the rep gene directs the synthesis of two distinct pro-teins, Rep and Rep’, using differentially-spliced transcripts. [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. 329. Copyright © Elsevier (2005), with permission.]

figure 13.3 Genome organization of chicken anemia virus (CAV). The unspliced CAV transcript (5-3) contains three partially overlapping ORFs, which are expressed in CAV-infected cells. The non-transcribed region possesses promoter-enhancer activity. Open reading frame (ORF) 1 (cap gene) encodes the capsid protein VP1; ORF2 encodes VP2, a protein phosphatase, and ORF3 encodes VP3 also known as apoptin. [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. 332. Copyright © Elsevier (2005), with permission.]

Chapter | 13 Circoviridae 239

T lymphocytes and is probably important to the pathogen-esis of infections in chickens.

These viruses are all very stable in the environment; they are not inactivated by heating at 60°C for 30 minutes, are resistant to many disinfectants, and may require long exposure to efficacious chemicals.

virus replication

The receptors responsible for cellular attachment of circoviruses are uncertain, but some circoviruses hemag-glutinate erythrocytes and thus they are likely to bind to sialic acid on the cell surface. Virus particles are taken up by endocytosis, although the specific mechanisms also are not well understood. Viral DNA replication occurs in the nucleus and requires cellular proteins and other components produced during the S phase of the cell cycle. Replication of the genome is believed to occur via a rolling circle that originates at a stem-loop structure. Three distinct proteins are produced during replication of chicken anemia virus.

A major feature of the circoviruses that determines their pathogenesis is the requirement for dividing cells to facilitate their DNA replication, thus virus replication typi-cally is maximized in actively dividing cells in the tissues of young animals. Similarly, replication of porcine circo-virus 2 in swine is enhanced during periods of immune stimulation that result in proliferation of lymphocytes in which the virus can replicate. Circoviruses typically cause persistent infections of their respective hosts, although the mechanisms responsible are poorly characterized, as the viruses persist despite apparently robust host antiviral immune responses. In the case of chicken anemia virus, virus replication in the oviduct of chickens may be regu-lated by estrogen, and hence is differentially stimulated, particularly during egg laying, to allow more efficient ver-tical transmission. The apoptin protein of chicken anemia virus may itself cause destruction of infected lymphocytes, and hence promote a relative immune suppression that favors virus persistence.

beAk And feAther diseAse virus

It had long been known that many species of Australian parrots undergo permanent loss of feathers and develop beak and claw deformities when in captivity. In 1984, thin-section electron microscopic examination of affected tis-sues from such birds revealed large numbers of virions that resembled the previously described porcine circovirus 1. More recently, similar viruses have been identified in most species of parrots and related psittacine birds, and viruses of this type or their DNA have been detected in many other birds, including canaries, ostriches, pigeons, ducks, geese, finches, gulls, ravens, and starlings.

Clinical features and epidemiology

Many infections with these circoviruses are mild or sub-clinical. Where it occurs, beak and feather disease is a debilitating disease of cockatoos, parrots, and budgerigars, although is principally a disease of cockatoos. Natural infection occurs primarily in birds less than 5 years of age, most often in young birds during first feather formation. Typical findings include feather loss, abnormal pin feath-ers (constricted, clubbed, or stunted), abnormal mature feathers (retention of sheaths, blood in shaft, fracture of rachis), and various beak abnormalities (Figure 13.4). The beaks of affected birds are variously described as being shiny, overgrown, or broken, exhibiting delaminations, or with palatine necrosis. Birds may have feather lesions, beak lesions, or both. Severe leukopenia and non-regenerative anemia have been reported in some parrots, but usually without feather lesions.

Pathogenesis and Pathology

The disease can be reproduced experimentally by expos-ing psittacine birds to homogenates of feather follicles from affected birds. The virus replicates in the basal epithelial layer of the feather follicles, beak, and claw. Basophilic intracytoplasmic (“botryoid”) inclusions occur in follicular epithelium, which by electron microscopy contain masses of virions. Inclusions also occur in macrophages and the epithelium of the cloacal bursa, but as a consequence of phagocytosis and not virus replication. Lymphoid depletion occurs, perhaps as a result of indirect effects of the infec-tion. The disease is progressive; some birds die after the first appearance of malformed feathers or beak abnormalities, whereas, if cared for, others may live for months or years in a featherless state. Infection can result in persistent immu-nosuppression, so that affected birds are often also affected by other (secondary) viral, fungal, or bacterial infections.

figure 13.4 Beak and feather disease in a cockatoo. (Courtesy of L. Lowenstine, University of California, Davis.)

PArt | ii Veterinary and Zoonotic Viruses240

diagnosis

Diagnosis of beak and feather disease is made on the basis of clinical signs and signalment, and the presence of char-acteristic basophilic intracytoplasmic inclusion bodies as determined by histopathologic examination of biopsy specimens of affected feather follicles. The presence of virus can be confirmed using electron microscopy, immu-nohistochemical staining with virus-specific antisera, or demonstration of circovirus genome by polymerase chain reaction (PCR), which can detect viral DNA in feather tips, blood, biopsy samples, or swabs.

immunity, Prevention, and Control

The contagious nature of beak and feather disease and its persistent, progressive course may lead to requests for euthanasia of infected birds. Beak and feather disease virus is highly prevalent as a consequence of subclinical infections in many birds and, as a result, eradication of the virus is difficult once it is present in a colony. Strict hygiene, screening protocols, and lengthy quarantines are used in cockatoo and other affected breeding colonies to prevent introduction of the virus. The virus persists and is shed by adult birds, and virus transmission can be via either vertical or horizontal routes. Antibodies are protec-tive, but vaccines are not available because the virus has yet to be propagated in cell-culture systems. However, experimental vaccines have been developed that uti-lize either preparations of virus recovered directly from affected birds or capsid protein alone expressed from recombinant baculoviruses.

other AviAn CirCoviruses

Circovirus infections have been reported in a variety of wild and domestic avian species, including pigeons, canar-ies, geese, ducks, and ostriches, typically causing immuno-suppression and developmental abnormalities. Most cases are in young birds. Infected pigeons may manifest poor performance, diarrhea and ill thrift, but feather lesions are rare in racing pigeons. In contrast, infected doves may exhibit feather loss. Atrophy of the cloacal bursa is com-mon and results in immunosuppression. Mulard ducks have feather dystrophy, growth retardation, and mortality throughout rearing. Canaries have abdominal distension and failure to thrive. Circoviruses are implicated in, but not proven to cause, a fading chick syndrome in ostriches, characterized by listlessness, anorexia, and diarrhea.

PorCine CirCoviruses

Porcine circovirus 1 was first isolated in Germany in 1974 from a pig kidney cell line (PK15) persistently infected

with this virus. Initial serologic studies suggested that the virus was widespread in all tested swine populations; how-ever, at least some of this apparent seropositivity to porcine circovirus 1 may represent cross-reactive antibodies to the replicase protein, which is highly conserved between por-cine circoviruses 1 and 2. More recent studies, using more specific serological tests, indicate that porcine circovirus 1 infection is not as highly prevalent in pigs as first thought. Of various animals tested, only domestic swine, mini-pigs, and wild boars have antibodies. Porcine circovirus 1 is con-sidered to be apathogenic in swine; however, it has been isolated from stillborn piglets. Porcine circovirus 2 is an antigenically distinct virus that was first isolated in France in 1997. Subsequent studies have clearly shown the virus was present in pigs long before that time, as determined by the presence of porcine circovirus 2 capsid-specific anti-bodies and the virus itself in archival tissues and sera. The virus has been isolated in most regions of the world where swine are raised, including North America, Asia, Europe, and Oceania. Global isolates of porcine circovirus 2 are quite similar (96% identical), and they are distinct (80% homology) from porcine circovirus 1, primarily on the basis of differences in the capsid proteins.

The potential pathogenic significance of porcine circo-virus 2 was quickly recognized following its initial iden-tification. Porcine circovirus 2 is associated with several disease syndromes, collectively designated porcine-circo-virus-associated disease, which occur most commonly in weanling piglets at 7–15 weeks of age, but sometimes also in adults. Two genetically distinct subgroups of porcine circovirus 2 are recognized, each of which can be further subdivided into subgroups based on their DNA sequences, which may or may not be associated with disease expres-sion. Although retrospective studies have clearly shown that porcine circovirus 2 infection has been present in swine populations for many years, for as yet undetermined reasons both the frequency and clinical severity of infec-tions appear to have increased dramatically since 1997.

Clinical features and epidemiology

Porcine circovirus 2 strains are widespread in most pig populations, and it is clear that infections are often sub-clinical or very mild. Transmission occurs through direct contact and fomite transmission, with virus being shed in the feces, respiratory secretions, and urine. Vertical trans-mission occurs in swine, although maternal antibodies pro-tect piglets against infection.

Porcine circovirus infections can be associated with substantial mortality (up to 50%) and disease occurrence in pig-rearing enterprises. Porcine circovirus 2 has been associated with a remarkable variety of disease syndromes, including postweaning multisystemic wasting syndrome, porcine dermatitis and nephropathy syndrome, porcine

Chapter | 13 Circoviridae 241

respiratory disease complex, reproductive failure, granu-lomatous enteritis, exudative epidermitis, and necrotiz-ing lymphadenitis. The precise role of porcine circovirus 2 infection in the pathogenesis of each of the porcine cir-covirus associated diseases remains to be clearly defined. The porcine respiratory disease complex, for instance, is typically manifest as bronchointerstitial pneumonia asso-ciated with combinations of pathogens, including Myco-plasma hyopneumoniae and other viral infections; however, abundant porcine circovirus 2 antigen can be detected in the lesions in some cases.

Pathogenesis and Pathology

The expression of clinical disease in pigs infected with por-cine circovirus typically involves secondary microbial infec-tions that may directly or indirectly influence the type of disease expressed. Infections that appear to enhance the repli-cation and pathogenicity of porcine circovirus 2 include por-cine parvovirus, swine influenza virus, porcine reproductive and respiratory syndrome virus, and M. hyopneumoniae, but other agents also might predispose. For example, torque teno viruses have recently been implicated as potentially contrib-uting to the pathogenesis of porcine circovirus associated dis-ease. It appears that the common feature of those infections is immune activation, which somehow enhances the replication of porcine circovirus 2 in a variety of target cells. Indeed, immune stimulation alone (without any associated infection) can promote the replication of porcine circovirus 2. However, immune suppression by corticosteroids may also result in increased expression of disease, thus the pathogenesis of por-cine circovirus associated diseases is highly complex.

The porcine circovirus associated disease identified as postweaning multisystemic wasting syndrome is characterized by individual to coalescing foci of granulomatous inflamma-tion in lymphoid tissues, lungs, liver, kidney, heart, and intes-tines, sometimes with prominent “botryoid” inclusion bodies

in virus-infected macrophages (Figure 13.5). Porcine der-matitis nephropathy syndrome has also been associated with porcine circovirus 2 infection, and is further characterized by infarctive (ischemic necrosis) skin lesions, particularly on the rear legs, and the kidneys of affected pigs exhibit vasculitis and glomerulonephritis; however, porcine circovirus 2 anti-gens or nucleic acid are rarely demonstrated in these lesions. The pathogenesis of the various porcine circovirus associated disease syndromes is not well characterized, including the role of co-infecting pathogens and immune-mediated mechanisms of tissue injury.

diagnosis

Because porcine circovirus 2 is widespread in pig popu-lations and often causes subclinical infections, diagnosis requires careful interpretation to determine the specific role of the virus in any diseases that occur. Assessment of the extent of infection in individual swine by quantitation of the number and distribution of virus-infected cells by immunohistochemistry, and/or viral load by quantitative PCR, is critical to interpretation.

immunity, Prevention, and Control

Control of porcine circovirus associated diseases should involve several approaches, including general manage-ment practices to limit both circovirus infections and those caused by other, presumably “secondary,” pathogens that can act as triggers for enhanced replication of porcine cir-covirus 2. Good nutrition and hygiene are critical, as is disinfection to prevent transmission of the virus between groups. Inactivated or baculovirus-expressed virus-like particles that include the capsid protein of the virus are available as vaccines, and new generation chimeric vac-cines have been developed that utilize the non-pathogenic porcine circovirus 1 as a genetic backbone for expression

(A) (B)

figure 13.5 Porcine circovirus infection. (A) Macrophages with “botryoid” inclusions. (B) Paracrystalline viral array in inclusions. (Courtesy of D. Imai, University of California, Davis.)

PArt | ii Veterinary and Zoonotic Viruses242

of the immunogenic capsid protein of porcine circovirus 2. Vaccines can be effective in reducing viral load and subse-quent shedding, and they can significantly reduce porcine circovirus 2 associated disease and mortality.

ChiCken AnemiA virus

Chicken anemia virus associated disease was first recog-nized in Japan in 1979, although it is not a new virus and had probably been present in chickens for many years. Infection occurs worldwide and is a problem in all coun-tries with industrial poultry industries. The virus is not known to infect birds other than chickens, and only a single serotype has been recognized, although low levels of vari-ation have been reported among virus isolates both within and between countries.

Clinical features and epidemiology

Chicken anemia virus is transmitted horizontally by direct contact and contaminated fomites. The virus is also trans-mitted vertically through the egg. Horizontal transmission is through inhalation or oral exposure, and virus is shed in feces and feather dander. Breeder flocks may become infected before they begin to lay fertile eggs, and virus subsequently is transmitted vertically for as long as the hen is viremic. If hens are seropositive, maternal antibody generally protects chicks from disease, but not from infection. Many flocks of otherwise specific-pathogen-free chickens carry chicken ane-mia virus, and it is often difficult to eradicate the virus once it is present.

Chicken anemia virus causes an acute, immunosuppres-sive disease of young chickens, characterized by anorexia, lethargy, depression, anemia, atrophy or hypoplasia of lym-phoid organs, cutaneous, subcutaneous, and intramuscular hemorrhages, and increased mortality. Disease occurs in chicks hatched to asymptomatically infected breeder hens that have been infected before egg laying. At 2–3 weeks of age the chicks become anorectic, lethargic, depressed, and pale. They are anemic, and develop bone marrow apla-sia and atrophy of the thymus, cloacal bursa, and spleen. Disease is most severe in chicks that are co-infected with other viruses such as avian reoviruses, avian adenoviruses, reticuloendotheliosis virus, Marek’s disease virus, or infec-tious bursal disease virus. There is usually no illness or loss of egg production when adult chickens are infected, but as the infected birds can become chronically or persist-ently infected, transmission can occur both horizontally and vertically.

Pathogenesis and Pathology

When 1-day-old susceptible chicks are inoculated with chicken anemia virus, viremia occurs within 24 hours and

virus can be recovered from most organs and rectal con-tents for up to 35 days. The virus infects hemocytoblasts, causing pancytopenia evident as anemia, leukocytopenia, and thrombocytopenia. Packed cell volumes are low, and blood smears often reveal anemia and leukopenia. Blood may be watery and clot slowly as a consequence of throm-bocytopenia. Mortality rates usually are low (10% or less), but may be higher than 50%. Histologically, there is deple-tion of lymphoid cells in all lymphoid organs and panmy-elophthisis of bone marrow. Secondary bacterial infection is common. Age resistance to disease (but not infection) begins at about 1 week of age and is complete by 2 weeks after hatching. However, protective effects of maternal anti-body and age resistance can be overcome where there is co-infection with other immunosuppressive viruses. Infection with chicken anemia virus also can suppress the immune system of chickens, and dual infections involving the virus and other avian pathogens are often more severe than would otherwise be expected.

diagnosis

Diagnosis of chicken anemia virus infection in chickens is based on history, clinical signs, and gross and microscopic pathologic findings. Viral DNA is readily detected by PCR and virus isolation can be done in MDCC-MSB1 cells (a lymphoblastoid cell line derived from Marek’s disease tumors), 1-day-old chicks, or chick embryos (which must be virus- and antibody-negative). Because the virus is non-cytopathic when first isolated, immunologic methods must be used to identify its presence. Methods for serological identification of chicken anemia virus infection include enzyme-linked immunosorbent assay, indirect immuno-fluorescence, and virus neutralization.

immunity, Prevention, and Control

Immunity to chicken anemia virus is complex. Neutralizing antibodies are protective against disease, but do not com-pletely protect chickens against infection or result in virus clearance. The presence of antibodies in breeders greatly reduces vertical as well as horizontal transmission. Several commercial vaccines are available and are mainly used in broiler breeders. Maternal antibodies and controlled exposure are primary methods for control in broilers.

Young breeder hens may be infected deliberately with wild-type virus by adding crude homogenates of tissues from affected chickens to drinking water. This ensures infection and seroconversion before hens begin to lay eggs, but is not recommended because it can maintain high levels of virus in the population. Because severe disease results from co-infection with immunosuppressive viruses such as Marek’s disease virus, control of these other pathogens also is important.