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Assessing the risk potential of porcine circoviruses for xenotransplantation: consensus primer-PCR-based search for a human circovirus.

BACKGROUND: An important issue with respect to virus safety in xenotransplantation is the search for human analogues of porcine viruses, because transmission of a porcine virus followed by recombination with a related human virus may lead to a new emerging virus of unknown pathogenicity, host range and virulence. In case of circoviruses, two types of porcine circovirus (PCV1 and PCV2) are described, but the existence of an analogous human circovirus has not yet been investigated. METHODS: This study describes the analysis of human samples with a consensus primer-PCR approach designed to amplify conserved regions from the rep gene of circoviruses from the genus Circovirus. DNA from human sera, lymph nodes, blood and urine was extracted and investigated with this method that has led previously to the identification of a new avian circovirus. RESULTS: By screening 1101 samples (there of 168 from immunocompromised patients), no evidence for the existence of a human circovirus related to the genus Circovirus was obtained. CONCLUSIONS: This result renders the existence of a human circovirus related to the porcine circoviruses more unlikely, nevertheless the presence of such a virus cannot be ruled out.

Animals↗

Sequence of porcine circovirus DNA: affinities with plant circoviruses.

The complete nucleotide sequence (1759 nt) of the ssDNA genome of porcine circovirus (PCV) was determined from a cloned dsDNA replicative form isolated from PCV-infected cells. Sequence analysis detected no significant nucleic acid or protein similarity with another animal circovirus, chicken anaemia virus (CAV) but, surprisingly, the highest protein similarity was obtained between the product of the largest predicted PCV ORF (ORF1; encoding a potential protein of 35.7 kDa) and a putative protein encoded by the plant circovirus banana bunchy top virus (BBTV). High protein similarity was also detected with the other plant circoviruses subterranean clover stunt virus (SCSV) and coconut foliar decay virus (CFDV). This region of protein identity corresponds with the putative plant circovirus replication-associated protein (Rep). The presence of a nonanucleotide sequence at the apex of a potential-stem loop structure, identical to that found in the plant circoviruses CFDV and SCSV and similar (one mismatch) to that found in the plant circovirus BBTV and in the geminiviruses, suggests that rolling-circle replication may operate during PCV DNA replication. These findings show that PCV is unique in that it bridges the gap between animal and plant circoviruses. The taxonomic relationship of PCV with other members of the Circoviridae is discussed.

Amino Acid Sequence↗

Avian circoviruses of the genus Circovirus: A potential trigger in Pigeon breeder's lung (PBL)/Bird fancier's lung (BFL).

Pigeon breeder's lung (PBL) or Bird fancier's lung (BFL) is one of the most common extrinsic allergic alveolitis or hypersensitivity pneumonitis. It is caused after prolonged inhalation of avian antigens and provokes a hypersensitivity reaction in the lungs of sensitised people. Although the pathogenic mechanism is unclear, the epidemiology of BFL shows that it occurs worldwide, and has been described in adults keeping birds and also in their children. Laboratory findings associated with the disease classified as a type III immunologic reaction that produces blood precipitin antibodies against birds' serum, feathers, intestinal mucin and/or faeces. In particular, the fine dust from pigeon feathers has strong antigenic properties. There is an interaction between host and antigen that seems to be influenced by both genetic and environmental factors. Avian circoviruses (ACV) of the genus Circovirus, has been detected in free-ranging and captive birds worldwide, such as pigeons, canaries, psittacines, Senegal doves, finches, gulls, Australian ravens and geese. T lymphocytes are the main target cells of the ACV and in the above avian species circovirus-like particles were detected in blood, macrophages, feathers, crop secretions, intestinal contents and/or faeces. Most of the ACV was demonstrated that are pantropic and viral antigen in pigeon tissues was most commonly detected in respiratory organs, including the trachea, pharynx and lung. The transmission of the circovirus between the birds usually occurs through inhalation of feathers dust. There is evidence that animal circoviruses may originate when vertebrates become "infected" with DNA from a plant nanovirus. So, it seems that further investigation for the avian circoviruses is needed to determine if they are host specific or not. This study attempts to demonstrate ACV or ACV-like particles as potential triggers in the BFL aetiology, and the possible involvement in BFL's pathogenic mechanism.

Agricultural Workers' Diseases↗

Nucleotide sequence analysis of a novel circovirus of canaries and its relationship to other members of the genus Circovirus of the family Circoviridae.

The circular, single-stranded DNA genome of a novel circovirus of canaries, tentatively named canary circovirus (CaCV), was cloned and sequenced. Sequence analysis indicated that the genome was 1952 nucleotides (nt) in size and had the potential to encode three viral proteins, including the putative capsid and replication-associated (Rep) proteins. The CaCV genome shared greatest sequence similarity (58.3% nt identity) with the newly characterized columbid circovirus (CoCV) and was more distantly related to the two porcine circovirus strains, PCV1 and PCV2, beak and feather disease virus (BFDV) and a recently isolated goose circovirus (GCV) isolate (46.8-50.9% nt identity). In common with other members of the Circovirus genus, several nt structures and amino acid motifs thought to be implicated in virus replication were identified on the putative viral strand. Phylogenetic analysis of both the capsid and Rep protein-coding regions provided further evidence that CaCV is more closely related to CoCV and BFDV and more distantly related to GCV, PCV1 and PCV2.

Amino Acid Sequence↗

Comparison of the structures of three circoviruses: chicken anemia virus, porcine circovirus type 2, and beak and feather disease virus.

Circoviruses are small, nonenveloped icosahedral animal viruses characterized by circular single-stranded DNA genomes. Their genomes are the smallest possessed by animal viruses. Infections with circoviruses, which can lead to economically important diseases, frequently result in virus-induced damage to lymphoid tissue and immunosuppression. Within the family Circoviridae, different genera are distinguished by differences in genomic organization. Thus, Chicken anemia virus is in the genus Gyrovirus, while porcine circoviruses and Beak and feather disease virus belong to the genus CIRCOVIRUS: Little is known about the structures of circoviruses. Accordingly, we investigated the structures of these three viruses with a view to determining whether they are related. Three-dimensional maps computed from electron micrographs showed that all three viruses have a T=1 organization with capsids formed from 60 subunits. Porcine circovirus type 2 and beak and feather disease virus show similar capsid structures with flat pentameric morphological units, whereas chicken anemia virus has stikingly different protruding pentagonal trumpet-shaped units. It thus appears that the structures of viruses in the same genus are related but that those of viruses in different genera are unrelated.

Animals↗

Beak and feather disease virus and porcine circovirus genomes: intermediates between the geminiviruses and plant circoviruses.

Circoviruses are a diverse group of animal and plant pathogens with undefined relationships to one another but for their non-geminate, non-enveloped capsids and circular, single-stranded DNA genomes. The sequences of the beak and feather disease virus and porcine circovirus genomic DNAs are presented and analyzed in the context of the other members of the family. Sequence comparisons, inferred phylogenies, and geographic occurrence suggest that the ambisense circoviruses, particularly the beak and feather disease virus, represent an evolutionary link between the geminiviruses and the plant circoviruses. We propose that the family members be reclassified into three groups: The family Circoviridae consists of the animal pathogens (beak and feather disease virus and porcine circovirus) that possess ambisense genomes with striking similarities to the geminiviruses. The BBTV-like viruses include the plant pathogens (coconut foliar decay virus, banana bunchy top virus, subterranean clover stunt virus) with a geminivirus-like stem-loop element in their DNAs, and single to multiple component genomes. The chicken anemia virus is an unassigned virus possessing unique characteristics bearing little similarity to the other ssDNA viruses.

Amino Acid Sequence↗

Cloning and sequencing of columbid circovirus (coCV), a new circovirus from pigeons.

The complete nucleotide sequence of columbid circovirus (CoCV) isolated from pigeons is described. CoCV was amplified using a consensus primer PCR approach directed against conserved sequences within the rep genes of vertebrate circoviruses. The genome of CoCV is circular and 2037 nt in size. It displays 55% homology to the genome of psittacine beak and feather disease virus and is more distantly related (< 40% homology) to porcine circovirus type 1 and 2. Two major open reading frames were identified, encoding the replicase and the putative capsid protein of CoCV. A region similar to the origin of replication of other circoviruses was found: it encompasses a stem-loop structure with the nonamer 5'-TAGTATTAC, conserved in circo-, nano- and geminiviruses. Phylogenetic analyses suggest classification of CoCV as member of the genus Circovirus of the virus family Circoviridae.

Amino Acid Sequence↗

Psittacine beak and feather disease virus nucleotide sequence analysis and its relationship to porcine circovirus, plant circoviruses, and chicken anaemia virus.

Cloning and sequencing of the circular, single-stranded DNA of one isolate of psittacine beak and feather disease virus (BFDV) demonstrate a genome composed of a circular molecule of 1993 nucleotide bases. An analysis of the assembled replicative form demonstrated seven open reading frames (ORFs) (three in the virion strand and four in the complementary strand), potentially encoding seven viral proteins of >8.7 kDa. High amino acid sequence similarity was demonstrated between a potential 33.3-kDa protein product of ORF1 of BFDV and the replicase-associated protein of porcine circovirus (PCV), subterranean clover stunt virus, and faba bean necrotic yellows virus. However, significant similarity in nucleotide or amino acid sequences was not present between BFDV and chicken anaemia virus. A potential stem-loop structure similar to that found in PCV and plant circoviruses was present in the putative encapsidated strand of the BFDV genome. At the top of this structure, a nonanucleotide motif (TAGTATTAC) similar to that of PCV, plant circoviruses, and geminiviruses also was recognised. Comparison of the deduced amino acid sequences of ORF2 of BFDV and PCV demonstrated 29.1% identity, and in both viruses, ORF2 is located on the complementary strand, beginning close to or within the hairpin stem. Our findings provide further evidence of a close relationship among BFDV, PCV, and plant circoviruses but not chicken anaemia virus.

Amino Acid Sequence↗

Infection studies on human cell lines with porcine circovirus type 1 and porcine circovirus type 2.

BACKGROUND: The lack of human donor organs in allotransplantation has led to a proposal for the use of porcine tissues and organs as alternative therapeutic material for humans. Besides immunological problems like graft rejection, one of the major concerns is the transmission of porcine microorganisms as viruses, bacteria and fungi to a human recipient. METHODS: Human cell lines have been infected with porcine circovirus type 1 (PCV1) and porcine circovirus type 2 (PCV2) to investigate whether PCV can infect and replicate in human epithelial cells and lymphocytes. Infection of PCV1 was observed with 293, Hela and Chang liver cells, infection with PCV2 only in Rd cells. In addition, religated viral DNA of PCV1 and PCV2 has been used to transfect adherent human cell lines. RESULTS: PCV1 persisted in most cell lines without causing any visible changes, while PCV2-transfected cells showed a cytopathogenic effect. Presence of PCV DNA was detected in cells and supernatant by PCR, expression of viral proteins by an indirect immune fluorescence assay. A replication assay showed that the replication of PCV DNA was initiated at the origin of replication. When virus-free cells were inoculated with the supernatant of PCV-infected human cells, the infection was not passed. CONCLUSION: Although PCV gene expression and replication took place in human cells, the infection is non-productive. Alteration of protein localization suggests that protein targeting may be disturbed in human cells.

Animals↗

Porcine circovirus antigens in PK-15 cell line (ATCC CCL-33) and evidence of antibodies to circovirus in Canadian pigs.

Permanent infection of a PK-15 (ATCC CCL-33) cell line by the porcine circovirus was demonstrated. This virus appears to be common in the Canadian swine population as indicated by the presence of antibodies detected by indirect immunofluorescence or immunoperoxidase tests in the serum of culled sows and sows in commercial herds. One source of the PK-15 cell line was found to be free of circovirus antigen.

Animals↗

Infectivity of porcine circovirus 1 and circovirus 2 in primary porcine hepatocyte and kidney cell cultures.

Infectivity of porcine circovirus (PCV) 1 and PCV2 was examined in primary porcine hepatocyte culture by comparing that of PCV in primary kidney cell culture. The virus titer of PCV2-infected hepatocyte cultures was higher than that of the PCV1-infected hepatocyte cultures and the PCV-infected kidney cell cultures. The number of virus-positive cells was most abundant in PCV2-infected hepatocyte cultures as determined by immunohistochemistry and/or in situ hybridization. The results of our data suggest that PCV2 preferably infects cultured hepatocytes as observed in the liver of pigs with postweaning multisystemic wasting syndrome.

Animals↗

Identification of a novel circovirus in Australian ravens (Corvus coronoides) with feather disease.

The complete genome of a novel Circovirus isolated from an Australian raven (Corvus coronoides) with feather lesions similar to those that occur in psittacine beak and feather disease is reported. Degenerate polymerase chain reaction primers were designed to amplify and sequence novel Circovirus DNA from affected feathers. Sequence analysis indicated that the tentatively named raven circovirus (RaCV) was 1898 nucleotides in size with two major open reading frames synonymous with other avian circoviruses, ORF C1 and ORF V1, likely to encode a putative capsid protein (Cap) and replicase-associated protein (Rep), respectively. In common with other circoviruses was the conservation of several nucleotide structures and amino acid motifs implicated in virus replication. Comparison with other members of the Circoviridae demonstrated that RaCV shares the greatest sequence homology with canary circovirus (CaCV) and pigeon circovirus (PiCV) and was more distantly related to the beak and feather disease virus, goose circovirus, duck circovirus and the two porcine circoviruses, PCV1 and PCV2. Phylogenetic analysis of the genome and the putative Cap and Rep proteins provided further evidence of the close relationship of RaCV with CaCV and PiCV.

Amino Acid Sequence↗

Genome of a novel circovirus of starlings, amplified by multiply primed rolling-circle amplification.

The genus Circovirus comprises small non-enveloped viruses with a circular single-stranded DNA genome. By using PCR with degenerate primers, a novel circovirus (starling circovirus, StCV) was detected in spleen samples of wild starlings (Sturnus vulgaris and Sturnus unicolor) found dead during an epidemic outbreak of septicaemic salmonellosis in northeastern Spain. Using a specific PCR, StCV was also detected in apparently healthy birds from the same population. The genome was amplified using multiply primed rolling-circle amplification and cloned. Open reading frames (ORFs) with similarities to the replication-associated protein and the capsid protein of circoviruses as well as an additional ORF encoding a protein of 106 aa were evident from the sequence. Phylogenetic analysis of circovirus genomes revealed the highest degree of similarity (67.1 %) between StCV and canary circovirus. A similar analysis of the evolutionarily conserved cytochrome b gene of the circovirus host species revealed a strict co-evolution of circoviruses with their hosts; however, the circoviruses showed about a threefold higher genetic divergence than their hosts.

Amino Acid Sequence↗

Circovirus-infected geese studied by in situ hybridization.

It has now been established that circovirus infection is common in farmed geese, but little is known about the clinicopathological significance of such infections. Ten clinically diseased geese suspected of being infected by circovirus were studied by in situ hybridization using a goose circovirus DNA probe. Circovirus DNA was demonstrated in the bursa of Fabricius (BF), spleen, thymus, bone marrow, liver, kidney, lung and heart, indicating that infection can be multisystemic. In some birds, virus DNA was present in very large quantities, most notably in the BF, liver and small intestine. With the exception of BF and thymus, there were no histological findings that would have suggested the presence of such quantities of circovirus DNA. In view of the very large quantities of virus DNA labelling present in some tissues, and by analogy to porcine circovirus type 2 infection and psittacine beak and feather virus infections, which are known to cause severe disease, and which have similar virus distribution to that found in our geese, it seems probable that the circovirus was important in the disease manifestations shown by the infected geese.

Animals↗

Genome sequence determinations and analyses of novel circoviruses from goose and pigeon.

The genomes of novel circoviruses from goose and pigeon, which were isolated using degenerate primer and inverse primer PCR methods, were cloned and sequenced. Comparative nucleotide (nt) sequence analyses showed that the goose circovirus (GCV) and pigeon circovirus (PiCV) possessed genomes which were 1821 and 2037 or 2036 nt, respectively, and which had features in common with the genomes of porcine circoviruses types 1 and 2 (PCV1, PCV2) and psittacine beak and feather disease virus (BFDV), such that they can now be assigned to the genus Circovirus of the family Circoviridae. Common features include the possession of (i) a potential stem-loop/nonanucleotide motif with which the initiation of rolling circle replication of the virus DNA is associated; (ii) two major ORFs, located on the virus (V1 ORF) and complementary (C1 ORF) strands, which encode the replication-associated protein (Rep) and capsid protein, respectively; (iii) high levels of amino acid identity (41.2--58.2%) shared with other circovirus Rep proteins; and (iv) direct/inverted repeat sequences within the putative intergenic region. On the basis of nt and amino acid sequence identities, GCV is substantially less closely related to BFDV than PiCV is to BFDV.

Amino Acid Sequence↗

Identification of pig circovirus type 2 in New Zealand pigs.

Interest in porcine circovirus has been stimulated by the recent emergence of postweaning multisystemic wasting syndrome (PMWS) in pigs and the potential use of pig organs for xenotransplantation in humans. Porcine circovirus type 1 (PCV1) is considered to be widespread in pigs but nonpathogenic. Circovirus type 2 (PCV2) is a similar virus but has been differentiated only recently as a separate type. High tissue concentrations of PCV2 are associated with lesions in PMWS cases, but the etiological role of this agent in the disease remains unclear. The presence of PCV1 in New Zealand pigs has been previously reported based on serological data. PMWS has been recently recorded in New Zealand pigs. The epidemiology of PCV2 in New Zealand pigs has not been examined. The purpose of the study was to look for evidence of circoviruses in New Zealand pig herds. Pig circovirus DNA was sought in various tissues using the polymerase chain reaction. Circovirus type 2 was found in New Zealand pig herds, without any evidence that PMWS has ever occurred in these herds. Newborn piglets were shown to have infection, suggesting vertical transmission of the virus.

Aging↗

Avian circovirus diseases: lessons for the study of PMWS.

The diseases associated with psittacine beak and feather disease virus (BFDV), pigeon circovirus (PiCV) and goose circovirus (GoCV), which can be classified with porcine circovirus type 2 (PCV2) as members of the genus Circovirus of the family Circoviridae, have clinico-pathological features in common with post-weaning multisystemic wasting syndrome (PMWS), with which PCV2 infection is causally associated. Intracytoplasmic botryoid inclusions within macrophages and depletion of T and B lymphocytes are common histopathological features, and, in each case, affected animals usually exhibit ill-thrift and a predisposition to secondary infections, that is suggestive of an underlying immunosuppression. Although these avian diseases have been the subjects of relatively little research, their study can provide directly applicable lessons in the areas of diagnosis, epidemiology, pathogenesis and disease control for those charged with investigating PMWS. In keeping with its taxonomic separation as the only member of the genus Gyrovirus, the disease caused by chicken anaemia virus (CAV) differs histopathologically from the other circovirus-associated diseases. Most notably, the target cells of CAV have been identified as haemocytoblasts and precursor T lymphocytes, with lymphocyte depletion, which affects T cells only, occurring in cells directly infected with the virus. Nonetheless, CAV is the best-researched circovirus and provides excellent examples of both virus-induced immunosuppression and virus-virus interactions. The study of CAV-induced disease can therefore provide valuable, if less directly applicable lessons.

Animals↗

Characterization of novel circovirus DNAs associated with wasting syndromes in pigs.

Porcine circovirus (PCV) was initially recognized as a contaminant of continuous pig kidney cell lines and was not thought to be pathogenic. Antibodies reactive to the cell culture isolate of PCV (PCV PK-15) are prevalent in the swine population worldwide. Recently, PCV PK-15-like antigen and nucleic acid were demonstrated in lesions associated with wasting syndromes in pigs in North America and Europe. Monoclonal antibodies raised to circoviruses isolated from pigs with wasting syndromes highlighted differences between these circoviruses and the PCV PK-15 cell culture isolate. This has led to speculation that a new pathogenic PCV may have emerged in the swine populations of several countries. We report the cloning and characterization of novel circovirus DNAs purified from virus isolates made from tissues of North American and European pigs with wasting syndromes. These North American and European circoviruses form a closely related group at the nucleotide sequence level (> 96% intra-group nucleotide sequence identity) but exhibit < 80% nucleotide sequence identity with the PCV PK-15 cell culture isolate. This report provides evidence for a new type of possibly pathogenic PCV. We propose that these new circoviruses should be referred to as PCV2 as opposed to the original PK-15 cell culture isolate, which should be referred to as PCV1.

Amino Acid Sequence↗