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.     

Molecular characterization of novel circoviruses from finch and gull.

The purpose of this study was to molecularly characterize circoviruses that infect finches and gulls. Circovirus-specific DNAs were isolated using polymerase chain reaction methods from bursa of Fabricius tissues from a Gouldian finch (Chloebia gouldiae) and a herring gull (Larus argentatus) that were known to be circovirus-infected. Nucleotide sequence determination and analysis of cloned genomic DNAs showed that these circoviruses represented novel members of the genus Circovirus of the family Circoviridae, and have been tentatively named Finch circovirus (FiCV) and Gull Circovirus (GuCV). Both new circoviruses shared genome organizational features with previously characterized circoviruses, such that both contained two major, inversely-arranged open reading frames encoding the putative replication-associated and capsid proteins, and both contained a potential stem-loop and nonanucleotide motif. Phylogenetic analyses based on genome nucleotide sequences and involving the seven additional genus members indicated that FiCV and GuCV were more closely related to canary circovirus, beak and feather disease virus and pigeon circovirus, and that FiCV and canary circovirus were the most closely related avian circoviruses. Pairwise comparisons showed that the capsid proteins of FiCV and GuCV shared highest amino acid identity values with those of canary circovirus (62.0%) and pigeon circovirus (40.6%), respectively. The 5' intergenic region of GuCV was longer (207 nucleotides) and contained more direct and inverse repeated sequences than those of other circoviruses, while the 3' intergenic region of FiCV was notable in being longer (307 nucleotides) than its counterparts in other circoviruses and in containing two long repeats of 77 nucleotides.     

Pigeon circoviruses from feral pigeons in Australia demonstrate extensive recombination and genetic admixture with other circoviruses

ABSTRACT

Like other avian circovirus species, Pigeon circovirus (PiCV) is known to be genetically diverse with a relatively small circular single-stranded DNA genome of 2?kb that encodes for a capsid protein (Cap) and a replication initiator protein (Rep). Recent paleoviral evidence hints towards a probable Gondwanan origin of avian circoviruses, paralleling the evolution and dispersal of their hosts. Limited availability of PiCV genome sequence data in Australia has hindered phylogeographic studies in this species, so we screened clinically normal rock doves (Columba livia) in regional New South Wales, and demonstrated a high prevalence (76%) of PiCV infection by PCR. We also recovered 12 complete novel PiCV genomes and phylogenetic analyses revealed that PiCV circulating in Australian feral pigeons formed two strongly supported monophyletic clades. One clade resided with PiCV genomes from Poland, Australia, United Kingdom, Belgium, China, and Japan, and another basal clade was more closely related to PiCV genomes from Poland. A novel more distantly-related PiCV rep gene formed a solitary clade with weak posterior support. So we further analysed all selected partial rep gene sequences to demonstrate a likely naturally occurring spillover infection from a passerine circovirus candidate. The findings suggest that there is a high degree of genetic variation within PiCV in Columbiformes with potential greater admixture between avian circoviruses within Australia than previously known.

RESEARCH HIGHLIGHTS

• Confirmed high prevalence rate of PiCV circulating in Australian wild pigeons.

• Highlighted extensive recombination events within Australian PiCV.

• Demonstrated a likely naturally occurring spillover infection from a passerine circovirus candidate.

     

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

Summary.  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. Accepted June 20, 2000 Received May 18, 2000     

Identification and whole-genome characterization of a novel Porcine Circovirus 3 subtype b strain from swine populations in Vietnam

Porcine circovirus 3 (PCV3) is a novel circovirus detected in pigs suffering from porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, and multisystemic infection. In this study, we identified PCV3 infection in aborted fetuses and reported the full-length genome sequence of a PCV3 strain identified from southern Vietnam. The complete genome of this PCV3 strain is 2000 nucleotides in length. We found that it shares 98.5–99.25% sequence identity with other reference sequences and that it clusters with the PCV3b subtype. Several specific mutated sites were found to be unique to this Vietnamese PCV3b strain, including I14M in the Rep protein and K139R, I150F, and P169T in the Cap protein. The sequence data that have been made publically available as part of this study will help investigators to better understand the molecular characteristics, genetic diversity, and evolutionary history of PCV3. Careful and in-depth investigations into the epidemiology, pathogenicity, and the evolution of this novel virus is a matter of urgent economic and agricultural interest in Vietnam.

     

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

Summary.  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. Received March 3, 1998 Accepted April 4, 1998     

Circoviruses: Immunosuppressive threats to avian species: A review

Circoviruses are small, non-enveloped, icosahedral viruses that are unique among animal viruses in having circular, single-stranded DNA genomes. Their genomes are also the smallest possessed by animal viruses. The circovirus family currently comprises three members, chicken anaemia virus, porcine circovirus, and psittacine beak and feather disease virus, with pigeon circovirus being classified as a tentative member. Infections with each of the four circoviruses are associated with potentially fatal diseases in which virus-induced damage to lymphoid tissue and immunosuppression are common features. Experience with other animal virus families suggests that additional animal species will be infected by, as yet undiscovered, circoviruses and that these may display similar tissue tropism and disease-causing potential. Recent reports describing the association of circovirus-like viruses with immunodeficiency-related diseases of geese and southern black-backed gulls suggest that circovirus infections of avian species may be more common than previously recognized, and prompt the question of whether novel circoviruses infect poultry to cause clinical and/or subclinical diseases that may be economically important. This review has three purposes. First, it is designed to summarize the currently available information about the classified circoviruses and viruses that are regarded as circovirus-like. Second, it aims to alert the readership to the possibility that other avian species, including commercial poultry, may be infected with novel circoviruses. Finally, possible methods for discovering novel circoviruses and for controlling infections by such viruses are suggested.     

Detection of a novel circovirus in mute swans (Cygnus olor) by using nested broad-spectrum PCR

Circoviruses are the causative agents of acute and chronic diseases in several animal species. Clinical symptoms of circovirus infections range from depression and diarrhoea to immunosuppression and feather disorders in birds. Eleven different members of the genus Circovirus are known so far, which infect pigs and birds in a species-specific manner. Here, a nested PCR was developed for the detection of a broad range of different circoviruses in clinical samples. Using this assay, a novel circovirus was detected in mute swans (Cygnus olor) found dead in Germany in 2006. Sequence analysis of the swan circovirus (SwCV) genome, amplified by multiply primed rolling-circle amplification and PCR, indicates that SwCV is a distinct virus most closely related to the goose circovirus (73.2% genome sequence similarity). Sequence variations between SwCV genomes derived from two different individuals were high (15.5% divergence) and mainly confined to the capsid protein-encoding region. By PCR testing of 32 samples derived from swans found dead in two different regions of Germany, detection rates of 20.0 and 77.3% were determined, thus indicating a high incidence of SwCV infection. The clinical significance of SwCV infection, however, needs to be investigated further.     

Comparison of three animal viruses with circular single-stranded DNA genomes

Summary No common antigenic determinants and no DNA sequence homologies were detected when three animal viruses, chicken anaemia agent (CAA), porcine circovirus (PCV), and psittacine beak and feather disease virus (PBFDV), all of which possess circular single-stranded DNA genomes, were compared. Negative contrast electron microscopy showed that PCV and PBFDV particles were 30% smaller than CAA particles and lacked the surface structure of CAA.     

Genomic characterization of a novel bat-associated Circovirus detected in European Miniopterus schreibersii bats

Circoviruses are small circular DNA viruses causing severe pig and poultry disease, recently identified in various bat species worldwide. We report the detection and full-genome molecular characterization of a novel bat-associated Circovirus identified in faecal samples of Miniopterus schreibersii bats (Schreiber’s bent-winged bats) from Sardinia, Italy. Full-genomic sequencing revealed a new putative member of Circoviridae family, with a genome size of 2063 nt. Sequencing allowed the characterization of the two major ORFs, inversely arranged, encoding replicase and capsid proteins, as well as the finding of a polythymidine tract within the genome, and highlighted phylogenetic relationships of the novel virus. This is the first report of circovirus in European bats. Giving the high level of genetic diversity of bat circoviruses, it is paramount to further investigate the relationships between these viruses and bats.

     

Interaction of the replication proteins and the capsid protein of porcine circovirus type 1 and 2 with host proteins

Porcine circovirus type 2 (PCV2) is an important pathogen in swine, whereas porcine circovirus type 1 (PCV1) is apathogenic. To analyze the interactions between PCV and its host, we have used a yeast two-hybrid assay to identify cellular proteins interacting with Cap and Rep proteins of both PCV genotypes. Six cellular proteins were found to interact with Cap (MKRN1, gC1qR, Par-4, NAP1, NPM1 and Hsp40) and three with Rep (ZNF265, TDG and VG5Q). These interactions were confirmed by co-immunoprecipitation. Investigation of the localisation of the proteins by immunofluorescence revealed in some cases only limited spatial overlapping with Cap, while in others a clear co-localisation and prominent protein redistribution was observed. The nine cellular proteins are associated with distinct aspects of viral lifecycle and our data is likely to support future research in the field of PCV2 pathogenesis.     

Analysis of the subcellular localization of the proteins Rep, Rep' and Cap of porcine circovirus type 1

Porcine circovirus type 1 (PCV1) encodes two major ORFs. The cap gene comprises the major structural protein of PCV, the rep gene specifies Rep and Rep', which are both essential for initiating the replication of the viral DNA. Rep corresponds to the full-length protein, whereas Rep' is a truncated splice product that is frame-shifted in its C-terminal sequence. In this study, the cellular localization of PCV1-encoded proteins was investigated by immune fluorescence techniques using antibodies against Rep, Rep' and Cap and by expression of viral proteins fused to green and red fluorescence proteins. Rep and Rep' protein co-localized in the nucleus of infected cells as well as in cells transfected with plasmids expressing Rep and Rep' fused to fluorescence proteins, but no signal was seen in the nucleoli. Rep and Rep' carry three potential nuclear localization signals in their identical N-termini, and the contribution of these motifs to nuclear import was experimentally dissected. In contrast to the rep gene products, the localization of the Cap protein varied. While the Cap protein was restricted to the nucleoli in plasmid-transfected cells and was also localized in the nucleoli at an early stage of PCV1 infection, it was seen in the nucleoplasm and the cytoplasm later in infection, suggesting that a shuttling between distinct cellular compartments occurs.     

Molecular characterisation of a novel cassava associated circular ssDNA virus

The application of sequence non-specific rolling circle amplification of circular single stranded (ss) DNA molecules to viral metagenomics has facilitated the discovery in various ecosystems of what is probably a diverse array of novel ssDNA viruses. Here we describe a putative novel ssDNA virus (at a genome level), cassava associated circular DNA virus (CasCV), isolated from cassava leaf samples infected with the fungi Collectotrichum and Plectosphaerella. CasCV has a circular ambisense genome and shares significant genome similarities with Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), Mosquito VEM virus SDBVL and Meles meles faecal virus (MmFV). The CasCV genome (2220 nt) has three large open reading frames. While it is probable that one of these encodes a capsid protein, the other two probably express a replication associated protein (Rep) following the removal of an intron such as that found in the Rep encoding genes of some geminiviruses. This Rep would contain four conserved rolling circle replication (RCR) related motifs that have previously been identified in geminivirus, circovirus and nanovirus Reps. Given both that the CasCV Rep and CP share 62.7% and 39.8% amino acid identity respectively with the Rep and CP of SsHADV-1, and that CasCV was discovered associated with cassava infecting fungi, we suggest that CasCV should be classified within the mycovirus taxonomic family. However, host range studies using infectious clones will be required to demonstrate the novel virus' likely origin and actual host species.     

Discovery and evolving history of two genetically related but phenotypically different viruses, porcine circoviruses 1 and 2

Porcine circoviruses (PCVs) belong to the genus Circovirus, family Circoviridae, and are the smallest non-enveloped, single stranded, negative sense, circular DNA viruses that replicate autonomously in mammalian cells. Two types of PCV have been characterised, PCV1 and PCV2 and these two viruses show 83% sequence identity at open reading frame (ORF) 1 and 67% identity at ORF2. PCV1 is a non-pathogenic virus of pigs. In contrast, PCV2 has emerged as a major pathogen of swine around the world. The discovery of PCV1 and how the subsequent studies on this virus eventually led to the recognition and characterisation of PCV2, and the disease scenarios associated with PCV2, serve as a model of how multidisciplinary collaboration among field veterinarians, diagnosticians and researchers can lead to the rapid characterisation and control of a globally important emerging disease.     

Development of a loop-mediated isothermal amplification method for rapid detection of pigeon circovirus

There are no effective antiviral treatments for pigeon circovirus (PiCV); thus, rapid diagnosis is critical for effective control of the disease caused by this virus. The recent development of a novel LAMP technique that amplifies nucleic acids rapidly with high specificity and sensitivity under isothermal conditions has overcome some of the deficiencies of nucleic-acid-based diagnostic tests. We established a LAMP method for rapid detection of PiCV using two pairs of primers that were designed from PiCV and compared its sensitivity and specificity with that of PCR. Amplification by LAMP was optimal at 63 °C for 60 min. The detection limit was nearly 0.5 pg of PiCV DNA, making it ten times more sensitive than PCR. There was no cross-reaction with porcine circovirus type 2 (PCV2), pigeon Trichomonas gallinae, or pigeon herpesvirus (PHV) under the same conditions. The assay also successfully detected the pathogen DNA in the tissues of infected pigeons. This is the first report indicating that LAMP is a valuable, rapid method of detecting PiCV with high sensitivity and specificity.     

Nucleotide sequence-based identification of a novel circovirus of canaries

Circovirus-like, spherical particles measuring 16 to 18 nm in diameter were detected in organ homogenates from adult canaries that had died after a short illness characterized by dullness, anorexia, lethargy and feather disorder. A polymerase chain reaction method, based on degenerate primers specific to conserved amino acid sequences in the circovirus replication-associated protein, was used to amplify DNA specific to a novel circovirus, tentatively named canary circovirus (CCV). Sequence analysis of a 510 nucleotide genomic fragment indicated that CCV exhibited 67.4, 64.9, 53.7 and 53.9% nucleotide identities and 70.0, 61.8, 40.4 and 40.1% amino acid identities with columbid (pigeon) circovirus (CoCV), beak and feather disease virus (BFDV), porcine circovirus type 1 and porcine circovirus type 2, respectively. CCV therefore represents a new avian virus of the genus Circovirus of the family Circoviridae, and is more closely related to CoCV than BFDV.The availability of nucleotide sequence data will facilitate the development of DNA-detecting diagnostics with which the prevalence of CCV infections can be assessed.     

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.