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Journal of Virology, April 2006, p. 3523-3531, Vol. 80, No. 7
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.7.3523-3531.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Characterization of Two Novel Polyomaviruses of Birds by Using Multiply Primed Rolling-Circle Amplification of Their Genomes

Reimar Johne,1* Walter Wittig,2 Daniel Fernández-de-Luco,3 Ursula Höfle,4 and Hermann Müller1

Institute for Virology, Faculty of Veterinary Medicine, University of Leipzig, Leipzig, Germany,1 Dresden, Germany,2 Department of Animal Pathology, Veterinary Faculty, University of Zaragoza, Zaragoza, Spain,3 Centro de Investigación Agropecuaria, El Deheson del Encinar, Oropesa, Spain4

Received 7 October 2005/ Accepted 13 January 2006


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ABSTRACT
 
Polyomaviruses are small nonenveloped particles with a circular double-stranded genome, approximately 5 kbp in size. The mammalian polyomaviruses mainly cause persistent subclinical infections in their natural nonimmunocompromised hosts. In contrast, the polyomaviruses of birds—avian polyomavirus (APV) and goose hemorrhagic polyomavirus (GHPV)—are the primary agents of acute and chronic disease with high mortality rates in young birds. Screening of field samples of diseased birds by consensus PCR revealed the presence of two novel polyomaviruses in the liver of an Eurasian bullfinch (Pyrrhula pyrrhula griseiventris) and in the spleen of a Eurasian jackdaw (Corvus monedula), tentatively designated as finch polyomavirus (FPyV) and crow polyomavirus (CPyV), respectively. The genomes of the viruses were amplified by using multiply primed rolling-circle amplification and cloned. Analysis of the FPyV and CPyV genome sequences revealed a close relationship to APV and GHPV, indicating the existence of a distinct avian group among the polyomaviruses. The main characteristics of this group are (i) involvement in fatal disease, (ii) the existence of an additional open reading frame in the 5' region of the late mRNAs, and (iii) a different manner of DNA binding of the large tumor antigen compared to that of the mammalian polyomaviruses.


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INTRODUCTION
 
Polyomaviruses are widely distributed among mammalian and avian species; to date, 14 different polyomaviruses infecting humans, monkeys, cattle, rabbits, rats, mice, hamsters, geese, and various bird species are known (3, 17, 19). The mammalian polyomaviruses typically cause subclinical infections in their natural hosts, with lifelong persistence, which may develop into disease after severe immunosuppression (28). Infection of newborn laboratory rodents causes tumor growth (4). In contrast, the two known avian polyomaviruses are the causative agents of fatal disease in birds (30, 31). Avian polyomavirus (APV) causes budgerigar fledgling disease characterized by ascites, hepatitis, and hydropericardium of young budgerigars, but other bird species are also susceptible to APV infection (14, 31, 43). Goose hemorrhagic polyomavirus (GHPV) is the causative agent of hemorrhagic nephritis and enteritis of geese, an acute disease of young geese with mortality rates of up to 67% (11, 21, 30, 35).

Members of the family Polyomaviridae are characterized by a nonenveloped icosahedral capsid and a circular double-stranded genome of approximately 5,000 bp (3). The genome is transcribed bidirectionally from a noncoding regulatory region for the expression of the early genes encoding two or three tumor antigens (T-Ags) and the late genes encoding the capsid proteins VP1, VP2, and VP3. In addition, the human viruses JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) as well as the monkey polyomavirus simian virus 40 (SV40) encode the so-called agnoprotein in the 5' region of the late mRNAs, which is a nonstructural multifunctional protein (5, 13, 33, 41). In the corresponding region, APV and GHPV encode proteins with no homology to the agnoproteins, designated VP4 (16) and open reading frame X (ORF-X) (17), respectively. VP4 is an additional structural protein and induces apoptosis in cell culture (15, 16).

Most of the polyomavirus species were discovered by the screening of tissue cultures for viral contaminants (4, 9). Recently, a novel polyomavirus was detected in the feces of a chimpanzee by using a nested broad-spectrum PCR (19). A sequence-independent strategy for the selective amplification of circular DNA genomes has been successfully used for the detection of novel papillomaviruses (38, 40) and anelloviruses (32). This technique, called multiply primed rolling-circle amplification (RCA), employs the DNA polymerase of bacteriophage {phi}29 for amplification of circular DNA using random hexamer primers. By strand displacement synthesis, a high-molecular-weight DNA is produced containing repeated copies of the complete genome, from which single genome units can be excised using a single cutting restriction enzyme (39). As the RCA technique is not dependent on specific primer sequences, it should be convenient for the amplification of any circular DNA genome.

To test the suitability of RCA for the amplification of the genomes of novel polyomaviruses, the technique was applied to samples of diseased birds that previously tested positive for polyomaviruses by using a broad-spectrum PCR. The genomes of the novel polyomaviruses were cloned, and the genome sequences were analyzed to assess their relationship to the known polyomaviruses. These data along with the clinical data obtained from the examination of the infected birds were used to group the polyomaviruses according to their phylogenetic and pathogenetic properties.


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MATERIALS AND METHODS
 
Organ samples. Liver samples of Eurasian bullfinches belonging to an East Asian race (Pyrrhula pyrrhula griseiventris) were obtained from nestlings which died within the first week after hatching in an aviary in Germany in 2004. Spleen samples of wild Eurasian jackdaws (Corvus monedula) were collected during a mortality incident in the wild bird population in northeastern Spain in January and February 2005 (2). A kidney of a goose (Anser anser) infected with GHPV (30) was included in control experiments. In all cases, the types of organs investigated were chosen based on inflammatory changes observed by histological examination.

Nested broad-spectrum PCRs. Detection of polyomavirus-specific DNA was performed by using nested broad-spectrum PCRs (19) with DNA isolated from the samples using a DNeasy tissue kit (QIAGEN, Germany). Taq DNA polymerase (PeqLab, Germany) and buffer Y (PeqLab, Germany) were used in these PCRs. A fragment of the VP1-encoding region was amplified with primers VP1-1f and VP1-1r (19), followed by nested PCR with primers VP1-2f and VP1-2r (19). Secondary PCR products with a length of approximately 250 bp were cloned into the vector pCR4-TOPO (Invitrogen, Germany) and sequenced. The sequences were aligned using the BLAST search program (1). Polyomavirus-positive samples were further subjected to PCR amplifying a fragment of the region encoding VP3/VP1 using the primers VP3-1f and VP3-1r (19), followed by nested PCR with primers VP3-2f and VP3-2r (19). Secondary PCR products with a length of approximately 400 bp were cloned, sequenced, and aligned as above.

Multiply primed RCA and long-range PCRs. DNA isolated from the samples was directly amplified by RCA (39) using a TempliPhi 100 amplification kit (Amersham Biosciences, United Kingdom). A total of 1 µl of DNA was mixed with 5 µl of TempliPhi sample buffer supplemented with a 450 µM concentration of each deoxynucleoside triphosphate, incubated at 95°C for 3 min, and subsequently cooled on ice. After the addition of 5 µl of TempliPhi reaction buffer and 0.2 µl of TempliPhi enzyme mix, the mixture was incubated at 30°C for 16 h and thereafter inactivated at 65°C for 10 min. For restriction enzyme analysis, 2-µl aliquots of the mixture were digested with EcoRI, PstI, and BamHI and subjected to electrophoresis on ethidium bromide-stained 0.8% agarose gels. For cloning, a total of 30 µl of an RCA reaction was digested with BamHI, and the resulting 5-kbp product was ligated with the BamHI-restricted vector pBluescript II SK(+) (Stratagene, Germany). Both strands of the insert were sequenced by the primer walking method.

Long-range PCRs were performed for amplification of the remaining genome fragments in the case of the sample of the Eurasian jackdaw. Primer sequences were delineated from the sequences of the secondary PCR products obtained by the nested broad-spectrum PCRs. A total of 1 µl of the DNA isolated from the sample or 1 µl of the respective RCA product was used as a template along with a FastStart High-Fidelity PCR System (Roche, Switzerland). The two fragments were amplified with the primer pair 5'-GGA TGG ACG GTC AGC CAA TGC A-3' and 5'-CCC ATG TTC TTG ATT TTC CAG G-3' or the pair 5'-TGC ATT GGC TGA CCG TCC ATC C-3' and 5'-CCT GGA AAA TCA AGA ACA TGG G-3', respectively. The cycling profile consisted of an incubation at 95°C for 5 min, 40 cycles with 94°C for 30 s, 56°C for 30 s, and 72°C for 4 min, followed by a final incubation at 72°C for 10 min. PCR products with a length of 4,173 bp and 948 bp were cloned into the vector pCR4-TOPO (Invitrogen, Germany), and both strands of the inserts were sequenced by primer walking.

Sequence analysis. The genome sequences of finch polyomavirus (FPyV) and crow polyomavirus (CPyV) were reassembled from the sequence fragments using the EditSeq module of the DNASTAR software package (Lasergene, Madison, WI). For sequence alignment and phylogenetic analyses, the genome sequences of 10 polyomaviruses were derived from the GenBank database (under the given accession numbers): APV, strain BFDV-1 (AF241168) (49); BKPyV (NC001538) (47); bovine polyomavirus (D13942) (46); GHPV (AY140894) (17); hamster polyomavirus ([HaPyV] M26281) (7); JCPyV (NC001699) (8); ß-lymphotropic polyomavirus, strain K38 (K02562) (36); murine polyomavirus (MPyV), strain Crawford small-plaque (K02737) (42); murine pneumotropic virus (MPtV), strain Kilham (M57473) (29); and SV40, strain K661 (AF038616) (23). Alignment was performed using the MegAlign module of the above-mentioned software package with the ClustalW method (50) and the "residue identity" weight table.

Virus isolation and recovery from cloned DNA. Attempts to isolate virus were made by inoculation of organ homogenates onto subconfluent cultures of primary chicken embryo (CE) cells. At 6 days after inoculation, the cultures were subjected to three cycles of freezing and thawing, and the supernatants were used for infection of fresh cultures. After 6 days, a third passage was performed analogously. The cells were harvested and subjected to immunoblotting using a rabbit antiserum directed against APV particles (49) and cross-reacting with VP1 proteins of several other polyomaviruses (44).

Attempts to recover infectious virus from cloned DNA were performed by transfection of CE cells as previously described (15). Briefly, the FPyV genome was recovered from the plasmids by digestion with BamHI and linearized with T4 DNA ligase (New England Biolabs, Germany), and 5 µg of DNA was transfected into CE cells using Lipofectamine reagent (Invitrogen, Germany). The plasmid pAPVinf (15) containing the genome of APV was used as a control. At 6 days after transfection, cells were treated by three cycles of freezing and thawing, and the supernatants were passaged two times as above. Analysis of the cells was performed by immunoblotting as above.

Nucleotide sequence accession numbers. The complete genome sequences of FPyV and CPyV have been deposited in the GenBank database under accession numbers DQ192571 and DQ192570, respectively.


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RESULTS
 
Detection of novel polyomaviruses in samples of diseased birds. Liver samples of Eurasian bullfinches and spleen samples of Eurasian jackdaws were tested by VP1-specific and VP3/VP1-specific nested broad-spectrum PCRs for the presence of polyomaviruses. A kidney of a GHPV-infected goose served as a positive control. The clinical symptoms observed in the birds prior to death are summarized in Table 1. In the bullfinch aviary, increased mortality of nestlings had been observed during a period of 3 years, and during 1 year several older birds died with signs of inflammation of the skin of the head. The wild jackdaws died during an epidemic outbreak of a disease in which Salmonella spp. had been detected repeatedly in many organs of the birds (2). In all cases, a coinfection with circoviruses was excluded by PCR as previously described (52; data not shown). Analysis of the PCR products of the polyomavirus-specific PCRs revealed bands with the appropriate size (Fig. 1A shows the results of a VP1-specific PCR). The sequence analysis of cloned PCR products revealed homologies but no identities to genome sequences of polyomaviruses (not shown). The two novel viruses were tentatively designated FPyV and CPyV.


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  		TABLE 1. Clinical symptoms and postmortem findings observed in birds infected with avian polyomaviruses


Figure 1
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  		FIG. 1. Detection of novel polyomaviruses by nested broad-spectrum PCR, amplification of their genomes by RCA and long-range PCR, and attempts to rescue infectious virus from the cloned genome of FPyV. The products were analyzed on ethidium bromide-stained agarose gels. The positions of the specific products are indicated by arrows; the sizes of molecular mass markers are specified next to the panels. (A) Secondary PCR products of the VP1-specific nested broad-spectrum PCR obtained with samples derived from Eurasian bullfinches (Bullfinches 1 to 3), a Eurasian jackdaw, a goose, or a negative control (–). (B) RCA products of the sample of Eurasian bullfinch 2 (Bullfinch), the goose, and the Eurasian jackdaw after digestion with EcoRI or PstI. (C) RCA products after pretreatment of the sample of Eurasian bullfinch 2 with BamHI (lin.) or without pretreatment (circ.) and analysis of the RCA products using BamHI. (D) PCR products obtained after long-range PCR with the sample of the Eurasian jackdaw either with (RCA+) or without (RCA–) preamplification by RCA, as well as a negative control (–). (E) Immunoblot analysis of CE cells transfected (T) with the excised and circularized genome of FPyV from plasmid 1 (FPyV1), plasmid 2 (FPyV2), a plasmid containing the APV genome (APV), or without DNA (–). P1 and P2, CE cells after one or two consecutive passages, respectively, of the supernatants of the transfected cells. A rabbit serum directed against APV particles and cross-reacting with other polyomavirus VP1 molecules was used.

Amplification of the viral genomes by RCA and long-range PCR. Attempts to isolate virus by inoculation of the organ homogenates onto cultures of CE cells remained unsuccessful as no cytopathic changes were evident, and no viral proteins could be detected in the cells by immunoblotting with a cross-reacting rabbit antiserum directed against VP1 (not shown). To further characterize the viruses, the samples of a bullfinch, a jackdaw, and a goose were subjected to RCA. After digestion of the RCA products with EcoRI and PstI, multiple bands were visible in the case of the samples derived from the bullfinch and the goose but not in the case of the jackdaw sample (Fig. 1B). Also, no band was visible after digestion of the jackdaw sample with BamHI, whereas BamHI digestion of the bullfinch sample resulted in a single band of approximately 5 kbp (Fig. 1C), which was subsequently cloned and sequenced. Analysis of the sequence revealed that the cloned fragment enclosed the sequence of the PCR product mentioned above, indicating that the FPyV genome was cloned. A PCR amplifying the region including the BamHI site did not reveal additional sequences (not shown), indicating that this restriction site was unique and that the whole genome was cloned.

To confirm the sequence of the FPyV genome and to assess the accuracy of the technique used, a second RCA was performed with the same bullfinch sample in an independent experiment, and the determined sequence of the cloned fragment was compared to that of the original clone. The sequences of both clones differed only at nucleotide position 2745 (A or T), which represents a silent mutation within the VP1-encoding region. To test whether a circular conformation of the FPyV genome in the sample was required for successful amplification by RCA, the sample was linearized overnight with BamHI before the RCA was applied as described above. A control sample was treated identically, however, without prior addition of the restriction enzyme. After analysis of the RCA products using BamHI, a band of approximately 5 kbp was visible only in the nonlinearized sample (Fig. 1C). Recovery of infectious virus from the cloned DNA was attempted by transfection of the excised and circularized genome sequences of the FPyV clones into CE cells and subsequent passaging of the supernatants. The cloned APV genome was used as a control. From immunoblot analysis of the cells, virus-specific bands were detected only in the case of APV, indicating that no infectious virus was recovered in the case of the FPyV clones (Fig. 1E).

In order to amplify the remaining parts of the CPyV genome, long-range PCRs were performed using primers with binding sites within the sequences obtained by the broad-spectrum PCRs. Using DNA directly isolated from the spleen tissue, no PCR products were detectable; however, after preamplification of the DNA by RCA, PCR products up to 4 kbp in length were obtained (Fig. 1D). Sequence analysis of the cloned PCR products revealed that the whole CPyV genome was cloned in two overlapping fragments.

Analysis of the genome sequences. The genomes of FPyV and CPyV have a length of 5,278 bp and 5,079 bp, respectively. ORFs encoding proteins with homologies to VP1 and VP2 as well as large and small T-Ags of polyomaviruses are found. The noncoding regulatory region has a length of 359 bp and 282 bp in FPyV and CPyV, respectively. Based on sequence homologies to APV (26) and GHPV (17), three introns were predicted in the late genome regions and one intron was predicted in the early genome regions of FPyV and CPyV. The removal of the two intron sequences in the 5' region of the late mRNA leads to an additional ORF, designated ORF-X. A scheme of the genome organization of FPyV and CPyV is presented in Fig. 2.


Figure 2
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  		FIG. 2. Genome organization of FPyV and CPyV. Coding regions for large T-Ag, small T-Ag, VP1, VP2, VP3, and ORF-X are marked by arrows. Intron sequences inferred from the DNA sequences as well as the restriction sites of the enzymes used in RCA analysis are indicated. NCR, noncoding regulatory region.

Phylogenetic analysis of the genome sequences of 12 polyomaviruses revealed the that FPyV is most closely related to APV (50.4% nucleotide sequence identity), whereas CPyV is most closely related to GHPV (56.0% nucleotide sequence identity). A phylogenetic tree constructed on the basis of polyomavirus genome sequences shows that all avian viruses cluster in a separate branch (Fig. 3A). Also, separate branches are formed by the three primate viruses SV40, JCPyV, and BKPyV, as well as by the rodent viruses HaPyV and MPyV. However, a general host-specific clustering could not be demonstrated as the primate virus B-lymphotropic polyomavirus and the rodent virus MPtV were found in additional separate branches.


Figure 3
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  		FIG. 3. Phylogenetic relationship of 12 polyomaviruses. The phylogenetic tree was established using the nucleotide sequences of the whole genomes (A) or the amino acid sequences of the large T-Ag (B) with the Clustal W method. Clusters of avian viruses (APV, CPyV, FPyV, and GHPV), primate viruses (BKPyV, JCPyV, and SV40), and rodent viruses (HaPyV and MPyV) are indicated. CPyV and FPyV are marked by arrows.

Analysis of the deduced amino acid sequences and the noncoding regulatory region. The sizes of the proteins deduced from the genome sequences of FPyV and CPyV and their calculated molecular weights are shown in Table 2. In all cases, the proteins of FPyV show the highest similarity to the APV proteins, and the CPyV amino acid sequences are most closely related to that of GHPV, which is indicated as the percentage of amino acid similarity in Table 2.


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  		TABLE 2. Proteins putatively encoded by the FPyV and CPyV genome

VP1 is the most conserved among the proteins encoded by the FPyV and CPyV genome, and functional sequences like the calcium-binding domain in the C terminus are evident. In contrast, the N-terminal sequences carrying the nuclear localization signal (NLS) in SV40 (KRKX8KKPK) (12) are not conserved in FPyV (KKX6R) and CPyV (KRXR). This has also been shown for the other avian viruses APV (KXKX4R) (18) and GHPV (KRXR) (17). A stretch of basic amino acid residues in the C-terminal region of VP2 that has been shown to represent its NLS in SV40 and APV is conserved in the VP2 sequences of FPyV and CPyV. The N-terminal consensus sequence MGX4S, which has been shown to mediate myristoylation in the APV VP2 (45), is also found in FPyV and slightly changed in CPyV into MGX4A; however, the latter sequence is also found in the VP2 of BKPyV, JCPyV, and MPtV. In polyomavirus genomes, VP3 usually is encoded from the ORF also encoding VP2, by using an internal initiation codon. In FPyV, the first methionine is at position 111 of VP2, which is considered to be the N-terminal amino acid of VP3. In CPyV, the first methionine of VP2 is found at position 77; however, by homology to other N termini of VP3, the second methionine at position 107 is more likely to represent the first amino acid of VP3.

The unspliced early mRNA of FPyV and CPyV encodes the small T-Ag. In contrast to the mammalian polyomaviruses, the small T-Ags of the avian viruses have no consensus sequence for binding of PP2A (CXCX2C). Also, no ORF encoding a middle T-Ag is present in the FPyV and CPyV genomes. Removal of the intron in the early mRNA creates the ORF encoding the large T-Ag. Conserved features (37) like the J domain carrying the highly conserved HPDKGG box and the pRB-binding motif LXCXE are also found in the FPyV and CPyV T-Ag sequences. The amino acids shown to mediate p53 binding in the large T-Ag of SV40 (P399, D402, C411, and P584) (24) and which are also present in BKPyV and JCPyV are not conserved in FPyV and CPyV; however, they are also not consistently found in the other polyomavirus large T-Ags.

A phylogenetic tree established for the amino acid sequences of the large T-Ags of 12 polyomaviruses shows a distinct grouping of avian and mammalian polyomaviruses in separate clades (Fig. 3B). A closer look at the sequences localizes the major region of difference of the two groups between amino acids (aa) 100 and 300. This region carries the NLS (KKKRK, aa 127 to 131 in SV40) (22) and is involved in sequence-specific binding of the large T-Ag to the origin of replication in SV40. It is evident from Fig. 4A that this NLS is truncated in the avian viruses (KR in APV and FPyV; K in GHPV and CPyV). In the region corresponding to the DNA-binding domain A of SV40 (aa 152 to 155) (46), no homologies are found between the avian and the mammalian viruses; however, the sequences of this region are well conserved within the two groups. In the region corresponding to DNA-binding domain B2 of SV40 (aa 204 to 208 (48), the mammalian viruses show the consensus sequence HRVSA, whereas the avian viruses have the sequence TR(I/V)ST.


Figure 4
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  		FIG. 4. Comparison of the DNA binding domain of large T-Ags of polyomaviruses and the corresponding target sequences within the noncoding regulatory region. (A) Alignment of the DNA-binding regions of 12 polyomavirus large T-Ags. The NLS and the DNA-binding domains A and B2 in SV40 are indicated by dotted lines. Basic amino acid residues within the NLS region are shaded gray. Sequences that are conserved in the avian viruses but not in the mammalian viruses are boxed. (B) Nucleotide sequence of an 80-bp fragment of the noncoding regulatory region of six polyomaviruses. The (putative) binding sequences for the large T-Ag are shaded gray; the orientation of the binding sequences is indicated by arrows. For MPyV, the complementary strand (c) is shown due to the inverse nucleotide numbering of its genome.

Corresponding to the differences in the DNA-binding motifs of the large T-Ags, the target sequences located within the noncoding regulatory region differ between mammalian and avian viruses. Whereas in mammalian viruses, such as SV40 and MPyV, the large T-Ag binds to the pentanucleotide GAGGC (51), in APV the palindromic sequence TCC(A/T)6GGA (25) has been identified as the binding sequence of the large T-Ag (Fig. 4B). Similar sequences are found in the noncoding regions of FPyV [TCC(A/T)6GGG, nucleotides 115 to 126] and CPyV [ACC(A/T)6GGG, nucleotides 126 to 137].

The additional ORF-X is located in the 5' region of the late mRNA of FPyV and CPyV. Limited homologies are found between VP4 of APV and ORF-X of FPyV as well as between the proteins encoded by ORF-X of GHPV and CPyV. This is also reflected by the branching of a phylogenetic tree established for these amino acid sequences (Fig. 5A). No homologous proteins are encoded by the mammalian polyomaviruses, and no significant similarities are found to protein sequences deposited in the GenBank database. It is evident from the amino acid sequences (Fig. 5B) that all of these proteins contain a relatively high percentage of proline, between 14.2% in APV and 16.0% in CPyV. A detailed sequence comparison shows structural homologies in the central regions of the APV VP4 and the FPyV ORF-X proteins with a typical coiled-coil motif indicated by regularly repeated hydrophobic amino acid residues. This motif is not present in the CPyV ORF-X and GHPV ORF-X proteins; however, both proteins have a region of nearly identical amino acid sequences in the C-terminal region.


Figure 5
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  		FIG. 5. Comparison of the amino acid sequences encoded by the additional ORFs in the 5' region of the late mRNA of the avian polyomaviruses. (A) Phylogenetic tree based on an alignment of the amino acid sequences of VP4 of APV and the ORF-X sequences of FPyV, CPyV, and GHPV. (B) Alignment of the respective amino acid sequences. The coiled-coil motifs in the APV VP4 and FPyV ORF-X proteins as well as a region of high similarity between the ORF-X sequences of CPyV and GHPV are indicated; conserved amino acid residues are shaded gray.


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DISCUSSION
 
Mammalian polyomaviruses are known to cause subclinical persistent infections in their natural hosts which may develop into diseases after severe immunosuppression (4). In contrast, APV and GHPV are primary etiologic agents of severe diseases with high mortality rates in birds (11, 20). In this communication, we describe two novel polyomaviruses detected in birds that died after disease, raising the question whether the polyomaviruses of birds generally are primary pathogens. Until now, the pathogenicity of FPyV and CPyV was not tested by experimental infection. However, the distinct clinical signs observed in the bullfinches are reminiscent of an APV infection in parrots (Table 1), and FPyV was the only pathogen that has been repeatedly detected in the diseased birds over a period of 3 years. The assessment of pathogenicity of CPyV is complicated by the fact that the jackdaws were coinfected with Salmonella spp., which alone may be able to induce a severe disease. Further testing of a larger number of samples derived from diseased and clinically healthy birds as well as experimental infection studies will be necessary to assess the pathogenicity of these viruses.

Attempts to isolate virus in cultures of CE cells were not successful. Although the reasons for that are unknown, a distinct host specificity of FPyV and CPyV may be responsible. Polyomaviruses are known to have a narrow host range for productive infection (4); GHPV could only be cultured on primary goose kidney cells, with a remarkably low replication efficiency (17). The unsuccessful attempt to recover infectious virus from the cloned FPyV genome by transfection of CE cells, which was readily successful using the cloned APV genome, may also indicate that chicken cells are not adequate host cells for FPyV. However, it cannot be completely excluded that two replication-defective clones of the FPyV genome have been cloned. Investigations with tissue cultures or experimental animals originating from other bird species, which were not available in this study, are necessary to assess the distinct host specificity of FPyV and CPyV.

To characterize the novel viruses and to uncover common features of the avian polyomaviruses that could explain the biological differences to mammalian polyomaviruses, their genomes were cloned, and the sequences were analyzed. The recently developed technique of RCA was successfully applied for genome amplification from the field samples, either alone or in combination with long-range PCR. The major advantages of RCA are the independence from sequence-specific primers and the single-step amplification of the whole genome, which ensures a fast characterization of viruses with even low homologies to known genome sequences (39). The proofreading activity of the bacteriophage {phi}29 DNA polymerase used in RCA should ensure a low error rate during amplification of the genome sequences. Using several biochemical assays, an error rate between 10–5 and 10–6 was assessed for the {phi}29 DNA polymerase (6), and the application of this enzyme in a technique for amplification of the human genome resulted in an estimated error rate of 9.5 x 10–6 (34). In the study presented here, one nucleotide exchange was found within a 5,000-bp fragment, corresponding to an error rate of 2 x 10–4; however, the reliability of this value is limited as only two clones have been sequenced, and the degree of homogeneity of the sequences within the sample was not known.

Although the {phi}29 DNA polymerase could be used for amplification of linear DNA (34), the amplification rate for circular papillomavirus genomes has been described to be 2.8 x 103-fold higher compared to linear genomic DNA of the host cells (39). The requirement of circular DNA for efficient amplification by RCA is also supported by the experiment presented here in which pretreatment of the bullfinch sample with a linearizing restriction enzyme resulted in no detectable band after analysis of the RCA products. The inability to directly clone the RCA product in the case of CPyV may, therefore, be explained by a low amount of circular viral DNA due to the poor quality of the sample. However, a combination of RCA and long-range PCR enabled amplification of the genome, which may be a general possibility for genome amplification in those cases.

The analysis of the genome sequences revealed two major differences between the avian and the mammalian polyomaviruses: (i) a different manner of DNA-binding of the large T-Ag and (ii) the presence of an additional ORF in the avian viruses, which is created by splicing of the 5' region of the late mRNA and which encodes a proline-rich protein. The DNA-binding mechanism of the SV40 large T-Ag has been well characterized, and the regions A and B2 of the large T-Ag have been shown to specifically interact with the pentanucleotide sequence GAGGC present in the noncoding regulatory region (27, 48, 53). The regions A and B2 are highly conserved among the mammalian polyomaviruses. In contrast, the avian polyomaviruses have a different sequence in the corresponding region which, again, is conserved among them. Also, the pentanucleotide sequence GAGGC is not consistently found in the noncoding region of the avian polyomaviruses, but the palindromic sequence NCC(A/T)6GGN is found at least once in this region in all avian polyomaviruses. As this sequence has been shown to be specifically bound by the large T-Ag of APV (25), a similar manner of large T-Ag DNA-binding may be common in all avian polyomaviruses. Because of the biological and structural differences between polyomaviruses of mammals and birds, a grouping into two different genera within the family Polyomaviridae should be considered.

Analysis of the amino acid sequences encoded by the additional ORF of the avian viruses revealed a high degree of heterogeneity. Two groups of proteins could be defined, with the APV VP4 and FPyV ORF-X proteins forming one group that is characterized by a central coiled-coil domain sequence as well as the GHPV ORF-X and CPyV ORF-X proteins without such a domain but with other sequence similarities. The distinct function of these proteins is not known; however, APV VP4 has been characterized as an additional structural protein of this virus (16), and it has been shown to induce apoptosis after expression in avian and insect cells (15). The latter finding led to the assumption that this protein may contribute to the pathogenicity of APV by destroying infected cells in the absence of an inflammatory response, which allows efficient virus release from the nucleus and rapid virus spread through the organism (15). Further investigations on the proteins encoded by the ORF-X of FPyV, CPyV, and GHPV will be necessary to assess the distinct functions of these proteins and to clarify their contribution to the pathogenicity of the polyomaviruses of birds.


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ACKNOWLEDGMENTS
 
This work was funded by a grant from the Deutsche Forschungsgemeinschaft (JO 369/3-2).


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute for Virology, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 29, D-04103 Leipzig, Germany. Phone: 49 341 9738204. Fax: 49 341 9738219. E-mail: johne{at}vetmed.uni-leipzig.de. Back


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Journal of Virology, April 2006, p. 3523-3531, Vol. 80, No. 7
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.7.3523-3531.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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