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Journal of General Virology (2006), 87, 21912201

DOI 10.1099/vir.0.81738-0

Avipoxvirus phylogenetics: identication of a PCR length polymorphism that discriminates between the two major clades
Susan Jarmin,13 Ruth Manvell,2 Richard E. Gough,2 Stephen M. Laidlaw13 and Michael A. Skinner13
Correspondence Michael A. Skinner m.skinner@imperial.ac.uk
1 2

Institute for Animal Health, Compton, Newbury, Berkshire RG20 7NN, UK Veterinary Laboratory Agency, New Haw, Addlestone, Surrey KT15 3NB, UK

Received 6 December 2005 Accepted 18 April 2006

Avipoxvirus infections have been observed in an extensive range of wild, captive and domesticated avian hosts, yet little is known about the genome diversity and host-range specicity of the causative agent(s). Genome-sequence data are largely restricted to Fowlpox virus (FWPV) and Canarypox virus (CNPV), which have been sequenced completely, showing considerable divergence between them. It is therefore proving difcult, by empirical approaches, to identify pan-genus, avipoxvirus-specic oligonucleotide probes for PCR and sequencing to support phylogenetic studies. A previous preliminary study used the fpv167 locus, which encodes orthologues of vaccinia virus core protein P4b (A3). PCR per se did not discriminate between viruses, but restriction-enzyme or sequence analysis indicated that the avipoxviruses clustered either with FWPV or with CNPV. Here, further study of the P4b locus demonstrated a third cluster, from psittacine birds. A newly identied locus, anking fpv140 (orthologue of vaccinia virus H3L), conrms the taxonomic structure. This locus is particularly useful in that viruses from the fowlpox-like and canarypox-like clusters can be discriminated by PCR on the basis of fragment size, whilst sequence comparison allows discrimination for the rst time between Pigeonpox virus and Turkeypox virus. Except within the psittacines, virus and avian host taxonomies do not show tight correlation, with viruses from the same species located in very different clades. Nor are all the existing recognized avipoxvirus species, dened primarily by avian host species (such as CNPV and Sparrowpox virus), resolved within the present structure.

INTRODUCTION
Avipoxvirus infections have been observed in more than 230 of the known 9000 species of bird, spanning 23 orders (Bolte et al., 1999), yet little is known about the genome diversity and host-range specicity of the causative agent(s). Avian poxviruses are all classied in the genus Avipoxvirus of the family Poxviridae within the subfamily Chordopoxvirinae. In common with other poxviruses, they contain a doublestranded DNA genome, ranging from 230 to >300 kbp, and replicate within the cytoplasm of the cells that they infect. Fowlpox virus (FWPV) is the prototypic member of the avipoxviruses. It is well known that the diseases present in two main forms, which may be the result of different routes of infection. The cutaneous form occurs following mechanical trauma, including bites of arthropods that serve as
3Present address: Department of Virology, Imperial College London, Faculty of Medicine, St Marys Campus, Norfolk Place, London W2 1PG, UK. The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are shown in Table 1.

vectors for mechanical transmission of the virus. Lesions normally occur on unfeathered areas around the eyes and nares, on the comb, wattle or legs. In poultry, this form is associated with low mortality and, frequently, subclinical infection. The diphtheritic form, following inhalation or ingestion of virus, involves mucous membranes of the mouth, pharynx, larynx and sometimes trachea. It is associated with a higher mortality rate, usually due to occlusion of the oropharynx. Avipoxvirus infections of canaries (Johnson & Castro, 1986) and nches can cause signicantly higher rates of mortality than those of chickens or turkeys. A third, pneumonia-like form of the disease has been described in canaries (Johnson & Castro, 1986). Conventional diagnosis of avipoxviruses is carried out by histopathological examination, electron microscopy, virus isolation on chorioallantoic membranes of embryonated chicken eggs, serological methods and restriction-fragment length polymorphism. These detection methods are being supplemented by the use of PCR using primers for a 578 bp product of fpv167, the orthologue of vaccinia virus A3L that encodes P4b (Huw Lee & Hwa Lee, 1997). [To distinguish
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S. Jarmin and others

genes or open reading frames (ORFs) from their encoded protein products, italicized lower case is used for genes and ORFs (e.g. fpv030) and plain text (with capitalized rst letter) for the products that they encode (e.g. Fpv030)]. Vaccines are available that utilize FWPV, Canarypox virus (CNPV), Pigeonpox virus (PGPV) or Quailpox virus (QUPV), but, due to new trends in avian farming and conservation, there is a demand for vaccines for use in other bird species, such as rare, and often expensive, falcons, eagles and ostriches. Vaccine application is by trial and error, with some viruses inducing cross-protection. For instance, vaccination with PGPV-based vaccines, but not those based on QUPV or Turkeypox virus (TKPV) (Wintereld & Reed, 1985), protects chickens from fowlpox. Such antigenic differences have served as the basis for avipoxvirus classication (Tripathy, 1984). Little is known about the host ranges of avipoxviruses, although they are generally assumed to be limited. For instance, a virus isolated recently from an Andean condor (Vultur gryphus) caused an aggressive, diphtheritic form of the disease in the condor, but produced only small, localized, cutaneous lesions in inoculated chickens, inducing no cross-protection against challenge with FWPV (Kim et al., 2003). Partial sequencing of the CNPV genome around the thymidine kinase locus (Amano et al., 1999) rst showed that it was surprisingly highly diverged from that of FWPV. This was conrmed when the complete genome sequence of CNPV was derived (Tulman et al., 2004), allowing it to be compared in its entirety with that of FWPV (Afonso et al., 2000). The extent of divergence is illustrated by the fact that CNPV and FWPV orthologues of P4b, a conserved 75?2 kDa virion core protein found in all poxviruses (Binns et al., 1989; Huw Lee & Hwa Lee, 1997), share only 64?2 % amino acid identity. Despite sequencing of the complete genomes of FWPV and CNPV, little is known about the phylogenetic relationship between these and other avipoxviruses and how this might relate to host range, pathogenesis and epidemiology. Concern has been raised that poxvirus infections are responsible for the decline in populations of native birds in Hawaii and the Canary Islands (Tripathy et al., 2000) and that they might threaten marginal species, such as the ightless, nocturnal New Zealand owl parrot or kakapo (Stone & Forbes, 20022003). Recent preliminary phylogenetic studies of avipoxvirus isolates using the fpv167 locus (Luschow et al., 2004; Weli et al., 2004) indicated that most of the isolates clustered around either CNPV or FWPV. Corroboration of the phylogenetics of the avipoxviruses will require data from additional loci. For diagnostic purposes, it would also be useful to have fragment-length polymorphisms that could be detected directly by PCR rather than having to resort to the restriction-enzyme digestion that was used at the fpv167 locus by Luschow et al. (2004).
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In an attempt to address these issues, a collection of 22 avipoxviruses, comprising isolates, vaccines and laboratory strains, was examined, initially at the fpv167 locus, then subjected to PCR at the loci of genes encoding the three immunodominant FWPV antigens Fpv140, Fpv168 and Fpv191 (Boulanger et al., 2002), using a variety of primers with sequences specic for targets conserved between FWPV and CNPV. Sequence data obtained successfully from the fpv140 locus, as well as from the fpv167 locus, were subjected to phylogenetic analysis. The implications of the results for avipoxvirus epidemiology, virus host range and nomenclature are considered.

METHODS
Viruses and cells. Twenty-two avipoxviruses were used in this study (Table 1). Five were commercial fowlpox vaccines, Diftosec CT (Rhone Merieux), Nobilis Variole W (Intervet), Chick-N-Pox, Websters FPV M (Salsbury Laboratories Inc.) and Poxine (Duphar). One was a canarypox vaccine, Canarypox V (Duphar). One was the attenuated FWPV FP9, the genome of which has been sequenced completely (Laidlaw & Skinner, 2004). The remaining 16 viruses were isolates obtained from outbreaks and samples collected at the Central Veterinary Laboratory (CVL) of the Veterinary Laboratory Agency (VLA), Weybridge, UK. These poxviruses from chicken (Gallus gallus), canary (Serinus canaria), pigeon (Columba livia), turkey (Meleagris gallopavo), sparrow (Passer domesticus), starling (Sturnus vulgaris), houbara bustard (Chlamydotis undulata), macaw (Ara spp.), parrot (Amazona spp.), falcon (Falco spp.) and albatross (Diomedea melanophis) were isolated from birds with clinical signs of infection. Most of the isolates had been passaged on chick embryo broblasts (CEFs) and/or specic-pathogen-free (SPF) embryonated chicken eggs between ve and 30 times.

Samples were propagated for PCR on CEFs under E199 medium (16 E199 with Earles salts, 10 % tryptose phosphate broth, 3 % sodium bicarbonate, 1 % L-glutamine, 0?1 % penicillin/streptomycin, 2?5 % nystatin, 2 % newborn calf serum). The samples were incubated for 4 days under 5 % CO2 at 37 uC or until cytopathic effects were observed.
Virus purication and DNA extraction. Virus DNA was isolated by using standard methods, as described previously (Boulanger et al., 1998). PCR amplication. Primers described previously (Huw Lee & Hwa

Lee, 1997) were used to amplify the FWPV P4b gene (fpv167). The sequences of the primers were M2925, 59-CAGCAGGTGCTAAACAACAA-39 (fpv167 nt 459478), and M2926, 59-CGGTAGCTTAACGCCGAATA-39 (complementary to nt 10161035). The 39 end of fpv139 through to the 59 end of fpv141 was amplied by PCR using primers designed on sequence conserved between CNPV and FWPV. M2904, 59-GAAGTAGAGTTACGGTTC-39, is identical to nt 171290171311 of FP9 (Laidlaw & Skinner, 2004) in fpv139, and M2912, 59-GGTGATCCATTTCCATTTC-39), is complementary to nt 173254173272 in fpv141. PCR amplication was performed essentially as described by Huw Lee & Hwa Lee (1997), but using Expand High Fidelity PCR Enzyme mix (2?6 U; Roche).
Electrophoresis, purication and sequencing of PCR product. PCR samples (5 ml) were loaded onto 1 % agarose gels [90 mA for

30 min in TBE buffer (0?1 M Tris, 0?09 M boric acid, 0?01 M EDTA)]. PCR products were puried by using a QIAquick PCR Purication kit (Qiagen) and resuspended in 50 ml H2O. These Journal of General Virology 87

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Table 1. Details of avipoxviruses used in this study


CVL, Central Veterinary Laboratories, Weybridge, UK. Virus name Acronym Host Nature Origin GenBank accession no. P4b locus Fowlpox HP1-444 (FP9) 174/4/04 Diftosec CT (Merial) Nobilis Variole W (Intervet) Chick-N-Pox (Fort Dodge) Poxine (Duphar; Fort Dodge) Mild (Websters; Fort Dodge) Canarypox 1445/97/33 (Duphar; Fort Dodge) Pigeonpox Peekham 19/11/75 950 24/3/77 Turkeypox 2/11/66 10/12/98 Sparrowpox 9037 31/5/66 /23 Starlingpox /27 Houbarapox 1252/98/42 1165/04 Macawpox 1305/86 Parrotpox 364/89 Falconpox 1381/96 Albatrosspox 353/87 FWPVFP9 FWPV174 FWPVD FWPVN FWPVC FWPVP FWPVM CNPV1445 CNPVV PGPVP PGPV950 TKPV66 TKPV98 SRPV9037 SRPV23 SLPV HOPV1252 HOPV1165 MCPV PRPV FLPV1381 ABPV Chicken Chicken Chicken Chicken Chicken Chicken Chicken Canary Canary Pigeon Pigeon Turkey Turkey Sparrow Sparrow Starling Houbara bustard Houbara bustard Macaw Parrot Falcon Black-browed albatross >400 CEF passages, plaquepuried Clinical isolate, CVL, UK Commercial vaccine Commercial vaccine Commercial vaccine Commercial vaccine Commercial vaccine Clinical isolate, CVL, UK Commercial vaccine Clinical isolate, CVL, UK Clinical isolate, CVL, UK Clinical isolate, CVL, UK Clinical isolate, CVL, UK Clinical isolate, CVL, UK Clinical isolate, CVL, UK Clinical isolate, CVL, UK Clinical isolate, CVL, UK Clinical isolate, CVL, UK Clinical isolate, CVL, UK Clinical isolate, CVL, UK Clinical isolate, CVL, UK Clinical isolate, CVL, UK Europe UK Europe Europe USA USA Australia UK AJ581527 AM050377 AM050380 AM050379 AM050378 AM050375 AM050384 AM050385 AM050386 AM050387 AM050388 AM050389 AM050390 AM050391 AM050381 AM050382 AM050383 AM050376 AM050392 fpv140 locus AJ581527 AM071393 AM071395 AM071394 AM071512 AM071389 AM071390 AM071391 Phylogenetics and discrimination of avipoxvirus clades AM071511 AM071510 AM071513 AM071515 AM071388

UK UK Italy UK UK

United Arab Emirates Morocco UK quarantine UK quarantine (S. America) United Arab Emirates Falkland Islands

S. Jarmin and others products were then sequenced on both strands, using the amplication primers and internal primers in an automated sequencer (Beckman CEQ 8000). For FWPV-like viruses, primers M3238 (59-GTTAGAAGAGGAGATGGAGATGG-39), M3239 (59-CATCATAGTTATTTAACTAAATC-39), M3242 (59-GTCTACAGACGTCAGAATAATAACC-39) and M3243 (59-GGTTATTATTCTGACGTCTGTAGAC-39) were used. For CNPV-like viruses, primers M3240 (59-ATAAGAAGAGGAGAAGGAGACGG-39), M3241 (59-GTATACGCTATTAAATGAACATTC39), M3244 (59-GCGATAGAATTTTTTGTTTC-39), M3245 (59-GGATGACAGATTCTATGACG-39), M3251 (59-GGAGAGCTTAGTTTCTCTATC-39), M3252 (59-GCATTGATTAAAATCTATATCACC-39), M3253 (59-GCACTAATATCATACTTTCCAAAC-39), M3254 (59GGTATAAATAGATATGTTATGAGTAACG-39), M3255 (59-CGCGTAAAAAAAGATACGGG-39), M3256 (59-GGTTTACAGTATCGGGTATATGG-39), M3257 (59-CGATACTAGTCATCTATTATTAG-39) and M3258 (59-GCCGAAATCAACACATTTAAATC-39) were used to extend the reads. Sequencing PCR was carried out in a total volume of 10 ml, containing 2 ml puried PCR product, 2 ml Quick Start Enzyme mix (Beckman), 0?5 ml buffer and 5 pmol primer. Samples were subjected to 30 cycles (20 s at 96 uC, 20 s at 40 uC, 4 min at 60 uC) in a TC-412 (Techne). The sequencing samples were run by using the LFR program (Beckman). Sequence analysis was carried out with STADEN (Staden, 1982) and alignments were made with CLUSTAL_X (Thompson et al., 1997), manipulated with Se-Al (Rambaut, 1996; http://evolve.zoo.ox.ac.uk/) and displayed with MacGDE (http://www.msu.edu/~lintone/macgde/). Phylogenetic analysis was performed by using PAUP, with approaches and methods as described by Hall (2001).

acknowledged that the single-protein trees are more susceptible to functional pressures upon the particular protein than are consensus trees. However, our main interest in the protein trees was to compare the relative divergence of avian and mammalian viruses (acknowledging that the rates of divergence may vary among viruses in these different classes of vertebrates, thereby preventing any conclusions about the timescales involved). Such comparison, by neighbourjoining and parsimony analysis, indicates that the extent of protein divergence between avipoxvirus clades is equivalent to that between the various genera of mammalian poxviruses (Fig. 2). Protein and DNA trees show the avipoxviruses forming two major clades (A and B), with strong support by neighbourjoining bootstrap, parsimony and (for DNA) maximum likelihood (Fig. 2). In protein and DNA trees, clade B diverges into two subclades, B1 and B2, also with strong support. B1 consists of the isolates from canaries, great tits, houbara, stone curlew and four of the ve sparrow isolates, whilst B2 comprises the starling isolate as well as two of the four viruses from pigeons. In protein and DNA trees, clade A comprises FWPV, TKPV, Falconpox virus (with one of the two, FLPV36202, as a proximal outlier), Ospreypox virus and Albatrosspox virus, as well as the remaining isolates from pigeons and sparrow. In the DNA trees, the clade A viruses are further resolved (with strong support by neighbourjoining bootstrap, parsimony and maximum likelihood). FLPV36202 still forms a proximal outlier (subclade A4) to clade A. Subclade A1, with strong support, comprises essentially the FWPVs plus two single isolates from a turkey and a sparrow. Subclade A2, with strong support, consists of the two (of four) isolates from pigeons, two viruses from turkeys (isolated 30 years apart in the UK and Italy) and an isolate from an osprey. Subclade A3, also with strong support, consists of an albatross virus and the second falcon virus. In the DNA trees, a third major clade, C, which consists of the psittacine viruses, branches proximal to the MOCV outgroup, with strong support by neighbour-joining bootstrap, parsimony and maximum likelihood. In the protein trees, however, placement of the psittacine group is more ambiguous. By neighbour joining, the psittacine group clusters (at a bootstrap value of 71 %) with FWPV and the FWPV-like viruses of clade A. By parsimony, it clusters with CNPV and the CNPV-like viruses of clade B in 75 % of trees but, as in the DNA trees, it forms a distinct clade, proximal to MOCV, in 25 % of trees. Detailed examination of the various coding sequences shows that a number of nucleic acid changes unique to the psittacine viruses are masked in the protein sequence, due to codon redundancy. We therefore conclude that the DNA trees represent a more accurate description of the status of the psittacine viruses as a distinct clade, in accord with in vivo cross-protection studies (Boosinger et al., 1982; Wintereld & Reed, 1985). In the DNA trees, clade A represents the clusters of FWPV and FWPV-like viruses seen previously [clusters IIII of Luschow et al. (2004)]. Clade B represents CNPV and the
Journal of General Virology 87

RESULTS
P4b gene (fpv167) locus Primers for fpv167 were used to amplify sequences from DNA extracted from isolates propagated on CEFs. These primers gave products of about 600 bp (predicted size, 578 bp) on all viruses tested (data not shown). The products were puried and sequenced; a common sequence of 450 nt was obtained from all of the isolates, allowing comparison with equivalent, previously published sequences in the databases (Table 2). The derived and aligned protein sequences (Fig. 1a) were used to generate phylogenetic trees for P4b, including viruses from other chordopoxvirus genera, by using neighbour-joining analysis (with bootstrapping) and parsimony. Trees were also derived for the P4b-encoding sequences of the avipoxviruses, with Molluscum contagiosum virus (MOCV) as an outgroup, using neighbour-joining analysis (with bootstrapping), maximum likelihood and parsimony. The grouping of the mammalian poxviruses in the P4b protein trees differs slightly from that in trees based on consensus sequences of many conserved proteins (McLysaght et al., 2003; Gubser et al., 2004). In the consensus trees, yatapoxviruses form the most proximal branch of the group containing leporipox-, suipox- and capripoxviruses after that groups divergence from the orthopoxviruses. In the P4b protein trees, the yatapoxviruses are in the same group, but are in a distal node with Swinepox virus. It must be
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Table 2. Details of poxvirus sequences obtained from GenBank


Genus/species Orthopoxvirus Camelpox virus Cowpox virus Monkeypox virus Vaccinia virus Leporipoxvirus Myxoma virus Rabbit broma virus Capripoxvirus Lumpy skin disease virus Suipoxvirus Swinepox virus Deerpoxvirus Deerpox virus Molluscipoxvirus Molluscum contagiosum virus Yatapoxvirus Yaba-like disease virus Yaba monkey tumor virus Parapoxvirus Orf virus Avipoxvirus Agapornispox virus Canarypox virus Fowlpox virus Strain Abbreviation GenBank accession no. AY009089 AF482758 AF380138 AY243312 AF170726 AF170722 AF409138 AF410153 AY689436 U60315 AJ293568 AY386371 AY38263 AY530311 AY318871 AY530309 AY453172 AF198100 AY530302 AY530306 AY453175 AY453173 AY453174 AY530305 AY453177 AY530303 AY530307 AY530308 AY453176 AY530310 AY530304

CMS Brighton Red Zaire-96-I-16 Western Reserve Lausanne

CMPV CPXV MPXV VACV MYXV SFV LSDV SWPV DPV MOCV YLDV YMTV

Neethling LW 1959 17077-99 DPV W-848-83 Subtype 1

IA 82 APIII ATCC VR-111 GB 724/01-22 VR250 USDA Standard Challenge HP-B GB 362/02 GTPV-256 GTPV-A310 GTPV-A311 GB 724/01-20 B7 TP-2 DD1258 GB 320/02 SPV-A468 GB 182/98 GB 134/01

ORFV AGPV CNPVATCC CNPV72401 FWPVVR250 FWPVUS FWPVHPB FLPV36202 GTPV256 GTPVA310 GTPVA311 OSPV PGPVB7 PGPVTP2 SRPVDD1258 SRPV32002 SRPVA468 SCPV18298 TKPV13401

Falconpox virus Great titpox virus

Ostrichpox virus Pigeonpox virus Sparrowpox virus

Stone curlewpox virus Turkeypox virus

CNPV-like viruses seen previously [cluster IV of Luschow et al. (2004)]. Branch V observed by Luschow et al. (2004) consisted of only one virus, an isolate from agapornis. We can now see that it is joined by viruses from two other psittacines, parrot and macaw, to form clade C. This clade is the least divergent of the three, although this may be due to it consisting of only three isolates. Branch IV observed by Luschow et al. (2004) is represented by subclade B1.
Clade A. Sequence analysis at the P4b locus revealed that

the FWPVs (FWPVFP9, FWPV174, FWPVD, FWPVHPB


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and FWPVN), in subclade A1, were identical to each other as well as to SRPVDD1258 and TKPV13401, both German clinical isolates (Fig. 1a). The subclade A1 viruses showed 95?3 % nucleic acid identity (95 % amino acid identity) to FLPV36202 in subclade A4. Two other TKPV isolates (66 and 98) could be distinguished from FWPV (91 % nucleic acid identity), but not from PGPVP and PGPVTP2, as they clustered on the same branch of subclade A2, with 100 % nucleic acid identity, as they did with OSPV 724/ 01-20. ABPV and FLPV138196 (subclade A3) show 99 % nucleic acid identity to the subclade A2 viruses.
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Fig. 1. (a) Fpv167 (P4b) amino acid sequences aligned by using CLUSTAL_X and displayed by using MacGDE and Se-Al. See Table 2 for GenBank accession numbers. FWPV-like: FWPVD, FWPVN, FWPVM, FWPV174, FWPVHPB, TKPV13401, SRPVDD1258, FWPVFP9, FWPVV250, PGPVTP2, OSPV, PGPVP, TKPV66, TKPV98, ABPV353 and FLPV138196; CNPVATCC-like: CNPVATCC, SCPV182, GTPVA311, GTV256, SPRV32002, HOPV1252 and SRPV9037. In the consensus line, strictly conserved residues are in upper case. (b) Fpv139 amino acid alignment. FWPV-like: FWPVFP9, FWPVD, FWPVN, FWPVM, FWPV174 and FWPVC; CNPVATCC-like: CNPVATCC, CNPV1445, HOPV1165, HOPV1252 and SRPV9037.

Clade B. Viruses in clade B, all with 98 % or more

nucleic acid identity, show about 80 % nucleic and amino acid identity to viruses in clades A and C.
Clade C. MCPV and PRPV have identical sequences and

show 99 % nucleic acid identity to AGPV; thus, these viruses form clade C, with 86 % amino acid identity to viruses in clade A1 and 80 % to those in clade B1. Although the low divergence within this clade may be attributable to the small number of isolates, the isolates were apparently well separated in terms of host species, location and time: MCPV and PRPV were isolated from the different species in UK quarantine facilities some 3 years apart and AGPV was isolated in Germany (Luschow et al., 2004). H3L gene (fpv140) locus PCR products for the H3L (fpv140) gene locus, amplied from primers in fpv139 and fpv141, were obtained for viruses from clades A (FWPV-like) and B (CNPV-like), but not C (psittacine viruses). A clade-specic fragment-length polymorphism was observed for the PCR product, with each virus producing a product of either 1800 bp (for clade A viruses) or 2400 bp (for clade B viruses). Comparison of the known genome sequences for FWPV and CNPV indicated that the polymorphism was probably due to the absence (from clade A viruses) or presence (in clade B viruses) of a gene, CNPV185 or its equivalents, inserted between the orthologues of fpv139 (CNPV184) and fpv140 (CNPV186; Fig. 3).
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Because of the presence of the insertion, sequences were determined from each end of the products by using internal primers in combination with the anking primers M2904 and M2912. Sequence analysis of the PCR products conrmed the anticipated correlation between sequence-based phylogeny and the size of PCR product. All clade A viruses, like FWPV, encode a product for fpv139 consisting of 190 aa, whereas clade B viruses encode products, like CNPV184, of 189 aa. Within clade A, genes fpv141 and fpv140 are translated from the same reading frame, the two ORFs being separated only by the stop codon of fpv141. In clade B, in contrast, the orthologous proteins are translated from different reading frames, the start codon of CNPV186 being separated from the stop codon of CNPV187 by 91 bp. The phylogenetic trees for fpv139 (Fig. 4) and fpv141/140 (data not shown) are essentially the same as each other and as those seen for clades A and B with P4b. However, this locus also provides additional resolution within subclade A2, allowing us for the rst time to discriminate between the turkey and the pigeon isolates from within that subclade. Bootstrap analysis indicates that the pigeon isolate available to us from that subclade, PGPVP, is now clearly distinct from the turkey isolates remaining in subclade A2, clustering either with (but proximal to) subclade A3 (in 5560 % of trees) or proximal to both subclades A2 and A3 (in 4045 % of trees).
Clade A. At this locus, viruses in subclade A1 all show 100 % amino acid identity to each other and 9798 %
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Fig. 2. Phylogenetic analysis of avipoxvirus P4b orthologues. Abbreviations are as in Tables 1 and 2. (a) Unrooted, neighbour-joining phylogram of partial protein sequences for all genera in the subfamily Chordopoxvirinae. Bootstrap values (1000 replicates) of >60 % are indicated. (b) Neighbour-joining phylogram of DNA sequences from genes encoding avipoxvirus and MOCV P4b orthologues, rooted on MOCV. Bootstrap values (1000 replicates) of >80 % are shown. Sequences derived as part of this study are indicated by a dot. Avipoxvirus clades AC and subclades are labelled. Isolates from the same genus of host located in different clades or subclades are as indicated: *, pigeons; +, falcons. SRPVDD1258 and TKPV13401 are not thus marked as it could not be excluded that they were really FWPV (Luschow et al., 2004).

138 FWPV
170768

139

140

141
175414

183 CNPV
217940

184

185

186

187

223609 M2904 M2912

Fig. 3. (a) Schematic (to scale) showing the arrangement of FWPV genes fpv138 fpv141 aligned with their CNPV orthologues. CNPV185 has no FWPV orthologue. Primers M2904 and M2912 are shown below the CNPV sequence to which they bind. (b) PCR amplication of the fpv140 gene from avipoxvirus isolates. M, lX174 DNA digested with HaeIII. See Table 1 for virus abbreviations.

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DISCUSSION
The phylogenetics and epidemiology of few large groups of viruses are understood as poorly as are those of the avian poxviruses. Yet this group of viruses has, by virtue of the lifestyle of its hosts, an ability to cross natural and political boundaries, reaching new hosts as their migration and habitation patterns change. This feature of avian viruses is well illustrated by recent developments with West Nile virus and avian inuenza virus strain H5N1. Without knowing more about the avipoxviruses, it is difcult to assess the threat to reintroduction programmes, such as those for the great bustard on Salisbury Plain, Wiltshire, UK. The risks involved with reintroduction programmes in the Middle East for the closely related houbara bustard have been recognized (Samour et al., 1996; Bailey et al., 2002; Smits et al., 2005). Most of our knowledge concerning the avipoxviruses is based on clinical isolates from diseased birds. The infectious history of such isolates is rarely clear. It is important, therefore, that large-scale surveys, such as that performed in the Canary Islands (Huw Lee & Hwa Lee, 1997), are also conducted to understand the nature of viruses carried, sometimes as subclinical infections, by natural populations. Comparison at the molecular level of viruses from these two situations could then help to identify novel threats faced by birds, whether captive, farmed or free-living, in response to natural or man-made changes to their habitats and avian contacts. Unfortunately, the divergence among avipoxviruses is, as reinforced by this study, so great that it is difcult to identify PCR primers, whether pan-genus or species-specic. As a consequence, the only PCR locus used until now has been the P4b locus. It is in this context that this study sought to obtain a better phylogenetic framework for the avipoxviruses, by surveying more viruses with the previously used P4b locus and to identify new loci, such as H3L, that would permit a more robust classication.

Fig. 4. Phylogenetic analysis of avipoxvirus and MOCV orthologues of fpv139. Abbreviations are as in Tables 1 and 2. Neighbour-joining phylogram of DNA sequences (480 bp) from avipoxvirus and MOCV genes, rooted on MOCV. All avipoxvirus sequences, except FP9 and CNPVATCC, were derived as part of this study. Avipoxvirus clades A1, A2, A3, B1 and B2 are labelled. Bootstrap values (1000 replicates) of >75 % are shown, as is the value (in parentheses) for PGPVP. It was found as shown in a node with, but proximal to, subclade A3 in 55 % of trees or, alternatively, proximal to both subclades A2 and A3 in 45 % of trees.

P4b gene (fpv167) locus PCR analysis of the highly conserved P4b gene was initially used as a diagnostic marker for FWPV infections, generating a 578 bp product (Tadese & Reed, 2003). Amplication of an overlapping 1409 bp product (by changing one primer) allowed subsequent discrimination of FWPV from PGPV and TKPV by EcoRV restriction-enzyme digestion prole (Luschow et al., 2004). MseI or EcoRV digestion of the original 578 bp P4b PCR product identied six different proles, broadly discriminating between FWPV-like viruses, CNPV-like viruses, SRPV, PGPV, FLPV and AGPV. After sequence and phylogenetic analysis, the viruses were clustered into three groups equivalent to our subclades A1, A2 and B1, with two more single-virus branches, equivalent to subclade A4 and clade C.
Journal of General Virology 87

amino acid identity to viruses in subclade A2. Within subclade A2, the TKPV isolates (66 and 98) are identical to each other, with 97?9 and 96?5 % amino acid identity to ABPV and PGPVP isolates, respectively. The latter show 97?4 % identity to each other.
Clade B. At this locus, viruses in subclade B1 show 86 87 % amino acid identity to those in clade A. Within subclade B1, divergence between viruses isolated from different species (canary, houbara and sparrow) is barely more than that observed within CNPV.
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A problem with the host species-based approach to the taxonomy of avipoxviruses is that sequences of isolates taken from particular species can be found in different subclades (e.g. FLPV) or even different clades (e.g. PGPV). Luschow et al. (2004) had already reported that one of two SRPVs that they had sequenced (SRPV32002) grouped with CNPV-like viruses (clade B), but that the other (SRPVDD1258) grouped with FWPV-like viruses (clade A). Two others, examined only by restriction-enzyme analysis, were CNPV-like. Both of our SRPV isolates (SRPV23 and SRPV9037), as well as one from another study (SRPV32002; Weli et al., 2004), also cluster with CNPV-like viruses. SRPVDD1258 is actually identical to all of the FWPV strains at the P4b locus. As the majority of SRPV isolates are therefore CNPV-like, it is not clear whether SRPVDD1258 represents (i) a rarer form of the disease in sparrows, (ii) a sporadic species jump, perhaps into an already sick bird, or (iii) an emerging disease. The phylogenetic analysis conducted by Luschow et al. (2004) included only one PGPV (PGPVTP2), placing it in a cluster with OSPV, although restriction-enzyme analysis would place two more PGPV isolates in the same cluster (subclade A2). Weli et al. (2004), however, obtained the sequence of a PGPV isolate (B7) that clusters with CNPVlike viruses in clade B. Of the two PGPV clinical isolates analysed in this study, one (Peekham) falls with PGPVTP2 in subclade A2 and the other (950) in subclade B2 with PGPVB7. It is therefore apparent that viruses from clades A and B can infect and cause disease in pigeons. It is not clear whether there are any signicant differences in the disease caused by the two different types of viruses. In this study, both a 1962 UK TKPV isolate and a 1998 Italian TKPV isolate cluster not with FWPV in subclade A1, but with PGPVTP2 and PGPVP, as well as with OSPV, in subclade A2. However, by examining more recent isolates, Luschow et al. (2004) showed that a 2001 TKPV isolate from Germany clusters with FWPV (in subclade A1). By restriction-enzyme analysis, two more German TKPV isolates (from 1999 and 2000) cluster similarly. This therefore reinforces the changing picture of poxvirus infections in turkeys, with FWPV as a recently emerging pathogen of turkeys. A feature of the host-based approach to the taxonomy of avipoxviruses is that the host genus or species in which a virus causes severe disease and, therefore, from which it is most likely to be isolated need not be the normal reservoir host. Such situations may be common amongst passeriform birds infected by clade B CNPV-like viruses. Indeed, canaries, in which poxviruses cause devastating disease, may not be the natural reservoir for CNPV. In support of this perspective, a survey of wild birds in the Canary Islands found that 50 % of short-toed larks (Calandrella rufescens) and 28 % of Bertholets pipits (Anthus berthelotti) had mild poxvirus lesions (Smits et al., 2005). Virus isolates from sparrow and stone curlew cluster with CNPV (subclade B1), which correlates with all of the hosts
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for this subclade belonging to the order of passeriform birds. HOPV is part of the same viral phylogenetic cluster and, taxonomically, houbara bustard (order Gruiformes) is grouped with the order Passeriformes. PGPV clusters with viruses in the FWPV-like cluster (subclade A1), even though pigeon (order Columbiformes) is classied separately from turkey and chicken (order Galliformes) as well as ostrich (order Struthioniformes). However, another group of PGPVs clusters loosely with the order Passeriformes, in subclade B2. Therefore, the evolutionary taxonomy of the host does not appear to be the primary factor in driving avipoxvirus evolution, in so far as samples and sequences used within this study can show. In this context, it is interesting to note a recent report (Adams et al., 2005) describing the closely related P4b sequences from a wide range of different birds presenting to a wildlife centre in Virginia, USA, in 20032004. The viruses form a tight cluster resembling our subclade B1, but distinct from it and B2, essentially forming a new subclade B3. The authors admit the possibility of cross-infection in the clinic, but, even excluding those birds that developed lesions during hospitalization, the hosts included birds as diverse as cardinal, blue-grey gnatcatcher, mockingbird, house nch and great blue heron. It will be interesting to see whether the observation is conrmed by other centres, showing whether the viruses are more widespread within the USA, whether they can be found in earlier epornitics and whether they persist over time. H3L gene (fpv140) locus The fpv140 locus PCR product allows easy distinction between clades A and B based solely on the size of the primary product, without further restriction-enzyme digestion. Sequence analysis of these products also allows better discrimination between viruses within subclade A2, in particular between the isolates from turkey and pigeon that lie within this subclade. These primers can therefore be used as diagnostic markers to distinguish between FWPV-like and CNPV-like isolates by PCR and to differentiate between isolates by sequence analysis. They provide a useful addition to the currently used primers for the gene encoding P4b orthologues. Unfortunately, the marker appeared to fail with clade C viruses and with the one subclade A3 virus available to us. There is, therefore, still a need for other pan-genus markers, such as the P4b locus. The difculties of nding such a marker for genomes of this size and diversity, in the absence of more extensive genome-sequence data, should not be underestimated. Implications for phylogeny and systematics of the poxviruses Not all currently recognized avipoxvirus species are represented in the current tree: Juncopox virus, Mynahpox virus and QUPV (as well as tentative species Crowpox virus,
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Peacockpox virus and Penguinpox virus) remain to be analysed. However, a recent study (Hsieh et al., 2005) demonstrated that an isolate from Chinese jungle mynah (Acridotheres cristatellus), the sequence of which is not available in the databases, grouped with that from a wood pigeon (GenBank accession no. AY453177, described as PGPVB7 in this manuscript). This is interesting, and consistent with the phylogenetic structure reported here, because our starling isolate (SLPV) also groups with PGPVB7 and because transmission of enzootic poxvirus from starlings to mynahs has been reported (Landolt & Kocan, 1976). It may be premature to suggest changes to the nomenclature of avipoxviruses specically and of poxviruses generally, particularly as most data are available for only the P4b gene/ fpv168 locus (although more limited data for the H3L gene/ fpv140 locus are consistent). However, it is appropriate to consider the existing taxonomic structure (Fauquet et al., 2005) and to question whether it can accommodate the emerging data without reorganization. As an interim approach to nomenclature, we recommend reducing the number of recognized species to correspond with the phylogenetic groups. These could be FWPV (subclade A1), TKPV (subclade A2), CNPV (subclade B1), SLPV (subclade B2) and PSPV (clade C). PGPV would not be used, as it remains ambiguous until it can be established whether the existing taxonomic unit represents viruses in subclades A2 or B2. SRPV would be subsumed by CNPV and Mynahpox virus by SLPV. It is relevant to any longer-term changes in nomenclature that the avipoxviruses are currently represented by only one genus (Avipoxvirus), whereas the mammalian poxviruses are represented by seven genera. It is clear that, for the P4b orthologues, the divergence between the avipoxvirus clades A, B and C is comparable to that seen between the existing genera of mammalian poxviruses. The family Poxviridae is divided into subfamilies of insect (Entomopoxvirinae) and vertebrate (Chordopoxvirinae) viruses. This division offers little scope for taxonomic nomenclature that would both distinguish mammalian poxviruses from avipoxviruses and allow systematic groupings of the avipoxviruses. Two alternative approaches would offer such scope. The rst would envisage replacing the two existing subfamilies with three: for the insecta, the diapsida (or archosauria) and the synapsida (or mammalia). The other alternative would envisage elevating the family Poxviridae to order status and the existing subfamily divisions to family level. This would allow groupings for the insecta, the diapsida (or archosauria) and the synapsida (or mammalia) at the subfamily level. A third approach would be to maintain the existing nomenclature for family and subfamilies, but to replace the existing genus Avipoxvirus with three (or more) genera based on the major clades, with appropriate nomenclature.
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The loss of the genus Avipoxvirus might be mitigated by the use of genus names that incorporate avipoxvirus, such as: Falconiform avipoxvirus (with or without the space), Psittacine avipoxvirus, Passeriform avipoxvirus, Galliform avipoxvirus or Collumbiform avipoxvirus. Such genus names would, of course, also be compatible with the earlier two approaches. We hope that the data presented here will encourage the acquisition of sequence data from a wider range of enzootic and epizootic isolates of this fascinating and neglected group of viruses. This will allow a rational and effective approach to more extensive genome sequencing of carefully selected members of the group, bringing more insight into the complex relationship between avian viruses and their hosts. We urge those responsible for collecting isolates to be as rigorous as possible in recording details of the host (genus and species, if and where possible supported by archiving of a small sample of host tissue for future typing), relevant epidemiology (date, location, whether captive or wild, any known disease contacts), as well as virus isolation and passage history.

ACKNOWLEDGEMENTS
This work was funded by the Biotechology and Biological Sciences Research Council.

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