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Short Communication |
1 Institute for Animal Health, Compton, Newbury RG20 7NN, UK
2 Veterinary Laboratory Agency (VLA), New Haw, Addlestone, Surrey KT15 3NB, UK
Correspondence
Michael A. Skinner
m.skinner{at}imperial.ac.uk
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AM396435–AM396442.
Present address: Department of Virology, Imperial College London, Faculty of Medicine, St Mary's Campus, Norfolk Place, London W2 1PG, UK. 
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Afonso et al., 2000). Recent phylogenetic analysis indicates that the avipoxviruses show considerable divergence, falling into three major groups, representing (i) Fowlpox virus (FWPV) or FWPV-like viruses, (ii) Canarypox virus (CNPV) or CNPV-like viruses, and (iii) psittacine poxviruses (Jarmin et al., 2006). The prototypic avipoxvirus, FWPV, causes disease in chickens and turkeys worldwide and is the most extensively studied member of the genus. The disease is characterized by the development of lesions on various parts of the skin, particularly unfeathered areas. The clinical signs in naturally infected birds vary depending on the virulence of the virus, susceptibility of the host and distribution and type of lesion in the birds (Tripathy & Reed, 2003; Skinner et al., 2005).
Vaccines have been available since the 1920s and have been an effective method for control of transmission of this disease. Commercial, live-attenuated vaccines against fowlpox, turkeypox, pigeonpox and quailpox are available and are used to reduce the economic impact of the respective diseases on the poultry industry. These vaccines are sometimes used against non-target species. One example is the accepted use of Pigeonpox virus to vaccinate against the closely antigenically related FWPV. Another example is their use in avian species for which no appropriate homologous vaccine exists, such as birds held in captivity in zoos and private collections, or in conservation and reintroduction programmes (Bailey et al., 2002). Problems with fowlpox persist in many parts of the world, with indications that variant viruses may be responsible (Back et al., 1995; Singh et al., 2000). There is, therefore, a need to characterize vaccine strains so that their persistence in the field may be monitored in comparison with the presence of field strains. The increasing field use of recombinant FWPV vaccines, such as those against avian influenza H5N1 (Swayne et al., 2000) and Newcastle disease, makes this imperative.
The genome sequences of two FWPVs are available. The first (FPV USDA; GenBank accession no. AF198100
[GenBank]
) was described in the publication (Afonso et al., 2000) only as a ‘pathogenic fowlpox virus’, but is described by the US Department of Agriculture (USDA), who supply it as a standard challenge virus, as ‘derived from a fowl pox vaccine manufactured by a commercial firm in the early 1960's' [USDA Animal and Plant Health Inspection Service Veterinary Services (APHIS VS)]. The second (FP9; GenBank accession no. AJ581527
[GenBank]
) is of a highly passaged and highly attenuated derivative (HP1-438 Munich FP9). FP9 shows multiple deletions relative to FPV USDA, totalling 22 kbp. We were interested in determining whether commercial vaccines show deletions similar to those found in FP9.
Five commercial fowlpox vaccines, Diftosec CT (Rhone Merieux), Nobilis Variole W (Intervet), Chick‘N’Pox (Salsbury Laboratories Inc.), FPV M (Cyanamid Webster; Boyle et al., 1997) and Poxine (Duphar) were studied. One pathogenic field isolate was HP1 Munich (Mayr & Malicki, 1966), the progenitor of FP9 (Laidlaw & Skinner, 2004). FP9 was derived by plaque purification from a mixed population of viruses derived by more than 400 serial passages of HP1 in chick embryo fibroblast (CEF) culture (Mayr & Malicki, 1966). Here, HP1 and FP9 were also compared with laboratory virus HP1-200, a mixed population of viruses derived from HP1 by 200 passages in CEFs (Mayr & Malicki, 1966). The other FWPV field isolate was FPV 174/04 (FPV 174), a pathogenic UK isolate collected by the VLA, New Haw, Addlestone, UK (Jarmin et al., 2006). Received samples were propagated on CEFs; virus purification and DNA extraction were performed as described previously (Jarmin et al., 2006).
Oligonucleotide primers used in this study (Table 1) were based on the FPV USDA sequence, GenBank accession no. AF198100
[GenBank]
(Afonso et al., 2000), and were designed to distinguish FP9 from its virulent precursor HP1 (Laidlaw & Skinner, 2004). PCR amplification was carried out as described previously (Jarmin et al., 2006). The mix was subjected to one cycle of 5 min at 94 °C, 30 cycles of denaturation for 30 s at 94 °C, annealing for 30 s at 40 °C and elongation for 5 min at 72 °C, then 72 °C for 10 min.
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), with the exception of the H9 locus, which produced a length heterogeneity (Fig. 1a). As expected, FP9 did not produce a PCR product for this locus, as primer M1411 is spanned by a deletion of 9334 bp (deletion 24) that removed fpv241–fpv247 inclusive (Laidlaw & Skinner, 2004). NB: to distinguish 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 first letter) for the products that they encode (e.g. Fp9.030 or Fpv030). Primer M1396 binds upstream of fpv241 (FPV USDA position 267357), whilst M1411 binds within fpv242 (FPV USDA position 268356); see Fig. 1(b, d). Not surprisingly, products of about 1 kbp were seen with Chick‘N’Pox and FPV M, compatible with the 999 bp product predicted for FPV USDA. It was, however, surprising to see larger-than-expected products of 2200 bp for both European field isolates (HP1 and FPV 174), passaged HP1 (HP1-200) and three of the vaccines: Nobilis Variole W (Nobilis), Diftosec CT (Diftosec) and Poxine. We had previously only confirmed that HP1 did not have FP9 deletion 24, using primers different from those shown in Table 1 (Laidlaw & Skinner, 2004).
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), with the same primers as were used for amplification. Sequence analysis was carried out with programs in STADEN (Staden, 1982), CLUSTAL_X (Thompson et al., 1997; Hall, 2001) and PAUP.
When sequenced and aligned, the larger 2.2 kbp products were found to contain fpv241 and fpv242 sequences on either side, but the middle section contained 1207 bp (1206 bp in the case of Nobilis) of ‘novel sequence’ (i.e. not observed previously and not found within the existing sequence databases, either within FWPV genomes or elsewhere). When the ‘novel sequence’ is present, the fpv241 ORF is longer than it is in FPV USDA (GenBank accession no. AF191800
[GenBank]
), encoding a protein of 186 aa, compared with 106 aa for FPV USDA. Although 10 residues shorter than the CNPV309-encoded orthologue (196 aa), the extended Fpv241 protein (Fpv241ext) shows similarity to that encoded by CNPV309 throughout its length. It therefore appears likely that fpv241ext represents an ancestral FWPV form of the gene, with more extensive similarity to CNPV309. This suggests that, rather than representing an insert, the ‘novel sequence’ represents sequence found in European field isolates and retained in some vaccines, but lost from others. ORFs fpv241ext and CNPV309 appear to encode potential orthologues of vaccinia virus M1, an ankyrin-repeat protein deleted from MVA, the attenuated, host-restricted vaccinia virus (Shisler & Jin, 2004).
Analysis of the next 830 bp of ‘novel sequence’ downstream of fpv241ext showed essentially no significant similarity at nucleotide (except for 41 bp, showing 90 % identity to mitochondrial DNA from the fungus Schizophyllum commune) or amino acid level (as determined by using BLASTX or TBLASTX) with any sequences in GenBank. Even though small ORFs do exist (Fig. 1b), they show no significant database similarity by TBLASTN.
The remaining 163 bp of the ‘novel sequence’ encodes a short ORF with similarity to part of the protein encoded by CNPV313 [the first of the four short CNPV313-like ORFs shown in Fig. 1(b, c)] and provides an N-terminal extension to the first of the three CNPV313-like ORFs found in FPV USDA [these are the second, third and fourth short CNPV313-like ORFs shown in Fig. 1(b, c), downstream of the right-hand breakpoint of the 1207 bp ‘insert’]. Taken together, the four short ORFs span most of a fragmented FWPV orthologue of CNPV313 (Fig. 1c), an immunoglobin-domain protein (Tulman et al., 2004). Fragmentation of this gene, therefore, appears to be species-specific rather than merely strain-specific. The adjacent gene in CNPV, CNPV314, is equivalent to a fusion of fpv242 and fpv243 (Afonso et al., 2000; Tulman et al., 2004).
Some minor differences are apparent between the viruses within the ‘novel sequence’: Nobilis has an A to C transversion (changing I to L in Fpv241ext) and deletion of a G, FPV 174 has a synonymous G to A transition and Diftosec has two neighbouring transitions (AA to GG). The single-base deletion in Nobilis disrupts the upstream CNPV313-like ORF fragment found in HP1, FPV 174, HP1-200, Poxine and Diftosec (Fig. 1b, c).
The provenance of the commercial vaccines is not documented. However, it is interesting that the ‘novel sequence’ was present in both European field isolates, FPV 174 and HP1, even though they were isolated more than 40 years apart in the UK and Germany, respectively. It is possible that the ‘novel sequence’ will be specific to European isolates; isolates from elsewhere were not available to us. The observed retention of sequence within HP1, HP1-200, FPV 174, Poxine, Diftosec and Nobilis, the partial deletion in FPV M, FPV USDA and Chick‘N’Pox and the extensive deletion in FP9 are indicative of a significant level of instability of the region. It is tempting to suppose that the large deletion in FP9 occurred between passages 200 and 438. However, although FP9 is a plaque-purified derivative, HP1-200 is not. It is almost certainly heterogeneous and so the possibility that a small amount of deletant virus was already present at passage 200 and that it was subsequently amplified cannot be excluded. Nor, indeed, is it certain that FP9 did not represent a minority population at this locus, although it was chosen as the plaque most representative of the unpurified population, as determined by Southern blot analysis (S. M. Laidlaw, unpublished observations).
The novel sequence described here may not represent the full extent of sequence existing in the fowlpox ‘virosphere’ at this locus or, indeed, even at other loci. The possibility cannot be excluded that additional sequence is present in field viruses, but is lost rapidly during any attempts at culture. We previously reported a mixture of parental and mutant sequences in HP1 for two deletions (11 and 22) found in FP9, even though the HP1 had been passaged only a few times through CEFs (Laidlaw & Skinner, 2004). It is apparent that the genome sequence of FPV USDA should not be regarded as that of a ‘wild-type’ virus, but as that of a vaccine strain of uncertain history, possibly with additional deletions relative to field isolates. The full genomic sequence of a bona fide pathogenic field isolate would clearly be useful in this regard.
It is intriguing that two commercial vaccines (FPV M and Chick‘N’Pox), as well as FPV USDA, which was ‘derived from a fowl pox vaccine manufactured by a commercial firm in the early 1960's' (USDA, APHIS, VS), all carry the identical deletion. It seems most likely that they share a common ancestry, unless there is strong selection for that particular deletion genotype. We do not know the extent of distribution of field viruses retaining the ‘novel sequence’. It is possible that field viruses that have fixed the 1.2 kbp deletion exist in nature, so that vaccines derived from them share this feature. These issues will be difficult to resolve without extensive sequencing of field viruses and exhaustive sequencing of the vaccines, given that the other 17 PCR loci revealed no obvious differences between the viruses.
It is possible that the 1.2 kbp sequence might play a significant role in virulence, although retention of the sequence in two extensively used commercial vaccines might argue against this likelihood. It is more likely that that the larger deletion in FP9 would play a role in attenuation of that virus. Both deletions (1.2 and 9.3 kbp) mainly target proteins containing ankyrin repeats, namely Fpv241/CNPV309, CNPV310 (equivalent to a fusion of Fpv242 and Fpv243), Fpv244, Fpv245 and Fpv246. Many such proteins are encoded by the avipoxviruses; smaller numbers are found in mammalian poxviruses. Their roles, except in a few specific cases, such as myxoma virus M-T5 or M150 and vaccinia virus K1 (Camus-Bouclainville et al., 2004; Bradley & Terajima, 2005; Johnston et al., 2005; Wang et al., 2006), as well as the need for so many members of this gene family, remain enigmatic. Recent identification of an F-Box-like domain in most poxvirus ankyrin-repeat proteins led to the suggestion that they might target a wide range of cellular proteins for ubiquitination (Mercer et al., 2005).
The H9 locus may prove useful for monitoring clinical isolates from fowlpox-infected birds or for monitoring the spread and persistence of vaccine strains, particularly when used alongside screens for the presence of full-length reticuloendotheliosis virus (REV) proviral sequences, found in the genome of many field isolates of FWPV, or for REV long terminal repeat sequences retained in many FWPV vaccines (Hertig et al., 1997; Singh et al., 2003).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 11 July 2006;
accepted 4 August 2006.
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