1 Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK
2 Viral Genomics and Bioinformatics Group, Department of Virology, and Department of Immunology & Molecular Pathology, University College London, 46 Cleveland Street, London W1T 4JF, UK
Correspondence
Geoffrey Smith
glsmith{at}imperial.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Recently, the number of poxvirus genome sequences has increased considerably (Table 1) (Upton et al., 2003
) and the sequences of two EnPVs and at least one member of each ChPV genus, except genus Parapoxvirus, are available for comparison. These data enable analysis of the evolutionary relationships of these viruses and the result of such an investigation is presented here. To study the evolutionary relationships of poxviruses, we have compared the size of virus genomes, the number of conserved and unique genes and their arrangement within the genome. The nucleotide or amino acid sequences of subsets of those genes were then used for phylogenetic analyses. Lastly, we have considered the OPV genus in more detail. The results showed that in addition to the close relationship of VAR and Camelpox virus (CMPV) that was noted previously (Gubser & Smith, 2002
), Monkeypox virus (MPV), which causes a disease with clinical similarity to smallpox, is divergent from all OPVs, and so are Ectromelia virus (ECT) and Cowpox virus (CPV) Brighton Red (BR). Data presented also suggest that the classification of CPV-BR and CPV-GRI-90 as two strains of the same species should be reassessed.
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METHODS |
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Alignment of multiple protein sequences.
Amino acid sequences of individual proteins were aligned by a method similar to that used previously (McGeoch et al., 2000). Sequences of each protein from the different viruses were aligned separately by each of the programs CLUSTALW (Thompson et al., 1994
), Dialign2 (Morgenstern, 1999
) and Multalin (Corpet, 1988
) using the default program parameters. For each set of data, combined alignments were produced by re-extracting the individual sequences from these three alignments, with retention of the gapping characters introduced by each program. Then a new alignment was made from this triple set of sequences using the program CLUSTALW. All positions in the combined alignment that had a gap in any sequence were then excised, thus deleting both unanimously placed gaps and sections where the three primary alignments were in conflict.
Phylogenetic analysis of multiple protein sequences.
The amino sequences of (i) 17 proteins that are conserved in all ChPVs or (ii) 12 proteins that are present in 12 OPVs were aligned individually and positions with gaps were excluded from the alignments as described above. The individual alignments were concatenated to form a single file of 10 451 (i) and 2316 (ii) amino acids, respectively. The most appropriate model of sequence evolution was determined using the program Treepuzzle. For both concatenated alignments, neighbour-joining trees (Saitou & Nei, 1987) were constructed using the programs Prodist and Neighbor from the PHYLIP package version 3.0 (Felsenstein, 1989
). These were then used as starting trees to construct the maximum-likelihood trees (Felsenstein, 1973
) using the program ProML according to the JonesTaylorThornton model (Jones et al., 1992
) with gamma distribution. Bootstrap analyses (Felsenstein, 1985
) were performed on both trees using the programs SEQBOOT (1000 random replicates, random number seed=133333), Protdist and CONSENSE.
Phylogenetic analysis of multiple OPV DNA sequences.
The nucleotide sequences of 12 genes present in the terminal region of 12 OPVs were aligned individually with the program CLUSTALW version 1.8 (Thompson et al., 1994). Positions with gaps were excluded from the alignment by manual inspection and individual alignments were concatenated to form a single file of 7233 (all genes), 4170 (genes present in the left end of the genome) and 3063 (genes present in the right end of the genome) nucleotides. For all concatenated alignments, neighbour-joining trees were constructed using the program PAUP* (Swofford, 2003
) and these were used as starting trees for the construction of maximum-likelihood trees (Felsenstein, 1973
) implemented using PAUP*. The model of nucleotide substitution used, as determined with Modeltest (Posada & Crandall, 1998
), was the General Time Reversible (GTR) model with gamma distribution and proportion of invariable sites (shape parameter of the gamma distribution=0·7082; proportion of invariable sites=0·5676). The robustness of trees was evaluated by bootstrap analysis of the neighbour-joining trees, with 1000 rounds of replication, using PAUP*.
Similarity analysis.
An analysis of the similarity of the terminal region of the CPV-BR and ECT-NAV genomes was done on the concatenated alignment used for phylogenetic alignment of OPV DNA sequences by using the program SimPlot (Ray, 1997). Genetic similarity was calculated according to the F84 model of evolution with a transition/transversion rate of 1·95.
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RESULTS |
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Fig. 1 also demonstrates that in the central region of ChPV genomes, the overall gene order and content are very well conserved between the the OPV, Leporipoxvirus, Yatapoxvirus, Capripoxvirus, Suipoxvirus and Molluscipoxvirus genera. A notable feature is the presence of a gene (C7L in VV-COP, Fig. 1
arrowheads) in the central region of the Leporipoxvirus, Yatapoxvirus, Capripoxvirus and Suipoxvirus genomes, but in the terminal region of OPVs. In some viruses, this gene has been triplicated. This indicates that the genera Leporipoxvirus, Yatapoxvirus, Capripoxvirus and Suipoxvirus form a subgroup that is distinct from the OPVs.
In contrast, the genomes of FPV (Avipoxvirus) and MCV (Molluscipoxvirus) are divergent from other ChPV genera and contain many unique genes. FPV also shows rearrangement of the conserved genes. Whereas all the other ChPV genera have a conserved gene order, blocks of FPV genes have been transposed and/or inverted. The blocks of VV genes that run left to right 1, 2, 3 and 4 are present in FPV in the order 3, 1, 2 and 4 with blocks 1 and 3 in inverted orientation (Afonso et al., 2000). This observation suggests that the FPV genome is most divergent compared to the other ChPVs.
Fig. 1 does not include the EnPVs because these are too divergent from ChPVs by genome size, gene arrangement and gene sequence similarity. Previous work showed that EnPVs are also divergent amongst themselves (Afonso et al., 1999
; Bawden et al., 2000
), which suggests that EnPVs diverged from ChPVs before the ChPVs evolved into distinct genera. These results also agree with previous suggestions that the orthopteran and lepidopteran members of genus B of EnPV might be split into separate genera (Afonso et al., 1999
; Bawden et al., 2000
). The overall amino acid identity of the 17 proteins we used for phylogenetic analysis of ChPVs (next section) is between 26·0 % and 29·9 % when comparing either EnPV with any ChPV, and only 55 % between the two EnPVs. For comparison, this value is 98·7 % between two members of the OPV genus (VV-COP and VAR-BSH), 94·8 % between the two leporipoxviruses (MYX and Shope fibroma virus (SFV)] and
94 % between different capripoxviruses (data not shown). Because EnPVs are too divergent to be compared to ChPVs, or even to provide a reliable root, they were omitted from further analysis.
Phylogenetic relationships
Previously, the evolutionary relationships of poxviruses had been investigated based largely on genome collinearity and the nucleotide or amino acid sequence alignment of a few genes or proteins. However, rigorous phylogenetic studies using DNA and protein sequences from multiple genes for each virus are lacking. To establish phylogenetic relationships for ChPVs, we have compared (i) multiple protein sequences that are conserved in all sequenced ChPVs, (ii) DNA and protein sequences from terminal regions of the genomes that are conserved in OPVs, and (iii) DNA sequences from the central 100 kb of OPV genomes.
To compare the different ChPVs we selected 17 out of the 49 proteins conserved in all poxviruses. These were aligned for one or more member of each genus, nine viruses in total: SWPV, YLDV, VV-COP, VAR-BSH, MYX, SFV, MCV, LSDV and FPV. The use of several protein sequences to produces a single tree is more likely to represent the species tree accurately than a tree constructed with any single sequence. Previously, phylogenetic trees for single OPV proteins gave variable topologies (Afonso et al., 2002b; Gubser & Smith, 2002
). Similarly, others reported inconsistent tree topologies using single genes from closely related species (Huelsenbeck & Bull, 1996
). The proteins chosen for analysis (VV-COP E9L, I7L, I8R, G9R, J3R, J6R, H2R, H4L, H6R, D1R, D5R, D6R, D11L, D13L, A7L, A16L and A24R) were selected to represent enzymes that are essential for transcription or DNA replication, and structural components of new virions (Table 2
). All selected proteins are of similar length in the different viruses and are well conserved. Alignments were made for individual proteins, these alignments were edited and the sequences were concatenated into a single file that was used to construct a maximum-likelihood tree (Methods; Fig. 2
). A tree drawn using the neighbour-joining method gave similar data (data not shown). The branch structure of the maximum-likelihood tree is unequivocal.
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After the eradication of smallpox, MCV remains the only endemic human-specific poxvirus and it is divergent from other ChPVs. MCV is well adapted to humans (it survives long term and causes little morbidity) and this is reflected by 70 unique proteins (including several immunomodulators) and the lack of most of the immunomodulators encoded by other poxviruses (Senkevich et al., 1996). Previous phylogenetic studies carried out using single MCV proteins resulted in different tree topologies depending on the gene and the method of tree construction, with MCV grouping individually in most cases but also together with FPV (Senkevich et al., 1996
). Data presented in Fig. 1
show that MCV and FPV are distinct ChPVs that have diverged from other ChPVs long ago. Like FPV, the conserved MCV proteins show a modest percentage amino acid identity with other ChPVs (range 61·7 % to 63·4 %) and MCV is no closer to any specific ChPV genus. MCV is the only ChPV not to contain a counterpart of VV-COP gene J2R (thymidine kinase).
The third and largest cluster of ChPVs includes the Yatapoxvirus (YLDV), Capripoxvirus (LSDV), Suipoxvirus (SWPV) and Leporipoxvirus (SFV and MYX) genera. Within this group, SFV and MYX, which cluster strongly together, also group with SWPV and LSDV, whereas YLDV is slightly more divergent. The genomes of all these viruses are relatively well conserved in gene content, gene arrangement (Fig. 1) and amino acid identity (data not shown). Notably, unlike OPVs, these viruses all contain at least one counterpart of VV gene C7L within the central region of their genomes (Fig. 1
, arrowheads) between counterparts of VV-COP genes J2R and J3R. For these four ChPV genera, the overall amino acid identity is highest between SWPV and LSDV (79·2 %). This is consistent with the tree topology (Fig. 2
) and suggests that SWPV and LSDV have evolved from a common ancestor.
The fourth ChPV group is genus OPV, illustrated by VV-COP and VAR-BSH, which group together tightly and separately from other ChPVs. The scale of the phylogenetic tree shows how closely related these viruses are compared to, for instance, the different leporipoxviruses and suggests that this group of viruses diverged more recently than members of other ChPV genera. Another feature that distinguishes the OPVs from other ChPVs is the presence of genes equivalent to VV-COP F14L, E7L and O2L within the central conserved region. ChPVs from outside the OPV genus lack these genes.
In summary, the comparison of poxviruses from different ChPV genera with each other using two different computational methods gave a robust phylogenetic tree. The only ChPV genus not represented here is Parapoxvirus, for which a complete genome sequence is awaited. The OPV genus is now considered in more detail.
Orthopoxvirus phylogeny
OPVs are the most intensively studied poxviruses. The reasons for this are largely historical: smallpox, caused by VAR, used to be a very serious disease of mankind; CPV is thought to have been used by Jenner in 1798 as the first human vaccine; and VV is the smallpox vaccine that was used in the modern era. Currently, there are 12 complete OPV genome sequences from 6 species (VAR, VV, CPV, MPV, ECT, CMPV) (Table 1). The origin and evolutionary relationships of these viruses are ill-defined, although it was demonstrated recently that CMPV-CMS and VAR are closely related (Gubser & Smith, 2002
).
To compare the phylogeny of OPVs we selected genes that are conserved in the terminal genome regions of these viruses, where genes have greater divergence between species. Within the terminal regions of 10 sequenced OPVs, only 12 out of 100 genes are present in every virus and this is due in part to mutations causing disruption of several genes in different OPVs. The conserved genes are VV-COP C7L (host range function; Perkus et al., 1990
), C6L (unknown), N1L (intracellular virulence factor; Bartlett et al., 2002
), K2L (serine proteinase inhibitor, SPI-3; Law & Smith, 1992
), F2L (dUTPase; McGeoch, 1990
), F4L (ribonucleotide reductase, small subunit; Slabaugh et al., 1988
), F6L (unknown), F8L (unknown), A56R (the haemagglutinin glycoprotein that forms part of the extracellular enveloped virus outer envelope; Shida, 1986
), B1R (protein kinase; Banham & Smith, 1992
), B5R (EEV glycoprotein; Engelstad et al., 1992
) and B15R (unknown). These genes are known or likely to have an important function.
The proteins encoded by the selected genes were aligned and used to construct a maximum-likelihood tree (Methods, Fig. 3). Several facts may be deduced from the tree. First, the close relationship of CMPV and VAR is confirmed. The three strains of VAR each cluster together, as do the two strains of CMPV, and the VAR and CMPV clusters are more closely related to each other than to any other OPV species. Second, CPV-BR, MPV and ECT do not group closely with other OPVs. Lastly, although the two VV strains cluster closely together, the two CPV strains do not and they show remarkable divergence for two strains of the same virus species. It will be interesting to determine if other CPVs group predominantly with CPV-BR or GRI-90 or even form further divergent groups. These results suggest that classification of CPV as a single species within the genus OPV needs reconsideration.
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From this alignment, a distance matrix was constructed and is shown in Table 3. As expected the genetic distances between OPVs are low (0·01500·0354) compared to the genetic distance for the same region of SFV and MYX, which was eightfold greater than between CMPV and VAR (Gubser & Smith, 2002
). This suggests that OPVs have diverged more recently than the leporipoxviruses from their common ancestor. When comparing different OPV species, the genetic distance was lowest between strains of CMPV and VAR (0·01510·0154), and highest between ECT and VAR (0·03520·0354) (Table 3
).
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DISCUSSION |
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The phylogenetic relationships of the sequenced poxviruses were examined. The genome organization, and percentage amino acid sequence identities, showed that the two sequenced EnPVs are distinct from ChPVs and quite divergent from each other so that they might be classified in separate genera (Afonso et al., 1999; Bawden et al., 2000
). Within the ChPVs, the most divergent virus is FPV (genus Avipoxvirus) followed by MCV (genus Molluscipoxvirus). This overall conclusion is reached by comparison of the size of these genomes, the number of unique genes, the gene arrangement (Fig. 1
) and phylogenetic analysis of the amino acid sequences of 17 conserved proteins (Fig. 2
). Avipoxviruses are the only ChPVs to infect birds and MCV is a strictly human pathogen; both viruses have evolved unique immunomodulatory proteins that enable them to counteract the immune system of their hosts.
The other ChPVs show two clusters: the first includes the genera Leporipoxvirus, Capripoxvirus, Suipoxvirus and Yatapoxvirus, within which SWPV and LSDV share a common ancestor; the second is genus OPV. The first group of viruses have smaller genomes [range 144 575 (YLDV) to 161 774 (MYX); Table 1] and few unique genes in the central region of the genome (Fig. 1
). These genomes also all have the orthologue of the VV-COP gene C7L within the central region of the genome, whereas in OPVs this is present near the left end. The relatively small size (139 kb) of the Orf virus genome (Mercer et al., 1987
) suggests genus Parapoxvirus might group within this cluster.
The OPVs represent a closely related group of viruses with larger genomes [177 923 (VV-MVA) to 224 501 (CPV-BR)]. Note that VV-MVA has lost about 30 kb compared to the parental Ankara strain (Meyer et al., 1991).
Overall, the phylogenetic analyses of poxvirus protein sequences give relationships between genera (Fig. 2) that are consistent with relationships deduced from comparisons of genome organization and gene content (Fig. 1
).
A more detailed analysis of OPVs revealed that the central regions of these genomes are very similar. Here the genes are collinear and over 100 kb CMPV-CMS and VAR-BSH differ in length by only 82 nucleotides. Because of the high degree of similarity of genes and proteins from this region of OPVs, we compared the phylogenetic relationships of these viruses by using the DNA and protein sequences of genes from the terminal regions of the genome (Figs 3
5). These analyses established phylogenetic relationships but also indicated that these viruses have undergone recombination during their evolution. Data presented show that CMPV and VAR are closely related species, as reported previously (Gubser & Smith, 2002
), but CPV-BR, MPV and ECT are divergent and do not group closely with other OPVs. For MPV, this is despite it causing a disease in man similar to smallpox. MPV occurs naturally in western and central Africa but is poorly transmitted from person to person and human infections tend to be limited local outbreaks. It has been proposed that rodents are the natural host for MPV (Fenner et al., 1988
).
The origin of the most extensively studied OPV, VV, is obscure. If Jenner used CPV as the first smallpox vaccine in 1796, sometime between then and 1939 when A. W. Downie reported that the available smallpox vaccine strains were a distinct OPV that became known as VV (Downie, 1939a, b
), CPV was replaced by VV as the smallpox vaccine. This probably had occurred by the late nineteenth century because the smallpox vaccine taken to the USA in 1856 and which became the New York City Board of Health Vaccine is VV not CPV. Similarly, pathologists who studied cells infected by smallpox vaccines used in the late nineteenth century reported the eosinophilic B type inclusion bodies made by VV and CPV, but failed to report the much more obvious A type inclusion bodies that are made by CPV but not VV. Therefore, by this time VV was probably already used as the smallpox vaccine. The possible origin of VV was discussed by Baxby (1981)
. He proposed VV was a distinct OPV species that was isolated from a species in which it was no longer endemic. Horsepox was one possibility. In support of this proposal, early vaccinators took vaccine from horses when the supply of CPV (a relatively rare disease) was scarce and one strain of VV (Ankara) was isolated from a horse. The recent demonstration that the VV-WR interferon-
receptor binds and neutralizes equine interferon-
(Symons et al., 2002
) is also consistent with this proposal. However, given the broad host range of VV and the broad species specificity of the VV interferon-
receptor, these observations might be interpreted only as VV being a zoonosis in horses and that its natural host lies elsewhere. Phylogenetic comparisons indicate VV is not derived recently from either VAR or CPV but that it is closer to CPV-GRI-90 than CPV-BR.
An interesting feature of OPV genomes is the presence of many genes that are intact in one virus but fragmented in another. Comparison between OPV genomic sequences reveals that fragmented ORFs (i) are located mainly within 50 kbp of either terminus, (ii) represent 33/206 ORFs (16 %) in CMPV (Gubser & Smith, 2002) and (iii) are fewest in CPV (Shchelkunov et al., 1998
). Many of these gene fragments are unlikely to encode functional protein, so their retention is surprising. This might reflect the relative stability of OPV genomes and the apparent lack of a stringent packaging limit on poxvirus DNA (Smith & Moss, 1983
; Perkus et al., 1991
). Another interpretation is that some OPVs are relatively recent pathogens of their respective hosts and have diverged from an ancestral virus recently in an evolutionary timescale, with disruption of some genes accompanying their divergence. The much closer relationship between OPVs than between leporipoxviruses is consistent with this interpretation (Fig. 2
, Table 3
). With time these viruses might have lost some of these non-essential gene fragments. In contrast to OPVs, within the leporipoxviruses SFV contains eight fragmented genes compared to MYX, but MYX contains only one fragmented gene compared to SFV (Cameron et al., 1999
; Willer et al., 1999
).
Finally, the two CPV strains are sufficiently divergent to justify their reclassification as different OPV species and it will be interesting to determine how other CPV strains isolated from different geographical locations compare with the two strains analysed to date. CPV-GRI-90 was proposed as a possible ancestral virus for OPVs because its genes in the terminal genome regions are mostly complete (Shchelkunov et al., 1998), whereas other OPVs possess broken fragments of these genes. By this criterion, CPV-BR also might be considered close to a possible ancestral virus.
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ACKNOWLEDGEMENTS |
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Received 8 August 2003;
accepted 3 October 2003.