1 Department of Biochemistry and Microbiology, University of Victoria, Ring Road, Petch Bldg, Rm 150, Victoria, BC, Canada V8W 3P6
2 Department of Molecular Microbiology and Immunology, St Louis University School of Medicine, St Louis, MO 63104, USA
3 Department of Microbiology, University of Alabama (Birmingham), Birmingham, AL 35294-2170, USA
Correspondence
C. Upton
cupton{at}uvic.ca
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number of the RPXV-UTR genome sequence is AY484669.
A full list of RPXV-UTR annotated ORFs is available as a supplementary table in JGV Online.
These authors contributed equally to this paper.
Present address: Genelux Corporation, 3030 Bunker Hill Street, Suite 310, San Diego, CA 92109, USA.
Present address: Department of Microbiology & Immunology, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA.
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INTRODUCTION |
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Rabbitpox virus (RPXV) was first isolated and reported in 1932 from one of a series of epidemics of a highly lethal, airborne infection of laboratory rabbits at the Rockefeller Institute in New York (Greene, 1933). At this time, the clinical, pathological and virological features of the disease were thoroughly studied by Greene (1934a
, b)
. In 1941 in Utrecht, the Netherlands, another outbreak occurred. An isolate from this outbreak was designated RPXV strain Utrecht (RPXV-UTR) (Fenner, 1994
; Esposito & Fenner, 2001
). VACV infections of the skin of rabbits by intradermal scarification (dermal VACV) or the respiratory tract with aerosol infectious doses as high as 1·3x104 p.f.u. failed to produce a fatal disease; however, similar aerosol infection with RPXV-UTR produces almost uniform fatalities with a dose of 15 p.f.u. per rabbit (Westwood et al., 1966
). Virological studies determined that dermal VACV and RPXV-UTR replicated to similar titres in the rabbit lung, but only RPXV-UTR showed consistent and significant titres in internal organs such as spleen, liver, kidney, gonads and brain, which suggests that dermal VACV is unable to disseminate from the lung (Westwood et al., 1966
). Since RPXV is spread via the respiratory tract, it may prove to be a good non-primate model for the study of smallpox. For many years it was suspected that RPXV may have been derived from VACV, as it arose at Rockefeller University in laboratories where work on VACV in rabbits was ongoing, and its genome is similar to VACV by restriction endonuclease analysis (Wittek et al., 1977
); however, the molecular basis of RPXV's enhanced virulence for the rabbit compared with VACV was unknown.
In this study we report the DNA sequence of the entire protein-coding region of the genome of RPXV-UTR and the identification of three RPXV-UTR genes that are conserved in VARV but not in sequenced VACV strains and thus may contribute to the enhanced virulence of RPXV and VARV over VACV. In addition, phylogenetic analysis of these genomes shows that VACV and RPXV are so closely related that they may be considered strains of the same virus.
Finally, knowledge of the protein-coding regions of the RPXV genome will facilitate the use of this virus in testing the efficacy of OPV antivirals and vaccines. Currently most antivirals and vaccines are tested in mouse-based OPV challenge models, and additional small animal (non-primate) models will be necessary for US Food and Drug Administration approval of smallpox prophylactics and therapeutics as described in the Animal Rule (FDA, 2002). Since RPXV-UTR encodes several virulence factors that are also present in VARV but absent from VACV, RPXV-UTR infections of rabbits may prove to be such a model.
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METHODS |
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Purification of RPXV genomic DNA, genome sequencing and assembly.
Standard procedures were used and have been described previously (Chen et al., 2003). With the exception of the terminal hairpin loops, the entire RPXV genome was divided into 19 overlapping fragments of an average size of 11 kb. Each fragment was amplified from genomic DNA and sequenced with a bank of sequencing primers. The final DNA consensus sequence represented on average a 3·7-fold redundancy, with each nucleotide being covered by at least one high-quality sequence read in each direction.
In order to improve reliability in the DNA sequence the following precautions were taken: (i) DNA templates pooled from multiple PCRs (typically 818) were used in sequencing reactions; (ii) a relatively large amount of template was used in the original PCRs; (iii) uncloned PCR products were used as sequencing templates; the Expand Long Template PCR System was chosen because it employs a high-fidelity DNA polymerase. We have sequenced two VACV isolates, separated by only a few passages in tissue culture, with this method and found no differences between the genomes (data not shown).
Annotation.
An open reading frame (ORF) was defined as a continuous stretch of DNA that translated into a polypeptide initiated by a methionine residue and extending for at least 60 amino acids prior to a termination codon (TGA, TAA or TAG). ARTEMIS (Mural, 2000), the Poxvirus Orthologous Clusters database (POCs) (Ehlers et al., 2002
; Upton et al., 2003
), BLASTP (Altschul et al., 1997
) and GeneStar software (Windows) (Burland, 2000
) were used to detect and annotate ORFs. In addition, for some ORFs, BLASTN, TBLASTN and BLASTP searches were carried out at the NCBI website (Altschul et al., 1990
). EMBOSS Showorf software (Rice et al., 2000
) and nucleotide-amino acid alignment program (NAP) (Huang & Zhang, 1996
) were used on a Linux platform to compare the nucleotide sequence of the fragmented ORF regions of RPXV against the corresponding longest protein sequence encoded by orthologous chordopoxvirus (ChPV) genes. Based on the results of Showorf and NAP, we constructed a physical map of the genome that includes the fragmented ORF regions IXII. Viral genome organizer (VGO) software (Upton et al., 2000
) was used to analyse the position and arrangement of genes.
Phylogeny analysis.
Phylogenetic analysis was carried out using a genomic nucleotide sequence alignment of the conserved central region of all OPV genomic sequences currently available in GenBank (see Sequence availability section below). The alignment extended from base 20352 to base 163510 of the RPXV-UTR genome. This alignment starts with ORF 13 and extends just past ORF 159 of RPXV-UTR and corresponds to the VACV-Copenhagen (VACV-COP) genes C7L to A51R (which is fragmented in RPXV-UTR). Sequence alignments were generated using a combination of the programs MAVID and multi-LAGAN (Brudno et al., 2003; Bray & Pachter, 2004
). The final computational alignment was then hand-edited extensively to optimize the alignment.
Phylogenetic inferences were generated using both maximum-parsimony and Bayesian inference methods. Maximum-parsimony trees were constructed using PAUP* version 4.0b10 (Swofford, 2003), while MrBayes version 3.1 (Ronquist & Huelsenbeck, 2003
) was used for Bayesian inference methods. Maximum-parsimony trees were constructed using the branch-and-bound search method and employed 1000 replicates for bootstrap resampling analysis. Bayesian inference using Markov chain Monte Carlo methods used a general time reversible (GTR) model of nucleotide substitution and allowed for gamma-distributed variation across sites with a proportion of invariable sites. Tree analysis was performed using 100 000 generations with a sampling frequency of 100. Standard deviation of split frequencies converged to 0 following 45 000 generations, resulting in one final tree with a probability of 99 %.
Sequence availability.
The RPXV-UTR genome sequence has been deposited in GenBank under accession number AY484669 and at http://www.poxvirus.org. The genomes used for comparison are: VACV-COP, M35027 (Goebel et al., 1990); VACV-TT, AF095689 (Q. Jin and others, unpublished); VACV-MVA, U94848 (Antoine et al., 1998
); VACV-WR, AY243312.1 (J. J. Esposito and others, unpublished); VARV-Bangladesh-1975 (VARV-BSH), L22579 (Massung et al., 1994
); VARV-India-1967 (VARV-IND), X69198 (S. N. Shchelkunov and others, unpublished); VARV-Garcia-1966 (VARV-GAR), Y16780 (S. N. Shchelkunov and others, unpublished); MPXV-Zaire-96-I-16 (MPXV-ZAI), AF380138 (Shchelkunov et al., 2001
); MPXV-WRAIR, AY603973 (Chen et al., 2005
); ectromelia virus-Moscow (ECTV-MOS), AF012825 (Chen et al., 2003
); cowpox virus-Brighton Red (CPXV-BR), AF482758 (D. J. Pickup, unpublished); CPXV-GRI-90, X94355 (S. N. Shchelkunov and others, unpublished); camelpox virus (CMLV)-M-96, AF438165 (Afonso et al., 2002
); and CMLV-CMS, AY009089 (Gubser & Smith, 2002
).
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RESULTS AND DISCUSSION |
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Phylogenetic analyses
Phylogenetic relationships among all available OPV genomes were inferred using two different computational methods to ensure that the final evolutionary tree was not dependent on the method used. Inferences based on maximum-parsimony analysis and Bayesian inference each produced a single tree that showed identical topologies and no significant differences in branch lengths (Fig. 2). While confidence values based on bootstrap analysis of the maximum-parsimony tree showed 100 % confidence for the majority of branch points, confidence was lower for a few of the lineages. In contrast, when assessing tree reliability using Bayesian inference, the final tree showed a probability of 99 % with the posterior probability of all bipartitions (branch points) equal to 1·0, providing a great deal of confidence in the final topology.
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Our analyses support the idea that RPXV-UTR is very closely related to VACV. How RPXV-UTR compares to the RPXV strain that arose at Rockefeller University is unknown, since we have been unable to locate any stocks of the Rockefeller Institute strain of RPXV. The clouded history of VACV together with the fact that it was passaged as a crude mixture in animals at the time RPXVs were isolated leaves the connection between these viruses unclear. However, one hypothesis is that RPXV was present as a natural variant in VACV crude stocks and was selected in vivo from this complex mixture after infection of rabbits.
Virulence genes
RPXV-UTR was compared with available sequences of strains of VACV and other OPVs to identify ORFs that may contribute to the enhanced virulence of RPXV-UTR over other VACV strains for rabbits. The VACV-MVA sequence was not used in this analysis as it contains a large number of deletions and grows poorly in mammalian cells (Antoine et al., 1998; Blanchard et al., 1998
; Drexler et al., 1998
). Furthermore, we have avoided relying heavily on the genome sequence of VACV-TT because our group, and others, has identified a number of errors in this sequence (Upton et al., 2003
). This analysis indicates that RPXV-UTR contains three genes that are not present in VACV-COP or VACV-WR; these genes encode a zinc RING finger protein (ORF 008), an ankyrin repeat-containing protein (ORF 180) and a chemokine-binding protein (ORF 001/184).
RPXV-UTR-008 is predicted to encode an orthologue of the ECTV strain Moscow 012 gene product (ECTV-MOS-012; also known as p28), a key ECTV virulence protein (Senkevich et al., 1994). Orthologues are also present in VARV, CPXV, MPXV, CMLV and a number of other ChPVs, but are deleted in VACV-COP and fragmented in VACV-TT (Upton et al., 2003
) and VACV-WR. The most prominent feature of the predicted ORF 008 protein is a C-terminal zinc-binding motif, known as the RING finger motif (PROSITE database PDOC00449; http://www.expasy.ch); similar motifs are present in a wide variety of proteins with diverse functions (Lovering et al., 1993
; Boddy et al., 1997
; Lyngso et al., 2000
; Kaiser et al., 2003
). This poxvirus protein localizes to the virus factory in the cytoplasm of infected cells (Upton et al., 1994
), reduces apoptosis in infected cells (Brick et al., 1998
) and has recently been found to have a ubiquitin ligase activity (Huang et al., 2004
; Nerenberg et al., 2005
). The predicted protein product of RPXV-UTR-008 is 96·4 % identical to ECTV p28, which is not required for virus multiplication in cell culture, but is an important determinant of ECTV pathogenicity. Disruption of this gene in ECTV increases the LD50 by several orders of magnitude (Senkevich et al., 1994
) and functional p28 protein was found necessary for ECTV replication in some primary murine macrophages (Senkevich et al., 1995
). By analogy, if a functional p28 protein is necessary for efficient virus replication in alveolar macrophages and subsequent spread to, and replication in, the draining hilar lymph node following a respiratory infection, this might explain the reduced dermal VACV titres compared with RPXV-UTR in the hilar lymph node and internal organs (Westwood et al., 1966
).
The protein product of RPXV-UTR-180, which is predicted to be a 791 aa orthologue of VARV-BSH-B18R, contains three ankyrin repeat motifs (PROSITE database PDOC50088; http://www.expasy.ch). This is an interesting protein because evidence is accumulating that ankyrin repeat motifs mediate proteinprotein interaction events, such as those between integral membrane and cytoskeletal proteins (Lambert et al., 1990; Lux et al., 1990
). Moreover, poxvirus proteins containing ankyrin repeat motifs are thought to influence virus host range and pathogenesis (Shchelkunov et al., 1993
, 1998
). Orthologues of RPXV-UTR-180 are present in many of the OPVs but are fragmented in VACV (Fig. 3
). A notable exception among the virulent OPVs is ECTV, which lacks an orthologue of RPXV-UTR-180 due to a series of small deletions that shift the reading frame. However, both ECTV and CPXV contain a paralogue of this gene at the left end of the genome that has approximately 46 % amino acid identity; thus it is possible that this ankyrin repeat motif-containing protein may still be important for virulence in RPXV and that ECTV-MOS-005 functions to complement the loss of the RPXV-UTR-180 orthologue in ECTV. The C-terminal region of RPXV-UTR-180 contains some similarity to the F-box motif (PROSITE database PDOC50181; http://www.expasy.ch). This protein motif is believed to play a general role in proteinprotein interactions and functions in the association of the Skp1cullinF-box protein ligase complexes; it is associated with ubiquitination and degradation of several proteins (Bai et al., 1996
). The RPXV-UTR-180 gene is also interesting because it contains 719 nucleotides (from 186989 to 187708; Fig. 3
) that are not present anywhere in the genomes of any of the four sequenced VACV strains; this encodes the C terminus of the RPXV-UTR-180 protein. Although an orthologous region is present in other sequenced OPVs, they possess significant minor differences that exclude the possibility that this is a contaminating laboratory sequence arising from the processes of DNA sequencing. This finding has implications for the evolutionary relationship of the OPVs, suggesting that RPXV is not a direct descendant of the known VACV strains without recombination with a second unknown OPV but could have been present in the uncloned population of VACV in use at that time.
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It is also interesting that RPXV-UTR-134, the orthologue of the OPV structural protein (P4c) that directs intracellular mature virus particles into A-type inclusions, is only full-length in the WR strain of VACV (McKelvey et al., 2002). The general conservation of this gene in a number of OPVs that do not produce a full-length A-type inclusion protein suggests both that the P4c proteins and the partial A-type inclusion proteins may be providing a selectable advantage to these viruses.
Fragmented regions
To be consistent with our previous work (Chen et al., 2000), in annotating the RPXV genome, we intentionally avoided including a number of small ORFs that are clearly fragments of larger genes present in other OPVs. However, we describe 12 regions that clearly correspond to disrupted ORFs and have named them fragmented regions I to XII (Table 1
; Fig. 1
). Mutations in these regions were mapped by comparing the RPXV DNA sequence to the protein sequence of the longest OPV orthologues using the NAP program. It is very unlikely that these 12 regions generate functional gene products, since the ORFs contained within them are all significantly truncated with respect to their orthologues. However, further work is required to confirm this loss of function.
Most of the fragmented regions in RPXV-UTR correspond to regions that are also fragmented in other VACV genomes. However, fragment region VII corresponds to a fragmented orthologue of VACV-COP-A51R; the function of this gene product is unknown but it is present in all other OPVs. The mutation that disrupts the orthologue of VACV-COP-A51R was very clear in the sequence trace data (data not shown); since DNA sequencing was performed in two directions using multiple pooled PCR products as the DNA sequencing template, we are confident that this and other mutations that disrupt coding sequences are not from PCR mutations or sequencing errors.
The fragmented haemagglutinin (HA) gene was annotated as RPXV-UTR-163 because it represents approximately the C-terminal two-thirds of the gene; however, this is additionally annotated as fragmented in the VOCs database (http://www.biovirus.org). The RPXV HA gene is frame-shifted because of an additional adenine residue after a run of six adenines; the gene was resequenced and analysed using two different sequencing machines and software packages to remove any systematic error. Fragmentation of the HA gene was also confirmed by visualizing the plaque phenotype of RPXV-UTR (data not shown), in which the cells fuse at the edges of the plaques (Ichihashi & Dales, 1971). Thus, although the gene encoding HA is conserved in all other OPVs, the sequencing and experimental data indicate that this isolate of RPXV is indeed HA negative.
In conclusion, we have identified three genes that are present in RPXV-UTR but absent from other VACVs. Each of these gene products has features that associate them with poxvirus virulence; some are better characterized than others. For example, knock-out experiments have clearly shown a role for the RING finger protein in ECTV infections of mice (Senkevich et al., 1994, 1995
) and orthologues of the RPXV-UTR-001 protein have been identified as binding host chemokines (Alcami et al., 1998a
; Lalani et al., 1998
, 1999
), but, since deletion of the chemokine gene did not attenuate RPXV in mice or rabbits (Martinez-Pomares et al., 1995
), it is more likely that one or both of the other genes are responsible for the enhanced RPXV virulence over VACV in rabbits. Although bioinformatics analysis of the RPXV genome cannot substitute for a thorough biochemical characterization of the contribution that each of these three genes makes to virus virulence, it may be prudent to ensure that all three of these genes are absent from any VACV strains that are engineered for human vaccines or therapeutics. It should also be noted that there are a large number of minor differences between the genomes that could affect virulence. For example, even single nucleotide changes in poxvirus promoters may significantly alter transcription levels and single amino acid changes in proteins can result in relatively major changes in the proteinprotein interactions required for a viral protein to bind a specific host cytokine.
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ACKNOWLEDGEMENTS |
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Received 11 July 2005;
accepted 12 August 2005.
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