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1 Bavarian Nordic GmbH, Fraunhoferstrasse 13, D-82152 Martinsried, Germany
2 Center for Biotechnology, University of Bielefeld, D-33594 Bielefeld, Germany
3 University of Zürich, Zürich, Switzerland
Correspondence
Jürgen Hausmann
juergen.hausmann{at}bavarian-nordic.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work. 
Present address: 9 Sing Crescent, Berwick, VIC 3806, Australia.
The GenBank accession number of the sequence reported in this paper is AM501482.
A supplementary table showing the comparison of CVA ORFs with orthologous ORFs in MVA, representative VACV strains and CPXV-GRI is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The VACV strains most widely used during this program were EM-63 in Russia, Lister/Elstree in Europe, New York City Board of Health (NYCBH)/Dryvax/Wyeth in the United States and Temple of Heaven in China (Fenner et al., 1988). These and additional VACV strains were used and characterized extensively in many laboratories worldwide. The most frequently used laboratory strain has been Western Reserve (WR), a derivative of the NYCBH strain adapted to the mouse by serial brain passages (Parker et al., 1941).
The chorioallantois vaccinia virus Ankara (CVA) strain of VACV, maintained by the Turkish vaccine institute in Ankara by donkey-calf-donkey passages, was used in Turkey as a smallpox vaccine (Mayr et al., 1975). The CVA-based vaccine was produced from skin lesion material after intracutaneous inoculation of calves and donkeys. A sample of this virus was brought to Germany in 1953, amplified on bovine skin and used as a smallpox vaccine in Germany (Mayr et al., 1975; Staib & Sutter, 2003). However, the use of CVA as smallpox vaccine in Germany was soon halted since it was associated with a high incidence of secondary lesions after primary vaccination (Herrlich & Mayr, 1957). The name CVA was coined because the virus was routinely passaged and titrated on the chorioallantois membrane of embryonated chicken eggs. The properties of CVA were extensively studied in various biological systems including embryonated chicken eggs, white mice, rabbits and cell culture in the 1950–1960s (Herrlich & Mayr, 1954, 1955; Mayr & Munz, 1964). Due to its wide host range and well-characterized biological properties, CVA was chosen as the starting virus for serial passaging experiments in chicken embryo fibroblasts (CEF) cells. The rationale was to reproduce the assumed evolution of poxviruses from an ancestral virus with broad host range to a poxvirus with very narrow host range, such as VARV. The virus that resulted after more than 500 passages on CEF cells did not productively replicate in most mammalian cell lines, including human cells, and had a highly attenuated phenotype in vivo (Mayr & Danner, 1978; Mayr et al., 1975; Carroll & Moss, 1997; Drexler et al., 1998). It was subsequently named MVA due to its novel properties (Mayr et al., 1975).
MVA was used as a priming vaccine prior to the administration of conventional smallpox vaccine in a two-step program without any significant adverse events in more than 120 000 primary vaccinees in Germany (Stickl et al., 1974; Mayr & Danner, 1978). Despite smallpox eradication, interest in a safe smallpox vaccine never ceased, due to the versatility of VACV and MVA as gene expression vectors, and interest was renewed due to the recent appreciation of the possibility of accidental or deliberate release of smallpox. Recent clinical studies using a third-generation vaccine MVA-BN as a standalone smallpox vaccine confirmed that its excellent safety profile is maintained even in immunosuppressed individuals (Harrer et al., 2005; Vollmar et al., 2006), and underscore its potential as a safe vaccine vector for human infections with various pathogens.
The first complete genomic sequence of a VACV was determined for the Copenhagen strain (VACV-COP; Goebel et al., 1990). The number of complete genomic sequences of VACV strains has increased in recent years and so has our understanding of the genetic basis of poxviral virulence. We describe here the complete coding sequence of CVA, which will permit further insights into virulence and host range determinants of poxviruses, particularly by direct comparisons with its descendant, MVA.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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DNA isolation, sequencing and genome assembly.
Genomic DNA of CVA was isolated with a commercially available kit (NucleoSpin Blood Quick Pure, Macherey-Nagel) using 107 TCID50 of crude viral stock suspension. Purified viral genomic DNA was used as template to amplify DNA fragments of approximately 5 kb. These fragments covered the complete coding sequence starting before ORF CVA001 and extending through ORF CVA229 with an overlap of approximately 500 bp each. Design of the 19–36mer PCR primers (MWG-Biotech) used for amplification of the overlapping 5 kb fragments was based on the DNA sequence of MVA. The sequences contained within the six deletions found in MVA were then amplified by primers located on both sides of each deletion site and sequenced by primer walking. Briefly, PCR fragments were amplified using the TripleMaster PCR system (Eppendorf) and purified with the QIAquick PCR purification kit (QIAGEN). The PCR fragments were directly sequenced (Eurofins Medigenomix GmbH) with an Applied Biosystems 3730 DNA Analyser using 10–14 custom-designed primers per PCR fragment. Contigs were assembled and analysed using SeqMan II (DNASTAR). The first nucleotide of the leftmost ORF representing the last nucleotide of the stop codon was arbitrarily designated base 1.
Annotation and sequence analysis.
The GenDB annotation system (Meyer et al., 2003), integrating the gene-finding programs GLIMMER (Delcher et al., 1999), CRITICA (Badger & Olsen, 1999), REGANOR (Linke et al., 2006) and GISMO (Krause et al., 2007), was used to identify ORFs. The programs employed predict genes based on both sequence compositional properties as well as sequence similarity searches using Pfam (Finn et al., 2006) or BLASTP (Altschul et al., 1997). A continuous DNA sequence stretch was considered an ORF if it encoded a polypeptide sequence defined by a 5' start codon (ATG) and a 3' termination codon (TGA, TAG, TAA) and met the criteria of the employed ORF identification tools for a coding sequence. No size limitation was defined for an ORF. Genes were numbered from the left to the right end of the genome. Numbering of protein fragments encoded by fragmented ORFs is from N terminus to C terminus of the full-length gene product. Accordingly, the most N-terminal protein fragment is always numbered as fragment 1 (f1). Since the nomenclature of VACV-COP ORFs is most common in poxvirus literature, it is used throughout this study, except where stated otherwise.
Phylogenetic analysis.
For phylogenetic analysis the indicated sequences were aligned using CLUSTAL W (Thompson et al., 1994), starting with the last base of the termination codon of the K2L orthologue in each genome and ending with the last base of the termination codon of the A50R orthologue. Aligned sequences were trimmed using GBlocks (Castresana, 2000) and maximum-likelihood trees were generated using the DNAML program from the PHYLIP program package (Felsenstein, 1997). Bootstrap analysis with 100 replicates was performed using SEQBOOT and CONSENSE from the PHYLIP package. All programs were run using default parameter settings.
Nucleotide sequence accession number.
The sequence of the complete coding region of CVA isolate BN has been deposited in GenBank under accession number AM501482.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The nucleotide sequence of CVA contains 229 predicted ORFs encoding polypeptides of 42–1286 aa, numbered consecutively from the left to the right end of the genome (Supplementary Table S1, available with the online version of this paper). All of the 229 predicted ORFs have orthologues in other VACV strains. Since 16 ORFs are duplicated in the ITRs, the CVA genome contains 213 unique ORFs (Supplementary Table S1). One of the unique ORFs (CVA136) that was originally defined as a minor ORF in VACV-COP probably does not represent a real gene (Goebel et al., 1990). Indeed, this ORF has recently been classified as non-coding based on purine skew and other analyses (Da Silva & Upton, 2005). All other ORFs identified in CVA correspond to major VACV ORFs and probably represent true coding regions. Of the 229 predicted ORFs, 51 are fragments of full-length OPV genes and presumably do not specify functional proteins. ORFs were considered full-length if they had the same or a very similar length as their CPXV strain GRI-90 (CPXV-GRI) orthologues. CPXV was found to contain the highest number of full-length genes among OPVs and thus CPXV genes were considered to be mostly complete (Shchelkunov et al., 1998; Gubser et al., 2004).
We identified 71 orthologous ORFs whose predicted gene products were identical between CVA and MVA (Supplementary Table S1). The remaining 124 of the 195 ORFs within the MVA genome encode gene products that contain at least one aa exchange or insertions/deletions compared with the gene products specified by the orthologous CVA ORFs. The gene products of nine of these 124 ORFs showed a 100 % identity in the local BLAST hit but had different lengths in CVA and MVA (Supplementary Table S1).
Phylogenetic relationships between CVA and other VACV strains
Phylogenetic analysis was used to substantiate a direct relationship between CVA and MVA and to identify the closest relatives of CVA among other VACV strains. First, three ORFs (C10L, A9L and VACV-WR016; see Supplementary Table S1) were identical only between CVA and MVA, but not between any other OPV genome and MVA. Secondly, three MVA ORFs, MVA008L, 016L, and 166R, share with their CVA counterparts exactly the same lengths, but lengths are not shared with any other orthologous OPV ORF (Supplementary Table S1). Thirdly, the number of ORFs with a local BLAST hit identity of 100 % was highest between CVA and MVA. Fourth, all ORFs of MVA except ORF 188R are shared with CVA. Taken together, these findings provide a strong indication of the descent of MVA from CVA.
To evaluate the overall similarities between CVA, MVA and selected VACV and OPV genomes, we chose a large portion of these genomes covering the highly conserved central region of OPVs and adjacent conserved genes. The DNA sequence used for comparison spanned ORFs K2L in the left terminal region to ORF A50R in the right terminal region. As expected, the DNA sequences of MVA and CVA show the closest phylogenetic relationship (Fig. 1), confirming that CVA is indeed the ancestor of MVA. The second most similar sequence is that of VACV-DUKE, which was isolated from a patient with progressive vaccinia after primary vaccination with Dryvax. The other three sequences of Dryvax-derived VACVs, including strain 3737 which is a clinical human isolate originating from a Dryvax vaccine-associated skin lesion, group closely together with VACV-DUKE and are also very similar to CVA and MVA (Fig. 1). Interestingly, HSPV shows a relatively high similarity to CVA within the chosen gene complement and appears to be more closely related to CVA than to most other VACV strains such as VACV-COP, VACV-WR and VACV-LC16mO (Fig. 1), which is a derivative of the Lister/Elstree VACV strain. Only the group of Dryvax-derived VACVs is more similar to HSPV than CVA. Again as expected, the genome of CPXV-GRI is most distantly related to CVA, together with the genome of RPXV strain Utrecht. Taken together, phylogenetic analyses indicated that CVA and MVA are most closely related to the Dryvax vaccine strain DUKE and the other Dryvax-derived VACV strains.
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). Six major deletions that were numbered with roman numerals I to VI had previously been identified (Meyer et al., 1991). Compared with CVA, the coding region of the MVA genome lacks 26 577 bp, corresponding to 13 % of the ancestral CVA genome. Loss of nucleotides within the six major deletions in MVA adds up to 24 668 bp. The remaining difference of 1909 lacking nucleotides is not clustered in an additional large deletion but these nucleotide deletions are distributed relatively evenly over the whole genome (data not shown). All major deletions occurred in the terminal regions of the genome that showed a considerably higher genetic divergence between OPVs than the central conserved genomic region.
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). This could be expected for deletions I and IV, which are located in highly variable genome regions of VACVs containing many fragmented ORFs, and for deletion VI that also mainly affects an already fragmented gene. In contrast, it is somewhat surprising for the deletions II, III and V that are located in more conserved areas of the terminal genomic regions. In these areas, genes that are usually conserved in VACVs are fragmented or truncated in CVA (Fig. 2).
Deletion I is located in the left terminal region of CVA and affects the orthologues of the three ORFs C12L, C15L and C16L. The former codes for a serine protease inhibitor (serpin) designated SPI-I which is involved in poxviral host range determination (Ali et al., 1994; Wyatt et al., 1998). ORFs C13L and C14L are lacking in CVA. All putative ORFs upstream of the C16L orthologue are fragmented or deleted, except the C23L orthologue encoding a putatively inactive version of the viral 35 kDa chemokine-binding protein (Supplementary Table S1) (Alcami et al., 1998).
Analysis of CVA ORFs contained in deletion VI showed that, apart from the four fragments of the CPXV ATI gene (CVA152–155, Fig. 1), the full-length ORF CVA156 encoding the OPV p4c protein was also affected by deletion VI and by an additional internal deletion of four nucleotides, resulting in a truncation of this ORF by more than 50 % in MVA (Supplementary Table S1 and Fig. 2). The p4c protein directs viral particles into A-type inclusion bodies (ATIs) and is not essential for viral replication in vitro (McKelvey et al., 2002). Its truncation in MVA was not noted upon comparison of MVA with VACV-COP, in which this gene is also truncated and fused to a short fragment of the CPXV ATI gene.
Deletion site IV is located in the right ITR region of the CVA genome and affects a total of nine ORFs (Fig. 2). Only four of these ORFs, corresponding to VACV-COP ORFs C10L, C11R, C12L and CPXV ORF C8L, are encoding full-length OPV proteins (Supplementary Table S1). Apart from the C12L ORF, these genes are still intact in the left part of the MVA genome corresponding to the former left ITR region of CVA. The occurrence of deletion IV resulted in the formation of a new, 70-aa-long ORF in MVA (188R). This ORF was created by the fusion of a short remnant of the largest known OPV ORF (CPXV-GRI B22R), encoding a putative 1933-aa-long 7-transmembrane G-protein-coupled receptor-like protein, to an in-frame start codon upstream of deletion site IV. ORFs similar to MVA188R have not been described in any other VACV strain. Thus, MVA188R represents a gene fragment that was incidentally restored due to occurrence of deletion IV.
Within deletion V, encompassing genes C1L–C5L, the C4L gene coding for a putative 189 aa polypeptide in the CVA version is truncated by 126–127 aa compared with other VACVs or CPXV-GRI (Fig. 2, Supplementary Table S1). A specific role for the C4L gene product in the poxviral life cycle or in poxvirus–host interactions has not been assessed. The C2 protein, encoded by the C2L gene within the same deletion, is shortened by 17 aa compared with the 512-aa-long full-length OPV version of C2. The CVA C2L orthologue is the shortest C2L gene among VACV and CPXV strains. The C2L gene product is involved in virulence regulation and induction of cytopathic effects in infected cells (Pires de Miranda et al., 2003), and belongs to the class of kelch-like proteins in the OPV genome. Deletion V also affects the N1L gene which encodes a major VACV virulence determinant. N1 inhibits intracellular signalling via the transcription factors NF
B and IRF-3 (DiPerna et al., 2004) and counteracts apoptosis of infected cells (Cooray et al., 2007). The MVA orthologue of N1L contains a frameshift mutation leading to a 4 aa shorter protein with a completely different C terminus of 23 aa, compared with the highly conserved OPV version of N1.
Deletion II is the third major deletion within the left terminal region of the CVA genome and affects the M1L and M2L genes as well as the poxviral host range gene K1L, resulting in its functional inactivation (Fig. 2, Supplementary Table S1) (Sutter et al., 1994). Interestingly, the well-conserved M1L gene is already fragmented in the CVA genome.
Within the right terminal region, the most interesting deletion site is deletion III, which comprises ORFs A51R to A55R (Fig. 1, Supplementary Table S1). A51R and A55R are already fragmented in CVA. However, A51R is nearly intact in MVA, except that the putative polypeptide encoded by A51R lacks the 25 C-terminal aa residues due to a frameshift caused by deletion III. This suggests that the CVA clone sequenced here is not the direct ancestor of MVA.
Major differences between CVA, MVA and other VACV strains outside the major deletion sites
In the central conserved region between ORFs F1L and A24R, MVA contains three fragmented genes, F5L, F11L and O1L, which are intact in CVA (Supplementary Table S1). Small block deletions of 4–9 aa are present in the gene products of MVA ORFs F1L, F3L, D3R, A4L and A12L, respectively, but not in the gene products of the corresponding CVA ORFs. In addition, the gene product of F7L was reported to contain a 12 aa deletion in MVA compared with VACV-COP (Antoine et al., 1998). However, VACV-COP is rather unique in having a 12 aa insertion of Lys-Asn repeats compared with almost all other OPVs, including CVA. Likewise, the gene product of ORF A22R is not divergent in MVA from most OPVs, but is annotated in the VACV-COP sequence as a protein with an 11 aa shorter N terminus.
The fragmented ORFs in the left terminal region of the CVA genome preceding ORF F1L are without exception fragmented or deleted in MVA. Compared with the genomes of most other VACV strains, the CVA genome is unique in that the three ORFs C9L, C4L and M1L are fragmented or truncated in this region. In addition, the well-conserved OPV ORF C2L encodes a 17 aa shorter protein in CVA, due to three internal block deletions of 7, 4 and 6 aa, respectively. The protein encoded by the MVA orthologue of C6L is 6 aa longer than in all other OPVs, including CVA, due to a mutation in the stop codon resulting in a 6 aa longer C terminus of the C6 protein of MVA.
Within the right terminal region, ORF A26L coding for the p4c protein is intact in CVA, but truncated in MVA and VACV-COP. Most other ORFs in the right terminal region of the genome that are of different lengths in CVA and MVA represent truncated or fragmented ORFs such as A57R, B2R/B3R, B9R, B10R, B20R and B13R/B14R (Supplementary Table S1). Most likely, these ORFs do not encode functional gene products and are fragmented or truncated in all other sequenced VACV strains (Supplementary Table S1 and data not shown). Exceptions are B13R and B14R, which represent fragments of a full-length gene that is present in VACV-WR (VACWR195, Supplementary Table S1) where it codes for a functional serine protease inhibitor (SPI-2) (Kettle et al., 1995). VACV strains IHD-J and IHD-W as well as CPXV and RPXV have been shown to express SPI-2 proteins of very similar or slightly reduced size compared with the VACWR195 gene product (Kettle et al., 1995).
The membrane protein encoded by the MVA orthologue of A36R, which is required for intracellular transport and egress of virus particles, is different from its CVA and OPV counterparts because of a 9 aa internal deletion and a 4 aa C-terminal truncation. Proteins encoded by ORFs A43R, B7R and B15R have small block deletions of 4, 5 and 6 aa, respectively, in MVA compared with CVA. The gene product of the MVA orthologue of A47L has two block deletions of 4 and 9 aa, respectively. Interestingly, the protein encoded by ORF B5R is present as a full-length version in MVA, whereas it shows a truncation of 9 aa in CVA.
Putative genetic basis of the restricted host range of MVA in cultured cells
Five major host range (hr) genes have been described for OPVs: C12L (SPI-1) (Ali et al., 1994), Chinese hamster ovary (CHO) cell host range gene CP77 (Gillard et al., 1985; Hsiao et al., 2006), C7L (Oguiura et al., 1993; Perkus et al., 1990), K1L (Perkus et al., 1990) and E3L (Beattie et al., 1996; Chang et al., 1995). The CHO host range protein CP77 from CPXV-GRI (ORF C9L) is fragmented in all known VACV strains, including CVA, as well as in the HSPV genome (Table 1). Thus, it does not play a role in MVA host range restriction. Of the remaining four known OPV hr genes, two ORFs, SPI-1 and K1L, are deleted or truncated in MVA. Deletion of these genes contributes to the limited MVA host range, but their reconstitution cannot completely reverse the MVA hr restriction (Sutter et al., 1994; Wyatt et al., 1998).
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Differences in the set of immunomodulator genes between CVA, MVA and other VACVs
Major differences in the set of immunomodulator genes between CVA and other members of the VACV species are limited to the fragmentation of the A39R gene, encoding a semaphorin-like protein, and substantial mutations in two kelch-like proteins encoded by the C2L and A55R genes (Table 1). The lack of a functional A39R gene is not unique to CVA and MVA, since a number of VACV strains also possess a fragmented A39R gene (Table 1). Kelch-like proteins C2 and A55 have been shown to contribute to the cytopathic effect of VACV infection in cell culture, and to modulate the local inflammatory response and local lesions in a mouse infection model (Pires de Miranda et al., 2003; Beard et al., 2006). Both genes are absent from the MVA genome (Fig. 2, Table 1). Interestingly, the protein encoded by the C2L ORF shows a unique 17 aa C-terminal truncation, and the A55R orthologue is fragmented in CVA. The ORF coding for kelch-like protein C5 is absent in MVA but intact in CVA. The kelch-like proteins encoded by the MVA orthologues of F3L and B10R have 4 and 8 aa internal deletions in MVA, whereas the CVA versions are highly conserved among VACVs. Thus, three of the five kelch-like proteins encoded by VACVs are deleted in MVA and the remaining two show mutations (Table 1). As pointed out above, B10R is most probably a non-functional ORF because it codes for a highly truncated protein compared with the gene product encoded by the orthologous ORF in CPXV-GRI (166 instead of 501 aa).
CVA contains intact genes for a soluble and cell-surface interferon-
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receptor (B19R) and a secreted TNF receptor (CrmC, A53R) whose orthologues code for a non-functional protein or are absent in the MVA genome. Interestingly, most VACV strains do not encode a functional CrmC homologue (Table 1 and Alcami et al., 1999). Small block deletions of 4–8 aa in the MVA orthologues of A42 (profilin-like protein), A45 (superoxide dismutase-like protein) and B7 (chemokine-binding domain-containing protein) are not present in the respective CVA gene products. Likewise, the CVA orthologue of A40R coding for a C-type lectin-like protein is well-conserved, whereas the MVA orthologue contains a 37 nt deletion leading to a frameshift mutation that is located 6 aa away from the C terminus and creates a new, 9 aa longer C terminus in the A40 protein. The remaining set of genes in CVA specifying known or predicted immunomodulators shows no major differences from other VACV strains.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Marker rescue experiments indicated that additional genetic traits must exist that govern the MVA host range (Wyatt et al., 1998). Exact knowledge of the genetic differences between MVA and its ancestor CVA is a prerequisite for the elucidation of the genetic basis of the restricted host range of MVA and to identify new host range genes.
There is no clear history of the parental CVA strain. Broad phylogenetic analysis and analysis of specific ORFs were chosen to address this question. CVA and MVA were most similar to VACVs derived from the Dryvax vaccine. The finding that CVA and MVA are close relatives of Dryvax vaccine strains is supported by a recent study which showed that the Dryvax-derived VACV-DUKE was most similar to MVA using a complement of 82 centrally located, highly conserved ORFs (Li et al., 2006). Apart from VACV-DUKE, MVA is also closely related to three other Dryvax-derived VACV strains, Clone3, ACAM2000 and strain 3737. Clone3 and ACAM2000 were obtained by multiple virus cloning steps in the laboratory (Monath et al., 2004). VACV strain 3737 is a clinical human isolate originating from a skin lesion following vaccination with the Dryvax vaccine. Analysis of the gene fragmentation pattern in CVA provides further examples for the close relation of the CVA and the Dryvax strains. First, fragmentation of the M1L gene observed in the CVA genome was never found in any other VACV genome except the Dryvax-derived strain Clone3 (Li et al., 2006). Second, the A51R gene is fragmented in CVA, which has so far been found only in the genomes of three Dryvax-derivatives (VACV-DUKE, VACV-3737 and VACV ACAM2000), and in VACV-LC16mO/m8. Third, ORF C4L, showing a truncation in CVA, is also truncated in Dryvax derivatives ACAM2000, Clone3 and in VACV strain Tian Tan, and is completely lacking in Dryvax variants 3737 and DUKE. It is therefore tempting to speculate that CVA and the VACVs contained in the Dryvax vaccine have a common ancestor.
The genome of CVA contains a small number of ORFs with mutations that are unique for CVA (L2R, A25L, A51R, B5R and B14R). The sequences of the corresponding ORFs in MVA that were derived from a plaque-purified virus isolate are identical to the consensus OPV versions of these ORFs. The original virus sample of CVA, obtained from A. Mayr, had no reported history of virus cloning. We subjected the original sample of CVA to three rounds of plaque purification on BHK-21 cells and to additional passages on BHK-21 and CEF cells to obtain the viral stock that was used for sequencing of the genomic DNA of CVA. This procedure either might have given rise to mutations in the CVA genome or might have selected a pre-existing variant within the original virus sample. The latter explanation appears more likely, since the original sample was not cloned and thus most probably contained a mixture of viral variants. VACV strains used as vaccines during the smallpox eradication campaign have been shown in at least two cases to represent broad mixtures of viruses. First, analysis of the genomes of the widely used Lister/Elstree vaccine and its derivatives LC16mO and LC16m8 revealed that the sequence of the parental, uncloned Lister virus contained nucleotide polymorphisms at more than 1200 sites in the genome (Morikawa et al., 2005). The second example is the Dryvax vaccine, which was subjected to cloning procedures to obtain a less virulent VACV-based smallpox vaccine (Weltzin et al., 2003; Monath et al., 2004). Two such clones, VACV-ACAM3 and VACV-ACAM2000, and the Dryvax-derived clinical isolate VACV-DUKE showed variations in ORFs M1L, A51R and C4L and near the right ITR affecting three genes, among them the B19R orthologue coding for a soluble and cell surface type I interferon receptor (Li et al., 2006). It was concluded that the original Dryvax vaccine represents a mixed population of viruses with significant variations in virulence in various hosts (Monath et al., 2004; Li et al., 2006). Although the possibility cannot be excluded that CVA variants which still contain intact versions of genes M1L, A51R or A55R might exist in uncloned virus preparations, the CVA isolate described here represents an excellent tool to analyse the genetic basis of the unique properties of MVA regarding host range, virulence and immunogenicity due to the close genetic relationship of these two viruses.
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ACKNOWLEDGEMENTS |
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Received 11 May 2007;
accepted 17 August 2007.
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