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1 Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK
2 Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 3P6, Canada
Correspondence
Linda K. Dixon
linda.dixon{at}bbsrc.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AM712239 and AM712240.
A table showing the ORFs encoded by the BA71V, Benin 97/1 and OURT88/3 isolates is available as a supplementary material with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Salas, 1999). In its natural hosts, warthogs (Phacochoerus aethiopicus), bushpigs (Potamochoerus porcus) and soft ticks of the Ornithodorous spp., ASFV causes a persistent but asymptomatic infection. In domestic pigs it causes an acute haemorrhagic infection with mortality rates up to 100 %, although, both moderately virulent and low virulent isolates have also been observed in domestic pigs.
The sequencing of the Badajoz 1971 Vero-adapted strain (BA71V) of ASFV (Yanez et al., 1995) was the first step in defining the complexity of the genome. The sequencing of the additional non-pathogenic and highly pathogenic isolates described in this and other studies (Dixon et al., 1994; Duarte, 2000; Yozawa et al., 1994) provides the data to perform comparative genomic analyses and allow prediction of the molecular aspects controlling both pathogenicity and cell tropism. Central to this aspect of ASFV are the multigene families (MGFs) present in the left and right hand variable regions of the genome. ASFV contains five such MGFs: MGF 100, 110, 300, 360 and 505/530, named after the average number of codons present in each gene. The N-terminal regions of members of MGFs 300, 360 and 505/530 share significant similarity with each other (Yozawa et al., 1994). Several genes in MGF 360 and 505/530 determine host range and virulence (Afonso et al., 2004; Neilan et al., 2002; Tulman & Rock, 2001). It has also been observed that repeated passage of field isolates of ASFV through tissue culture results in the loss of members of the MGF 110 family (Almendral et al., 1990; de la Vega et al., 1990; Pires et al., 1997). In this study we compared the genomic coding sequences, apart from the inverted terminal repeats (ITRs) and terminal cross-links, of a pathogenic isolate from Benin 97/1, and a non-pathogenic isolate from Portugal OURT88/3, with that of the tissue culture-adapted strain BA71V. The results provide valuable information on the coding information of these isolates and will help to direct future studies aimed at understanding the molecular basis for the differences in pathogenesis.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Both viruses were used to infect pig bone marrow cells and virus-containing cell supernatants were collected at 4 days post-inoculation. Cell debris was pelleted by centrifugation at 12 000 g for 15 min.
Purification of virus DNA.
Virus from culture supernatants was treated with DNase I (Sigma) 50 µg ml–1 for 1 h to digest contaminating cellular DNA. Virus was pelleted by centrifugation through a 30 % sucrose cushion at 53 000 g for 45 min at 4 °C. Virus pellets were dissolved and treated with proteinase K and SDS prior to the extraction of DNA.
Sequence determination and analysis.
The viral genome was amplified from 100 ng viral DNA using the Repli-G method according to the manufacturer's instructions (Qiagen). This method uses an isothermal multiple displacement amplification with a processive DNA polymerase capable of replicating up to 100 kbp. The DNA polymerase has a 3'
5' exonuclease proofreading activity to maintain high fidelity. The sequence was determined using ASFV-specific primers designed from the complete ASFV BA71V isolate genome sequence and from a partial sequence of the ASFV E70 isolate genome. Reactions were carried out (Qiagen) using the ABI prism Big Dye Terminator Cycle Sequencing Ready Reaction kit according to the manufacturer's recommendations. The sequences were compiled into a single contig using the Staden software version 4.5 (Staden, 1996). The estimated error rate was 5 nt or less per genome. The restriction enzyme site map for the OURT88/3 isolate generated in silico closely matched that obtained by analysis of the virus genome (Boinas et al., 2004).
The ASFV genomes were first annotated using the Genome Annotation Transfer Utility (GATU) (Tcherepanov et al., 2006) software and then a database of all ASFV genomes was created with Virus Orthologous Clusters (VOCs) (Upton et al., 2003). Open reading frames (ORFs) identified had a minimum length of 180 bp and did not substantially overlap with other larger ORFs. This ASFV database is available at the online bioinformatics resource Viral Bioinformatics – Canada (http://athena.bioc.uvic.ca/database.php?db=asfarviridae). Analysis of genome sequences and orthologous protein families was carried out using various programs available at Viral Bioinformatics – Canada (Brodie et al., 2004a, b; Syed & Upton, 2006; Upton et al., 2000). This analysis included searches against the most recent databases using BLAST and against protein motif databases including Pfam. GenBank accession numbers for the genome sequences are AM712239; for Benin 97/1, and AM712240 for OURT/88/3.
The nomenclature of ORFs is based on that used for the BA71V isolate genome (Yanez et al. 1995), except in the case of MGF families where we have introduced a new nomenclature system to take into account the presence of copies not found in the BA71V genome (Table 2). This system describes the orthologous cluster by the family to which it belongs, its direction of transcription and its relative position in the genome when reading from left to right. Table 3 and Supplementary Table S1 (available in JGV Online) give this nomenclature as well as previous nomenclature used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Oura et al., 2005). The genomes of these isolates were compared with the tissue culture-adapted Spanish isolate BA71V (Yanez et al., 1995) (see Table 1). The BA71V, OURT88/3 and Benin 97/1 isolates are closely related to each other and fall into the same genotype, Genotype I, as defined by partial sequencing of the gene encoding VP72 (Nix et al., 2006). Annotation was previously available only for the BA71V isolate. Therefore, the other genomes were first annotated using the GATU software (Tcherepanov et al., 2006). This program first transfers annotations from a closely related reference genome to the target genome by searching for common genes and then allows the user to search for additional genes unique to the target genome. We also annotated the sequences from seven isolates that were available in GenBank without annotation. These were from domestic pigs, warthogs or ticks in national parks in eastern and southern Africa, including the Kenyan isolate obtained in 1950, the Tengani isolate from Malawi in 1962, the Malawi isolate 1983, the Pretoruskup isolate from South Africa in 1996, the Mkuzi isolate from Zululand in 1979, the Warthogs isolate from Namibia in 1980 and Warmbaths isolate from South Africa in 1987. The sequences obtained contained variable lengths of the ITRs (see Table 1), which are only complete for the BA71V isolate (Gonzalez et al., 1986; Yanez et al., 1995). Comparing the unique sequences obtained showed that the shortest genomes are the tissue culture-adapted BA71V isolate (165 795 bp) and the non-pathogenic OURT88/3 isolate (171 701 bp), whereas the Benin 97/1 isolate unique sequence was 180 971 bp. The unique sequences available for the other isolates vary in size between 184 767 bp for the Tengani 62 isolate and 191 036 bp for the Kenyan isolate. The complete BA71V isolate genome encodes 151 ORFs, the sequence obtained for the Benin 97/1 isolate encodes 156 and for the OURT88/3 isolate encodes 157 ORFs. The BA71V isolate contains additional ORFs (three or four) within the ITR that are absent from the other genomes since not all of the ITR sequences were obtained. One hundred and sixty-seven ORFs are encoded by the sequence available for the longest genome from the Kenyan isolate. The ORFs from these genomes have been clustered into orthologous families and are available on the website (http://athena.bioc.uvic.ca/database.php?db=asfarviridae). Use of the VOCs database system allowed us to quickly identify orthologous genes present in the different isolates. This analysis found 109 non-duplicated ORFs that are conserved between all 10 virus isolates. The predicted protein sequences were searched against recent databases using the BLAST and the Pfam protein motifs database. This identified ORFs encoding proteins, including enzymes involved in DNA replication and repair, mRNA transcription and processing, other enzymes, structural proteins and proteins involved in evading host defence systems (see Supplementary Table S1). Comparison of the sequences of these proteins between the Benin 97/1, OURT88/3 and BA71V genomes showed that most share between 99 and 100 % amino acid identity and are closely conserved in length between these three isolates (see Supplementary Table S1).
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and 2 and Supplementary Table S1). Previously, restriction enzyme site mapping identified gene deletions at the left end of the OURT88/3 isolate compared with other field isolates and the position of these corresponds with the results we obtained (Boinas et al., 2004). At the left end of the genome, the BA71V isolate encodes two ORFs within the ITRs, KP86R and KP93L. Copies of these ORFs are encoded in the ITRs at the right end of the BA71V genome. A partial sequence of the KP93L ORF truncated at the C-terminal end was obtained from the OURT88/3 isolate and Benin 97/1 isolates. Since the Benin 97/1 and OURT88/3 sequences do not include the complete terminal repeat sequences we do not have information as to whether the KP86R ORF is encoded.
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and Table 2b). The Benin 97/1 and BA71V isolates both encode five copies of MGF 110, whereas the OURT88/3 genome encodes seven copies. Of these only one, MGF 110-1L, which is the furthest left of MGF 110 on the genome, is present in all 10 isolates. This and ORF BA71V-69R are present in all three genomes but a number of genome rearrangements have occurred between the positions of these ORFs (positions 6172–12 922 on the Benin 97/1 genome) in the different isolates. Comparing copies of MGF 110 shows that three members of MGF 110 are present downstream from MGF 110-1L in both the BA71V and OURT88/3 genomes (MGF 110-2L, MGF 110-4L and MGF 110-5L; Fig. 1). The MGF 110-6L is present only in the BA71V genome, MGF 110-9L is present only in the OURT88/3 genome and MGF 110-11L and MGF 110-12L are present only on the genome of the Benin 97/1 isolate. Two members of this family (MGF 110-13L and MGF 110-14L) are encoded by both the OURT88/3 and Benin 97/1 isolates but not the BA71V isolate. The Tengani 62 isolate has seven copies, but the genomes of all the other isolates encode between 11 and 13 copies of MGF 110 (see Table 2b).
Two ORFs of unknown function, 285L and 86R, and one belonging to MGF 100 (MGF 100-1L) are encoded by the OURT88/3 isolate, but not the other isolates (Fig. 1a and Tables 2 and 3). These are located between MGF 110-5L and MGF 110-9L. Downstream from the final MGF 110-14L, the OURT88/3 and Benin 97/1 genomes both encode a copy of MGF 360 (MGF 360-4L) and the Benin 97/1 isolate encodes an additional MGF 360 (MGF 360-6L) downstream from this. The BA71V genome contains a truncated MGF 360 gene encoding only the N-terminal 60 aa (MGF 360-5L). The MGF 360-4L gene is present in genomes of all other isolates except for Tengani 62, whereas, as well as in Benin 97/1, the MGF 360-6L gene is present in the Kenyan, Malawi and Mkuzi genomes. The MGF 360-5L is present in the Pretoriskop, Tengani and Warthogs genomes (see Table 2);.
Variation is also observed in members of MGF 300 (see Fig. 1b and Table 2c). MGF 300-1L and MGF 300-2R are present in Benin 97/1, OURT88/3 and BA71V genomes. The MGF 300-4L (J182L) ORF is also present in all three isolates, but in the BA71V isolate it is interrupted to produce one ORF encoding the C-terminal 104 aa (MGF 300-3L) and another of 182 aa (MGF 300-4L).
At positions further downstream, both the BA71V and OURT88/3 isolates have large deletions from a genome region encoding members of MGF 360 and 530 (position 17 495 on the BA71V genome and 20 351 on the OURT88/3 genome) (see Fig. 2 and Table 2d, e). Compared to the Benin 97/1 genome, the BA71V isolate has a deletion of 8250 bp, which extends between positions 18 873 and 27 123 bp on the Benin 97/1 genome. A slightly larger deletion of 10 077 bp is observed in the OURT88/3 genome. This extends between positions 19 448 and 29 525 on the Benin 97/1 genome. These deletions result in the truncation of one copy of MGF 360 (MGF 360-9L) on the BA71V genome. In addition, the following ORFs, in sequence from left to right, are deleted from the genomes of both the OURT88/3 and BA71V isolate genomes, MGF 360-10L, MGF 360-11L, MGF 505-1R, MGF 360-10L, MGF 360-11L, MGF 360-12L and MGF 360-13L. The MGF 505-2R is also deleted from the OURT88/3 isolate. These ORFs are present in the genomes of the seven other isolates. Directly downstream the MGF 505-3R ORF is present in all genomes but is truncated by 178 aa at the N terminus in the OURT88/3 isolate and therefore may not be expressed. The MGF 505-4R and MGF 505-5R ORFs are conserved in all three isolates but the Benin 97/1 isolate has an additional copy of MGF 505 (MGF 505-6R) not found in the other isolates. The OURT88/3 isolate also has an additional MGF 505 member (MGF 505-8R) not present in the other isolates. The proteins encoded by both MGF 505-6R and MGF 505-8R are quite similar to the MGF 505-7R protein. Thus, it is likely that these ORFs are derived following a relatively recent duplication of MGF 505-7R. Downstream from these ORFs an additional two copies of MGF 505 (MGF 505-9R and MGF 505-10R) are conserved in all three isolates.
Central conserved genome region
The central part of the ASFV genome, from the start of ORF BA71V-224L, which encodes the IAP homologue (positions 41 455 Benin 97/1 isolate, 29 120 BA71V isolate and 31 275 OURT88/3 isolate) to the ORF BA71V-I196L (positions 169 573 Benin 97/1 isolate, 158 175 BA71V isolate and 160 573 OURT88/3 isolate) is well conserved. Of the 108 ORFs present, all are conserved in 10 isolates, apart from ORFs EP402R and EP153R, which encode the CD2-like protein and C-type lectin protein, respectively. Although these ORFs are present on the OURT88/3 genome, they contain frameshift mutations close to the N terminus that introduce in-frame stop codons. In the OURT88/3 isolate a second methionine codon is in-frame with part of the coding sequence of the EP402R protein. If this protein was expressed, it would start 14 aa downstream of the authentic initiation codon and would thus lack the signal peptide. An additional frameshift mutation in the sequence encoding the cytoplasmic domain would result in the final 215 aa not being translated. The EP153R ORF is also interrupted by a frameshift mutation close to the start codon, which brings a stop codon into frame. The interruptions in these ORFs indicate that functional proteins (CD2-like and C-type lectin) are very unlikely to be expressed.
Two ORFs vary in length due to the presence of sequences encoding variable numbers of amino acid repeats. Variation in the repeats encoded by the B602L ORF has been used to genotype virus isolates (Nix et al., 2006; Lubisi et al., 2007). The B407L ORF encodes a protein that contains between 9 and 11 copies of a 5 aa repeat. Seven different types of this repeat sequence are encoded in the three isolates. The C122R ORF has a frame-shift mutation after the codon for residue 85, in the BA71V isolate, which results in an extension that encodes an additional 18 aa compared with the other isolates.
Variation is observed between genomes due to mutations at the first ATG codon of orthologous genes in the Benin 97/1 and several other isolates genomes. These ORFs include A238L, F334L, B117L, G1211R, CP204L, CP312R and R298L. Usually these would result in the protein sequence starting less than eight residues downstream. The exceptions are the B117L ORF, which would start 25 residues downstream in the Benin 97/1, Malawi LIL 20/1 and Mkuzi isolates and the R298L ORF, which would start 35 residues downstream in the Benin 97/1 isolate. The N-terminal 35 residues of R298L are not part of domains involved in the kinase activity of the protein.
Thirty-five ORFs encode proteins similar to enzymes or factors involved in nucleotide metabolism, mRNA transcription and processing, DNA replication or DNA repair. These have been described previously (see references in Supplementary Table S1). Four other conserved enzymes, a prenyl transferase, ubiquitin conjugating enzyme, protein kinase and nudix hydrolase, may either be involved in regulating the virus replication cycle or in modulating host cell function (Alejo et al., 1997; Baylis et al., 1993a; Cartwright et al., 2002; Hingamp et al., 1992). Eight conserved ORFs encode proteins involved in regulating host cell activities. These include the A238L protein, which inhibits activation of the host transcription factor NF-
B and inhibits calcineurin phosphatase activity (Powell et al., 1996; Miskin et al., 1998). Two proteins with similarity to host apoptosis inhibitory proteins, a Bcl2 homologue and IAP homologue, are conserved (Afonso et al., 1996; Chacon et al., 1995; Nogal et al., 2001). One conserved ORF encodes a protein containing an FTS J-like methyl transferase domain and another is similar to the NifS protein.
Twenty-two ORFs conserved between all isolates encode either structural proteins or other proteins involved in virus assembly. A further 18 conserved ORFs encode proteins that contain predicted transmembrane domains and/or signal peptides.
Although the EP402R CD2-like protein and EP153R C-type lectin-like protein are identical in sequence between the BA71V and Benin 97/1 isolates, they share between 43 and 100 % identity with the proteins encoded by the ORFs in other genomes making these amongst the most divergent orthologous clusters.
Right variable genome region
From the 3' end of the I196L gene (positions 160 578 on the OURT88/3 isolate, 169 640 on the Benin 97/1 isolate and 158 175 on the BA71V isolate) to the right end of the sequences, genome rearrangements are observed that result in gain or loss of ORFs (see Fig. 2 and Tables 2 and 3.). The first of the variable ORFs, DP69R, is only encoded by the BA71V isolate. One copy of MGF 505 (MGF 505-9R) and two copies of MGF 100 (MGF 100-2R and -3R), located downstream from DP69R, are conserved between all three isolates. Downstream from this, the BA71V isolate genome has a deletion of 3530 bp compared with the Benin 97/1 isolate and OURT88/3 isolates. This deletion starts at position 163 861 bp on the BA71V genome and results in the loss of five ORFs (l7L, l8L, l9R, l10L and l11L). The proteins encoded by l7L and l8L ORFs share between 52 % identity. The l10L ORF is similar to the KP177R ORF, which is located close to the left genome end, and encodes the p22 structural protein. The amino acid identity of the l10L proteins compared with the p22 structural protein encoded at the left genome end is quite low (40 %) but the l10L proteins of Benin 97/1 and OURT88/3 isolates are 100 % conserved. Comparison of the other seven genomes, shows that all contain the five ORFs that are deleted from the BA71V isolate. In addition, isolates Warthog, Kenya, Mkuzi, Pretoriskup and Tengani encode an extra copy of the l10L ORF (l10L-2) located upstream of the l10L ORF. The proteins encoded by these ORFs are quite divergent with amino acid identities between l10L and l10L-2 proteins varying between 56 and 60 %. Likewise, comparing the proteins between different isolates shows that the l10L proteins share between 53 and 100 % identity and the l10L-2 proteins share between 76 and 100 % identity. However, the predicted N-terminal transmembrane domain is conserved in all the l10L and l10L-2 proteins. One extra ORF, 112R, is only encoded by the Malawi LIL20/1 isolate.
Downstream from the l11L ORF, the Benin 97/1 isolate encodes a copy of MGF 360 (MGF 360-18R) of 254 aa. The OURT88/3 isolate encodes the N-terminal 72 aa of this ORF, which is then interrupted by a frame shift mutation. We have designated this as a separate ORF (MGF 360-17R). Another ATG codon 107 aa residues downstream is in-frame with the C-terminal 148 aa of the MGF 360-18R ORF. The BA71V isolate does not encode a MGF 360-17R ORF since the ATG codon of this ORF is included in the deletion from the BA71V genome. The same ATG codon for the MGF 360-18R ORF as in the OURT88/3 is in-frame with the C-terminal part of the MGF-18R protein and encodes 148 aa. Examination of this region from other genome sequences available, shows that the Mkuzi isolate encodes a protein of the same length as the Benin MGF 360-18R. In the Malawi isolate, an ATG codon 39 aa downstream is in-frame with the C-terminal part of MGF 360-18R and in all other isolates the first ATG codon is the same as in the OURT88/3 isolate.
The virus encoded DP71L protein, is similar to the herpes simplex virus-encoded neurovirulence factor ICP34.5 and the DNA damage response gene GADD34/myd116. Most of the genomes encode a 71 or 72 aa protein but in the Malawi isolate an additional 74 aa are encoded in the centre of the protein (Rivera et al., 2007). Downstream from this the DP96R ORF and a copy of MGF 360 (MGF 360-19R) are conserved between all three isolates. Closer to the genome end a truncated ORF, which encodes 42 aa from the N terminus of MGF 360 (MGF 360-20R), is present in the genomes of the BA71V and Benin 97/1 isolates. An ORF, DP60R, is encoded by the BA71V isolate. This sequence is also present in the Benin 97/1 isolate but without an ATG codon and is missing from the OURT88/3 isolate. The final ORFs encoded at the right genome end are DP93R and DP86L, which are copies of the KP93L and KP86R ORFs at the left genome end. A truncated version of the DP93R ORF encoding the N-terminal 60 aa is present in the Benin 97/1 sequence but neither ORF is included in the available sequence of OURT88/3 isolate. As discussed previously the sequences from the Benin 97/1 and OURT88/3 isolates do not include the complete sequence of the ITRs.
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DISCUSSION |
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) and the pathogenic isolate from Benin 97/1 West Africa. These sequences were compared with that of the tissue culture-adapted isolate BA71V in order to gain information to probe the molecular basis for the differences in pathogenicity. We also annotated the genome sequences available in the database for a further seven ASFV isolates and made these available on a public bioinformatics resource. This enabled comparison of our data with a wider range of field isolates. Amongst the factors that may account for the difference in pathogenicity between the OURT88/3 isolate and Benin 97/1 isolate are variation in the amino acid sequences of proteins encoded by all isolates, the presence or absence of ORFs or differences in non-coding sequences, which might alter transcription or replication.
Conserved ORFs
The coding sequences of the conserved non-duplicated ORFs are closely related in sequence and length between the Benin 97/1, OURT88/3 and BA71V isolates. More variation is observed between the B602L and B407L proteins due to the presence of amino acid repeats that vary in number and sequence. The B602L ORF encodes a chaperone required for assembly of the major capsid protein into virus particles (Cobbold et al. 2001). Variation was observed in the first start codon used in seven ORFs. However, the transcription start sites for these ORFs have not been mapped and therefore the translation start codon used is not known. The high amino acid identity suggests that sequence variation between these conserved ORFs is unlikely to explain the observed differences in phenotype of these virus isolates, although this cannot be excluded without further investigation involving manipulation of the genome.
Non-conserved ORFs
Most of the variable ORFs are present close to the genome termini, within approximately 30 000 bp from the left genome end and 10 000 bp from the right genome end. Many of the genome rearrangements include members of MGFs. The largest and most variable MGFs are MGF 110 and MGF 360. Fourteen members of MGF 110 and 22 members of MGF 360 can be distinguished based on the amino acid identity of encoded proteins when compared with the genomes of ASFV isolates. The degree of sequence variability between different members of the same MGF would suggest that at least some of the genes must have different functions. For example, the different MGF 360 family members from the Benin 97/1 isolate encode proteins that share between 12.6 and 61.8 % amino acid identity although all contain some conserved residues. In contrast, other MGFs (MGF 100, 300 and 505/530) have fewer members and show less variation in the presence and absence of different members between isolates.
Close to the left genome end rearrangements involve members of MGF 110. Of these only one, MGF 110-1L, is present in all 10 isolates. Previous work using the BA71V isolate has shown that MGF 110-1L is an immediate-early gene (Almazan et al., 1992). Potentially, variation in members of MGF 110 encoded by Benin 97/1 compared to OURT88/3 and BA71V isolates might play a role in the differing properties of these isolates since each isolate encodes at least one unique member of this family. However, as yet variation in members of MGF 110 has not been linked with virus pathogenesis. In one study, loss of all copies of MGF 110 from the left end of the genome was shown not to reduce the virulence of the virus for pigs (Aguero et al., 1990).
The proteins encoded by different MGF 110 members all contain potential signal peptides and a highly conserved central cysteine-rich domain, suggesting that they operate in an oxidizing environment such as that found in the endoplasmic reticulum (ER) (Netherton et al., 2004). Two of the MGF 110-encoded proteins, MGF 110-4L and MGF 110-6L, have an ER retention motif at the C terminus. MGF 110-6L contains the classic motif, KDEL, whereas, MGF 110-4L contains the related KEDL motif. As anticipated MGF 110-6L is localized to the ER; however, MGF 110-4L is localized to the post-ER–pre-Golgi structures (Netherton et al., 2004). The MGF 110 members encoding these two proteins are present in all the isolates except Benin 97/1, which contains neither of them, and OURT 88/3, which only has MGF 110-4L.
In the left variable genome region that includes MGF 110 members, some additional variable genes are encoded. These include three ORFs encoded only by the OURT88/3 isolate (285L, 86R and MGF 100-1L), one encoded only by Benin 97/1 (MGF 360-6L) and one encoded by both OURT88/3 and Benin 97/1 isolates but not by BA71V (MGF 360-4L). One MGF 300 member (MGF 300-4L) is truncated in the BA71V isolate compared with other isolates. Thus, potentially these genome rearrangements may also be involved in explaining differences in pathogenicity between the different isolates.
An important finding is that both the BA71V and OURT88/3 isolates are missing a sequence of 8 or 10 kbp, respectively, from a region that begins at either 17 or 20 kbp from the left end of the sequence. This sequence is present in all eight other genomes and contains the same six copies of the MGF 360 (MGF 360-9L, 10L, 11L, 12L, 13L, 14L) and one or two copies of MGF 505 (MGF 505-1R and 2R). Deletion of these six MGF 360 and two MGF 505/530 members from the Pretoriskup isolate markedly reduced viral growth in macrophages and virulence in pigs. The insertion of the same ORFs restored the ability of the tissue culture-adapted BA71V isolate to grow in macrophages (Zsak et al., 2001). The recombinant Pretoriskup isolate with these ORFs deleted (Pr4
35) also induced a strong type I interferon (IFN) response not elicited by the parental wild-type strain. This suggests that these genes also play a role in suppressing IFN responses (Afonso et al., 2004). The removal of only three of these MGF 360 ORFs, (MGF 360-12L, 13L and 14L) reduced viral titres by 100- to 1000-fold in infected Ornithodorous ticks (Burrage et al., 2004). The deletion of these genes from the OURT88/3 and BA71V genomes seems likely to provide one explanation for the attenuation of these virus isolates. Within the left variable genome region the Benin 97/1 isolate contains one extra copy of MGF 505-8R not present in either BA71V or OURT88/3 isolates, and OURT88/3 contained a copy, MGF 505-6R, not present in either of the other isolates.
Strikingly, the BA71V isolate has a deletion close to the right genome terminus that encodes either five or six ORFs compared with the other isolates. The ORFs encoded in this genome region in the Benin 97/1 and OURT88/3 isolates include one ORF l10L, similar to an ORF close to the left genome end that encodes the p22 structural protein (Camacho & Viñuela, 1991). Comparison with the genomes of other isolates shows that some genomes encode two copies of the p22-related ORF. The sequences of proteins encoded by these p22-related ORFs are quite divergent. Although it is not yet known if the p22-related proteins encoded by the l10L ORFs are also incorporated into the virus structure, it is tempting to speculate that expression of divergent forms of this structural protein may have an important role in either generating antigenic variation to help the virus evade the host's immune system or in helping the virus to replicate in its different hosts. The other ORFs deleted from the BA71V genome have no similarity to known proteins, although two of them (l7L and l8L) are similar to each other. The replication of BA71V in porcine macrophages has been reported to be inefficient, although there are conflicting reports in the literature (Bustos et al., 2002; Zsak et al., 2001). It is possible that the genes deleted from close to the right genome end of the BA71V isolate may have a role in enhancing virus replication in macrophages.
Of interest is the finding that the ORFs EP153R and EP402R are interrupted in the genome of the OURT88/3 isolate. These ORFs encode proteins with similarity to the host CD2 protein (EP402R) and a C-type lectin (EP153R). The CD2-like protein is required for the binding of red blood cells to infected cells and to virus particles (Borca et al., 1994; Rodriguez et al., 1993). Thus, the interruption of this ORF in the OURT88/3 isolate genome explains the non-haemadsorbing phenotype of this isolate. Deletion of this gene encoding the CD2-like protein from the genome of the Malawi isolate delayed the onset of viraemia and virus dissemination in infected pigs, although all infected pigs died. Deletion of the gene abrogated the ability of ASFV infection to inhibit the proliferation of bystander lymphocytes in vitro. This suggests that the protein may be involved in impairment of lymphocyte activity (Borca et al., 1998). It is possible that disruption of this gene may lead to an enhanced immune response in infected pigs and could help explain why the OURT88/3 isolate induces effective protective immunity.
ORFs conserved between all 10 isolates include 109 non-duplicated ORFs, six copies of MGF 360, seven copies of MGF 530, two of MGF 100, two of MGF 300 and one of MGF 110 as well as one copy of the ORF encoding p22. This may indicate a strong selection to maintain these ORFs in the virus genome.
Our comparison of the genomes of three ASFV isolates from the same genotype that cause very different pathogenesis in pigs, provides the information to further probe the molecular basis for observed differences in pathogenesis of these viruses by genome manipulation. This will aid the rational development of attenuated virus vaccines. Comparison of these three genome sequences with an additional seven ASFV genomes has helped to define the core genes required for virus replication.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 30 July 2007;
accepted 20 October 2007.
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