| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Southeast Poultry Research Laboratory, Agricultural Research Service, United States Department of Agriculture, Athens, GA 30605, USA
2 Institute for Animal Health, Compton, Berkshire RG20 7NN, UK
Correspondence
Stephen J. Spatz
sspatz{at}seprl.usda.gov
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
) indicate that CVI988 ORFs are generally under purifying selection (<1), whereas UL39, UL49, UL50, RLORF6 and RLORF7 (Meq) appear to evolve under relaxed selective constraints. No CVI988 ORF was found to be under positive evolutionary selection (>>1). The GenBank/EMBL/DDBJ accession number for the CVI988-BAC genome described in this study is DQ530348.
A supplementary table and figure are available in JGV Online.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
). The causative agent of the disease is a mardivirus, Marek's disease virus (MDV-1), a member of the subfamily Alphaherpesvirinae in the family Herpesviridae. MDV is by far the most oncogenic herpesvirus known and represents the first of three neoplastic diseases (including hepatocellular carcinoma, caused by Hepatitis B virus, and cervical carcinoma, caused by Human papillomavirus) for which an effective vaccine has been developed (Churchill et al., 1969; Pagliusi & Teresa Aguado, 2004; Prince, 1996). The disease is largely controlled through mass vaccination of chick embryos or day-old chicks with live-attenuated strains of MDV-1 (Witter, 2001b). Since their introduction in the 1970s, MDV vaccines have reduced economic losses from MD dramatically, by more than 99 % (Witter, 2001a). However, over the last 40 years of intense vaccination, virulent strains have reappeared, necessitating the continued introduction of new vaccines at fairly regular intervals (Witter, 1997). In the 1970s, MD was controlled by a vaccine derived from herpesvirus of turkeys (HVT) (Witter et al., 1970), only to be replaced in the 1980s with a bivalent vaccine consisting of SB-1 (serotype 2 MDV) and HVT (Schat & Calnek, 1978). Since the 1990s, the disease has been largely controlled by vaccination with the attenuated serotype 1 strain CVI988 (Rispens et al., 1972). Despite its global usage and exceptional protective efficacy, there are signs of further increases in MDV virulence, as shown by the isolation of strains with higher pathogenicity even in birds vaccinated with the CVI988 strain (Davison & Nair, 2005; Nair, 2005). Because of this, there is great concern that CVI988-based vaccination programmes may eventually fail to protect poultry in the same way that previous generations of MD vaccines have lost their usefulness. The emergence of so-called breakthrough variants may have devastating effects on the poultry industry in the future.
In order to prevent this scenario, a better understanding of the genes involved in virulence is needed. Although many unique MDV genes, such as the viral lipase homologue (vLIP), virus-encoded telomerase RNA (vTR), pp38 and Meq, have been demonstrated to play a role in virulence or oncogenicity through studies on knockout mutants, it still remains to be determined whether vaccine strains have alterations in the genes encoding these factors or possibly in other genes (Fragnet et al., 2003; Gimeno et al., 2005; Kamil et al., 2005; Lupiani et al., 2004). To this end, we have embarked on a comparative genome analysis of vaccine and oncogenic strains in the hope that it will provide some clue to the multigenic nature of MDV virulence and identify CVI988 ORFs that have evolved under positive Darwinian selection as a result of immune evasion.
The genome organization of linear MDV has been reviewed elsewhere (Osterrieder & Vautherot, 2004; Silva et al., 2001). In this paper, we present the sequence of the full-length bacterial artificial chromosome (BAC) clone of the CVI988 genome, designated pCVI988-BAC (Petherbridge et al., 2003). The genomic structure and gene content will be compared with those of three oncogenic strains of MDV: GA, Md5 and Md11 (Lee et al., 2000a; Niikura et al., 2006; Tulman et al., 2000).
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
). Briefly, CVI988 genomic DNA and pDS-pHA1 were cotransfected into secondary chicken embryo fibroblasts by the calcium phosphate precipitation procedure. After selection in medium containing mycophenolic acid, xanthine and hypoxanthine, genomic DNA from infected cells was electroporated into Escherichia coli (DH10B) cells (Invitrogen) and plated onto agar dishes containing chloramphenicol.
DNA sequencing.
DNA from E. coli DH10B cells harbouring pCVI988-BAC was isolated by Midiprep alkaline lysis according to the manufacturer's instruction (Qiagen). Sequencing of pCVI988-BAC was initially carried out commercially. This involved the construction of a random shotgun library of pCVI988-BAC DNA in the pGEM-T vector (Promega) and sequencing of sufficient clones to obtain a sixfold coverage of the genome. To obtain the sequence of the US region of the CVI988 genome replaced by the pDS-PHA1 vector, the corresponding region was amplified by PCR using Pfu DNA polymerase (Promega) and wild-type CVI988 DNA. PCR products from the pCVI988-BAC DNA were also used for gap closure of the initial contigs using primers derived from the sequence. DNA sequence data were obtained from recombinant plasmids by using BigDye Terminator cycle sequencing and a model ABI-3730 XL DNA Analyzer (Applied Biosystems). Problematic regions were sequenced from clones containing inserts generated by PCR using Platinum Taq DNA polymerase (Invitrogen) and numerous custom primers. Regions in the genome containing highly repetitive elements (a-like sequences) were sequenced from clones by using random transposon-mediated mutagenesis (Epicentre Technologies) and transposon-specific primers. The diverse regions of the UL36 and UL48 genes were sequenced from PCR products generated in amplification reactions containing whole-genome DNA isolated from two additional CVI988 strains, Intervet p27 and ADOL BP5.
Copy-number determination of a-like sequences.
Anchor PCR and restriction analysis were used to determine the number of a-like sequences within the IRL/IRS and TRS/TRL junctions. Amplification reactions contained 100 ng pCVI988-BAC, a primer specific for repeat short sequences that flanked the a-like sequence and a primer specific for either the 5' or 3' end of the unique long sequence (Fig. 1). PCR products (
16 kb) were digested with SmaI and resolved on 1x TAE agarose gels.
|
), NCBI Entrez and other web-based tools. Novel ORFs were identified based on codon usage and investigated by using Spin (Staden) and CUSP (EMBOSS) programs (Staden et al., 2000). Homology searches were conducted by using the NCBI programs BLAST (BLASTP) and PSI-BLAST (Schaffer et al., 2001) with default settings, as well as Pairwise FLAG (BMEC/ITRI). Published mRNA and cDNA data were compared with the CVI988 genome by using PROT_MAP (SoftBerry). Multiple protein sequences were aligned by using MultAlin, CLUSTAL_W and MAFFT (Corpet, 1988; Katoh et al., 2005). Repeat elements were investigated by using the Tandyman program (Los Alamos National Laboratory, NM, USA). Phylogenetic trees were constructed by using MAFFT and MEGA version 3 (Kumar et al., 2004) with full-length genomic sequences corresponding to the available Md11 sequencing data. Evolutionary adaptation estimations based on synonymous and non-synonymous substitutions were calculated by using a model that includes allelic mixtures and the Jukes–Cantor correction for back mutations (Syn-SCAN) (Gonzales et al., 2002; Jukes & Cantor, 1969). |
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
, large PCR products with the predicted sizes of 16.1 and 16.2 kb were produced with primers specific for the IRL and TRL regions, respectively. A SmaI restriction digestion of these products generated a nearly identical restriction profile. Differences were only noticeable for the bands (C) that contained unique long and repeat long sequences. The B bands, which contained the a-like sequences, were the same size for both digestions. These results and the inability to generate PCR products containing two a-like copies indicated that only one copy was present at the IRL/IRS and TRS/TRL junctions. Single copies of the a-like sequence within these junctions have also been reported for the Md11-BAC construct (Niikura et al., 2006). As it is impossible to determine the boundaries of the a-like sequence in a circular construct containing only one copy at each junction, the combined lengths of the internal repeat regions (long and short) of CVI988-BAC, including the a-like sequences, are 26 639 bp. The lengths of the terminal long (TRL) and short (TRS) repeats (14 476 and 12 055 bp, respectively) were determined based on the assumption that cleavage of concatameric replicative intermediates occurs within telomeric repeats bracketed by pac1 and pac2 sites.
In comparison to other MDV isolates, the genomic regions of CVI988-BAC are most similar to those of Md11. Overall, the sizes of the unique regions (UL and US) were comparable among all of the strains. Size differences among the genomes can be attributed to the junctions between the unique short and the repeat short regions, as well as tandem reiterations found in the repeat long regions. An evolutionary tree of the four strains of MDV-1 is presented in Fig. 2, based on the nucleotide alignment of the TRL, UL, IRL, IRS regions and part of the US region, and illustrates that both CVI988 and GA are related more distantly to a common ancestor than are Md5 and Md11.
|
50 aa. Those of significance for a genomic comparison between pathotypes are presented in Table 1 and are numbered according to the decimal system (Tulman et al., 2000). Novel ORFs, predicted based on the codon usage of well-characterized mardivirus genes, were named according to their first 4 aa (Table 1b). A homology comparison of each ORF from the four strains is also presented in Table 1, along with the putative functions of many of these ORFs based on protein binding partners and similarities to homologues belonging to other alphaherpesviruses (Niikura et al., 2004; Nishiyama, 2004). Overall, identity is typically >98 % at the amino acid level for non-novel ORFs (Table 1a). Because of frameshift mutations, a large number of genes (LORF11, RLORF1, UL6, UL7, UL12, UL15, UL16, UL17, UL18, UL19, UL24, UL26, UL29, UL37 and UL52) within the GA genome have a lower degree of identity than homologues found in other mardiviral genomes.
|
). These mutations were exclusive to CVI988 and corresponding substitutions were identical in all three of the virulent strains.
|
). To investigate this further and to determine whether evolutionary pressures are acting upon individual ORFs, the non-synonymous to synonymous substitution ratio (=dN/dS) was determined for ORFs containing non-synonymous mutations (see Supplementary Table S1 in JGV Online). Most of the genes examined had a mean score of <1.0, indicating purifying (negative) selection or conservation of functional amino acids. For example, the UL49.5 gene in the CVI988 genome, encoding a 95 aa protein, is under strong selection for the synonymous mutations Ile6, Val10 and Thr36, with a score of 0.10. If GA ORFs that contain frameshift mutations are disregarded (e.g. LORF11, UL26, 29, 37 and 52), then only two genes, UL50 (dUTPase) and RLORF7 (Meq), can be considered to have neutral evolution, with mean scores of >0.95. Interestingly, UL49.5 has been implicated in the repression of major histocompatibility complex (MHC) class I presentation by inhibiting TAP, and both dUTPase and Meq have been demonstrated to encode virulence factors in studies involving knockout mutants (Jons et al., 1997; Koppers-Lalic et al., 2005; Lipinska et al., 2006; Lupiani et al., 2004; Tischer et al., 2002). With our limited dataset, no ORF was discovered to be under positive selection, i.e. with a dN/dS ratio>>1.0.
Differences in ORFs within the unique regions
Most CVI988-BAC ORFs from the unique regions were found to be virtually identical (>98 % amino acid identity) to homologous ORFs from other MDV-1 strains. These genes are generally most similar to those in the genomes of Md5 and Md11 and show a reduced level of homology to those of the GA strain. Based largely on its size, the majority (>85 %) of non-synonymous amino acid substitutions occur within ORFs mapping to the unique long region and occur frequently within ORFs encoding structural proteins, mainly tegument proteins. ORF49 (UL36), which encodes the large tegument protein (McNabb & Courtney, 1992) of CVI988-BAC, is particularly interesting due to the fact that it contains small deletions and the largest number of nucleotide polymorphisms. Its herpes simplex virus type 1 (HSV-1) orthologue has been shown to be involved in protein–protein interactions with other tegument proteins (Klupp et al., 2002; Vittone et al., 2005) and, recently, a ubiquitin (Ub)-specific cysteine protease function has been mapped to the hightly conserved amino terminus of both HSV-1 and MDV-1 orthologues (Kattenhorn et al., 2005; Schlieker et al., 2005). Most of the non-synonymous substitutions in UL36 of MDV-1, however, occur in the carboxyl terminus, which is extremely heterogeneous both between isolates of MDV-1 and within other members of the subfamily Alphaherpesvirinae (Gomi et al., 2002). As shown in Fig. 4, we have determined the sequence of this region for two additional isolates of the vaccine strain CVI988 and, in comparison to CVI988-BAC and other strains, have found this region to contain reiterations of two sequences: KPPPPDPDFKS/TPAPKP and KPPPA/TPDSKPSPAPKP. Similar reiterations of proline-rich motifs have been found to be important in protein–protein interactions via SH3, WW and EHV1 domains (Kay et al., 2000; Zarrinpar et al., 2003).
|
50 aa) that are present in the genomes of oncogenic strains, but absent in the CVI988-BAC genome. In the genomes of the oncogenic strains, ORF49.1 encodes a protein of 93 aa (Md5 and Md11) or 94 aa (GA), but in the genome of CVI988-BAC, a truncated protein of 34 aa is predicted, due to a point mutation. It is important to note that this is a silent mutation with respect to the UL36 ORF. Comparative genomic studies with other MDV strains have indicated that the genomes of the CVI988 vaccine strains from Intervet (p27) and ADOL (BP5) also encode the 34 aa derivative, whereas virulent strains RB1B, JM/102W, 549a, 571, 584a, 595, 648a and 686, representing the virulent pathotypes, encode the full-length protein (unpublished results). A BLAST search for proteins similar to ORF49.1 failed to generate any believable hits. The role of this ORF in the pathogenesis of MDV remains unknown and will require additional investigation.
Another gene within the unique long region that differs among attenuated and oncogenic strains is UL49 (ORF62). This gene encodes the tegument protein VP22 and has been shown to be indispensable for virus propagation in cell culture (Dorange et al., 2000, 2002). A short deletion of 18 bp was found in this gene within the CVI988-BAC genome. This deletion corresponds to a domain that is rich in serine and threonine residues in the oncogenic homologues. Close inspection of the multiple alignment (Fig. 3) suggests a domain deletion (KSERT). In addition to CVI988-BAC, this deletion was also found in other vaccine derivatives of CVI988 from Intervet (p27) and ADOL (BP5). There is little sequence resemblance between this region and corresponding regions in the VP22 homologues of HSV and varicella-zoster virus (VZV). However, like its HSV-1 counterpart, VP22 (MDV-1) is a phosphorylated protein with DNA-binding activity and displays similar functional properties: intracellular spreading and nuclear-targeted protein transporting (Blaho et al., 1994; Dorange et al., 2000; Elliott et al., 1999; Phelan et al., 1998).
|
), CVI988-BAC is more similar to Md11 and GA and lacks ORF97. Overall, the gene organization in the US region of the four sequenced strains is almost identical, with the exception of ORF97 in the Md5 genome. What is remarkable is the lack of sequence conservation in the inverted-repeat regions, which flank the unique short region.
Differences in ORFs within the inverted-repeat regions
This region is by far the most interesting, well-characterized region in the MDV genome and many of its ORFs have been defined functionally (Lupiani et al., 2001; Osterrieder & Vautherot, 2004). These ORFs are largely mardiviral-specific, some even serotype 1-specific, and homologues are not generally found in other members of the subfamily Alphaherpesvirinae. The protein products encoded by these ORFs include the oncoprotein MEQ (ORF5/ORF76) (Jones et al., 1992), the 23 kDa protein (ORF4/ORF77) (Peng & Shirazi, 1996b) and viral interleukin-8 (vIL-8; ORF3/ORF78) (Liu et al., 1999; Parcells et al., 2001).
Within the repeat long region, the ORFs of CVI988-BAC show a mean of 97.2, 94.5 and 91.7 % identity to those of Md5, Md11 and GA, respectively. This subdued level of similarity is largely due to proteins that are truncated or contain insertions, most notably the gene products of ORF4/ORF77, ORF5/ORF76 and ORF5.1/ORF77.5, encoding the 23 kDa protein, MEQ and RLORF6, respectively (Jones et al., 1992; Lee et al., 2000a; Peng & Shirazi, 1996b). As illustrated in Fig. 4, the overlapping genes encoding the 23 kDa, MEQ and RLORF6 proteins of CVI988-BAC all contain 177 bp insertions. Other researchers have previously reported insertions in the Meq loci of CVI988 (Chang et al., 2002b; Lee et al., 2000b; Shamblin et al., 2004). In the original paper by Lee et al. (2000b), a 178 bp insertion within the coding region of Meq (CVI988), specifically in the proline-rich domain, was reported to result in a frameshift. Petherbridge et al. (2003) reported previously, and we confirm, that the genes encoding the MEQ proteins of CVI988-BAC contain insertions of only 177 bp that are in frame. This is also in agreement with research examining Meq mutations not only in CVI988, but also in other attenuated strains of MDV-1 (Chang et al., 2002a; Petherbridge et al., 2003; Shamblin et al., 2004). The general conclusion is that most attenuated strains of MDV-1 contain in-frame insertions (59 aa) in the Meq ORF, the probable result of domain duplication (see boxed regions in Fig. 4). One notable exception is the attenuated strain R2/23, in which an additional thymidine residue within the Meq locus causes a frameshift mutation (Spatz & Silva, 2007). Interestingly, all of the insertion mutations within the Meq genes of attenuated strains occur in the same region, the proline-rich domain, which has been shown to be essential for the trans-activation of MEQ-responsive promoters (Liu & Kung, 2000; Qian et al., 1996). This domain, however, when used by itself in trans-activation analysis, represses transcription and behaves as a dominant-negative mutant. In comparing the trans-activation/repression abilities of MEQ variants from attenuated and virulent strains, Chang et al. (2002c) reported that those containing the duplicated proline-rich motifs (coined L-MEQ) exhibit a higher level of trans-repression than do MEQ variants containing the single proline-rich motif (S-MEQ) found in virulent strains.
In addition to insertional mutations in the Meq genes of CVI988-BAC, three point mutations were discovered at aa 71, 77 and 385 (Table 2). This places the sequence of CVI988-BAC in the IV allelic group described by Laurent et al. (2004). Other strains of MDV-1 classified as virulent (e.g. BC-1, JM102, 567, 617A, 637, 571, 573) have been reported to contain mutations at either position 71 or 77 (Shamblin et al., 2004). Only one other strain, CU-2, which has a mild virulent phenotype, contained the double mutations at position 71 and 77, similar to CVI988-BAC. The Ile385 mutation is exclusive to the CVI988-BAC MEQ protein. The SNP responsible for Ile385, when examined in the overlapping ORF (RLORF6), encodes either leucine or phenylalanine. RLORF6 of CVI988-BAC contains Phe228; in contrast, all oncogenic strains were found to contain Leu169.
Within the repeat long region of CVI988-BAC, three ORFs (ORF3/ORF78, ORF5.5/ORF75.91 and ORF74) encode truncated proteins relative to those encoded by the genomes of the three oncogenic strains. Because of an opal (TGA) mutation, 20 aa are missing in the putative proteins of ORF5.5/ORF75.91 (CVI988-BAC). An extensive homology search using BLAST programs provided few clues to the possible function of these proteins. The function of the ORF3/ORF78 gene products, however, is far more defined. These ORFs are spliced genes with similarity to genes encoding the CxC chemokine IL-8 and hence termed vIL-8. We have discovered that, in the CVI988-BAC genome, these ORFs encode a truncated version of vIL-8 that is 11 and 13 aa shorter than those of Md5 and Md11/GA, respectively. Although the function of the carboxyl-terminal residues in the mammalian IL-8 orthologue has been reported to be non-essential for neutrophil binding, little information is available about the function of these residues in vIL-8 or avian IL-8 (Kaiser et al., 1999). In fact, avian IL-8 contains only 103 aa, thus to some extent resembling CVI988-BAC vIL-8. As vIL-8-null MDV-1 mutants have been reported to be mildly virulent and to have a lower incidence of tumour formation, truncated versions of vIL-8 may also contribute to an attenuated phenotype (Cortes & Cardona, 2004; Cui et al., 2004).
One ORF (ORF74 or RLORF12) within the internal repeat long region is predicted to encode a truncated protein of 67 aa. Unexpectedly, its diploid counterpart (ORF7) within the terminal repeat long region encodes a protein of 115 aa. All sequenced oncogenic strains of MDV encode RLORF12 homologues containing 115 aa; however, we have found recently that some attenuated strains (i.e. RM-1, CVI988-BP5 and JM/102W) contain deletions within this ORF and encode similar truncated proteins (Spatz & Silva, 2007). This could be significant, as these deletions occur at the origin of replication and may affect the binding of the DNA-binding proteins (UL9 and UL8.5) involved in replication, as well as other proteins demonstrated to bind the RLORF12 protein, such as the growth-related translationally controlled tumour protein (TCTP) (Niikura et al., 2004).
Within the repeat long regions, only one pair of diploid ORFs (ORF6.2/ORF75.6) was absent in the CVI988 genome, due to a point mutation in the start codon. In virulent strains, these ORFs are predicted to encode short polypeptides of 66 aa. Because of their small size, no significant similarity was found to any protein in GenBank. Whether these genes actually encode proteins or represent exons of uncharacterized proteins remains to be determined. In support of the latter notion, splicing and alternative splicing have been shown to occur within this region, as evidenced by the spliced gene products vIL-8 (Liu et al., 1999), 14 kDa A and 14 kDa B (Hong & Coussens, 1994), as well as alternatively spliced gene products involving exons 2 and 3 of vIL-8 RNA designated Meq/vIL-8, RLORF5a/vIL-8 and RLORF4/vIL-8 (Anobile et al., 2006; Jarosinski & Schat, 2007; Peng & Shirazi, 1996a; Peng et al., 1995). Given the complex transcription patterns and alternative splicing that occur in this region, it is quite possible that additional spliced gene products exist.
The repeat short regions of CVI988-BAC, like those of oncogenic strains, largely encode the major immediate-early protein ICP4 (Anderson et al., 1992). Over 57 % of the repeat short regions are devoted to encoding this trans-activator. These proteins are >98 % identical among strains and, although there are differences, especially with ICP4 (GA), no polymorphisms were discovered that could differentiate attenuated versus oncogenic strains collectively. Thirty-four substitutions (23 non-synonymous and 11 synonymous) were discovered in the ICP4 gene of the GA strain relative to CVI988. Only three and six substitutions were found in ICP4 of Md5 and Md11, respectively, again suggesting a close evolutionary relationship between CVI988 and these two strains. Whether these substitutions are significant for attenuation remains to be determined, particularly in light of research demonstrating that ICP4 of a vaccine strain of VZV (Oka) has a lower trans-activation capacity than its parental counterpart and this contributes to its attenuated phenotype (Cohrs et al., 2006; Gomi et al., 2002). What can be hypothesized is that the trans-activation potential of these two alphaherpesviruses seems to play a role in attenuation. Therefore, as few mutations were noted between ICP4 proteins of Md5 and CVI988, it seems likely that the trans-repression capability of L-MEQ and not the ICP4 protein itself is important in attenuation of CVI988. It is interesting to note that, although MEQ is expressed predominantly in the latent state, it is also expressed early in lytic infection and is capable of repressing the transcription of the ICP4 promoter through direct binding as a MEQ homodimer (Jones et al., 1992; Levy et al., 2003).
Conclusions
In recent years, the use of MDV BAC recombinants and overlapping cosmid constructs has been instrumental in determining the function of various genes in the pathogenesis of MD. Five genes (RLORF4, vLIP, vTR, vIL-8 and the Meq/23 kDa locus) have been demonstrated to encode virulence factors (Cui et al., 2005; Jarosinski et al., 2005; Kamil et al., 2005; Lupiani et al., 2004; Trapp et al., 2006). Of these, our data support a role for the MEQ oncoprotein in the attenuation of CVI988. No genetic changes were found in the genes encoding RLORF4, lipase (vLIP) or telomerase (vTR) of CVI988 compared with those found in Md5, Md11 and GA. It is likely that additional ORFs contribute to the attenuated phenotype of CVI988. This study has identified eight ORFs [49, 49.1, 62 and the diploid ORFs: Meq, RLORF6, 23 kDa, 5.5/75.91 and 3/78 (vIL-8)] in the CVI988-BAC genome that differ (are expanded or truncated) from those in the oncogenic strains, as well as one ORF (6.2/75.6) that is exclusively present in the genomes of the virulent strains. Only a short deletion in the carboxyl terminal of vIL-8 (CVI988) was discovered, but its significance is doubtful, due to the fact that other isolates of CVI988 contain genes encoding full-length vIL-8. Seventy non-synonymous amino acid substitutions were identified within the CVI988-BAC ORFs. These mutations were exclusive to CVI988 and corresponding substitutions were identical in all of the three virulent strains. Most of these occurred in tegument proteins, especially UL36, and will be useful in future genotyping studies. The ratios of non-synonymous to synonymous mutations were examined in order to understand the selective pressures acting on various ORFs. Only two genes (UL50 and RLORF7) had dN/dS ratios >0.95, indicating a lack of selective pressure to conserve functional amino acids. All other genes examined with variations in non-synonymous and synonymous substitutions were under purifying selection.
Future comparative genomic studies examining a larger number of avirulent or less virulent strains in order to identify positively selected genes will undoubtedly contribute to our overall understanding of the genes involved in virulence. This information will be essential to engineer novel MDV-1 vaccines capable of protecting chickens against continuously evolving very virulent plus (VV+) strains.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Anobile, J. M., Arumugaswami, V., Downs, D., Czymmek, K., Parcells, M. & Schmidt, C. J. (2006). Nuclear localization and dynamic properties of the Marek's disease virus oncogene products Meq and Meq/vIL8. J Virol 80, 1160–1166.
Besemer, J. & Borodovsky, M. (2005). GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res 33, W451–W454.
Blaho, J. A., Mitchell, C. & Roizman, B. (1994). An amino acid sequence shared by the herpes simplex virus 1 alpha regulatory proteins 0, 4, 22, and 27 predicts the nucleotidylylation of the UL21, UL31, UL47, and UL49 gene products. J Biol Chem 269, 17401–17410.
Calnek, B. W. (2001). Pathogenesis of Marek's disease virus infection. Curr Top Microbiol Immunol 255, 25–55.[Medline]
Chang, K. S., Lee, S. I., Ohashi, K., Ibrahim, A. & Onuma, M. (2002a). The detection of the meq gene in chicken infected with Marek's disease virus serotype 1. J Vet Med Sci 64, 413–417.[CrossRef][Medline]
Chang, K. S., Ohashi, K. & Onuma, M. (2002b). Diversity (polymorphism) of the meq gene in the attenuated Marek's disease virus (MDV) serotype 1 and MDV-transformed cell lines. J Vet Med Sci 64, 1097–1101.[CrossRef][Medline]
Chang, K. S., Ohashi, K. & Onuma, M. (2002c). Suppression of transcription activity of the MEQ protein of oncogenic Marek's disease virus serotype 1 (MDV1) by L-MEQ of non-oncogenic MDV1. J Vet Med Sci 64, 1091–1095.[CrossRef][Medline]
Churchill, A. E., Payne, L. N. & Chubb, R. C. (1969). Marek's disease immunization against Marek's disease using a live attenuated virus. Lancet i, 610–611.
Cohrs, R. J., Gilden, D. H., Gomi, Y., Yamanishi, K. & Cohen, J. I. (2006). Comparison of virus transcription during lytic infection of the Oka parental and vaccine strains of varicella-zoster virus. J Virol 80, 2076–2082.
Corpet, F. (1988). Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16, 10881–10890.
Cortes, P. L. & Cardona, C. J. (2004). Pathogenesis of a Marek's disease virus mutant lacking vIL-8 in resistant and susceptible chickens. Avian Dis 48, 50–60.[CrossRef][Medline]
Cui, X., Lee, L. F., Reed, W. M., Kung, H. J. & Reddy, S. M. (2004). Marek's disease virus-encoded vIL-8 gene is involved in early cytolytic infection but dispensable for establishment of latency. J Virol 78, 4753–4760.
Cui, X., Lee, L. F., Hunt, H. D., Reed, W. M., Lupiani, B. & Reddy, S. M. (2005). A Marek's disease virus vIL-8 deletion mutant has attenuated virulence and confers protection against challenge with a very virulent plus strain. Avian Dis 49, 199–206.[CrossRef][Medline]
Davison, F. & Nair, V. (2005). Use of Marek's disease vaccines: could they be driving the virus to increasing virulence? Expert Rev Vaccines 4, 77–88.[CrossRef][Medline]
Dorange, F., El Mehdaoui, S., Pichon, C., Coursaget, P. & Vautherot, J.-F. (2000). Marek's disease virus (MDV) homologues of herpes simplex virus type 1 UL49 (VP22) and UL48 (VP16) genes: high-level expression and characterization of MDV-1 VP22 and VP16. J Gen Virol 81, 2219–2230.
Dorange, F., Tischer, B. K., Vautherot, J. F. & Osterrieder, N. (2002). Characterization of Marek's disease virus serotype 1 (MDV-1) deletion mutants that lack UL46 to UL49 genes: MDV-1 UL49, encoding VP22, is indispensable for virus growth. J Virol 76, 1959–1970.
Elliott, G., O'Reilly, D. & O'Hare, P. (1999). Identification of phosphorylation sites within the herpes simplex virus tegument protein VP22. J Virol 73, 6203–6206.
Fragnet, L., Blasco, M. A., Klapper, W. & Rasschaert, D. (2003). The RNA subunit of telomerase is encoded by Marek's disease virus. J Virol 77, 5985–5996.
Gimeno, I. M., Witter, R. L., Hunt, H. D., Reddy, S. M., Lee, L. F. & Silva, R. F. (2005). The pp38 gene of Marek's disease virus (MDV) is necessary for cytolytic infection of B cells and maintenance of the transformed state but not for cytolytic infection of the feather follicle epithelium and horizontal spread of MDV. J Virol 79, 4545–4549.
Gomi, Y., Sunamachi, H., Mori, Y., Nagaike, K., Takahashi, M. & Yamanishi, K. (2002). Comparison of the complete DNA sequences of the Oka varicella vaccine and its parental virus. J Virol 76, 11447–11459.
Gonzales, M. J., Dugan, J. M. & Shafer, R. W. (2002). Synonymous-non-synonymous mutation rates between sequences containing ambiguous nucleotides (Syn-SCAN). Bioinformatics 18, 886–887.
Hong, Y. & Coussens, P. M. (1994). Identification of an immediate-early gene in the Marek's disease virus long internal repeat region which encodes a unique 14-kilodalton polypeptide. J Virol 68, 3593–3603.
Jarosinski, K. W. & Schat, K. A. (2007). Multiple alternative splicing to exons II and III of viral interleukin-8 (vIL-8) in the Marek's disease virus genome: the importance of vIL-8 exon I. Virus Genes 34, 9–22.[CrossRef][Medline]
Jarosinski, K. W., Osterrieder, N., Nair, V. K. & Schat, K. A. (2005). Attenuation of Marek's disease virus by deletion of open reading frame RLORF4 but not RLORF5a. J Virol 79, 11647–11659.
Jones, D., Lee, L., Liu, J.-L., Kung, H.-J. & Tillotson, J. K. (1992). Marek's disease virus encodes a basic-leucine zipper gene resembling the fos/jun oncogenes that is highly expressed in lymphoblastoid tumors. Proc Natl Acad Sci U S A 89, 4042–4046.
Jons, A., Gerdts, V., Lange, E., Kaden, V. & Mettenleiter, T. C. (1997). Attenuation of dUTPase-deficient pseudorabies virus for the natural host. Vet Microbiol 56, 47–54.[CrossRef][Medline]
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
Kaiser, P., Hughes, S. & Bumstead, N. (1999). The chicken 9E3/CEF4 CXC chemokine is the avian orthologue of IL8 and maps to chicken chromosome 4 syntenic with genes flanking the mammalian chemokine cluster. Immunogenetics 49, 673–684.[CrossRef][Medline]
Kamil, J. P., Tischer, B. K., Trapp, S., Nair, V. K., Osterrieder, N. & Kung, H. J. (2005). vLIP, a viral lipase homologue, is a virulence factor of Marek's disease virus. J Virol 79, 6984–6996.
Katoh, K., Kuma, K., Toh, H. & Miyata, T. (2005). MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Res 33, 511–518.
Kattenhorn, L. M., Korbel, G. A., Kessler, B. M., Spooner, E. & Ploegh, H. L. (2005). A deubiquitinating enzyme encoded by HSV-1 belongs to a family of cysteine proteases that is conserved across the family Herpesviridae. Mol Cell 19, 547–557.[CrossRef][Medline]
Kay, B. K., Williamson, M. P. & Sudol, M. (2000). The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains. FASEB J 14, 231–241.
Klupp, B. G., Fuchs, W., Granzow, H., Nixdorf, R. & Mettenleiter, T. C. (2002). Pseudorabies virus UL36 tegument protein physically interacts with the UL37 protein. J Virol 76, 3065–3071.
Koppers-Lalic, D., Reits, E. A., Ressing, M. E., Lipinska, A. D., Abele, R., Koch, J., Marcondes Rezende, M., Admiraal, P., van Leeuwen, D. & other authors (2005). Varicelloviruses avoid T cell recognition by UL49.5-mediated inactivation of the transporter associated with antigen processing. Proc Natl Acad Sci U S A 102, 5144–5149.
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform 5, 150–163.
Laurent, S., Esnault, E. & Rasschaert, D. (2004). Single-nucleotide polymorphisms in two Marek's disease virus genes (Meq and gD): application to a retrospective molecular epidemiology study (1982–1999) in France. J Gen Virol 85, 1387–1392.
Lee, L. F., Wu, P., Sui, D., Ren, D., Kamil, J., Kung, H. J. & Witter, R. L. (2000a). The complete unique long sequence and the overall genomic organization of the GA strain of Marek's disease virus. Proc Natl Acad Sci U S A 97, 6091–6096.
Lee, S. I., Takagi, M., Ohashi, K., Sugimoto, C. & Onuma, M. (2000b). Difference in the meq gene between oncogenic and attenuated strains of Marek's disease virus serotype 1. J Vet Med Sci 62, 287–292.[CrossRef][Medline]
Levy, A. M., Izumiya, Y., Brunovskis, P., Xia, L., Parcells, M. S., Reddy, S. M., Lee, L., Chen, H. W. & Kung, H. J. (2003). Characterization of the chromosomal binding sites and dimerization partners of the viral oncoprotein Meq in Marek's disease virus-transformed T cells. J Virol 77, 12841–12851.
Lipinska, A. D., Koppers-Lalic, D., Rychlowski, M., Admiraal, P., Rijsewijk, F. A., Bienkowska-Szewczyk, K. & Wiertz, E. J. (2006). Bovine herpesvirus 1 UL49.5 protein inhibits the transporter associated with antigen processing despite complex formation with glycoprotein M. J Virol 80, 5822–5832.
Liu, J. L. & Kung, H. J. (2000). Marek's disease herpesvirus transforming protein MEQ: a c-Jun analogue with an alternative life style. Virus Genes 21, 51–64.[CrossRef][Medline]
Liu, J. L., Lin, S. F., Xia, L., Brunovskis, P., Li, D., Davidson, I., Lee, L. F. & Kung, H. J. (1999). MEQ and V-IL8: cellular genes in disguise? Acta Virol 43, 94–101.[Medline]
Lupiani, B., Lee, L. F. & Reddy, S. M. (2001). Protein-coding content of the sequence of Marek's disease virus serotype 1. Curr Top Microbiol Immunol 255, 159–190.[Medline]
Lupiani, B., Lee, L. F., Cui, X., Gimeno, I., Anderson, A., Morgan, R. W., Silva, R. F., Witter, R. L., Kung, H. J. & Reddy, S. M. (2004). Marek's disease virus-encoded Meq gene is involved in transformation of lymphocytes but is dispensable for replication. Proc Natl Acad Sci U S A 101, 11815–11820.
McNabb, D. S. & Courtney, R. J. (1992). Analysis of the UL36 open reading frame encoding the large tegument protein (ICP1/2) of herpes simplex virus type 1. J Virol 66, 7581–7584.
Nair, V. (2005). Evolution of Marek's disease – a paradigm for incessant race between the pathogen and the host. Vet J 170, 175–183.[Medline]
Niikura, M., Liu, H. C., Dodgson, J. B. & Cheng, H. H. (2004). A comprehensive screen for chicken proteins that interact with proteins unique to virulent strains of Marek's disease virus. Poult Sci 83, 1117–1123.
Niikura, M., Dodgson, J. & Cheng, H. (2006). Direct evidence of host genome acquisition by the alphaherpesvirus Marek's disease virus. Arch Virol 151, 537–549.[CrossRef][Medline]
Nishiyama, Y. (2004). Herpes simplex virus gene products: the accessories reflect her lifestyle well. Rev Med Virol 14, 33–46.[CrossRef][Medline]
Osterrieder, K. & Vautherot, J.-F. (2004). The genome content of Marek's disease-like viruses. In Marek's Disease: an Evolving Problem, pp. 17–31. Edited by F. Davison & V. Nair. Oxford: Elsevier.
Pagliusi, S. R. & Teresa Aguado, M. (2004). Efficacy and other milestones for human papillomavirus vaccine introduction. Vaccine 23, 569–578.[CrossRef][Medline]
Parcells, M. S., Lin, S. F., Dienglewicz, R. L., Majerciak, V., Robinson, D. R., Chen, H. C., Wu, Z., Dubyak, G. R., Brunovskis, P. & other authors (2001). Marek's disease virus (MDV) encodes an interleukin-8 homolog (vIL-8): characterization of the vIL-8 protein and a vIL-8 deletion mutant MDV. J Virol 75, 5159–5173.
Peng, Q. & Shirazi, Y. (1996a). Characterization of the protein product encoded by a splicing variant of the Marek's disease virus Eco-Q gene (Meq). Virology 226, 77–82.[CrossRef][Medline]
Peng, Q. & Shirazi, Y. (1996b). Isolation and characterization of Marek's disease virus (MDV) cDNAs from a MDV-transformed lymphoblastoid cell line: identification of an open reading frame antisense to the MDV Eco-Q protein (Meq). Virology 221, 368–374.[CrossRef][Medline]
Peng, Q., Zeng, M., Bhuiyan, Z. A., Ubukata, E., Tanaka, A., Nonoyama, M. & Shirazi, Y. (1995). Isolation and characterization of Marek's disease virus (MDV) cDNAs mapping to the BamHI-I2, BamHI-Q2, and BamHI-L fragments of the MDV genome from lymphoblastoid cells transformed and persistently infected with MDV. Virology 213, 590–599.[CrossRef][Medline]
Petherbridge, L., Howes, K., Baigent, S. J., Sacco, M. A., Evans, S., Osterrieder, N. & Nair, V. (2003). Replication-competent bacterial artificial chromosomes of Marek's disease virus: novel tools for generation of molecularly defined herpesvirus vaccines. J Virol 77, 8712–8718.
Phelan, A., Elliott, G. & O'Hare, P. (1998). Intercellular delivery of functional p53 by the herpesvirus protein VP22. Nat Biotechnol 16, 440–443.[CrossRef][Medline]
Prince, A. M. (1996). Prevention of liver cancer and cirrhosis by vaccines. Clin Lab Med 16, 493–505.[Medline]
Qian, Z., Brunovskis, P., Lee, L., Vogt, P. K. & Kung, H. J. (1996). Novel DNA binding specificities of a putative herpesvirus bZIP oncoprotein. J Virol 70, 7161–7170.
Rispens, B. H., van Vloten, H., Mastenbroek, N., Maas, H. J. & Schat, K. A. (1972). Control of Marek's disease in the Netherlands. I. Isolation of an avirulent Marek's disease virus (strain CVI 988) and its use in laboratory vaccination trials. Avian Dis 16, 108–125.[CrossRef][Medline]
Schaffer, A. A., Aravind, L., Madden, T. L., Shavirin, S., Spouge, J. L., Wolf, Y. I., Koonin, E. V. & Altschul, S. F. (2001). Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements. Nucleic Acids Res 29, 2994–3005.
Schat, K. A. & Calnek, B. W. (1978). Characterizations of an apparently non-oncogenic Marek's disease virus. J Natl Cancer Inst 60, 1075–1082.[Medline]
Schlieker, C., Korbel, G. A., Kattenhorn, L. M. & Ploegh, H. L. (2005). A deubiquitinating activity is conserved in the large tegument protein of the herpesviridae. J Virol 79, 15582–15585.
Shamblin, C. E., Greene, N., Arumugaswami, V., Dienglewicz, R. L. & Parcells, M. S. (2004). Comparative analysis of Marek's disease virus (MDV) glycoprotein-, lytic antigen pp38- and transformation antigen Meq-encoding genes: association of meq mutations with MDVs of high virulence. Vet Microbiol 102, 147–167.[CrossRef][Medline]
Silva, R. F., Lee, L. F. & Kutish, G. F. (2001). The genomic structure of Marek's disease virus. Curr Top Microbiol Immunol 255, 143–158.[Medline]
Spatz, S. J. & Silva, R. F. (2007). Polymorphisms in the repeat long regions of oncogenic and attenuated pathotypes of Marek's disease virus 1. Virus Genes (in press). doi:10.1007/s11262-006-0024-5
Staden, R., Beal, K. F. & Bonfield, J. K. (2000). The Staden package, 1998. Methods Mol Biol 132, 115–130.[Medline]
Tischer, B. K., Schumacher, D., Messerle, M., Wagner, M. & Osterrieder, N. (2002). The products of the UL10 (gM) and the UL49.5 genes of Marek's disease virus serotype 1 are essential for virus growth in cultured cells. J Gen Virol 83, 997–1003.
Trapp, S., Parcells, M. S., Kamil, J. P., Schumacher, D., Tischer, B. K., Kumar, P. M., Nair, V. K. & Osterrieder, N. (2006). A virus-encoded telomerase RNA promotes malignant T cell lymphomagenesis. J Exp Med 203, 1307–1317.
Tulman, E. R., Afonso, C. L., Lu, Z., Zsak, L., Rock, D. L. & Kutish, G. F. (2000). The genome of a very virulent Marek's disease virus. J Virol 74, 7980–7988.
Vittone, V., Diefenbach, E., Triffett, D., Douglas, M. W., Cunningham, A. L. & Diefenbach, R. J. (2005). Determination of interactions between tegument proteins of herpes simplex virus type 1. J Virol 79, 9566–9571.
Witter, R. L. (1997). Increased virulence of Marek's disease virus field isolates. Avian Dis 41, 149–163.[CrossRef][Medline]
Witter, R. L. (2001a). Marek's disease virus vaccines – past, present and future (chicken vs. virus – a battle of the centuries). In Current Progress on Marek's Disease Research. Proceedings of the 6th International Symposium on Marek's Disease, pp. 1–9. Edited by K. A. Schat, R. M. Morgan, M. S. Parcells & J. L. Spencer. Kennett Square, PA: American Association of Avian Pathologists.
Witter, R. L. (2001b). Protective efficacy of Marek's disease vaccines. Curr Top Microbiol Immunol 255, 57–90.[Medline]
Witter, R. L., Nazerian, K., Purchase, H. G. & Burgoyne, G. H. (1970). Isolation from turkeys of a cell-associated herpesvirus antigenically related to Marek's disease virus. Am J Vet Res 31, 525–538.[Medline]
Zarrinpar, A., Bhattacharyya, R. P. & Lim, W. A. (2003). The structure and function of proline recognition domains. Sci STKE 2003, RE8.
Received 27 September 2006;
accepted 4 December 2006.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
