The genome of herpesvirus of turkeys: comparative analysis with Marek’s disease viruses

Brewster F. Kingham1, Vladimir Zelnik2, Juraj Kopácek2, Vladimir Majerciak2, Erik Ney1 and Carl J. Schmidt1

Department of Animal and Food Sciences, University of Delaware, Newark, DE 19717, USA1
Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 842 45 Bratislava, Slovak Republic2

Author for correspondence: Carl Schmidt. Fax +1 302 831 8177. e-mail schmidtc{at}udel.edu


   Abstract
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
The complete coding sequence of the herpesvirus of turkeys (HVT) unique long (UL) region along with the internal repeat regions has been determined. This allows completion of the HVT nucleotide sequence by linkage to the sequence of the unique short (US) region. The genome is approximately 160 kbp and shows extensive similarity in organization to the genomes of Marek’s disease virus serotypes 1 and 2 (MDV-1, MDV-2) and other alphaherpesviruses. The HVT genome contains 75 ORFs, with three ORFs present in two copies. Sixty-seven ORFs were identified readily as homologues of other alphaherpesvirus genes. Seven of the remaining eight ORFs are homologous to genes in MDV, but are absent from other herpesviruses. These include a gene with similarity to cellular lipases. The final, HVT-unique gene is a virus homologue of the cellular NR-13 gene, the product of which belongs to the Bcl family of proteins that regulate apoptosis. No other herpesvirus sequenced to date contains a homologue of this gene. Of potential significance is the absence of a complete block of genes within the HVT internal repeat that is present in MDV-1. These include the pp38 and meq genes, which have been implicated in MDV-1-induced T-cell lymphoma. By implication, other genes present in this region of MDV-1, but missing in HVT, may play important roles in the different biological properties of the viruses.


   Introduction
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
Herpesvirus of turkeys (HVT) provided one of the first vaccines for avian lymphoma (Purchase et al., 1971 ). HVT is used to prevent Marek’s disease, which is characterized by T-cell lymphoma and severe immune suppression in commercial chicken flocks (Calnek & Witter, 1997 ) and is caused by Marek’s disease virus serotype 1 (MDV-1). A third serologically related avian herpesvirus is termed MDV-2 (Schat & Calnek, 1978 ); however, it is non-oncogenic and is also used to control Marek’s disease in some vaccine formulations. Immunization with HVT and MDV-2 does not prevent productive infection by MDV-1 and vaccinated flocks still shed active MDV-1. Instead, HVT vaccination prevents the onset of both T-cell lymphoma and immune suppression caused by uncontrolled MDV-1 infection.

HVT and MDV are both avian alphaherpesviruses. Their genomes consist of two distinct regions, L and S, that are composed of unique long (UL) and unique short (US) sequences bounded by inverted repeats. The genomic organization of UL has been remarkably conserved during the evolution of these herpesviruses. The US region, while containing obvious homologues conserved in the different alphaherpesviruses, is more variable in both its organization and gene content.

We have completed the DNA sequence of the UL region, the long internal repeat (IRL) and the inverted repeats of the short region (complete IRS and partial TRS) of HVT. As sequences of the complete MDV-1 genome and the UL and US regions of MDV-2 are available (Brunovskis & Velicer, 1995 ; McKie et al., 1995 ; Jang et al., 1998 ; Lee et al., 2000 ; Tulman et al., 2000 ), our data provide a tool for comparative analysis of all three serologically related avian herpesviruses. The gene organization within the HVT UL and US regions is clearly conserved relative to other alphaherpesviruses. In addition, HVT encodes seven genes that are found in both MDV-1 and HVT (MDV-2 shares six of them), but are absent from the sequenced genomes of other alphaherpesviruses. Among these genes is one encoding a product with similarity to cellular phospholipases. Another gene, present in HVT but absent from MDV-1, encodes a product that is similar to the quail anti-apoptotic protein NR-13. A striking feature is the different genetic organization within the repeat regions.

Partial sequences of the HVT genome have been reported previously (Kato et al., 1989 ; Martin et al., 1989 ; Scott et al., 1989 , 1993 ; Coussens et al., 1990 ; Zelník et al., 1993 ; Smith et al., 1995 ; Kopácek et al., 1997 ). However, these sequences represent only a small portion of the HVT genome. The data presented in this paper compile the complete encoding potential of HVT, enabling subsequent expression and functional analysis of its genetic elements.


   Methods
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Abstract
Introduction
Methods
Results and Discussion
References
 
{blacksquare} DNA sequence determination.
Transposons (Nag et al., 1988 ) were used to insert priming sites randomly within the cloned BamHI fragments of HVT strain Fc126 (Igarashi et al., 1987 ). The DNA sequence was determined by using transposon-specific primers and the dye terminator method (ABI Prism Dye Terminator) on an ABI 377 sequencer (Perkin Elmer). Nested deletion clones of the BamHI-A and -O fragments served as templates for manual sequencing using the T7 sequencing kit (Pharmacia Biotech) and [{alpha}-35S]dATP (Amersham Biotech). The sequences of the BamHI fragments were linked by using specific primers to amplify across the junctions by PCR. DNA sequences of these linking products were then determined by using the specific primers. All sequences were determined in both directions and each nucleotide was read at least five times.

{blacksquare} Bioinformatics.
DNA sequences were aligned by using the GCG software package. Further analysis was performed with the programs OPEN READING FRAME, BLAST and PSI-BLAST (Altschul et al., 1997 ). Additional analysis was conducted with the web-based programs GRAIL (Uberbacher & Mural, 1991 ), MOTIF (Bucher & Bairoch, 1994 ), PSORT, ProDom and DIALIGN 2 (Morgenstern et al., 1996 ).

{blacksquare} Source of sequences.
The accession numbers of the sequences included in the analysis were as follows: MDV-1, AF147806; MDV-2, AB049735; herpes simplex virus type 1 (HSV-1; human herpesvirus-1), NC 001806; varicella-zoster virus (VZV), NC 001348; bovine herpesvirus-1 (BHV-1), NC 001847; equine herpesvirus-1 (EqHV-1) UL45, P36323; quail NR-13 homologue, Q90343; chicken Bcl-x, A47537; pseudorabies virus (PRV) gC, AAF28243; EqHV-1 UL54 homologue, Q05906.


   Results and Discussion
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
Overall genomic structure
From the sequence data, the overall size of the HVT genome is approximately 160673 bp, 13400 bp smaller than the MDV-1 genome (for comparison see also Table 1). Precise numbers of nucleotides in both viruses cannot be determined, as there are regions with variable length (e.g. a-like sequence). Seventy-five potential ORFs and genes were identified in the HVT genome. Three of these are present in two copies, as they are located in repeat regions flanking the UL and/or US regions. Human herpesvirus-1 (HSV-1) nomenclature was adopted for those HVT genes that are homologous to HSV-1 genes. HVT-specific ORFs are referred to as LORFs, RLORF, RSORFs and SORFs, depending on the location of the initiation codon (UL, RL, RS or US). Within the UL region of HVT, homologues of alphaherpesvirus genes corresponding to HSV-1 UL1–UL55 (McGeoch et al., 1988 ; Roizman & Sears, 1996 ) were readily recognized (Fig. 1a). The organization of the genes UL1–UL54 is collinear with HSV-1. In HVT, a unique gene (HVT LORF3) of unknown function is inserted between UL54 and UL55. This insertion is also seen in MDV (Tsushima et al., 1999 ; Lee et al., 2000 ). Five genes are found either within or proximal to the UL repeats of HVT and are absent from other herpesviruses (LORF1, LORF2, LORF3, LORF5 and LORF6; see Fig. 1a). HVT LORF4 homologues are found in MDV-1, VZV, BHV-1 and EqHV-1, but not in HSV-1 or MDV-2. HVT also contains a gene, RSLORF1, that is absent from MDV-1 and other herpesviruses. In addition, an entire region of MDV-1 containing genes that are implicated in oncogenesis has no homologous counterpart in HVT. This region of MDV-1 contains 11 ORFs spanning from pp38 to RLORF1 (Fig. 1b). The partial pp38 reading frame in HVT and MDV-2 suggests that this region may have been present in an ancestral virus common to both MDV and HVT. Deletion of this region may have been an important step in the evolution of both HVT and the non-oncogenic MDV-2 strains. The genetic organization of the HVT US region was described previously (Zelník et al., 1993 ).


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Table 1. Comparison of the sizes of the genomic elements of HVT, MDV-1 and MDV-2

 


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Fig. 1. Comparison of the genomic structure of MDV-1, MDV-2 and HVT UL (a) and US and repeat regions (b). Genes pictured in blue have alphaherpesvirus homologues. Those in yellow are found in MDV and HVT, but have not been identified in other herpesviruses. Finally, those in red have only been identified in the virus shown. Abbreviations: hom., homologue; pol., polymerase; prot., protein; rep, repeat; RR, ribonucleotide reductase; sub., subunit; TK, thymidine kinase.

 
Gene-function predictions
The HVT genome contains at least 75 genes that are predicted to provide both essential and ancillary functions (Table 2). While few of these genes have been examined for their expression and function (Bandyopadhyay, 1989 ; Martin et al., 1989 ; Zelník et al., 1994 ), many roles can be predicted on the basis of similarity to other herpesviruses, including HSV-1 (Roizman & Sears, 1996 ) and MDV-1. Comparisons reveal potential structural proteins such as those of the capsid, tegument and envelope of the mature virus. In addition, HVT contains the complete set of genes that have been implicated in HSV-1 DNA replication, nucleotide metabolism and DNA packaging. Some of these homologues belong to the family of core genes that have been found in all alpha-, beta- and gammaherpesviruses. These putative functional assignments are indicated in Table 2. Some characteristics revealed by inspection of the HVT coding regions are discussed below.


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Table 2. ORFs identified in the HVT genome

 
Glycoproteins
HVT has conserved homologues of 10 of the 12 glycoproteins found in HSV-1. Absent from HVT are coding regions for glycoprotein G and glycoprotein J. HVT retains several glycoproteins that function as heterodimers. Both gL (HVT UL1) and gH (HVT UL22) genes are found and these two glycoproteins have been shown in other herpesviruses to form heterodimeric complexes and function in virus particle fusion with the host cell (Milne et al., 1998 ; Roop et al., 1993 ). gI (HVT US7) and gE (HVT US8) homologues are also found in HVT and this dimer may play an important role in cell-to-cell transfer of virus particles (Dingwell et al., 1994 , 1995 ). Also, both gM (HVT UL10) and gN (HVT UL49.5) homologues are recognized in the HVT sequence. The herpesvirus gN has been shown to form a disulphide cross-link to gM (Jons et al., 1998 ; Wu et al., 1998 ). Alignment of gN sequences from several different herpesviruses indicated that only one cysteine residue, corresponding to HVT gN residue 45, is conserved completely (not shown). If cross-linking to gM is one of the conserved functions of gN, this cysteine may be the functional residue linked to gM. Additional putative glycoprotein genes include homologues of HSV-1 gD (HVT US6), gB (HVT UL27), gC (HVT UL44) and gK (HVT UL53). One of these, gD, has been shown to be dispensable in MDV (Anderson et al., 1998 ).

In herpesviruses that infect mammals, gC plays an important role in binding heparan sulphate, an initial step in virus infection (Shieh et al., 1992 ; Spear et al., 1992 ; WuDunn & Spear, 1989 ). In addition, gC binds and inhibits complement C3b, which may be important for immune evasion (Lubinski et al., 1998 ). The infectious laryngotracheitis virus (ILTV) gC protein lacks a heparan sulphate-binding domain, and heparan sulphate does not appear to play a role in the initial stages of infection by ILTV (Kingsley & Keeler, 1999 ). Comparison of HVT gC with those of PRV and HSV-1, which have mapped heparan sulphate-binding domains, suggests that HVT may also lack a heparan-binding domain (Fig. 2a).



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Fig. 2. Comparison of herpesvirus glycoproteins. (a) Comparison of the region of HSV-1 and PRV gC containing heparan sulphate-binding domains (boxed). These domains do not appear to be conserved in this region of HVT, MDV-1 or MDV-2 strains. (b) Comparison of glycoprotein B dimerization domains (boxed) from HVT, MDV-1, MDV-2 and HSV-1.

 
gB of other herpesviruses functions in the fusion of the virus particle membrane with the host cell. Comparison of HVT glycoproteins with those from both MDV and HSV-1 (see Table 2) reveals that gB exhibits the greatest level of conservation among the glycoproteins. In particular, domains involved in HSV-1 gB oligomerization (Claesson-Welsh & Spear, 1986 ; Sarmiento et al., 1979 ), which is important for fusion with host cells (Laquerre et al., 1996 ), may be conserved in HVT gB (Fig. 2b).

Additional predicted membrane-associated proteins
Homologues of HSV-1 genes UL20, UL34, UL43 and UL45 are found in HVT. Based upon sequence similarity, it is predicted that the HVT genes UL20, UL34 and UL43 are orthologues of the respective genes found in HSV-1 (McGeoch et al., 1988 ; Baines et al., 1991 ). In contrast to these predicted membrane proteins, the HVT gene UL45 shows little similarity to HSV-1 UL45 (Table 2) but strong similarity to MDV UL45 (Lee et al., 2000 ; Izumiya et al., 1998 ) and EqHV-1 gene 15 (Telford et al., 1992 ) (not shown).

Regulatory proteins
The HVT genome is predicted to encode two protein kinases, UL13 and US3; both gene products contain motifs associated with kinase activity of other homologous proteins (Ng et al., 1994 ; Purves & Roizman, 1992 ; Purves et al., 1992 ).

The HVT UL41 gene product is similar to other herpesvirus proteins that are responsible for inhibition of host protein synthesis (Kwong & Frenkel, 1989 ). Inhibition appears to occur by UL41 promoting the degradation of host mRNA (Pak et al., 1995 ). PSI-BLAST analysis revealed weak similarity of the HVT UL41 gene product to the RAD13 and FEN-1 endonucleases (not shown). However, it is not certain whether UL41 homologues function directly as nucleases or modulate a cellular ribonuclease (Elgadi et al., 1999 ; Zelus et al., 1996 ).

When compared with HSV-1, HVT lacks the ICP0 gene, which plays a role in modulating HSV-1 mRNA levels. In contrast, the MDV-1 RLORF1 gene product displays weak similarity to HSV-2 ICP0 (Lee et al., 2000 ). The HVT US1 gene is much smaller than those homologues found in other herpesviruses. Three of the major gene-regulatory proteins identified in HSV-1, VP16 (UL48), ICP27 (UL54) and ICP4, are encoded in the HVT genome. The HSV-1 proteins encoded by the genes UL46, UL47 and UL49 all interact with VP16 (Elliott et al., 1995 ; Zhang et al., 1991 ). HSV-1 VP16 is responsible for induction of the early genes of the virus (Campbell et al., 1984 ; Dalrymple et al., 1985 ) and the HSV-1 UL46 and UL47 gene products are believed to modulate VP16 activity (Zhang et al., 1991 ). The significant difference between HVT VP16 and HSV-1 VP16 is the absence of a carboxy-terminal acid-rich region that functions in gene activation. This region is also absent from the MDV-1 VP16 homologue (Yanagida et al., 1993 ). The domain of HSV-1 VP16 involved in interaction with the HSV-1 UL49 gene product (VP22) has been mapped to this acid-rich region (Elliott et al., 1995 ), suggesting that HVT VP16 and HVT VP22 may not interact. However, it should be noted that HVT VP16 displays more similarity to ORF10 of VZV, which contains an activation domain at the amino terminus of the protein (Kopá<cek et al., 1997 ).

HSV-1 ICP27 controls gene expression at the level of mRNA processing (Sandri-Goldin, 1998 ). HVT ICP27 contains multiple nuclear-localization signals consistent with the nuclear role of other herpesvirus UL54 products (not shown). Furthermore, the carboxy-terminal zinc finger domain (CCCH class) of HSV-1 ICP27 has been implicated in its regulatory functions (Zhi et al., 1999 ), and this domain is also conserved in HVT ICP27 (Fig. 3a). These characteristics suggest that HVT ICP27 plays a role in nuclear post-transcriptional gene regulation.



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Fig. 3. (a) UL54 carboxy-terminal zinc finger domain (CCCH type) (boxed). (b) Comparison of the amino termini of the HVT, MDV-1 and MDV-2 ‘long’ ICP4 ORFs.

 
The HVT ICP4 gene encodes a protein that contains an additional 809 amino acids at the amino terminus when compared with HSV-1 and other herpesvirus ICP4 proteins. The carboxy-terminal portion of the HVT ICP4 protein is similar to those of other herpesviruses. Hence, it is likely that this HVT protein is responsible for modulating the expression of both early and late genes during infection (Roizman & Sears, 1996 ). The amino-terminal extension contains no significant region of similarity to any known protein and its function remains unclear. This potential amino-terminal extension can be also found in MDV-1 and MDV-2. Alignment of amino acid sequences immediately downstream of the initiation methionine of ‘long’ ICP4 ORFs reveals a high level of similarity (Fig. 3b). Synthesis of the relevant ‘long’ ICP4 protein products remains to be verified, although data are available on the presence of transcripts spanning both the MDV-1 and HVT ‘long’ ICP4 ORFs (Cantello et al., 1997 ; V. Zelník, unpublished results).

Novel ORFs
In addition to the conserved genes shared by other alphaherpesviruses, HVT contains at least nine ORFs that are not common to all herpesviruses. Homologues of seven of these genes are readily identified in MDV-1 and MDV-2. One of the genes described below, specific for HVT and encoded by RSORF1 (ORF 65), is a homologue of a cellular gene that regulates apoptosis.

HVT LORF1.
The product of this ORF appears to be encoded by a spliced transcript, with a homologous gene at a similar position in MDV. The homologous MDV-1 gene is transcribed and undergoes splicing to yield a single processed transcript (Becker et al., 1994 ). Database analysis did not reveal homologues in any herpesviruses other than MDV-1 and MDV-2 (LORF2 and ORF759, respectively). However, these protein sequences show similarity to gene products found in some avian adenoviruses, including fowl adenovirus type 8 (Fig. 4). The HVT product of the spliced LORF1 mRNA shows similarity to proteins involved in lipid metabolism and transport, including triacylglycerol lipase, phospholipase A1, lipoprotein lipase and insect yolk proteins. The active site of lipases contains a catalytic triad of amino acids responsible for hydrolysis of lipids (Dodson et al., 1992 ). Inspection of the HVT LORF1 alignment revealed that only one of the three residues, corresponding to the HVT LORF1 serine residue at position 198, is conserved between lipases and the HVT gene product. In addition, similarly to lipid-associating proteins, the HVT-encoded protein contains one predicted trans-membrane domain, between amino acids 21 and 35. This combined analysis leads us to speculate that HVT LORF1 may encode a membrane protein that either binds or metabolizes lipids.



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Fig. 4. Herpesvirus lipase homologues. Comparison of HVT LORF1, MDV-1 LORF2, MDV-2 ORF759, Musca yolk protein 2, chicken lipase (LIPL CHICK), human triacylglycerol lipase (LIPL HUMAN TAGL) and fowl adenovirus lipase homologue (FA8). The line labelled ‘Lipase active site’ indicates the residues found at the positions indicated that form part of the catalytic triad in functional lipases.

 
HVT LORF2.
This ORF encodes a protein with significant similarity to MDV-1 LORF3 and MDV-2 ORF4. HVT LORF2 contains a serine/arginine-rich domain in the carboxy-terminal coding region (amino acids 175–400) similar to that found in many RNA-binding proteins (Blencowe et al., 1999 ). Also, a shorter stretch of amino acids (125–250) exhibits similarity to eukaryotic steroid co-activator proteins. These factors modulate the ability of steroid receptors to regulate transcription (Leo & Chen, 2000 ). Taken together, these similarities suggest that the product of the HVT LORF2 gene may regulate either transcription or processing of mRNAs.

HVT LORF4.
Homologues of this gene are found in MDV-1 (MDV LORF10), BHV-1 (circ protein, IE R1.5), EqHV-1 (KyA strain ORF1) and VZV (ORF2), but not in HSV-1 or MDV-2. MOTIF analysis predicted that HVT LORF4 encodes an amino-terminally myristoylated protein. Myristoylation would be predicted to localize the encoded protein to either intracellular membranes or the plasma membrane (McCabe & Berthiaume, 1999 ). This potential amino-terminal myristoylation site is conserved in BHV-1 and EqHV-1 but absent from VZV.

HVT LORF3, LORF5 and LORF6.
The only clear homologues to these putative ORFs are found in MDV-1 and MDV-2 (Table 2). MOTIF analysis yielded no similar motifs or domains currently deposited in the public databases. The coding region of HVT LORF5 exhibits 47% identity to HVT LORF3, suggesting a specific duplication of this gene in HVT during the evolution of these herpesviruses.

HVT RSORF1 (NR-13 homologue).
The product of this ORF is encoded by a spliced mRNA and displays significant similarity to the quail anti-apoptotic gene NR-13 (Fig. 5) (Gillet et al., 1995 ; Mangeney et al., 1996 ). The MDV-1 genome does not contain a relative of this gene. BLAST and MOTIF analysis showed clear similarity of the encoded product to other proteins that regulate apoptosis, including Bcl-2 and Bcl-x (reviewed by Chinnaiyan, 1999 ). The BH1 and BH2 domains can both be identified in the encoded HVT NR-13 protein sequence. These two domains are essential for the anti-apoptotic properties of members of the Bcl family (Reed, 1996 ). In addition, a region of partial similarity to the BH3 domain of apoptosis-regulating proteins is also found. In contrast to BH1 and BH2, the BH3 domain typically promotes apoptosis (Inohara et al., 1997 ). Finally, the carboxy terminus of the HVT NR-13 protein is predicted to contain a membrane-association domain. This is consistent with the membrane attachment of other Bcl-like proteins (Chen-Levy et al., 1989 ; Hockenbery et al., 1990 ; Gillet et al., 1995 ). These observations suggest that the HVT NR-13 gene encodes a membrane-associated protein that might regulate apoptosis. The presence of this gene product may explain the ability of HVT, but not MDV-1 strains, to inhibit apoptosis in the DT40 B cell line (Ewert & Duhadaway, 1999 ).



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Fig. 5. Comparison of the HVT NR-13 gene product (RSORF1) with quail NR-13 and chicken Bcl-x proteins. The various domains indicated are boxed.

 
Repeat region ORFs absent from HVT
Although an ORF encoding a homologue of the MDV-1 pp38 protein could be identified, the HVT pp38 (RLORF1) is truncated relative to MDV-1 pp38 (Smith et al., 1995 ). An apparent deletion of genomic material relative to MDV-1 has occurred within the long repeat regions.

In addition to the truncation of pp38, HVT lacks several genes identified in the repeat regions of MDV. Of potential importance is the absence of genes implicated in oncogenesis. The meq gene, which belongs to the fos/jun class of transcriptional activators and may play a key role in MDV-induced T-cell lymphomas (Jones et al., 1992 ; Liu et al., 1998 ; Xie et al., 1996 ), is clearly absent from HVT. In addition, at least nine other ORFs identified in MDV GA strain (Lee et al., 2000 ) are also absent from HVT. These include the vIL-8 homologue, which has been speculated to recruit cells for infection or to diminish IL-8-mediated responses (Lee et al., 2000 ). However, other genes identified within RL and RS may also be important for MDV-1 oncogenesis.

Evolution of HVT
Individual genes from the HVT UL region and other herpesviruses were compared by using CLUSTAL W (Higgins & Sharp, 1988 ) to align the protein products and to generate trees. The genes analysed included the small (UL40) and large (UL39) subunits of ribonucleotide reductase, exonuclease (UL12), thymidine kinase (UL23), dUTPase (UL50), DNA polymerase (UL30), UL8, UL42 and UL5. Fig. 6 shows one such tree generated by using the DNA polymerase genes. The results for DNA polymerase were typical of those obtained for other HVT genes. In this analysis, we included the mouse DNA polymerase {delta} as an outgroup. The alpha-, beta- and gammaherpesvirus DNA polymerase genes each formed individual phyletic groups. An initial branching event led to the alpha- and beta- plus gammaherpesvirus families, with subsequent divergence of the beta- and gammaherpesviruses. Within the alphaherpesviruses, ILTV appears to have arisen earliest. Subsequently, a branch leading to HVT and MDV-1 and -2 splits off, followed by clustering of the remaining mammalian herpesvirus DNA polymerases. The combined results support the evolution of HVT and MDV early in the alphaherpesvirus radiation, as has been noted in other analyses of herpesvirus evolution (Karlin et al., 1994 ; McGeoch & Cook, 1994 ; McGeoch et al., 1995 ; McGeoch & Davison, 1999 ).



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Fig. 6. Rooted phylogenetic tree of herpesvirus DNA polymerase proteins (including HVT UL30). Mouse DNA polymerase {delta} was included in this analysis as an outgroup to provide the root. Analysis was conducted using the DIALIGN 2 program. Viruses included in the analysis and not abbreviated elsewhere were human herpesvirus 8 (HHV-8), Epstein–Barr virus (EBV), human herpesvirus 6 (HHV-6) and human cytomegalovirus (CMV).

 
The sequence data provided here will allow additional understanding of the evolution of the herpesvirus family and the analysis of coding content helps to formulate additional hypotheses of gene functions. Finally, the comparison of HVT with MDV-1 reveals differences that may be significant in the differing pathology of these viruses.


   Acknowledgments
 
We would like to thank Dr Akiko Tanaka for providing the HVT BamHI fragments, Dr Robin Morgan for her help and encouragement and Dr Mark Parcells for critically reading the manuscript. Funding from the University of Delaware Center for Agricultural Biotechnology, the Delaware Biotechnology Institute and partially grant no. 2/6075/20 of the VEGA Grant Agency of the Slovak Republic supported this work. This is publication number 1686 from the Delaware Agricultural Experiment Station.


   Footnotes
 
Sequence data reported in this paper will appear in GenBank under the accession number AF282130.


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Received 9 October 2000; accepted 7 December 2000.