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1 Department of Nutrition & Health Science, Faculty of Human Environmental Science, Fukuoka Woman's University, Fukuoka 813-8529, Japan
2 Division of Dermatology, Sakura Hospital, Faculty of Medicine, Toho University, Sakura 285-8741, Japan
3 Division of Human Molecular Genetics, Center for Genetic Information, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan
Correspondence
Kenichi Umene
umene{at}fwu.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AB329728–AB329753.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). HSV infections occur most commonly on the genitalia and oral mucosa, whilst lesions can occur at almost all visceral and mucocutaneous sites.
The HSV-1 genome is a 152 kb, linear, duplex DNA molecule composed of two covalently linked components, L and S (Fig. 1) (McGeoch et al., 1988). The short sequence a is repeated directly at both ends of the genome and is present in inverse orientation at the L–S junction (Fig. 1) (Davison & Wilkie, 1981; McGeoch et al., 1988; Mocarski & Roizman, 1981, 1982; Umene, 1998). One to several copies of the a sequence are present at the end of the L component and at the L–S junction, but only one copy is present at the end of the S component. The a sequence encodes several cis-acting sites involved in (i) cleavage of the unit-length HSV-1 DNA from concatemeric forms generated by rolling-circle replication and encapsidation of the excised molecule (cleavage/packaging system) (Adelman et al., 2001; Baines & Weller, 2005; Deiss et al., 1986; Hodge & Stow, 2001; Mocarski & Roizman, 1982), (ii) the circularization of viral DNA after infection (Baines & Weller, 2005; Mocarski & Roizman, 1982), (iii) recombination relating to the L–S inversion (Chou & Roizman, 1985; Mocarski et al., 1980; Smiley et al., 1981, 1990) and (iv) the expression of a mRNA extending from the a sequence (Chou & Roizman, 1986; Martin & Weber, 1998; Sarisky & Weber, 1994). The a sequence contains unique (U) and directly repeated (DR) sequence elements, and is made up of DR1, Ub, a DR2 array, a DR4 stretch and Uc (Fig. 2).
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). Linear unit-length HSV-1 DNA present in the viral particle is generated by cleavage at a specific site in DR1 shared by two neighbouring a sequences (Fig. 1c) (Baines & Weller, 2005; Mocarski & Roizman, 1982). The pac1 (present in Ub) and pac2 (present in Uc) sequences, regions of close homology among the a sequences of diverse herpesvirus genomes, are candidates for signals that direct the site-specific cleavage (Fig. 2a) (Baines & Weller, 2005; Deiss et al., 1986; Hodge & Stow, 2001; Smiley et al., 1990; Varmuza & Smiley, 1985). The DR2 array consists of a number of DR2 elements, and the copy number of DR2 elements is variable among HSV-1 isolates (Davison & Wilkie, 1981; Mocarski & Roizman, 1981; Umene, 1991). The length of the DR2 element reported so far is 11 or 12 bp. DR4 of the 37 bp sequence that contains one copy of DR2 was originally defined in strain F, in which the DR4 sequence was reiterated three times (Mocarski & Roizman, 1981). Sequences corresponding to DR4 of strain F were not reiterated in other HSV-1 strains, and DR4 of strain F could be regarded as one copy of the DR2 element plus a DR4 stretch. Several types of DR4 stretch have been identified (Davison & Wilkie, 1981; Mocarski & Roizman, 1981; Umene, 1991, 1993, 1998; Varmuza & Smiley, 1985).
Present-day herpesviruses are regarded as descendants of a common ancestor supposed to have existed in the past and are assumed to have diversified through three processes: (i) acquisition of other DNA sequences, (ii) DNA rearrangements and (iii) nucleotide substitutions (Bowden & McGeoch, 2006; Bowden et al., 2004; Davison & McGeoch, 1995; Gentry et al., 1988; McGeoch & Cook, 1994; McGeoch et al., 1995; Norberg et al., 2004; Umene & Sakaoka, 1999; Umene, 1998, 1999). Genetic variation of a virus enables the differentiation and classification of isolates, thus paving the way for studies concerning (i) evolution, (ii) mode of transmission and (iii) the relationship between isolate-specific genomic characteristics, biological characteristics and clinical manifestations (Bowden & McGeoch, 2006; Bowden et al., 2004, 2006; Nahmias et al., 2006; Norberg et al., 2004; Sakaoka et al., 1994; Umene, 1998; Umene & Kawana, 2003; Umene & Sakaoka, 1999; Umene et al., 2007). Variations in restriction endonuclease cleavage patterns between HSV-1 isolates have been analysed and a number of restriction fragment length polymorphisms (RFLPs) detected (Norberg et al., 2006; Sakaoka et al., 1994; Umene & Yoshida, 1993). An RFLP is usually due to a nucleotide substitution that causes the gain or loss of a restriction endonuclease cleavage site. HSV-1 isolates have been classified into genotypes based on the condition of a set of RFLPs distributed widely over the whole genome of HSV-1 (Umene & Yoshida, 1993). In the present study, nucleotide sequences of the a sequence of a number of HSV-1 isolates were determined and variations in the nucleotide sequence of the a sequence were compared with the genotypes and RFLPs of these HSV-1 isolates. A close connection of the DR4 stretch of the a sequence with the state of RFLPs was inferred from statistical analyses; thus, the DR4 stretch was assumed to indicate the derivation of HSV-1 isolates. New variations on the DR2 array were discovered and the generation of these variations could largely be explained by supposing recombination events entailing DR2 elements; hence, the tendency for DR2 elements to be involved in recombination is suggested.
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METHODS |
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and 2) (Umene & Yoshida, 1993). The nucleotide sequences of the a sequence of four HSV-1 isolates (K52, K57, Ty154 and K41) have been reported in previous studies (Umene, 1991, 1993), and those of the other 26 HSV-1 isolates were determined in the present study. HSV-1 isolates prefixed with ‘Ty’ were separated in Tokyo and those with ‘K’ in Osaka.
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; Umene & Yoshida, 1993).
Molecular cloning and sequencing.
DraI DNA fragments or BamHI DNA fragments containing the a sequence of HSV-1 isolates were cloned into plasmid DNA (Sambrook & Russell, 2001; Umene, 1991). HSV-1 DNA was cleaved with DraI to isolate DraI DNA fragments containing the unit-length a sequence, as the a sequence usually has a unique cleavage site for DraI (Fig. 2b). HSV-1 DNA cleaved with DraI was separated in a 5 % polyacrylamide gel and DraI fragments containing the a sequence were extracted from the gel. An extracted DraI DNA fragment corresponding to the unit-length a sequence was cloned into the SmaI site of plasmid pUC19. HSV-1 DNA cleaved with BamHI was separated in a 1 % agarose gel, and BamHI fragments containing the a sequence were extracted from the gel. An extracted BamHI DNA fragment containing the a sequence was cloned into the BamHI site of plasmid pUC19. The a sequence cloned into the plasmid was sequenced using an automated sequencer with universal primers and custom-made primers.
The nucleotide sequences of the a sequences of 26 HSV-1 isolates were submitted to GenBank/EMBL/DDBJ under accession numbers AB329728 [GenBank] –AB329753 [GenBank] as follows: isolate Ty89, AB329728 [GenBank] ; isolate K79, AB329729 [GenBank] ; isolate K93, AB329730 [GenBank] ; isolate K81, AB329731 [GenBank] ; isolate Ty44, AB329732 [GenBank] ; isolate Ty106, AB329733 [GenBank] ; isolate Ty25, AB329734 [GenBank] ; isolate K78, AB329735 [GenBank] ; isolate Ty127, AB329736 [GenBank] ; isolate K59, AB329737 [GenBank] ; isolate K90, AB329738 [GenBank] ; isolate Ty43, AB329739 [GenBank] ; isolate K84, AB329740 [GenBank] ; isolate Ty161, AB329741 [GenBank] ; isolate Ty1, AB329742 [GenBank] ; isolate K60, AB329743 [GenBank] ; isolate Ty179, AB329744 [GenBank] ; isolate Ty3, AB329745 [GenBank] ; isolate K95, AB329746 [GenBank] ; isolate K76, AB329747 [GenBank] ; isolate K64, AB329748 [GenBank] ; isolate K56, AB329749 [GenBank] ; isolate Ty109, AB329750 [GenBank] ; isolate K26, AB329751 [GenBank] ; isolate Ty21, AB329752 [GenBank] ; isolate Ty128, AB329753 [GenBank] .
Statistical analysis.
The data files analysed in this study were created and edited using Microsoft Access 2000 on the basis of the relational database model (Mata-Toledo & Cushman, 2000). The statistical software JMP6 (SAS Institute Inc.) was used for statistical analyses (Lehman et al., 2005; Sall et al., 2005).
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RESULTS |
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) (Davison & Wilkie, 1981; Mocarski & Roizman, 1981; Umene, 1998). A recognition sequence of DraI, TTTAAA, is usually present in Ub of the a sequence (Fig. 2b); hence, the unit-length a sequence, of which both ends are generated by cleavage with DraI, was produced. This unit-length a sequence was cloned into the SmaI site of plasmid pUC19 and nucleotide sequences were determined (Sambrook & Russell, 2001). Attempts to clone the unit-length a sequence of several HSV-1 isolates (e.g. Ty127, K90 and Ty179) using DraI were unsuccessful, as the DraI site was absent from the a sequence (Table 2). As an alternative method for cloning the a sequence, a BamHI fragment from the L–S junction of HSV-1 DNA (containing the a sequence) was cloned into the BamHI site of pUC19 and nucleotide sequences were determined.
Comparison of nucleotide sequences of the a sequence among HSV-1 isolates
The HSV-1 isolates have been classified previously into genotypes based on the state of RFLPs located at various positions along the genome (Table 1) (Umene & Yoshida, 1993). Two genotypes (F1 and F35) were predominant, and genotype F1 was the most common of these. Thirty HSV-1 isolates of 16 genotypes were analysed in this study (Tables 1–6).
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) (Deiss et al., 1986; Hodge & Stow, 2001): (i) the pac1 proximal GC element; (ii) the pac1 T element, which is flanked by sequences CnGm (e.g. C5G6) from the pac1 proximal GC element and Gk (e.g. G8) from the pac1 distal GC element, and has been proposed to represent the functional pac1 signal; and (iii) the pac1 distal GC element. The length of Ub analysed in the present study was 76–79 bp, depending on the copy number (C0–2) of the C residue situated in the region corresponding to nt 1 and 2 of Ub of isolate Ty89 and that of the G residue (G8–10) at nt 51–60 (Fig. 2b, Table 2). The region of the pac1 T element and flanking sequences of C5G6 and G8–10 (corresponding to nt 32–60 of Ub of isolate Ty89) was preserved in the a sequence of the 30 HSV-1 isolates analysed in the present study (Fig. 2b, Table 2). The DraI recognition sequence of TTTAAA (placed in the region corresponding to nt 13–18 of Ub of isolate Ty89) was changed to TTAAAA in the isolates Ty127, K90 and Ty179 (Fig. 2b, Table 2).
Uc.
The following conserved pac2 motifs are thought to be situated in Uc (Fig. 2a) (Deiss et al., 1986; Hodge & Stow, 2001): (i) the pac2 consensus (CGCCGCG) next to the DR4 stretch; (ii) the pac2 T element; and (iii) the pac2 GC element that adjoins the DR1. The length of Uc of the HSV-1 isolates analysed was constant at 58 bp (Table 3). Residue C, corresponding to nt 6 of Uc of isolate Ty89 (equivalent to nt 6 of the pac2 consensus), was changed to T in isolates of genotype F35 (Fig. 2, Table 3). Nucleotide sequences corresponding to the pac2 T element were unchanged.
DR1.
The nucleotide sequences of DR1 in 26/30 isolates analysed (87 %) were the same (major type) (Tables 4 and 5). Nucleotide substitution was revealed in the DR1 of isolates K93, Ty44 and Ty1 (Table 4). The DR1 of isolates Ty44 (18 bp) and K76 (17 bp) was shorter than the major type of DR1 (20 bp).
DR2 array.
The DR2 array consists of directly repeated DR2 (also called reiteration I) elements, and the DR2 elements reported previously were 11 or 12 bp in the form CGCTCCT(C)n (n=4 or 5) (Davison & Wilkie, 1981; Mocarski & Roizman, 1981; Umene, 1991, 1993, 1998; Varmuza & Smiley, 1985). The DR2 array is thought to be composed usually of one kind of DR2 element, whilst that of strain KOS consists of DR2 elements of 11 and 12 bp (Varmuza & Smiley, 1985) and that of isolate Ty154 had a mutated DR2 element, CGCTTCT(C)4 (corresponding to B11 in Table 4), as well as the standard DR2 elements of 11 bp (Umene, 1991). The DR2 array in 23/30 isolates analysed (77 %) in the present study consisted of one kind of DR2 element of 11 or 12 bp of the form CGCTCCT(C)n (n=4 or 5) (Table 6). DR2 elements of 9 or 13 bp of CGCTCCT(C)n (n=2 or 6) (corresponding to A9 and A13 in Table 4) were discovered in isolates Ty44, Ty161 and K76. Each DR2 array of isolates Ty44, Ty3, K76 and K56 was made up of DR2 elements of different lengths (Table 6). A mutated DR2 element of CGCTCCG(C)5 of 12 bp (corresponding to C12 in Table 4) was found in isolate K90. Elements of CGCTCCTGCGG(C)n (n=1 or 3) (corresponding to D12 and D14 in Table 4) that were the same as a part of the DR4 stretch (DR4t2) of isolate K56 were detected on the DR2 array of isolate K56 (Tables 4 and 6).
DR4 stretch.
Three kinds of DR4 stretch of DR4t (renamed DR4t1 in the present study), DR4n (renamed DR4n1 in the present study) and DR4n2 have been reported previously (Davison & Wilkie, 1981; Mocarski & Roizman, 1981; Umene, 1991, 1993, 1998; Varmuza & Smiley, 1985). Six other kinds of DR4 stretch of DR4j1 (Ty44, Ty106, Ty25 and K78), DR4j2 (K76, K64, K26 and Ty128), DR4t2 (Ty161 and K56), DR4t3 (Ty127 and K59), DR4t4 (Ty179) and DR4t5 (K90) were revealed in the present study (Tables 4 and 5). The DR4 stretches so far identified could be classified into three groups, the DR4n group (DR4n1 and DR4n2), DR4j group (DR4j1 and DR4j2) and DR4t group (DR4t1, DR4t2, DR4t3, DR4t4 and DR4t5) (Table 5).
Statistical analyses of the connection between RFLPs and nucleotide sequences of the a sequence
A method for multivariate statistical analysis, discriminant analysis, can be used to determine the group to which an individual belongs, based on the characteristics of that individual (Huberty, 1994; Sharma, 1996; Umene et al., 2007). Variables for the characteristics of individuals are called predictor (or independent) variables, and a group membership variable is referred to as a criterion (or dependent) variable. Suppose we have measurements of the characteristics (e.g. 20 RFLPs) of each sample of individuals (e.g. 30 HSV-1 isolates) and we know that each individual belongs to one group (e.g. classification based on nucleotide sequences of the a sequence); discriminant analysis attempts to maximize the probability of correct allocation of individuals (e.g. 30 HSV-1 isolates) based on predictor variables (e.g. 20 RFLPs). The accuracy of classification reflects the association between predicted and actual group membership.
The DR2 arrays (corresponding to the criterion variable) analysed in the present study were classified into three groups: (i) group 1, composed of DR2 elements of 11 bp (16 isolates); (ii) group 2, composed of DR2 elements of 12 bp (nine isolates); and (iii) group 3, other than groups 1 and 2 (five isolates) (Table 6). Predictor variables were 20 RFLPs, which are distributed widely throughout HSV-1 DNA (Table 1). Discriminant analysis was conducted using JMP6 statistical discovery software (Lehman et al., 2005; Sall et al., 2005). The ‘correct classification rate’ gives the percentage of grouped cases correctly classified and that of the DR2 array was 93 % (28/30). The classification results show how well group membership (coming under the criterion variable) can be predicted using RFLPs (coming under predictor variables). Misclassified cases were K84 (genotype F17) and Ty44 (genotype F35). DR2 arrays of two isolates of genotype F17 (isolates K84 and Ty161) were classified into different groups: the DR2 array of isolate K84 was group 1 and that of isolate Ty161 was group 3 (Table 6); therefore, at least one of two isolates of genotype F17 had to be misclassified. The DR2 arrays of four isolates of genotype F35 (Ty44, Ty106, Ty25 and K78) were classified into two groups: the DR2 array of three isolates (Ty106, Ty25 and K78) was group 1 and that of isolate Ty44 was group 3, indicating that at least one of four isolates of genotype F35 had to be misclassified. Except for two cases of inevitable misclassification (isolates K84 and Ty44), HSV-1 isolates were correctly classified into groups of the DR2 array on the basis of the state of RFLPs, suggesting a connection between variation in the DR2 array and the diversification of HSV-1 isolates.
The DR4 stretches (corresponding to the criterion variable) analysed in the present study were classified into three groups: DR4n, DR4j and DR4t (Table 5). Predictor variables were 20 RFLPs and discriminant analysis was conducted using JMP6. The ‘correct classification rate’ of the DR4 stretch was 100 % (30/30), indicating a close relationship between the DR4 stretch and the differentiation of HSV-1 isolates.
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DISCUSSION |
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; Baines & Weller, 2005; Davison & Wilkie, 1981; Mocarski & Roizman, 1981; Mocarski et al., 1985; Umene, 1991, 1993, 1998; Varmuza & Smiley, 1985). The nucleotide sequences of the a sequence determined in the present study conformed to the consensus structure, whilst the length of Ub of several HSV-1 isolates was 79 bp (Table 2); therefore, added significance was given to the consensus structure, and the length of Ub of the consensus structure was required to be revised from 64–78 bp to 64–79 bp.
Cleavage event in the a sequence generating authentic termini of HSV-1 DNA
The sequence-specific cleavage event at a site on DR1 (generating authentic HSV-1 genomic termini) is assumed to be promoted by pac1 and pac2 elements, located in Ub and Uc, respectively (Fig. 2a) (Adelman et al., 2001; Baines & Weller, 2005; Deiss et al., 1986; Hodge & Stow, 2001; Varmuza & Smiley, 1985). The cleavage reaction is presumed to involve two site-specific breaks at defined distances from the pac1 and pac2 signals (Figs 1 and 2). Conserved pac1 and pac2 motifs were preserved in the a sequences analysed in the present study (Tables 2 and 3); hence, the importance of pac1 and pac2 elements for functioning of the a sequence was confirmed.
Nt 18–20 on DR1 of 20 bp become termini of unit-length linear HSV-1 DNA after cleavage in DR1 of concatemeric DNA, generating one partial DR1 copy of 18.5 bp [with a single nucleotide (G) extended 3'] of the L-component terminus and another partial DR1 copy of 1.5 bp [with a single nucleotide (C) extended 3'] of the S-component terminus (Fig. 1) (Mocarski & Roizman, 1982). Upon HSV-1 infection of a cell, partial copies of DR1 at the L (18.5 bp) and S (1.5 bp) termini of a linear HSV-1 DNA are conjoined in an intact DR1 of 20 bp, yielding a monomeric circular DNA molecule. Variations of nt 18–20 of DR1 were revealed in three isolates of K93, Ty44 and K76, whilst variation of nucleotides other than nt 18–20 was found in one isolate of Ty1 (Tables 4 and 5); thus, nt 18–20 appeared to be more liable to mutation than other nucleotides. Cleavage and joining events, which take place frequently at nt 18–20 during HSV-1 DNA replication, were assumed to be related to the occurrence of mutation around nt 18–20, probably through the degradation of DNA termini followed by repair (MacLean et al., 1991).
Association of the DR2 element with the generation of DNA variation
Almost all DR2 elements had the standard structure of CGCTCCT(C)n and most DR2 elements were CGCTCCT(C)n (n=4 or 5) (A11 or A12; Tables 4 and 6) (Davison & Wilkie, 1981; Mocarski & Roizman, 1981; Umene, 1991, 1998; Varmuza & Smiley, 1985). The DR2 arrays known so far are usually made up of one kind of DR2 element of A11 or A12. Therefore, a mechanism is presumed to work to maintain the DR2 array with one kind of DR2 element of A11 or A12, probably by removing DR2 elements other than A11 or A12 and equalizing the DR2 element in a DR2 array (Umene, 1991, 1998).
The DR2 array of isolate K56 contained DR2 elements of CGCTCCTGCGG(C)n (n=1 and 3) (D12 and D14), which differed from the standard DR2 element at three residues (indicated by underlining) (Tables 4 and 6). A sequence the same as that of D14 was present on a part of the DR4 stretch [nt 1–14 of the DR4 stretch (DR4t2) of isolate K56]. In isolate K56, nt 7 of the DR4 stretch (DR4t2) was T and that of the DR4 stretch other than DR4t2 was C, whilst nt 7 of the standard DR2 element was T, indicating that DR4t2 was more similar to the standard DR2 element than were DR4t stretches other than DR4t2. A recombination event between the standard DR2 element and DR4 stretch, which involved homologies of DNA sequences, was assumed to have occurred in an ancestor of isolate K56, incorporating part of the DR4 stretch into the DR2 array as an element of DR2, such as D12 or D14.
The array of six copies of a 24 bp sequence (corresponding to another direct repeat, DR3.5) was first identified in the a sequence of strain USA-8 (Davison & Wilkie, 1981), and a 23 bp sequence reiterated twice in the a sequence of strain Justin was later identified as DR3.5 (Mocarski et al., 1985). DR3.5 can be regarded as a 13 bp sequence of GTCTGTGGGTGGG plus the DR2 element (11 bp in strain USA-8, 10 bp in strain Justin). DR4 of the 37 bp sequence, which was originally identified in the a sequence of strain F and had the structure of DR2 plus the DR4 stretch, was reiterated three times (Mocarski & Roizman, 1981). Reiteration of a subfragment of the a sequence (such as the DR2 array, DR3.5 and DR4) was associated with the presence of the DR2 element as part of the subfragment. Therefore, the DR2 element was assumed to have the potential to promote recombination, which gives rise to (i) the reiteration of a subfragment of the a sequence, (ii) incorporation of part of the DR4 stretch into the DR2 array (e.g. D12 and D14 of isolate K56), (iii) variations in the length of DR2 elements (e.g. A9, A11, A12 and A13) and (iv) variation of the copy number of the DR2 element in a DR2 array.
Grouping of DR4 stretches
DR4 stretches were classified into three groups, DR4n, DR4j and DR4t (Tables 4 and 5). The three groups differed in the region corresponding to nt 1–16 of DR4n1 (DR2-side region), whilst the other regions corresponding to nt 17–35 of DR4n1 (Uc-side region) were mostly the same (Table 4). The structure of DR4n2 could be generated from DR4n1 by removing the region corresponding to nt 1–7 or 5–11 of DR4n1. A difference of 1 bp in the length of the DR2-side region was seen between DR4j1 (CCG) and DR4j2 (CG). Sequences of DR4t2 and DR4t3 could be made from DR4t1 by nucleotide substitution at nt 7 (from C to T) and 22 (from C to A) of DR4t1, respectively. The structures of DR4t4 and DR4t5 could be formed from DR4t3 by duplication of nt 1–14 and by removal of nt 7–10 of DR4t3, respectively. DR4 stretches belonging to the same group were closely related, whilst DR4 stretches of different groups were remarkably dissimilar. Variations in the DR2-side region of the DR4 stretch responsible for the classification of DR4 stretches into three groups were presumed to be generated by the occurrence of DNA rearrangement with DNA recombination during the diversification of HSV-1 isolates, and to have been preserved in isolates of each HSV-1 lineage.
Variations on the a sequence during evolution of HSV-1
HSV-1 isolates were classified into genotypes defined on the basis of the state of a set of 20 RFLPs (Table 1) (Umene & Yoshida, 1993), which is supposed to reflect the condition of the HSV-1 isolate in the diversification of HSV-1. HSV-1 isolates classified into the same genotype had a DR4 stretch of the same group (Table 5). The ‘correct classification rate’ of discriminant analysis using the DR4 stretch as a criterion variable and 20 RFLPs as predictor variables was 100 %; therefore, the distribution pattern of variations in the DR4 stretch among HSV-1 isolates conformed to that of the set of 20 RFLPs. The DR2 elements of HSV-1 isolates classified into the same genotype were the same length, except for genotypes F35 and F17 (Table 6). The ‘correct classification rate’ of discriminant analysis using the DR2 array as a criterion variable and 20 RFLPs as predictor variables was 93 %. Thus, the length of the DR2 element seemed to be closely related to the diversification of HSV-1 isolates, albeit to a lesser degree than the DR4 stretch. Some variations on the a sequence were assumed to have been maintained during the diversification of HSV-1 and could be used as markers for tracing the source of HSV-1 isolates.
Bowden et al. (2004) determined the nucleotide sequence of three segments of approximately 2 kb in HSV-1 clinical isolates, and the rate of recombination was estimated to be comparable to that of mutation; hence, the importance of co-infection by genetically distinct strains for HSV-1 epidemiology and evolution was suggested, as recombination requires the co-existence of two viral genomes. A high level of recombination between genetically distinct strains in infection is thought to make virus genealogy a complex, non-tree-like structure, whilst all segments of DNA along the genome are assumed to share the same history in a clonal evolutionary process (Bowden & McGeoch, 2006). Norberg et al. (2004, 2007) performed DNA sequencing of genes encoding glycoproteins E, G and I for clinical HSV-1 isolates: the gene sequence for each glycoprotein was separated into three genetic groups and the phylogenetic classification of the isolates into different genetic groups revealed that recombination is an important feature in the evolution of the HSV-1 genome. The HSV-1 genome contains several tandem repeat (TR) regions, including the DR2 array of the a sequence, and the copy number of TR sequences is prone to vary, probably through recombination (Bowden & McGeoch, 2006; Umene, 1998). The glycoprotein I (gI) gene contains a TR region. The TR region of gI was shown to be polymorphic, varying from two to six or eight blocks, each containing 21 nt, and was assumed to be introduced into the gI gene after the separation of HSV-1 from HSV-2 (Norberg et al., 2004, 2007). No association was found between the number of repeated blocks of the gI gene and classification into phylogenetically separated groups, suggesting that the TR region evolved separately from and faster than the remaining part of the gene. Association of the copy number of DR2 elements with the genotype was not evident in the present study (Table 6), as has been reported in the TR region of the gI gene by Norberg et al. (2004, 2007).
RFLPs of HSV-1 isolates from geographically separate countries have been analysed, and HSV-1 isolates were presumed to have evolved independently in geographically separated human beings (Bowden & McGeoch, 2006; Sakaoka et al., 1994; Umene & Sakaoka, 1997, 1999). DNA sequences from selected genomic regions of HSV-1 isolates from different countries were determined and HSV-1 populations were assumed to be much more highly differentiated than human populations (Bowden et al., 2006). The greater differentiation that exists among populations of HSV-1 than among human populations was explained by the supposition that genetic drift was faster in HSV-1 than in humans. Because of this greater differentiation, HSV-1 variability was suggested to be more informative about processes over short timescales than human genetic data, validating HSV-1 as a system for studying human populations and origins (Bowden et al., 2006). A human population associated with HSV-1 genotype F1 (having the DR4 stretch of group DR4n) is supposed to be different from another human population with HSV-1 genotype F35 (having the DR4 stretch of group DR4j) (Bowden & McGeoch, 2006; Umene & Sakaoka, 1997, 1999). Therefore, the determination of nucleotide sequences of the a sequence is assumed to contribute to the study of human populations.
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ACKNOWLEDGEMENTS |
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Received 21 September 2007;
accepted 9 December 2007.
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