Future assay methods

New approaches to the detection and quantitation of HHV-6 DNA in clinical specimens include non-PCR-based gene amplification or detection methods such as branched DNA (bDNA) hybridization (Urdea et al., 1987) and isothermal amplification (Guatelli et al., 1990; Walker et al., 1992; Ihira et al., 2004).

Fig. 1 Alignment of HHV-6A and HHV-6B. A: Inter-variant alignment (HHV-6A U1102 and HHV-6B Z29). B: Intra-variant alignment (HHV-6B HST and HHV-6B Z29). Shown is a visual overview of the overall genetic conservation between (panel A) and within (panel B) variants of HHV-6, based on available genomic sequences (HHV-6A U1102, HHV-6B Z29 and HHV-6B HST; respectively, GenBank Accession Numbers X83413, AF157706 and AB021506). The alignments are color-coded; dark regions represent areas of greatest sequence divergence and numbers represent the genomic location (in base pairs). Gapped regions are indicated by darkly shaded semicolons (:); these correspond to regions where there are sequence insertions or deletions in one genome, relative to another. It can be readily appreciated that sequence divergence between virus variants (panel A) is much greater than sequence divergence within a single variant (i.e., between different strains of HHV-6B, panel B; note that comparable data are not available for HHV-6A since there is to date only a single genomic sequence for HHV-6A). Global nucleotide sequence identity between variants of HHV-6 is approximately 90% but this is unevenly distributed across the viral genome (panel A). The central region of the HHV-6 genome is highly conserved between virus variants and contains sequence blocks found in other herpesviruses, whereas the terminal direct repeats (TDR) and right end of the unique region of the genome are more divergent both between (panel A) and within (panel B) variants (Dominguez et al., 1999; Isegawa et al., 1999; Mori et al., 2003). The TDR encompasses roughly 8-9 kb of largely non-coding sequence, while the segment of the unique region that is most divergent between variants is only about 72% identical between HHV-6A and HHV-6B, and spans the open reading frames (ORFs) U86-U100 (panel A) (Dominguez et al., 1999; Isegawa et al., 1999; Mori et al., 2003). Among these ORFs, the IE transactivator U89/90 (IE1) and the gQ-encoding ORF U100 are especially divergent while only the adeno-associated virus (AAV) rep-homolog, U94, is conserved (Dominguez et al., 1999; Isegawa et al., 1999; Mori et al., 2003). As panel B shows, the extent of intra-variant divergence is lower within this region, but some striking differences do exist—including the presence of a putative ORF (HN1) that is unique to the HST strain and absent in Z29, as well as significant divergence among viral transactivators (e.g., U89 from HST is only 92% identical to the corresponding gene product from Z29, at the amino acid level; Isegawa et al., 1999). It is important to add that two distinct groups of variant B isolates have been described, of which Z29 and HST form the prototype members; sequences derived from viruses representative of each individual group exhibit a very high level sequence conservation with one another (99% nucleotide identity or greater), which greatly exceeds the identity between viruses from different groups (Isegawa et al., 1999). Methods: the alignments shown were kindly generated by Dr. Vasily Tcherepanov and Dr. Chris Upton of the University of Victoria, BC, Canada, using the base-by-base alignment algorithm (Brodie et al., 2004). This algorithm can be downloaded freely via the WWW (http://athena.bioc.uvic.ca/ ). Alignments were initially generated with ClustalW 1.83.1, using a fast alignment with the following parameters: gap penalty = 3, K-tuple(word) size = 1, number of top diagonals = 5, window size = 5. The alignment from ClustalW was then loaded into Base-by-Base multiple alignment editor (BBB) and adjusted manually.

Microarray technology offers the potential to screen a single clinical specimen for many different pathogens (Wang et al., 2002a; Bryant et al., 2004; Foldes-Papp et al., 2004; Striebel et al., 2004). HHV-6-specific gene sequences will likely be included in these new arrays, yet it will take some time before this approach becomes adopted in the clinic. Practical concerns include sensitivity, cost and the complex data analysis and interpretation.

Biosensors based on quartz crystal microbalance technology (using antibodies to provide specificity) or highly sensitive nucleic acid hybridization assays, as well as living cell-based immunosensors (Cooper et al., 2001; Rider et al., 2003; Vernon et al., 2003) are at early stage of development, and its clinical utility remains unknown.

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