The method is based on the fact that herpesvirus genomes contain highly conserved genes, which are present in all because they code for proteins essential for viral growth. One of the most conserved is the herpesvirus DNA polymerase (DPOL) gene. Investigators aligned the DPOL genes of all herpesviruses for which sequence data were available, in order to identify short blocks of greatest amino acid identity that encode domains essential for DPOL function. This procedure allowed the design of five degenerate primers that were used in a nested PCR -three primers in the first round and two primers in the second round (Figure 1). The region amplified in between the primers displayed a lower degree of conservation. This allowed investigators to assess (1) whether the detected herpesvirus was known or novel, and (2) to which herpesvirus subfamily the novel species could be assigned. In some cases, even a tentative assignment to a virus genus was possible.
The primers are degenerate in their 3' part and nonde-generate in their 5' part. As an example, the sense primer (TGV) of the second round is shown in Figure 2. The degenerate 3' part enables it to bind universally to the DPOL gene of higher vertebrate herpesviruses in the first PCR cycle. The nondegeneracy of the 5' part ensures amplification efficiency in subsequent cycles. The product amplified in the second round has a length of—210-235 bp, depending on the virus. Only HCMV and chimpanzee cytomegalovirus, as well as some herpesviruses of insecti-voral shrews, give rise to significantly larger amplification products of >300bp (Figure 3).
Several variations of the original method were subsequently published, using (1) deoxyinosine-substituted primers, (2) a nondegenerate consensus sequence as 5'-part of the primer (also called consensus-degenerate hybrid oligonucleotide primers (CODEHOPs)), or (3) other conserved primer binding sites in the DPOL gene. Also, targeting of other highly conserved herpesvirus genes such as the glycoprotein B (gB) gene or the terminase gene was attempted. Degenerate consensus primers were also used to amplify a larger number of conserved genes (n = 8) of a novel porcine herpesvirus (porcine lymphotropic herpesvi-rus 3, PLHV-3). These sequence 'islands' were then connected with the help of long-distance PCR to yield a contiguous sequence of —100 kbp (accession AY170316). This is the longest part of a herpesvirus genome of unknown sequence ever determined directly from primary viruspositive organ material solely through PCR (i.e., without virus cultivation).
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