CODEHOP-mediated PCR – A powerful technique for the identification and characterization of viral genomes
© Rose; licensee BioMed Central Ltd. 2005
Received: 08 January 2005
Accepted: 15 March 2005
Published: 15 March 2005
Consensus-Degenerate Hybrid Oligonucleotide Primer (CODEHOP) PCR primers derived from amino acid sequence motifs which are highly conserved between members of a protein family have proven to be highly effective in the identification and characterization of distantly related family members. Here, the use of the CODEHOP strategy to identify novel viruses and obtain sequence information for phylogenetic characterization, gene structure determination and genome analysis is reviewed. While this review describes techniques for the identification of members of the herpesvirus family of DNA viruses, the same methodology and approach is applicable to other virus families.
Only a very small fraction of the vast number of viral species belonging to the different virus families have been identified and characterized to date. The majority of these uncharacterized viral species are found in host organisms which have not been targeted in biomedical, plant or animal research. However, recent reports have noted an increase in the occurrence of viral diseases, not only in humans, but in animals and plants as well. While some of this rise may reflect more effective surveillance techniques, disease outbreaks caused by novel cross-species infections and/or subsequent virus recombination events have occurred . Therefore, the development of tools for the detection of viruses, the characterization of their genomes and the study of their evolution, becomes important, not only for basic scientific study, but also for the protection of public health and the well-being of the plant and animal life that surrounds us.
We have developed a novel technology to identify and characterize distantly related gene sequences based on consensus-degenerate hybrid oligonucleotide primers (CODEHOPs). CODEHOPs are designed from amino acid sequence motifs that are highly conserved within members of a gene family, and are used in PCR amplification to identify unknown related family members. We have developed and implemented a computer program that is accessible over the World Wide Web to facilitate the design of CODEHOPs from a set of related protein sequences . This site is linked to the Block Maker multiple sequence alignment site  on the BLOCKS WWW server  hosted at the Fred Hutchinson Cancer Research Center, Seattle, WA.
We have utilized the CODEHOP technique to develop novel assays to detect previously unknown viral species by targeting sequence motifs within stable housekeeping genes that are evolutionarily conserved between different members of virus families. Using CODEHOPs derived from conserved motifs within retroviral reverse transcriptases, we have previously identifed a diverse family of retroviral elements in the human genome , as well as a novel endogenous pig retrovirus , and a new retrovirus in Talapoin monkeys . We have also developed assays to detect unknown herpesviruses by targeting conserved motifs within herpesvirus DNA polymerases. Using this approach, we have identified fourteen previously unknown DNA polymerase sequences from members of the alpha, beta and gamma subfamilies of herpesviruses , and have discovered three homologs of the Kaposi's sarcoma-associated herpesvirus in macaques [9, 10]. We have also used the CODEHOP technique to clone and characterize the entire DNA polymerase gene from these new viruses  and to obtain sequences for larger regions of viral genomes containing multiple genes, targeting the divergent locus B of macaque rhadinoviruses . The sequence information obtained from the amplified gene and genomic fragments from these studies has allowed informative phylogenetic characterization of the new viral species, and has provided critical information regarding the gene structure and genetic content of these unknown viral genomes.
In this review, the CODEHOP methodology and its utilization in the identification and characterization of novel viral genomes using the herpesvirus family as an example is described. Published CODEHOP assays that we have previously used to identify new herpesviruses are discussed and the latest refined assays and their utility are provided. The use of the CODEHOP methodology for the analysis of larger regions of viral genomes is presented along with the general application of this technology for the identification of viral species and their genes in other virus families. Finally, the software and Web site that we have developed to derive CODEHOP PCR primers from blocks of multiply aligned protein sequences are described.
General CODEHOP Design and PCR Strategy
Conserved amino acid motifs used for CODEHOP design are identified by alignment of related proteins from a targeted gene family using computer programs such as the Clustal W multiple alignment program . Optimal blocks contain 3–4 highly conserved amino acids with restricted codon multiplicity from which the 3' degenerate core is derived; the presence of serines, arginines and leucines are not favored due to the presence of six possible codons for each amino acid. In addition, optimal blocks contain 5 or more conserved amino acids from which the 5' consensus clamp is derived. These blocks of conserved amino acid sequences should be situated in close enough proximity to allow efficient PCR amplification between blocks yet distant enough to flank a region of significant sequence information.
CODEHOPs developed for herpesvirus screens targeting the DNA polymerase
5'>3' Sequence(degenerate codons are in lower case)3
"TVG-IYG" Assay 4
All HV (IHV, HHV6,7)
All HV (IHV, HHV6,7)
All HV (IHV, AlHV1, RRV)
"DFASA-GDTD1B" Assay 7
All HV (IHV, HHV6,7)
All HV (IHV)
"QAHNA" Assay 7
αHV γHV (IHV, βHV)
"SLYP" Assay 8
All HV (CMV, EHV2)
CMV (All other HV)
CODEHOP Predicted 9
All HV (IHV)
All HV (IHV)
CODEHOP PCR Amplification, Product Cloning and Sequence Analysis
CODEHOP PCR amplification has been performed using classical and touch-down approaches with a hot-start initiation . More recently, thermal gradient PCR amplification has been used to empirically determine optimal annealing and amplification conditions for the pool of primers . Different buffers, salt concentrations, and enzymes have been employed with varying success due to differences in DNA template preparation and the unknown nature of the targeted sequence. PCR products are either sequenced directly or after TA-cloning.
In this review, sequences were compared by BLAST analysis  and multiple alignment using Clustal W . Phylogenetic analysis of the multiply aligned sequences was performed using protein distance and neighbor-joining analysis implemented in the Phylip analysis package . Bootstrap analysis was also performed with 100 replicates and a consensus phylogenetic tree was determined. For the phylogenetic analysis, positions in the multiple alignment containing gaps due to insertions or deletions within the sequence blocks were eliminated.
The "TGV-IYG" CODEHOP assay to detect novel herpesviruses
We have shown the utility of this CODEHOP PCR primer strategy by identifying and characterizing14 previously unknown DNA polymerase sequences from members of the alpha, beta and gamma subfamilies of herpesviruses . Since this original publication, more than 21 additional "TGV-IYG" DNA polymerase sequences from previously uncharacterized herpesviruses have been obtained by other investigators using this CODEHOP primer strategy (see Additional File 1; "TGV-IYG" assay). In some cases, PCR amplification was performed with modified deoxyinosine-substituted primers .
Parameters for refinement of the "TVG-IYG" assay
Limiting degeneracy to increase sensitivity
While the "TVG-IYG" herpesvirus assay demonstrated the ability to detect disparate herpesvirus species in high titer virus cultures in vitro, the detection of limiting amounts of virus in tissue samples in vivo was problematic. This was especially true in sections obtained from formalin-fixed, paraffin-embedded tissue blocks which contained small amounts of degraded DNA. The degeneracy of the primer pool, ie. the number of different primers necessary to encode all codon possibilities for the specified block of conserved amino acids, plays a direct role in the sensitivity of the PCR amplification. Whereas highly degenerate primers consisting of pools of hundreds or thousands of primers with different DNA sequences may allow amplification of DNA templates present in high copy number, as found in cultured virus stocks, they are less successful in amplifying low copy numbers of DNA templates found in virus infected tissues in vivo, especially in formalin-fixed tissue. As the degeneracy increases, the concentration of the primer or primers that will participate in the desired amplification reaction decreases and can become suboptimal. Conversely, the vast excess of primers not participating in the amplification of the targeted gene can cause non-specific amplification which can, in turn, inhibit or mask the amplification of the desired target.
As indicated in Table 1, the degeneracy of the primers utilized in the "TVG-IYG" assay ranged from 48–1024. This level of degeneracy was driven by the number of nucleotide possibilities encoding the targeted amino acids at each position as well as by the number of amino acid positions allowed to be degenerate. Figure 5A shows the DFA/DFAS/QAHN sequence block produced by Block Maker from multiple alignments of 11 different herpesvirus polymerase sequences. Figure 5C shows the consensus amino acids at each position, as determined by the CODEHOP algorithm, which are boxed and bolded with the alternate amino acids positioned above. The original primer manually derived from this motif, "DFA" is, in fact, completely degenerate, with multiple codons provided for each amino acid position, except the ultimate proline (P) residue, yielding a pool of 512 different primers . Because the performance of this primer was consistently suboptimal in samples with limiting template, the overall structure and degeneracy of the primer was altered by designing a PCR primer "DFASA" from the same sequence motif using the CODEHOP methodology. This primer had an 11 bp 5' consensus region and a 3' degenerate core containing multiple codons at 5 amino acid positions resulting in a pool of 256 different primers (Figure 5C). The "DFASA" primer was successfully used to amplify extremely low amounts of viral DNA in a background of genomic DNA from paraffin-embedded formalin-fixed tissue in the discovery of the macaque homolog of Kaposi's sarcoma-associated herpesvirus, called retroperitoneal fibromatosis herpesvirus (RFHV) . Subsequent estimates of virus copy number using real-time quantitative PCR indicated a level of RFHV DNA in the available samples that was 1/100–1/1000 of a single copy cellular gene (unpublished observations). The "DFASA" primer has been successfully used to identify a number of novel alpha-, beta- and gammaherpesviruses in a wide variety of host organisms (see Additional File 1: "DFASA-GDTD1B assay").
Due to the presence of a highly conserved leucine (L) at block position 7 within the "DFAS" motif (Figure 5) which significantly increased the degeneracy of the primer pool with its six possible codons, an additional CODEHOP was designed from the "QAHN" motif immediately downstream of "DFAS" to further decrease degeneracy. The "QAHNA" primer had an 11 bp 5'consensus region and a 3' degenerate core containing multiple codons at 4 amino acid positions resulting in a pool of 48 different primers (Figure 5C). This CODEHOP has been successfully used to identify several primate rhadinoviruses related to KSHV in tissue samples with limiting amount of viral DNA [10, 19], see also Additional File 1.
Primer bias and specificity
The "DFA" and "DFASA" primer pools were originally designed using only the alanine (A) codon at block position 5 in the "DFAS" motif and did not include the glutamine (Q) codon found in that position of the motif in HHV6 and HHV7, "DFQS" (highlighted, Figure 5A, B). The nucleotide mismatches in this region are shown in Figure 8. While the "DFA" and "DFASA" primers are biased by design against HHV6 and HHV7, they have been used successfully to detect betaherpesviruses related to HHV6 and HHV7 . This suggests that mismatches 13–14 nucleotides from the 3' end of the primer, do not have major affects on the utility of the primers, especially when viral template is not limiting.
More significant bias against HHV6- and HHV7-like herpesviruses was present in the "TGV" primer used in conjunction with the "IYG" primer in the secondary nested PCR reaction in the "TGV-IYG" assay (see Figure 2B). The "TGV" primer contains the partial valine (V) codon "GT" at its 3' end (Block position 11; Figure 3C). Since both HHV6 and HHV7 contain alanine (A) (codon = GCN) at this position (highlighted in Fig. 3A, B), the "TGV" primer would mismatch at the 3' terminal nucleotide with both HHV6- and HHV7-like sequences. This mismatch occurs at the 3' end of the "TGV" primer and is predicted to significantly impair polymerase extension. To remove this bias, the "TGV" primer was redesigned as the "VYGA" primer removing the 3' terminal "GT" of the valine codon and the terminal degenerate position of the glycine (G) codon. The "TGV" primer contained an additional bias against amplification of HHV6-like sequences due to the use of only the phenylalanine (F) codons (TTY) (Block position 8) at a position encoding valine (V) in both HHV6 and HHV7 (highlighted in Figure 3A and 3B). To remove this bias, "VYGA" was designed to include both the valine (V) and (F) codons at this position. The total degeneracy of the "TGV" and "VYGA" primer pools remained the same, with 256 different primers, due to the loss of the degenerate codon position in the glycine, block position 10 in "TGV" and the gain of the degenerate codon positions in the valine, block position 8 in "VYGA".
The subsequent cloning and sequence analysis of new herpesvirus DNA polymerases from the rhadinoviruses, rhesus rhadinovirus (RRV) and alcelaphine herpesvirus 1 (AlHV1) [20, 21], revealed mismatches with the downstream "IYG" primer of the "TVG-IYG" herpesvirus assay. The "IYG" primer (a reverse orientation primer) includes the codons (ATH) for isoleucine (I) at its 3' end (Block position 1; Figure 4C). Both RRV and AH1 contain a valine (V) codon (GTN) at this position (highlighted in Figure 4A). Thus, "IYG" is biased against RRV-like or AH1-like rhadinoviruses due to a T-C mismatch at the 3' end of the primer. To eliminate this bias, the "IYG" primer was redesigned as "GDTD1B" to remove the isoleucine position within the 3' degenerate core (Figure 4C) and, in addition, the length of the 5' consensus clamp was increased.
Decrease in size of the amplification products
Because typical tissue samples especially paraffin-embedded formalin-fixed tissue contain degraded DNA with sizes averaging near 300–500 bp in length, we decided to decrease the maximal amplification product size of the herpesvirus assay. The initial amplification product of the "TGV-IYG" assay (DFA-KG1) was ~800 bp (Fig. 2B). To reduce the initial amplification product size, a hemi-nested PCR assay was developed in which the newly designed downstream anti-sense primer "GDTD1B" targeting the highly conserved "YGDT" motif was used in a primary PCR amplification with the new upstream primer "DFASA". This amplification yields an approximate 500 bp PCR product (Figure 2B). This initial PCR product is then used as template in a secondary PCR amplification using the nested primer "VYGA" with the downstream anti-sense primer "GDTD1B". This amplification yields a PCR product of approximately 200 bp (see Figure 2B). These modifications produce amplification products close to the average size of degraded DNA present in fixed tissue.
The "DFASA/QAHNA-GDTD1B" herpesvirus assay: a refinement of the "TGV-IYG" assay
Alpha- and Betaherpesviruses identified and/or characterized using CODEHOP-based PCR assays targeting the herpesvirus DNA polymerase
Caretta caretta HV
Florida loggerhead turtle
Chelonia mydas HV-Florida
Florida green turtle
Chelonia mydas HV-Hawaii
Hawaiin green turtle
Infectious laryngotracheitis virus (Gallid HV-1)
Marek's disease virus (Gallid HV-3)
Lepidochelys olivacea HV
Olive ridley turtle
S. American squirrel monkey
Tursiops truncatus HV-1
Tursiops truncatus HV-2
African elephant endotheliolytic virus
Asian elephant endotheliolytic virus
Not Deposited (60aa)
Chlorocebus aethiops cytomegalovirus (Cercopithecine HV-5)
African green monkey
Mandrill HV β
Gammaherpesviruses identified and/or characterized using CODEHOP-based PCR assays targeting the herpesvirus DNA polymerase (see legend to Table 2)
Bovine lymphotrophic HV
Rhesus lymphocryptovirus-1 (cercopithecine HV-15)
This study AF091053
This study Unpublished
HV papio (cercopithecine HV-12)
Ovine HV 2
Porcine lymphotrophic virus-1a
Porcine lymphotrophic virus-1b
S. American squirrel monkey
Zalophus californianus HV
Caprine lymphotropic HV
Deer malignant catarrhal fever virus
S. American spider monkey
African green monkey
African green monkey
Gorilla rhadinoherpesvirus 1
Kaposi's sarcoma-associated HV (HHV8)
Macaque fascicularis rhadinovirus-2 (Macaque fascicularis gamma virus)
Macaque nemestrina rhadinovirus-2
Pan troglodytes rhadinoherpesvirus-1a
Pan troglodytes rhadinoherpesvirus-1b
Retroperitoneal fibromatosis HVMm
Retroperitoneal fibromatosis HVMn
Rhesus rhadinovirus (Macaque mulatta gamma virus)
Tapirus terrestris HV
Equus somalicus HV
Equus zebra HV
The "SLYP1A-GDTD1B" herpesvirus assay: a general herpesvirus detection assay
We designed additional primers from the DFAS/QAHN sequence motif using the CODEHOP strategy to develop further assays to detect new herpesviruses. The primer "SLYP1A" was one such primer designed to eliminate bias in the 3' degenerate core of "DFA" and "DFASA" primers against HHV6 and HHV7, described above. The "SLYP1A" primer overlaps the "DFA" and "DFASA" primers and extends further downstream in a region very well conserved across the different herpesvirus species including HHV6 and HHV7 (Block positions 8–12; Figure 5C) . Primer design across this region was based on the similarities in the first two positions for the codons for isoleucine (I) – (ATA, ATC, ATT) and methionine (M) – (ATG). These two amino acids are conserved in two positions within this sequence block in all herpesvirus species, including IHV (Block positions 11,12; Figure 5) and provide the penultimate and ultimate 3' codons for the primer. Also, the SLYP1A primer was designed with only one of the two codon types utilized for serine (S) – (AGY) to minimize degeneracy in the 3' degenerate core (Block position 10; Figure 5C). Serine at this position (Block position 10; Figure 8) is encoded by AGY-type codons in all herpesvirus species, except for CMV-like herpesviruses which use TCN-type codons and EHV2 which contains a codon for threonine. A second related primer, SLYP2A was also designed from this region with an identical sequence except that the other serine codons (TCN) were used in the third position. Although this primer was biased for CMV-like sequences, we have successfully amplified KSHV which contains an AGT codon (unpublished results).
Using the CODEHOP strategy to determine the complete sequence of novel viral genes
CODEHOP and gene-specific primers developed for cloning the complete DNA polymerase gene of novel macaque rhadinoviruses.
5'>3' Sequence (degenerate codons are in lower case)1
Using the CODEHOP strategy to characterize genomic regions within novel viral genomes
CODEHOP and gene-specific primers developed for cloning the divergent region B within the RFHV genome
5'>3' Sequence (degenerate codons are in lower case)3
All cellular and viral TS
All cellular and viral TS
CODEHOP-mediated PCR – a general approach to identify novel viral genes
Using the web-based software to design CODEHOP PCR primers to a conserved viral gene
Other examples of CODEHOP PCR primers designed from multiple alignments of the herpesvirus DNA polymerase sequences using the Web-based CODEHOP software are shown in Figures 3, 4, 5, 6. The VYG1A primer designed from the conserved VYG motif shown in Figure 3 is aligned with the original manually designed "TGV" and "VYGA" primers. The computer-predicted "YGDTB" primer designed from the conserved GDTD motif is aligned with the original "IYG" and "GDTD1B" primers (Figure 4). In the prediction of this primer, the conserved sequence block identified by BlockMaker from the sequences shown in Figure 4A, extended only from amino acid position 1 – 10, which was the limit of the conserved sequence block determined by BlockMaker. The CODEHOP software indicated the necessity to add additional nucleotides to the 5' end of the "YGDTB" primer to obtain the minimal length for the 5' consensus region of the primer. As such, the amino acid sequences of block positions 11–13 were obtained manually and compared in order to derive the eight terminal nucleotides for "YGDTB" (overlined in Figure 4C).
In this review, the utility of CODEHOP-mediated PCR for the identification of novel viruses and the characterization of new viral genes and genomic regions is presented. While the focus of this study was on the herpesvirus family, other virus families can be easily targeted using analogous approaches. We have previously developed successful CODEHOP assays targeting the reverse transcriptase genes of retroviruses and lentiviruses [2, 6]. Recently, the CODEHOP strategy has been used to develop assays to detect novel papillomaviruses targeting the highly conserved L1 protein . With the CODEHOP strategy, molecular sequence data can be readily obtained for comprehensive virus phylogenies and tracing of evolutionary pathways. Furthermore, comparison of multiple representatives of homologous viral proteins can be of importance for understanding the protein structure and function and provided insight into virus-host relationships.
List of Abbreviations
consensus-degenerate hybrid oligonucleotide primer
polymerase chain reaction
retroperitoneal fibromatosis herpesvirus
Kaposi's sarcoma-associated herpesvirus.
The author would like to thank Emily Schultz, Greg Bruce, Lin Bennet, Brian Raden, Jon Ryan, and Kurt Strand for their help in developing the CODEHOP PCR strategy, Jorja and Steve Henikoff, of the Fred Hutchinson Cancer Research Center, for the creation and maintenance of the CODEHOP software and website, and Jeannette Stahli for editing advice.
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