Abstract
PCR-based variant-specific hybridization (VSH) and single-strand conformational polymorphism (SSCP) analyses were compared for their capacities to detect mixed human papillomavirus type 16 (HPV-16) variant infections within clinical specimens. The SSCP assay used in this comparison targets a 682-bp fragment that spans nucleotides 7445 to 222 within the HPV-16 genome. This fragment includes portions of the HPV-16 long control region and the E6 open reading frame and identifies three categories of SSCP patterns: those identical to the patterns of prototype HPV-16 (P), those identical to the patterns of Caski-derived HPV-16 (C), or those that are different from the P and C HPV-16 patterns and that are therefore classified as belonging to novel (N) HPV-16 variants. VSH targets the entire HPV-16 E6-coding region (nucleotides 56 to 640) and distinguishes previously described variant nucleotides at positions 109, 131, 132, 143, 145, 178, 286, 289, 350, 403, and 532. Clinical samples used in VSH and SSCP analyses were subjected to multiple independent amplification reactions. The resultant amplicons were cloned, and 14 to 78 clones per clinical specimen were evaluated by VSH. VSH detected an HPV-16 variant that represented at least 20% of the amplified HPV-16 variant population. In contrast, SSCP analysis detected HPV-16 variants that represented 36% of the amplified HPV-16 population. Comparison studies were conducted with mixed HPV-16 variant laboratory constructs. Again, VSH had a higher sensitivity than SSCP analysis in detecting mixed HPV-16 variant infections in these constructed amplicon targets. Accurate detection of HPV-16 variants may enhance our understanding of the natural history of HPV-16 infections.
Human papillomaviruses (HPVs) are a group of over 100 distinct viral genotypes. Approximately 30 of these HPV types have been associated with cervical neoplasia (7, 13, 15, 17, 18, 20). HPV type 16 (HPV-16) is most often linked with invasive cervical cancers (ICC) worldwide and it is detected in approximately 50% of specimens from patients with ICC (5). For all HPV types studied to date, intratypic variants have been identified (11, 12, 24, 32, 33). Although it has been recognized that individuals may be simultaneously infected with multiple HPV types, the population distribution of intratypic HPV variant infections and the prevalence of multiple intratypic HPV infections have not been adequately explored (14). Recently, HPV-16 E6 and E7 variants have been shown to differ in their abilities to alter in vitro transformation of keratinocyte differentiation (22, 25). HPV variants may differ functionally and may represent risk factors for the development of cervical (6, 10, 16, 31, 34) or anal (28) dysplasia. Therefore, it is important to develop and characterize assays that can facilitate their detection.
Phylogenetic relationships between HPV-16 variants have been established in earlier studies that have analyzed more than 100 kb of DNA sequence information including the long control region (LCR), E6, L1, and L2 genomic segments. More recent studies have demonstrated that the E6-coding region identified distinct HPV-16 subclasses not identified by marker nucleotides within the LCR (27, 32, 33). Additionally, nucleotide changes that occur within the MY09/11 region of the L1-coding segment as well as within other genomic segments were linked to nucleotide changes within the E6-coding segment (8, 27, 32, 33). On the basis of this evidence, variant-specific hybridization (VSH) analysis targeted the E6 region, a short continuous sequence of the HPV-16 genome, to distinguish HPV-16 lineage-specific variants. In the present study, single-strand conformational polymorphism (SSCP) analysis targets the LCR and the 5′ portion of the E6-coding region, and thus, the specificity of HPV-16 variant assignments by both methods shares partial sequence relatedness.
Our study sought to compare methods of HPV variant analysis for HPV-16, the most common HPV type present in normal cervical epithelia (20) and in ICCs (3–5, 26). SSCP analysis was initially performed with clinical samples containing HPV-16 DNAs that were then subsequently selected for comparison studies by VSH. Experiments that attempted to increase the sensitivity of VSH were conducted. In addition, laboratory constructs prepared from previously characterized clinical specimens were compared by VSH and SSCP analyses.
MATERIALS AND METHODS
Preparation of clinical specimens.
Specimens that contained HPV-16 variants were obtained from prospective studies conducted at the University of Washington, Seattle, and are designated WA (28–31). Briefly, cervical and vulvovaginal specimens were collected from women attending a sexually transmitted disease or a university health clinic at the time of study enrollment and subsequently at times ranging from 4 to 32 months after collection of the first sample. Anal swab specimens were collected from men presenting to the Department of Public Health AIDS Prevention Project at the time of study enrollment and subsequently at times ranging from 8 to 12 months after collection of the first sample. Additional specimens were obtained from studies performed in Portland, Oreg. (27), and the United Kingdom (25a). These specimens contained both single and mixed HPV-16 variants and were designated OR and UK, respectively.
PCR-based SSCP analysis.
SSCP analysis was performed as described previously (29). Briefly, DNA amplification was conducted for 35 cycles in a total volume of 10 μl in a Perkin-Elmer 9600 Thermal Cycler (Perkin-Elmer Cetus, Norwalk, Conn.). Each cycle consisted of denaturation (94°C, 25 s), annealing (62°C, 25 s), and extension (72°C, 50 s). Incorporation of [α-33P]dATP (Du Pont NEN Research Products, Boston, Mass.) into the PCR products occurred during the amplification with a pair of type-specific primers, primers C and D (29), that target 682 bp from nucleotides 7445 to 222 in the HPV-16 noncoding region and the 5′ E6-coding region. PCR products were cleaved into three fragments of 318, 166, and 198 bp from 5′ to 3′ by digestion with the restriction endonuclease DdeI. The digested products (3 μl) were mixed with 27 μl of loading buffer, and the mixture was incubated at 97°C for 10 min and was then rapidly chilled on ice. Samples (4 μl) that corresponded to 0.4 μl of the original PCR products were electrophoresed in a 5% polyacrylamide gel containing 10% glycerol for 6 h at 750 V and 4°C. The gel was dried on filter paper and was exposed to X-ray film overnight at room temperature.
The SSCP patterns of the three fragments were compared individually with the reference patterns from prototype plasmid HPV-16 (pHPV-16) DNA (21) and Caski cellular DNA (2) and with the patterns of isolates from other clinical specimens. The patterns for each of the three fragments were designated as follows: P, a pattern identical to the prototype pattern C; a pattern identical to the Caski cellular DNA pattern; or N (N1, N2, etc.), a novel pattern not the same as pattern P or C (31). Two isolates were regarded as different HPV-16 variants as long as any one of the three fragments showed different electrophoretic mobility polymorphisms. The predominant variant was defined as the one with the most intensive banding pattern. The presence of multiple HPV-16 variants in a single specimen was interpreted when, in addition to the predominant SSCP patterns, one or more bands were present (30). If the extra bands fit a precharacterized SSCP pattern, the pattern of the minor variant would be assigned. Otherwise, the presence of a minor HPV-16 variant was interpreted only as suggestive.
PCR for E6 VSH.
Thermus aquaticus-based PCRs were performed as described previously (27). Briefly, 100-μl amplification reaction mixtures contained 10 mM Tris (pH 8.5), 50 mM KCl, 200 μM (each) deoxynucleoside triphosphate (dATP, dCTP, dGTP, and dTTP), 2.5 mM MgCl2, 2.5 U of Taq DNA polymerase (Perkin-Elmer, Foster City, Calif.) and 0.1 or 20 pmol (each) of sense and antisense E6 primers for the outer and the inner reactions, respectively. Two microliters of crude DNA digests from the original clinical samples was added to each amplification reaction mixture (100 μl). Four independent one-tube nested PCRs (27) were performed for each clinical specimen.
PCR tubes were always opened with sterile gauze on a sterile surface, and gloves were exchanged for processing of each clinical specimen. Potential contamination was evaluated by interspersing amplification reaction mixtures with no DNA between every four clinical specimens. PCR product contamination was reduced by the use of our one-tube nested PCR method developed for the E6-coding region. All resultant amplification products were evaluated by agarose gel electrophoresis in 0.5× Tris-borate-EDTA running buffer. Gels were stained with ethidium bromide for subsequent visualization of PCR products by UV transillumination.
Ligation and transformation of PCR-generated E6 amplicons.
The products from the amplification reactions, four from each clinical specimen (n = 8), were cloned by ligating 5 μl of each HPV-16 E6 PCR product into 10 ng of pCRTM2.1 Vector (Invitrogen, San Diego, Calif.) with T4 DNA ligase. The ligation reaction was conducted overnight at 14°C and subsequently frozen until the transformation experiments were performed. For the transformation experiments, 2 μl of each ligation reaction mixture was mixed with 50 μl of INVαF′ One Shot competent cells (Invitrogen) with 2 μl of β-mercaptoethanol per 50 μl of cells. Transformations were conducted according to the manufacturer's instructions.
Colony screening of cloned E6 amplicons.
Positive transformants selected by their ampicillin resistance and the lack of beta-galactosidase production were screened by colony PCR amplification with the same E6-specific primers used previously for the inner reaction of the one tube-nested PCR. Sterile toothpicks were used to isolate individual colonies, which were subjected to amplification reactions with 50-μl reaction mixtures. Inner reaction sense (ACCGGTTAGTATAAAAG) and antisense (GCTCATAACAGTAGAG) primers (20 pmol) were used. The amplification program consisted of 40 cycles of 94°C for 30 s, 45°C for 30 s, and 72°C for 1 min. Again, a 10-min 72°C extension period completed the amplification program. The amplified products were evaluated by agarose gel electrophoresis in 0.5× Tris-borate-EDTA running buffer and then ethidium bromide staining. PCR products that displayed the expected molecular size were reamplified as detailed above. Two microliters of the 50-μl amplification reaction mixture was used as a template for the subsequent reactions with 100-μl reaction mixtures.
Dot blotting for VSH.
The HPV-16 E6 amplicons that resulted from the DNAs from the original clinical specimens or from individual cloned molecules were treated as follows. Eighty microliters of each amplification reaction mixture was mixed in 1.1 ml of denaturation solution (0.4 M NaOH containing 25 mM EDTA) for 10 min at room temperature. The mixture (70 μl) was then aliquoted to replicate Biotrans nylon membranes (Pall for ICN, East Hills, N.Y.) by using 96-well dot blot manifolds (Bio-Rad Laboratories, Hercules, Calif.). This resulted in the application of 4 μl of the original amplified sample to each well. Each denatured sample was completely aspirated under vacuum and was rinsed with 400 μl of 20× SSPE (3.6 M NaCl, 0.2 M NaH2PO4 · H2O, 0.11 M NaOH, and 0.02 M disodium EDTA · 2H2O adjusted to pH 7.4). The vacuum was reapplied to completely remove the wash solution. Nylon membranes were removed from the manifold under complete vacuum, and the bound DNA was immediately cross-linked to the membranes with a Stratalinker UV cross-linker on the autolink setting (Stratagene, San Diego, Calif.). The membranes were then stored in 2× SSPE at room temperature until subsequent hybridization.
The products of the amplification reactions with the original clinical samples and cloned HPV-16 E6 targets were evaluated by previously reported hybridization methods (27). Only oligonucleotide probes that distinguish HPV-16 E6 nucleotide positions 109, 131, 132, 143, 145, 178, 286, 289, 350, and 403 were used in these studies. Control amplicons were prepared by subjecting previously characterized clinical specimens (27, 32, 33) to amplification in the E6 PCR system. Control representatives from HPV-16 class variants and HPV-16 subclass variants (E, E-A176T, E-A178T, E-C188T, E-As, Af1-a, Af2-c, Af1-e, Af2-b, NA1, and AA) were included on each membrane for the E6 hybridization assays. DNA sequence information had previously been obtained for the complete E6-coding sequences in each control specimen (27, 32, 33).
VSH data analysis.
The results of the E6 hybridization assays were interpreted from the chemiluminescent signals recorded on X-ray film. The results were entered into a database file containing fields for all probes under study. Data entry was performed manually. An analysis program facilitated the linkage of results for individual oligonucleotide probes and was used to establish an overall hybridization pattern for the targeted HPV-16 E6 regions in each sample examined (27). Combined hybridization patterns were assigned on the basis of the predicted HPV-16 class, subclass, or minor class variants observed previously (32, 33). These assignments are capable of distinguishing Asian, African, Asian-American, and European variants.
Semiquantitative dot blot studies of VSH sensitivity.
Ninety-five clinical specimens that contained HPV-16 DNA that had previously been characterized by either VSH or DNA sequencing (19), or both, were amplified by use of 2 μl of each original clinical specimen in a one-tube nested E6 amplification system (27). These samples were selected since they contained one or more of the HPV-16 variants observed in four previous studies involving HPV-16 variants identified in U.S. and European populations (n = 700). The distribution of variants from these 95 selected samples was as follows: E-P 350T, n = 23; E-P C109, n = 10; E-G131, n = 13; E-P 350G, n = 17; AA, n = 18; Af1, n = 4; Af2, n = 6; and As, n = 4. Specimens from study subjects with cervical or anal dysplasia were included, as were specimens from controls with normal Pap smear results. Three clinical samples (samples OR.1783, OR.4072, and UK.63550) previously identified as having mixed HPV-16 variant infections were included in this set of 95 specimens as controls.
In addition, a set of 16 laboratory constructs was created to simulate mixed variant infections. Clinical specimens containing non-European variants of HPV-16 including variants of the Af-1, Af-2, and AA classes were combined with an E-P variant in these constructs. The 16 laboratory constructs were created by combining one of four separate clinical specimens that contained non-European HPV-16 variants with a single clinical specimen that contained a European HPV-16 variant at four ratios (vol/vol): 1:4, 3:7, 2:3, or 1:1. Group 1 consisted of an Af1 and E-P variant, group 2 consisted of an AA and E-P variant, and groups 3 and 4 consisted of an Af2 and an E-P variant. The variants were tested at each of the ratios noted above. These 16 constructs as well as three clinical samples previously identified as having mixed variant infections (27, 33) were analyzed by both SSCP analysis and VSH.
The PCR products derived from the 95 clinical specimens, 16 laboratory constructs, and known mixed variant samples were used to prepare two separate Biotrans membranes in replicate. The PCR products were divided such that one set of membranes received 8 μl of each original PCR product and the other set of membranes received 80 μl of each original PCR product. These volumes of PCR products represent increases of approximately 2 and 20 times, respectively, when compared with the volumes used in our previously reported procedures (27). An additional membrane that contained laboratory constructs and samples known to contain mixed variants was generated with 4 μl of each original PCR product to represent the 1× concentration, consistent with our previous reports (27).
The VSH analysis for this portion of our studies was limited to probes capable of detecting variants at nucleotide positions 335 and 350. These particular variant nucleotide positions were selected on the basis of their ability to distinguish between European and non-European variants (27, 31–33). The hybridization assays were performed as described previously (27), with the following modifications. Hybridization was conducted at 43°C for 2 h, followed by washes at 47°C for the 335T- and 335C-specific probes and 48°C for the 350G- and 350T-specific probes. It should be noted that these wash temperatures are higher than those previously reported (27) for these probes. The temperature increase was necessary to accommodate the nonspecific background levels of hybridization associated with the increased amount of DNA loaded onto the solid-phase nylon support. Appropriate hybridization and wash conditions were adjusted for these studies following optimization reactions with precharacterized samples and different amounts of PCR products (data not shown).
The chemiluminescence signals recorded on X-ray films were compared to those of standards to determine relative intensities, as follows: +++++, a very strong hybridization signal; ++++, a strong signal, +++, a moderate signal; ++, a weak signal; +, a very weak signal; and −, no detectable signal. The laboratory workers who performed SSCP and VSH analyses were blinded to the HPV-16 variant designations for the variants in specimens previously analyzed in these comparison studies.
RESULTS
SSCP and VSH analyses of original clinical specimens.
Specimens from longitudinal studies were screened for the presence of HPV-16 variants by SSCP analysis, and then selected samples (i.e., WA.20066A and WA.20066C, WA.20133A and WA.20133C, WA.20195A and WA.20195C, and WA.40084A and WA.40084B) were subjected to VSH (Table 1). Two clinical specimens were obtained from each study subject at times that ranged from 4 to 32 months after collection of the first sample.
TABLE 1.
Analysis of multiple variants
| Specimen no.a | Variant found in original clinical specimens
|
Variant (%) found by VSH of cloned ampliconsb
|
No. of clones | ||
|---|---|---|---|---|---|
| SSCP analysisc | VSH | Major variant | Minor variant | ||
| WA.20066A (9/1990) | CPP | Mixed | E-350T (80) | AA-NA1 (20) | 63 |
| WA.20066C (11/1991) | C(N)P(N)P(N) | Mixed | AA-NA1 (64) | E-350T (36) | 50 |
| WA.20133A (10/1990) | NdPN | E-C109T | E-C109T (100) | 25 | |
| WA.20133C (6/1993) | NdPN | E-C109T | E-C109T (100) | 14 | |
| WA.20195A (2/1992) | PNP | E-350T | E-350T (100) | 55 | |
| WA.20195C (12/1993) | PN(P)P | E-350T | E-350T (100) | 57 | |
| WA.40084A (3/1990) | CCCd | E-G131G | E-G131G (95.6) | E-350G (2,9), E-G131T (1.5) | 68 |
| WA.40084B (7/1990) | CCCd | E-G131G | E-G131G (96.1) | E-350G (2.6), E-G131T (1.3) | 78 |
The date (month/year) that specimens were obtained from the study subject is given in parentheses.
Results determined by VSH of individual PCR-derived E6 molecules cloned from each clinical specimen.
The pattern for a minor variant is shown in parentheses.
Suggestive of the presence of multiple variants and the pattern of the minor variant could not be discerned.
SSCP analysis suggested the presence of multiple variants in six of the specimens (WA.20066C, WA.20133A, WA.20133C, WA.20195C, WA.40084A, and WA.40084B). In two of these specimens (WA.20066C and WA.20195C) the patterns of the minor variants were definitively N and P, respectively. The presence of a mixed HPV-16 variant infection was suggested in both specimens from study subjects WA.20133 and WA.40084 by SSCP analysis on the basis of the presence of one or more extra bands, but the patterns of the minor variants could not be unequivocally assigned.
When the same specimens were analyzed by VSH, only one study subject (WA.20066) was identified as having a persistent mixed HPV-16 variant infection according to the results for both samples obtained 14 months apart. Persistent single HPV-16 variant infections were detected in the remaining three study subjects (WA.20133, WA.20195, and WA.40084) at both clinical visits.
VSH analysis of cloned specimens.
The same clinical specimens that were evaluated by SSCP and VSH analyses as described above were also amplified in four independent one-tube nested PCRs (33), and the resultant amplicons were cloned. Clones were screened by colony PCR and were subjected to VSH analysis. A total of 410 positive clones containing the HPV-16 E6 PCR product were evaluated by hybridization with 17 oligonucleotide probes.
VSH analysis of cloned amplicons detected mixed HPV-16 variant infections in two study subjects (WA.20066 and WA.40084) at both clinical visits. Study subject WA.40084 was infected with two minor HPV-16 variants that comprised 1.3 to 1.5% (E-G131G) and 2.6 to 2.9% (E-G350T) of the clones analyzed at two visits, respectively, over a 4-month time period, as shown in Table 1. Twenty and 36% of the clones from study subject WA.20066 collected at two clinical visits, respectively, over a 14-month time period were minor variants. In the population of cloned amplicons from sample WA.20066A, E-350T and AA-NA1 were identified as the major and minor variants, respectively. Conversely, HPV-16 variant AA-NA1 was identified as the major variant and E-350T was defined as the minor variant in cloned amplicons derived from the clinical sample obtained 14 months later (sample WA.20066C). These cloning studies demonstrated that a range of major to minor HPV-16 variant ratios were detected by VSH.
Semiquantitative dot blot studies.
In the semiquantitative dot blotting portion of our investigation, we found that 14 of 95 clinical specimens were potentially infected with mixtures of HPV-16 variants. These samples were not representative of samples from a random population but, rather, were selected to represent a spectrum of samples containing HPV-16 variants. The samples were from study subjects with a variety of clinical diagnoses and were obtained from numerous studies conducted by our groups to date. Thus, it is inappropriate to use these samples to estimate the prevalence of infections with mixtures of HPV-16 variants within the general population.
The certainty of the results of semiquantitative dot blotting was considered in the context of a standard of graded hybridization intensities. The major HPV-16 variant refers to the variant with the greatest load and was assigned by the variant identification by the initial VSH analysis. The minor or secondary variant refers to the variant with the smaller load and was assigned by the lack of significant detection in previous studies.
In the current study, it was not possible to distinguish minor variants beyond the level of European versus non-European variants as neither of the nucleotide positions examined (E6 nucleotide positions 335 and 350) provide the level of detail necessary to make further designations. The results for these 14 samples are summarized in Table 2 along with results obtained from studies with membranes prepared with 4 μl of PCR product (representative of a 1× volume) for comparative purposes.
TABLE 2.
Semiquantitative dot blot results for 14 clinical samples containing multiple HPV-16 variantsa
| Sample no. | Signal atb:
|
Variants present by VSHc | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Nucleotide position 335
|
Nucleotide position 350
|
||||||||||||
| 1×
|
2×
|
20×
|
1×
|
2×
|
20×
|
||||||||
| C | T | C | T | C | T | T | G | T | G | T | G | ||
| OR.5392 | +++ | − | +++++ | − | +++++ | − | − | ++++ | + | +++++ | +++ | +++++ | E-G/T |
| OR.6428 | ++++ | − | +++++ | − | +++++ | − | − | ++++ | +++ | ++++++ | +++++ | +++++ | E-G/T |
| OR.9887 | ++++ | − | +++++ | − | +++++ | − | − | ++++ | + | +++++ | ++ | +++++ | E-G/T |
| UK.41484 | ++++ | − | +++++ | − | +++++ | − | − | ++++ | − | +++++ | ++ | +++++ | E-G/T |
| UK.11902 | ++++ | − | +++++ | − | +++++ | − | ++++ | − | +++++ | + | +++++ | +++ | E-G/T |
| OR.3127 | ++++ | − | +++++ | − | +++++ | − | ++++ | − | +++++ | ++ | +++++ | +++ | E-G/T |
| OR.1783d | ++ | − | ++++ | +++++ | +++++ | +++++ | +++ | ++++ | +++++ | +++++ | +++++ | +++++ | E/NE |
| OR.4072d | + | ++ | ++++ | +++++ | +++++ | +++++ | ++ | ++++ | +++++ | +++++ | +++++ | +++++ | E/NE |
| WA.166 | − | ++++ | + | +++++ | +++ | +++++ | − | ++++ | ++ | +++++ | +++ | +++++ | E/NE |
| WA.74 | − | ++++ | ++ | +++++ | ++++ | +++++ | − | ++++ | ++++ | +++++ | +++++ | +++++ | E/NE |
| WA.101 | − | ++++ | + | +++++ | ++ | +++++ | − | ++++ | + | +++++ | ++ | +++++ | E/NE |
| WA.201 | + | ++++ | ++ | +++++ | ++++ | +++++ | − | ++++ | ++++ | +++++ | ++++ | +++++ | E/NE |
| WA.153 | − | +++ | − | +++++ | ++ | +++++ | − | ++++ | − | +++++ | ++ | +++++ | E/NE |
| WA.188 | − | +++ | + | +++++ | ++ | +++++ | − | ++++ | + | +++++ | + | +++++ | E/NE |
| UK.40090 | ++++ | − | +++++ | − | +++++ | − | − | ++++ | ++ | +++++ | ++++ | +++++ | E-G/T |
| WA.40084A | ++++ | − | +++++ | − | +++++ | − | − | ++++ | − | +++++ | ++ | +++++ | E-G/T |
| WA.272d | ++++ | ++ | +++++ | ++++ | +++++ | +++++ | ++++ | +++ | +++++ | +++++ | +++++ | +++++ | E/NE |
As described in Materials and Methods, only probe pairs for HPV-16 nucleotide positions 335 (C versus T) and 350 (T versus G) were used in these particular studies. Thus, HPV-16 variant class designations could be defined only as either European (E) or non-European (NE).
The relative volumes of resultant amplification reaction mixtures are designated 1×, 2×, and 20×. Chemiluminescent signal intensities recorded on X-ray film are as follows: +++++, very strong hybridization signal; ++++, a strong signal; +++, a moderate signal; ++, a weak signal; +, a very weak signal; and −, no detectable signal. Positive chemiluminescent signals for both possible nucleotides at either one or both nucleotide positions (position 335 or 350) indicated mixed HPV-16 variant infections (i.e., sample WA.201 gave signal intensities of + with the 335C probe and ++++ with the 335T probe with a PCR product volume of 1×, indicating the potential presence of both European and non-European variants). These results were confirmed with the 2× and 20× PCR product volumes.
The variant designations are E-G/T (European 350G mixed with European 350T) and E/NE (European variant mixed with a non-European variant, i.e., Af-1, Af-2, AA, or NA-1).
Samples previously identified as having mixed HPV-16 variant infections by VSH and included in this study as controls.
The probe pair used to detect variation at E6 nucleotide position 335 demarcates European and non-European variants (335C is characteristically observed in isolates containing European E6 variants, and 335T is observed in isolates containing non-European E6 variants). These probes identified nine samples which potentially harbored minor HPV-16 variants. We were able to detect eight of the nine minor variants using data generated for nucleotide position 335 by using the membranes with volumes that were increased two times (2× membranes). In one sample (WA.153) the minor variant was detected only on the membranes with volumes that were increased 20 times (20× membranes).
The probe pair for nucleotide position 350 identified a total of 14 samples that potentially harbor minor variants, including the 9 isolates identified with the probes for nucleotide 335. The nucleotide 350 probe set detected 11 of these minor variants with the 2× membranes. Three samples (UK.41484, WA.153, and WA.40084A) exhibited the presence of a minor variant only on the 20× membranes. The reproducibilities of these semiquantitative dot blotting results were determined in replicate experiments. Replication included reamplification of crude DNA from the original clinical sample, preparation of new membranes, and an independent series of hybridizations (data not shown).
Laboratory construction of HPV-16 variant mixes: detection by VSH and SSCP analysis.
A subset of our previously published probe battery (27) was used to distinguish both major and minor HPV-16 variants and included probes specific for HPV-16 nucleotide positions 131/2, 143/5, 286/9, 335, and 350. VSH analysis consistently detected mixed variant infections for all laboratory constructs as well as three clinical samples previously identified by VSH as being infected with mixtures of variants. The results obtained were identical for all four constructs within each of the four groups; therefore, the hybridization data for nucleotide positions 335 and 350 are provided by group only in Table 3.
TABLE 3.
VSH and SSCP analysis results for laboratory constructs and three clinical samples previously identified as having mixed HPV-16 variant infectionsa
| PCR product vol | Sample Id no. | Signal at:
|
Variants detected by VSH | SSCP analysis resultsb | |||
|---|---|---|---|---|---|---|---|
| Nucleotide position 335
|
Nucleotide position 350
|
||||||
| C | T | T | G | ||||
| 1× | Group 1 | ++++ | +++ | +++++ | − | E-350T/Af | s/o multiple |
| Group 2 | +++ | +++ | +++ | +++++ | E-350T/AA | Single | |
| Group 3 | − | ++++ | +++++ | − | E-350T/Af | s/o single | |
| Group 4 | +++++ | ++ | +++++ | − | E-350T/Af | Multiple | |
| OR.1783 | ++ | − | ++ | +++++ | E-350T/AA | Single | |
| OR.4072 | + | ++ | ++ | +++++ | E-350T/AA | Single | |
| UK.63550 | +++++ | − | +++++ | +++++ | E-G131G/T/AA | Multiple | |
| 2× | Group 1 | +++++ | +++++ | +++++ | − | E-350T/Af | s/o multiple |
| Group 2 | ++++ | +++++ | ++++ | +++++ | E-350T/AA | Single | |
| Group 3 | + | +++++ | +++++ | − | E-350T/Af | s/o single | |
| Group 4 | +++++ | +++ | +++++ | − | E-350T/Af | Multiple | |
| OR.1783 | ++++ | +++ | ++++ | +++++ | E-350T/AA | Single | |
| OR.4072 | ++++ | ++++ | ++++ | +++++ | E-350T/AA | Single | |
| UK.63550 | +++++ | − | +++++ | +++++ | E-G131G/T/AA | Multiple | |
| 20× | Group 1 | +++++ | +++++ | +++++ | − | E-350T/Af | s/o multiple |
| Group 2 | +++++ | +++++ | +++++ | +++++ | E-350T/AA | Single | |
| Group 3 | ++ | +++++ | +++++ | − | E-350T/Af | s/o single | |
| Group 4 | +++++ | +++++ | +++++ | − | E-350T/Af | Multiple | |
| OR.1783 | +++++ | +++++ | +++++ | +++++ | E-350T/AA | Single | |
| OR.4072 | +++++ | +++++ | +++++ | +++++ | E-350T/AA | Single | |
| UK.63550 | +++++ | + | +++++ | +++++ | E-G131G/T/AA | Multiple | |
Chemiluminescent signal intensities are as described in footnote b of Table 2. Positive chemiluminescent signals for both possible nucleotides at either one or both nucleotide positions (position 335 or 350) indicated mixed HPV-16 variant infections. 1×, VSH results obtained with a PCR product volume of 4 μl per dot. This is essentially equivalent to the standard volume reported previously (27). 2×, VSH results obtained with a PCR product volume of 8 μl per dot, which is approximately twice the standard volume reported previously (27). 20×, VSH results obtained with a PCR product volume of 80 μl per dot, approximately 20 times the standard volume reported previously (27). Designations of both the major and minor HPV-16 variants present were determined by using probes specific for nucleotide positions 131, 132, 143, 145, 286, and 289 (data not shown), in addition to those listed.
SSCP analyses used a volume of 0.4 μl of PCR product per sample. Results of SSCP analyses are for only a single experiment; however, these results are repeated for each volume to facilitate comparison with VSH results at each volume. SSCP analysis results are defined as follows: Multiple, the presence of multiple HPV-16 variants; s/o multiple, suggestive of the presence of multiple HPV-16 variants; Single, only a single HPV-16 variant was detected; and s/o single, ambiguous results but the presence of only a single HPV-16 variant is suggested.
The laboratory constructs and three clinical specimens were assayed by SSCP analysis without knowledge of variant status. SSCP analysis positively identified only the group 4 constructs as containing mixtures of HPV-16 variants and suggested the presence of mixtures of variants in the group 1 constructs. Group 2 constructs were negative for mixtures of HPV-16 variants, and the results for group 3 constructs were somewhat ambiguous but group 3 was interpreted as being negative for mixtures of variants.
In the three clinical specimens (OR.1783, OR.4072, and UK.63550), VSH consistently detected mixed HPV-16 variant infections and identified the major class of both HPV-16 variants present in the sample when a PCR product volume of 20× was used. SSCP analysis positively identified only one sample as having a mixed HPV-16 variant infection and found the other two samples to be negative for a secondary variant.
DISCUSSION
Neither the prevalence nor the significance of intratypic HPV variants in clinical specimens has been well defined. Both direct DNA sequencing of pooled PCR products and the analysis of cloned PCR molecules suffer different biases in distinguishing infections caused by mixtures of HPV-16 variants and those caused by only single variants. Therefore, it is important to evaluate and improve existing methods available for the detection of infections caused by HPV-16 variants, especially in the context of large-scale epidemiological investigations. We conducted an investigation to evaluate the performances of VSH and SSCP analyses for the detection of mixed HPV-16 variant infections within single clinical specimens. Both VSH and SSCP analyses detected mixed variant infections, but they did so with different sensitivities.
VSH analysis of cloned E6 amplimers did not detect mixed HPV-16 variant infections in three clinical specimens, as suggested by SSCP analysis (Table 1). In these cloning experiments, significant numbers of individual molecules cloned from four separate amplification reactions for each clinical specimen should have reduced the likelihood that the amplification reaction itself could account for amplification-based artifacts. Nonetheless, the proportions of mixed HPV-16 variants in specimens from patients with cervical infections may vary over time, and further studies are warranted.
It is important to acknowledge that the number of clones obtained from specimens WA.20133A and WA.20133C compared to those obtained from specimens from other study subjects was significantly less by VSH analysis of cloned amplicons. Given that a minor variant in these specimens may account for only a small proportion of the total viral load, a lack of detection of the presence of minor variants by chance may not be surprising. Furthermore, since the regions targeted by VSH and SSCP analyses differ, it is possible that certain nucleotides identified by SSCP analysis are not linked to the E6 nucleotide changes in the samples observed at different clinical visits. It is also notable that the SSCP analysis of study subject WA.20066 suggests the persistence of a dominant HPV-16 variant infection during the time between the two clinical visits, with a minor HPV-16 variant infection emerging at the time of the second clinical visit. In contrast, VSH detected a mixed variant infection at both clinical visits in study subject WA.20066, but the ratio of the major and minor HPV-16 variants differed at each visit.
The semiquantitative dot blot studies reported here indicate that the VSH assay can be modified to detect mixed HPV-16 variant infections in which the secondary variant can be approximately 3% of the total HPV-16 variant population (see Table 1 and Table 2, sample WA.40084A). This increased sensitivity was achieved by the application of a larger volume of the PCR product onto the hybridization membrane. Assay parameters are particularly essential in the modified VSH analysis and may preclude routine application. We observed that the amount of DNA bound to the membrane clearly affects the specificity of probe-target interactions under the conditions used in these experiments. Thus, it is evident that the sensitivity of VSH is influenced by the amount of amplicon placed onto the hybridization membrane.
Semiquantitative dot blot studies detected potential multiple HPV-16 variant infections in less than 15% of the HPV-16-containing clinical specimens selected for study. These results may be confounded by background hybridization due to the very high concentration of DNA present on the 20× membranes. Alternatively, the few samples (n = 3) for which HPV-16 was detected only on the 20× membranes may be infected with a minor HPV-16 variant that represents an extremely low proportion of the total viral load. Resolution of this ambiguity would require the sequencing of several hundred clones from each sample in order to detect what may be as few as 2 or 3% of the total HPV-16 population. It should be noted that HPV-16 mixed variant infections have been characterized by a predominant variant phenotype. Rarely are two HPV-16 variants detected in semiequivalent proportions (27, 29, 30). The biological significance of this is unclear, but it may reflect immunologic influences or other possible host–pathogen-related interactions.
Laboratory construction of HPV-16 variant mixes demonstrated the ability of VSH to consistently detect mixed HPV-16 variant infections and identify the classes of both HPV-16 variants present within the sample. VSH can detect commonly identified signature nucleotides of HPV-16 variants distinguished by DNA sequencing (27, 32, 33). In the region targeted by the PCR primers used in this study, additional nucleotide changes have been reported at various positions (1, 16, 31). SSCP analysis possesses the theoretical ability to distinguish nucleotide changes not targeted by the currently designed battery of VSH probes. However, incorporation of additional probe pairs or sets into the hybridization procedures could easily provide information on nucleotide changes identified within the targeted region.
In comparing the methods and their practical usefulness, each of the techniques (SSCP and VSH analyses) has advantages and disadvantages, which we discuss below. In our comparison study we have identified three important areas in which the methods differ, namely, in the variety of HPV-16 variants that each method can detect, the labor intensity of each method, and the ability of each method to accurately identify mixed HPV-16 variant infections. SSCP analysis is able to identify some nucleotide changes not limited to those that are predefined. SSCP analysis is less sensitive than VSH for the identification of mixed HPV-16 variant infections. An advantage of SSCP analysis over VSH is that it is less arduous than VSH, but SSCP analysis must be performed by experienced laboratory technicians who have knowledge of the expected gel-based mobility patterns and associated HPV-16 sequence variations. By comparison, accurate and broad-spectrum variant designations can be achieved by VSH, although the labor required to achieve these results is considerable.
In order to reduce the labor intensity of VSH, analysis can be limited to the distinction of only major HPV-16 variant classes through the use of a limited number of selected probes. For example, probes specific for E6 nucleotide position 335 can distinguish HPV-16 European variants from non-European variants (i.e., Af-1, Af-2, and AA), and the probe specific for nucleotide positions 14/5 can distinguish Asian-American and North American variants from other HPV-16 variants. Therefore, these probes alone have great utility in defining four major classes of HPV-16 variants. Use of the 350G/T probes would provide the maximum likelihood of detection of two HPV-16 variants infecting a single individual since strains with variations at HPV-16 nucleotide position 350 are distributed within the North American population in an approximate ratio of 40% with the G variation and 60% with T variation (27).
While the accuracy of determining proportions of specific mixed HPV-16 variants within individual clinical specimens by either VSH or SSCP analysis may be questionable, assignment of the variants that cause mixed HPV-16 variant infections by SSCP analysis can often be made immediately on the basis of gel mobility. Specific identification of HPV-16 variants within a presumed mixed infection by VSH may not be entirely possible since VSH identifies mixed HPV-16 infections only via positive hybridization with multiple probes at single or multiple targeted nucleotide positions. The linkage of specific nucleotides within these mixed hybridization patterns cannot be assigned and may even represent novel or uncharacterized nucleotide combinations. However, amplimers from mixed HPV-16 infections detected by VSH can subsequently be subjected to restriction fragment length polymorphism analysis or sequencing for further potential designations. For example, E-G131 can be identified within an amplicon population when the amplicons are subjected to restriction enzyme digestion with MspI (6).
For both SSCP and VSH analysis methods it may be possible to modify the amplified targets and to reduce the size of the amplicons required for HPV-16 variant distinction. This is supported by the observed linked nucleotide changes across HPV-16 variant genomes that provide somewhat redundant lineage-specific information. Modifications that target shorter HPV-16 amplicons would make either method amenable to the analysis of archival tissue specimens (9).
In summary, the results of these studies suggest that both SSCP and VSH analyses can be used as tools for detection of the presence of HPV-16 variant infections in clinical samples. Either method can be considered favorable, depending upon the study's focus. Both methodologies can be improved as suggested above. Future interlaboratory studies should more rigorously consider the specificities of these assays by comparison of the tests under blinded test conditions.
To date, several investigators have reported that the risk for detecting cervical and anal disease is not the same for all HPV-16 variants (6, 10, 16, 23, 25, 28, 30, 31, 34). In addition, in vitro studies have demonstrated differences in biochemical and biological properties between HPV-16 variants which may result in differences in pathogenicity (25). The studies reported here provide information pertinent to consideration of laboratory methods for future epidemiological studies of HPV variants.
ACKNOWLEDGMENTS
This work was supported by grants to C. M. Wheeler, L. A. Koutsky, and N. B. Kiviat from the National Institutes of Health (grants AI/CA32917, AI 38383, CA 34493, and CA 55488).
The Molecular Analysis Shared Facility at the University of New Mexico School of Medicine was used in our studies.
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