There are more than 100 known types of human papillomavirus (HPV), of which at least 13 seem to confer high risk (HR) for cervical carcinogenesis including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68.1 In most cases, HPV infection can be cleared within 2 years by the woman’s immune system, although some infections persist and can lead to neoplastic progression.2 Both persistence of HPV infection and genotype of the infecting virus correlate with subsequent occurrence of cervical intraepithelial neoplasia (CIN),2,3 as well as progression from CIN to cervical cancer (CC).4 Approximately 70% of cervical cancers worldwide are associated with HPV 16 and 18 infections, among which type 16 accounts for more than half and type 18 for approximately 18% of all cases.5 One large prospective cohort study demonstrated that women with normal cytologic results who were infected with HPV 16 or 18 had a higher 10-year cumulative incidence rate of CIN 3 or greater than women with other HR HPV types or who tested HPV negative.6 Generally, HPV 16 predominates in squamous cell carcinoma (SCC), whereas HPV 18 has been associated with a more aggressive form of CIN, invasive CC (adenocarcinoma and adenosquamous carcinomas [ICCs]), higher genome integration rate, and a greater likelihood of cancer recurrence and lymph node metastasis.7
The nucleotide variability of HPV 16 and 18 has been extensively studied, and several variants have been described based on their geographical distribution.8–10 Human papillomavirus 18 variants coevolved with 3 major phylogenetic human branches of Africans, whites, and Asians and were clustered into 3 distinct groups as European (E), Asian-American (AA), and African (Af).10
To date, intratype variations within the HPV-18 E6, E7, and L1 regions have been studied. Several reports suggest that some variants of HPV 18 are associated with the risk of cervical neoplasia. Diversity in oncogenic potential of HPV 18 seems to vary geographically, possibly owing to the different prevalence of the predominant variants in a given region but also with the ethnic origin of the populations under study.11,12 However, no data about HPV 18 intratype variations within strains from Chinese women at Northeast China have been reported so far. The purpose of the study was to identify intratype variations within E6, E7, and L1 genes and to find prevalent and novel variants of HPV 18 in Northeast China.
MATERIALS AND METHODS
Between July 2007 and June 2011, a total of 3528 cervical swabs were collected from patients who referred to the Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University for routine gynecological detections. The median age of the studied populations was 39 years (range, 18–66 years) at the time the cervical scrapings were obtained. Patients were enrolled under the following criteria: (1) not pregnant when cervical swabs were collected, (2) no history of immunodeficiency disease, (3) no history of cervical biopsy or treatment and pelvic radiation or chemotherapy, and (4) willing to undergo HPV testing. The study was approved by the Institutional Review Board of the Affiliated Shengjing Hospital of China Medical University. Informed written consent was obtained from all participants.
Collection of Cervical Specimens and Histological Diagnosis
Cervical swabs were obtained from the participants using a Cervex-Brush (Rovers Medical Devices BV, USA). The cells were resuspended in 20 mL of PreservCyt solution (Thin prep liquid Pap vial; Cytyc Corporation, USA). Liquid-based cytology specimens were processed according to the manufacturer’s protocol and stained by the Papanicolaou method. The Bethesda System 2001 was used to classify our observations: normal limits, cervicitis, CIN 1, CIN 2 to 3, or invasive cervical carcinoma (ICC). All cytologic specimens were collected before any cervical treatment.
For HPV-infected patients, histological diagnoses were available within 3 months after the cervical swabs were obtained. The diagnostic examinations were cervical punch biopsies, loop electrosurgical excision procedures, cone biopsies by colposcopy, cervical cone biopsies, or hysterectomies. Histological assessments were made by a pathologist who was unaware of the cytologic and HPV results.
Viral DNA was amplified and viral types were detected using the HPV GenoArray test kit (HPV GenoArray test kit; Hybribio Ltd, Hong Kong) according to the manufacturer’s instructions and described in a previous study.13 The method could classify 21 HPV subtypes including 13 HR types, 5 low-risk types, and 3 common subtypes for Chinese women.
Analyses of HPV-18 E6, E7, and L1 Variations and Variants
The E6 (477 base pairs [bp]) and E7 (318 bp) regions of HPV 18 strains were amplified from HPV18–positive samples by half nest polymerase chain reaction using 2 pairs of primers. One set of the primers flanks the extreme ends of the 2 genes, and the other locates within the genes. The sequence of the E6/E7 forward primer of the first set of primers is 5′-taacaattgtagtatataaaaa-3′ and that of the E6/E7 reverse primer is 5′-aatagcttgtacataaaaccag-3′. The forward primer of the second set of primers is 5′-aagatttatttgtggtgta-3′, and the second reverse primer is 5′-catacaacatacaacaacaaccat-3′. The polymerase chain reaction amplicons were sequenced using Big Dye Terminator version 3.1 Cycle sequencing kit and automated sequencer (Perkin Elmer ABI 310 Prism, Applied Biosystems, USA). The same primers used in the second round amplifications were also used for sequencing.
To amplify and sequence the HPV 16 L1 gene (1707 bp), 3 sets of primers were designed and used, resulting in 3 overlapping fragments. The names and sequences of the first set of primers are L1-F 5′-cactatatcttctgcctcttccta-3′ and L1-770R 5′-atctaccatatcaccatctt-3′. Those of the second set of primers are L1-647F 5′-agttatgtattttgggctgtg-3′ and L1-1381R 5′-acaccaaagttccaatcctctaa-3′. In addition, those of the third set of primers are L1-1271F 5′-agtatagcagacatgttgaggaa-3′ and L1-R 5′-catacaacatacaacaacaaccat-3′. After amplification, the 3 overlapping fragments were sequenced separately. To build up the whole L1 sequence, sequences of the 3 overlapping fragments were compiled into a single contiguous sequence. The sequence data are shown only for those that could be sequenced fully.
The software used was DNA Baser for simple and batch DNA sequence assembly, Bioedit (T.A. Hall Software) for editing alignments of nucleotide and amino acid sequence. To search nucleotide mutations, the E6, E7, and L1 sequences were lined up alongside the consensus HPV 18 sequences from GenBank (GenBank accession no. X05015)10 by using the multiple sequence alignment algorithm as implemented in the Bioedit software. To identify whether these nucleotide mutations can cause some changes in the amino acid sequence of the proteins, the sequences were theoretically translated (in silico) into corresponding amino acid sequences; and the amino acid changes were identified by multiple sequence alignment algorithm. Phylogenetic analysis was done by multiple sequence alignment with DNASTAR (Lasergene, USA).
Based on the E6 sequences, HPV 18 variants were grouped into E, Af-1, Af-2, or AA lineages according to the lineage category as previously reported by Ong et al10 and Schlecht et al.14
The distribution variables of identified variants among women with different degrees of neoplasia were assessed using the linear-by-linear association (P trend) and the Pearson χ2 test (P) or the Fisher exact test (P). Risk associations were examined by comparing odds ratios. Two-tailed P < 0.05 was considered to be statistically significant.
Samples from patients with multiple HPV infections were excluded to avoid confounding the results. Among 3528 samples collected at the present study, 70 samples were positive for HPV 18 only. A total of 65 sequences of E6 and E7 and 36 sequences of L1 were finally obtained from strains in the cervical samples of HPV 18–infected women. The histologic diagnoses of the women whose infected strains were sequenced successfully for the E6 and E7 genes were as follows: 10 normal cases (15.4%), 46 cervicitis (70.8%), 3 CIN 1 (4.62%), 2 CIN 2 to 3 (3.08%), and 4 ICC (6.15%) (Table 1); those for L1 gene were as follows: 6 normal cases (16.6%), 23 cervicitis (63.9%), 3 CIN 1 (8.33%), 2 CIN 2 to 3 (5.56%), and 2 ICC (5.56%) (Table 2).
With respect to HPV 18, an algorithm was used to distinguish the 4 major HPV 18 variant groups detected in our population, based on departures from the AA primary reference sequence at 3 positions in the E6 region: 317C, 548G, and 549A. All HPV 18 E variants carried a C-A substitution at position 549, whereas Af variants carried an additional A-G substitution at position 548. Variants of Af could be further divided into Af-1 and Af-2 subgroups, based on a T-C substitution at position 317.
According to the aforementioned criteria, only AA and E variants were detected in our studied strains. The most common HPV 18 variant was AA (81.5% [53/65]), followed by E (18.5% [12/65]) (Table 1). No Af variant was found in our population. There were no statistical differences between the lineages and risk for cervical neoplasia.
HPV-18 E6 and E7 Sequence Variations
As shown in Table 1, 8 nucleotide variations in E6 gene and 1 in the E7 gene were synonymous mutations as follows: A92G, T104C, C153T, C287G, T482C, T485C, C519A, and C549A in E6 and C751T in E7. A C-G transversion at position 287 was observed in all strains. The substitution T497C found in E6 of 2 strains from patients with cervicitis led to an H131N amino acid change. Among the variations, C287G, T482C, and C519A in E6 and C751T in E7 had never been identified in previous studies.
HPV-18 L1 Sequence Variations
The HPV-18 L1 sequence was obtained from 36 of the 65 studied strains. Representative variations of the L1 sequence are shown in Table 2. Of the 36 sequenced strains, 28 strains (77.8%) belonged to the AA variant and 8 strains (22.2%) belonged to the E variant. It should be noted that none of the HPV 18 strains contains exactly the same L1 sequence of the HPV 18 reference strain reported previously. Compared to the HPV 18 reference sequence, strains in the AA branch showed 6 to7 nucleotide changes, and those in the E branch had 7 to 9 nucleotide changes. The L1 gene of all the analyzed HPV 18 strains had 4 C-G transversions at positions 5701, 6460, 6625, and 6842 and a G-A transition at position 5503. The characteristic nucleotide changes of strains in the AA branch were T6383G, A6431T, G6987A, and A7045C (Table 2); whereas those of the E branch were C5875A, C5920T, A6401G, and A6430C (Table 2). Six novel nucleotide variations of C5920T, T6383G, A6430C, A6431T, G6987A, and A7045C were found in the HPV-18 L1 gene.
At protein level, L1-encoded proteins of all strains showed 4 aa changes at residues R25Q (G5503A), P91Q (C5701G), P344R (C6460G), and P399R (C6625G). Besides the 4 aa changes, another 3 aa changes at residues T149N (C5875A), A164V (A5920T), and Q334P/H (A6430C/A6431T) were found in the E variants (Table 2), and one aa change at P520N (G6987A) was observed in the AA variants. The aa substitutions of A164V (A5920T), Q334P/H (A6430C/A6431T), and D520N (G6987A) have never been reported before (Table 2). The new variations identified in the study did not show particular association with specific histopathological findings.
Oncogenic HPV infection is the necessary cause of cervical cancer. Approximately 73% of invasive cervical cancer cases worldwide are associated with either HPV 16 (57% of cases) or HPV 18 (16% of cases) infections.15 Several studies have provided evidence demonstrating that specific intratype HPV 16 and HPV 18 genome variations may be relevant to virus infectivity and pathogenicity.16,17 These variations may influence viral persistence and progression to invasive cancer. Amino acid substitutions resulting from mutations in viral genome sequences may affect viral assembly, carcinogenic potential, and host immune responses. Moreover, it is still not clear whether immunity to one HPV variant can protect infection by another variant. Therefore, identification of HPV genetic diversity in specific clinical settings may be important for the rational design of diagnostic, therapeutic, and vaccine strategies.18 In the present study, the E6, E7, and L1 sequences of HPV 18 strains from women in Northeast China were analyzed.
Similar to the analysis of HPV 16 variants reported before,19 nucleotide changes in the HPV-18 E6, E7, and L1 genes were phylogenetically compatible. Compared with HPV 16, HPV 18 was less heterogeneous with a small group of variants accounting for most HPV 18 strains. Of special note, Schlecht et al14 found that a small number of key nucleotide positions could be used to distinguish all of the major variant lineages and perhaps even their subgroups. With respect to HPV 18, the relevant nucleotide positions were 317, 548, and 549 in the E6 gene.14 As previous analyses based on these nucleotide positions, 2 major variants of HPV 18, AA (81.5%) and E (18.5%), were detected in our population. The examined strains in the present study did not seem to be the products of genetic recombination between viruses of different HPV 18 lineages. The fact that recombination between variants of HPV 18 is rare or nonexistent implies that nucleotide changes in one region can be used as markers of changes found in other regions within the same lineage.19 Thus, the HPV 18 variants were classified as proposed for the E6 region and extended this classification to the E7 and L1 regions.10 In addition to the E6, variations in the E7 and L1 regions did not change the clustering of the HPV 18 variants (Table 2).
Specific HPV 18 variants may increase oncogenic potential and may represent an additional factor contributing to the disproportionately high burden of cervical cancer in different regions.20,21 In a previous study of HPV 18, a higher risk of high-grade cervical lesion was observed to associate with E and AA variants.12 Sichero et al11 reported that infection by HPV 18 E variants was more prone to persist. Furthermore, it has been shown that HPV-18 AA variants presented an increased ability of inducing tumor formation in vivo.22 However, Villa et al23 found that infection with non–E variants of HPV 18 had a general tendency to persist more frequently and to be associated with preinvasive lesions. Although these data are controversial, it was already demonstrated that HPV 18 intratypic variants play a different role in the pathogenesis of cervical cancer.21
In the present study, new nucleotide substitutions were found in the HPV-18 E6 and E7 regions. Variation at nucleotide 104 has been reported to be associated with a higher activity of the E6/ E7 promoter by modulating a previously unrecognized Yin Yang (YY1) transcription factors.24 It was also verified that individuals with T104C variation were less likely to present tumor recurrence than those with the reference sequence (HPV18-X05015).24 E6 gene analysis in our strains revealed only one amino acid alteration of H131N in HPV-18 E variants.
The HPV major capsid protein encoded by the L1 open reading frame is the target of neutralizing antibody responses, and naturally occurring L1 antibodies react almost exclusively with conformation-dependent epitopes. L1 hypervariable regions lie on the outward-facing surface of the pentamer25; thus, a challenge is the determination of whether intratype HPV 18 variants are relevant to HPV vaccine strategies.
Changes in the L1 gene may compromise the fitness of the virus and may be important in discriminating the infectious potential of different variants and in defining epitopes relevant to vaccine design.26,27 The previously described substitutions of R25Q, P91Q, P344R, and P399R of L1 were observed in all HPV 18 strains analyzed here, whereas the aa substitutions of A164V, Q334P/H, and D520N were novel mutations. These results extended the data about nucleotide and aa changes in the HPV-18 L1 region.
The L1 pentamer has 5 elbowlike lateral projections that are composed of the approximately 100 residues at the C-terminus. Each projected elbow consists of an α-helix (helix 4 [h4]) anchored to the core structure through 2 other helices, h2 and h3. Pentamers are linked through strong hydrophobic interactions between h4 on one pentamer and h2 and h3 of a neighbor. The remaining C-terminal residues return to the L1 core to form a fifth helix, h5. The structural elements h2 (aa384–393), h3 (aa396–403), and h4 (aa4127–428) near the C-terminal end of L1 are indispensable for the assembly of papillomaviruses into particles.27 The mutation of P399R observed in the L1 gene of our studied strains is located in the h3 region; and the substitution of L1 T149N in HPV-18 E lineage found in our study is located near a linear epitope, which is present in the surface of the capsid and is common among many HPV genotypes.28 Whether these mutations have some effects on its immune response needs to be studied in the future.
In summary, the main HPV 18 variants found in women from Northeast China were AA (81.5%) followed by E (18.5%) branch. Moreover, our data provide several novel HPV 18 nucleotide variations in the E6, E7, and L1 genes. Although a significant correlation between HPV 18 variants and the results from cervical cytopathologic tests was not observed in this study, these data may form a basis for considering HPV 18 sequence variations in E6-, E7-, and L1-based oncogenic potential and vaccine strategies.
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Keywords:Copyright © 2012 by IGCS and ESGO
HPV 18; E6/E7/L1 genes; Genetic diversity