Human papillomavirus (HPV) 18 genetic variants and cervical cancer risk in Taizhou area, China

Accepted Manuscript Human papillomavirus (HPV) 18 genetic variants and cervical cancer risk in Taizhou area, China

Hui-Hui Xu, Ling-Zhi Zheng, Ai-Fen Lin, Shan-Shan Dong, ZeYing Chai, Wei-Hua Yan PII: DOI: Reference:

S0378-1119(18)30045-3 https://doi.org/10.1016/j.gene.2018.01.037 GENE 42487

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Please cite this article as: Hui-Hui Xu, Ling-Zhi Zheng, Ai-Fen Lin, Shan-Shan Dong, ZeYing Chai, Wei-Hua Yan , Human papillomavirus (HPV) 18 genetic variants and cervical cancer risk in Taizhou area, China. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), https://doi.org/ 10.1016/j.gene.2018.01.037

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ACCEPTED MANUSCRIPT Human papillomavirus (HPV) 18 genetic variants and cervical cancer risk in Taizhou area, China Hui-Hui Xu1, Ling-Zhi Zheng2, Ai-Fen Lin3, Shan-Shan Dong3, Ze-Ying Chai2,*, Wei-Hua Yan1,*

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Province, Wenzhou Medical University, Linhai, Zhejiang, China

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1 Laboratory of Gynecologic Oncology, Medical Research Center, Taizhou Hospital of Zhejiang

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2 Department of gynecology and obstetrics, Taizhou Hospital of Zhejiang Province, Wenzhou Medical University, Linhai, Zhejiang, China

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3 Human Tissue Bank, Taizhou Hospital of Zhejiang Province, Wenzhou Medical University,

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Linhai, Zhejiang, China

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Correspondence: Ze-Ying Chai, MD, Department of Gynecology and Obstetrics, E-mail:

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[email protected]; Wei-Hua Yan, PhD, MD, Medical Research Center, Taizhou Hospital of

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Zhejiang Province, Wenzhou Medical University, Linhai, Zhejiang 317000, People’s Republic of China, E-mail: [email protected]

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ABSTRACT Human papillomavirus (HPV) type 18 is predominantly associated with the development of cervical adenocarcinomas, whereas data on HPV18 genetic variability in China are limited. HPV18 genetic variants were formed phylogenetic tree, including lineages A, B, and C. We aimed

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to evaluate the diversity of HPV18 genetic variants by sequencing the entire E6, E7 and L1 genes.

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Between 2012 and 2015, a total of 138 (0.8%, 138/17669) women with single HPV18 infection

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were selected in this study. Finally, we observed 122 HPV18 isolates of the complete E6-E7-L1 sequences, and obtained 36 distinct variation patterns which the accession GenBank numbers as

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KY457805-KY457840. Except KY457805, KY457813, KY457819, KY457827, KY457829, the

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rest of HPV18 isolates (81.1%, 31/36) are novel variants. All of HPV18 variants belong to lineage A, while no lineage B, and C was found in our population of Taizhou region, Southeast China.

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Sublineage A1 was the most common variants (85.2%, 104/122), followed by sublineage A4, A3

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and A5, while no sublineage A2 was obtained. Based on the tree topologies, there were three newly identified candidates’ sublineages A6-A8. Out of 122 women, 67 (54.9%) had diagnosed by

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biopsy, including 49 women who diagnosed with cervicitis, 12 with cervical intraepithelial

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neoplasia (CIN)1, 4 with CIN2/3, and 2 with adenocarcinomas, respectively. Nevertheless, there was no association between HPV18 (sub) lineages and CIN1 or worse (CIN1+) lesions comparing with normal biopsies

(P=0.469).

In conclusion, knowledge of the distribution of

geographic/ethnical HPV18 genetic diversity provides critical information for developing diagnostic probes, epidemiologic correlate of cervical cancer risk and design of HPV vaccines for targeted populations. Keywords: HPV18, Genetic variants, Cervical lesion, Gynecological cancer

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1. Introduction

Cervical cancer is the second most common female malignancy worldwide. In China, approximately 98,900 new cases have been reported in 2015 which accounts for 18.7% of the

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global incidence (Chen et al., 2016). Presence of carcinogenic genotypes of papillomavirus HPV was recognized as a necessary factor in the development of cervical cancer. HPV16 and HPV18

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are well-known as “high-risk” because of their strong carcinogenic potentials (Crosbie et al.,

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2013). HPV18, which was first described in 1984 (Boshart et al., 1984), is widely accepted as the

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second most carcinogenic genotype after HPV16 since they are responsible for approximately 10-15% and 55-65% of all invasive cervical cancers (ICC) worldwide, respectively (Guan et al.,

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2012). Moreover, HPV18 is significantly over-represented in glandular lesions or in adenocarcinomas (ADC) in comparison to squamous cell carcinomas (SCC) (Burk et al., 2003;

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Clifford et al., 2008). These findings suggest HPV18 share a phylogenetic trait that denotes a greater tendency to cause ADC in comparison to other HPV genotypes. Previous study revealed

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that different variants of HPV18 coevolved with the three main phylogenetic human branches of Africans, Whites, and Asians which were clustered into three distinct groups as African (Af),

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European (E), and Asian-American (As/Ai) lineages (Ong et al., 1993). Currently, based on the phylogenetic tree the new nomenclature for HPV18 genetic variants, three lineages have been described: lineages A (that includes previously called As/Ai and E lineages), B and C (these two included previous Af lineage) (Chen et al., 2009). HPV18, which belongs to the alpha genus and the A7 species, is a small double-stranded DNA virus with 8 kb genomes containing early expressed genes (E1, E2, E4, E5, E6, and E7), late genes (L1, L2) and a long control region (Bernard et al., 2013). E6 and E7 are major oncogenes, which 3

ACCEPTED MANUSCRIPT are highly expressed in tumors and related to induce cellular immortalization, malignant transformation, and carcinogenesis. The L1 open reading frame (ORF) encodes the major capsid protein that assemble into virus-like particles (VLPs) are the component of the HPV prophylactic vaccines (Doorbar et al., 2012).

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In our previous study, we found that the natural prevalence of HPV18 was 1.5% among the

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general population of Taizhou area, which is located in the southeast China (Xu et al., 2016).

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Among women diagnosed with cervical intraepithelial neoplasia (CIN) grade 2 or worse (CIN2+), the prevalence of HPV18 was increased to 5.6% (Xu et al., 2016), but far lower than that of 13.0%

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reported in a global meta-analysis (Guan et al., 2012). HPV18 genetic variants have been reported

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to differ in their virus assembly, pathogenic potential, and host immune responses (Xi et al., 2006; Xi et al., 2007; de Araujo Souza et al., 2009), but data on its evolutionary history and genetic

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variability in China are limited. The aim of the present study was: 1) to analyze the nucleotide

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variants in the E6, E7, and L1 viral genes of HPV18, 2) to assess the prevalence of HPV18 variants in our region, and 3) to explore the relationship between HPV18 genetic variants and the

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risk for cervical cancer.

2. Materials and Methods 2.1 Ethics Statement

This study was approved by the Institutional Medical Ethics Review Board of Taizhou Hospital of Zhejiang Province (approval # MERB-2016-047). Informed consent was obtained from all of the participants. Confidentiality was ensured during the data collection process, which was completed by Taizhou Hospital. Data were analyzed anonymously.

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2.2 Study population Between December 2012 and April 2015, a total of 17669 consecutive women (median age 41.3 years; range 16-89) were underwent cervical cancer screening in gynecological clinic at Taizhou

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Hospital of Zhejiang Province, and 3923 (22.2%) of them were HPV positive (Xu et al., 2016). All

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cervical exfoliated cell samples for HPV genotyping were performed with a standard protocol as

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previously described (Xu et al., 2016). Of the 264 (1.5%) women with HPV18 infection, a single infection was detected in 138 (0.8%) women (median age 39.7 years; range 22-70) who were

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selected in the present study. Genomic DNA was extracted using the DNeasy Blood and Tissue kit

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(QIAGEN) according to the manufacturer’s instructions. DNA was extracted from 200μl of each sample and collected in 50μl elution buffer and stored at -20°C until amplification. According to

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the 2012 ASCCP consensus guidelines for the abnormal cervical cancer screening, women with

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HPV18 positive should be referred directly to colposcopy (Massad et al., 2013). Histological diagnoses were adjudicated by two pathologists and classified as normal, CIN grade 1, 2, 3 or

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ICC.

2.3 PCR amplification and sequencing PCR amplifications were performed with 20ng genomic DNA by the following conditions: 1×PCR buffer, 2mM MgCl2, 0.2mM 2′-deoxyribonucleotide 5′-triphosphate mix, 1mM of each primer and 2.0U Taq DNA polymerase (QIAGEN) in a final volume of 50μl. PCR reactions were pre-heated for 5min at 95°C, followed by 35 repeated cycles of 94°C for 30s, 55/60°C for 30s, 72°C for 30s, and a final extension step at 72°C for 10min (Thermo Hybaid, Waltham, MA).

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ACCEPTED MANUSCRIPT Amplification of HPV18 E6, E7 and L1 genes were performed using type-specific primers, which were shown in Table 1. All data were confirmed twice at least by repeat PCR amplification and sequence analysis. PCR products were visualized by 1.5% agarose gel, and then sequenced (BigDye™ Terminator v3.1

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Cycle Sequencing Kit) by Beijing Genomics Institute and Shanghai Genomics Institute (ABI 3730,

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Applied Biosystems, Foster City, CA, USA). The same forward and reverse primers were used

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2.4 Variant analysis and phylogenetic tree construction

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separately in cycle sequencing in order to sequence both sense and antisense strands.

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HPV18 prototype reference sequence (GenBank accession number NC001357), which belongs to the A1 sublineage, was used as the standard for comparisons and nucleotide position numbering

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in this study. The nucleotide sequences that contained complete E6, E7 and L1 genes were aligned

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and compared to the HPV 18 prototype sequence, using NCBI Blast, BioEdit Sequence Alignment Editor, and Clustal X 1.83. The nucleotide sequences were translated into proteins using MEGA

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version 5.05 for determination of amino acid changes caused by nucleotide changes.

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Other entire E6-E7-L1 sequences were downloaded from GenBank (A1 sublineage: EF202143EF202145, A2 sublineage: EF202146, A3 sublineage: EF202147-EF202149, A4 sublineage: EF202150-EF202151, A5 sublineage: GQ180787; B1 sublineage: EF202153-EF202155, B2 sublineage: KC470224-KC470225, B3 sublineage: EF202152; and C lineage: KC470229KC470230). Maximum likelihood tree was constructed using the MEGA version 5.05 with 1000 bootstrapped replicates and values above 50% were considered significant.

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3. Results 3.1 Characteristics of the Population 138 women with HPV18 single-infection were obtained from our previous TZHPV Study (Xu et al., 2016). 131 (94.9%) E6-E7 sequences and 124 (89.9%) L1 sequences from HPV18 isolates

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were successfully amplified by PCR. Other sequences were excluded because of virus DNA

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integration or PCR failure. In total, the complete E6-E7-L1 genes of 122 (88.4%) HPV18 isolates

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were sequenced for variant analysis. Out of 122 women, 67 (54.9%) had diagnosed by biopsy, including 49 women who diagnosed with cervicitis, 12 with CIN1, 4 with CIN2/3, and 2 with

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adenocarcinomas, respectively.

3.2 Nucleotide variants

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All sequences used in this analysis were submitted to GenBank, and the GenBank accession

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numbers were KY457805-KY457840. All of HPV18 variants belong to lineage A, while no lineage B, and C was found in our population of Taizhou region, Southeast China. Compared to

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the HPV18 prototype sequence (NC001357), we detected 64 nucleotides substitutions, with 37/64

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(57.8%) being novel substitution and 30/64 (46.9%) being non-synonymous substitution. A summary of nucleotide and amino acid sequence variation throughout the HPV18 E6-E7-L1 region is shown in Figure 1. In the E6 gene, eleven nucleotide variants were detected. Five of them were specific to the A1 sublineage: two synonymous substitutions T149C, A482C and three non-synonymous substitutions G189C (E29Q), G222A (E40K), T382G (L93R). Four synonymous substitution C153T, A377G, T485C, C549A and one non-synonymous substitution T314G (D70E) was found

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ACCEPTED MANUSCRIPT to be specific to the A3-A8 sublineages, while T485C and C549A was only detected among the European lineage (except A6 sublineage). In our study, synonymous substitution C287G was observed in all HPV18 isolates. E7 gene genetic variability analysis revealed four nucleotides substitutions. One synonymous

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substitution C860T was detected in A1 sublineage. One synonymous substitution C751T and two

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non-synonymous substitutions T734G (L49V), A770C (M61L) was found to be specific for the

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A3-A5, and A8 sublineages. To our knowledge, the base substitutions of G189C, G222A, T314G, T382G in E6 and T734G, A770C, C860T in E7 had never been reported in previous studies.

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L1 sequence analysis revealed forty-nine nucleotides substitutions and demonstrated the

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presence of substitutions G5503A (R25Q), C5701G (P91R), C6460G (P344R), C6625G (P399R), and C6842G in all our isolates. One non-synonymous substitution C5920T (A164V) was detected

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in lineage A. Seventeen substitutions were specific to the A1 sublineage: seven synonymous

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substitutions C5478T, T5801C, A5832C, A5924C, G6071A, C6170A, G6400A and ten non-synonymous substitutions C5496T (H23Y), T5570G (I47M), A5580G (R51G), G5671A

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(R81K), G5952A (G175S), G5991C (D188H), A6943C (K505T), A7045C (K539T), T7092G

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(S555A), G7129C (R567T). Seventeen synonymous substitutions A5741G, T5810G, A5825G, A5828C, A5852C, T5942G, T5966C, C6014T, A6059G, G6089A, G6143C, T6182G, A6638G, G6929A, T6983A, G7022A, G7130A and nine non-synonymous substitutions G5446A (R6Q), A5497C (H23P), T5619A (L64M), A5796G (I123V), C5875A (T149N), A6401G (R324K), C6427A (P333H), A6430C (Q334P), A6923C (E498D) was found to be specific for the European lineage. To our knowledge, most of them are novel nucleotide substitutions detected in our study except for C5496T, A5497C, G5503A, T5619A, C5701G, C5875A, C5920T, T5942G, G6089A,

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ACCEPTED MANUSCRIPT G6400A, A6401G, A6430C, C6460G, C6625G, C6842G, A6943C, G7022A, A7045C, and G7130A in L1.

3.3 HPV18 variants

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We obtained 36 distinct variation patterns denoted as 18CNTZ01-18CNTZ36, and the accession

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GenBank numbers was KY457805-KY457840. 31 (81.1%, 31/36) novel HPV18 variants were

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detected in our study (Figure 1). 18CNTZ01, which was reported in 2009 (GenBank accession number EF202143) (Chen et al., 2009), is the most common variants (60.7%, 74/122) in our

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population. Four variants 18CNTZ09, 18CNTZ15, 18CNTZ23, and 18CNTZ25 were reported in

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previous studies (KU721790 (unpublished), EF202145 (Chen et al., 2009), KU707802 (King et al. 2016), KU707808 (King et al. 2016), respectively). The novel variant 18CNTZ18 was the second

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most common (6.6%, 8/122) in our population. Followed by 18CNTZ16 (2.5%, 3/122),

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18CNTZ25 (2.5%, 3/122), 18CNTZ11 (1.6%, 2/122), 18CNTZ23 (1.6%, 2/122), and the remaining HPV18 variants (0.8%, 1/122).

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In addition to previously described lineages and sublineages (A1 to A5, B1 to B3, and C) (Burk

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et al., 2013; Chen et al., 2013), we saw evidence of three candidate A6-A8 sublineages according to the phylogenetic tree of our study (Figure 2). Lineage A was further divided into eight sublineages A1-A8 based on tree topologies, of which A1, A2 belong to the Asian-American branch and A3-A8 belong to the European branch. Isolates of all Asian-American variants from our population mapped to the A1 sublineage contains 21 variants (58.3%) including 18 novel variants (50.0%). HPV18 prototype was also assigned to the A1 sublineage. There was no HPV18 variant found in A2 sublineage. The A3 and A4 sublineages contain 15 variants (41.7%) including

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ACCEPTED MANUSCRIPT 13 novel variants (36.1%). One variant 18CNTZ28 represented A5 sublineage, which appeared to be uniquely defined by the substitutions A5741G, A5796G (I123V), A6059G, G6089A, G6143C, A6923C (E498D), and G7130A in L1. However, 18CNTZ28 variant sequence is not consistent with the previously reported A5 whole-genome sequence GQ180787 from Thailand (Chen et al.,

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2013; Lurchachaiwong et al., 2010), which has 149C, 153C, 770A, 5790A, 5914T, 6053A, and

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6917A. In addition, we observed nine variants representing ten samples that appeared to form

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three new candidates, A6-A8 sublineages. A6 sublineage appeared to be uniquely defined by the C6014T in L1 and A7 to be uniquely defined by the T5810G, in L1 which were first detected in

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our study. 18CNTZ32 variant was found to be recombinant, which classified as European branch

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but in E6 region belonged to the Asian-American branch. Furthermore, 18CNTZ22 and 18CNTZ23 represented A8 sublineage appeared to be uniquely defined by the T5942G, G7022A in

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L1, but differ with the A3-A5 sublineages, which has G6401A (R324K).

3.4 Genetic variants and cervical lesion

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Out of 122 women, 67 (54.9%) had diagnosed by biopsy, including 49 women with cervicitis,

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12 with CIN1, 4 with CIN2/3, and 2 with adenocarcinomas, respectively (Table 2). We detected the presence of the 18CNTZ01 variant in both cases of adenocarcinomas. 18CNTZ01, which was reported in previous study in 2009 (GenBank accession number EF202143) (Chen et al., 2009), was the most common variant (60.7%, 74/122) in our population. Four cases of CIN2/3 were detected three A1 sublineage (two 18CNTZ01 and one 18CNTZ18) and one European variant 18CNTZ22, which nucleotides substitutions of T314G (D70E) in E6 and T734G (L49V) in E7. Our results suggest that the two sites may affect E6 and E7 function. Twelve cases of CIN1 were

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ACCEPTED MANUSCRIPT detected nine A1 sublineage (five 18CNTZ01, one 18CNTZ11, one 18CNTZ14, one 18CNTZ18, one 18CNTZ19) and three European lineages (one 18CNTZ23, one 18CNTZ31, one 18CNTZ35). Nevertheless, there was no association between HPV18 (sub) lineages and CIN1+ lesions comparing with the normal biopsies (P=0.469).

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Previous studies confirmed that different HPV 18 variants were associated with progression of

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cervical neoplasia dependent on geography and ethnicity (Villa et al., 2000; Sichero et al., 2007).

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Therefore, nucleotides substitutions may play an important role in HPV 18 variants obtained from our population. Substitutions C287G in E6, and G5503A, C5701G, C6460G, C6625G, C6842G in

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L1 were found in all HPV18 isolates. Furthermore, seven novel nucleotides substitutions G189C,

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G222A, T314G, T382G in E6 and T734G, A770C, C860T in E7 have never been reported in previous studies and six of them were non-synonymous and lead to amino acid changes, which

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may interfere with the viral oncogenic potential.

4. Discussion

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Human papillomavirus (HPV) variants differ in geographic origins, evolutionary dynamics, and

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pathogenicity. The HPV18 is associated with a higher proportion of ADC in comparison with other HPV (Burk et al., 2003; Clifford et al., 2008), whereas data on HPV18 genetic variability in China are limited. Moreover, identification of HPV genetic diversity in specific clinical settings may be important for the rational design of diagnostic, and vaccine strategies (Li et al., 2011). In this study, we analyzed the genetic variants in HPV18 E6, E7, and L1 viral genes, constructed phylogenetic trees of all HPV18 variants, and explored the relationship between HPV18 genetic variants and the risk for cervical cancer in our population.

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ACCEPTED MANUSCRIPT In this study, we observed 122 cases of HPV18 variants in the complete E6-E7-L1 genes, which is the first comprehensive study to explore HPV18 evolutionary history and genetic variants in Southeast China. The changes of the amino acid sequence may influence the transforming activity of the E6, and E7 genes, which integrate into host genome, resulting in inhibit Rb and p53

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functions and induce cellular immortalization and transformation (Bernard et al., 2013; Doorbar et

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al., 2012). L1 gene encodes the viral major capsid protein which is the primary target for

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prophylactic vaccination (Erickson et al., 2014). Substitution C287G, G5503A, C5701G, C6460G, C6625G, and C6842G were found in all HPV18 isolates in our population of Taizhou region,

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Southeast China. The synonymous substitution C287G may be a common nucleotides substitution

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worldwide (Arroyo et al., 2012; Sun et al., 2012; Shen et al., 2013). In China, the substitution C287G were found in all HPV18 isolates in Northeast (Sun et al., 2012) and Southeast region, but

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the frequent in Southwest region was 48.2% (Shen et al., 2013). The frequent of G5503A, C5701G,

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C6460G, C6625G, and C6842G were found in all HPV18 isolates in Southeast region, but was about 40% in Southwest region (Shen et al., 2013). Thus, it is worthy to evaluate the L1 gene in

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geographical region and racial characteristics of the study population. The AS04-adjuvanted

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HPV16/18 vaccine (Cervarix®, GlaxoSmithKline Biologicals, Belgium), which has been licensed in China in 2015, is a prophylactic vaccine containing a mixture of virus-like particles derived from the L1 proteins of HPV16 and 18 (McKeage et al., 2011; Zhu et al., 2017). In addition, our data about the genetic diversity of HPV18 variants in China may be helpful to design the second-generation prophylactic HPV vaccine strategies and implement a feasible nationwide vaccination programs. The distribution of HPV18 variants (sub)lineages in Southeast China were confirmed to be

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ACCEPTED MANUSCRIPT geographically/ethnically specific (Xi et al., 2006; de Araujo Souza et al., 2009; Chen et al., 2013). Consistent with previous nomenclature of A1/A2 branch as Asian-American lineage (Burk et al., 2013), our results shown the A1 variants account for the majority (85.2%) but no A2 variants in Southeast China. Additionally, our results also show that there are no B and C branches, which are

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specific for Africa (Ong et al., 1993; Chen et al., 2013; Schlecht et al., 2005). In our study, the

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18CNTZ28 variant was classified as A5 branch; its sequence is not consistent with the previously

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reported A5 whole-genome sequence GQ180787 from Thailand (Chen et al., 2013; Lurchachaiwong et al., 2010), which has 149C, 153C, 770A, 5790A, 5914T, 6053A, and 6917A.

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Of note, the A5 branch was found in a higher proportion of HPV18 isolates from northern Africa

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than elsewhere (Chen et al., 2015). Moreover, the 18CNTZ32 variant was found to be recombinant, which classified as European (A6) branch but in E6 region belonged to the Asian-American (A1)

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branch. Our data provided new evidence of recombination in HPV, which is an important

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molecular evolutionary mechanism that could impact pharmacogenomics and vaccine development (Angulo et al., 2007).

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Previously studies reported the risk of developing high-grade CIN to be significantly higher in

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non-European variants (Villa et al., 2000; Arroyo et al., 2012). Xi et al (Xi et al., 2007) reported that Asian-American/European variants were found to be statistically signigicant more associated with pre-invasive lesions than African variants. In contrast, other studies showed that no significant difference in pre-invasive lesions risk was observed between the variant lineages (A, B, and C) (Chen et al., 2015; Schiffman et al., 2010; Arias-Pulido et al., 2005). Furthermore, there was not a statistically significant association between HPV18 (sub)lineages level and cervical pre-invasive lesions risk in Southeast China(P=0.469). This result may have influenced by the

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ACCEPTED MANUSCRIPT limited sample size of case-matched HPV18-related cervical lesions in this study. A1 variants account for the majority in eastern Asia (Chen et al., 2015; Sun et al., 2012), but significantly underrepresented in South/Central Asia (Chen et al., 2015). Such regional differences should be taken into account in assessing pre-invasive lesions risk as described above. Whatever the

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underlying cause, such apparent regional difference reveals the inherent complexity of HPV

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variants and cervical cancer risk studies, and gives warnings about the extent to which data can be

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pooled across countries/regions.

Our study was limited by the number of HPV18-poistive samples, due to the low natural

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incidence of HPV18 in our region. However, the number of HPV18 positive samples obtained

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from our previous TZHPV study (2012-2015) (Xu et al., 2016) was by far the largest studied in China. In this study, we reported 31 novel HPV18 variants that will ensure sequencing of the

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whole genomes in order to strengthen the full picture of HPV18 genetic evolution in our ensuing

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studies.

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5. Conclusion

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In summary, our present study provides a practical approach for phylogenetic classification for use in epidemiological studies of the natural history and carcinogenicity of HPV18 genetic variants in China. Data about the geographic/ethnical HPV18 genetic diversity distribution may be helpful to design of diagnostic probes, epidemiologic correlate of cervical cancer risk and design of HPV vaccines for targeted populations in China.

Competing interests

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ACCEPTED MANUSCRIPT The authors declare that they have no competing interests.

Acknowledgments This work was supported by grants from the National Natural Science Foundation of China

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(31370920, 81372247), the Science and Technology Bureau of Zhejiang Province (2016C33231),

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the Health Bureau of Zhejiang Province (2015KYB438) and the Zhejiang Provincial program for

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the cultivation of high-level innovative health talents.

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Authors’ contributions

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HHX, AL, and WHY designed the experiments, performed analysis and drafted the manuscript. LZZ, ZYC, and SSD carried out the sample collection, performed laboratory testing. All authors

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read and approved the final manuscript.

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ACCEPTED MANUSCRIPT Legends: Figure 1. Genetic variability of HPV18 E6-E7-L1 sequences in Taizhou area, Zhejiang province. Numbering refers to the first nucleotide of the HPV18 prototype reference sequence (GenBank accession number NC001357). Each row indicates the isolate identification and the

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nucleotide sequence alignment compared to the reference. Isolates EF202143-EF202155 are HPV

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18 known variant sequences which belong to lineage A, B and C. Novel HPV18 variants are

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highlighted in bold and novel nucleotide substitutions are highlights in gray.

Figure 2. Phylogenetic tree of the HPV18 variants. Maximum likelihood analysis (with MEGA

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5.05 program) of E6-E7-L1 nucleotide sequences. Numbers below branches indicate bootstrap

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Table 1. Polymerase chain reaction characteristics for Human papillomavirus 18.

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Table 2. Distribution of HPV18 (sub) lineages in grade of cervical lesion.

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Table 1. Polymerase chain reaction characteristics for Human papillomavirus 18 Site**

Annealing Temp/Cycles

Amplicon size

GGAGTRACCRAAAACGGTYG CCTTCTGRATCAGCCATTGTT GTMTCTGCYACGGRGGACAA CACAGCTGCCAGGTGAAGC CTGGATATGGTGCCAYGGRC CTCACYAGGGCGCAACCACAT

36-55 909-930 5258-5277 6462-6480 6220-6239 7308-7328

55°C /35 cycles

895 bp

60°C /35 cycles

1223bp

60°C /35 cycles

1109bp

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18E6/7 F 18E6/7 R 18L1 F1 18L1 R1 18L1 F2 18L1 R2

Primer Sequence(5′ - 3′) *

EF202143-EF202155, KU298886, KC470208-KC470230, GQ180784-GQ180792);

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* The degenerate primers were matched with complete genomes of the HPV18 from the Nucleotide Database (accession number

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** Position numbering refers to the first nucleotide of the HPV18 reference genome (accession number NC001357).

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Table 2. Distribution of HPV18 (sub)lineages in grade of cervical lesion CIN2/3

Adenocarcinomas

38.6 ± 8.8 42 0 0 3 1 1 2 0 49

40.1 ± 11.3 9 0 0 0 0 1 1 1 12

40.1 ± 8.5 3 0 0 0 0 0 0 1 4

59.0 ± 5.7 2 0 0 0 0 0 0 0 2

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CIN1

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Ages A1 A2 A3 A4 A5 A6 A7 A8 Total

Cervicitis

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ACCEPTED MANUSCRIPT Highlights  The data on HPV18 genetic variability in Southeast China are limited.  We obtained 36 distinct variation patterns denoted as 18CNTZ01-18CNTZ36 (GenBank accession numbers were KY457805-KY457840)，and 31 (86.1%) are novel.  All of HPV18 variants belong to lineage A, while no lineage B, and C was found in our

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population of Taizhou region, Southeast China.  There was no association between HPV18 (sub) lineages and CIN1+ lesions comparing with

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the normal biopsies.

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ACCEPTED MANUSCRIPT Abbreviations

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ADC:adenocarcinomas Af:African As/Ai:Asian-American CIN: cervical intraepithelial neoplasia E:European HPV:Human papillomavirus ICC:invasive cervical cancers ORF:open reading frame SCC:squamous cell carcinomas VLPs:virus-like particles

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Figure 1

Figure 2