A1 Sublineages Research Articles

Identification of HPV16 Lineages in South African and Mozambican Women with Normal and Abnormal Cervical Cytology.

Human papillomavirus 16 (HPV16) is an oncogenic virus responsible for the majority of invasive cervical cancer cases worldwide. Due to genetic modifications, some variants are more oncogenic than others. We analysed the HPV16 phylogeny in HPV16-positive cervical Desoxyribonucleic Acid (DNA) samples collected from South African and Mozambican women to detect the circulating lineages. Polymerase chain reaction (PCR) amplification of the long control region (LCR) and 300 nucleotides of the E6 region was performed using HPV16-specific primers on HPV16-positive cervical samples collected in women from South Africa and Mozambique. HPV16 sequences were obtained through Next Generation Sequencing (NGS) methods. Geneious prime and MEGA 11 software were used to align the sequences to 16 HPV16 reference sequences, gathering the A, B, C, and D lineages and generating the phylogenetic tree. Single nucleotide polymorphisms (SNPs) in the LCR and E6 regions were analysed and the phylogenetic tree was generated using Geneious Prime software. Fifty-eight sequences were analysed. Of these sequences, 79% (46/58) were from women who had abnormal cervical cytology. Fifteen SNPs in the LCR and eight in the E6 region were found to be the most common in all sequences. The phylogenetic analysis determined that 45% of the isolates belonged to the A1 sublineage (European variant), 34% belonged to the C1 sublineage (African 1 variant), 16% belonged to the B1 and B2 sublineage (African 2 variant), two isolates belonged to the D1-3 sublineages (Asian-American variant), and one to the North American variant. The African and European HPV16 variants were the most common circulating lineages in South African and Mozambican women. A high-grade squamous intraepithelial lesion (HSIL) was the most common cervical abnormality observed and linked to European and African lineages. These findings may contribute to understanding molecular HPV16 epidemiology in South Africa and Mozambique.

Distribution of Human Papillomavirus Genotypes among the Women of South Andaman Island, India.

Human Papillomavirus (HPV) causes various types of cancer in both men and women. Woman with HPV infection has a risk of developing invasive cervical cancer. Globally, HPV 16 and 18 were predominant. This study aims to find the distribution of various HPV types in South Andaman. A cross-sectional study was conducted among women in South Andaman, where cervical scrapes were collected after collecting written informed consent. Detection of HPV genotypes was carried out by using a PCR assay. Further, sequencing analysis was performed using MEGA11 to identify various genotypes in this territory. Of these 1000 samples, 32 were positive for HR-HPV 16, and four were positive for HR-HPV 18. Fifteen HPV genotypes were detected using molecular evolutionary analysis. Six cases were identified with multiple genotypes. The most prevalent genotype is HPV 16 which belongs to Lineage-A and sub-lineage A2. HPV 18 identified in South Andaman belonged to the lineage A1 to A5. Various HPV types were identified among women in South Andaman. Global burden of cervical cancer associated with various HPV sub-lineages. HPV-16 A1 sub-lineage was globally widespread, whereas sub-lineages A1, A2 and D1 prevailed in South Andaman. HR-HPV identified in this study enlightens the importance of HPV vaccination among women in remote places. These findings will help to strengthen public health awareness programs and prevention strategies for women in remote areas.

Genetic diversity and functional implication of the long control region in human papillomavirus types 52, 58, and 16 from Central China.

High-risk human papillomavirus (HR-HPV) is a main reason for cervical cancer. The long control region (LCR) of the genome plays a variety of roles in the transcription of the virus. LCR sequences were amplified by polymerase chain reaction (PCR) and confirmed by DNA sequencing. MEGA 11.0 software and NCBI blast were used to analyze the sequences and construct the Neighbor-Joining tree. In addition, the JASPAR database was used to predict the potential transcription factor binding sites (TFBS). For HPV-52 LCR, 68 single nucleotide polymorphisms (SNPs), 8 deletions, and 1 insertion were found, 17 of which were novel variations. Most of the variants were clustered in B2 sub-lineage (96.22%). For HPV-58 LCR, 25.43% of samples were prototype. 49 SNPs, 2 deletions, and 1 insertion were observed in the remaining samples. A1 sub-lineage was the most frequent (64.16%). For HPV-16 LCR, 75 SNPs and 2 deletions were identified, 13 of which were newly identified. A total of 55.68% of the variants were distributed in A4 sub-lineage. The JASPAR results suggested that multiple variations occurred in TFBSs, which might affect the function of transcription factors. This study provides experimental data for further studies on the epidemiology and biological function of LCR. Various LCR mutational data may prove useful for exploring the carcinogenic mechanism of HPV.

Genotypic distribution of human papillomavirus and phylogenetic analysis of E6 and E7 gene of HR-HPV variants isolated from Pakistani population

High-risk-human papillomavirus (HR-HPV)-induced cervical cancer is the second most common cause of death among females worldwide. HPV16 is the most prevalent HR-HPV infection worldwide. This study found the genotypic distribution of HR-HPV in the local population and investigated the sequence variations among the E6 and E7 oncogenes of the local HPV16 genotype to the E6 and E7 oncogenes of the foreign HPV16 genotypes and constructed a phylogenetic relationship based on nucleotide sequence comparison among the variants identified in our study along with previously reported isolates that were obtained from different regions of the world. The samples were collected from patients with cervical cancer. Genomic DNA was extracted, and HR-HPV genotypes were determined using real-time PCR. The HPV16 E6 and E7 genes were amplified and sequenced. A HPV16 phylogenetic tree was constructed using the maximum likelihood method with MEGA 7. HPV16 was the most prevalent human papillomavirus (HPV) type identified in the present study. HPV16 isolates belonged to the A1 sublineage of the European branch. Twenty-one nucleotide sequences were included in this analysis. The first, second, and third codon positions are also included. The final dataset included 776 positions.

Characterization of the Human Papillomavirus 16 Oncogenes in K14HPV16 Mice: Sublineage A1 Drives Multi-Organ Carcinogenesis.

The study of human papillomavirus (HPV)-induced carcinogenesis uses multiple in vivo mouse models, one of which relies on the cytokeratin 14 gene promoter to drive the expression of all HPV early oncogenes. This study aimed to determine the HPV16 variant and sublineage present in the K14HPV16 mouse model. This information can be considered of great importance to further enhance this K14HPV16 model as an essential research tool and optimize its use for basic and translational studies. Our study evaluated HPV DNA from 17 samples isolated from 4 animals, both wild-type (n = 2) and HPV16-transgenic mice (n = 2). Total DNA was extracted from tissues and the detection of HPV16 was performed using a qPCR multiplex. HPV16-positive samples were subsequently whole-genome sequenced by next-generation sequencing techniques. The phylogenetic positioning clearly shows K14HPV16 samples clustering together in the sub-lineage A1 (NC001526.4). A comparative genome analysis of K14HPV16 samples revealed three mutations to the human papillomaviruses type 16 sublineage A1 representative strain. Knowledge of the HPV 16 variant is fundamental, and these findings will allow the rational use of this animal model to explore the role of the A1 sublineage in HPV-driven cancer.

Prevalence and persistence of HPV-16 molecular variants in the anal canal of men: The HIM study

BackgroundHPV-16 causes approximately 90% of anal canal (AC) cancers worldwide. This study aimed to evaluate the prevalence and persistence of HPV-16 genetic variants in the AC of men from three different countries (Brazil, Mexico and United States) and to further identify sociodemographic and behavioral factors associated with these infections. MethodsParticipants from the multinational prospective HPV Infection in Men (HIM) Study who had at least one HPV-16 positive AC swab were included. Characterization into HPV-16 genetic variants was successfully performed by PCR-sequencing in 95.6% (217/227) samples and these were classified into HPV-16 lineages and sublineages. ResultsWe observed higher prevalence of lineage A variants, mainly from A1 sublineage, in all countries. Non-A lineage variants were mostly detected in men from Brazil, where higher diversity of sublineage variants was detected during follow-up. Compare to men detected with Non-A HPV-16 lineage variants, men infected with lineage A reported a higher lifetime number of female sexual partners. Finally, a significantly higher prevalence of Non-A lineage variants was observed among men who have sex with men (MSM) with a transient HPV-16 AC infection (p = 0.033), but no significant differences regarding variants lineages and persistence status were observed when stratified by country, self-reported ethnicity or age. ConclusionsOur data extend previous reports which indicate that globally HPV-16 variants are unevenly distributed, and contribute further to studies of the natural history of AC HPV infections in men.

Lineage analysis of human papillomavirus type 39 in cervical samples of Iranian women

BackgroundThe data with regards to the regional variants of distinct HPV types is of great value. Accordance with this, this study aimed to investigate the sequence variations of E6 gene and long control region of HPV 39 among normal, premalignant and malignant cervical samples in order to characterize the frequent HPV 39 variants circulating in Tehran, Iran.MethodsIn total, 70 cervical samples (45 normal, 16 premalignant, and 9 malignant samples) infected with HPV 39 were analyzed by nested-PCR and sequencing.ResultsOur results revealed that all samples belonged to A lineage. Almost all sequences (98.6%) were classified in A1 sublineage and only one sample (1.4%) was A2 sub lineage.ConclusionsOur findings showed that lineages A, sublineage A1, is dominant in Tehran, Iran. However, the small sample size was the most important limitations of this study. Further studies with larger sample size from different geographical regions of Iran are necessary to estimate the pathogenicity risk of HPV 39 variants in this population.

Abstract PO-087: Anal cancer and its association with HPV16 lineage B in African Americans

Abstract Background: Human papillomavirus type 16 (HPV16) is one of the most common and carcinogenic HPV types associated with high risk of anal, vagina, vulva, penis and cervical neoplastic transformations. However, many genetic variants exist within this virus and not all seem to have the same carcinogenic potential. Aim: To determine HPV16 lineages and their association with risk of high-grade anal lesions in African Americans in an inner-city hospital. Methods: We reviewed medical records of 370 African Americans with anal lesions from Jan. 2007 to Dec. 2015. This study was approved by Howard University Institutional Review Board. Demographic, clinical and pathological data including HPV, HIV, HCV (hepatitis C virus), diabetes mellitus, hypertension and body mass index (BMI) were collected. DNA was extracted from a subset of HPV16-positive patients with FFPE tissue samples (72 patients, 111 samples) and used for HPV16 whole-genome sequencing. We assessed HPV16 variant lineages and associations with disease stage. Statistical analyses were performed using Chi-square tests, Student’s t-tests, and logistic regression. Odds ratios (OR) and p-values were calculated for comparisons of normal/condyloma/high-grade dysplasia (HGD) vs. squamous cell carcinoma (SCC) and for normal/condyloma vs. HGD. The most common HPV16 A1 sublineage was used as a reference in these comparisons. Results: Males represented 75% of the patients (n=276), with a median age of 44 years and BMI of 25.8 kg/m2. The frequency of condyloma, high-grade dysplasia, SCC and adenocarcinoma was 191 (52%), 26 (7%), 31 (8%) and 8 (2%), respectively. The frequency of HPV, HIV, and HCV was 231 (68%), 147 (43%) and 42 (12%), respectively. HPV and HIV were risk factors for condyloma and dysplasia (P<0.05). All four main lineages of HPV16 (A,B,C,D) were detected in our specimens, with sublineage A1 most common. Lineage B, also named the African-1 HPV16 lineage because it is most common in Africa, had the strongest association with SCC (OR=10.5) whether alone or in combination with lineages A4 and D (OR=10.5), although with a lower statistical significance (0.054 vs. 0.009). As for HGD, lineage B along with A4, C and D only gave an OR of 1.4. Conclusion: We show that the majority of patients with anal lesions are young males with HPV and HIV co-infections. HPV16 lineage B was associated with a high risk of SCC development. Citation Format: Hassan Ashktorab, Adeyinka Laiyemo, Lisa Mirabello, Hassan Brim. Anal cancer and its association with HPV16 lineage B in African Americans [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-087.

Abstract 2027: Anal cancer among African Americans associate with HPV16 lineage B and HIV

Abstract Background: Human papillomavirus type 16 (HPV16) is one of the most common and carcinogenic HPV types associated with high risk of anal, vagina, vulva, penis and cervical neoplastic transformations. However, many genetic variants exist within this virus and not all seem to have the same carcinogenic potential. Aim: To determine HPV16 lineages and their association with risk of high-grade anal lesions in African Americans in an inner-city hospital. Methods: We reviewed medical records of 370 African Americans with anal lesions from Jan. 2007 to Dec. 2015. This study was approved by Howard University Institutional Review Board. Demographic, clinical and pathological data including HPV, HIV, HCV (hepatitis C virus), diabetes mellitus, hypertension and body mass index (BMI) were collected. DNA was extracted from a subset of HPV16-positive patients with FFPE tissue samples (72 patients, 111 samples) and used for HPV16 whole-genome sequencing. We assessed HPV16 variant lineages and associations with disease stage. Statistical analyses were performed using Chi-square tests, Student's t-tests, and logistic regression. Odds ratios (OR) and p-values were calculated for comparisons of normal/condyloma/high-grade dysplasia (HGD) vs. squamous cell carcinoma (SCC) and for normal/condyloma vs. HGD. The most common HPV16 A1 sublineage was used as a reference in these comparisons. Results: Males represented 75% of the patients (n=276), with a median age of 44 years and BMI of 25.8 kg/m2. The frequency of condyloma, high-grade dysplasia, SCC and adenocarcinoma was 191 (52%), 26 (7%), 31 (8%) and 8 (2%), respectively. The frequency of HPV, HIV, and HCV was 231 (68%), 147 (43%) and 42 (12%), respectively. HPV and HIV were risk factors for condyloma and dysplasia (P<0.05). All four main lineages of HPV16 (A,B,C,D) were detected in our specimens, with sublineage A1 most common. Lineage B, also named the African-1 HPV16 lineage because it is most common in Africa, had the strongest association with SCC (OR=10.5) whether alone or in combination with lineages A4 and D (OR=10.5), although with a lower statistical significance (0.054 vs. 0.009). As for HGD, lineage B along with A4, C and D only gave an OR of 1.4. Conclusion: We show that the majority of patients with anal lesions are young males with HPV and HIV co-infections. HPV16 lineage B was associated with a high risk of SCC development. Citation Format: Hassan Brim, Lisa Mirabello, Ali Afsari, Muneer Abbas, Meredith Yeager, Joseph Boland, Sara Bass, Mia Steinberg, Michael Cullen, Adeyinka Laiyemo, Tammy Naab, Babak Shokrani, Edward Lee, Mehdi Nouraie, Hassan Ashktorab. Anal cancer among African Americans associate with HPV16 lineage B and HIV [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2027.

Impact of naturally occurring variation in the human papillomavirus (HPV) 33 capsid proteins on recognition by vaccine-induced cross-neutralizing antibodies.

We investigated naturally occurring variation within the major (L1) and minor (L2) capsid proteins of human papillomavirus (HPV) genotype 33. Pseudoviruses (PsV) representing HPV33 lineages A1, A2, A3, B and C exhibited comparable particle-to-infectivity ratios and morphology but demonstrated a decreased sensitivity (A2, A3, B and C) to cross-neutralization by HPV vaccine antibodies compared to the A1 sublineage. Chimeric PsVs demonstrated that these differences in sensitivity were due to polymorphisms in the L1 protein, with little or no influence from variation within the L2 protein. Site-directed mutagenesis of the L1 gene identified the DE loop residue 133 and the FG residue 266 as being critical for conferring this differential sensitivity. The use of HPV33 homology models based upon the HPV16 crystal structure suggested that they are likely to act independently on more than one antibody epitope. These data improve our understanding of the potential impact of natural capsid variation on recognition by vaccine antibodies.

Whole-Genome Analysis of Human Papillomavirus Types 16, 18, and 58 Isolated from Cervical Precancer and Cancer Samples in Chinese Women

Human papillomavirus (HPV) types 16, 18 and 58 are ranked the top three high-risk HPV types for cervical intraepithelial neoplasia (CIN) and invasive carcinoma. We aimed to evaluate the diversity of HPV16, HPV18, and HPV58 genetic variants by HPV capture technology combined with next generation sequencing. 295, 73, and 148 variations were observed in 51 HPV16, 7 HPV18, and 11 HPV58 genomes, respectively. HPV16 isolates were predominantly of the A variant lineage, and sublineage A4 (Asian) was the most common. However, there were no significant differences in the distribution of HPV16 A1–3 and A4 variants between CIN1-, CIN2/3, and cervical cancer groups. The 7 HPV18 genomes were assigned to the A3/A4 and A1 sublineages. Of the 11 HPV58 genomes, the most predominant variant sublineages were A2, followed by A1 and B2. The majority of HPV16/18 samples containing contiguous genomic deletions were found to harbor HPV integration. Some T-cell epitope sequences in HPV16 E6 and E7 showed considerable divergence from the prototype NC_001526, suggesting their importance in immunotherapy of HPV-associated carcinomas. In conclusion, sequence diversity and phylogenies of HPV16, 18, and 58 provide the basis for future studies of discrete viral evolution, epidemiology, pathogenicity, and the differences in response to vaccines.

Naturally Occurring Major and Minor Capsid Protein Variants of Human Papillomavirus 45 (HPV45): Differential Recognition by Cross-Neutralizing Antibodies Generated by HPV Vaccines

We investigated naturally occurring variation within the major (L1) and minor (L2) capsid proteins of human papillomavirus genotype 45 (HPV45). Pseudoviruses (PsVs) representing HPV45 sublineages A1, A2, A3, B1, and B2 exhibited comparable particle-to-infectivity ratios and morphologies but demonstrated both increased (A2, A3, and B1) and decreased (B2) sensitivities to cross-neutralization by HPV vaccine antibodies compared to that of the A1 sublineage. Mutant PsVs identified HI loop residue 357 as being critical for conferring this differential sensitivity.

Human papillomavirus 33 worldwide genetic variation and associated risk of cervical cancer

Human papillomavirus (HPV) 33, a member of the HPV16-related alpha-9 species group, is found in approximately 5% of cervical cancers worldwide. The current study aimed to characterize the genetic diversity of HPV33 and to explore the association of HPV33 variants with the risk for cervical cancer. Taking advantage of the International Agency for Research on Cancer biobank, we sequenced the entire E6 and E7 open reading frames of 213 HPV33-positive cervical samples from 30 countries. We identified 28 HPV33 variants that formed 5 phylogenetic groups: the previously identified A1, A2, and B (sub)lineages and the novel A3 and C (sub)lineages. The A1 sublineage was strongly over-represented in cervical cases compared to controls in both Africa and Europe. In conclusion, we provide a classification system for HPV33 variants based on the sequence of E6 and E7 and suggest that the association of HPV33 with cervical cancer may differ by variant (sub)lineage.