Infection, Genetics and Evolution

The E6 gene polymorphism of Human papillomavirus 16 in relation to the risk of cervical cancer in Tunisian women

Human papillomavirus type 16 (HPV-16) is the most prevalent HPV type worldwide and in Tunisia and the major carcinogenic HPV type found in cervical precancers and cancers. Previous studies have reported that genetic diversity of HPV16-E6 oncoprotein might be associated with cervical intraepithelial neoplasia progression. In this study we aimed to investigate the prevalence of HPV-16 E6 variants in precancerous lesions in Tunisian population to assess potential correlation with disease severity. Positive HPV cervical samples were obtained from the Laboratory of Anatomy Pathology of Pasteur Institute of Tunis. Cytological study was performed to identify cervical precancerous lesions. HPVs were typed using Reverse Line Hybridization. Only samples with HPV-16 single infection were selected for HP16-E6 genetic diversity investigation. HPV-16 E6 gene amplification was performed by PCR using specific primers and sequenced by Sanger Sequencing. The multiple alignment of generated sequences was performed using MEGAX software. Phylogenetic tree was constructed using Maximum Likehood method. The ternary complex of E6, E6AP and p53 core domain was used to perform in silico point mutations and thermodynamic calculations to assess stability and binding affinity. Genetic analysis of Tunisian E6-HPV16 sequences showed the presence of three lineages: European (A), African (C) and Asian American (D). Interestingly, the EUR variants were identified as the dominant lineage of HPV-16 and HPV-16 E6 350 G (L83V) was the most detected mutation in precancerous lesions. Modelling data showed that African variants induced the largest destabilizing effect on E6 structure and decreasing thereby in the affinity toward E6AP. Therefore, women infected with European variants are associated with low and high intraepithelial lesions. The findings give useful information for personalized decision algorithms of intra-epithelial cervical neoplasia in Tunisian women.

Human papillomavirus and ovarian cancer (review of literature and meta-analysis)

Many factors are involved in carcinogenesis of the ovary, such human genetic and physiological characteristics as lifestyle, existing diseases of the reproductive system, and, as suggested, the human papillomavirus (HPV). It is well known that the human papillomavirus virus of high carcinogenic risk (HCR) plays a crucial role in the onset and development of cervical cancer, as well as cases of HPV positive breast cancer and endometrial cancer. The data on the presence of HPV in ovarian cancer are ambiguous: the researchers claim that there is no complete effect of the virus on the development of this type of cancer, and the detection of HPV in 60-80% of tumors. In this regard, there is a need to systematize the currently available research results on this controversial issue and conduct a meta-analysis of the association of HPV infection with the risk of ovarian cancer.

Viral integration and fusion transcript characteristics of possibly high-risk HPV in cervical cancer

HPV34, HPV66, HPV73, and HPV82 are classified by the International Agency for Research on Cancer as possibly high-risk HPV (pHR-HPV) types. Despite their relatively lower pathogenicity, some cervical cancer (CC) patients have been found to be infected with pHR-HPV, though the underlying pathogenic characteristics remain unclear. Using viral integration detection and RNA sequencing in 8 pHR-HPV

Molecular epidemiology of human papillomavirus 18 infections in Japanese Women

Among the oncogenic genotypes of human papillomavirus (HPV), HPV18 is the second most common type detected in cervical cancer worldwide and is primarily involved in the generation of cervical adenocarcinoma. Although HPV intra-type variants confer different risks of cervical carcinogenesis, there is little information on the genetic diversity of HPV18 compared to the most prevalent type, HPV16. In this study, we investigated the genetic variation of HPV18 in cervical specimens obtained from Japanese women with normal cervices or cervical cancers and precancers. Of the 101 HPV18-positive samples analyzed, viral whole genome amplification followed by next-generation sequencing led to the determination of viral complete genome sequences of 18 samples. Phylogenetic analysis of these HPV18 whole genome sequences identified a distinct variant cluster consisting of only Japanese samples (n = 7) belonging to sublineage A1. Viral genome sequences were also analyzed for the E6/E7 (n = 66) and E2 (n = 27) genes by Sanger sequencing. Phylogenetic analyses of these regions showed that the variant distribution among Japanese women was strongly biased toward sublineage A1 (72 of 87; 82.8%). No significant differences were observed in the prevalence of specific sublineages between cervical cancer/precancer cases and controls, and between squamous cell carcinoma and adenocarcinoma cases. These data contribute to our understanding of the genetic diversity of HPV18 in Japanese women.

Characteristics of Cervical Cancer Caused by the Human Papillomavirus 18 and Its Genetic Variations in Vietnamese Women

The involvement of HPV18 in cervical cancer pathogenesis, as well as its high oncogenic potential and influence on the variation of cervical cancer distribution in different geographical regions, makes assessing the characteristics of cervical cancer and its variants the basis for considering potential carcinogenic HPV18 sequence variations and vaccine strategies. A prospective study was conducted at Vietnam Central Obstetrics Hospital from January 1, 2019 to December 31, 2020. HPV18 infection was confirmed in cervical cancer patients using molecular diagnostics. Nucleotide sequences of the HPV18 E6, E7, and L1 genes were used to analyze genetic variations. The demographic, clinical, and laboratory data of the patients were collected and statistically analyzed. Among 48 patients with HPV18-infected cervical cancer, 79.2% were between the ages of 35-54; while only 20.8% were  54 years old. 100% of patients have been pregnant at some point in their lives, with ≥3 pregnancies accounting for 83.3%. Patients with cervical cancer caused by HPV18 infection were predominantly in stages 0 and I, with no patients in stages II, III, or IV. A single HPV18 infection generates much more cervical cancer cases than multiple HPV18 infections. Symptoms such as lower abdomen pain, unusual anginal discharge, and vaginal bleeding were observed in both stages 0 and I; however, vaginal bleeding after sex was only detected in women with stage I cervical cancer. Cervicitis, cervical ectropion, and ulcers are reported in cervical status stages 0 and I; however, warts and ulcers were only present in stage I. Magnetic resonance imaging produces far superior outcomes than ultrasound. All cytology and pathology tests confirmed L/HSIL, SCC, AC, and CIS. On the other hand, a single HPV18 infection was associated with a significantly higher risk of L/HSIL, SCC, AC, and CIS than multiple HPV18 infections. Nulceotide sequences of the E6, E7, and L1 genes revealed 20 mutations, including three (E6), five (E7), and twelve (L1) mutations. High-frequency mutations (95.8%-100% of HPV18 samples had mutations) occur at the following positions: C287G - P61P (E6 gene), G5503A - R25Q, C5701G - P91R, C6460G - P344R, C6625G - P399R, and C6842G - P471R (L1 gene). A phylogenetic tree based on the E6/E7/L1 gene sequence revealed that 100% belonged to A lineage, with 97.9% belonging AA (Asian Amerindian - A1) and 2.1% belonging to the E (European - A5). Patients with a single HPV18 infection have a higher risk of cervical cancer than those infected with HPV18 and other high-risk strains simultaneously. HPV18 single-infection, on the other hand, had considerably higher incidences of L/HSIL, SCC, AC, and CIS than HPV18 co-infection. The HPV18 strain that was found in Vietnam belonged to lineage A (A1 and A5), which contains several oncogene mutations.

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.

Identification of human leukocyte antigen in precancerous and cancerous cervical lesions from Ecuadorian women

Cervical cancer is the fourth most common type of cancer in women. Worldwide, it is a public health problem with around 604,127 women diagnosed per year and 341,831 deaths. Cervical cancer and persistent high-risk human papillomavirus (HPV) infection are highly associated. However, other factors are also involved, such as viral load, HPV variants, sexual behavior, and genetic factors. The host immune response against HPV has been widely studied and it has shown associations with development of cervical cancer. The human leukocyte antigen (HLA) genes are related to the persistence of HPV infection and progression to cervical cancer because of their role in controlling T-cell mediated immune response to clear the infection. In Ecuador, there is scarce information about HLA and HPV infection with high-risk genotypes in the population. This study aimed to identify host-specific HLA alleles in women with cervical intraepithelial neoplasia (CIN) II and III, and cancer infected with HPV-16, 58, and 52. In this study, we included 51 samples previously identified as positive for HPV-16, 58, and 52 from 12 Ecuadorian provinces. As a result, we found that HLA-A*02, HLA-B*35, HLA-C*04, HLA-DRB1*04, and HLA-DQB1*03 alleles were the most frequent, these alleles have been associated with cervical cancer in previous studies; nevertheless, we did not find a statistically significant association between HLA alleles, HPV genotype, and histopathological lesion. This is a baseline study to uncover possible relationships between HLA and HPV to elucidate why this virus can develop a persistent infection in some women leading to the development of cervical cancer.

Bioinformatics analysis of HPV-68 E6 and E7 oncoproteins for designing a therapeutic epitope vaccine against HPV infection

The incidence and mortality of cervical cancer, which mainly results from the infection of human papillomavirus (HPV) is significantly increasing in Xinjiang. According to the previous research, the incidence of HPV-68 in cervical cancer patients in Xinjiang is significantly higher than in other parts of China. HPV E6 and E7 oncoproteins play a crucial role in cervical cancer, and can be used as ideal targets for therapeutic vaccines. Therefore, we analyzed and identified the possible T-cell and B-cell dominant epitopes and various aspects of HPV-68 E6 and E7 oncoproteins, including the physicochemical properties, secondary and tertiary structures using a bioinformatic approach, which provided a basis for designing an effective HPV infection therapeutic vaccine. The results showed that E6 oncoproteins was an unstable and hydrophilic protein, while E7 oncoproteins was unstable and hydrophilic protein. The secondary structure of the E6 oncoproteins consisted of 45.57% alpha helixes, 14.56% extended strands, 4.43% beta turns and 35.44% random coils. The secondary structure of E7 oncoproteins consisted of 35.45% alpha helixes, 17.27% extended strands, 0.91% beta turns and 46.36% random coils. Moreover, our results identified 5 dominant T-cell epitopes and 6 dominant B-cell epitopes in the E6 oncoproteins structure and 5 dominant T-cell epitopes and 3 dominant B-cell epitopes in E7 oncoproteins. In conclusion, this study provides comprehensive biological information about the HPV-68 E6 and E7 oncoproteins, which will lay a theoretical foundation for multi-epitope vaccines against HPV infection.

Assessing genetic evolution and detecting human papillomavirus by matching two complementary highly sensitive approaches, nested-qPCR and sequencing

Becoming armed with an appropriate strategy to isolate the minimum number of human papillomaviruses (HPV), regardless of DNA extraction method, can be a huge step in preventing false negative; it has a significant effect on the management and control of HPV infection among women's population. This study was conducted in Qom province, considering the risk factors associated with HPV. It was able to analyze genetic evolution in its genotypes and evaluated the limit of detection by a new diagnostic approach. Totally, 486 Pap smear samples were tested; then, the HPV DNA was developed by a semi-nested quantification PCR. Positive samples were sequenced and submitted to the GenBank (MG825048-MG825061). After alignment, phylogenetic and polymorphism analyses were performed on the sequenced samples with a number of GenBank sequences. The overall HPV prevalence among all women in Qom was 11.7%. HPV6 (43.24%) and HPV16 (6.75%) were the most frequent LR and HR genotypes, respectively. Although the Tajima's D of all genotypes was positive, it was negative individually. The position of genotypes 6, 11, and 73 was controversial on phylogenetic trees. Limit of detection (LOD) was obtained as about 10-100 copies per reaction in various genotypes of HPV by semi-nested qPCR. The nature of HPV could be preserved during natural selection. This research, through innovative usage of the primers, could detect different genotypes of the HPV, and inform the women society of the probable risk through its prevalence determination.