Background: Long control region (LCR) of human papillomavirus (HPV) has shown multiple functions on regulating viral transcription. The variations of LCR related to different lineages/sub-lineages have been found to affect viral persistence and cervical cancer progression differently. In this study, we focused on gene polymorphism of HPV16/ 18/58 LCR to assess the effect variations caused on transcription factor binding sites (TFBS) and provided more data for further study of LCR in Southwest China. Methods: LCR of HPV16/18/58 were amplified and sequenced to do polymorphic and phylogenetic anlysis. Sequences of each type were aligned with the reference sequence by MEGA 6.0 to identify SNPs. Neighbor-joining phylogenetic trees were constructed using MEGA 6.0. Transcription factor binding sites were predicted by JASPAR database. Results: The prevalence of these three HPVs ranked as HPV16 (12.8%) > HPV58 (12.6%) > HPV18 (3.5%) in Chengdu, Southwest China. 59 SNPs were identified in HPV16-LCR, 18 of them were novel mutations. 30 SNP were found in HPV18-LCR, 8 of them were novel. 55 SNPs were detected in HPV58-LCR, 18 of them were novel. Also, an insertion (CTTGTCAGTTTC) was detected in HPV58-LCR between position 7279 and 7280. As shown in the neighbor-joining phylogenetic trees, most isolates of HPV16/18/58 were clustered into lineage A. In addition, one isolate of HPV16 was classified into lineage C and 3 isolates of HPV58 were classified as lineage B. JASPAR results suggested that TFBS were potentially influenced by 7/6 mutations on LCR of HPV16/18. The insertion and 5 mutations were shown effects in LCR of HPV58. Conclusion: This study provides more data for understanding the relation among LCR mutations, lineages and carcinogenesis. It also helps performing further study to demonstrate biological function of LCR and find potential marker for diagnosis and therapy.

Background Variations in human papillomavirus (HPV) E6 and E7 have been shown to be closely related to the persistence of the virus and the occurrence and development of cervical cancer. Long control region (LCR) of HPV has been shown multiple functions on regulating viral transcription. In recent years, there have been reports on E6/E7/LCR of HPV-16 and HPV-58, but there are few studies on HPV-52, especially for LCR. In this study, we focused on gene polymorphism of the HPV-52 E6/E7/LCR sequences, assessed the effects of variations on the immune recognition of viral E6 and E7 antigens, predicted the effect of LCR variations on transcription factor binding sites and provided more basic date for further study of E6/E7/LCR in Chengdu, China. Methods LCR/E6/E7 of the HPV-52 were amplified and sequenced to do polymorphic and phylogenetic analysis. Sequences were aligned with the reference sequence by MEGA 7.0 to identify SNP. A neighbor-joining phylogenetic tree was constructed by MEGA 7.0, followed by the secondary structure prediction of the related proteins using PSIPRED 4.0. The selection pressure of E6 and E7 coding regions were estimated by Bayes empirical Bayes analysis of PAML 4.9. The HLA class-I and II binding peptides were predicted by the Immune Epitope Database server. The B cell epitopes were predicted by ABCpred server. Transcription factor binding sites in LCR were predicted by JASPAR database. Results 50 SNP sites (6 in E6, 10 in E7, 34 in LCR) were found. From the most variable to the least variable, the nucleotide variations were LCR > E7 > E6. Two deletions were found between the nucleotide sites 7387–7391 (TTATG) and 7698–7700 (CTT) in all samples. A deletion was found between the nucleotide sites 7287–7288 (TG) in 97.56% (40/41) of the samples. The combinations of all the SNP sites and deletions resulted in 12 unique sequences. As shown in the neighbor-joining phylogenetic tree, except for one belonging to sub-lineage C2, others sequences clustered into sub-lineage B2. No positive selection was observed in E6 and E7. 8 non-synonymous amino acid substitutions (including E3Q and K93R in the E6, and T37I, S52D, Y59D, H61Y, D64N and L99R in the E7) were potential affecting multiple putative epitopes for both CD4+ and CD8+ T-cells and B-cells. A7168G was the most variable site (100%) and the binding sites for transcription factor VAX1 in LCR. In addition, the prediction results showed that LCR had the high probability binding sites for transcription factors SOX9, FOS, RAX, HOXA5, VAX1 and SRY. Conclusion This study provides basic data for understanding the relation among E6/E7/LCR mutations, lineages and carcinogenesis. Furthermore, it provides an insight into the intrinsic geographical relatedness and biological differences of the HPV-52 variants, and contributes to further research on the HPV-52 therapeutic vaccine development.

Background Human papillomavirus type 39 associated with genital intraepithelial neoplasia and invasive cancers, has a high prevalence in Southwest China. HPV E6, E7 are two main papillomavirus oncoproteins, closely relate to the function of HPV immortalization, cell transformation, and carcinogenesis. L1 is the major capsid protein, can reflect the replication status of the virus in cells and the progression of cervical lesions. The purpose of this study is to reveal the prevalence of HPV 39 and the genetic polymorphisms of HPV39 based on E6, E7 and L1 gene in southwest China. Methods Cell samples were collected by cervical scraped for HPV detecting and typing, and HPV39 positive samples were selected out. Important E6, E7 and L1 genes of HPV39 were sequenced and analyzed for the study of HPV39 genetic polymorphisms. Phylogenetic trees were constructed by Maximum-likelihood and Kimura 2-parameters methods in Molecular Evolutionary Genetics Analysis version 6.0. The selection pressures of E6, E7 and L1 genes were estimated by Datamonkey web server. The secondary and three-dimensional structure of HPV39 E6, E7 proteins were created by sopma server and SWISS-MODEL software. Results 344 HPV39 positive samples were selected from 5718 HPV positive cell samples. Among HPV39 E6-E7 sequences, 20 single nucleotide mutations were detected, including 10 non-synonymous and 10 synonymous mutations; 26 single nucleotide mutations were detected in HPV39 L1 sequences, including 7 non-synonymous and 19 synonymous mutations respectively. 11 novel variants of HPV39 E6-E7 (5 in E6 and 6 in E7) and 14 novel variants of HPV39 L1 were identified in this study. A-branch was the most frequent HPV39 lineage in southwest China during our investigation. Selective pressure analysis showed that codon sites 26, 87, 151 in E6 and 75, 180, 222, 272, 284, 346, 356 in L1 were positively selected sites, as well as codon sites 45, 138, 309, 381 were negative selection sites in L1 gene, E7 has neither positive selection sites nor negative selection sites. A certain degree of secondary and three-dimensional structure dislocation was existed due to the non-synonymous mutations. Conclusions Amino acid substitution affected the secondary and three-dimensional structure of HPV39, and resulting in the differences of carcinogenic potential and biological functions as well as the immune response due to the antigen epitopes difference, the antigen epitopes with stronger adaptability in Southwest will be screened out based on the above research results for the later vaccine development. And gene polymorphism of HPV39 in Southwest China may improve the effectiveness of clinical test and vaccine design, specifically for women in Southwest China.

Background:Cervical cancer is one of the malignant tumors threatening women's health worldwide, only second to breast cancer. 99.7% cervical cancer was found to be associated with high-risk HPV (HR-HPV) persistent infections. HPV68 is a common HR-HPV, closely related to cervical cancer.Methods:Cell samples were collected by cervical scraped for HPV detecting and typing, and HPV 68 positive samples were selected out. Important E6, E7 genes of HPV 68 were sequenced and analyzed for the study of HPV 68 genetic polymorphisms. Phylogenetic tree of E6-E7 was constructed by Maximum likelihood method of MEGA v7.0 software. The selection pressure sites of HPV68 E6, E7 were predicted by codeml in the PAML 4.8. The secondary structure and three-dimensional structure of HPV68 E6, E7 were analyzed by PSIPRED server and SWISS-MODEL respectively. T-cell and B-cell antigen epitopes of HPV68 E6, E7 were predicted by immune epitope Database Analysis (IEDB) resource and ABCpred server respectivelyResults:a total of 10650 cell samples were collected for detecting and typing, and 2939 (27.60%, 2939/10650) positive samples were detected, 174 (5.92%, 174/2939) were HPV68; 150 HPV68 E6-E7 were successful amplified and analyzed, 32 nucleotide mutations were observed in this study, 50% (16/32) were non-synonymous mutations; among them, 4 non-synonymous mutations were detected in E6 gene (one in the Coil and three in the Strand), 12 were in E7 gene (four in the alpha helix, two in the Coil and six in the Strand). Phylogenetic analysis of HPV68 E6-E7 suggested that C was the most frequent HPV68 lineage in Sichuan China. 82-90SESVYATTL, 85-93VYATTLETI, 13-21KLPDLCRTL and 67-81-SCIKFYAKIRELRYY, 68-82CIKFYAKIRELRYYS, 66-80QSCIKFYAKIRELRY were the most potential HPV68 E6 HLA-Ⅰ, HLA-Ⅱepitopes respectively; and B-cell epitopes were 138-153CRHCWTSKREDRRRTR, 82-97SESVYATTLETITNTK. 92-101LLFMDSLNFV, 45-53AVNHHQHQL and 20-35EIEPVDLVCHEQLGDS were the most potential HPV68 E7 HLA-Ⅰand B-cell epitopes. 3 HPV68 E6 positive selection sites and 5 HPV68 E7 were detected, and they all had a certain influence on the proteins structure and epitopes affinity.Conclusion:Non-synonymous mutations of HPV 68 located in positive selection sites resulted in differences in protein structure and epitopes affinity, that may affect the pathogenicity and adaptability of HPV68 to the environment. HPV68 data enrichment is of great significance for understanding the inherent geographical and biological differences of HPV68 in china; and targeting potential epitopes for therapeutic vaccines may improve the effective of vaccines design for specific populations.

Idiopathic asthenozoospermia, a common factor in male infertility, is characterized by altered sperm motility function in fresh ejaculate. Although the β-defensin 126 ( DEFB126 ) protein is associated with asthenozoospermia, DEFB126 gene polymorphisms have not been extensively studied. Therefore, the association between DEFB126 gene polymorphisms and asthenozoospermia requires further investigation. Screening was performed by semen analysis, karyotype analysis, and Y microdeletion detection, and 102 fertile men and 106 men with asthenozoospermia in Chengdu, China, were selected for DEFB126 gene sequence analyses. Seven nucleotide mutations and two nucleotide deletions in the DEFB126 gene were detected. rs11467417 (317–318 del/del ), rs11467497 (163–166 wt/del ), c.152T>C, and c.227A>G were significantly different between the control and asthenozoospermia groups, likely representing high-risk genetic factors for asthenozoospermia among males. DEFB126 expression was not observed in sperm with rs11467497 homozygous deletion and was unstable in sperm with rs11467417 homozygous deletion. The rs11467497 four-nucleotide deletion leads to truncation of DEFB126 at the carboxy-terminus, and the rs11467417 binucleotide deletion produces a non-stop messenger RNA (mRNA). The above deletions may be responsible for male hypofertility and infertility by reducing DEFB126 affinity to sperm surfaces. Based on in silico analysis, the amino acids 51M and 76K are located in the highly conserved domain; c.152T>C (M51T) and c.227A>G (K76R) are predicted to be damaging and capable of changing alternative splice, structural and posttranslational modification sites of the RNA, as well as the secondary structure, structural stability, and hydrophobicity of the protein, suggesting that these mutations are associated with asthenozoospermia.