Epidemiologic Characterization Of High-risk Human Papillomavirus Infection And Genetic Variability Evaluation Of Human Papillomavirus Type52in Rural Chaozhou

Cervical cancer is the second most common malignant tumors in Chinese women,about130000new cases were detected annually. Therefore, the social consequencesof this disease are still tremendous.Certain types of human papillomavirus (HPV) are now recognized as the maincause of cervical carcinoma. At present, more than150HPV genotypes have beenmolecularly characterized. According to previous report, HPV-16,-18,-31,-33,-35,-39,-45,-51,-52,-56,-58,-59,-68,-69,-73and-82were classified as "high-risktypes", which are known to infect the genital tract and tightly associated with cervicalcancer.The development of prophylactic vaccine against HPV infections now appears tobe the most promising way of controlling cervical neoplasia. There are two licensedHPV vaccines; Gardasil and Cervarix, mainly against the two major cancer-causingHPV types16and18. Since limited cross-protection could be offered between HPVtypes, heterogeneity in HPV type-specific distribution from different populationsshould be taken into account when predicting the effect of current prophylacticvaccines and forming the basis for the second-generation vaccines targeted to specificregions.To the best of our knowledge, no epidemiologic data on HPV genotypes ingeneral female population from rural area was reported from Guangdong Province,southern China, which highlights the need for timely population-based study in thisregion. From December2009to September2010, an epidemiologic screening forHPV infections was organized by Chaozhou municipal government, with thecooperation of gynaecological practitioners in13hospitals. We investigated thecervical HPV infection in asymptomatic, general female population to understand the overall, type-specific, age-specific high-risk human papillomavirus (HR-HPV)prevalence and extent of multiple infections in rural Chaozhou. Considering thatHPV-52is the most frequent HPV type in this area, we investigated its geneticvariability to understand the different carcinogenicity between HPV-52variants, andto collect molecular information for prophylactic vaccine invention in the future.1. Prevalence of HR-HPV DNAAmong48559eligible rural Chaozhou women, real time-PCR revealed that3830cases were positive for HR-HPV DNA, and the overall prevalence was7.89%, whichis similar with many reports in southern China.1.1Age-specific prevalence of HR-HPV DNAThe peak HR-HPV prevalence was observed in the oldest women (age from56-60years), which is hypothesized the results of reactivation of latent infections dueto impaired immune response.1.2Area-specific prevalence of HR-HPV DNAThe lower HR-HPV prevalence was observed in women who lived in the plain,which is suggested the results of regular living style and seldom works outside.Specific age-related prevalence of overall HPV infection was also observed inwomen who lived in the plain and mountain area, but not in those lived in thesuburban and seashore. The HR-HPV prevalence of women who lived in the seashoreincreased from35to50years of age, and decreased in the elders. While a "U" shapedage-related HR-HPV prevalence was found in women who lived in the suburban.1.3Type-specific prevalence of HR-HPV1.3.1The most common HR-HPV typesConsistent with some previous reports in eastern Asia, type52was the mostcommon HR-HPV type in rural Chaozhou, the other5most common HR-HPV typesin descending order, were types16,58,33,68and18. The high prevalence ofHPV-52, rather than HPV-16, may be due to the geographical and biological interplaybetween HPV types and host immunogenic factors. 1.3.2The prevalence of HPV-16and/or-18and HPV-52and/or-58Because HPV-16and-18are target types of current prophylactic vaccines, andHPV-52and-58are most frequent HPV types in eastern Asia, the prevalence of type16plus18and type52plus58are discussed independently.The combined prevalence of HPV types16and18accounted for28.52%of totalHR-HPV infected cases. However, type52plus58presented48.23%of totalHR-HPV infectors. Interestingly, the prevalence of HPV type16and/or18decreasedslowly with age increasing, on the contrary, the type52and/or58increased with ageincreasing.1.3.3The prevalence of multiple infectionsHR-HPV typically infected with a single type, no more than one quarterHR-HPV carriers presented multiple infections. The most common infection typeparticipated in the most common multiple infection state, for example, the type52combined with type16was the most common double infections. Furthermore, wefound out that the constituent ratio of multiple infections increased with age, and therewas a linear regression relationship, the Square R was0.783, and the linear regressionequation was: Y∧=0.054+0.006X2Risk factors for cytology abnormalitiesOur results suggest that living area, HR-HPV infection and viral load, single ormultiple infection and HPV type were all important determinants of abnormalcytology incidence.2.1Area-specific prevalence of cytology abnormalitiesThe peak abnormal cytology incidence was found in women who lived in thesuburban, following was those lived in the plain, fewer women who lived in themountain area or seashore presented cytology abnormalities. Suburban people livednearby the city, they are heavily influenced by city people, whose sexual behaviour isgreat different from rural females. In addition, the suburban women may contact withmore carcinogens, since well-developed industries in their surroundings. 2.2The predictable effect of HPV infection on abnormal cytologyCoincident with many related reports previously, the percentage of abnormalcytology in HPV positive population was extremely higher than that in negative ones.The former had almost16fold of odd ratio (OR) than the latter for abnormal cytology.Furthermore, the former had almost20fold of OR than the latter for high gradecytology lesions.2.3Single infection vs. multiple infectionsThe women who presented multiple infections had higher OR for cytologyabnormalities and high grade cytology lesions, which have been suggested thecooperative carcinogenesis between HPV types. However, the operation mechanism isstill blurry.2.4The predictable effect of HPV types on abnormal cytologyFor single infection, the women who infected with HPV types16,31or33hadhigher risk for cytological abnormalities compared with type52carriers. And thosewho infected with HPV type16or33had almost9and8fold of OR for high gradecytology lesions compared with type52carriers, respectively. Thus, we believe thatHPV-16and-33were most dangerous HPV type in rural Chaozhou.In addition, the women who infected with type16and/or18had almost4fold ofOR compared with type52and/or58carriers for high grade cytology lesions. While,no significant difference was found between their abnormal cytology and low gradecytology lesions incidence. These results suggest that the HPV-16and-18carriershave higher risk for cervical cancer, but not for cervical intraepithelial neoplasiacompared with types52and58carriers.2.5The predictable effect of HPV load on abnormal cytologyAs reported previously, with the increasing of viral load, the grade of cytologylesions increased correspondingly. The viral load is another important determinant forcytology abnormalities. 3. The high risk population for cervical caner and prevention3.1The high risk population for cervical caner in rural ChaozhouTaken all of mentioned above into consideration, the high risk population forcervical caner in rural Chaozhou should include those who (1) age55or older;(2) livein the suburban;(3) present HR-HPV infection. The HR-HPV infectors who areinfected with multiple HPV types, with higher viral load or with types16or33havemuch higher risk for cervical cancer development.3.2Cervical caner prevention in rural ChaozhouConsidering the limitation of licensed HPV vaccines, most high risk populationis not competent for vaccination. We believe that the cytology test should beconsidered regular screening and should be prolonged for this cohort. However, theHPV vaccine could provide perfect prevention for youngsters, because HPV-16is oneof most common and dangerous HPV type, and we assume that the second-generationvaccines including HPV-52and-58may provide higher protection for female in ruralChaozhou.4. Genetic variability of HPV-52in rural ChaozhouBased on the mismatches on E6and L1genes, totally21variants were identifiedin73specimens.18of them could be divided into Asian lineage and the other3wasphylogenetically related to European lineage.11nucleotide and2encoded aminoacids were variable across the E6gene, and31nucleotide and6encoded amino acidswere variable across the L1gene. The maximum nucleotide diversity (1.1%) wasobserved between isolates CZ52A375and CZ52E429.4.1Genetic variability of Asian lineages variantsFor18Asian lineages variants,6nucleotides and1encoded amino acid werevariable across the E6gene, and20nucleotide and5encoded amino acids werevariable across the L1gene. The isolate CZ52A105represented the most commonvariant in Asian lineage, and it located at the bifurcation of Asian branch, at aminoacid level,83.6percents (61/73) variants were identical with CZ52A105. Moreover,its E6and L1sequences were completely matched with some isolates that reports in southeast Asian countries. Taken all above mentioned into consideration, we suggestthat the isolate CZ52A105represents the most common and ancient HPV-52variantin southeastern Asia.4.2Genetic variability of European lineages variantsFor3European lineages variants,3nucleotides were variable across the E6gene,but no encoded amino acid was changeable;8nucleotides and1encoded amino acidwere variable across the L1gene. The isolate CZ52E429was the most frequentvariant, while isolate CZ52E928represented the most widely spread variant. Thesame E6and L1sequences were also found in other3continents besides Asia. Inaddition, it located at the bifurcation of European branch. We speculate that thisvariant may derived from the worldwide migration of the European hosts.4.3Variants pathogenicity assessment and HRM analysis application4.3.1Pathogenicity assessment between Asian and European lineages variantsA preliminary pathogenicity comparison between HPV-52variants wasperformed in this study. No significant difference was found between the pathogenicrisk of Asian and European lineages variants. However, there are some limitations forthis conclusion, because there is a great unbalance between the cases numbers of the2lineages variants. In order to validate this conclusion, it is necessary to get a largersampling.4.3.2Distinguish Asian and European lineages variants with HRM analysisThe HRM analysis provides us a more economic and rapid method to deal withlarge scale screening HPV-52variants. As a preliminary attempt, an E6gene reliedHRM analysis was used to group identified HPV-52variants. We found that the Asianand European variants could be distinguished by HRM analysis with a completeaccuracy.4.4Multiple variants infectionsIn this study, we found that6specimens involved2kinds of HPV-52variants.The restriction enzyme test confirmed the existence of this novel phenomenon. Webelieve that this phenomenon might be similar to the multiple type infections. Once a woman is singly infected with HPV-52, she has an almost equal opportunity of furtherinfected with other HPV types or infected with type52again, the former events leadto multiple infections, and the latter possibly lead to2kinds of HPV-52variantsinfections.