Typing of Human Papilloma Virus (HPV) and molecular variants of identified types

Uvod: U novije vreme sve veći broj studija sugeriše da se genotipske varijante humanih papiloma virusa (HPV), uprkos filogentskoj srodnosti, mogu razlikovati u patogenosti i različito doprineti razvoju cervikalnih neoplazija. Cilj ovog rada je bio da se definiše zastupljenost različitih onkogenih tipova HPV kod ispitanica sa područja Južnobačkog okruga (AP Vojvodina) i odredi genotipska varijabilnost najprevalentnijih genotipova HPV, analizom DNK sekvenci i RFLP metodom. Materijal i metode: Istraživanje je sprovedeno u periodu od januara 2014. do novembra 2015. godine. Studija je obuhvatila 564 osobe ženskog pola, starosti od 18 do 69 godina. Genotipizacija 12 visokorizičnih tipova HPV izvršena je upotrebom komercijalnog kita HPV High Risk Typing Real-TM (Sacace Biotechnologies, Italy, CE, IVD). Automatsko sekvenciranje amplifikovanih fragmenata L1 gena HPV rađeno je na automatskom sekvenatoru ABI Prism BigDye 3.1 (PE Applied Biosystems, Foster, CA, SAD). Bioinformatičkom analizom utvrđene su genetičke distance i filogenetski odnosi HPV izolata dobijenih u AP Vojvodini u odnosu na izolate iz drugih geografskih područja za tipove: 16, 18, 31 i 33. PCR-RFLP metodom ispitivana je heterogenost unutar HPV tipa 51, na nivou E1, L1 i L2 gena 11 odabranih HPV DNK pozitivnih izolata, primenom restrikcionih enima DraI, TaqI i PstI. Rezultati: Prevalencija HPV infekcije kod testiranih žena je iznosila 51,8%. Učestalost različitih tipova vema je varirala, pri čemu su najprevalentniji bili: HPV16 (34,5%); HPV 31 (20,5%); HPV 51 (13%); HPV33 (11,6%); HPV52 (11%) i HPV18 (11%). HPV tipovi 16 i 33 su najčešće dijagnostikovani u starosnoj grupi od 31-40 godina (25,5% i 6,21%), dok su HPV18 (8,16%), HPV31 (7,04% ) i HPV51 (12,3%) najčešće dokazani kod žena mlađih od 30 godina. Određene su genetičke distance i filogenetski odnosi u okviru populacije izolata iz Južnobačkog okruga, AP Vojvodine, u odnosu na druge izolate čije su genomske sekvence dostupne u GenBank bazi podataka. Filogenetska analiza nukleotidnih sekvenci HPV tipa 16 je potvrdila da se najveći broj izolata iz područja AP Vojvodine grupiše u liniju A, (podlinije A3 i A4 ) dok se jedan izolat izdvojio u liniju D (podlinija D1). Tri izolata HPV tipa 18 su grupisana u liniju A, a dva izolata kao posebna grupa. Utvrđeno je da genotipske varijante HPV 31 iz našeg područja pripadaju linijama A, B i C. Izolati HPV tipa 33 pripadali su liniji A (podlinije A1 i A2). Dobijene nukleotidne distance su pokazale da je razlika između analiziranih i referentnih sekvenci manja od 2%, što potvrđuje sličnost na nivou varijante. Na osnovu PCR-RFLP analize u odabranim DNK pozitivnim na HPV tip 51, restrikcionom analizom PCR produkata na nivou L1, L2 i E1 gena, utvrđeno je da su prisutni samo genotipovi koji odgovaraju liniji A sa podlinijom A1. Zaključak: Rezultati studije predstavljaju prve dostupne podatke o rasprostranjenosti 12 visokorizičnih tipova HPV, kao i podatke o genomskoj arijabilnosti i filogenetskoj srodnosti najprevalentnijih HPV tipova kod žena sa područja Južnobačkog okruga. Najprevalentniji HPV tipovi našeg regiona su pokazali usklađenost sa evropskim izolatima, ali su nađene i ne-evropske varijante. Nizak procenat genetičkih distanci u okviru istog HPV tipa je u skladu sa niskom stopom mutacija kod ovih virusa. Iako se u današnje vreme RFLP metoda primenjuje u manjoj meri zbog uspona tehnologija sekvenciranja DNK, ova tehnika se može koristiti kao brza i jeftinija metoda za određivanje pojedinih linija ovih virusa. S obzirom da je u većini kliničkih laboratorija sekvenciranje DNK nedostupno,razvijena metoda se može koristiti za diferenciranje dve glavne linije HPV tipa 51, koje imaju različit onkogeni potencijal. Rezultati predstavljaju izuzetan doprinos epidemiološkoj proceni realnih potreba uvođenja rutinske imunizacije protiv visokoprevalentnih genotipova prisutnih u prekanceroznim promena na grliću materice inficiranih žena sa našeg područja. Tipizacija HPV virusa primenjenim metodama može biti od velike koristi u ranom otkrivanju maligne transformacije inficiranih ćelija i prevenciji karcinoma grlića materice.Introduction: In recent times increasing number of researches suggests that genotypic variants of human papillomavirus (HPV), in spite of phylogenic relations, could differ in virulence and contribute the development of cervical neoplasia. The aim of this work was to define the partition of different oncogenic HPV types in examined women from the area of Juznobacka district (AP Vojvodina) and to determine genotypic variability of the most prevalent HPV genotypes, by DNA sequences’ analysis and RFLP method. Material and methods: The research was conducted in period from January 2014 till November 2015. Study included 564 female persons, age from 18 to 69. The genotypization of 12 high-risk HPV types was performed with use of commercial kit HPV High Risk Typing Real-TM (Sacace Biotechnologies, Italy, CE, IVD). The automatic sequencing of amplified fragments of L1 HPV gene was performed on automatic sequencer ABI Prism BigDye 3.1 (PE Applied Biosystems,Foster, CA, USA). By bioinformatic analysis genetic distances, as well as phylogenic relations of HPV isolates in AP Vojvodina were determined in relation with isolates from other regions for types 16,18, 31 and 33. By PCR-RFLP, the heterogeneity within HPV type 51, at the level of E1, L1 and L2 genes 11 selected HPV DNA positive isolates, by use of restriction enzymes DraI, TaqI and PstI. Results: The prevalence of HPV infection in tested women was 51,8%. The frequency of different types varied considerably, where the most prevalent types were: HPV16 (34,5%); HPV 31 (20,5%); HPV 51 (13%); HPV33 (11,6%); HPV52 (11%) and HPV18 (11%). The HPV types 16 and 33 are diagnosed the most in age group from 31-40 (25,5% and 6,21%), while HPV 18 (8,16%), HPV31 (7,04% ) and HPV51 (12,3%) are the most diagnosed in women younger than 30. The genetic distances and phylogenic relations within samples from population of Južnobacka region, AP Vojvodina, are determined in relation to other samples whose genomic sequences are available in GenBank data base. Phylogenetic analysis of nucleotide sequences of HPV 16 confirmed that the most of isolates from the area of AP Vojvodina are grouped in line A (sublines A3 and A4), while one isolate separated to the line D (subline D1). Three isolates of HPV type 18 are grouped in line A, and two isolates grouped as separate group. It was confirmed that genotypic variants of HPV 31 from our region belong to lines A, B and C. Isolates of HPV type 33 belonged to line A (sublines A1 and A2). Gained nucleotide distances showed that difference between analyzed and referent sequences is lower than 2%, which confirme the similarity at the level of variant. Based on PCR-RFLP analysis in selected DNA of persons positive on HPV type 51, by restriction analysis of PCR products on the level of L1, L2 and E1 genes, it is confirmed that only genotypes that correspond to line A, subline A1 are present . Conclusions: The results of study present the first available data on abundance of 12 high-risk types, as well as data on genomic variability and phylogenic relations of the most prevalent HPV types in females from the Juznobacka region. The most prevalent HPV types of our region showed concordance with European isolates, but non-european variants were also found. Low percentage of genetic distances within the same HPV type is in concordance with low mutation rate of these viruses. Although today RFLP method is applied at lower scale because of rise of DNA technologies,this technique could be used as fast and efficient method for determination of particular lines of these viruses. Regarding that DNA sequencing is unavailable to the most of clinical laboratories,developed technique could be used for discrimination of two main lines of HPV type 51 that have different oncogenic potential. The results present considerable contribution to the epidemiological assessment of real needs for implementation of routine immunization against the high prevalent genotypes, present in precancerous alterations on the cervix of infected women from our area. Typization of HPV viruses by applied methods could be of great benefit in early detection of malignant transformations of infected cells and prevention of cancer of cervix