Molekularna opredelitev, klinični pomen in tkivni tropizem novega človeškega papilomavirusa, HPV-204 in sorodnih genotipov

Šterbenc, Anja (avtor), Poljak, Mario (mentor)

Človeške papilomaviruse (HPV) etiološko povezujemo z nastankom številnih benignih in malignih sprememb kože in sluznic pri ljudeh. Po podatkih Mednarodnega referenčnega centra za HPV je bilo do novembra 2017 uradno priznanih 206 genotipov HPV, nedavno pa so dokazali, da naj bi obstajalo še vsaj 400 potencialno novih, še neopredeljenih genotipov HPV. Naša raziskovalna skupina je leta 2014 v vzorcu analnega brisa dokazala prisotnost novega genotipa HPV, HPV-204, ki smo ga uvrstili v rod Mupapillomavirus (Mu-PV), kamor sta bila do tedaj uvrščena le dva genotipa HPV (HPV-1 in HPV-63). V doktorski nalogi smo želeli natančno filogenetsko in molekularno opredeliti HPV-204. Poleg tega smo na podlagi testiranja reprezentativne zbirke kliničnih vzorcev želeli opredeliti tkivni tropizem in klinični pomen novega genotipa HPV-204 ter sorodnih genotipov HPV-1 in HPV-63. V zadnjem delu doktorske naloge smo želeli opredeliti genetsko raznolikost genotipov iz rodu Mu-PV s pomnoževanjem in neposrednim določanjem nukleotidnega zaporedja nekodirajočega genomskega področja (LCR). S pomočjo več programov in prosto dostopnih spletnih aplikacij smo v prvem delu doktorske naloge opredelili značilna genomska področja celotnega virusnega genoma HPV-204. Celotno nukleotidno zaporedje HPV-204 je sestavljeno iz 7.227 baznih parov (bp) s 37,8% deležem bp gvanin-citozin. HPV-204 izkazuje organizacijo genoma, ki je značilna za predstavnike rodu Mu-PV, saj vsebuje zapis za šest zgodnjih (E6, E7, E1, E2, E8 in E4) in dve pozni (L1 in L2) virusni beljakovini, vendar nima zapisa za zgodnjo beljakovino E5. Na podlagi filogenetske analize gena L1 vseh uradno priznanih genotipov HPV smo potrdili preliminarno uvrstitev HPV-204 v rod Mu-PV, in sicer v novo vrsto Mu-3, pri čemer lahko sklepamo, da je HPV-204 najbolj soroden s HPV63, s katerim izkazuje 66,9% skladnost v genu L1, medtem ko s HPV-1 izkazuje 66,1% skladnost v genu L1. Tkivni tropizem in klinični pomen okužb z genotipi iz rodu Mu-PV smo opredelili s testiranjem reprezentativne zbirke kliničnih vzorcev (n=1.006), za kar smo razvili in uvedli analitično zelo občutljive kvantitativne HPV-204, HPV-1 in HPV-63 tipsko-značilne reakcije pomnoževanja s polimerazo v realnem času (RT-PCR). Odkrili smo 11 neodvisnih izolatov HPV-204, in sicer v brisu materničnega vratu (1/116; 0,8%), vzorcu kožne bradavice (1/43; 2,3%), brisih analnega kanala (4/110; 3,6%) in brisih penisa (5/110; 4,5%). V sklopu doktorske naloge smo skupno odkrili 27 neodvisnih izolatov HPV-1, ki smo jih odkrili v brisih nosnega dela žrela (2/110; 1,8%), dlačnem mešičku obrvi (1/110; 0,9%), vzorcih kožnih bradavic (21/43; 48,8%) in vzorcih anogenitalnih bradavic (3/40; 7,5%), ter 19 neodvisnih izolatov HPV-63, ki smo jih odkrili v brisih nosnega dela žrela (3/110; 2,7%), brisih ustne votline (2/105; 1,9%), dlačnih mešičkih obrvi (4/110; 3,6%), vzorcih kožnih bradavic (9/43; 20,9%) in brisu analnega kanala (1/110; 0,9%). Na podlagi testiranja arhivskih vzorcev benignih in malignih sprememb ter klinično nespremenjene kože in sluznic smo dokazali, da vsi genotipi iz rodu Mu-PV izkazujejo dvojni tkivni tropizem. V vzorcu kožne bradavice, v katerem smo dokazali HPV-204, je bilo virusno breme HPV-204 izjemno nizko (7,4 x 10-7 virusnih kopij/celico), kar nakazuje na latentno, klinično nepomembno okužbo. Čeprav je bilo virusno breme HPV-1 večinoma nizko (razpon 5,9 x 10-6 – 1,8 x 10-2 virusnih kopij/celico), je bil HPV-1 najverjetnejši povzročitelj treh kožnih bradavic, v katerih smo dokazali visoko virusno breme HPV-1 (razpon 8,5 x 103 – 5,2 x 104 virusnih kopij/celico). Virusno breme HPV-63 je bilo podobno kot pri HPV-1 v večini vzorcev nizko (razpon 3,2 x 10-6 – 1,1 x 10-2 virusnih kopij/celico), HPV-63 pa je predstavljal najverjetnejšega povzročitelja le v vzorcu kožne bradavice z visokim virusnim bremenom (3,9 x 102 virusnih kopij/reakcijo). Z opredelitvijo virusnega bremena HPV-204, HPV-1 in HPV-63 smo potrdili, da je okužba s temi genotipi HPV večinoma klinično neaktivna, pri čemer sta HPV-1 in HPV-63 etiološka povzročitelja zgolj posameznih kožnih bradavic, medtem ko HPV-204 nismo uspeli povezati z nastankom kožnih bradavic. V zadnjem delu doktorske naloge smo na osnovi analize nukleotidnega zaporedja nekodirajočega genomskega področja LCR genotipov iz rodu Mu-PV odkrili eno podtipsko različico HPV-204, ki je vsebovala eno točkasto mutacijo, šest podtipskih različic HPV-1, ki so vsebovale 1-19 nukleotidnih zamenjav oziroma izpadov, ter dve podtipski različici HPV-63, pri čemer je prva podtipska različica vsebovala 13, druga pa šest nukleotidnih zamenjav oziroma izpadov. Predvidevamo, da opredeljene podtipske različice posameznih genotipov HPV niso povezane z značilno klinično sliko ali anatomskim področjem okužbe. Izsledke raziskave, ki so predstavljeni v doktorski nalogi, smo objavili kot znanstveni raziskovalni članek v reviji, ki jo citira Science Citation Index (SCI) (IF2016=2,806): Šterbenc A, Hošnjak L, Chouhy D, Bolatti EM, Oštrbenk A, Seme K, Kocjan BJ, Luzar B, Giri AA, Poljak M. Molecular characterization, tissue tropism, and genetic variability of the novel Mupapillomavirus type HPV204 and phylogenetically related types HPV1 and HPV63. PLoS One 2017;12:e0175892.

• Jezik: Slovenski jezik
• Ključne besede: človeški papilomavirusi, HPV, HPV-204, Mupapillomavirus, nov genotip HPV, molekularna opredelitev, filogenetska analiza, klinični pomen, tkivni tropizem, podtipske različice
• Vrsta gradiva: Doktorsko delo/naloga
• Organizacija: MF - Medicinska fakulteta
• Leto izida: 2018
• PID: 20.500.12556/RUL-99466
• COBISS.SI-ID: 293606656
• Datum objave v RUL: 25.01.2018

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Molecular characterization, clinical significance and tissue tropism of a novel human papillomavirus, HPV-204, and related types

Izvleček

Human papillomaviruses (HPV) are etiologically linked with the development of various benign and malignant neoplasms in humans. As of November 2017, a total of 206 HPV types were officially recognized by the HPV Reference center at the Karolinska Institute in Sweden, although it has recently been shown that there are still at least 400 novel HPV types that have not yet been identified. In 2014, our research group identified a novel HPV, HPV-204, in an anal canal swab sample. HPV-204 was preliminarily classified into the genus Mupapillomavirus (Mu-PV) that consisted of only two HPV types: HPV-1 and HPV-63. In this study, our aim was to perform comprehensive phylogenetic and molecular characterization of HPV-204. In the second part of the study, we wanted to determine the tissue tropism and potential clinical significance of Mu-PVs by testing a representative collection of clinical specimens. Finally, we wanted to evaluate the genetic variability of Mu-PVs by amplifying and sequencing the long control region (LCR) of each Mu-PV. Using several programs and freely available online applications, we determined characteristic genomic regions of HPV-204. The 7,227 base pair (bp) long genome of HPV-204 with a GC ratio of 37.8% exhibits typical genomic organization of Mu-PVs with eight open reading frames (ORFs) (E6, E7, E1, E2, E8, E4, L2, and L1), while lacking the E5 ORF. Based on phylogenetic analysis of the L1 gene of all officially recognized HPV types, HPV-204 clusters within the Mu-PV genus, species Mu-3. HPV-204 is most similar to HPV-63, since the L1 gene of HPV-204 exhibits 66.9% nucleotide sequence identity with the L1 gene of HPV-63 and 66.1% with the L1 gene of HPV-1. To evaluate the tissue tropism and potential clinical relevance of Mu-PVs, we tested a representative collection of clinical samples (n=1,006) using three newly developed and highly sensitive quantitative type-specific real-time polymerase chain reactions (RT-PCRs) for detection of HPV-204, HPV-1, and HPV-63. A total of 11, 27, and 19 HPV-204, HPV-1, and HPV-63 isolates were obtained, respectively. HPV204 was detected in cervical swabs (1/116; 0.8%), cutaneous wart (1/43; 2.3%), swabs of the anal canal (4/110; 3.6%), and swabs of the penile surface (5/110; 4.5%). HPV-1 was detected in nasopharyngeal swabs (2/110; 1.8%), eyebrow hair follicle (1/110; 0.9%), cutaneous warts (21/43; 48.8%), and anogenital warts (3/40; 7.5%), while HPV-63 was detected in nasopharyngeal swabs (3/110; 2.7%), swabs of the oral cavity (2/105; 1.9%), eyebrow hair follicles (4/110; 3.6%), cutaneous warts (9/43; 20.9%), and swab of the anal canal (1/110; 0.9%). We have confirmed that all Mu-PVs exhibit dual tissue tropism, since HPV-204, HPV-1, and HPV-63 were detected in cutaneous, mucosal, and mucocutaneous epithelia. HPV-204 viral load was extremely low in a single HPV-204-positive cutaneous wart (7.4 × 10−7 viral copies/cell). Hence, etiological association between infection with HPV-204 and the development of cutaneous warts could not be confirmed. Although the majority of HPV-1-positive samples contained low number of viral copies (range 5.9 x 10-6 – 1.8 x 10-2 viral copies/cell), HPV-1 was the most likely causative agent of three cutaneous warts with high HPV-1 viral load (range 8.5 x 103 – 5.2 x 104 viral copies/cell). Similarly, HPV-63 viral load was low in the majority of HPV-63-positive tissue samples (range 3.2 x 10-6 – 1.1 x 10-2 viral copies/cell). However, HPV-63 was the most likely causative agent of one common wart, where it was present in high viral copy numbers (3.9 x 102 viral copies/cell). Thus, we have confirmed that the majority of infections with Mu-PVs are clinically latent, whereas HPV-1 and HPV-63 may be the causative agents of sporadic cases of common warts. In contrast, we were not able to establish a causative association between the presence of HPV-204 and the development of common warts. Finally, we evaluated the genetic variability of Mu-PVs by sequencing complete LCR genomic regions of HPV-204, HPV-1, and HPV-63. We identified one genetic variant of HPV-204 that contained a single nucleotide polymorphism (SNP), six genetic variants of HPV-1 that contained 1-19 SNPs and/or nucleotide deletions and two genetic variants of HPV-63 that contained 6-13 SNPs and/or nucleotide deletions. Based on the obtained data, none of the genetic variants could be reliably associated with specific clinical manifestations or anatomical location of infection. Part of the doctoral thesis was published as a scientific paper in a journal cited by the Science Citation Index (SCI) (IF2016=2.806): Šterbenc A, Hošnjak L, Chouhy D, Bolatti EM, Oštrbenk A, Seme K, Kocjan BJ, Luzar B, Giri AA, Poljak M. Molecular characterization, tissue tropism, and genetic variability of the novel Mupapillomavirus type HPV204 and phylogenetically related types HPV1 and HPV63. PLoS One 2017;12:e0175892.

• Ključne besede: Key words: human papillomaviruses, HPV, HPV-204, Mupapillomavirus, novel HPV type, molecular characterization, phylogenetic analysis, clinical significance, tissue tropism, genetic variants