Genome announcement : complete genome sequence of a novel Mupapillomavirus, HPV204

Human papillomaviruses (HPVs) are small, non-enveloped viruses with a circular double-stranded DNA genome, etiologically associated with various benign and malignant neoplasms of the skin and mucosa. As of May 30, 2015, 201 different HPV types had been completely sequenced and officially recognized and divided into five PV-genera: Alpha-, Beta-, Gamma-, Mu-, and Nupapillomavirus. The Mupapillomavirus genus currently consists of only two HPV types: HPV1 and HPV63, identified in 1980 and 1993, respectively, both associated with sporadic cases of cutaneous warts. In this preliminary study, we announce the complete genome sequence of a novel HPV type, now officially recognized as HPV204. Based on preliminary data, the genome of HPV204 comprises a total of 7,227 bp and contains five early open reading frames (E1, E2, E4, E6, and E7) and two late ORFs (L1 and L2). No E5 ORF could be identified. Preliminary HPV204 clusters to the Mu-PV genus, species Mu-3.Fil: Kocjan, Boštjan J. University of Ljubljana. Faculty of Medicine. Institute of Microbiology and Immunology; SloveniaFil: Šterbenc, Anja. University of Ljubljana. Faculty of Medicine. Institute of Microbiology and Immunology; SloveniaFil: Hošnjak, Lea. University of Ljubljana. Faculty of Medicine. Institute of Microbiology and Immunology; SloveniaFil: Chouhy, Diego. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área de Virología; ArgentinaFil: Bolatti, Elisa María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área de Virología; ArgentinaFil: Giri, Adriana Angélica. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área de Virología; ArgentinaFil: Poljak, Mario. University of Ljubljana. Faculty of Medicine. Institute of Microbiology and Immunology; Sloveni

Molecular characterization, clinical significance and tissue tropism of a novel human papillomavirus, HPV-204, and related types

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 (1/1160,8%), vzorcu kožne bradavice (1/432,3%), brisih analnega kanala (4/1103,6%) in brisih penisa (5/1104,5%). V sklopu doktorske naloge smo skupno odkrili 27 neodvisnih izolatov HPV-1, ki smo jih odkrili v brisih nosnega dela žrela (2/1101,8%), dlačnem mešičku obrvi (1/1100,9%), vzorcih kožnih bradavic (21/4348,8%) in vzorcih anogenitalnih bradavic (3/407,5%), ter 19 neodvisnih izolatov HPV-63, ki smo jih odkrili v brisih nosnega dela žrela (3/1102,7%), brisih ustne votline (2/1051,9%), dlačnih mešičkih obrvi (4/1103,6%), vzorcih kožnih bradavic (9/4320,9%) in brisu analnega kanala (1/1100,9%). 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 201712:e0175892.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 (1/1160.8%), cutaneous wart (1/432.3%), swabs of the anal canal (4/1103.6%), and swabs of the penile surface (5/1104.5%). HPV-1 was detected in nasopharyngeal swabs (2/1101.8%), eyebrow hair follicle (1/1100.9%), cutaneous warts (21/4348.8%), and anogenital warts (3/407.5%), while HPV-63 was detected in nasopharyngeal swabs (3/1102.7%), swabs of the oral cavity (2/1051.9%), eyebrow hair follicles (4/1103.6%), cutaneous warts (9/4320.9%), and swab of the anal canal (1/1100.9%). 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&#87227 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 201712:e0175892

Usefulness of rapid antigen testing for SARS-CoV-2 screening of healthcare workers : ǂa ǂpilot study

Background. Identification of infected healthcare workers (HCWs) is an important step in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) transmission control. Rapid antigen tests (RATs) are considered an important addition to molecular tests in diagnosing coronavirus disease 2019 (COVID-19), mainly because of their fast turnaround time, easier analytical procedure and lower price. However, real-life studies on the usefulness of such testing for screening of HCWs are limited. Methods. Physicians, nurses and hospital attendants currently working at the University Clinic of Respiratory and Allergic Diseases Golnik were invited to participate in the pilot study. Nasopharyngeal swabs were obtained three times per week for two consecutive weeks and tested with a point-of-care RAT and reverse transcription polymerase chain reaction (RT-PCR). Serum samples were obtained at the beginning of the study and 2 weeks after the last swab was collected to evaluate the serological status. Results. A total of 191 nasopharyngeal swabs from 36 HCWs were obtained. None of the samples tested was positive for the presence of SARS-CoV-2 antigen, whereas two HCWs tested positive on RT-PCR. Of these, one HCW had a newly identified SARS-CoV-2 infection, whereas RT-PCR probably detected a previous but recent infection in the other HCW. Conclusio.n Based on the results of this pilot study, it is unlikely that RAT will reliably detect novel SARS-CoV-2 infections among asymptomatic HCWs despite serial sampling. Although RT-PCR-based screening of HCWs may not be feasible due to high sample volume, molecular methods may identify SARS-CoV-2-infected HCWs already during the presymptomatic stage

Prevalence of the Helicobacter pylori babA2 Gene in Children Mainly Depends on the PCR Primer Set Used

Various polymerase chain reaction- (PCR-) based methods with varying positivity rates were designed to detect the Helicobacter pylori babA2 gene. To compare different primer sets, babA2 prevalence was determined in 279 H. pylori-positive pediatric samples using the 832 bp, 139 bp, and 271 bp PCR primer sets, resulting in 34.0%, 51.3%, and 79.6% prevalence of the babA2 gene, respectively. The babA2 status determined using the 832 bp and 139 bp PCR primer sets significantly correlated with bacterial density and activity of inflammation, whereas no such correlations were found using the 271 bp PCR primer set. The 139 and 832 bp PCR primer sets concordantly detected the babA2 gene in 93 cases; however, in comparison to the 832 bp PCR primer set, the 139 bp PCR primer set detected additional 50 babA2 cases, whereas only two 832 bp positive cases were missed. The 271 bp PCR primer set missed 32 babA2 cases that were 832 bp and/or 139 bp PCR positive, but tested solely positive in 109 cases. Interestingly, cloning of a subset of 271 bp PCR positive samples revealed amplification of the babA/B gene chimera. Hence, in our opinion, the 271 bp PCR protocol is not a reliable diagnostic tool for detecting the babA2 gene in children. Our results reaffirm previous observations that the use of certain babA2 PCR primer sets can significantly impact estimation of the prevalence and clinical relevance of the H. pylori babA2 gene in children, suggesting babA2 detection methods should be carefully selected

Identification of a Novel Human Papillomavirus, Type HPV199, Isolated from a Nasopharynx and Anal Canal, and Complete Genomic Characterization of Papillomavirus Species <i>Gamma</i>-12

<div><p>The novel human papillomavirus type 199 (HPV199) was initially identified in a nasopharyngeal swab sample obtained from a 25 year-old immunocompetent male. The complete genome of HPV199 is 7,184 bp in length with a GC content of 36.5%. Comparative genomic characterization of HPV199 and its closest relatives showed the classical genomic organization of Gammapapillomaviruses (<i>Gamma</i>-PVs). HPV199 has seven major open reading frames (ORFs), encoding five early (E1, E2, E4, E6, and E7) and two late (L1 and L2) proteins, while lacking the E5 ORF. The long control region (LCR) of 513 bp is located between the L1 and E6 ORFs. Phylogenetic analysis additionally confirmed that HPV-199 clusters into the <i>Gamma</i>-PV genus, species <i>Gamma</i>-12, additionally containing HPV127, HV132, HPV148, HPV165, and three putative HPV types: KC5, CG2 and CG3. HPV199 is most closely related to HPV127 (nucleotide identity 77%). The complete viral genome sequence of additional HPV199 isolate was determined from anal canal swab sample. Two HPV199 complete viral sequences exhibit 99.4% nucleotide identity. To the best of our knowledge, this is the first member of <i>Gamma</i>-PV with complete nucleotide sequences determined from two independent clinical samples. To evaluate the tissue tropism of the novel HPV type, 916 clinical samples were tested using HPV199 type-specific real-time PCR: HPV199 was detected in 2/76 tissue samples of histologically confirmed common warts, 2/108 samples of eyebrow hair follicles, 2/137 anal canal swabs obtained from individuals with clinically evident anal pathology, 4/184 nasopharyngeal swabs and 3/411 cervical swabs obtained from women with normal cervical cytology. Although HPV199 was found in 1.4% of cutaneous and mucosal samples only, it exhibits dual tissue tropism. According to the results of our study and literature data, dual tropism of all <i>Gamma</i>-12 members is highly possible.</p></div

Phylogenetic analysis of HPV199.

<p>The complete HPV genome sequences of 75 officially recognized HPV types belonging to the genus <i>Gamma</i>-PV and all putative <i>Gamma</i>-PV types were obtained and aligned and a maximum likelihood phylogenetic tree was constructed. The nucleotide sequences of two <i>Beta</i>-PVs, HPV5 and HPV8, were used to root the tree. The numbers in each branch are bootstrap support values and are given as percentages. Species <i>Gamma</i>-12 is indicated by a black box.</p

Molecular characterization, tissue tropism, and genetic variability of the novel Mupapillomavirus type HPV204 and phylogenetically related types HPV1 and HPV63.

HPV204 is the only newly identified Mupapillomavirus (Mu-PV) type in more than a decade. To comprehensively characterize HPV204, we performed a detailed molecular analysis of the viral genome and evaluated its clinical relevance in comparison to the other Mu-PVs, HPV1 and HPV63. The 7,227-bp long genome of HPV204 exhibits typical genomic organization of Mu-PVs with eight open reading frames (ORFs) (E6, E7, E1, E2, E8, E4, L2, and L1). We developed three type-specific quantitative real-time PCRs and used them to test a representative collection (n = 1,006) of various HPV-associated benign and malignant neoplasms, as well as samples of clinically normal cutaneous, mucosal, and mucocutaneous origins. HPV204, HPV1, and HPV63 were detected in 1.1%, 2.7%, and 1.9% of samples tested, respectively, and were present in skin and mucosa, suggesting dual tissue tropism of all Mu-PVs. To evaluate the etiological role of Mu-PVs in the development of HPV-associated neoplasms, Mu-PV viral loads per single cell were estimated. HPV1 and HPV63 were present in high viral copy numbers in 3/43 and 1/43 cutaneous warts, respectively, and were identified as the most likely causative agents of these warts. HPV204 viral load was extremely low in a single HPV204-positive cutaneous wart (7.4 × 10-7 viral copies/cell). Hence, etiological association between HPV204 and the development of cutaneous warts could not be established. To the best of our knowledge, this is the first study to evaluate the genetic variability of Mu-PVs by sequencing complete LCR genomic regions of HPV204, HPV1, and HPV63. We detected several nucleotide substitutions and deletions within the LCR genomic regions of Mu-PVs and identified two genetic variants of HPV204 and HPV63 and five genetic variants of HPV1