Razvoj amplikonov za sekvenciranje celotnega genoma ortohantavirusa Dobrava v Sloveniji

Rak, Karin (author), Korva, Miša (mentor)

Naravni gostitelji ortohantavirusov so glodavci (Rodentia), rovke (Soricidae) in netopirji (Chiroptera). Ortohantavirusi, ki povzročajo bolezni pri ljudeh, se v naravi ohranjajo v glodavcih in rovkah. V diplomski nalogi smo se osredotočili na vrsto Orthohantavirus dobravaense (virus Dobrava; DOBV), ki je bil odkrit v Sloveniji v rumenogrli miši (Apodemus flavicollis), ki pri ljudeh povzroči hemoragično mrzlico z renalnim sindromom [1]. V diplomskem delu smo načrtali začetne oligonukleotide, s katerimi smo določili celoten genom za človeka patogenega DOBV. Delali smo z virusno RNA, izolirano iz celične kulture virusa Vero E6 in iz tkiva okužene rumenogrle miši. Ortohantavirusi imajo segmentiran genom (segmenti S, M in L), zato smo razvili 3 amplikonske sheme, za vsak segment posebej. Za določitev amplikonov smo poravnali vsa do sedaj znana zaporedja DOBV in identificirali ohranjene regije ter s programskim orodjem PrimalScheme in VarVamp načrtali začetne oligonukleotide. Te smo preverili tako, da smo iz izolirane RNA z reverzno transkripcijo sintetizirali cDNA ter jo pomnožili z izbranimi začetnimi oligonukleotidi. Amplikone smo preverili z gelsko elektroforezo in sekvencirali na platformi Oxford Nanopore. Delo je zajemalo tudi analizo z bioinformacijskimi orodji. Rezultati diplomske naloge vključujejo načrtovanje začetnih oligonukleotidov, celovito analizo PCR-produktov na agaroznem gelu, določitev nukleotidnega zaporedja vzorcev, pridobljenih z različnimi metodami ter podrobno analizo prileganja sekvenčnih odčitkov in konsenznih zaporedij genoma DOBV. S prvimi amplikonskimi shemami smo uspešno načrtali začetne oligonukleotide za segmenta L in S. Za segment M smo pripravili novo shemo, ki pa še ni dosegla optimalne učinkovitosti, zato je cilj nadaljnjih raziskav njena izboljšava. Validacija metode na tkivnem vzorcu miši je potrdila zanesljivost in učinkovitost razvite metode. Poleg tega je uporaba metode SISPA-A jasno pokazala, zakaj je za natančno določitev virusnega genoma ključno skrbno načrtovanje amplikonskih shem. Na koncu smo pridobili skupna zaporedja vseh treh segmentov genoma DOBV, kar predstavlja pomemben korak v nadaljnjem raziskovanju genetske raznolikosti ortohantavirusov.

• Language: Slovenian
• Keywords: amplikoni, ortohantavirus Dobrava, sekvenciranje genoma, sekvenciranje naslednje generacije
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2025
• PID: 20.500.12556/RUL-172685
• COBISS.SI-ID: 257621507
• Publication date in RUL: 10.09.2025

Secondary language

• Language: English
• Title: Development of amplicons for whole genome sequencing of Dobrava orthohantavirus in Slovenia

Abstract

The natural hosts of orthohantaviruses are rodents (Rodentia), shrews (Soricidae), and bats (Chiroptera). One associated with human disease are maintained in nature in rodents and shrews. In this thesis, we focused on the Orthohantavirus dobravaense (Dobrava virus; DOBV) species, which was discovered in Slovenia in the yellow-necked mouse (Apodemus flavicollis) and causes hemorrhagic fever with renal syndrome in humans [1]. We designed primers, which were used to determine the entire genome of the human pathogen DOBV. We worked with viral RNA, isolated from viral culture and tissue of infected, yellow-necked mouse. Orthohantaviruses have a segmented genome (segments S, M, and L), so we developed three amplicon schemes, one for each segment. To design the amplicons, we aligned all currently known DOBV sequences and identified conserved regions. Furtheron, we used PrimalScheme and VarVamp software packages to design the primers. We verified the designed primers by synthesizing cDNA from isolated RNA using reverse transcription and amplifying it with the selected primers. We analyzed the amplicons by gel electrophoresis and sequenced them on the Oxford Nanopore platform. The work also included analysis with bioinformatics tools. The results of the thesis include a design of primer pairs, comprehensive analysis of PCR products by agarose gel electrophoresis, sequencing samples obtained by various methods, as well as a detailed analysis of reads, alignments and consensus sequences of the DOBV genome. With the initial amplicon schemes, we successfully amplified complete L and S segments. For the M segment, a new scheme was developed. However, it has not yet reached optimal efficiency, thus further improvements are necessary. Validation of the method on a tissue sample of infected mouse confirmed the reliability and effectiveness of the developed sequencing approach. Furthermore, the application of the SISPA-A method clearly demonstrated why careful design of amplicon schemes is crucial for accurate determination of viral genomes. In the end, consensus sequences for all three segments of the DOBV genome were obtained, marking an important step forward in the research of genetic diversity of orthohantaviruses.

• Keywords: amplicons, Dobrava virus, next-generation sequencing, whole genome sequencing