Varijabilnost genoma humanog virusa Parvovirus B19 (Parvoviridae, Erythrovirus)

Abstract

Uvod: Infekcija parvoB19 virusom koja se prenosi respiratornim i vertikalnim putem, ali i preko zaražene krvi i krvnih derivata, može predstavljati ozbiljan zdravstveni problem za rizične grupe pacijenata. Ovaj, po dimenzijama mali DNK virus bez omotača je vrlo otporan na standardne tehnike inaktivacije virusa u proizvodnji derivata krvi, a pouzdanu dijagnostiku otežava varijabilnost genomske sekvence virusa. Određivanje karakteristika nukleotidnih sekvenci izolata B19 virusa od značaja je za razvoj dijagnostičkih metoda i daje uvid u puteve nastanka i održavanja genomske varijabilnosti B19 virusa. Cilj ovog rada bio je da se odredi učestalost B19 virusa kod dobrovoljnih davalaca krvi i kod pacijenata sa sumnjom na B19 infekciju i da se na osnovu molekularno genetskih karakteristika dobijenih genomskih sekvenci, a u poređenju sa genomskim sekvencama dostupnim u bazama podataka, odredi evolutivna dinamika varijabilnosti parvoB19 virusa i filogenetski i filogeografski odnosi ispitivanih B19 izolata. Metode: Uzorci plazme dobrovoljnih davalaca i plazme i krvi pacijenata sa sumnjom na B19 infekciju analizirani su na prisustvo B19 DNK primenom lančane reakcije polimeraze (PCR), kako metodama iz literature tako i razvijenom internom metodom. Za uzorke sa detektovanim B19 genomom, direktnim nukleotidnim sekvenciranjem određena je genomska sekvenca. Bioinformatičkom analizom u odnosu na genomske sekvence B19 virusa dostupne u bazama podataka određen je genotip izolovanih virusnih genoma, evolutivni pritisci na osnovu odnosa nesinonimnih i sinonimnih zamena (dN/dS) za nukleotidne sekvence koje kodiraju virusne proteine, kao i genetičke distance i filogenetski odnosi B19 izolata dobijenih u Srbiji u odnosu na izolate iz drugih geografskih regija. Rezultati: Za 5 izolata iz Srbije, utvrđena je sekvenca B19 genoma u regiji kompletne cDNK. Iz baze podataka izdvojeni su i analizirani izolati sa poznatom sekvencom u regiji kompletne ili skoro kompletne cDNK, kojih je do 2012. godine bilo ukupno 146, od toga 117 genotipa 1. Analizom odnosa dN/dS za virusne proteine na osnovu većeg broja nukleotidnih sekvenci u odnosu na dosadašnje literaturne podatke procenjen je uticaj pozitivne i negativne selekcije na varijabilnost virusnog genoma...

Abstract: Background: Parvovirus B19 infection, being transmitted via the respiratory route, vertically from mother to fetus as well as through blood-derived products administered parenterally, can represent a serious health problem for risk patients groups. This small non-enveloped DNA virus is extremely resistant to standard viral inactivation techniques, while a reliable diagnosis is challenged by the virus genome sequence variability. Characterization of B19 isolates nucleotide sequences is valuable for development of diagnostic methods, and it gives an insight into the genesis and maintenance of the B19 virus genome variability. Aim of this thesis was to determine the frequency of B19 viremia in voluntary blood donors and in patients with the B19 infection symptoms, investigate the level and evolutionary dynamics of the B19 genome variability as well as to examine phylogenetic and phylogeographic relations of the B19 genomes obtained in Serbia with the isolates from the other parts of the world on the bases of the almost complete B19 cDNA sequences. Methods: Plasma samples of the voluntary blood donors, and plasma and blood samples of the B19 infection suspected patients were analysed for the B19 DNA presence by the polymerase chain reaction (PCR), using the methods described in the literature as well as internally developed method. For the B19 genome positive samples genome sequences were determined by direct sequencing. Bioinformatic analysis was performed by using the generated data as well as the database information in order to determine a genotype of the isolated genomes, evolutionary pressures (based on dN/dS ratio) for virus protein coding sequences, and genetic distances and phylogenetic relations of the B19 isolates from Serbia compared to the other geographic regions. Results: For 5 isolates from Serbia, the B19 genome sequence was determined for the complete cDNA. Database isolates with the known, complete or almost complete cDNA, available until the end of 2012 (146 isolates in total, out of which 117 were genotype 1) were chosen and used for the analysis. Positive and negative selection influence for the virus protein coding sequences was determined by the dN/dS ratio analysis by using larger data set than described in the literature so far...