Molecular and epidemiological characterisation of the novel mammalian orthoreovirus variants

Mikuletič, Tina (author), Steyer, Andrej (mentor)

Recently, the mammalian orthoreovirus (MRV) isolate SI-MRV01 with high similarity to newly described bat MRVs was first observed in a child with severe diarrhoea. Furthermore, a closely related MRVs were described in a dog with haemorrhagic enteritis and pigs with diarrhoea. The aim of this doctoral dissertation was to determine the prevalence of MRVs in bats, dogs and hospitalised children with acute gastroenteritis to reveal the occurrence and genetic variability of these viruses. In addition, MRV seroprevalence in dogs and children was investigated. The particular focus was on the occurrence of SI-MRV01 variants in target populations. To determine MRV prevalence, archive bat guano samples, stool samples of hospitalised children with diarrhoea and prospective dog stool samples were analysed with a broad-spectrum RT-PCR and a specific real time RT-PCR, targeting a novel bat MRV. Subsequently, MRV isolates were propagated in cell line and whole-genomes were obtained with NGS sequencing. For MRV seroprevalence study, dog sera and sera samples from children were analysed with serum-neutralisation test on MRV serotype reference strains (Reo1TL, Reo2TJ, Reo3TD) and bat isolate SI-MRV01. A novel bat MRV variant with 99% whole genome identity to isolate SI-MRV01 is present in Slovenian bats. Overall, SI-MRV01 prevalence was at 9.1%. Five independent strains were successfully isolated in cell culture following whole genome sequence determination from the archived bat guano samples. In addition to SI-MRV01-like variant four further genetically distant bat MRV variants were identified. Acute MRV infection was detected in one hospitalised child with diarrhoea (0.2%) and in zero dogs (0%). Neutralising antibodies against SI-MRV01 were detected in 7.5% children‘s sera and in 81.3% dogs‘ sera. MRVs are frequently present in bats, indicating bats as possible reservoir of circulating MRVs. Serology analysis of dog sera indicates possible route of infection via pets.

• Language: Slovenian
• Keywords: mammalian orthoreovirus, bat, RT-PCR, real-time RT-PCR, next-generation sequencing, phylogenetic analysis, serum-neutralisation test, virus isolation, cell culture
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2021
• PID: 20.500.12556/RUL-134155
• COBISS.SI-ID: 128012291
• Publication date in RUL: 24.12.2021

Secondary language

• Language: English
• Title: Molekularna in epidemiološka opredelitev novih različic sesalčjega ortoreovirusa

Abstract

Nedavno smo pri otroku s hudo drisko odkrili sesalčji ortoreovirus (MRV), poimenovan SI-MRV01, ki je izjemno soroden netopirskim MRV, na novo odkritim pri netopirjih v Italiji in Nemčiji. Poleg tega so bili sorodni MRV opisani tudi pri psu s hemoragičnim enteritisom in prašiču z drisko. Cilj te doktorske naloge je bil določiti pogostnost pojavljanja MRV pri netopirjih, psih in hospitaliziranih otrocih z akutnim gastroenteritisom. Želeli smo odkriti pojavljanje in genetsko raznolikost teh virusov. Poleg tega smo raziskali seroprevalenco MRV pri psih in otrocih. Posebno pozornost smo posvetili pojavljanju različic SI-MRV01. Za določanje pogostnosti pojavljanja MRV smo arhivske netopirske vzorce gvana, vzorce blata pri hospitaliziranih otrocih in prospektivno zbrane pasje iztrebke analizirali s široko-spektralnim RT-PCR in SI-MRV01 specifičnim RT-PCR v realnem času. Izolate MRV smo namnožili na celični liniji in analizirali njihove celotne genome s sekvenciranjem naslednje generacije. Za seroprevalenčno študijo MRV smo pasje in otroške serume analizirali s serum-nevtralizacijskim testom, pri čemer smo uporabili referenčne seve MRV (Reo1TL, Reo2TJ, Reo3TD) in netopirski izolat iz otroka SI-MRV01. Pri slovenskih netopirjih smo dokazali nove netopirske različice MRV z 99 % podobnostjo na nivoju celotnega genoma z izolatom SI-MRV01. Celokupna pogostnost pojavljanja SI-MRV01 je 9,1 %. Iz arhivskih vzorcev netopirskega gvana smo na celični kulturi uspešno osamili 5 neodvisnih izolatov in jim določili nukleotidno zaporedje celotnega genoma. Poleg različic SI-MRV01 smo določili 4 genetsko bolj oddaljene netopirske različke MRV. Akutno okužbo z MRV smo določili pri enem hospitaliziranem otroku z drisko (0,2 %) in pri nobenem psu (0 %). Nevtralizirajoča protitelesa proti izolatu SI-MRV01 smo določili pri 7,5 % otroških serumih in pri 81,3 % pasjih serumih. MRV so pogosto prisotni pri netopirjih, ki predstavljajo možen vir krožečih MRV. Serološka analiza pasjih serumov nakazuje možno okužbo z MRV preko domačih ljubljenčkov.

• Keywords: sesalčji orthoreovirus, netopir, RT-PCR, RT-PCR v realnem času, sekvenciranje naslednje generacije, filogenetska analiza, serum-nevtralizacijski test, osamitev virusa, celična kutura