High similarity of novel orthoreovirus detected in a child hospitalized with acute gastroenteritis to mammalian orthoreoviruses found in bats in Europe

Abstract

Mammalian orthoreoviruses (MRVs) are known to cause mild enteric and respiratory infections in humans. They are widespread and infect a broad spectrum of mammals. We report here the first case of an MRV detected in a child with acute gastroenteritis, which showed the highest similarity to an MRV reported recently in European bats. An examination of a stool sample from the child was negative for most common viral and bacterial pathogens. Reovirus particles were identified by electron microscopic examination of both the stool suspension and cell culture supernatant. The whole-genome sequence was obtained with the Ion Torrent next-generation sequencing platform. Prior to sequencing, the stool sample suspension and cell culture supernatant were pretreated with nucleases and/or the convective interaction medium (CIM) monolithic chromatographic method to purify and concentrate the target viral nucleic acid. Whole-genome sequence analysis revealed that the Slovenian SI-MRV01 isolate was most similar to an MRV found in a bat in Germany. High similarity was shared in all genome segments, with nucleotide and amino acid identities between 93.8 to 99.0% and 98.4 to 99.7%, respectively. It was shown that CIM monolithic chromatography alone is an efficient method for enriching the sample in viral particles before nucleic acid isolation and next-generation sequencing application.

Fig 1

Electron micrographs of reoviruses in stool suspension (A) (magnification, 100,000×) cell culture supernatant (B) (magnification, 100,000×), and ultrathin section of LLC-MK2 cells (C) (magnification, 10,000×) infected with the SI-MRV01 orthoreovirus strain. Arrows in Fig. 1C indicate reovirus particles.

Fig 2

Elution peaks obtained in the chromatographic runs involving samples 1 (stool, Benzonase, CIM one-step elution), 2 (cell culture, Benzonase, CIM one-step elution), and 3 (cell culture, CIM gradient elution). mAU, milliabsorbance units. Fraction analysis for 18S rRNA (as indicative of eukaryotic RNA presence) and reovirus EM detection is shown below the graphs. In the case of sample 1, the presence of reovirus had to be confirmed by classic quantitative RT-PCR (RT-qPCR) (shown to the left below). NTC, negative template control; NT, not tested; undet, undetermined.

Fig 3

Phylogenetic analysis of the L1, M1, S1, and S2 genome segments for the Slovenian strain and most related whole-genome strains from GenBank. The neighbor-joining algorithm was used for the construction of the phylogenetic tree with bootstrap values of 1,000 replicates shown at the branches. The scale bar represents the p-distance. ●, Slovenian MRV isolate SI-MRV01; △, Italian MRV strain identified in a bat; ▲, German MRV isolate 342/08 identified in a bat.

Fig 4

Similarity plot analysis of the whole-genome nucleotide sequence comparing the Slovenian orthoreovirus strain with some of the most related strains from GenBank.