Genomic Characteristics and Pathogenicity of Novel Reassortant Mammalian Orthoreoviruses From Sheep, China

Abstract

Mammalian orthoreoviruses (MRVs) have a wide geographic distribution worldwide and have been detected from humans and a variety of animal species. This study represents the first isolation of MRV from sheep rectal swabs in China, with analyses of its molecular and pathogenicity characteristics. MRV-positive samples were inoculated into Madin-Darby bovine kidney (MDBK) cells, resulting in stable cytopathic effects (CPEs) after three generations of blind passage. Two isolates were isolated and confirmed as MRV, named MRV-XJ23 and MRV-sheep/SY13, through reverse-transcription polymerase chain reaction (RT-PCR), transmission electron microscopy, and indirect immunofluorescence assay (IFA). The viruses exhibited broad cellular tropism. Whole-genome sequences were obtained and subjected to homology and evolutionary analyses, revealing that MRV-XJ23 and MRV-sheep/SY13 belong to the MRV-1 serotype. Phylogenetic analyses demonstrated that MRV-XJ23 is a reassortant virus containing gene segments from three MRVs that infected humans, bovines, and bats, with nucleotide homology exceeding 94.56%. The gene segments of MRV-sheep/SY13 were derived from five strains-Osaka2005, BatMRV-2/SNU1/Korea/2021, T1/human/Netherlands/1/84, IND/MZ/3013814/reo, and B/03-with nucleotide homology exceeding 95.47%. Animal experiments demonstrated that MRV-sheep/SY13 infection induced significant pathological changes in the respiratory and digestive tracts of mice. In sheep, MRV-sheep/SY13 caused respiratory infections, but no obvious lesion was observed from the digestive tract. This study expands our understanding of the MRV host range, reveals the potential public health risk of MRV transmission across species and zoonotic transmission, and underscores the necessity of further studies on epidemiology, reassortment patterns, and pathogenicity of MRV in sheep and domestic animals.

Keywords: mammalian orthoreoviruses (MRVs); pathogenicity; reassortment; sheep; virus isolation and identification.

Figure 1

CPE of MRV-XJ23 infection. (A) Control MDBK cells incubated with PBS. (B) CPE was observed in MDBK cells incubated with MRV. (C) Electron micrograph of MRV particles. CPE, cytopathic effect; MDBK, Madin–Darby bovine kidney; MRV, mammalian orthoreovirus; PBS, phosphate-buffered saline.

Figure 2

MRVs exhibit broad cell tropism. Three days postinfection with MRV-XJ23, HRT-18, MA104, Vero, F81, IPEC-J2, and MDBK cells were subjected to indirect immunofluorescence analysis. Sheep MRV antigens were stained with rabbit anti-MRV polyclonal antibody as the primary antibody, followed by fluorescein isothiocyanate-labeled mouse anti-rabbit IgG (green). Cell nuclei were counterstained with DAPI (blue). Scale bar: 200 μm. DAPI, 4′,6-diamidino-2-phenylindole; MDBK, Madin–Darby bovine kidney; MRVs, mammalian orthoreoviruses.

Figure 3

Phylogenetic and genomic analyses of MRV-XJ23 and MRV-sheep/SY13. (A) Phylogenetic tree and pairwise genetic distance heatmap of MRV-XJ23 and MRV-sheep/SY13 based on the complete nucleotide coding sequences of the S1 fragments. The tree was constructed using the neighbor-joining method in MEGA 11 with bootstrap values from 1000 replicates and was refined using https://www.chiplot.online/tvbot.html. Different MRV lineages and countries of origin are indicated by color blocks, and MRV-XJ23 and MRV-sheep/SY13 are marked with black triangles. The pairwise genetic distance heatmap was created using Megalign software. (B and C) Alignment of deduced amino acid sequences of the δ1 protein. Comparison of the δ1 protein sequences from MRV-XJ23, MRV-sheep/SY13, and closely related MRV-1 strains. (B) Alignment for MRV-XJ23. (C) Alignment for MRV-sheep/SY13. MRV, mammalian orthoreovirus.

Figure 4

Phylogenetic and genomic analyses of MRV-XJ23 and MRV-sheep/SY13. Phylogenetic tree and pairwise genetic distance heatmap of MRV-XJ23 and MRV-sheep/SY13 based on the complete nucleotide coding sequences of the S2–S4, M1–M3, and L1–L3 segments. The tree was constructed using the neighbor-joining method in MEGA 11 with bootstrap values from 1000 replicates and was refined using https://www.chiplot.online/tvbot.html. Different MRV lineages and countries of origin are indicated by color blocks, and MRV-XJ23 and MRV-sheep/SY13 are marked with black triangles. The pairwise genetic distance heatmap was created using Megalign software. MRV, mammalian orthoreovirus.

Figure 5

Schematic summary of the genomic compositions of novel MRV isolates and related strains. Segments with close relationships were identified based on nucleotide identity and phylogenetic analysis, with each distinguished by a unique color. Dashed lines indicate unidentified segments of the T1/human/Netherlands/1/84 strain. (A) Indicates MRV-sheep/SY13 strain. (B) Indicates MRV-XJ23 strain. MRV, mammalian orthoreovirus.

Figure 6

Tissue analysis and pathological examination of MRV-infected mice. (A) Genome copy number of MRV in various mouse tissues. (B–E) HE staining results. (B) Lungs of the control group. (C) Lungs of the infected group. (D) Small intestine of the control group. (E) Small intestine of the infected group. HE, hematoxylin and eosin; MRV, mammalian orthoreovirus.

Figure 7

Tissue analysis and pathological examination of MRV-infected sheep. (A) Genome copy number of MRV in various sheep tissues. (B–E) HE staining results. (B) Lungs of the control group. (C) Lungs of the infected group. (D) Trachea of the control group. (E) Trachea of the infected group. HE, hematoxylin and eosin; MRV, mammalian orthoreovirus.