Detection and molecular characterization of porcine type 3 orthoreoviruses circulating in South Korea

Abstract

Orthoreoviruses infect virtually all mammalian species, causing systemic infections including mild gastrointestinal and respiratory illnesses. However, little is known about the prevalence or genetic diversity of porcine orthoreoviruses in South Korea. We examined 237 diarrheic fecal samples collected from 78 pig farms around the country. RT-PCR utilizing primers specific for the L1 gene of mammalian orthoreoviruses showed that 45 (19.0%) samples were positive. The 10 strains isolated from orthoreovirus-positive samples formed typical perinuclear cytoplasmic inclusion bodies and had an atypical hemagglutination pattern; these are characteristics of type 3 orthoreovirus. Phylogenetic analysis of the S1 gene in these 10 Korean and other strains showed that type 3 orthoreoviruses could be divided into four lineages; the 10 Korean strains were included in porcine lineage IV, along with T3/porcine/Sichuan/2006. Sequence analysis showed that strains in lineage IV had nucleotide identities of 97.0-98.1% and deduced amino acid identities of 96.4-98.2%. Sequence analysis of the σ1 protein, a viral attachment protein, revealed that the amino acid sequences associated with neurotropism (amino acids 198-204, 249I, 350D, and 419E) were highly conserved among the Korean strains, confirming that neural tropism was present. In conclusion, our findings suggest that porcine orthoreovirus infections are endemic in pig farms in South Korea and that the 10 novel Korean porcine orthoreoviruses belong to porcine lineage IV of type 3 orthoreovirus. In addition, sequence analysis of S1 genes encoding the σ1 protein showed that the 9 of 10 Korean porcine orthoreoviruses exhibited neural tropism.

Fig. 1

Characterization of the 10 isolated Korean porcine orthoreovirus strains. (A) Histopathologic examination, showing intracytoplasmic inclusion bodies in the perinuclear region (arrow) of TF-104 cells. Diff-Quick stain. Bar = 100 μm. (B) Immunofluorescence, showing that a positive reaction (green) was confined to the cytoplasm. Bar = 50 μm. (C) Transmission electron micrograph, showing icosahedral, non-enveloped virus particles. Negative staining with 2% (w/v) sodium phosphotungstate at pH 6.8. Bar = 200 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 2

Phylogenetic tree of the σ1 protein of mammalian orthoreovirus strains.

Fig. 3

Deduced amino acid sequences of the σ1 protein of the 10 Korean porcine orthoreovirus strains, and the T3D/55 and T3S06 strains. The predicted sialic acid-binding domain is underlined; the sequence associated with sensitivity to cleavage by intestinal proteases is shown in a light-grey box; the sequences associated with neuronal tropism are in black boxes (Tyler et al., 2004).