Exploring the virome of diseased horses

Abstract

Metagenomics was used to characterize viral genomes in clinical specimens of horses with various organ-specific diseases of unknown aetiology. A novel parvovirus as well as a previously described hepacivirus closely related to human hepatitis C virus and equid herpesvirus 2 were identified in the cerebrospinal fluid of horses with neurological signs. Four co-infecting picobirnaviruses, including an unusual genome with fused RNA segments, and a divergent anellovirus were found in the plasma of two febrile horses. A novel cyclovirus genome was characterized from the nasal secretion of another febrile animal. Lastly, a small circular DNA genome with a Rep gene, from a virus we called kirkovirus, was identified in the liver and spleen of a horse with fatal idiopathic hepatopathy. This study expands the number of viruses found in horses, and characterizes their genomes to assist future epidemiological studies of their transmission and potential association with various equine diseases.

Fig. 1.. (a) Diagram of the two RNA segments of picobirnavirus (GenBank accession numbers NC_007026 and NC_007027). (b) Fusion genome and putative ORFs of PBV Equ4. The junction sequence was confirmed by nested PCR (nPCR). The distribution of sequence coverage obtained by next-generation sequencing is shown. RdRp, RNA-dependent RNA polymerase; Cap, capsid. (c) Un-rooted phylogenetic analysis of all picobirnavirus RdRp protein sequences >450 aa available in GenBank using the neighbour-joining method with p distance and 1000 bootstrap replications. Genogroups I, II and III are labelled. Proposed genogroups IV, V and VI are highlighted by the grey boxes. Bar, amino acid substitutions per position.

Fig. 2.. Un-rooted phylogenetic analysis of horse parvovirus CSF, generated with the near-complete NS1 protein (∼500 aa) using the neighbour-joining method with p distance and 1000 bootstrap replications. Bar, amino acid substitutions per position. Bootstrap values for each node are shown if >70  %.

Fig. 3.. (a) Genome organization of TTV Equ1. (b) Un-rooted phylogenetic analysis of the complete ORF1 protein using the neighbour-joining method with p distance and 1000 bootstrap replications. Bar, amino acid substitutions per position. Bootstrap values for each node are shown if >60 %. All 11 known anellovirus genera are shown and the new genus proposed by this study is highlighted.

Fig. 4.. (a) Genome organization of CyCV Equ1, KirV Equ1 and Po-Circo-like virus genomes. (b) Phylogenetic analysis of Rep proteins using the neighbour-joining method with p distance and 1000 bootstrap replications. Bar, amino acid substitutions per position. Bootstrap values for each node are shown if >70 %.

Fig. 5.. Pathological findings in the horse with severe hepatopathy. (a) Gross view of the liver, cut section. Diffuse enhancement of the reticular pattern characterized by a delicate tan/pale meshwork that delineates the periphery of the hepatic lobules is shown. In many lobules a similar tan/pale discoloration is evident in the centrilobular areas. The parenchyma between the portal and centrilobular areas is dark brown to red. The walls of the portal veins and hepatic arteries/arterioles are yellowish (icterus). Bar, 2 cm. (b) Liver histopathology, haematoxylin and eosin stain. Subgross microscopic view of the enhanced lobular pattern shown in Fig. 1(a), with marked diffuse congestion and haemorrhage in the midzonal areas. The hepatic lobules have an irregular instead of hexagonal shape, a consequence of parenchymal collapse. Bar, 500 μm. (c) Liver histopathology, haematoxylin and eosin stain. Higher magnification of a hepatic lobule showing a portal tract (upper right corner) and a centrilobular vein (^*). There is severe diffuse panlobular hepatocellular loss with few remaining hepatic cords in the periportal region (arrow), and marked congestion and haemorrhage in the midzonal and periportal areas. The centrilobular and periportal areas are hypercellular due to inflammatory cell infiltrates. Portal inflammation and fibrosis are also observed. Inset: closer view of a mitotic figure in a hepatocyte in the midzonal area (regenerative attempt). Bar, 100 μm. (d) Liver histopathology, haematoxylin and eosin stain. Centrilobular area and central vein. There is almost complete hepatocellular loss with very few remaining degenerate (arrowheads) and necrotic hepatocytes, although the centrilobular area is hypercellular due to infiltration of abundant histiocytes, fewer lymphocytes, and rare neutrophils. Numerous histiocytes/Kupffer cells contain intracytoplasmic haemosiderin granules, and occasional bi-/tri-nucleated cells contain intracytoplasmic red blood cells (erythrophagocytosis, arrow). Semicircular perivenous fibrosis is also shown (^*). Bar, 20 μm.