A Novel Squirrel Respirovirus with Putative Zoonotic Potential

Abstract

In a globalized world, the threat of emerging pathogens plays an increasing role, especially if their zoonotic potential is unknown. In this study, a novel respirovirus, family Paramyxoviridae, was isolated from a Sri Lankan Giant squirrel (Ratufa macroura), which originated in Sri Lanka and deceased with severe pneumonia in a German zoo. The full-genome characterization of this novel virus, tentatively named Giant squirrel respirovirus (GSqRV), revealed similarities to murine (71%), as well as human respiroviruses (68%) with unique features, for example, a different genome length and a putative additional accessory protein. Congruently, phylogenetic analyses showed a solitary position of GSqRV between known murine and human respiroviruses, implicating a putative zoonotic potential. A tailored real-time reverse transcription-polymerase chain reaction (RT-qPCR) for specific detection of GSqRV confirmed a very high viral load in the lung, and, to a lesser extent, in the brain of the deceased animal. A pilot study on indigenous and exotic squirrels did not reveal additional cases in Germany. Therefore, further research is essential to assess the geographic distribution, host range, and zoonotic potential of this novel viral pathogen.

Keywords: Paramyxoviridae; Sri Lankan Giant squirrel; isolate; novel respirovirus; pneumonia; potential zoonosis.

Figure 1

Histopathological findings in the lung of the deceased Sri Lankan Giant squirrel. (a) Hemorrhagic-necrotizing pneumonia with group cell necrosis of pneumocytes (circles), acute hemorrhages (plus), and infiltration of neutrophil granulocytes (arrow heads), as well as mononuclear inflammatory cells (arrows); (b) eosinophilic intranuclear inclusion bodies in pneumocytes (arrow heads).

Figure 2

Electron micrograph of Giant squirrel respirovirus (GSqRV) with liberated ribonucleoprotein complex.

Figure 3

Schematic representation of the coding sequences (CDS) located in the P gene including the new open reading frame (ORF) Y0 in the GSqRV genome. The P mRNA transcript serves as template for the synthesis of the phosphoprotein and the accessory proteins located in the +1 ORF, accessible through a ribosomal scanning mechanism. The superscript numbers indicate the position of the first base of the start codon relative to the 5′ end of the mRNA; blue: murine respirovirus (MRV), strain Nagoya (NC_001552), black: GSqRV (this study), orange: human respirovirus 1 (HRV1), strain Washington/1964 (NC_003461); underlined: protein expression was verified by others [39,41,42], lower case: non-start codons at the corresponding position.

Figure 4

Similarity analysis of the GSqRV genome to MRV and HRV1 using SimPlot. The sequence identity within a sliding window of 200 nt centered on the plotted position is depicted, with a step size of 20 nt.

Figure 5

Classification of GSqRV in the genus Respirovirus by phylogenetic analysis of selected representatives of each species with the main host depicted in black silhouettes (a) and phylogenetic analysis of murine, human, and porcine respirovirus 1 with bovine respirovirus 3 (NC002161) included as outgroup (b). Selected were available complete genome sequences within the species excluding clones and revertants. Sequences were aligned with MAFFT and phylogenetically analyzed using RAxML with the model GTR GAMMA and 1000 replicates for bootstrap support. Annotation includes strain/isolate and accession number.

Figure 5

Classification of GSqRV in the genus Respirovirus by phylogenetic analysis of selected representatives of each species with the main host depicted in black silhouettes (a) and phylogenetic analysis of murine, human, and porcine respirovirus 1 with bovine respirovirus 3 (NC002161) included as outgroup (b). Selected were available complete genome sequences within the species excluding clones and revertants. Sequences were aligned with MAFFT and phylogenetically analyzed using RAxML with the model GTR GAMMA and 1000 replicates for bootstrap support. Annotation includes strain/isolate and accession number.