Novel Filoviruses, Hantavirus, and Rhabdovirus in Freshwater Fish, Switzerland, 2017

Abstract

European perch (Perca fluviatilis) are increasingly farmed as a human food source. Viral infections of European perch remain largely unexplored, thereby putting farm populations at incalculable risk for devastating fish epizootics and presenting a potential hazard to consumers. To address these concerns, we applied metatranscriptomics to identify disease-associated viruses in European perch farmed in Switzerland. Unexpectedly, in clinically diseased fish we detected novel freshwater fish filoviruses, a novel freshwater fish hantavirus, and a previously unknown rhabdovirus. Hantavirus titers were high, and we demonstrated virus in macrophages and gill endothelial cells by using in situ hybridization. Rhabdovirus titers in organ samples were low, but virus could be isolated on cell culture. Our data add to the hypothesis that filoviruses, hantaviruses, and rhabdoviruses are globally distributed common fish commensals, pathogens, or both. Our findings shed new light on negative-sense RNA virus diversity and evolution.

Keywords: European perch; Switzerland; filovirus; freshwater fish; hantavirus; rhabdovirus; viruses.

Figure 1

Identifying a novel rhabdovirus in European perch. A) Schematic representation of the EGLV genome organization; open reading frames are indicated by colored arrows. B) Maximum-likelihood phylogenetic tree of the nucleotide sequence of the EGLV L gene (bold blue) and representative classified and unclassified members of the genus Perhabdovirus. Numbers near nodes on the trees indicate bootstrap values. Branches are labeled by GenBank accession number and virus name. Names of unclassified likely perhabdoviruses are placed in quotation marks. Scale bar indicates number of substitutions per site, reflected by branch lengths. EGLV, Egli virus; G, glycoprotein gene; HTS, high-throughput sequencing; L, large protein gene; M, matrix protein gene; N, nucleoprotein gene; P, phosphoprotein gene; RACE, rapid amplification of cDNA ends.

Figure 2

Identifying 3 novel filoviruses in European perch. A) Schematic representation of the genome organization of Fiwi virus, Oberland virus, and Kander virus compared with Huángjiāo virus (HUJV). Open reading frames (ORFs) are indicated by colored arrows. ORFs encoding HUJV-like proteins are depicted by the same color and sequence similarities are indicated as percentages. Undetermined ORF starts and ends are shown as stripes. B, C) Maximum-likelihood phylogenetic trees of the new filovirus genome sequences (bold blue) generated by using coding-complete and near-complete genome sequences (B) or only L gene sequences (C) of representative members of the family Filoviridae. Numbers near nodes on the trees indicate bootstrap values. Branches are labeled by GenBank accession number, and virus name. Scale bar indicates number of substitutions per site, reflected by branch lengths. GP, glycoprotein gene; L, large protein gene; NP, nucleoprotein gene; VP30, transcriptional activator gene; VP35, polymerase cofactor gene.

Figure 3

Identifying a novel hantavirus in European perch. A) Schematic representation of the 3 genome segments of Bern perch virus; open reading frames are indicated as colored arrows. Coverage plots of high-throughput sequencing reads are shown for each segment, and maximum-read coverages are indicated on the right. B) Alignment of the terminal sequences (11 nt) of the 3 segments. The terminal 8 nucleotides (gray box) are complementary within and conserved among segments. C) Maximum-likelihood phylogenetic tree of the Bern perch virus RNA-directed RNA-polymerase amino-acid sequence (bold blue) with RNA-directed RNA-polymerase amino-acid sequences of representative members of the family Hantaviridae. Numbers near nodes on the trees indicate bootstrap values. Branches are labeled by GenBank accession number, and virus name. Scale bar indicates number of substitutions per site, reflected by branch lengths. GP, glycoprotein gene; L, large; M, medium; S, small; NP, nucleocapsid protein gene.

Figure 4

Histopathologic lesions and viral RNA in European perch infected with Bern perch virus. A) Histopathologic lesions in gills (hematoxylin and eosin stain) showing epithelial hypertrophy and hyperplasia, multifocally leading to lamellar fusion (stars) and multifocal epithelial lifting due to edema (closed arrowheads). Scale bar indicates 25 μm. B) In situ hybridization detection of Bern perch virus RNA in gills (brown labeling): positive macrophages, more pronounced in proliferated areas, and endothelial cells. Inset: higher magnification showing positive macrophages (open arrowheads) and endothelial cells (arrows with open heads). Scale bar indicates 50 μm.