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

Melanie M Hierweger,Jens H Kuhn,Piet Maes,Rémy Bruggmann,Michel C Koch,Melanie Rupp,Heike Schmidt-Posthaus,Torsten Seuberlich,Nicholas Di Paola

doi:10.3201/eid2712.210491

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.

Highlights

Reads were quality-trimmed using trimmomatic v. 0.36 (28), and host-derived sequences were removed by aligning reads to the European perch genome (UTU_Pfluv_1.1, Bioproject PRJNA450919) using STAR v. 2.6.0c (1)
In Situ hybridization (ISH) process controls consisted of brain tissue sections of animals with bovine astrovirus CH13 (BoAstV CH13) infection and a BoAstV CH13-specific RNAscope probe [#406921] tested in parallel to each ISH experiment (6)
Maximum-likelihood phylogenetic trees of the nucleotide sequences of Egli virus (EGLV; bold blue) genes with those of viruses belonging to representative members of the genus Perhabdovirus. a) nucleoprotein gene (N), b) phosphoprotein gene (P) c) matrix protein gene (M), and d) glycoprotein gene (G)

Summary

Methods

Reads were quality-trimmed using trimmomatic v. 0.36 (28), and host-derived sequences were removed by aligning reads to the European perch genome (UTU_Pfluv_1.1, Bioproject PRJNA450919) using STAR v. 2.6.0c (1). 0.36 (28), and host-derived sequences were removed by aligning reads to the European perch genome (UTU_Pfluv_1.1, Bioproject PRJNA450919) using STAR v. 2.7.1+ (3) against viral nucleotide sequences in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and DIAMOND v. To fill gaps between HTS scaffolds, we reverse-transcribed extracted RNA to cDNA with SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) and performed PCR assays with Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Ipswhich, MA, USA) and scaffold-specific primers (Appendix Table 3) according to the manufacturers’ instructions. We performed 3' and 5' RACE, as described previously on RNA extracted from pooled organs and CNS as well as cell culture supernatants (5). ISH process controls consisted of brain tissue sections of animals with bovine astrovirus CH13 (BoAstV CH13) infection and a BoAstV CH13-specific RNAscope probe [#406921] tested in parallel to each ISH experiment (6)

Amino acid alignment

Comment RACE RACE RACE RACE