Molecular characterization of a novel orthomyxovirus from rainbow and steelhead trout (Oncorhynchus mykiss)

Abstract

A novel virus, rainbow trout orthomyxovirus (RbtOV), was isolated in 1997 and again in 2000 from commercially-reared rainbow trout (Oncorhynchus mykiss) in Idaho, USA. The virus grew optimally in the CHSE-214 cell line at 15°C producing a diffuse cytopathic effect; however, juvenile rainbow trout exposed to cell culture-grown virus showed no mortality or gross pathology. Electron microscopy of preparations from infected cell cultures revealed the presence of typical orthomyxovirus particles. The complete genome of RbtOV is comprised of eight linear segments of single-stranded, negative-sense RNA having highly conserved 5' and 3'-terminal nucleotide sequences. Another virus isolated in 2014 from steelhead trout (also O. mykiss) in Wisconsin, USA, and designated SttOV was found to have eight genome segments with high amino acid sequence identities (89-99%) to the corresponding genes of RbtOV, suggesting these new viruses are isolates of the same virus species and may be more widespread than currently realized. The new isolates had the same genome segment order and the closest pairwise amino acid sequence identities of 16-42% with Infectious salmon anemia virus (ISAV), the type species and currently only member of the genus Isavirus in the family Orthomyxoviridae. However, pairwise comparisons of the predicted amino acid sequences of the 10 RbtOV and SttOV proteins with orthologs from representatives of the established orthomyxoviral genera and a phylogenetic analysis using the PB1 protein showed that while RbtOV and SttOV clustered most closely with ISAV, they diverged sufficiently to merit consideration as representatives of a novel genus. A set of PCR primers was designed using conserved regions of the PB1 gene to produce amplicons that may be sequenced for identification of similar fish orthomyxoviruses in the future.

Keywords: Fish virus; Oncorhynchus mykiss; Orthomyxovirus; Rainbow trout.

Fig. 1

Cytopathic effect, at 20× magnification, produced by SttOV-1 in monolayers of Chinook salmon embryo cells (CHSE-214) incubated at 15 °C. (A) Normal cells. (B) Infected cells.

Fig. 2

(A) Virion stained using 1% phosphotungstic acid and (B) cells stained using 2% uranyl acetate and 0.4% lead citrate. Scale bars = 200 nm. Images were processed with Adobe Photoshop and included application of the unsharp mask filter in Image 2B.

Fig. 3

Pairwise comparisons of representative orthomyxoviruses of different genera in the PB1, PB2, PA, HA-HE, M1, M2, NP, NS1, NEP, and NA-F proteins. Each number represents the pairwise amino acid identities between the corresponding species. RbtOV = rainbow trout orthomyxovirus, SttOV = steelhead trout orthomyxovirus, ISAV = infectious salmon anemia virus, FLUA = influenzavirus A, FLUB = influenzavirus B, FLUC = influenzavirus C, THOG = thogotovirus, and QRFV = Quaranfil virus.

Fig. 4

RbtOV and ISAV genome segments with each 3′ leader and 5′ trailer terminal regions shown as genomic sense RNA. The 5′ terminus is in reverse complement orientation for comparison. Underlined nucleotides UAC indicate the site of the first AUG methionine codon for protein translation. In the consensus sequences, M, W, K, R refer to variable sites according to the convention used by the International Union of Pure and Applied Chemistry. Bold letters indicate nucleotides that do not match with the consensus sequence. Numbers following sequence are nucleotide matches with the consensus 24 nucleotides.

Fig. 5

Conserved motifs in PB1 proteins of Influenzavirus A/PR/8/34 (H1N1) aligned with PB1 amino acid sequences of RbtOV-1 and ISAV-Glesvaer. Locations of amino acid motifs on Influenzavirus A are: I aa 303–306, II aa 403–412, III aa 438–450, and IV aa 474–484. Dots indicate amino acid identity with FLUAV.

Fig. 6

Phylogenetic comparison of RbtOV and SttOV PB1 amino acid sequences with those of representative orthomyxoviruses. Two representatives of each established genus were as follows: ISAV-Glesvaer/2/1990 (ISAV_Nor; ADR77506), ISAV-Canada/CCBB (ISAV_Can; AAL67962), Influenzavirus A/PR/8/1934 H1N1 (FluA34; ABO21706), Influenzavirus A/swine/Texas/4199-2/1998 H3N2 (FluA98; AEK70339), Influenzavirus B/Lee/1940 (FluB40; AAA43767), Influenzavirus B/Yamagata/16/1988 (FluB88; ABL77264), Influenzavirus C/Ann Arbor/1/1950 (FluC50; BAD24938), Influenzavirus C/Johannesburg/1/1966 (FluC66; AAF89738), Influenzavirus D/swine/Oklahoma/1334/2011 (FluD11; AFJ19019), Influenzavirus D/bovine/France/2986/2012 (FluD12; CEE50061), Quaranfil/1953 (QRFV53; ACY56282), Johnston Atoll/1964 (JAV64; ACY56284), Thogotovirus (Thogoto; YP_145794), Dhori virus/1313/61 (Dhori; ADF56030), RbtOV-1/1997 (RbtOV1; KX882062), and SttOV-1/2014 (SttOV1; KX882070).