Ultra-Deep Pyrosequencing of Partial Surface Protein Genes from Infectious Salmon Anaemia Virus (ISAV) Suggest Novel Mechanisms Involved in Transition to Virulence

Abstract

Uncultivable HPR0 strains of infectious salmon anaemia viruses (ISAVs) infecting gills are non-virulent putative precursors of virulent ISAVs (vISAVs) causing systemic disease in farmed Atlantic salmon (Salmo salar). The transition to virulence involves two molecular events, a deletion in the highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) gene and a Q266→L266 substitution or insertion next to the putative cleavage site (R267) in the fusion protein (F). We have performed ultra-deep pyrosequencing (UDPS) of these gene regions from healthy fish positive for HPR0 virus carrying full-length HPR sampled in a screening program, and a vISAV strain from an ISA outbreak at the same farming site three weeks later, and compared the mutant spectra. As the UDPS data shows the presence of both HE genotypes at both sampling times, and the outbreak strain was unlikely to be directly related to the HPR0 strain, this is the first report of a double infection with HPR0s and vISAVs. For F amplicon reads, mutation frequencies generating L266 codons in screening samples and Q266 codons in outbreak samples were not higher than at any random site. We suggest quasispecies heterogeneity as well as RNA structural properties are linked to transition to virulence. More specifically, a mechanism where selected single point mutations in the full-length HPR alter the RNA structure facilitating single- or sequential deletions in this region is proposed. The data provides stronger support for the deletion hypothesis, as opposed to recombination, as the responsible mechanism for generating the sequence deletions in HE.

First field evidence of the evolution from a non-virulent HPR0 to a virulent HPR-deleted infectious salmon anaemia virus

“…Recently, ISAV quasispecies were demonstrated in farmed Atlantic salmon [53]. The authors identified low-frequency HPR-deleted variants in HPR0-positive screening samples from healthy fish as well as lowfrequency HPR0 variants in subsequent HPR-deleted samples from ISA diseased fish [53]. Similar findings of low-frequency HPAIV in LPAIV and vice versa have been documented in samples from a natural outbreak of AI [40].…”

“…RNA viruses are characterized by a high mutation rate and rapid evolutionary potential because of the intrinsic error-prone nature of virus RNA polymerases generating viral quasispecies to maximize diversity and adaptability [51,52]. Recently, ISAV quasispecies were demonstrated in farmed Atlantic salmon [53]. The authors identified low-frequency HPR-deleted variants in HPR0-positive screening samples from healthy fish as well as lowfrequency HPR0 variants in subsequent HPR-deleted samples from ISA diseased fish [53].…”

Immersion challenge with low and highly virulent infectious salmon anaemia virus reveals different pathogenesis in Atlantic salmon, Salmo salar L.

“…; Markussen et al . , ). In addition, a mutation within genomic segment 5, coding for the fusion protein, in all virulent strains has also been suggested as a possible virulence marker (Markussen et al .…”

A systematic surveillance programme for infectious salmon anaemia virus supports its absence in the Pacific Northwest of the United States

“…ISAV, as an RNA virus lacking polymerase proof reading capability, is prone to genetic shift and drift, and as a segmented genome is also capable of re‐assortment (Markussen et al., ). Consequently, ISAV exists as a number of different genotypes, the HPR0 grouping of which is thought to be both non‐virulent and a putative progenitor of virulent forms (Cook‐Versloot, Griffiths, Cusack, McGeachy, & Ritchie, ; Godoy et al., ).…”