Abstract

Piscine orthoreovirus (PRV) is ubiquitous in farmed Atlantic salmon (Salmo salar) and the cause of heart and skeletal muscle inflammation. Erythrocytes are important target cells for PRV. We have investigated the kinetics of PRV infection in salmon blood cells. The findings indicate that PRV causes an acute infection of blood cells lasting 1–2 weeks, before it subsides into persistence. A high production of viral proteins occurred initially in the acute phase which significantly correlated with antiviral gene transcription. Globular viral factories organized by the non-structural protein µNS were also observed initially, but were not evident at later stages. Interactions between µNS and the PRV structural proteins λ1, µ1, σ1 and σ3 were demonstrated. Different size variants of µNS and the outer capsid protein µ1 appeared at specific time points during infection. Maximal viral protein load was observed five weeks post cohabitant challenge and was undetectable from seven weeks post challenge. In contrast, viral RNA at a high level could be detected throughout the eight-week trial. A proteolytic cleavage fragment of the µ1 protein was the only viral protein detectable after seven weeks post challenge, indicating that this µ1 fragment may be involved in the mechanisms of persistent infection.

Highlights

  • Piscine orthoreovirus (PRV) belongs to the genus Orthoreovirus in the family Reoviridae [1,2].The orthoreoviruses are ubiquitous in various animal species, but only found to be of pathogenic significance in poultry and recently in fish [3,4,5,6,7]

  • The viral factories in PRV-infected cells resemble the globular structures observed for the mammalian orthoreovirus (MRV) type 3 Dearing (T3D) strain, in contrast to the filamentous-like viral factories generated by MRV Type 1 Lang (T1L) [19]

  • A PRV positive population of blood cells was observed from 4 wpc as a marked shift in the histograms compared to negative samples

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Summary

Introduction

Piscine orthoreovirus (PRV) belongs to the genus Orthoreovirus in the family Reoviridae [1,2]. Structures resembling viral factories have been observed in PRV-infected erythrocytes, and recombinant expression of the protein in fish cell lines indicate that PRV μNS has an analogous role in factory formation [16,19,31]. The viral factories in PRV-infected cells resemble the globular structures observed for the MRV type 3 Dearing (T3D) strain, in contrast to the filamentous-like viral factories generated by MRV Type 1 Lang (T1L) [19]. The latter is considered the most common morphology type of orthoreoviral factories [21,32]. We hypothesized that PRV causes an acute infection in blood cells correlating with innate antiviral gene expression, before the infection subsides to a low persistent level

Materials and Methods
Construction and Expression of Recombinant PRV λ1
Protein Purification
Immunization of Rabbits
Specificity of Antisera
Experimental Challenge of Salmon
Flow Cytometry
Immunofluorescence Microscopy
2.14. Computational Analysis
2.15. Statistical
Viral RNA Load in Blood Cells
Piscine
Expression of Innate Antiviral Genes in PRV Infected Blood Cells
Flow Cytometry Indicates a Transient Peak in Blood Cells
Presence
PRV Protein Levels Display a Transient Peak in Blood Cells
(Figures and
Discussion
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