Molecular Basis for Antigenic Diversity of Genus Betanodavirus.

Betanodaviruses are the causative agents of viral nervous necrosis (VNN), a devastating disease for the Mediterranean mariculture. Four different betanodavirus species are recognized, Striped jack-, Redspotted grouper-, Tiger puffer-, and Barfin flounder nervous necrosis virus (SJNNV, RGNNV, TPNNV and BFNNV), but there is little knowledge on their antigenic properties. In order to describe the serological relationships among different betanodavirus genotypes, serum neutralization assays were performed using rabbit polyclonal antisera against eight fish nodaviruses that cover a wide species-, temporal-, spatial- and genetic range. The results indicate that the SJNNV and RGNNV are antigenically distinct, constituting serotypes A and C, respectively. The TPNNV and BFNNV, the latter representing cold-water betanodaviruses, are antigenically related and cluster within serotype B. The reassortant viruses RGNNV/SJNNV and SJNNV/RGNNV group within serotypes A and C, respectively, indicating that the coat protein encoded by RNA2 acts as major immunoreactivity determinant. Immunostaining of in vitro expressed wild type and chimeric capsid proteins between the RGNNV and the SJNNV species indicated that the C-terminal part of the capsid protein retains the immunoreactive portion. The amino acid (aa) residues determining RGNNV and SJNNV antigenic diversity were mapped to aa residues 217-256 and aa 257-341, respectively. Neutralization of reverse genetics derived chimeric viruses indicated that these areas determine the neutralizing epitopes. The data obtained are crucial for the development of targeted serological tests for the diagnosis of VNN, and informative for development of cross-protective vaccines against various betanodavirus genotypes

Piscine orthoreovirus (PRV) infects Atlantic salmon erythrocytes

International audiencePiscine orthoreovirus (PRV) belongs to the Reoviridae family and is the only known fish virus related to the Orthoreovirus genus. The virus is the causative agent of heart and skeletal muscle inflammation (HSMI), an emerging disease in farmed Atlantic salmon (Salmo salar L.). PRV is ubiquitous in farmed Atlantic salmon and high loads of PRV in the heart are consistent findings in HSMI. The mechanism by which PRV infection causes disease remains largely unknown. In this study we investigated the presence of PRV in blood and erythrocytes using an experimental cohabitation challenge model. We found that in the early phases of infection, the PRV loads in blood were significantly higher than in any other organ. Most virus was found in the erythrocyte fraction, and in individual fish more than 50% of erythrocytes were PRV-positive, as determined by flow cytometry. PRV was condensed into large cytoplasmic inclusions resembling viral factories, as demonstrated by immunofluorescence and confocal microscopy. By electron microscopy we showed that these inclusions contained reovirus-like particles. The PRV particles and inclusions also had a striking resemblance to previously reported viral inclusions described as Erythrocytic inclusion body syndrome (EIBS). We conclude that the erythrocyte is a major target cell for PRV infection. These findings provide new information about HSMI pathogenesis, and show that PRV is an important factor of viral erythrocytic inclusions

Phylogenetic tree of the betanodavirus isolates used in the present study.

<p>Partial RNA2 sequences related to the betanodaviral strains under investigation were aligned and compared with reference sequences available in GenBank. The phylogenetic tree was inferred using the maximum likelihood method (ML) available in the RaxML program, incorporating the GTR model of nucleotide substitution with the CAT model of rate heterogeneity among sites [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158814#pone.0158814.ref048" target="_blank">48</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158814#pone.0158814.ref049" target="_blank">49</a>]. To assess the robustness of individual nodes, 100 bootstrap replicates were performed. Betanodavirus isolates used in the present study are highlighted in bold. *Viral isolates used for rabbit hyperimmune sera production and serological classification of fish nodaviruses. **Unknown field isolates used for blind evaluation of the SN test.</p

Negative staining TEM of recombinant viruses obtained through the reverse genetics technology.

<p>Pictures were taken at 28x magnification.</p

Molecular Basis for Antigenic Diversity of Genus <i>Betanodavirus</i> - Fig 2

<p><b>EPC cells expressing RGNNV, SJNNV and chimeric capsid proteins A, B, C, D, E and F.</b> Cells were immunostained with serum anti-283.2009 (RGNNV) and anti-484.2.2009 (SJNNV). Images were taken at 20x magnification.</p

Clustering of viruses by pattern plots of the principal component (PC).

<p>The principal components analysis is based on the serum neutralization titres expressed as GMT of the isolates tested. In each graphic, the three PCs are plotted one against the other. Viruses are grouped into three different clusters named A, B and C.</p

Molecular Basis for Antigenic Diversity of Genus <i>Betanodavirus</i> - Fig 5

<p><b>Genetic structure of the RNA1 and the RNA2 inserts cloned for the expression of recombinant coat proteins in EPC cells (A) and for the generation of reverse genetics viruses (B).</b> Thick bars represent RNA1 and RNA2 open reading frames (ORF), and thin bars indicate the 5’-UTR and 3’-UTR regions. Light grey designates nucleotide sequence of the RGNNV genotype (strain 283.2009), while dark grey indicates genetic sequence of the SJNNV genotype (strain 484.2.2009). Numbers specify the nucleotide position of the chimeric junction between the RGNNV and the SJNNV sequences. Numbers refer to the full length RNA2 nucleotide sequence related to strain 484.2.2009 (GenBank accession number JN189919).</p