Limited Interference at the Early Stage of Infection between Two Recombinant Novirhabdoviruses: Viral Hemorrhagic Septicemia Virus and Infectious Hematopoietic Necrosis Virus▿ †

The genome sequence of a hypervirulent novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) French strain 23-75, was determined. Compared to the genome of the prototype Fil3 strain, a number of substitutions, deletions, and insertions were observed. Following the establishment of a plasmid-based minigenome replication assay, recombinant VHSV (rVHSV) was successfully recovered. rVHSV exhibits wild-type-like growth properties in vitro as well as in vivo in rainbow trout. The dispensable role of NV for the novirhabdovirus replication was confirmed by generating rVHSV-ΔNV, in which the NV gene was deleted. This deletion mutant was shown to be as debilitated as that previously described for infectious hematopoietic necrosis virus (IHNV), a distantly related novirhabdovirus (S. Biacchesi, M. I. Thoulouze, M. Bearzotti, Y. X. Yu, and M. Bremont, J. Virol. 74:11247-11253, 2000). Recombinant VHSV and IHNV expressing tdTomato and GFPmax reporter genes, respectively, were generated, demonstrating the potential of these rhabdoviruses to serve as viral vectors. Interestingly, rIHNV-GFPmax could be recovered using the replicative complex proteins of either virus, whereas rVHSV-Tomato could be recovered only by using its own replicative complex, reflecting that the genome signal sequences of VHSV are relatively distant from those of IHNV and do not allow their cross-recognition. Moreover, the use of heterologous protein combinations underlined the importance of strong protein-protein interactions for the formation of a functional ribonucleoprotein complex. The rIHNV-GFPmax and rVHSV-Tomato viruses were used to simultaneously coinfect cell monolayers. It was observed that up to 74% of the cell monolayer was coinfected by both viruses, demonstrating that a limited interference phenomenon exists during the early stage of primary infection, and it was not mediated by a cellular antiviral protein or by some of the viral proteins

Zebrafish STING is a strong antiviral protein.

<p>EPC cells were transfected with 2 µg of pcDNA-STING, and pcDNA-MAVS or an empty vector (pcDNA) as positive and negative controls, respectively. At 48 h posttransfection, EPC cells were infected with a recombinant rVHSV-Tom expressing the tdTomato fluorescent protein at an MOI of 1 and incubated at 15°C. Cell monolayers were visualized under a UV-visible light microscope at 24 h postinfection (A) and then stained with crystal violet 4 days postinfection (B). The culture supernatants from cells infected with rVHSV-Tom were collected at 0, 24 and 96 h postinfection and the viral titer was determined by plaque assay on EPC cells (C). Each time point was represented by three independent experiments, and each virus titration was done in duplicate. Means are shown. The standard errors were calculated and the error bars are shown. Asterisks indicate significant difference (*p<0.01 and **p<0.001) as determined by Student’s <i>t</i> test.</p

STING and MAVS mediate RIG-I induction of interferon.

<p>EPC cells were transfected with 1 µg of pIFNproLUC reporter in combination with various plasmid constructs (1 µg each, except for dominant-negative mutants of MAVS and STING combinations where 0.5 µg of each plasmid was used) as mentioned under each histogram. An empty vector (pcDNA) was added in some experiments to keep the total amount of transfected DNA constant (3 µg total DNA for 5×10⁶ cells). In the condition were pRIG-I Nter-eGFP was not present in the transfection mixture, a peGFP vector was added. At 24 h and 48 h posttransfection, eGFP and luciferase signals were determined. Values of luciferase activities were normalized to the levels of eGFP fluorescence. The fold induction was calculated as the ratio of stimulated versus unstimulated (pcDNA alone) samples. Means of three independent experiments are shown together with the standard errors. Student’s <i>t</i>-test was used for statistical analysis, and asterisk indicate significant differences (*<i>p</i><0.05 and **<i>p</i><0.01).</p

Overexpression of STING induced IFN and ISG expression.

<p>EPC cells were transfected with 2 µg of pcDNA vector encoding STING, or MAVS as a positive control, or an empty vector (pcDNA) as a negative control. At 48 h posttransfection, EPC were infected or not with VHSV at an MOI of 1 and incubated at 15°C for 24 h before total RNA extraction. Quantitative Real-time RT-PCR was conducted using primers targeting IFN (A), RIG-I (B) and Viperin (C). The β-actin gene was used as an internal control to normalize the cDNA template and to do real-time PCR calculations. SD of triplicate experiments has been calculated. Asterisks indicate significant difference (*p<0.05, **p<0.01 and ***p<0.001) as determined by Student’s <i>t</i> test.</p

STING antiviral response is mediated by IRF3.

<p>EPC cells were co-transfected with 2 µg of pcDNA-IRF3-Cter encoding a dominant-negative mutant of IRF3, and 2 µg of pcDNA-STING, pcDNA-MAVS or an empty vector. At 48 h posttransfection, EPC cells were infected with a recombinant rVHSV-Tom expressing the tdTomato fluorescent protein at an MOI of 1 and incubated at 15°C. The culture supernatants were collected at 0, 24 and 96 h postinfection and the viral titer was determined by plaque assay on EPC cells. Cell monolayers were then stained with crystal violet either at 96 h postinfection. Each time point was represented by three independent experiments, and each virus titration was done in duplicate. Means are shown together with the standard errors. “ns” indicate non-significant difference (p>0.05) as determined by Student’s <i>t</i> test.</p

Overexpression of STING induces an antiviral immunity against a DNA virus.

<p>EPC cells were transfected with 2 µg of pcDNA-STING, and pcDNA-MAVS or an empty vector (pcDNA) as positive and negative controls, respectively. At 48 h posttransfection, EPC cells were infected with a DNA virus of the Iridoviridae family, i.e., EHNV, at MOI of 1, 0.1 and 0.01 (A and C). The culture supernatants were collected at 0, 24, 48 and 72 h postinfection and the viral titer was determined by plaque assay on EPC cells (B and D). Cell monolayers were then stained with crystal violet either at 24 h postinfection (A) or 4 days postinfection (C) depending to the MOI used, as indicated. Each time point was represented by three independent experiments, and each virus titration was done in duplicate. Means are shown together with the standard errors. Asterisks indicate significant difference (*p<0.01 and **p<0.001) as determined by Student’s <i>t</i> test.</p

Localization of zebrafish STING to endoplasmic reticulum.

<p>peGFP-STING or peGFP-MAVS and peGFP, as negative controls, were transfected together with a plasmid encoding the Red Fluorescent protein fused to a reticulum endoplasmic location signal (RFP-KDEL) into EPC cells (A). The mitochondria were <i>in vivo</i> stained with a red MitoTracker (B). The cells were imaged by microscopy 24 h post-transfection. The yellow staining in the overlay image indicates colocalization of STING and RFP-KDEL (A) or MAVS and MitoTracker (B).</p

Recombinant viral hemorrhagic septicemia virus with rearranged genomes as vaccine vectors to protect against lethal betanodavirus infection

The outbreaks of viral hemorrhagic septicemia (VHS) and viral encephalopathy and retinopathy (VER) caused by the enveloped novirhabdovirus VHSV, and the non-enveloped betanodavirus nervous necrosis virus (NNV), respectively, represent two of the main viral infectious threats for aquaculture worldwide. Non-segmented negative-strand RNA viruses such as VHSV are subject to a transcription gradient dictated by the order of the genes in their genomes. With the goal of developing a bivalent vaccine against VHSV and NNV infection, the genome of VHSV has been engineered to modify the gene order and to introduce an expression cassette encoding the major protective antigen domain of NNV capsid protein. The NNV Linker-P specific domain was duplicated and fused to the signal peptide (SP) and the transmembrane domain (TM) derived from novirhabdovirus glycoprotein to obtain expression of antigen at the surface of infected cells and its incorporation into viral particles. By reverse genetics, eight recombinant VHSVs (rVHSV), termed NxGyCz according to the respective positions of the genes encoding the nucleoprotein (N) and glycoprotein (G) as well as the expression cassette (C) along the genome, have been successfully recovered. All rVHSVs have been fully characterized in vitro for NNV epitope expression in fish cells and incorporation into VHSV virions. Safety, immunogenicity and protective efficacy of rVHSVs has been tested in vivo in trout (Oncorhynchus mykiss) and sole (Solea senegalensis). Following bath immersion administration of the various rVHSVs to juvenile trout, some of the rVHSVs were attenuated and protective against a lethal VHSV challenge. Results indicate that rVHSV N2G1C4 is safe and protective against VHSV challenge in trout. In parallel, juvenile sole were injected with rVHSVs and challenged with NNV. The rVHSV N2G1C4 is also safe, immunogenic and efficiently protects sole against a lethal NNV challenge, thus presenting a promising starting point for the development of a bivalent live attenuated vaccine candidate for the protection of these two commercially valuable fish species against two major diseases in aquaculture

STING sequences through vertebrate evolution.

<p><b>A... </b><b>Multiple alignment of STING sequences from zebrafish and other vertebrates.</b> Identical positions are boxed in black, conservative positions in grey and block of similar residues in light grey. The residues of the 5 putative transmembrane (TM) regions and the putative RXR ER retention motifs found in zebrafish STING sequence are boxed. Zebrafish (HE856619, this study), <i>Danio rerio</i>; human (NP_938023), <i>Homo sapiens</i>; mouse (NP_082537), <i>Mus musculus</i>; chicken (E1C7U0), <i>Gallus gallus</i>; xenopus (NP_001106445), <i>Xenopus tropicalis</i>. <b>B... </b><b>NJ phylogenetic tree of vertebrate STING.</b> The tree was based on multiple alignments of full-length and partial STING amino-acid sequences from fish and other vertebrates. The tree is drawn to scale. Full-length sequence accession numbers are the following: EPC (HE856620, this study), <i>Pimephales promelas</i>; goldfish (JF970229), <i>Carassius auratus</i>, the others are listed above and partial STING amino-acid sequences were deduced from the following EST sequences: salmon (GE786872), <i>Salmo salar</i>; weather loach (BJ827384), <i>Misgurnus anguillicandatus</i>. <b>C... </b><b>Conserved synteny around the </b><b><i>TMEM173</i></b><b> gene in zebrafish, mouse and human.</b> The location of the different markers and the chromosomes involved are indicated for the different species.</p