Long-Term Protection and Serologic Response of European Sea Bass Vaccinated with a Betanodavirus Virus-Like Particle Produced in Pichia pastoris.

Viral Nervous Necrosis (VNN) causes high mortality and reduced growth in farmed European sea bass (Dicentrarchus labrax) in the Mediterranean. In the current studies, we tested a novel Pichia-produced virus-like particle (VLP) vaccine against VNN in European sea bass, caused by the betanodavirus "Red-Spotted Grouper Nervous Necrosis Virus" (RGNNV). European sea bass were immunized with a VLP-based vaccine formulated with different concentrations of antigen and with or without adjuvant. Antibody response was evaluated by ELISA and serum neutralization. The efficacy of these VLP-vaccine formulations was evaluated by an intramuscular challenge with RGNNV at different time points (1, 2 and 10 months post-vaccination) and both dead and surviving fish were sampled to evaluate the level of viable virus in the brain. The VLP-based vaccines induced an effective protective immunity against experimental infection at 2 months post-vaccination, and even to some degree at 10 months post-vaccination. Furthermore, the vaccine formulations triggered a dose-dependent response in neutralizing antibodies. Serologic response and clinical efficacy, measured as relative percent survival (RPS), seem to be correlated with the administered dose, although for the individual fish, a high titer of neutralizing antibodies prior to challenge was not always enough to protect against disease. The efficacy of the VLP vaccine could not be improved by formulation with a water-in-oil (W/O) adjuvant. The developed RGNNV-VLPs show a promising effect as a vaccine candidate, even without adjuvant, to protect sea bass against disease caused by RGNNV. However, detection of virus in vaccinated survivors means that it cannot be ruled out that survivors can transmit the virus.

Steps of the Replication Cycle of the Viral Haemorrhagic Septicaemia Virus (VHSV) Affecting Its Virulence on Fish.

The viral haemorrhagic septicaemia virus (VHSV), a single-stranded negative-sense RNA novirhabdovirus affecting a wide range of marine and freshwater fish species, is a main concern for European rainbow trout (Oncorhynchus mykiss) fish farmers. Its genome is constituted by six genes, codifying five structural and one nonstructural proteins. Many studies have been carried out to determine the participation of each gene in the VHSV virulence, most of them based on genome sequence analysis and/or reverse genetics to construct specific mutants and to evaluate their virulence phenotype. In the present study, we have used a different approach with a similar aim: hypothesizing that a failure in any step of the replication cycle can reduce the virulence in vivo, we studied in depth the in vitro replication of VHSV in different cell lines, using sets of strains from different origins, with high, low and moderate levels of virulence for fish. The results demonstrated that several steps in the viral replication cycle could affect VHSV virulence in fish, including adsorption, RNA synthesis and morphogenesis (including viral release). Notably, differences among strains in any step of the replication cycle were mostly strain-specific and reflected only in part the in vivo phenotype (high and low virulent). Our data, therefore, support the need for further studies aimed to construct completely avirulent VHSV recombinants targeting a combination of genes rather than a single one in order to study the mechanisms of genes interplay and their effect on viral phenotype in vitro and in vivo.

VHSV Single Amino Acid Polymorphisms (SAPs) Associated With Virulence in Rainbow Trout.

The Viral Hemorrhagic Septicemia Virus (VHSV) is an OIE notifiable pathogen widespread in the Northern Hemisphere that encompasses four genotypes and nine subtypes. In Europe, subtype Ia impairs predominantly the rainbow trout industry causing severe rates of mortality, while other VHSV genotypes and subtypes affect a number of marine and freshwater species, both farmed and wild. VHSV has repeatedly proved to be able to jump to rainbow trout from the marine reservoir, causing mortality episodes. The molecular mechanisms regulating VHSV virulence and host tropism are not fully understood, mainly due to the scarce availability of complete genome sequences and information on the virulence phenotype. With the scope of identifying in silico molecular markers for VHSV virulence, we generated an extensive dataset of 55 viral genomes and related mortality data obtained from rainbow trout experimental challenges. Using statistical association analyses that combined genetic and mortality data, we found 38 single amino acid polymorphisms scattered throughout the complete coding regions of the viral genome that were putatively involved in virulence of VHSV in trout. Specific amino acid signatures were recognized as being associated with either low or high virulence phenotypes. The phylogenetic analysis of VHSV coding regions supported the evolution toward greater virulence in rainbow trout within subtype Ia, and identified several other subtypes which may be prone to be virulent for this species. This study sheds light on the molecular basis for VHSV virulence, and provides an extensive list of putative virulence markers for their subsequent validation.

Sequential Immunization With Heterologous Viruses Does Not Result in Attrition of the B Cell Memory in Rainbow Trout.

Long-term immunity is of great importance for protection against pathogens and has been extensively studied in mammals. Successive heterologous infections can affect the maintenance of immune memory, inducing attrition of T memory cells and diminishing B cell mediated protection. In fish, the basis of immune memory and the mechanisms of immunization to heterologous pathogens remain poorly understood. We sequentially immunized isogenic rainbow trout with two immunologically distinct viruses, VHSV and IPNV, either with one virus only or in combination, and analyzed the antibody responses and repertoires. Neutralizing antibodies and ELISPOT did not reveal an effect of heterologous immunization. Using a consensus read sequencing approach that incorporates unique barcodes to each cDNA molecule, we focused on the diversity expressed by selected responding VH/C combinations. We identified both public and private responses against VHSV and/or IPNV in all groups of fish. In fish immunized with two viruses, we registered no significant reduction in the persistence of the response toward the primary immunization. Similarly, the response to the second immunization was not affected by a prior vaccination to the other virus. Our data suggest that heterologous immunization does not enforce attrition of pre-existing antibody producing cells, which may impair the protection afforded by multiple successive vaccinations. These observations are potentially important to improve vaccination strategies practiced in aquaculture.

Presence and genetic variability of Piscine orthoreovirus genotype 1 (PRV-1) in wild salmonids in Northern Europe and North Atlantic Ocean.

Piscine orthoreovirus genotype 1 (PRV-1) is widespread in farmed Atlantic salmon (Salmo salar L.) populations in northern Europe, Canada and Chile. PRV-1 occurs in wild fish in Norway and Canada; however, little information of its geographical distribution in wild populations is currently available, and the effect of PRV-1 infection in wild populations is currently unknown. In this study, we present the findings of a survey conducted on 1,130 wild salmonids sampled in Denmark, Sweden, Ireland, Faroe Islands, France, Belgium and Greenland between 2008 and 2017. PRV-1 is reported for the first time in wild salmonids in Denmark, Sweden, Faroe Island and Ireland. The annual PRV-1 prevalence ranged from 0% in France, Belgium and Greenland to 43% in Faroe Islands. In total, 66 samples tested positive for PRV-1, including Atlantic salmon broodfish returning to spawn and Atlantic salmon collected at the feeding ground north of Faroe Islands. The phylogenetic analysis of S1 sequences of the PRV-1 isolates obtained in this survey did not show systematic geographical distribution. This study sheds light on the spread and genetic diversity of the virus identified in populations of free-living fish and provides rationale for screening wild broodfish used in restocking programmes.

Partial validation of a TaqMan real-time quantitative PCR for the detection of ranaviruses.

Ranaviruses are globally emerging pathogens negatively impacting wild and cultured fish, amphibians, and reptiles. Although conventional and diagnostic real-time PCR (qPCR) assays have been developed to detect ranaviruses, these assays often have not been tested against the known diversity of ranaviruses. Here we report the development and partial validation of a TaqMan real-time qPCR assay. The primers and TaqMan probe targeted a conserved region of the major capsid protein (MCP) gene. A series of experiments using a 10-fold dilution series of Frog virus 3 (FV3) MCP plasmid DNA revealed linearity over a range of 7 orders of magnitude (107-101), a mean correlation coefficient (R2) of >0.99, and a mean efficiency of 96%. The coefficient of variation of intra- and inter-assay variability ranged from <0.1-3.5% and from 1.1-2.3%, respectively. The analytical sensitivity was determined to be 10 plasmid copies of FV3 DNA. The qPCR assay detected a panel of 33 different ranaviral isolates originating from fish, amphibian, and reptile hosts from all continents excluding Africa and Antarctica, thereby representing the global diversity of ranaviruses. The assay did not amplify highly divergent ranaviruses, members of other iridovirus genera, or members of the alloherpesvirus genus Cyprinivirus. DNA from fish tissue homogenates previously determined to be positive or negative for the ranavirus Epizootic hematopoietic necrosis virus by virus isolation demonstrated a diagnostic sensitivity of 95% and a diagnostic specificity of 100%. The reported qPCR assay provides an improved expedient diagnostic tool and can be used to elucidate important aspects of ranaviral pathogenesis and epidemiology in clinically and sublinically affected fish, amphibians, and reptiles.

Genomic Sequencing of Ranaviruses Isolated from Edible Frogs (Pelophylax esculentus).

Ranaviruses were isolated from wild edible frogs (Pelophylax esculentus) during epizootics in Denmark and Italy. Phylogenomic analyses revealed that these isolates are closely related and belong to a clade of ranaviruses that includes the Andrias davidianus ranavirus (ADRV), common midwife toad ranavirus (CMTV), Testudo hermanni ranavirus (THRV), and pike-perch iridovirus (PPIV).

Genomic Sequencing of Ranaviruses Isolated from Turbot (Scophthalmus maximus) and Atlantic Cod (Gadus morhua).

Ranaviruses have been isolated from Atlantic cod (Gadus morhua) and turbot (Scophthalmus maximus) in Denmark. Phylogenomic analyses revealed that these two ranaviruses are nearly identical and form a distinct clade at the base of the ranavirus tree branching off near other fish ranaviruses.

Phylogeny of the Viral Hemorrhagic Septicemia Virus in European Aquaculture.

One of the most valuable aquaculture fish in Europe is the rainbow trout, Oncorhynchus mykiss, but the profitability of trout production is threatened by a highly lethal infectious disease, viral hemorrhagic septicemia (VHS), caused by the VHS virus (VHSV). For the past few decades, the subgenogroup Ia of VHSV has been the main cause of VHS outbreaks in European freshwater-farmed rainbow trout. Little is currently known, however, about the phylogenetic radiation of this Ia lineage into subordinate Ia clades and their subsequent geographical spread routes. We investigated this topic using the largest Ia-isolate dataset ever compiled, comprising 651 complete G gene sequences: 209 GenBank Ia isolates and 442 Ia isolates from this study. The sequences come from 11 European countries and cover the period 1971-2015. Based on this dataset, we documented the extensive spread of the Ia population and the strong mixing of Ia isolates, assumed to be the result of the Europe-wide trout trade. For example, the Ia lineage underwent a radiation into nine Ia clades, most of which are difficult to allocate to a specific geographic distribution. Furthermore, we found indications for two rapid, large-scale population growth events, and identified three polytomies among the Ia clades, both of which possibly indicate a rapid radiation. However, only about 4% of Ia haplotypes (out of 398) occur in more than one European country. This apparently conflicting finding regarding the Europe-wide spread and mixing of Ia isolates can be explained by the high mutation rate of VHSV. Accordingly, the mean period of occurrence of a single Ia haplotype was less than a full year, and we found a substitution rate of up to 7.813 × 10-4 nucleotides per site per year. Finally, we documented significant differences between Germany and Denmark regarding their VHS epidemiology, apparently due to those countries' individual handling of VHS.

Genomic Sequence of a Ranavirus Isolated from Short-Finned Eel (Anguilla australis).

The short-finned eel ranavirus (SERV) was isolated from short-finned eel imported to Italy from New Zealand. Phylogenomic analyses revealed that SERV is a unique member of the genus Ranavirus, family Iridoviridae, branching at the base of the tree near other fish ranaviruses.

Recommended reporting standards for test accuracy studies of infectious diseases of finfish, amphibians, molluscs and crustaceans: the STRADAS-aquatic checklist.

Complete and transparent reporting of key elements of diagnostic accuracy studies for infectious diseases in cultured and wild aquatic animals benefits end-users of these tests, enabling the rational design of surveillance programs, the assessment of test results from clinical cases and comparisons of diagnostic test performance. Based on deficiencies in the Standards for Reporting of Diagnostic Accuracy (STARD) guidelines identified in a prior finfish study (Gardner et al. 2014), we adapted the Standards for Reporting of Animal Diagnostic Accuracy Studies-paratuberculosis (STRADAS-paraTB) checklist of 25 reporting items to increase their relevance to finfish, amphibians, molluscs, and crustaceans and provided examples and explanations for each item. The checklist, known as STRADAS-aquatic, was developed and refined by an expert group of 14 transdisciplinary scientists with experience in test evaluation studies using field and experimental samples, in operation of reference laboratories for aquatic animal pathogens, and in development of international aquatic animal health policy. The main changes to the STRADAS-paraTB checklist were to nomenclature related to the species, the addition of guidelines for experimental challenge studies, and the designation of some items as relevant only to experimental studies and ante-mortem tests. We believe that adoption of these guidelines will improve reporting of primary studies of test accuracy for aquatic animal diseases and facilitate assessment of their fitness-for-purpose. Given the importance of diagnostic tests to underpin the Sanitary and Phytosanitary agreement of the World Trade Organization, the principles outlined in this paper should be applied to other World Organisation for Animal Health (OIE)-relevant species.

Virulence of viral haemorrhagic septicaemia virus (VHSV) genotype III in rainbow trout.

In general, viral haemorrhagic septicaemia virus (VHSV) isolates from marine fish species in European waters (genotypes GIb, GII and GIII) are non- to low virulent in rainbow trout. However, a VHSV isolation was made in 2007 from a disease outbreak in sea farmed rainbow trout in Norway. The isolate, named NO-2007-50-385, was demonstrated to belong to GIII. This isolate has attracted attention to assess which of the viral genome/proteins might be associated with the virulence in rainbow trout. In this study, we describe the difference of virulence in rainbow trout between the NO-2007-50-385 and 4p168 isolates as representatives of virulent and non-virulent GIII isolates, respectively. Rainbow trout were bath challenged with VHSV NO-2007-50-385 for 1 and 6 h, resulting in cumulative mortalities of 5 and 35%, respectively. No mortality was observed in the rainbow trout groups immersed with the genotype III VHSV isolate 4p168 for 1 and 6 h. The viral titre in organs from fish challenged with NO-2007-50-385 for 6 h increased more rapidly than those exposed for 1 h. By in vitro studies it was demonstrated that the final titres of VHSV DK-3592B (GI), NO-2007-50-385 and 4p168 inoculated on EPC cells were very similar, whereas when inoculated on the rainbow trout cell line RTG-2 the titre of the non-virulent 4p168 isolate was 3-4 logs below the two other VHSV isolates. Based on a comparative analysis of the entire genome of the genotype III isolates, we suggest that substitutions of amino acids in positions 118-123 of the nucleo-protein are candidates for being related to virulence of VHSV GIII in rainbow trout.