Revisiting the Classification of Percid Perhabdoviruses Using New Full-Length Genomes.

Perhabdoviruses are a threat to some freshwater fish species raised in aquaculture farms in Europe. Although the genetic diversity of these viruses is suspected to be high, the classification of isolates is still in its infancy, with just one full-length genome available and only partial sequences for a limited number of others. Here, we characterized a series of viruses isolated from percids in France from 1999 to 2009 by sequencing the nucleoprotein (N) gene. Four main clusters were distinguished, all related at varying levels of similarity to one of the two already-recognized species, namely Perch perhabdovirus and Sea trout perhabdovirus. Furthermore, we obtained the complete genome of five isolates, including one belonging to Sea trout rhabdovirus. The analysis of the complete L genes and the concatenated open reading frames confirmed the existence of four main genetic clusters, sharing 69 to 74% similarity. We propose the assignation of all these viral isolates into four species, including two new ones: Perch perhabdovirus 1, Perch perhabdovirus 2, Sea trout perhabdovirus 1 and Sea trout perhabdovirus 2. In addition, we developed new primers to readily amplify specific portions of the N gene of any isolate of each species by conventional PCR. The presence of such genetically diverse viruses in France is likely due to divergent viral populations maintained in the wild and then introduced to experimental facilities or farms, as well as via trade between farms across the European continent. It is now urgent to improve the identification tools for this large group of viruses to prevent their unchecked dissemination.

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.

Molecular investigations of outbreaks of Perch perhabdovirus infections in pike-perch.

In 2016, a total of 5 massive mortality episodes each affecting hundreds of thousands of pike-perch Sander lucioperca larvae occurred at 2 sites in 2 Western European countries. For each episode, perhabdoviruses related to the perch rhabdovirus (PRV) were detected in samples, using either PCR or cell culture combined with PCR. The sequences of the glycoprotein (g), phosphoprotein (p) and nucleoprotein (n) genes of these samples demonstrated that 2 different genotypes were present at 1 site, each associated with 1 of the 3 episodes. At the other site, a single genotype was associated with the 2 outbreaks. Furthermore, this genotype was strictly identical to 1 genotype involved in the outbreaks of the first site, strongly suggesting a common origin for these 2 viruses. The common origin was confirmed a posteriori because some larvae introduced to both sites had exactly the same geographic origin in Eastern Europe. Taken together, the molecular and epidemiological data suggest that both horizontal and vertical transmission of 2 distinct strains of perhabdoviruses were involved in the various outbreaks affecting pike-perch.

Rational development of an attenuated recombinant cyprinid herpesvirus 3 vaccine using prokaryotic mutagenesis and in vivo bioluminescent imaging.

Cyprinid herpesvirus 3 (CyHV 3) is causing severe economic losses worldwide in common and koi carp industries, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open reading frame 134 (ORF134), we unexpectedly obtained a clone with additional deletion of ORF56 and ORF57. This triple deleted recombinant replicated efficiently in vitro and expressed an in vivo safety/efficacy profile compatible with use as an attenuated vaccine. To determine the role of the double ORF56-57 deletion in the phenotype and to improve further the quality of the vaccine candidate, a series of deleted recombinants was produced and tested in vivo. These experiments led to the selection of a double deleted recombinant lacking ORF56 and ORF57 as a vaccine candidate. The safety and efficacy of this strain were studied using an in vivo bioluminescent imaging system (IVIS), qPCR, and histopathological examination, which demonstrated that it enters fish via skin infection similar to the wild type strain. However, compared to the parental wild type strain, the vaccine candidate replicated at lower levels and spread less efficiently to secondary sites of infection. Transmission experiments allowing water contamination with or without additional physical contact between fish demonstrated that the vaccine candidate has a reduced ability to spread from vaccinated fish to naïve sentinel cohabitants. Finally, IVIS analyses demonstrated that the vaccine candidate induces a protective mucosal immune response at the portal of entry. Thus, the present study is the first to report the rational development of a recombinant attenuated vaccine against CyHV 3 for mass vaccination of carp. We also demonstrated the relevance of the CyHV 3 carp model for studying alloherpesvirus transmission and mucosal immunity in teleost skin.

Sensitivity and permissivity of Cyprinus carpio to cyprinid herpesvirus 3 during the early stages of its development: importance of the epidermal mucus as an innate immune barrier.

Cyprinid herpesvirus 3 (CyHV-3) causes a lethal disease in common and koi carp (Cyprinus carpio). The present study investigated the ability of CyHV-3 to infect common carp during the early stages of its development (from embryos to fingerlings) after inoculation by immersion in water containing the virus. Fish were inoculated at different times after hatching with a pathogenic recombinant CyHV-3 strain expressing luciferase. The sensitivity and permissivity of carp to CyHV-3 were investigated using in vivo bioluminescence imaging. The susceptibility of carp to CyHV-3 disease was investigated by measuring the survival rate. Carp were sensitive and permissive to CyHV-3 infection and susceptible to CyHV-3 disease at all stages of development, but the sensitivity of the two early developmental stages (embryo and larval stages) was limited compared to later stages. The lower sensitivity observed for the early developmental stages was due to stronger inhibition of viral entry into the host by epidermal mucus. In addition, independent of the developmental stage at which inoculation was performed, the localization of light emission suggested that the skin is the portal of CyHV-3 entry. Taken together, the results of the present study demonstrate that carp are sensitive and permissive to CyHV-3 at all stages of development and confirm that the skin is the major portal of entry after inoculation by immersion in infectious water. The results also stress the role of epidermal mucus as an innate immune barrier against pathogens even and especially at the early stages of development.

Feeding Cyprinus carpio with infectious materials mediates cyprinid herpesvirus 3 entry through infection of pharyngeal periodontal mucosa.

Cyprinid herpesvirus 3 (CyHV-3), also known as Koi herpesvirus, is the etiological agent of a mortal disease in common and koi carp. Recently, we investigated the entry of CyHV-3 in carp using bioluminescence imaging and a CyHV-3 recombinant strain expressing luciferase (LUC). We demonstrated that the skin is the major portal of entry after inoculation of carp by immersion in water containing CyHV-3. While this model of infection mimics some natural conditions in which infection takes place, other epidemiological conditions could favour entry of virus through the digestive tract. Here, we investigated whether ingestion of infectious materials mediates CyHV-3 entry through the digestive tract. Carp were fed with materials contaminated with the CyHV-3 LUC recombinant (oral contamination) or immersed in water containing the virus (contamination by immersion). Bioluminescence imaging analyses performed at different times post-infection led to the following observations: (i) the pharyngeal periodontal mucosa is the major portal of entry after oral contamination, while the skin is the major portal of entry after contamination by immersion. (ii) Both modes of inoculation led to the spreading of the infection to the various organs tested. However, the timing and the sequence in which some of the organs turned positive were different between the two modes of inoculation. Finally, we compared the disease induced by the two inoculation modes. They led to comparable clinical signs and mortality rate. The results of the present study suggest that, based on epidemiological conditions, CyHV-3 can enter carp either by skin or periodontal pharyngeal mucosal infection.

Skin mucus of Cyprinus carpio inhibits cyprinid herpesvirus 3 binding to epidermal cells.

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a mortal and highly contagious disease in common and koi carp. The skin is the major portal of entry of CyHV-3 in carp after immersion in water containing the virus. In the present study, we used in vivo bioluminescence imaging to investigate the effect of skin mucus removal and skin epidermis lesion on CyHV-3 entry. Physical treatments inducing removal of the mucus up to complete erosion of the epidermis were applied on a defined area of carp skin just before inoculation by immersion in infectious water. CyHV-3 entry in carp was drastically enhanced on the area of the skin where the mucus was removed with or without associated epidermal lesion. To investigate whether skin mucus inhibits CyHV-3 binding to epidermal cells, tail fins with an intact mucus layer or without mucus were inoculated ex vivo. While electron microscopy examination revealed numerous viral particles bound on the fins inoculated after mucus removal, no particle could be detected after infection of mucus-covered fins. Finally, anti-CyHV-3 neutralising activity of mucus extract was tested in vitro. Incubation of CyHV-3 with mucus extract reduced its infectivity in a dose dependent manner. The present study demonstrates that skin mucus removal and epidermal lesions enhance CyHV-3 entry in carp. It highlights the role of fish skin mucus as an innate immune protection against viral epidermal entry.

Cyprinid herpesvirus 3.

The recently designated cyprinid herpesvirus 3 (CyHV-3) is an emerging agent that causes fatal disease in common and koi carp. Since its emergence in the late 1990s, this highly contagious pathogen has caused severe financial losses in common and koi carp culture industries worldwide. In addition to its economic role, recent studies suggest that CyHV-3 may have a role in fundamental research. CyHV-3 has the largest genome among viruses in the order Herpesvirales and serves as a model for mutagenesis of large DNA viruses. Other studies suggest that the skin of teleost fish represents an efficient portal of entry for certain viruses. The effect of temperature on viral replication suggests that the body temperature of its poikilotherm host could regulate the outcome of the infection (replicative vs. nonreplicative). Recent advances with regard to CyHV-3 provide a role for this virus in fundamental and applied research.