Nearly complete genome sequence of a Perhabdovirus isolated on European perch (Perca fluviatilis).

We report a nearly full-length genome of a Perhabdovirus isolated in 2022 on perch on a French farm. This virus is genetically related to virus 20/43, which was associated with an outbreak of perch on a farm in France in 2019. Both viruses represent a specific lineage of perch rhabdovirus.

Further spread of perch rhabdovirus on European percid farms.

Variants of perch rhabdovirus (PRV) circulate across European percid farms via the fish trade. To trace their circulation, they are usually isolated by cell culture and subsequently identified genetically by sequencing partial or complete genes. Here, a newly developed nested PCR-based method was used to amplify and sequence the complete N and P genes directly from clinical samples obtained during an outbreak on a farm as well as from four batches of fish sampled from two other farms in another country. In an attempt to trace the origin of the five detected viruses, their N and P sequences were concatenated and compared with related viruses. One virus found in pike-perch was highly related to a virus isolated in 2016 in Belgium. Two other viruses detected on a single farm were distinct from one another, with one being almost identical to another virus isolated in 2016 in Belgium and the other being more closely related to a subgroup with different origins, France and Belgium. Two other viruses found in perch from a third farm were identical and were more related to a subgroup of viruses isolated in France. Identifying variants by a direct PCR approach will help to prevent further dissemination in farms.

A New Lineage of Perch Rhabdovirus Associated with Mortalities of Farmed Perch.

A perhabdovirus was isolated from a mortality episode affecting a fish farm in 2019 in Western Europe. This virus was produced in cell culture and was readily detected by a species-specific real-time PCR assay. The near-complete sequence of the virus obtained showed some relatedness with viruses of the species Perhabdovirus perca. However, it was distinct enough from these viruses to form a separate genetic lineage. Multiple substitutions along the genome caused non-detection using a range of conventional PCRs previously shown to target four known genogroups of perhabdoviruses. However, various generic PCRs efficiently detected the isolated virus. The origin of this virus remains to be elucidated. It may have been introduced into the farm via wild genitors. This finding provides new evidence of the high genetic diversity of percid perhabdoviruses and the potential of new genotypes to emerge as threats for fish farming. Efforts to improve the existing diagnostic methods and control this large group of viruses are still needed.

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.

A Case Report of a Botulism Outbreak in Beef Cattle Due to the Contamination of Wheat by a Roaming Cat Carcass: From the Suspicion to the Management of the Outbreak.

We report a botulism outbreak in Charolais cattle fed with wheat flour contaminated by Clostridium botulinum type C and the management of the outbreak at each step from the clinical suspicion to the cleaning and disinfection operations. Diagnosis was based on typical suggestive clinical signs and detection of C. botulinum type C using real-time PCR in samples collected from three young affected bulls. All young exposed bulls and cows (18 animals) eventually died, but three young bulls and one cow were recovering when it was decided to euthanize them. C. botulinum type C was detected in the liver of these four animals. Analysis of the ration components demonstrated that wheat flour, wheat, and the mill used to make flour were positive for C. botulinum type C. A dead cat positive for C. botulinum type C was discovered in the silo where wheat grain was stored and was considered the source of contamination. The cat's entire body was found mummified, well preserved, and not rotting in the silo. Specific measures, in particular, vaccination of the rest of the herd and cleaning and disinfection operations, were implemented to prevent any recurrence of the outbreak. The presence of wild animal carcasses in feed harboring anaerobic conditions like silage, in particular during harvesting, are known to be at risk for the initiation of a botulism outbreak. This outbreak is a reminder that the presence of an animal carcass in feed, regardless of the kind of feed and whenever the contamination occurs, either during harvesting or storage, is sufficient to induce a botulism outbreak.

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.

RANAVIRUS CAUSES MASS DIE-OFFS OF ALPINE AMPHIBIANS IN THE SOUTHWESTERN ALPS, FRANCE.

Pathogenic fungi and viruses cause mortality outbreaks in wild amphibians worldwide. In the summer of 2012, dead tadpoles and adults of the European common frog Rana temporaria were reported in alpine lakes in the southwestern Alps (Mercantour National Park, France). A preliminary investigation using molecular diagnostic techniques identified a Ranavirus as the potential pathogenic agent. Three mortality events were recorded in the park, and samples were collected. The amphibian chytrid fungus Batrachochytrium dendrobatidis was not detected in any of the dead adult and juvenile frogs sampled (n=16) whereas all specimens were positive for a Ranavirus. The genome sequence of this Ranavirus was identical to previously published sequences of the common midwife toad virus (CMTV), a Ranavirus that has been associated with amphibian mortalities throughout Europe. We cultured virus from the organs of the dead common frogs and infecting adult male common frogs collected in another alpine region where no frog mortality had been observed. The experimentally infected frogs suffered 100% mortality (n=10). The alpine die-off is the first CMTV outbreak associated with mass mortality in wild amphibians in France. We describe the lesions observed and summarize amphibian populations affected by Ranaviruses in Europe. In addition, we discuss the ecologic specificities of mountain amphibians that may contribute to increasing their risk of exposure to and transmission of Ranaviruses.