Carp edema virus-related mortality in wild adult common carp (cyprinus carpio) in Italy.

Mortality in wild fish populations represents a challenging issue for public fish health inspectors. When a single fish species is involved, an infective aetiology is frequently suspected, with focus on viral notifiable diseases. However, other viral agents not subjected to regulation and causing mortality in common carp have been reported such as carp edema virus (CEV). In mid-June 2020, a severe common carp mortality was observed in an artificial lake in north-east of Italy. Sleepy fish were noted some days before the beginning of the mortality itself, which lasted several days and involved over 340 adult specimens. During the outbreak, water temperature was around 15°C, water quality was normal, and no adverse meteorological events were reported in the area. Four specimens, which showed severe cutaneous hyperaemia and increased mucus production on skin and gills, were tested by bacteriological methods and virological analysis targeting the main carp pathogens. Molecular analysis performed on gills, kidney and brains from all the fish analysed resulted positive for CEV, which, based on anamnestic information and laboratory findings, was considered the responsible for the mortality event herein described.

Detection and characterization of a rhabdovirus causing mortality in black bullhead catfish, Ameiurus melas.

This study fully describes a severe disease outbreak occurred in 2016 in black bullhead catfish farmed in Italy. Affected fish showed nervous clinical signs as well as emaciations and haemorrhagic petechiae on the skin at the fin bases, abdomen and gills. Viral isolation in cell culture allowed the subsequent identification of a rhabdovirus, tentatively named ictalurid rhabdovirus (IcRV), through electron microscopy, immunofluorescence and whole genome sequencing (WGS). The newly isolated virus, together with 14 additional viral strains stored in our repository and detected during similar mortality episodes in the period 1993-2016, was phylogenetically analysed on the basis of the nucleoprotein and the glycoprotein nucleotide and amino acid sequences. The genetic distances among Italian IcRV strains were also estimated. Our results show that all the IcRV strains belong to the genus Sprivivirus and are closely related to the tench rhabdovirus (TenRV). Italian catfish production is constantly decreasing, mainly due to viral infections, which include the newly characterized IcRV. Data presented in this work will assist to investigate the molecular epidemiology and the diffusive dynamics of this virus and to develop adequate surveillance activities.

Tilapia lake virus: A structured phylogenetic approach.

Tilapia Lake Virus (TiLV), also known as Tilapia tilapinevirus, is an emerging pathogen affecting both wild and farmed tilapia (Oreochromis spp.), which is considered one of the most important fish species for human consumption. Since its first report in Israel in 2014, Tilapia Lake Virus has spread globally causing mortality rates up to 90%. Despite the huge socio-economic impact of this viral species, to date the scarce availability of Tilapia Lake Virus complete genomes is severely affecting the knowledge on the origin, evolution and epidemiology of this virus. Herein, along with the identification, isolation and complete genome sequencing of two Israeli Tilapia Lake Virus deriving from outbreaks occurred in tilapia farms in Israel in 2018, we performed a bioinformatics multifactorial approach aiming to characterize each genetic segment before carrying out phylogenetic analysis. Results highlighted the suitability of using the concatenated ORFs 1, 3, and 5 in order to obtain the most reliable, fixed and fully supported tree topology. Finally, we also attempted to investigate the presence of potential reassortment events in all the studied isolates. As a result, we report a reassortment event detected in segment 3 of isolate TiLV/Israel/939-9/2018 involved in the present study, and confirmed almost all the other events previously reported.

Expression of Mytilus immune genes in response to experimental challenges varied according to the site of collection.

Mussels live in diverse coastal environments experience various physical, chemical and biological conditions, which they counteract with functional adjustments and heritable adaptive changes. In order to investigate possible differences in immune system capabilities, we analyzed by qPCR the expression levels of 4 immune genes (defensin, mytilin B, myticin B, lysozyme) and HSP70 in the Mediterranean mussel, Mytilus galloprovincialis collected in 3 European farming areas {Atlantic Ocean-Ría de Vigo-Spain (RV), French Mediterranean Gulf of Lion-Palavas-Prévost lagoon (PP) and Northern Adriatic Sea-Venice-Italy (VI)} in response to one injection of one of the 3 bacterial species (Vibrio splendidus LGP32, Vibrio anguillarum, Micrococcus lysodeikticus), and to heat shock or cold stress. We confirmed that the 5 genes are constitutively expressed in hemocytes, defensin being the less expressed, myticin B the highest. As suspected, the same gene resulted differently expressed according to mussel group, with the biggest difference being for HSP70 and lysozyme and lowest expression of all the 5 genes in mussels from RV. In addition, gene expression levels varied according to the challenge. Most frequent effect of bacterial injections was down-regulation, especially for mytilin B and myticin B. Heat shock enhanced transcript levels, particularly in mussels from RV, whereas cold stress had no effect. In situ hybridization of labelled probes on mussel hemocytes indicated that bacterial injections did not change the mRNA patterns of defensin and myticin B whereas mytilin B mRNA almost disappeared. In conclusion, these results demonstrated that constitutive level, nature and intensity of immune gene expression regulations strongly depended from mussel group, and support the concept of gene-environment interactions.

Genomic Sequence of a New Alphavirus Detected in Comber (Serranus cabrilla).

The comber alphavirus was isolated from a fish cell line from the brain of an apparently healthy Serranus cabrilla specimen collected during wild fish surveillance in southern Italy. The comber alphavirus is a new member of the genus Alphavirus, family Togaviridae.

Low evolutionary rate of infectious pancreatic necrosis virus (IPNV) in Italy is associated with reduced virulence in trout.

Infectious pancreatic necrosis virus (IPNV) is a naked double-stranded RNA virus with a bi-segmented genome that is classified within the family Birnaviridae, genus Aquabirnavirus. IPNV was first detected in Italian trout farms in the late 1970s and ultimately became endemic. To characterize the evolution of IPNV circulating in Italy, particularly whether there is a link between evolutionary rate and virulence, we obtained and analyzed the VP1 (polymerase) and the pVP2 (major capsid protein precursor) sequences from 75 IPNV strains sampled between 1978 and 2017. These data revealed that the Italian IPNV exhibit relatively little genetic variation over the sampling period, falling into four genetic clusters within a single genogroup (group 2 for VP1 and genogroup V for pVP2) and contained one example of inter-segment reassortment. The mean evolutionary rates for VP1 and pVP2 were estimated to be 1.70 and 1.45 × 10-4 nucleotide substitutions per site, per year, respectively, and hence significantly lower than those seen in other Birnaviruses. Similarly, the relatively low ratios of non-synonymous (dN) to synonymous (dS) nucleotide substitutions per site in both genes indicated that IPNV was subject to strong selective constraints, again in contrast to other RNA viruses infecting salmonids that co-circulate in the same area during the same time period. Notably, all the Italian IPNV harbored a proline at position 217 (P217) and a threonine at position 221 (T221) in pVP2, both of which are associated with a low virulence phenotype. We therefore suggest the lower virulence of IPNV may have resulted in reduced rates of virus replication and hence lower rates of evolutionary change. The data generated here will be of importance in understanding the factors that shape the evolution of Aquabirnaviruses in nature.

Viral nervous necrosis in gilthead sea bream (Sparus aurata) caused by reassortant betanodavirus RGNNV/SJNNV: an emerging threat for Mediterranean aquaculture.

Viral nervous necrosis (VNN) certainly represents the biggest challenge for the sustainability and the development of aquaculture. A large number of economically relevant fish species have proven to be susceptible to the disease. Conversely, gilthead sea bream has generally been considered resistant to VNN, although it has been possible to isolate the virus from apparently healthy sea bream and sporadically from affected larvae and postlarvae. Unexpectedly, in 2014-2016 an increasing number of hatcheries in Europe have experienced mass mortalities in sea bream larvae. Two clinical outbreaks were monitored over this time span and findings are reported in this paper. Despite showing no specific clinical signs, the affected fish displayed high mortality and histological lesions typical of VNN. Fish tested positive for betanodavirus by different laboratory techniques. The isolates were all genetically characterized as being reassortant strains RGNNV/SJNNV. A genetic characterization of all sea bream betanodaviruses which had been isolated in the past had revealed that the majority of the strains infecting sea bream are actually RGNNV/SJNNV. Taken together, this information strongly suggests that RGNNV/SJNNV betanodavirus possesses a particular tropism to sea bream, which can pose a new and unexpected threat to the Mediterranean aquaculture.

Molecular Evolution and Phylogeography of Co-circulating IHNV and VHSV in Italy.

Infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV) are the most important viral pathogens impacting rainbow trout farming. These viruses are persistent in Italy, where they are responsible for severe disease outbreaks (epizootics) that affect the profitability of the trout industry. Despite the importance of IHNV and VHSV, little is known about their evolution at a local scale, although this is likely to be important for virus eradication and control. To address this issue we performed a detailed molecular evolutionary and epidemiological analysis of IHNV and VHSV in trout farms from northern Italy. Full-length glycoprotein gene sequences of a selection of VHSV (n = 108) and IHNV (n = 89) strains were obtained. This revealed that Italian VHSV strains belong to sublineages Ia1 and Ia2 of genotype Ia and are distributed into 7 genetic clusters. In contrast, all Italian IHNV isolates fell within genogroup E, for which only a single genetic cluster was identified. More striking was that IHNV has evolved more rapidly than VHSV (mean rates of 11 and 7.3 × 10(-4) nucleotide substitutions per site, per year, respectively), indicating that these viruses exhibit fundamentally different evolutionary dynamics. The time to the most recent common ancestor of both IHNV and VHSV was consistent with the first reports of these pathogens in Italy. By combining sequence data with epidemiological information it was possible to identify different patterns of virus spread among trout farms, in which adjacent facilities can be infected by either genetically similar or different viruses, and farms located in different water catchments can be infected by identical strains. Overall, these findings highlight the importance of combining molecular and epidemiological information to identify the determinants of IHN and VHS spread, and to provide data that is central to future surveillance strategies and possibly control.