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

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 vivoThe project has been funded under the ERANET. The content of this article reflects only the authors’ views, and the ERANET Consortium is not liable for any use that may be made of the information contained thereinS

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

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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

Inter and intra-population variability of the migratory behaviour of a short-distance partial migrant, the Eurasian Stone-curlew Burhinus oedicnemus (Charadriiformes, Burhinidae)

Migratory behaviour in birds shows a remarkable variability at species, population and individual levels. Short-distance migrants often adopt a partial migratory strategy and tend to have a flexible migration schedule that allows a more effective response to extreme environmental variations. Weather seasonality and environmental heterogeneity have been reported as significant factors in the diversification of migratory behaviour for Mediterranean migrants, but relatively few studies investigated the migration patterns of non-passerine birds migrating within the Mediterranean basin. In this study, we investigated the migratory strategy of 40 Eurasian Stone-curlews Burhinus oedicnemus tagged with geolocators and GPS-GSM tags and belonging to continental and Mediterranean populations of the Italian peninsula. The proportion of migrants was higher in continental populations, but we observed a significant variability also within Mediterranean populations. All birds spent the winter within the Mediterranean basin. Continental Stone-curlews departed earlier in spring and later in autumn and covered longer distances than those from Mediterranean areas. The speed of migration did not change between seasons for continental birds, while Mediterranean individuals migrated faster in spring. The likelihood of departure for autumn migration of GPS-tagged birds increased when temperatures were near or below 0 °C suggesting that Stone-curlews tend to delay departure until weather conditions worsen abruptly. As a consequence of global warming in the Mediterranean, the frequency of migratory birds in the considered populations might decrease in the near future. This could affect the distribution of species throughout the year and should be taken into account when targeting conservation measures

Phylogeny of the Viral Hemorrhagic Septicemia Virus in European Aquaculture

<p>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.</p

Heterogeneity of Early Host Response to Infection with Four Low-Pathogenic H7 Viruses with a Different Evolutionary History in the Field

Once low-pathogenic avian influenza viruses (LPAIVs) of the H5 and H7 subtypes from wild birds enter into poultry species, there is the possibility of them mutating into highly pathogenic avian influenza viruses (HPAIVs), resulting in severe epizootics with up to 100% mortality. This mutation from a LPAIV to HPAIV strain is the main cause of an AIV's major economic impact on poultry production. Although AIVs are inextricably linked to their hosts in their evolutionary history, the contribution of host-related factors in the emergence of HPAI viruses has only been marginally explored so far. In this study, transcriptomic sequencing of tracheal tissue from chickens infected with four distinct LP H7 viruses, characterized by a different history of pathogenicity evolution in the field, was implemented. Despite the inoculation of a normalized infectious dose of viruses belonging to the same subtype (H7) and pathotype (LPAI), the use of animals of the same age, sex and species as well as the identification of a comparable viral load in the target samples, the analyses revealed a heterogeneity in the gene expression profile in response to infection with each of the H7 viruses administered

Protective Efficacy of H9N2 Avian Influenza Vaccines Inactivated by Ionizing Radiation Methods Administered by the Parenteral or Mucosal Routes

H9N2 viruses have become, over the last 20 years, one of the most diffused poultry pathogens and have reached a level of endemicity in several countries. Attempts to control the spread and reduce the circulation of H9N2 have relied mainly on vaccination in endemic countries. However, the high level of adaptation to poultry, testified by low minimum infectious doses, replication to high titers, and high transmissibility, has severely hampered the results of vaccination campaigns. Commercially available vaccines have demonstrated high efficacy in protecting against clinical disease, but variable results have also been observed in reducing the level of replication and viral shedding in domestic poultry species. Antigenic drift and increased chances of zoonotic infections are the results of incomplete protection offered by the currently available vaccines, of which the vast majority are based on formalin-inactivated whole virus antigens. In our work, we evaluated experimental vaccines based on an H9N2 virus, inactivated by irradiation treatment, in reducing viral shedding upon different challenge doses and compared their efficacy with formalin-inactivated vaccines. Moreover, we evaluated mucosal delivery of inactivated antigens as an alternative route to subcutaneous and intramuscular vaccination. The results showed complete protection and prevention of replication in subcutaneously vaccinated Specific Pathogen Free White Leghorn chickens at low-to-intermediate challenge doses but a limited reduction of shedding at a high challenge dose. Mucosally vaccinated chickens showed a more variable response to experimental infection at all tested challenge doses and the main effect of vaccination attained the reduction of infected birds in the early phase of infection. Concerning mucosal vaccination, the irradiated vaccine was the only one affording complete protection from infection at the lowest challenge dose. Vaccine formulations based on H9N2 inactivated by irradiation demonstrated a potential for better performances than vaccines based on the formalin-inactivated antigen in terms of reduction of shedding and prevention of infection

SARS-CoV-2 infection and replication in human gastric organoids

COVID-19 typically manifests as a respiratory illness, but several clinical reports have described gastrointestinal symptoms. This is particularly true in children in whom gastrointestinal symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. These observations raise the question of whether the virus can replicate within the stomach. Here we generate gastric organoids from fetal, pediatric, and adult biopsies as in vitro models of SARS-CoV-2 infection. To facilitate infection, we induce reverse polarity in the gastric organoids. We find that the pediatric and late fetal gastric organoids are susceptible to infection with SARS-CoV-2, while viral replication is significantly lower in undifferentiated organoids of early fetal and adult origin. We demonstrate that adult gastric organoids are more susceptible to infection following differentiation. We perform transcriptomic analysis to reveal a moderate innate antiviral response and a lack of differentially expressed genes belonging to the interferon family. Collectively, we show that the virus can efficiently infect the gastric epithelium, suggesting that the stomach might have an active role in fecal-oral SARS-CoV-2 transmission.Several clinical reports have described gastrointestinal symptoms for COVID-19, though whether the virus can replicate within the stomach remains unclear. Here the authors generate gastric organoids from human biopsies and show that the virus can efficiently infect gastric epithelium, suggesting that the stomach might have an active role in fecal-oral transmission