Development and validation of a real-time TaqMan PCR assay for the detection of betanodavirus in clinical specimens

Development and validation of a real-time TaqMan PCR assay for the detection of betanodavirus in clinical specimens Panzarin V, Patarnello P, Mori A, Rampazzo E, Cappellozza E, Bovo G, Cattoli G. Betanodaviruses are the causal agents of viral encephalo-retinopathy, an infectious disease affecting more than 40 marine fish species, characterized by high morbidity and mortality. Because of its severe impact, robust diagnostic tools are required. The aim of this work was to develop and validate a real-time TaqMan PCR assay to detect betanodaviruses in clinical specimens by amplifying a conserved region of the RNA2 strand. The method proved to be specific and sensitive, being capable of detecting as low as 10 TCID(50)/ml. For clinical validation, samples from 100 marine fish were collected during a natural outbreak of disease and tested by three distinct laboratory methods, namely real-time TaqMan PCR, RT-seminested PCR and virus isolation. The results indicated optimal agreement between tests. The assay that was developed is capable of detecting members of all of the betanodavirus genetic groups currently described and can be considered a valid alternative to the time-consuming and contamination-prone nested PCR

Bottlenecks in diagnostics of Mediterranean fish diseases

Three independent Horizon 2020 projects are prioritising a common task of solving diagnostic issues using different approaches and strategies. PerformFISH focuses its research objectives on diagnostic methods, MedAID has prepared a diagnostic manual for the most devastating fish diseases of the Mediterranean and ParaFishControl has prepared a diagnostic manual on parasitic diseases of European aquaculture species. Currently, the projects are investing large collaborative efforts to reinforce their tasks and to deliver mutual and cutting-edge material on the subject. Nevertheless, further efforts for the harmonisation, standardisation and validation of diagnostic methods for infectious diseases in Mediterranean aquaculture are needed, involving as many relevant stakeholders and experts as possible. The main goal of the workshop (WS) “Bottlenecks in diagnostics of Mediterranean fish diseases”, organised during the 19th International EAFP conference on Diseases of Fish and Shellfish, was to present results already obtained from the projects and discuss the bottlenecks in diagnostic procedures of target groups of pathogens. The WS provided an ideal environment and opportunity to exchange and discuss experiences and data with international experts in the diagnostic field attending the conference. Interactive discussions on diagnostics for a specific group of pathogens have resulted in highlighting new initiatives needed in the management of diseases in Mediterranean aquaculture. Furthermore, the WS emphasised a need for the establishment of this strong interactive network which will collaborate to receive, collect, compile and analyse all information gathered from different parts of the region.info:eu-repo/semantics/publishedVersio

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

Molecular tracing of viral diseases in aquaculture

Molecular Tracing of Viral Diseases in Aquaculture = Traçage Moléculaire des Maladies Virales en Aquaculture : Colloque, Montpellier (FRA), 2015/01/27-29International audienc

Isolation and molecular characterization of viral encephalopathy and retinopathy virus from gilthead seabream larvae (Sparus aurata) showing mass mortalities

Gilthead seabream (Sparus aurata, L.1578) has been reared since several years in close connection with the European seabass (Dicentrarchus labrax) often affected by severe losses caused by viral encepahalopathy and retinopathy (VER) without showing any clinical sign or mortality. This observation led to consider Sparus aurata a species completely resistant to betanodavirus infection. In 2001, Castric et al., during a VER outbreak affecting a farmed seabass population, succeeded to obtain a VER isolate from asymptomatic seabream, collected in the same farm. In the following years several rumours about seabream larvae mortalities caused by betanodavirus infections have circulated among farmers and in the scientific world but very few investigations have been performed. In 2007, Beraldo et al. described the occurrence of a betanodavirus outbreak in sea bream larvae showing mass mortalities. Clinical signs, histological and immunohistochemistry findings were described. Frozen samples belonging to the same outbreak were accidentally made available later on by the farmer and processed for virus isolation and characterization. In the present study we describe the isolation of the causative agent in SSN-1 cells, its identification by PCR and molecular characterization as reassortant virus containing the genome from RGNNV (RNA1) and SJNNV(RNA2) genotypes. Our findings corroborate the preliminary results described by Beraldo et al. and definitively demonstrate that gilthead seabream must be included in the list of VER susceptible species at least during larval stage. References Beraldo P., G. De Nigris, F. Rogato and M. Galeotti (2007). Histological and immunohistochemistry findings of viral encephalopathy-retinopathy in gilthead seabream larvae (Sparus aurata, L. 1578) reared in Italy. Book of abstract 13th international conference of the EAFP, diseases of fish and shellfish . Grado, 17-22 September 2007

Viral encephalopathy and retinopathy outbreak in freshwater fish farmed in Italy.

Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a neuropathological condition affecting > 40 species of fish. Although VER affects mainly marine fish, the disease has also been detected in certain species reared in freshwater environments. There are relatively few reports concerning the disease in freshwater species, and there is not much information on clinical signs. Nevertheless, the most common clinical findings reported from affected freshwater species are consistent with the typical signs observed in marine species. In this paper we describe the main clinical signs and the laboratory results associated with the detection of a betanodavirus in hybrid striped bass x white bass (Morone saxatilis x Morone chrysops) and largemouth bass Micropterus salmoides, reared in a freshwater environment. We also detected the virus by real-time PCR and isolated it in cell culture from a batch of pike-perch Sander lucioperca farmed in the same system