SARS-CoV-2 in a Mink Farm in Italy: Case Description, Molecular and Serological Diagnosis by Comparing Different Tests.

This study described a SARS-CoV-2 infection in minks on an Italian farm. Surveillance was performed based on clinical examination and a collection of 1879 swabs and 74 sera from dead and live animals. The farm was placed under surveillance for 4.5 months, from the end of July 2020, when a man working on the farm tested positive by RT-PCR, till mid-December 2020 when all the animals were sacrificed. Clinical examination revealed no clinical signs or increased mortality rates attributable to SARS-CoV-2, while diagnostic tests detected only four weak PCR-positive samples, but 100% of sera were positive for SARS-CoV-2 anti-S antibodies. The phylogenetic analysis of two SARS-CoV-2 sequences from two minks and the sequence of the worker showed that they belonged to different clades. It could be therefore assumed that two distinct introductions of the virus occurred on the farm, and that the first introduction probably occurred before the start of the surveillance period. From the data collected, and especially from the detection of specific antibodies through the combination of different tests, it can be postulated that syndromic surveillance combined with genome detection by PCR may not be sufficient to achieve a diagnosis in asymptomatic animals. In particular, the serological approach, especially when using tests directed towards the S protein, may be useful for improving the traceability of virus circulation in similar environments.

Organic Electrochemical Transistors as Versatile Tool for Real-Time and Automatized Viral Cytopathic Effect Evaluation.

In-vitro viral studies are still fundamental for biomedical research since studying the virus kinetics on cells is crucial for the determination of the biological properties of viruses and for screening the inhibitors of infections. Moreover, testing potential viral contaminants is often mandatory for safety evaluation. Nowadays, viral cytopathic effects are mainly evaluated through end-point assays requiring dye-staining combined with optical evaluation. Recently, optical-based automatized equipment has been marketed, aimed at the real-time screening of cell-layer status and obtaining further insights, which are unavailable with end-point assays. However, these technologies present two huge limitations, namely, high costs and the possibility to study only cytopathic viruses, whose effects lead to plaque formation and layer disruption. Here, we employed poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (Pedot:Pss) organic electrochemical transistors (OECTs) for the real-time, electrical monitoring of the infection of cytolytic viruses, i.e., encephalomyocarditis virus (EMCV), and non-cytolytic viruses, i.e., bovine coronavirus (B-CoV), on cells. OECT data on EMCV were validated using a commercially-available optical-based technology, which, however, failed in the B-CoV titration analysis, as expected. The OECTs proved to be reliable, fast, and versatile devices for viral infection monitoring, which could be scaled up at low cost, reducing the operator workload and speeding up in-vitro assays in the biomedical research field.

Fast and real-time electrical transistor assay for quantifying SARS-CoV-2 neutralizing antibodies

Due to the SARS-CoV-2 pandemic renewed attention has been directed towards viral neutralization assays and neutralizing antibodies quantification, for vaccine pre-clinical trials and determining vaccine efficacy over time. The gold standard to assess antibody titer is the plaque reduction neutralization test, an end-point assay which evaluates the highest serum antibody dilution that neutralizes viral replication, by inspecting the cytopathic effect induced on cell cultures. Here, we use planar, PEDOT:PSS-based organic electrochemical transistors for real-time, remote-controlled, reliable and fast electrical monitoring of the cytopathic effect induced by SARS29 CoV-2 on Vero E6 cell lines, allowing the quantification of serum neutralizing titer. Our low-cost and scalable device has the potential to speed-up large-scale viral neutralization screening without the need for cancerous staining or highly specialized operators. Finally, the technology could be easily transferred to assess neutralizing antibody response towards different viruses in their permissive cell substrates.The COVID-19 pandemic highlights the importance of tests for assessing antibody titer, such as for determining vaccine efficacy. Here, a fast-operating organic electrochemical transistor is shown to assess the cytopathic effect caused by the SARS CoV-2 virus on Vero E6 cells in real-time.

SARS-CoV2 vertical transmission with adverse effects on the newborn revealed through integrated immunohistochemical, electron microscopy and molecular analyses of Placenta.

BACKGROUND: . The occurrence of trans-placental transmission of severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) infection remains highly debated. Placental positivity for SARS-CoV-2 has been reported in selected cases, but infection or virus-associated disease of fetal tissues or newborns remains to be demonstrated. METHODS: We screened for SARS-CoV-2 spike (S) protein expression placentas from 101 women who delivered between February 7 and May 15, 2020, including 15 tested positive for SARS-CoV-2 RNA, 34 tested negative, and 52 not evaluated as they did not meet testing criteria (32), or delivered before COVID-19 pandemic declaration (20). Immunostain for SARS-CoV-2 nucleocapsid (N) was performed in the placentas of all COVID-19 positive women. One placenta resulted positive for the SARS-CoV-2 S and N proteins, which was further studied by RNA-in situ hybridization and RT-PCR for S transcripts, and by electron microscopy. A comprehensive immunohistochemical and immunofluorescence analysis of the placental inflammatory infiltrate completed the investigations. FINDINGS: SARS-CoV-2 S and N proteins were strongly expressed in the placenta of a COVID-19 pregnant woman whose newborn tested positive for viral RNA and developed COVID-19 pneumonia soon after birth. SARS-CoV-2 antigens, RNA and/or particles morphologically consistent with coronavirus were identified in villous syncytiotrophoblast, endothelial cells, fibroblasts, in maternal macrophages, and in Hofbauer cells and fetal intravascular mononuclear cells. The placenta intervillous inflammatory infiltrate consisted of neutrophils and monocyte-macrophages expressing activation markers. Absence of villitis was associated with an increase in the number of Hofbauer cells, which expressed PD-L1. Scattered neutrophil extracellular traps (NETs) were identified by immunofluorescence. INTERPRETATION: We provide first-time evidence for maternal-fetal transmission of SARS-CoV-2, likely propagated by circulating virus-infected fetal mononuclear cells. Placenta infection was associated with recruitment of maternal inflammatory cells in the intervillous space, without villitis. PD-L1 expression in syncytiotrophoblast and Hofbaeur cells, together with limited production of NETs, may have prevented immune cell-driven placental damage, ensuring sufficient maternal-fetus nutrient exchanges.

Eco-Virological Preliminary Study of Potentially Emerging Pathogens in Hedgehogs (Erinaceus europaeus) Recovered at a Wildlife Treatment and Rehabilitation Center in Northern Italy.

The Western European Hedgehog (Erinaceus europaeus) is one of the four hedgehog species belonging to the genus Erinaceus. Among them, E. amurensis is extant in East Asia's areas only, whereas E. europaeus, E. roumanicus and E. concolor are mainly found in Europe. E. europaeus is endemically distributed from western to central and southern Europe, including Italy. Western European hedgehogs' ecological and feeding habits, along with their high population densities, notable synanthropic attitudes, frequent contacts with sympatric wild and domestic species, including humans, implicate the possible involvement of E. europaeus in the ecology of potentially emerging viruses, such as coronaviruses, influenza A and influenza D viruses, canine distemper virus, pestiviruses and Aujeszky's disease virus. We examined 24 E. europaeus individuals found injured in urban and rural areas of Northern Italy. Of the 24 fecal samples collected and tested for the above-mentioned pathogens by both PCR-based and virus isolation methods, 14 were found PCR-positive for betacoronaviruses belonging to lineage C and related to the known Erinaceus coronaviruses (EriCoVs), as determined by partial sequencing of the virus genome. Our findings suggest that hedgehogs could be considered natural reservoirs of CoVs, and also act as chronic shedding carriers of these potentially emerging RNA viruses.