Characterisation of SARS-CoV-2 Lentiviral Pseudotypes and Correlation between Pseudotype-Based Neutralisation Assays and Live Virus-Based Micro Neutralisation Assays

The recent outbreak of a novel Coronavirus (SARS-CoV-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against SARS-CoV-2 spike protein and to evaluate the serological immunity in humans. Since the accessibility of live virus microneutralisation (MN) assays with SARS-CoV-2 is limited and requires enhanced bio-containment, the approach based on "pseudotyping" can be considered a useful complement to other serological assays. After fully characterising lentiviral pseudotypes bearing the SARS-CoV-2 spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an Italian sero-epidemiological study. The results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of the pseudotype-based neutralisation and MN results. The overall results lead to the conclusion that the pseudotype-based neutralisation assay is a valid alternative to using the wild-type strain, and although this system needs to be optimised and standardised, it can not only complement the classical serological methods, but also allows serological assessments to be made when other methods cannot be employed, especially in a human pandemic context

SARS-CoV-2 epidemiological trend before vaccination era: a seroprevalence study in Apulia, Southern Italy, in 2020

AimThe present study aimed at assessing the prevalence of antibodies against SARS-CoV-2 in the general population in the province of Bari (Apulia region, Southern Italy) during the year 2020.Subject and methodsIn this study, 1325 serum samples collected from January to December 2020 were tested for the presence of IgM and IgG antibodies against whole-virus SARS-CoV-2 antigen by commercial ELISA. Positive samples were further tested by in-house ELISA for the detection of anti-receptor binding domain (RBD) IgM and IgG antibodies and by micro-neutralization (MN) assay for the detection of neutralizing antibody.ResultsOne hundred (7.55%) samples had the presence of at least one antibody class against SARS-CoV-2 by commercial ELISA, of which 88 (6.6%) showed IgG and 19 (1.4%) showed IgM antibodies. The proportion of samples with IgG antibodies increased from 1.9% in January-February to 9.6% in November-December, while no significant increase was observed for IgM. When tested by in-house ELISA and MN assay, 17.0% and 31.6% were found positive to RBD IgG and RBD IgM, respectively, while 12.0% showed neutralizing antibody.ConclusionThe proportion of samples with SARS-CoV-2 IgG antibodies increased during 2020, especially in the second half of the year, consistent with data reported by the routine epidemiological surveillance of SARS-CoV-2 cases. Despite the high number of reported cases, the seroprevalence values are relatively low, and only a small proportion of samples had neutralizing antibodies

Role of the Macrophage Migration Inhibitory Factor (MIF) in the survival of first trimester human placenta under induced stress conditions

Macrophage Migration Inhibitory Factor (MIF) is a multifunctional molecule highly secreted by human placenta mainly in the early phases of pregnancy. Studies in different cells show that MIF is a pro-survival factor by binding to its receptor CD74. By using the in vitro model of placental explants from first trimester pregnancy, we investigated the role of MIF in the survival of placental cells under induced stress conditions that promote apoptosis or mimic the hypoxia/re-oxygenation (H/R) injury that placenta could suffer in vivo. We demonstrated that recombinant MIF (rMIF) treatment was able to reduce caspase-3 activation when cultures were challenged with the apoptosis-inducer Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) while, in the cultures exposed to H/R, the treatment with rMIF did not show any effect. However, a significant increase in caspase-3 and caspase-8 activation was found when H/R-exposed cultures, were treated with anti-MIF or anti-CD74 antibody. We also observed that under H/R, a significant amount of endogenous MIF was released into the medium, which could account for the lack of effect of rMIF added to the cultures. Our results demonstrate for the first time that the MIF/CD74 axis contributes to maintain trophoblast homeostasis, by preventing abnormal apoptotic death

Comparative analyses of SARS-CoV-2 binding (IgG, IgM, IgA) and neutralizing antibodies from human serum samples

A newly identified coronavirus, named SARS-CoV-2, emerged in December 2019 in Hubei Province, China, and quickly spread throughout the world; so far, it has caused more than 49.7 million cases of disease and 1,2 million deaths. The diagnosis of SARS-CoV-2 infection is currently based on the detection of viral RNA in nasopharyngeal swabs by means of molecular-based assays, such as real-time RT-PCR. Furthermore, serological assays detecting different classes of antibodies constitute an excellent surveillance strategy for gathering information on the humoral immune response to infection and the spread of the virus through the population. In addition, it can contribute to evaluate the immunogenicity of novel future vaccines and medicines for the treatment and prevention of COVID-19 disease.The aim of this study was to determine SARS-CoV-2-specific antibodies in human serum samples by means of different commercial and in-house ELISA kits, in order to evaluate and compare their results first with one another and then with those yielded by functional assays using wild-type virus. It is important to identify the level of SARS-CoV-2-specific IgM, IgG and IgA antibodies in order to predict human population immunity, possible cross-reactivity with other coronaviruses and to identify potentially infectious subjects.In addition, in a small sub-group of samples, a subtyping IgG ELISA has been performed. Our findings showed a notable statistical correlation between the neutralization titers and the IgG, IgM and IgA ELISA responses against the receptor-binding domain of the spike protein. Thus confirming that antibodies against this portion of the virus spike protein are highly neutralizing and that the ELISA Receptor-Binding Domain-based assay can be used as a valid surrogate for the neutralization assay in laboratories that do not have biosecurity level-3 facilities

Placental responses to maternal perturbations: altered glucose homeostasis and Bisphenol A exposure

Glucose is one of the main nutrients for fetal development. Nevertheless, a significant gluconeogenesis ability by the fetus has not been reported, therefore the fetal glucose homeostasis depends on the ability of the placenta to carry out glucose from the maternal blood and to transport it to fetal tissues by means of specific transport molecules, known as GLUTs. Several conditions contribute to an imbalance in fetal glucose homeostasis, including incorrect mother feeding, maternal obesity, maternal type II diabetes and/or pregnancy complications. These conditions can affect the normal development of the fetus by affecting glucose transfer through the placenta. Nowadays it is known that environmental contaminants are able to impair placental glucose homeostasis compromising fetal development. These substances include synthetic chemicals with estrogen-like behavior referred as to Endocrine Disrupting Chemicals (EDCs), of which Bisphenol A represents one of the most important for its wide distribution in many daily-use products. However, in which way these two possible maternal perturbations (altered glucose supply and BPA exposure) are able to interfere with placental glucose uptake and how this interference may reflect on an incorrect fetal development has to be elucidated. For this purpose, this study aimed to clarify the effect of such perturbations on human placental glucose homeostasis. The studies were conducted in vitro, in human placental cell line, and in vivo, in rat placenta and fetal heart. The results showed that both, altered glucose supply and BPA exposure, influence human and rat placental glucose transport. Moreover, the in vivo study revealed that BPA disruption on placental glucose homeostasis compromise fetal rat heart development

Prenatal Nutrition Containing Bisphenol A Affects Placenta Glucose Transfer: Evidence in Rats and Human Trophoblast

This work aims to clarify the effect of dietary supplementation with Bisphenol A (BPA), a chemical widely present in beverage and food containers, on placental glucose transfer and pregnancy outcome. The study was performed on female Sprague Dawley rats fed with a diet containing BPA (2.5, 25 or 250 μg/Kg/day) for a period of a month (virgin state) plus 20 days during pregnancy. Western blot analysis and immunohistochemistry were performed in placental tissues for glucose type 1 transporter (GLUT1). Furthermore, human trophoblast, HTR8-SV/neo cells, were used to evaluate the effect of BPA on glucose transport and uptake. Studies in rats showed that food supplementation with BPA, produces a higher fetal weight (FW) to placenta weight (PW) ratio at the lowest BPA concentration. Such low concentrations also reduced maternal weight gain in late pregnancy and up-regulated placental expression of GLUT1. Treatment of HTR8-SV/neo with the non-toxic dose of 1 nM BPA confirmed up-regulation of GLUT1 expression and revealed higher activity of the transporter with an increase in glucose uptake and GLUT1 membrane translocation. Overall, these results indicate that prenatal exposure to BPA affects pregnancy and fetal growth producing changes in the placental nutrients-glucose transfer

Multiple mechanisms of Rottlerin toxicity in A375 melanoma cells

Rottlerin is a cytostatic and cytotoxic drug in a variety of cancer cells. Our previous experience demonstrated that depending upon the genetic/biochemical background of cancer cells, rottlerin is able to induce both apoptotic and autophagic cell death, or dramatically disturb protein homeostasis leading to lethal cellular atrophy. In the current study, we investigated the cytotoxic effects and mechanisms of rottlerin against human amelanotic A375 melanoma cells. In this cell line, rottlerin exhibits its main and newest cytotoxic properties, that is, growth arrest, apoptosis induction, and translation shutoff. In fact, the drug, time-, and dose-dependently, markedly inhibited cell proliferation through cyclin D1 downregulation and induced apoptotic cell death as early as after 18 h treatment. Mechanistically, rottlerin triggered apoptosis by both intrinsic and extrinsic pathways. Both pathways are likely activated by the downregulation of the antiapoptotic B-cell lymphoma 2 (Bcl-2) protein, which simultaneously affects mitochondrial and endoplasmic reticulum (ER) membranes stability. Concomitantly to extrinsic apoptosis induction, the rottlerin-activated ER stress/eukaryotic initiation factor 2 (eIF2) alpha axis blocked the translational apparatus. The altered proteostasis precluded the complete cells' rescue from death in the presence of apoptosis inhibitors

Pathogenic mechanisms and current epidemiological status of HEV infection in asymptomatic blood donors and patients with chronic diseases.

In recent years, the seroprevalence of anti-hepatitis E virus immunoglobulins (HEV) has increased in European countries with significant variability among the different geographical areas. HEV infection is spread in a wide range of animal species of which domestic pigs and wild boar represent the main reservoirs of genotype 3 and 4 (the genotypes present also in Europe). European citizens are incidental hosts, mainly infected by direct contact or consumption of foods derived from undercooked or insufficient hygiene handling infected pork products or wild boar meat. Epidemiologically, the HEV incidence is low in humans but serological data show a high proportion of subclinical infection caused by genotypes 3 or 4. In the general population, asymptomatic infection represents a high potential risk in particular subjects such as blood component recipients or occupationally exposed workers. This review offers a landscape of the current epidemiological status of HEV infection (genotypes 1, 2, 3, 4, 7) both in European asymptomatic subjects, patients with chronic diseases, and domestic pig impact on humans. We also underline advantages/disadvantages of high sensitivity and specificity tests using for detecting viral RNA or anti-HEV antibodies

Potential in-vitro antiviral activity of MV1035 on SARS-CoV-2 wild type viruses

Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense, single stranded RNA virus, and is responsible for the pandemic outbreak called COVID-19. The pandemic, still ongoing, had presented unprecedented challenges in terms of finding appropriate pharmacological treatments. Methods Starting from the recent literature that demonstrates how ALKBH5 inhibitors could be used as a new strategy to reduce SARS-CoV-2 replication, we decided to repurpose our newly discovered ALKBH5 inhibitor MV1035, previously tested and proved effective against glioblastoma, for its putative antiviral activity against SARS-CoV-2. We demonstrated a reduction in SARS-CoV-2-induced CPE after 72 h incubation using MV1035 (50 µM), for SARS-CoV-2 wild type (Wuhan strain) and South African variant. Results and conclusion The results show how MV1035 seems to be able to reduce SARS-CoV-2 replication through an indirect mechanism of action, which might involve an interaction with the host cell rather than with a virus protein