Diffusion aluminide coatings for hot corrosion and oxidation protection of nickel-based superalloys. Effect of fluoride-based activator salts

The influence of two different fluoride-based activator salts (NH4F and AlF3) was studied for diffusion aluminide coatings obtained via pack cementation on a Ni-based superalloy (René 108DS). The resistance to oxidation and hot corrosion was assessed as a function of the concentration of activator salts used during the synthesis process by means of pack cementation. Two different concentrations were selected for activator salts (respecting the equimolarity of fluoride in the synthesis) and the obtained diffusion coatings were compared in terms of morphology, thickness and composition, as well as in terms of microstructural evolution after high temperature exposure. Isothermal oxidation tests were conducted at 1050 C in air for 100 h in a tubular furnace. The oxidation kinetics were evaluated by measuring the weight variation with exposure time. The microstructural evolution induced by the high temperature exposure was investigated by SEM microscopy, EDS analysis and X-ray diffraction. Results showed that the coatings obtained with AlF3 activator salt are thicker than those obtained using NH4F as a consequence of different growth mechanism during pack-cementation. Despite this evidence, it was found that the NH4F coatings show a better oxidation resistance, both in terms of total mass gain and of quality of the microstructure of the thermally grown oxide. On the other hand, coatings produced with high concentration of AlF3 exhibited a better resistance in hot corrosion conditions, showing negligible mass variations after 200 h of high temperature exposure to aggressive NaCl and Na2SO4 salts

Farmers as data sources: Cooperative framework for mapping soil properties for permanent crops in South Tyrol (Northern Italy)

Abstract Detailed knowledge of agricultural soil properties is a key element for high-quality food production. However, high-resolution soil data covering a large agricultural region are generally unavailable. This study explores a demand-driven cooperative framework for soil data sourcing that connects individual farmers to several stakeholders by means of a centralised database containing more than 16,000 records of soil information collected within the framework of an integrated production program for intensively managed permanent crops in the Adige/Etsch and Venosta/Vinschgau valleys in South Tyrol, Italy. Data for soil pH, soil organic matter (SOM), and soil texture were used to produce digital soil maps with a RMSE of 0.21, 1.25% and a cross-validation of 43%, respectively. Spatialisation was conducted using either regression-kriging or multinomial logistic regression. Collaboration among farmers, public administrators, and researchers provided a successful cooperative framework for digital soil mapping. The maps highlight the complex interplay of the postglacial evolution of these valleys due to the presence of a cluster of large alluvial fans and the anthropogenic influences of intense farming on pH, SOM, and soil texture. This study regarded a subset of the available soil properties, which can be dealt with using the geostatistical approaches presented herein. Thus, a long-term soil monitoring program and the combination of all available variables will allow digital assessment of the spatial patterns of nutrient availability, ecological risk assessments, change detection studies, and an overall long-term plan for soil security at larger spatial scales

Use of Equine Herpesvirus 1 glycoprotein pseudotyped lentiviral particles for the development of serological tests and assessment of lyophilisation for transport and storage

Equine herpesviruses (EHVs) are enveloped DNA viruses predominantly infecting members of the Equidae family. EHVs primarily cause respiratory disease, however EHV-1 can produce cases of a neurological disease, abortion and neonatal death. Thus, these viruses represent a welfare issue for the equine industry and scientific focus for researchers. EHV-1 exhibits a complex array of 12 glycoproteins on its surface envelope, but it is unclear precisely which are important for virus cell entry and the role of each in host immune response. In order to investigate the contribution of these glycoproteins, pseudotype viruses (PVs) could provide a useful study tool. We have successfully generated functional EHV-1 pseudotyped lentiviruses bearing four glycoproteins, gB, gD, gH and gL (sequences derived from an aborted foetus during a large EHV1 outbreak strain in Normandy, France). PVs were employed in a pseudotype virus neutralisation test (PVNT) to measure levels of specific neutralising antibodies serum samples (n=52) taken longitudinally from experimentally infected ponies, compared with uninfected controls. PVs routinely require -80oC for long term storage and a dry ice cold-chain during transport which can impede dissemination and utilisation in other laboratories. Consequently, we further investigated whether freeze-drying (lyophilisation) of EHV-1 PV could address this issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions, sampling at different timepoints. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests

Studying longitudinal neutralising antibody levels against Equid herpesvirus 1 in experimentally infected horses using a novel pseudotype based assay

Infection with equid herpesvirus 1 (EHV-1), a DNA virus of the Herpesviridae family represents a significant welfare issue in horses and a great impact on the equine industry. During EHV-1 infection, entry of the virus into different cell types is complex due to the presence of twelve glycoproteins (GPs) on the viral envelope. To investigate virus entry mechanisms, specific combinations of GPs were pseudotyped onto lentiviral vectors. Pseudotyped virus (PV) particles bearing gB, gD, gH and gL were able to transduce several target cell lines (HEK293T/17, RK13, CHO-K1, FHK-Tcl3, MDCK I & II), demonstrating that these four EHV-1 glycoproteins are both essential and sufficient for cell entry. The successful generation of an EHV-1 PV permitted development of a PV neutralisation assay (PVNA). The efficacy of the PVNA was tested by measuring the level of neutralising serum antibodies from EHV-1 experimentally infected horses (n = 52) sampled in a longitudinal manner. The same sera were assessed using a conventional EHV-1 virus neutralisation (VN) assay, exhibiting a strong correlation (r = 0.82) between the two assays. Furthermore, PVs routinely require -80 ◦C for long term storage and a dry ice cold-chain during transport, which can impede dissemination and utilisation in other stakeholder laboratories. Consequently, lyophilisation of EHV-1 PVs was conducted to address this issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions for different time periods. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests. Results indicated that lyophilisation could be used to stably preserve such complex herpesvirus pseudotypes, even after weeks of storage at room temperature, and that reconstituted EHV-1 PVs could be successfully employed in antibody neutralisation tests

Coronavirus Pseudotypes for All Circulating Human Coronaviruses for Quantification of Cross-Neutralizing Antibody Responses.

The novel coronavirus SARS-CoV-2 is the seventh identified human coronavirus. Understanding the extent of pre-existing immunity induced by seropositivity to endemic seasonal coronaviruses and the impact of cross-reactivity on COVID-19 disease progression remains a key research question in immunity to SARS-CoV-2 and the immunopathology of COVID-2019 disease. This paper describes a panel of lentiviral pseudotypes bearing the spike (S) proteins for each of the seven human coronaviruses (HCoVs), generated under similar conditions optimized for high titre production allowing a high-throughput investigation of antibody neutralization breadth. Optimal production conditions and most readily available permissive target cell lines were determined for spike-mediated entry by each HCoV pseudotype: SARS-CoV-1, SARS-CoV-2 and HCoV-NL63 best transduced HEK293T/17 cells transfected with ACE2 and TMPRSS2, HCoV-229E and MERS-CoV preferentially entered HUH7 cells, and CHO cells were most permissive for the seasonal betacoronavirus HCoV-HKU1. Entry of ACE2 using pseudotypes was enhanced by ACE2 and TMPRSS2 expression in target cells, whilst TMPRSS2 transfection rendered HEK293T/17 cells permissive for HCoV-HKU1 and HCoV-OC43 entry. Additionally, pseudotype viruses were produced bearing additional coronavirus surface proteins, including the SARS-CoV-2 Envelope (E) and Membrane (M) proteins and HCoV-OC43/HCoV-HKU1 Haemagglutinin-Esterase (HE) proteins. This panel of lentiviral pseudotypes provides a safe, rapidly quantifiable and high-throughput tool for serological comparison of pan-coronavirus neutralizing responses; this can be used to elucidate antibody dynamics against individual coronaviruses and the effects of antibody cross-reactivity on clinical outcome following natural infection or vaccination

Comparison of lentiviral and vesicular stomatitis virus core SARS-CoV-2 pseudotypes and generation of a stable cell line for use in antibody neutralisation assays

Betacoronavirus SARS-CoV-2, the causative agent of COVID19, is a single stranded positive sense RNA virus. Since its emergence there has been great efforts to identify correlates of protection,which is crucial for vaccine evaluation studies. However, handling SARS-CoV-2 requires BSL-3 containment facilities slowing research efforts. Pseudotype viruses (PV) are a safe alternative to authentic virus that can be handled at low containment. PVs are chimeric viruses containing the core of a virus where its genome has been completely or partially replaced by a reporter gene, displaying a correctly folded SARS-CoV-2 spike on its surface. We developed lentiviral and vesicular stomatitis virus (VSV) core PVs alongside a stable A549 cell line expressing receptor ACE2 and protease TMPRSS2 responsible for S protein priming, for use in neutralization assays. Lentiviral PVs were generated by transfection with plasmids encoding the spike, HIV-1 gag-pol and a luciferase reporter. For VSV PVs, producer cells pre-transfected with the spike were infected with recombinant VSV expressing luciferase,before harvesting. The stable A549 cell line was generated by sequential infection of VSV-G PVs bearing lentiviral vectors encoding ACE2 and TMPRSS2 genes followed by antibiotic selection, before being tested in neutralization assays. We compared lentiviral and VSV PV platforms using monoclonal antibodies and convalescent sera with our stable A549 cells or HEK293T cells pre-transfected with plasmids encoding ACE2 and TMPRSS2. Antibody titres showed equivalence however VSV had the advantage of a shorter incubation therefore enabling a higher throughput. PVs offer a robust platform for future seroepidemiology and vaccine evaluation studies

Genetic characterization of a new candidate hemagglutinin subtype of influenza A viruses

Avian influenza viruses (AIV) have been classified on the basis of 16 subtypes of hemagglutinin (HA) and 9 subtypes of neuraminidase. Here we describe genomic evidence for a new candidate HA subtype, nominally H19, with a large genetic distance to all previously described AIV subtypes, derived from a cloacal swab sample of a Common Pochard (Aythya ferina) in Kazakhstan, in 2008. Avian influenza monitoring in wild birds especially in migratory hotspots such as central Asia is an important approach to gain information about the circulation of known and novel influenza viruses. Genetically, the novel HA coding sequence exhibits only 68.2% nucleotide and 68.5% amino acid identity with its nearest relation in the H9 (N2) subtype. The new HA sequence should be considered in current genomic diagnostic AI assays to facilitate its detection and eventual isolation enabling further study and antigenic classification

Analysis of Antibody Neutralisation Activity against SARS-CoV-2 Variants and Seasonal Human Coronaviruses NL63, HKU1, and 229E Induced by Three Different COVID-19 Vaccine Platforms

Coronaviruses infections, culminating in the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic beginning in 2019, have highlighted the importance of effective vaccines to induce an antibody response with cross-neutralizing activity. COVID-19 vaccines have been rapidly developed to reduce the burden of SARS-CoV-2 infections and disease severity. Cross-protection from seasonal human coronaviruses (hCoVs) infections has been hypothesized but is still controversial. Here, we investigated the neutralizing activity against ancestral SARS-CoV-2 and the variants of concern (VOCs) in individuals vaccinated with two doses of either BNT162b2, mRNA-1273, or AZD1222, with or without a history of SARS-CoV-2 infection. Antibody neutralizing activity to SARS-CoV-2 and the VOCs was higher in BNT162b2-vaccinated subjects who were previously infected with SARS-CoV-2 and conferred broad-spectrum protection. The Omicron BA.1 variant was the most resistant among the VOCs. COVID-19 vaccination did not confer protection against hCoV-HKU1. Conversely, antibodies induced by mRNA-1273 vaccination displayed a boosting in their neutralizing activity against hCoV-NL63, whereas AZD1222 vaccination increased antibody neutralization against hCoV-229E, suggesting potential differences in antigenicity and immunogenicity of the different spike constructs used between various vaccination platforms. These data would suggest that there may be shared epitopes between the HCoVs and SARS-CoV-2 spike proteins

Production, Titration, Neutralisation, Storage and Lyophilisation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Lentiviral Pseudotypes

This protocol details a rapid and reliable method for the production and titration of high-titre viral pseudotype particles with the SARS-CoV-2 spike protein (and D614G or other variants of concern, VOC) on a lentiviral vector core, and use for neutralisation assays in target cells expressing angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). It additionally provides detailed instructions on substituting in new spike variants via gene cloning, lyophilisation and storage/shipping considerations for wide deployment potential. Results obtained with this protocol show that SARS-CoV-2 pseudotypes can be produced at equivalent titres to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudotypes, neutralised by human convalescent plasma and monoclonal antibodies, and stored at a range of laboratory temperatures and lyophilised for distribution and subsequent application