Genome-Wide DNA Methylome and Transcriptome Analysis of Porcine Testicular Cells Infected With Transmissible Gastroenteritis Virus

Transmissible gastroenteritis virus (TGEV) is a porcine pathogen causing highly communicable gastrointestinal infection that are lethal for suckling piglets. In an attempt to delineate the pathogenic mechanism of TGEV-infected porcine testicular cells (ST cells), we conducted a whole genome analysis of DNA methylation and expression in ST cells through reduced bisulfate-seq and RNA-seq. We examined alterations in the methylation patterns and recognized 1764 distinct methylation sites. 385 differentially expressed genes (DEGs) were enriched in the viral defense and ribosome biogenesis pathways. Integrative analysis identified two crucial genes (EMILIN2, RIPOR3), these two genes expression were negatively correlated to promoter methylation. In conclusion, alterations in DNA methylation and differential expression of genes reveal that their potential functional interactions in TGEV infection. Our data highlights the epigenetic and transcriptomic landscapes in TGEV-infected ST cells and provides a reliable dataset for screening TGEV resistance genes and genetic markers.

A novel approach to concentrate human and animal viruses from wastewater using receptors-conjugated magnetic beads

Viruses are present at low concentrations in wastewater, and therefore an effective concentration of virus particles is necessary for accurate wastewater-based epidemiology (WBE). We designed a novel approach to concentrate human and animal viruses from wastewater using porcine gastric mucin-conjugated magnetic beads (PGM-MBs). We systematically evaluated the performances of the PGM-MBs method (sensitivity, specificity, and robustness to environmental inhibitors) with six viral species including Tulane virus (a surrogate for human norovirus), rotavirus, adenovirus, porcine coronavirus (transmissible gastroenteritis virus or TGEV), and two human coronaviruses (NL63 and SARS-CoV-2) in influent wastewater and raw sewage samples. We determined the multiplication factor (the ratio of genome concentration of the concentrated over that of the initial solution) for the PGM-MBs method, which ranged from 1.3 to 64.0 depending on the viral species. Because the recovery efficiency became significantly higher when calculated based on virus titers than genome concentration, the PGM-MBs method could be an appropriate tool for assessing the risk due to wastewater contaminated with infectious enteric viruses. PCR inhibitors were not concentrated by PGM-MBs, suggesting this tool will be successful for use with environmental samples. The PGM-MBs method is cost-effective (0.43 USD/sample) and fast turnaround (3 hours from virus concentration to genome quantification), and thus this method can be implemented for high throughput facilities. Based on good performance, intrinsic characteristics of targeting the infectious virus, robustness to wastewater, and adaptability to high throughput systems, we are confident that the PGM-MBs method can be applied for successful WBE and ultimately provides valuable public health information.Graphical abstract

Genome-scale CRISPR screen identifies TMEM41B as a multi-function host factor required for coronavirus replication

Emerging coronaviruses (CoVs) pose a severe threat to human and animal health worldwide. To identify host factors required for CoV infection, we used α-CoV transmissible gastroenteritis virus (TGEV) as a model for genome-scale CRISPR knockout (KO) screening. Transmembrane protein 41B (TMEM41B) was found to be a bona fide host factor involved in infection by CoV and three additional virus families. We found that TMEM41B is critical for the internalization and early-stage replication of TGEV. Notably, our results also showed that cells lacking TMEM41B are unable to form the double-membrane vesicles necessary for TGEV replication, indicating that TMEM41B contributes to the formation of CoV replication organelles. Lastly, our data from a mouse infection model showed that the KO of this factor can strongly inhibit viral infection and delay the progression of a CoV disease. Our study revealed that targeting TMEM41B is a highly promising approach for the development of broad-spectrum anti-viral therapeutics.

The effect of vitamin and mineral supplements on infection of pigs with porcine transmissible gastroenteritis

The article describes studies to study the effect of vitamin and mineral supplements (VMS) on the survival of piglets with porcine transmissible gastroenteritis (TGS). The studies were carried out on animals of different age groups on the basis of pig breeding complexes with 2- and 3-phase systems of keeping, in farms disadvantaged by TGS. Studies have shown that in case of TGS, the abolition of VMS in the rearing groups can contribute to both a decrease and an increase in piglet mortality. There is a direct dependence of their death on the number of viruses in the external environment. A significant decrease in the mortality of piglets after stopping the intake of vitamins in comparison with control animals, in which VMS was not canceled, was proved. Shown is the effect of VMS on the survival rate of the livestock, depending on the system of keeping animals. With a 2-phase system of keeping, when the virus is constantly circulating in the room, the abolition of VMS provides a decrease in the mortality of piglets, and with a 3-phase systemof keeping, where the virus is periodically destroyed during the sanitization of premises before the next settlement, the abolition of VMS contributes small increase in piglet death. These data indicate a positive effect of the use of vitamins in the absence of the pathogen, and in the presence of it, the use of vitamins, on the contrary, enhances the development of the disease, which is fully consistent with studies on the reproduction of the virus in cell culture, when the addition of VMS to the growth medium stimulated the reproduction of the virus.

Delta Variant of Covid-19 Study, and Why it is a Concern: An Overview

COVID-19 is caused by the virus SARS-CoV-2 that belongs to the Coronaviridae groups. The subgroups of the coronavirus families are α , β , γ , and δ coronavirus (the four general human coronaviruses). Representative coronaviruses consist of NL63 coronavirus (human) and porcine transmissible gastroenteritis from the Alphacoronavirus genus; mouse hepatitis coronavirus (MHV), bovine coronavirus (BCoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV); avian infectious bronchitis virus (IBV); and porcine δ -coronavirus (PdCoV). This work exhibits, δ -coronavirus spikes are fundamentally and evolutionally more similar related to α -coronavirus spikes than to β -coronavirus or γ -coronavirus spikes due to the receptor recognition, membrane fusion phenomenon, and immune evasion behavior.

Hypericin Inhibit Alpha-Coronavirus Replication by Targeting 3CL Protease

The porcine epidemic diarrhea virus (PEDV) is an Alphacoronavirus (α-CoV) that causes high mortality in infected piglets, resulting in serious economic losses in the farming industry. Hypericin is a dianthrone compound that has been shown as an antiviral activity on several viruses. Here, we first evaluated the antiviral effect of hypericin in PEDV and found the viral replication and egression were significantly reduced with hypericin post-treatment. As hypericin has been shown in SARS-CoV-2 that it is bound to viral 3CLpro, we thus established a molecular docking between hypericin and PEDV 3CLpro using different software and found hypericin bound to 3CLpro through two pockets. These binding pockets were further verified by another docking between hypericin and PEDV 3CLpro pocket mutants, and the fluorescence resonance energy transfer (FRET) assay confirmed that hypericin inhibits the PEDV 3CLpro activity. Moreover, the alignments of α-CoV 3CLpro sequences or crystal structure revealed that the pockets mediating hypericin and PEDV 3CLpro binding were highly conserved, especially in transmissible gastroenteritis virus (TGEV). We then validated the anti-TGEV effect of hypericin through viral replication and egression. Overall, our results push forward that hypericin was for the first time shown to have an inhibitory effect on PEDV and TGEV by targeting 3CLpro, and it deserves further attention as not only a pan-anti-α-CoV compound but potentially also as a compound of other coronaviral infections.

L-Leucine Promotes STAT1 and ISGs Expression in TGEV-Infected IPEC-J2 Cells via mTOR Activation

L-leucine (Leu), as one of the effective amino acids to activate the mTOR signaling pathway, can alleviate transmissible gastroenteritis virus (TGEV) infection. However, the underlying mechanism by which Leu alleviates the virus infection has not been fully characterized. In particular, how Leu impacts TGEV replication through mTOR signaling has yet to be elucidated. In the present study, we found that TGEV proliferated efficiently in intestinal porcine epithelial cells (IPEC-J2 cells) as evidenced by the increase in viral contents by flow cytometry, the inhibition of cell proliferation by CCK-8 assay as well as the reduction of PCNA level by western blot. Besides, western blot analysis showed that STAT1 expression was markedly reduced in TGEV-infected cells. The results of ELISA revealed the inhibition of ISGs (ISG56, MxA, and PKR) expressions by TGEV infection. TGEV-induced mTOR and its downstream p70 S6K and 4E-BP1, STAT1 and ISGs downregulation were blocked by an mTOR activator-MHY1485 but not by an mTOR inhibitor-RAPA. Concurrently, mTOR activation by MHY1485 reduced the contents of TGEV and vice versa. Furthermore, Leu reversed the inhibition of STAT1 and ISGs by activating mTOR and its downstream p70 S6K and 4E-BP1 in TEGV-infected cells. Our findings demonstrated that Leu promoted the expressions of STAT1 and ISGs via activating mTOR signaling in IPEC-J2 cells, aiming to prevent TGEV infection.

Coating with Hypertonic Saline Improves Virus Protection of Filtering Facepiece Manyfold—Benefit of Salt Impregnation in Times of Pandemic

Recently, as is evident with the COVID-19 pandemic, virus-containing aerosols can rapidly spread worldwide. As a consequence, filtering facepieces (FFP) are essential tools to protect against airborne viral particles. Incorrect donning and doffing of masks and a lack of hand-hygiene cause contagion by the wearers’ own hands. This study aimed to prove that hypertonic saline effectively reduces the infectious viral load on treated masks. Therefore, a hypertonic salt solution´s protective effect on surgical masks was investigated, specifically analyzing the infectivity of aerosolized Alphacoronavirus 1 in pigs (Transmissible Gastroenteritis Virus (TGEV)). Uncoated and hypertonic salt pre-coated FFPs were sprayed with TGEV. After drying, a defined part of the mask was rinsed with the medium, and the eluent was used for the infection of a porcine testicular cell line. Additionally, airborne microorganisms´ long-term infectivity of sodium-chloride in phosphate-buffered saline comprising 5% saccharose was investigated. In the results from an initial Median Tissue Culture Infectious Dose, infection rate of TGEV was minimally reduced by untreated FFP. In contrast, this could be reduced by a factor of 104 if FFPs were treated with hypertonic salt solutions. Airborne pathogens did not contaminate the growth medium if salt concentrations exceeded 5%. We conclude that hypertonic saline is a vital tool for anti-virus protection, exponentially improving the impact of FFPs.