Identification of African Swine Fever Virus Inhibitors through High Performance Virtual Screening Using Machine Learning

African swine fever virus (ASFV) is a highly contagious virus that causes severe hemorrhagic viral disease resulting in high mortality in domestic and wild pigs, until few antiviral agents can inhibit ASFV infections. Thus, new anti-ASFV drugs need to be urgently identified. Recently, we identified pentagastrin as a potential antiviral drug against ASFVs using molecular docking and machine learning models. However, the scoring functions are easily influenced by properties of protein pockets, resulting in a scoring bias. Here, we employed the 5′-P binding pocket of AsfvPolX as a potential binding site to identify antiviral drugs and classified 13 AsfvPolX structures into three classes based on pocket parameters calculated by the SiteMap module. We then applied principal component analysis to eliminate this scoring bias, which was effective in making the SP Glide score more balanced between 13 AsfvPolX structures in the dataset. As a result, we identified cangrelor and fostamatinib as potential antiviral drugs against ASFVs. Furthermore, the classification of the pocket properties of AsfvPolX protein can provide an alternative approach to identify novel antiviral drugs by optimizing the scoring function of the docking programs. Here, we report a machine learning-based novel approach to generate high binding affinity compounds that are individually matched to the available classification of the pocket properties of AsfvPolX protein

Generation of a persistently infected MDBK cell line with natural bovine spongiform encephalopathy (BSE).

Bovine spongiform encephalopathy (BSE) is a zoonotic transmissible spongiform encephalopathy (TSE) thought to be caused by the same prion strain as variant Creutzfeldt-Jakob disease (vCJD). Unlike scrapie and chronic wasting disease there is no cell culture model allowing the replication of proteinase K resistant BSE (PrPBSE) and the further in vitro study of this disease. We have generated a cell line based on the Madin-Darby Bovine Kidney (MDBK) cell line over-expressing the bovine prion protein. After exposure to naturally BSE-infected bovine brain homogenate this cell line has shown to replicate and accumulate PrPBSE and maintain infection up to passage 83 after initial challenge. Collectively, we demonstrate, for the first time, that the BSE agent can infect cell lines over-expressing the bovine prion protein similar to other prion diseases. These BSE infected cells will provide a useful tool to facilitate the study of potential therapeutic agents and the diagnosis of BSE

Evaluation of Antiviral Effect against SARS-CoV-2 Propagation by Crude Polysaccharides from Seaweed and Abalone Viscera In Vitro

Crude polysaccharides, extracted from two seaweed species (Hizikia fusiforme and Sargassum horneri) and Haliotis discus hannai (abalone) viscera, were evaluated for their inhibitory effect against SARS-CoV-2 propagation. Plaque titration revealed that these crude polysaccharides efficiently inhibited SARS-CoV-2 propagation with IC50 values ranging from 0.35 to 4.37 μg/mL. The crude polysaccharide of H. fusiforme showed the strongest antiviral effect, with IC50 of 0.35 μg/mL, followed by S. horneri and abalone viscera with IC50 of 0.56 and 4.37 μg/mL, respectively. In addition, immunofluorescence assay, western blot, and quantitative RT-PCR analysis verified that these polysaccharides could inhibit SARS-CoV-2 replication. In Vero E6 cells, treatment with these crude polysaccharides before or after viral infection strongly inhibited the expression level of SARS-CoV-2 spikes, nucleocapsid proteins, and RNA copies of RNA-dependent RNA-polymerase and nucleocapsid. These results show that these crude marine polysaccharides effectively inhibit SARS-CoV-2 propagation by interference with viral entry

Evidence of Exposure to SARS-CoV-2 in Dogs and Cats from Households and Animal Shelters in Korea

The COVID-19 pandemic was caused by the zoonotic SARS-CoV-2. A variety of animals involved in human life worldwide have been investigated for infection. As the degree of infection increased, extensive monitoring in animals became necessary to determine the degree of infection in animals. The study was conducted on a sample of dogs and cats, which were randomly sampled according to the number of confirmed cases in the region. Animals from both COVID-19-confirmed households and generally disease-negative families and animal shelters were included. Tests included real-time qPCR tests for SARS-CoV-2 antigens, ELISA for antibodies, and plaque reduction neutralization tests (PRNT) for neutralizing antibodies. As a result, SARS-CoV-2 viral RNA was detected in 2 cats out of 1018 pets (672 dogs and 346 cats). A total of 16 dogs (2.38%) and 18 cats (5.20%) tested positive using ELISA, and 14 dogs (2.08%) and 17 cats (4.91%) tested positive using PRNT. Antigens of- and/or antibodies to SARS-CoV-2 were detected in the animals regardless of whether the companion family was infected; this was the case even in animal shelters, which have been regarded as relatively safe from transmission. In conclusion, continuous viral circulation between humans and animals is inevitable; therefore, continuous monitoring in animals is required

Immunofluorescence and Immunoblotting detection of bovine cellular prion protein (PrPC) in transduced/non-transduced Madin-Darby bovine kidney (MDBK; ATCC CCL-22) with infectious recombinant lentivirus.

<p>Detection of cell surface PrP^C in non-transduced MDBK (A), transduced MDBK with only pLEX vector (B) and MDBK C1–2F with bovine PRNP inserted into pLEX vector (C) were assayed by immunofluorescence using 4% paraformaldehyde fixation and anti-PrP mAb 6H4. (D) This photo shows an immunoblot of non-transduced MDBK (lane 1) and transduced MDBKs with infectious recombinant virus which had pLEX vector without and with bovine PRNP (lane 2, 3). Housekeeping protein, GAPDH (glyceraldehydes-3-phosphate dehydrogenase) was used as a control of comparative protein concentration. The positions of molecular marker proteins (M) are presented in kilodaltons.</p

Comparison of the mobilities of PK-resistant PrP derived from BSE-infected bovine brain (original cell inoculate) and M2B cell.

<p>Mobility phases of nonglycosylated PrP^BSE from the non-infected bovine brain (lane 1), BSE-infected bovine brain (lane 2), non-infected transduced MDBK (MDBK C1–2F, passage (p) 7: lane 3), and persistently PK-resistant PrP^BSE infected M2B cells (p17: lane 4; p48: lane 5 and p70: lane 6) in Western blotting after sequential treatments with PK and PNGase F. Molecular mass marker (M) in kilodaltons (kDa) is shown on the left.</p