Bis-Benzylisoquinoline Alkaloids Inhibit Porcine Epidemic Diarrhea Virus by Disrupting Virus Entry.

The porcine epidemic diarrhea virus (PEDV), belonging to the α-coronavirus, is the causative agent of porcine epidemic diarrhea (PED). Presently, protection from the existing PEDV vaccine is not effective. Therefore, anti-PEDV compounds should be studied. Berbamine (BBM), Fangchinoline (FAN), and (+)-Fangchinoline (+FAN), are types of bis-benzylisoquinoline alkaloids that are extracted from natural medicinal plants. These bis-benzylisoquinoline alkaloids have various biological activities, including antiviral, anticancer, and anti-inflammatory properties. In this study, we found that BBM, FAN, and +FAN suppressed PEDV activity with a 50% inhibitory concentration of 9.00 µM, 3.54 µM, and 4.68 µM, respectively. Furthermore, these alkaloids can decrease the PEDV-N protein levels and virus titers in vitro. The time-of-addition assay results showed that these alkaloids mainly inhibit PEDV entry. We also found that the inhibitory effects of BBM, FAN, and +FAN on PEDV rely on decreasing the activity of Cathepsin L (CTSL) and Cathepsin B (CTSB) by suppressing lysosome acidification. Taken together, these results indicated that BBM, FAN, and +FAN were effective anti-PEDV natural products that prevented PEDV entry and may be considered novel antiviral drugs.

Development and application of a TaqMan-based real-time PCR method for the detection of the ASFV MGF505-7R gene.

African swine fever virus (ASFV), the etiological agent of African swine fever (ASF), causes deadly hemorrhagic fever in domestic pigs. ASF's high mortality and morbidity have had disastrous effects on the world's swine industry. In recent years, the number of African swine virus strains has increased and presented new challenges for detecting classical ASFV-p72-based viruses. In this study, we observed that the ASFV MGF505-7R gene, a member of the multigene family that can enhance ASFV virulence and pathogenesis, has the potential to be a candidate for vaccine formulations. We also developed a real-time PCR assay based on the ASFV MGF505-7R gene and validated it in multiple aspects. The results indicated that the approach could detect standard plasmids with a sensitivity and a specificity of up to 1 × 101 copies/µL. Moreover, the assay had no cross-reactions with other porcine viruses. In laboratory and clinical settings, the assay can detect ASFV-infected samples at an early stage (4 hpi) and show a consistency of 92.56% when compared with classical ASFV detection in clinically ASFV-infected materials. This study's results also indicated that the TaqMan-based quantitative real-time PCR assay we developed for detecting the ASFV MGF505-7R gene is both sensitive and specific. This assay can provide a quick and accurate method for detecting ASFV and has the potential to be used as an optional tool for screening and monitoring ASF outbreaks.

Genetic analysis of genome sequence characteristics of two lumpy skin disease viruses isolated from China.

BACKGROUND: Lumpy skin disease (LSD) is an acute or subacute infectious disease caused by lumpy skin disease virus (LSDV) of genus Capripoxvirus. The outbreaks of LSD were confirmed in the Yili area of the Xinjiang autonomous region in August 2019 and the Fujian province in June 2020. We detected LSDV in our daily monitoring work, then isolated, identified and sequenced the virus, and analyzed the whole genome characteristics of the isolated strain. RESULTS: Whole genome sequencing revealed that the strains isolated were all LSDV and were named as LSDV XJ201901 and LSDV FJ2019. The results showed that the identity based on whole genome sequences between LSDV XJ201901 and LSDV FJ2019 was 100% and the identity based on whole genome sequences between the two isolated strains and the global LSDV strains was 97.28%-99.99%, with the strain LSDV72/PrachuapKhiriKhan/Thailand/2021 (99.99%) having the highest sequence identity. Analysis of potential recombination events revealed that a total of 18 potential recombination events were identified in strains LSDV XJ201901 and LSDV FJ2019. The two strains are a recombination of Neethling vaccine LW 1959 (GeneBank: AF409138.1) with KSGP 0240 (GeneBank: KX683219.1). It was observed that Neethling vaccine LW 1959 (11/18) and KSGP 0240 (10/18) are involved in most of the potential recombination events. CONCLUSIONS: The virus isolate in this study was LSDV and was identified as a vaccine recombinant strain. The most likely potential parent strains of the two strains in this study are Neethling vaccine LW 1959 and KSGP 0240. The strains in this study are very similar to those isolated in East and Southeast Asia since 2019.

Quantitative real-time PCR detection and analysis of a lumpy skin disease outbreak in Inner Mongolia Autonomous Region, China.

Lumpy skin disease (LSD) is a severe disease of bovine characterized by nodules on the skin, mucous membranes, and profuse nasal discharge which causes severe economic losses. In October 2020, an LSD outbreak case was found in Inner Mongolia Autonomous Region, China. A total of 1,206 cattle were sold from the same imported animal quarantine field to 36 farms after the quarantine period finished, and over 30 farmers reported symptoms such as skin scabs found in newly arrived cattle shortly after that. A large-scale LSD outbreak investigation was launched after laboratory diagnosis confirmed LSD. The clinical samples of 1,206 cattle from 36 farms, including 1,206 whole blood, 1,206 oral and nose swabs, and 355 scabs, were collected for the qRT-PCR test. The result showed that 51 whole blood samples (4.23%), 580 swab samples (48.09%), and 350 skin scabs (98.59%) were lumpy skin disease virus (LSDV) positive, 33 of 36 farms were affected. This study aims to provide a basis for LSD epidemiological traceability, movement control, and measures for prevention and control.

Effects of porcine STC-1 on cell metabolism and mitochondrial function.

Stanniocalcin (STC-1), a kind of glycoprotein hormone, was first found in fish and mainly regulates calcium/phosphorus metabolism in the body. To explore the biological function of the porcine STC-1 gene, the effects of changes in stanniocalcin expression on cellular metabolism and mitochondrial function were studied. A vector overexpressing the STC-1 gene and an siRNA silencer of the STC-1 gene were transfected into porcine kidney epithelial PK15 cells. After the STC-1 gene expression level was induced to change, STC-1 protein- and mitochondrial function-related proteins such as PMP70, OPA, DRP, Mfn and STC-1-related acetylated protein were detected by Western blotting. Cell apoptosis, mitochondrial membrane potential, reactive oxygen species (ROS), and ATP were detected using flow cytometry methods. Transmission electron microscopy was used to observe the changes in mitochondrial structure and morphology. The results showed that overexpression of the STC-1 gene could significantly upregulate the levels of PMP70, OPA, DRP and Mfn. STC-1 gene expression, which could decrease the apoptosis rate and reactive oxygen species production to significantly increase the cell membrane potential and reduce the formation of intracellular ATP, which also affected the morphology and number of mitochondria. The results were reversed when the STC-1 gene expression was silenced. The results suggested that the porcine STC-1 gene is closely related to cell growth metabolism and mitochondrial function, which influence the mitochondrial function-related proteins. The present study is useful for further understanding STC-1 gene function and provides a theoretical basis for improving the production characteristics of domestic pigs.