ABSTRACT
Swine acute diarrhea syndrome coronavirus (SADS-CoV), a newly discovered enteric coronavirus, is the etiological agent that causes severe clinical diarrhea and intestinal pathological damage in piglets. In this study, Vero E6 and IPI-2I cells were pretreated with different concentrations of glycyrrhizin (GLY) for 2 hours, and then infected with different concentrations of SADSCoV, aiming to investigate the inhibitory effect of GLY on SADS-CoV. Western blot and TCID50 results revealed a significantly decreased N protein expression and viral titer, indicating that GLY can inhibit the infection of SADS-CoV. Vero E6 and IPI-2I cells were pretreated with different concentrations of GLY for 2 hours and infected with SADS-CoV. Western blot results showed that when the concentration of GLY was 0.8 mmol/L, the expression of N protein decreased significantly, indicating that GLY inhibited the invasion of the virus. At first, cells were treated with 0.4 mmol/L GLY, and cell samples were collected at 2 hours, 6 hours and 12 hours after being infected with SADS-CoV for analysis, and the expression of N protein were found to be significantly reduced at all points, indicating that GLY had a significant inhibitory effect on the replication of the virus. GLY is a competitive inhibitor of high mobility group box 1 (HMGB1), and the receptors of HMGB1 mainly include TLR4 and RAGE. Based on this fact, the mutant plasmid at the key sites of HMGB1 (C45S, C106S, C45/106S) and the siRNA of the RAGE receptor were transfected to Vero E6 cells and infected with SADS-CoV, and the cell supernatant and samples were harvested. The western blot and TCID50 results showed that the expression of N protein and the virus titer were decreased, suggesting that GLY exerts its function by affecting the binding of HMGB1/TLR4/RAGE during SADS-CoV infection. To further explore the signaling pathway through which GLY functions, Vero E6 and IPI-2I cells were inoculated with SADS-CoV, and cell samples were harvested, western blot was used to detect the changes of MAPK proteins. The results showed that the protein expression levels of p-p38, p-JNK and p-ERK were up-regulated in the early and late stages, indicating that the MAPK pathway was activated by SADS-CoV infection. Vero E6 and IPI-2I were pretreated with different concentrations of GLY and TLR4 inhibitor TAK for 2 hours and infected with SADS-CoV. Protein samples were harvested and analysed by western blot which showed a decreased p-JNK and N proteins, while other proteins showed no significant changes. These results indicated that GLY and TAK regulated the phosphorylation of JNK but did not regulate the phosphorylation of p38 and ERK. Also, Vero E6 cells were treated with HMGB1 antibody, the siRNA of HMGB1 and HMGB1 mutants plasmid, and infected with SADS-CoV. Protein samples were harvested, western blot results showed that phosphorylation of JNK decreased, indicating that HMGB1 affected JNK phosphorylation. Finally, Vero E6 and IPI-2I cells were pretreated with different concentrations of JNK inhibitor SP600125 to infect SADS-CoV, western blot, TCID50 and IFA results showed that the expression of N protein and virus titer, as well as virus replication were reduced, indicating that SP600125 inhibited virus replication. In conclusion, our results revealed that GLY can inhibit in vitro replication of SADS- CoV, mainly through the HMGB1/TLR4/JNK signaling pathway. The discovery of this pathway provides theoretical support for the research of novel anti-SADS-CoV drugs.
ABSTRACT
The COVID-19 pandemic caused by the SARS-CoV-2 virus has spread worldwide since the end of 2019. This virus infects humans and has also been found to infect animals. Viruses enter the human body through the ACE2 receptor on apical epithelial cells. ACE2 receptors are also present in animal epithelial cells, so the possible effects of cell damage due to SARS-CoV-2 infection in humans can also occur in animals. However, studies on the long - term effects of SARS-CoV-2 infection in animals, especially fertility, have not been widely reported. Therefore, this literature review aims to identify and analyze the long-term effects of SARS-CoV-2 virus infection on the function of the reproductive system of animals, especially protected wildlife. The data were obtained from literature published in PubMed-gov with selected keywords and several other filters activated. The study result s indicate that there is a possibility that animals infected with the SARS-CoV-2 virus could cause infertility, so the application of health protocols is important for people who are in close contact with animals, especially wild animals.
ABSTRACT
This study aimed to explore the protective mechanism of baicalein against porcine deltacoronavirus (PDCoV) infection. The targets of baicalein were obtained through Pharmamapper, Pubchem, STITCH, TCMSP and Swiss Targer Prediction databases, and the targets of PDCoV infection were obtained according to the proteomics data from our previous study. The targets of baicalein-PDCoV interaction were obtained and analyzed by STRING database and Cytoscape 3.8.2 software to construct a network diagram of "baicalein-PDCoV-targets". The CytoNCA was used to analyze network topology and core network construction. Metascape database was used for GO and KEGG analysis of core network genes. The expression levels of genes in the predicted signaling pathways were detected in vitro. A total of 268 potential targets of baicalein were screened out. There were 75 potential targets of baicalein-PDCoV infection. GO enrichment results showed that baicalein was mainly involved in the formations of membrane raft, spindle and mitochondrial membrane, cell cycle and MAPK signaling pathways. A total of 277 signaling pathways (P < 0.01) were screened out by KEGG enrichment. The PI3K-Akt, Ras and MAPK signaling pathways were the main pathways that involved in the protective effects of baicalein against PDCoV infection. The results showed that compared with the cellular control groups, the mRNA expressions of PI3K, AKT and NF-B significantly increased in the PDCoV infection group. Compared with the PDCoV group, treatment of baicalein significantly decreased the mRNA expressions of PI3K, AKT and NF-B (P < 0.05). The effect of baicalein on PDCoV infection has the characteristics of multi-targets and multi-pathways, through the intervention of AKT1, HSP90AA1, SRC, EGFR, CASP3, MAPK, STAT3 and other core genes in regulating PI3K-Akt signaling pathway, Ras signaling pathway and MAPK signaling pathway, apoptosis, and virus infection. These results suggested that baicalein could be a potential therapeutic drug against PDCoV infection for further study.
ABSTRACT
Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging zoonotic pathogen which causes high mortality rate in humans. Dromedary camels may play a central role in virus transmission to humans. Dipeptidyl peptidase-4 (DPP4), a transmembrane protein located on the cell surface of many epithelial and endothelial tissues was identified as the receptor for MERS-CoV. The current study investigated the possibility that bacterial stimulation of camel blood could affect the expression level of DPP4 on camel leukocyte subpopulation, which in turn may contribute to the higher susceptibility of camels with bacterial infection to MERS-CoV infection. DPP4 expression was evaluated by membrane immunofluorescence and flow cytometry. Stimulation of camel blood with the bacterial species S. aureus or E. coil resulted in the upregulation of DPPV on both monocytes and granulocytes, while S. agalactiae did not significantly modulate DPPV expression on either of the immune cells (p > 0.05). None of the bacterial species could induce a change in DPPV expression on lymphocytes from stimulated blood. Collectively, the present study showed an enhancing effect of bacterial stimulation on DPPV expression on camel monocytes and granulocytes.
ABSTRACT
This article describes the differences between the influenza pandemic and the Covid-19 pandemic and the immunological and virus-host cell characteristics of SARS-CoV-2.
ABSTRACT
Long noncoding RNA (lncRNA) is a type of non-coding RNA molecule longer than 200 nt, which plays vital roles in biological events. Our previous results demonstrated that the host's lncRNA expression profile was significantly changed after porcine epidemic diarrhea virus (PEDV) infection. In this study, one of the lncRNAs, lncRNA9606, was selected to investigate its impact on PEDV replication. First, the kinetics of lncRNA9606 expression in IPEC-J2 cells were examined at different time points after PEDV infection. The results confirmed that PEDV infection significantly upregulated the expression of lncRNA9606. The lncRNA9606 expression levels in different cells or tissues were evaluated and the results showed that the amount of lncRNA9606 in Peyer's patches and peripheral blood mononuclear cells were significantly higher than that in small intestinal epithelial cell lines. It was mainly localized in the nucleus. Further investigations indicated that over expression of lncRNA in LLC-PK1 cells significantly inhibited PEDV replication. In conclusion, lncRNA9606 can suppress the PEDV replication in LLC-PK1 cells.
ABSTRACT
HEK293 cells were used as the cell model to investigate the role of human aminopeptidase N (hAPN) in the invasion of porcine deltacoronavirus (PDCoV) into human cells. The proliferation of PDCoV on HEK293 cells was firstly identified by RT-qPCR/RT-PCR. And then, hAPN knockout cell line was constructed by CRISPR/Cas9 technology and cell viability of HEK293 hAPN knockout and wild-type cells was verified by CCK-8 assay. Effect of hAPN knockout and overexpression on PDCoV replication was detected by RT-qPCR and Western blot. Meanwhile, interaction of PDCoV S protein and hAPN protein was analyzed by homology modeling and molecular docking. Results showed that PDCoV virus copies rapidly increased at 12-36 h and reached peak level at 36 h, it could propagate at least for passage 2 on HEK293 cells. There was no significant difference in cell viability between hAPN knockout cells and wild-type cells. Knockout of hAPN inhibit PDCoV replication and overexpression of hAPN enhance PDCoV replication. Homology modeling and molecular docking analysis showed S1 protein could bind hAPN domain II. Residues TYR92, THR51, THR48, PHE16 and MET14of S1 protein receptor binding motif 1 (RBM1) can form hydrogen bonds with residues PHE490, GLN531, ARG528 and SER529 of hAPN. This study indicates that hAPN plays a critical role in HEK293 cells during PDCoV infection, which provides new theoretical evidence for further studies on the mechanism of PDCoV entry into host cells and cross-species transmission.
ABSTRACT
In January 2020, Guangdong Province, China imported several suspected cases with SARS-CoV-2 from Wuhan City, Hubei Province. China, which were detected as SARS-CoV-2 positive in laboratory. To further understand the SARS-CoV-2 virulence, as well as drug development and epidemic prevention and control needs, we established a SARS-CoV-2 isolation procedure. Vero-E6 cells were infected with the positive bronchoalveolar-lavage sample. The cells were monitored daily for cytopathic effects using light microscopy. The presence of viral nucleic acid in the supernatant was detected by RT-PCR. RNA extracted from culture supernatants were used as a template to clone and sequence the genome. We used Illumina sequencing to characterize the virus genome and results showed that the isolated virus was SARS-CoV-2.
ABSTRACT
As a member of the family Picornaviridae, porcine sapelovirus (PSV) is often infected with porcine epidemic diarrhea virus, teschovirus and so on. In recent years, PSV has been isolated from porcine in many provinces of China. It suggests that it is necessary to strengthen the research on PSV. In this study, according to the sequence of PSV HuN2 strain, VP1 gene was inserted into the pGEX-6 P-1 vector, and expressed the recombinant protein. BALB/c mice aged 6-8 weeks were immunized according to the standard procedure. After the third immunization, the mouse orbital blood was collected to identify the antibody level. The highly positive mouse spleen cells were selected for cell fusion. The positive hybridoma cells and two subclones were screened by IFA method, and then a PSV VP1 monoclonal antibody was obtained, named as 33-2 A. The results of IFA showed that PSV could be recognized by 33-2 A MAb, and specific green fluorescence appeared in the cytoplasm;The results of WB and IP showed that PSV infected porcine cell could specifically bind to 33-2 A, and there was a specific band at 32 ku. We also identified the B-cell antigen epitope of 33-2 A, it was at amino acids 40-46 of PSV VP1 protein, and the polypeptide sequence was 40PALTAAE46. The results showed that the monoclonal antibody can react with PSV VP1 protein. The epitope was analyzed with the PSV sequences uploaded in NCBI, 33-2 A antibody can react with most PSV strains and has a certain universal to PSV. This study laid a foundation for the study of the etiology and pathogenesis of PSV.
ABSTRACT
The pandemic of the new coronavirus infection caused by the SARS-CoV-2 virus is a challenge to the health system around the world. At the moment, we have more information about this disease, which is manifested mainly by symptoms of a respiratory infection, from mild manifestations of ARVI to severe lung damage. Also, coronavirus infection often manifests itself as symptoms of a gastrointestinal disease, in the form of vomiting, diarrhea and abdominal pain. In addition, over a year of observation, starting from the very beginning of the pandemic in China, liver dysfunctions of various origins have been described in patients with coronavirus infection. Possible reasons include the direct cytopathic effect of the virus capable of binding to ACE2beta receptors of the hepato-biliary system, immune-mediated damage to hepatocytes, including during a "cytokine storm" and hepatotoxicity of drugs used in coronavirus infection. In addition to these mechanisms of liver damage, there is also a reactivation of chronic persistent infections. In particular, we are talking about the reactivation of chronic hepatitis B. In addition to the burden of infection in the manifest period, there are no less severe consequences for convalescents or those who have suffered a mild illness, which we must remember in order to take the necessary measures in the near and distant period of recovery after COVID -19. In this article, we present our own observations of the reactivation of chronic hepatitis in 4 patients who underwent manifest COVID-19.
ABSTRACT
Winter dysentery (WD) is a highly contagious disease characterized by gastrointestinal disorders in cattle. Bovine coronavirus (BCoV) has been recognized as the etiological agent of this syndrome. In Cuba, it appeared for the first time in adult cattle in 2004, and later between January 2008 and February 2009. In 2020, diarrheal outbreaks with clinical and epidemiological characteristics similar to WD occurred in units from Mayabeque province. Of eight stool samples collected, the presence of BCoV was confirmed in seven of them by reverse transcription assays coupled to endpoint polymerase chain reaction (RT-PCR), which confirmed 87.5% positivity. Virus was isolated in cell cultures and its characteristic cytopathic effect was observed on the fifth day after inoculation. The results of the present study confirmed that BCoV is the causative agent of diarrheas in the bovine herds studied, and confirmed the epizootic mode of presentation of this disease in them.
ABSTRACT
[Objective] To explore whether porcine deltacoronavirus (PDCoV) can infect and proliferate in different animal species-derived cell lines. [Methods] The Sichuan isolate CHN-SC2015of PDCoV was inoculated in twelve cell lines derived from hamster,poultry,monkey, human and swine. After at least five blindly passages in each cell line, the virus was identified by RT-PCR,RT-q PCR, indirect immunofluorescence assay (IFA), and sequencing. [Results] PDCoV caused distinct cytopathic effect (CPE) in Vero,PAM,PK15,ST, and LLC-PK1 cells at the 1st passage (P1) and proliferated to various degrees in PAM,PK15,ST, and LLC-PK1 cells, while the CPE gradually disappeared during subsequent passages in Vero and PAM cells. Except that in the three susceptible cell lines (PK15,LLC-PK1, and ST), the viral copies of the infected cell lines gradually decreased with the increase in passages, and PDCoV could not be detected at P4 or P5 of DEF,Marc-145,HEK-293,ZYM-SIEC02, and PAM cells. PCR results showed that PDCoV could be detected only in CEF and Vero cells at P5. The IFA results showed that PDCoV could infect other cell lines except BHK-21 and ZYM-SIEC02, and specific immunofluorescence was observed in PK15,LLC-PK1, and ST cells at P1,P3, and P9. Therefore, only three cell lines (PK15,LLC-PK1, and ST) were suitable for serial passage, with the virus titers up to 107.11,107.00, and 107.37 TCID50/mL at P9,respectively. After passage in different cell lines,CHN-SC2015 accumulated 14 nucleotide mutations corresponding to 12 amino acid mutations. [Conclusion] This study indicates that PDCoV can infect a variety of cells in vitro, suggesting that it may have the potential of cross-species transmission.