Novel interacting proteins identified by tandem affinity purification and mass spectrometry associated with IFITM3 protein during PDCoV infection.

Interferon-induced transmembrane 3 (IFITM3) is a membrane-associated protein that exhibits antiviral activities against a wide range of viruses through interactions with other cellular and viral proteins. However, knowledge of the mechanisms of IFITM3 in Porcine deltacoronavirus (PDCoV) infection has been lacking. In this study, we demonstrate that IFN-α treatment induces the upregulation of IFITM3 activity and thus attenuates PDCoV infection. PDCoV replication is inhibited in a dose-dependent manner by IFITM3 overexpression. To clarify the novel roles of IFITM3 during PDCoV infection, proteins that interact with IFITM3 were screened by TAP/MS in an ST cell line stably expressing IFITM3 via a lentivirus. We identified known and novel candidate IFITM3-binding proteins and analyzed the protein complexes using GO annotation, KEGG pathway analysis, and protein interaction network analysis. A total of 362 cellular proteins associate with IFITM3 during the first 24 h post-infection. Of these proteins, the relationship between IFITM3 and Rab9a was evaluated by immunofluorescence colocalization analysis using confocal microscopy. IFITM3 partially colocalized with Rab9a and Rab9a exhibited enhanced colocalization following PDCoV infection. We also demonstrated that IFITM3 interacts specifically with Rab9a. Our results considerably expand the protein networks of IFITM3, suggesting that IFITM3 participates in multiple cellular processes during PDCoV infection.

Bone Regeneration and Angiogenesis by Co-transplantation of Angiotensin II-Pretreated Mesenchymal Stem Cells and Endothelial Cells in Early Steroid-Induced Osteonecrosis of the Femoral Head.

Mesenchymal stem cells (MSCs) have been shown to exert a positive impact on osteonecrosis of the femoral head (ONFH) in preclinical experiments and clinical trials. After the femoral head suffers avascular necrosis, the transplanted MSCs undergo a great deal of stress-induced apoptosis and senescence in this microenvironment. So, survival and differentiation of MSCs in osteonecrotic areas are especially important in ONFH. Although MSCs and endothelial cells (ECs) co-culture enhancing proliferation and osteogenic differentiation of MSCs and form more mature vasculature in vivo, it remains unknown whether the co-culture cells are able to repair ONFH. In this study, we explored the roles and mechanisms of co-transplantation of angiotensin II (Ang II)-MSCs and ECs in repairing early ONFH. In vitro, when MSCs and ECs were co-cultured in a ratio of 5:1, both types of cells managed to proliferate and induce both osteogenesis and angiogenesis. Then, we established a rabbit model of steroid-induced ONFH and co-transplantation of Ang II-MSCs and ECs through the tunnel of core decompression. Four weeks later, histological and Western blot analyses revealed that ONFH treated with Ang II-MSCs and ECs may promote ossification and revascularization by increasing the expression of collagen type I, runt-related transcription factor 2, osteocalcin, and vascular endothelial growth factor in the femoral head. Our data suggest that co-transplantation of Ang II-MSCs and ECs was able to rescue the early steroid-induced ONFH via promoting osteogenesis and angiogenesis, which may be regarded as a novel therapy for the treatment of ONFH in a clinical setting.

miR-615 facilitates porcine epidemic diarrhea virus replication by targeting IRAK1 to inhibit type III interferon expression.

Porcine epidemic diarrhea virus (PEDV) in the Coronavirus family is a highly contagious enteric pathogen in the swine industry, which has evolved mechanisms to evade host innate immune responses. The PEDV-mediated inhibition of interferons (IFNs) has been linked to the nuclear factor-kappa B (NF-κB) pathway. MicroRNAs (miRNAs) are involved in virus-host interactions and IFN-I regulation. However, the mechanism by which the PEDV regulates IFN during PEDV infection has not yet been investigated in its natural target cells. We here report a novel mechanism of viral immune escape involving miR-615, which was screened from a high-throughput sequencing library of porcine intestinal epithelial cells (IECs) infected with PEDV. PEDV infection altered the profiles of miRNAs and the activities of several pathways involved in innate immunity. Overexpression of miR-615 increased PEDV replication, inhibited IFN expression, downregulated the NF-κB pathway, and blocked p65 nuclear translocation. In contrast, knockdown of miR-615 enhanced IFN expression, suppressed PEDV replication, and activated the NF-κB pathway. We further determined that IRAK1 is the target gene of miR-615 in IECs. Our findings show that miR-615 suppresses activation of the NF-κB pathway by suppressing the IRAK1 protein and reducing the generation of IFN-IIIs, which in turn facilitates PEDV infection in IECs. Moreover, miR-615 inhibited PEDV replication and NF-κB pathway activation in both IECs and MARC-145 cells. These findings support an important role for miR-615 in the innate immune regulation of PEDV infections and provide a novel perspective for developing new treatments.

MiR-140 inhibits classical swine fever virus replication by targeting Rab25 in swine umbilical vein endothelial cells.

Classical swine fever virus (CSFV) is one of the most important viral pathogens leading worldwide threats to pig industry. MicroRNAs (miRNAs) play important roles in regulating virus replication, but whether miRNAs affect CSFV infection is still poorly understood. In previous study, we identified four miRNAs that were down-regulated by CSFV in swine umbilical vein endothelial cells (SUVEC). In this study, miR-140, one of the most potently down-regulated genes was investigated. We found that the miRNA expression was significantly inhibited by CSFV infection. Subsequent studies revealed that miR-140 mimics significantly inhibited CSFV replication, while the inhibition of endogenous miR-140 enhanced CSFV replication. By using bioinformatics prediction, luciferase reporter system, real-time fluorescence quantitative PCR (RT-qPCR) and Western blot assays, we further demonstrated that miR-140 bind to the 3' UTR of Rab25 mRNA to regulate its expression. We also analyzed the expression pattern of Rab25 in SUVECs after CSFV infection. The results showed that CSFV infection induced Rab25 expression. Finally, Rab25 was found to promote CSFV replication. In conclusion, this study demonstrated that CSFV inhibits miR-140 expression and miR-140 inhibits replication by binding to host factor Rab25.

Antiviral activity of ISG15 against classical swine fever virus replication in porcine alveolar macrophages via inhibition of autophagy by ISGylating BECN1.

Interferons (IFNs) induce the expression of interferon-stimulated genes (ISGs) for defense against numerous viral infections, including classical swine fever virus (CSFV). However, the mechanisms underlying the effect of ISGs on CSFV infection are rarely reported. In this study, we demonstrate that IFN-α treatment induces upregulation of ISG15 and thus attenuates CSFV replication. To determine whether ISG15 is critical for controlling CSFV replication, we established porcine alveolar macrophages (PAMs) with stable overexpression or knockdown of ISG15. Overexpression of Flag-ISG15 significantly prevented CSFV replication, whereas loss of ISG15 led to abnormal proliferation of CSFV. Furthermore, upregulated ISG15 promoted beclin-1 (BECN1) ISGylation and dysfunction and subsequently inhibited autophagy, which is indispensable for CSFV replication. In addition, HECT and RLD domain containing E3 ubiquitin protein ligase 5 (HERC5), which functions to catalyze conjugation of ISG15 protein, was confirmed to interact with BECN1. Collectively, these results indicate that IFN-α restricts CSFV replication through ISG15-mediated BECN1 ISGylation and autophagy inhibition, providing insight into the mechanism of CSFV replication control by type I IFN. This mechanism may not be the only antiviral mechanism of ISG15; nonetheless, this study may contribute to the development of CSFV treatment and prevention strategies.

Antiviral Role of IFITM Proteins in Classical Swine Fever Virus Infection.

The proteins IFITM1, IFITM2, and IFITM3 are host effectors against a broad range of RNA viruses whose roles in classical swine fever virus (CSFV) infection had not yet been reported. We investigated the effect of these proteins on CSFV replication in mammalian cells. The proteins were overexpressed and silenced using lentiviruses. Confocal microscopy was used to determine the distribution of these proteins in the cells, and immunofluorescence colocalization analysis was used to evaluate the relationship between IFITMs and the CSFV endosomal pathway, including early endosomes, late endosomes, and lysosomes. IFITM1, IFITM2, or IFITM3 overexpression significantly inhibited CSFV replication, whereas protein knockdown enhanced CSFV replication. In porcine alveolar macrophages (PAMs), IFITM1 was mainly located at the cell surface, whereas IFITM2 and IFITM3 were mainly located in the cytoplasm. Following CSFV infection, the distribution of IFITM1 changed. IFITM1, IFITM2, and IFITM3 colocalization with Lamp1, IFITM2 with Rab5 and Rab7, and IFITM3 with Rab7 were observed in CSFV-infected cells. Collectively, these results provide insights into the possible mechanisms associated with the anti-CSFV action of the IFITM family.

MicroRNA-221-5p Inhibits Porcine Epidemic Diarrhea Virus Replication by Targeting Genomic Viral RNA and Activating the NF-κB Pathway.

MicroRNAs (miRNAs) are a class of noncoding RNAs involved in posttranscriptional regulation of gene expression and many critical roles in numerous biological processes. Porcine epidemic diarrhea virus (PEDV), the etiological agent of porcine epidemic diarrhea, causes substantial economic loss in the swine industry worldwide. Previous studies reported miRNA involvement in viral infection; however, their role in regulating PEDV infection remains unknown. In this study, we investigated the regulatory relationship between miRNA-221-5p and PEDV infection, finding that miR-221-5p overexpression inhibited PEDV replication in a dose-dependent manner, and that silencing endogenous miR-221-5p enhanced viral replication. Our results showed that miR-221-5p directly targets the 3' untranslated region (UTR) of PEDV genomic RNA to inhibit PEDV replication, and that miR-221-5p overexpression activates nuclear factor (NF)-κB signaling via p65 nuclear translocation, thereby upregulating interferon (IFN)-ß, IFN-stimulated gene 15, and MX1 expression during CH/HBTS/2017 infection. We subsequently identified NF-κB-inhibitor α and suppressor of cytokine signaling 1, negative regulators of the NF-κB pathway, as miR-221-5p targets. These results demonstrated the ability of miR-221-5p to inhibit PEDV replication by targeting the 3' UTR of the viral genome and activating the NF-κB-signaling pathway. Our findings will aid the development of preventive and therapeutic strategies for PEDV infection.

A novel complex valued cuckoo search algorithm.

To expand the information of nest individuals, the idea of complex-valued encoding is used in cuckoo search (PCS); the gene of individuals is denoted by plurality, so a diploid swarm is structured by a sequence plurality. The value of independent variables for objective function is determined by modules, and a sign of them is determined by angles. The position of nest is divided into two parts, namely, real part gene and imaginary gene. The updating relation of complex-valued swarm is presented. Six typical functions are tested. The results are compared with cuckoo search based on real-valued encoding; the usefulness of the proposed algorithm is verified.

Characterization of extended-spectrum ß-lactamase genes found among Escherichia coli isolates from duck and environmental samples obtained on a duck farm.

In this study, we focused on evaluating the occurrence of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in fecal samples of healthy ducks and environmental samples from a duck farm in South China. Duck cloacal swabs and pond water samples were cultivated on MacConkey agar plates supplemented with ceftiofur. Individual colonies were examined for ESBL production. Bacteria identified as E. coli were screened for the presence of ESBL and plasmid-borne AmpC genes. The genetic relatedness, plasmid replicon type, and genetic background were determined. Of 245 samples analyzed, 123 had E. coli isolates with ceftiofur MICs higher than 8 µg/ml (116 [50.4%] from 230 duck samples and 7 [46.7%] from 15 water samples). bla(CTX-M), bla(SHV-12), bla(CMY-2), and bla(DHA-1) were identified in 108, 5, 9, and 1 isolates, respectively. The most common bla(CTX-M) genes were bla(CTX-M-27) (n = 34), bla(CTX-M-55) (n = 27), bla(CTX-M-24e) (n = 22), and bla(CTX-M-105) (n = 20), followed by bla(CTX-M-14a), bla(CTX-M-14b), bla(CTX-M-24a), and bla(CTX-M-24b). Although most of the CTX-M producers had distinct pulsotypes, clonal transmission between duck and water isolates was observed. bla(CTX-M) genes were carried by transferable IncN, IncF, and untypeable plasmids. The novel CTX-M gene bla(CTX-M-105) was flanked by two hypothetical protein sequences, partial ISEcp1 upstream and truncated IS903D, iroN, orf1, and a Tn1721-like element downstream. It is suggested that the horizontal transfer of bla(CTX-M) genes mediated by mobile elements and the clonal spread of CTX-M-producing E. coli isolates contributed to the dissemination of bla(CTX-M) in the duck farm. Our findings highlight the importance of ducks for the dissemination of transferable antibiotic resistance genes into the environment.

Prevalence and characterisation of CTX-M ß-lactamases amongst Escherichia coli isolates from healthy food animals in China.

The impact of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae of food animal origins on human health has caught considerable attention worldwide. Intestinal Escherichia coli obtained from healthy food animals (pigs, cattle and poultry) in China were tested for the presence of ESBL genes. CTX-M-producing isolates were further characterised by pulsed-field gel electrophoresis (PFGE), phylogenetic grouping, genetic environment analysis, conjugation and plasmid replicon typing. A total of 127 of the 896 E. coli isolates showed reduced susceptibility to cefotaxime (minimal inhibitory concentration≥2 µg/mL). bla(CTX-M) genes were detected in 111 of the 127 isolates. The most common CTX-M types were CTX-M-14 (n=40), CTX-M-55 (n=29) and CTX-M-65 (n=22), followed by CTX-M-27, -15, -98, -24, -3, -102 and -104. CMY-2 was detected in two isolates. High clonal diversity was found amongst CTX-M-producing isolates. Insertion sequence ISEcp1 was observed 42 bp upstream of the start codon of all CTX-M-9 group genes, whereas the spacer region between the right inverted repeats and CTX-M-1 group genes varied from 45 bp to 127 bp. Most bla(CTX-M) genes were transferable by conjugation. IncFII, IncI1, IncFIB, IncN and IncA/C replicons were detected in 28, 21, 7, 5 and 1 of the 70 transconjugants carrying bla(CTX-M), respectively. This study demonstrates that commensal E. coli from healthy food animals can be important reservoirs of bla(CTX-M) genes and may contribute to the dissemination and transfer of these ß-lactamase genes throughout China.

Dissemination of the rmtB gene carried on IncF and IncN plasmids among Enterobacteriaceae in a pig farm and its environment.

OBJECTIVES: To investigate the prevalence and characterization of 16S rRNA methylase-producing bacteria in a pig farm and its environment in East China. METHODS: Enterobacteriaceae isolates and metagenomic DNA from 102 pig faecal samples from a pig farm and 97 soil samples taken in or around the farm were screened for the presence of 16S rRNA methylase genes. The clonal relationships of 16S rRNA methylase-positive isolates, plasmid content and other associated resistance genes were also characterized. RESULTS: Fifty-six rmtB-positive Enterobacteriaceae isolates, including 54 Escherichia coli, 1 Morganella morganii and 1 Proteus mirabilis, were recovered from 55 pig faecal samples. Nineteen rmtB-positive bacteria, including 13 E. coli, 2 M. morganii, 2 Leclercia adecarboxylata, 1 Enterobacter aerogenes and 1 Enterobacter cloacae, were recovered from 16 soil samples. Among the 75 rmtB-positive isolates, 31 and 25 also carried the qepA and bla(CTX-M) genes, respectively. The qepA gene co-localized with rmtB on the F2:A-:B1 plasmids and the bla(CTX-M-65) gene co-localized with rmtB on the F33:A-:B- plasmids. The rmtB gene was also found to be associated with the IncN plasmids. Clonal transmission of rmtB-positive E. coli isolates was observed between different pig groups and soil samples. CONCLUSIONS: Both horizontal gene transfer and clonal spread could be responsible for the dissemination of the rmtB gene in the pig farm and its environment. To our knowledge, this study is the first report of rmtB-positive bacteria from farmland soils and indicates that these antibiotic-resistant bacteria and/or resistance genes could be acquired by humans through the food chain.

F33:A-:B- and F2:A-:B- plasmids mediate dissemination of rmtB-blaCTX-M-9 group genes and rmtB-qepA in Enterobacteriaceae isolates from pets in China.

This study investigated the prevalence of 16S rRNA methylase genes in 267 Enterobacteriaceae isolates collected from pets. The rmtB gene was detected in 69 isolates, most of which were clonally unrelated. The coexistence of the rmtB gene with the bla(CTX-M-9) group genes and/or qepA within the same IncFII replicons was commonly detected. The two dominant types of IncF plasmids, F2:A-:B-, carrying rmtB-qepA, and F33:A-:B-, carrying the rmtB-bla(CTX-M-9) group genes (and especially bla(CTX-M-65)), shared restriction patterns within each incompatibility group.