ST Cells Research Articles

Efficient and Safe Editing of Porcine Endogenous Retrovirus Genomes by Multiple-Site Base-Editing Editor.

Gene-modified miniature pigs serve as alternative tissue and organ donors for xenotransplantation to alleviate the shortage of human allogenic organs. However, the high copy number of porcine endogenous retrovirus (PERV) genomes integrates with the porcine genome, which has a potential risk of cross-species transmission and hinders the clinical practice of xenotransplantation. Recently, CRISPR/Cas9 has been used to inactivate PERVs. However, Cas9 also triggers severe DNA damage at multiple integrated PERV sites in the porcine genome, which induces senescence and apoptosis of porcine cells. In this study, the cytosine base editor (CBE), an efficient and safe editor that does not cause DNA double strand breaks (DSBs), was used for PERV editing to reduce cytotoxic effects. Seven sgRNAs were set to target gag and pol loci of PERVs to induce premature stop codons. We found that approximately 10% of cell clones were completely inactivated for PERVs in pig ST cells, and the plasmid that was used for editing the PERVs did not integrate into host genome and influence the karyotype of the modified cells. Our studies offer a powerful and safe strategy for further generating PERV-knockout pigs using base editors.

Construction and characterization of the full-length cDNA of an infectious clone of emerging porcine teschovirus-2.

Porcine teschovirus (PTV) is a causative agent of polioencephalomyelitis, encephalomyelitis, reproductive disorders and gastrointestinal and respiratory diseases in swine. In the present study, the PTV2 GX/2020 strain was isolated from pig intestinal tissue through the use of ST cells. Phylogenetic analysis of VP1 nucleotide sequences indicated that the GX/2020 isolate is closely related to PTV2. Furthermore, the full-length cDNA of an infectious GX/2020 clone was constructed using seamless ligation technology. The genome sequence of the rescued virus is largely consistent with the sequence of the parental virus, and it exhibits viral growth properties. The PTV2 virus was successfully isolated in the present study, and the reverse-genetic platform provides a foundation for studies of the pathogenic mechanisms of porcine teschovirus.

Genome-wide identification of functional enhancers and their potential roles in pig breeding

BackgroundThe pig is an economically important livestock species and is a widely applied large animal model in medical research. Enhancers are critical regulatory elements that have fundamental functions in evolution, development and disease. Genome-wide quantification of functional enhancers in the pig is needed.ResultsWe performed self-transcribing active regulatory region sequencing (STARR-seq) in the porcine kidney epithelial PK15 and testicular ST cell lines, and reliably identified 2576 functional enhancers. Most of these enhancers were located in repetitive sequences and were enriched within silent and lowly expressed genes. Enhancers poorly overlapped with chromatin accessibility regions and were highly enriched in chromatin with the repressive histone modification H3K9me3, which is different from predicted pig enhancers detected using ChIP-seq for H3K27ac or/and H3K4me1 modified histones. This suggests that most pig enhancers identified with STARR-seq are endogenously repressed at the chromatin level and may function during cell type-specific development or at specific developmental stages. Additionally, the PPP3CA gene is associated with the loin muscle area trait and the QKI gene is associated with alkaline phosphatase activity that may be regulated by distal functional enhancers.ConclusionsIn summary, we generated the first functional enhancer map in PK15 and ST cells for the pig genome and highlight its potential roles in pig breeding.

The Probiotic Attributes and Anti-pseudorabies Virus Evaluation of Lactobacillus Isolates.

The emergence of pseudorabies virus (PRV) variants brings serious harm to the swine industry, and its effective treatments are limited at present. As one of the probiotics, the Lactobacillus species have beneficial characteristics of regulating the balance of intestinal flora, inhibiting the growth of pathogenic bacteria and viruses' proliferation, and improving self-immunity. In this study, Lactobacillus plantarum HN-11 and Lactobacillus casei HN-12 were selected and identified through morphology observation, Gram stain microscopy, 16S rRNA sequencing analysis, and specific amplification of the recA gene and pheS gene. All tested isolates exhibited rapid adaptation to the different conditions, excellent acid, and bile tolerance, and sensitivity to Salmonella, Staphylococcus aureus, and Escherichia coli. The antibiotic susceptibility assay displayed the isolates sensitive to most antibiotics and resistant to Lincomycin and Norfloxacin. Moreover, the supernatants of HN-11 and HN-12 inhibited PRV proliferation in ST cells. The results of animal experiments showed that supplementing the challenged mice with the supernatants of Lactobacillus isolates in advance delayed the course of the disease. PRV was detected in the heart, liver, spleen, lung, kidney, and brain tissues of dead mice in the test groups, and its copies in the lungs were significantly decreased compared with the control mice (P < 0.05). These findings proved the advantages of L. plantarum and L. casei as potential probiotic cultures, which could provide a basis for its application in microecological preparations and functional formulations.

The antagonistic effect of melatonin on TBBPA-induced apoptosis and necroptosis via PTEN/PI3K/AKT signaling pathway in swine testis cells.

Tetrabromobisphenol A (TBBPA) is a widely used industrial brominated flame retardant, which can endanger animal and human health, including cytotoxicity, endocrine disruption, reproductive toxicity and so on. Melatonin (MT) is a noteworthy free radical scavenger and an antioxidant to alleviate oxidative stress. To investigate the cytotoxic of TBBPA on swine testis cells (ST cells), as well as the antagonistic effect of MT, we established TBBPA exposure and MT antagonistic models, used flow cytometry and AO/EB staining methods to detect apoptosis and necroptosis, used DCFH-DA method to examine the content of reactive oxygen species (ROS) and investigated the expression of associated genes using RT-PCR and Western blot. According to our findings, TBBPA exposure induced cell death in ST cells. TBBPA increased ROS levels, thus increasing PTEN expression and decreasing PI3K and AKT expression. Apoptosis-related factors (Caspase-3, Bax, Cyt-c, and Caspase-9) and necroptosis-related factors (RIPK1, RIPK3, and MLKL) were considerably elevated, in addition to the reduced expression of BCL-2 and Caspase-8. We also found that MT inhibited apoptosis and necroptosis in TBBPA-induced ST cells and effectively resolved the abnormal expression of related signaling pathways. In summary, the above results indicate that MT alleviates the disorder of PTEN/PI3K/AKT signaling pathway via inhibiting ROS overproduction, thereby mitigating apoptosis and necroptosis caused by TBBPA. This research provides a theoretical basis for further understanding of the toxicity of TBBPA and the detoxification of MT against environmental toxics.

A198 COLONIC MUC2 MUCIN IS STABILIZED BY FCGBP TO RESIST SALMONELLA TYPHIMURIUM INVASION IN GOBLET CELLS

BackgroundThe colonic mucus bilayer is the first line of innate defense against pathogen invasion in the gut by forming a physical barrier between the lumen and the underlying single layer of mucosal epithelial cells. While MUC2 mucin is the primary component of the mucus layer, it also contains several mucus associated proteins of which FCGBP is of significant interest due to its structural similarities to MUC2 with unknown functions. Here we elucidated the functions of FCGBP in MUC2 mucin in response to S. typhimurium ( St) adherence and invasion in human colonic goblet cells.AimsHypothesis: FCGBP is coordinately produced with MUC2 mucin and plays an important role in innate host defense. The specific aims are:1. To determine if FCGBP alters the structural integrity of the mucus layer2. To determine the role of FCGBP in St adherence and invasion of goblet cellsMethodsTo investigate whether FCGBP impaired mucus barrier functions, wildtype LS174T (WT) MUC2 mucus-producing goblet cells and LS174T cells with a missense mutation in FCGBP (FCGBP MS) were used. To determine if FCGBP MS led to loss in barrier function, St adherence and invasion, and 0.2, 1, and 2 μM fluorescent bead penetration through the mucus monolayers were quantified. MUC2 and FCGBP mRNA and protein expression induced by St were analysed by RT-PCR and Western blotting. St-induced pro-inflammatory cytokines responses were analyzed by RT-PCR and human focus 15-plex cytokine and chemokine array. Cell killing was enumerated by LDH assay.ResultsFCGBP MS cells exhibited significant loss in MUC2 mucus barrier function as quantified by fluorescent beads penetration closer towards the epithelial cell surface temporally as compared to WT cells independent of bead size. Adherence of St was significantly increased in FCGBP MS cells and induced robust MUC2 mRNA and protein expressions in a time-dependent manner. Similarly, St elicited robust expression of pro-inflammatory cytokine mRNA and protein release in FCGBP MS as compared to WT cells. FCGBP MS were readily invaded by St that resulted in increased cell death as compared to WT cells.ConclusionsLoss of function by FCGBP MS, but not in WT cells, showed increased penetration of fluorescent beads through the mucus layer that resulted in increased St adherence, invasion, and cell death. In WT cells, FCGBP and MUC2 were coordinately upregulated in response to St. The concurrent increase in pro-inflammatory cytokine expression in FCGBP MS cells in response to St suggests that bacteria directly interacted with the cell surface indicative of an impaired mucus layer. The overall trend for increased bacterial invasion, pro-inflammatory response and cell death in FCGBP MS demonstrates that FCGBP was critical in providing structural integrity and protective functions of the mucus layer.Funding AgenciesCIHR

The Cellular Behavior, Intracellular Signaling Profile and Nuclear-Targeted Potential Functions of Porcine Growth Hormone (pGH) in Swine Testicular Cells.

Porcine growth hormone (pGH) has many important biological functions and roles, and the biological activity of pGH is closely related with its cell behavior and characteristics. However, so far, the behavior of pGH in swine testicular cell remains unclear. For this, in the current work, the swine testicular cell line (ST) was used as an in vitro model, and CLSM (Confocal laser scanning microscope), IFA (Indirect immunofluorescence assay), FCM (Flow cytometry) and WB (Western-blotting) were used to explore the pGH's cell behivior and function, and the results showed that pGH and GHR could internalize into ST cell and transported to the nucleus. Furthermore, we studied the internalization kinetics of pGH and GHR on ST cell, and found that pGH and GHR internalizes into ST cell in a time-dependent manner. More importantly, we also investigated the potential molecular functions of pGH-GHR after it entered into the cell nuclei. The results indicated that nuclear-localized GHR could participate in cell proliferation by regulating the signal intensity of STAT5. In summary, our current research shows that the nuclear-localized pGH-GHR participates in the cell proliferation of ST cell, which lays a solid foundation for further research on the regulatory effect of pGH on testicular tissue.

Progesterone differentially affects the transcriptomic profiles of cow endometrial cell types

BackgroundThe endometrium is a heterogeneous tissue composed of luminal epithelial (LE), glandular epithelial (GE), and stromal cells (ST), experiencing progesterone regulated dynamic changes during the estrous cycle. In the cow, this regulation at the transcriptomic level was only evaluated in the whole tissue. This study describes specific gene expression in the three types of cells isolated from endometrial biopsies following laser capture microdissection and the transcriptome changes induced by progesterone in GE and ST cells.ResultsEndometrial LE, GE, and ST cells show specific transcriptomic profiles. Most of the differentially expressed genes (DEGs) in response to progesterone are cell type-specific (96%). Genes involved in cell cycle and nuclear division are under-expressed in the presence of progesterone in GE, highlighting the anti-proliferative action of progesterone in epithelial cells. Elevated progesterone concentrations are also associated with the under-expression of estrogen receptor 1 (ESR1) in GE and oxytocin receptor (OXTR) in GE and ST cells. In ST cells, transcription factors such as SOX17 and FOXA2, known to regulate uterine epithelial-stromal cross-talk conveying to endometrial receptivity, are over-expressed under progesterone influence.ConclusionsThe results from this study show that progesterone regulates endometrial function in a cell type-specific way, which is independent of the expression of its main receptor PGR. These novel insights into uterine physiology present the cell compartment as the physiological unit rather than the whole tissue.

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.

In Vitro Infection Dynamics of Japanese Encephalitis Virus in Established Porcine Cell Lines.

Japanese encephalitis virus (JEV) is a zoonotic mosquito-borne pathogen that regularly causes severe neurological disease in humans in Southeast Asia and the Western Pacific region. Pigs are one of the main amplifying hosts of JEV and play a central role in the virus transmission cycle. The objective of this study was to identify in vitro cell systems to investigate early effects of JEV infection including viral replication and host cell death. Here, we demonstrate the susceptibility of several porcine cell lines to the attenuated genotype III JEV strain SA14-14-2. Monolayers of porcine nasal turbinate (PT-K75), kidney (SK-RST), testis (ST), and monocyte-derived macrophage (C2+) cells were infected with SA14-14-2 for up to five days at a multiplicity of infection (MOI) of 0.1. The hamster kidney cell line BHK-21, previously shown to be susceptible to SA14-14-2, was used as a positive control. Culture supernatants and cells were collected between 0 and 120 h post infection (hpi), and monolayers were observed for cytopathic effect (CPE) using brightfield microscopy. The number of infectious virus particles was quantified by plaque assay and cell viability was determined using trypan blue staining. An indirect immunofluorescence assay was used to detect the presence of JEV NS1 antigens in cells infected at 1 MOI. All four porcine cell lines demonstrated susceptibility to SA14-14-2 and produced infectious virus by 12 hpi. Virus titers peaked at 48 hpi in C2+, BHK-21, and SK-RST cells, at 72 hpi in PT-K75, and at 120 hpi in ST cells. CPE was visible in infected C2+ and BHK-21 cells, but not the other three cell lines. The proportion of viable cells, as measured by trypan blue exclusion, declined after 24 hpi in BHK-21 and 48 hpi in C2+ cells, but did not substantially decline in SK-RST, PT-K75 or ST cells. At 48 hpi, JEV NS1 was detected in all infected cell lines by fluorescence microscopy. These findings demonstrate several porcine cell lines which have the potential to serve as useful research tools for investigating JEV infection dynamics and host cell mechanisms in a natural amplifying host species, such as pigs, in vitro.

Antimicrobial, Cytotoxic, and α-Glucosidase Inhibitory Potentials Using the One Strain Many Compounds Technique for Red Sea Soft Corals Associated Fungi’ Secondary Metabolites and Chemical Composition Correlations

Aspergillus unguis SPMD-EGY (A. unguis) and Aspergillus sp. 2C1-EGY (Aspergillus sp.) were isolated from the soft corals Sinularia and Lobophyton sp in the Hurghada coast, Red Sea, Egypt. One strain many compounds (OSMAC) was applied to trigger fungal silent biosynthetic genes. The results revealed that the A. unguis static filtrate extracts (St F) of all tested media exhibited the highest antimicrobial activity (inhibition zones 18.5-30.5 mm) against S. aureus, P. aeruginosa, and C. albicans. The A. unguis shake cell extracts of media A and C exhibited the highest α-glucosidase inhibitory activity (Sh Cell, 78 and 82 %). A high degree of acetylcholinesterase enzyme inhibition was obtained from A. unguis (Sh F, 61-66 %) shake filtrate extracts. The A. unguis extracts (A St F and D St Cell) demonstrated the highest cytotoxic activity against cancer cells, namely, HepG2, MCF-7, and HCT-116 (%I: 62-95; IC50: 7.9-90.8 µg/mL). The A. unguis extracts (A St F&D St F) exhibited high cytotoxic activities against HCT116 cancer spheroids compared with cisplatin (42.2 % and 51 %, respectively, vs 32.6 % for cisplatin). The biological activities of the extracts were due to the presence of various secondary metabolites detected by GC/MS analysis. Further pharmacological studies may lead to the isolation of new antitumor compounds besides the prospective antimicrobial and α-glucosidase inhibition capacity of the active extracts.

The Stem-Loop I of Senecavirus A IRES Is Essential for Cap-Independent Translation Activity and Virus Recovery.

Senecavirus A (SVA) is a picornavirus that causes vesicular disease in swine and the only member of the Senecavirus genus. Like in all members of Picornaviridae, the 5′ untranslated region (5’UTR) of SVA contains an internal ribosome entry site (IRES) that initiates cap-independent translation. For example, the replacement of the IRES of foot-and-mouth disease virus (FMDV) with its relative bovine rhinitis B virus (BRBV) affects the viral translation efficiency and virulence. Structurally, the IRES from SVA resembles that of hepatitis C virus (HCV), a flavivirus. Given the roles of the IRES in cap-independent translation for picornaviruses, we sought to functionally characterize the IRES of this genus by studying chimeric viruses generated by exchanging the native SVA IRES with that of HCV either entirely or individual domains. First, the results showed that a chimeric SVA virus harboring the IRES from HCV, H-SVA, is viable and replicated normally in rodent-derived BHK-21 cells but displays replication defects in porcine-derived ST cells. In the generation of chimeric viruses in which domain-specific elements from SVA were replaced with those of HCV, we identified an essential role for the stem-loop I element for IRES activity and recombinant virus recovery. Furthermore, a series of stem-loop I mutants allowed us to functionally characterize discrete IRES regions and correlate impaired IRES activities, using reporter systems with our inability to recover recombinant viruses in two different cell types. Interestingly, mutant viruses harboring partially defective IRES were viable. However, no discernable replication differences were observed, relative to the wild-type virus, suggesting the cooperation of additional factors, such as intermolecular viral RNA interactions, act in concert in regulating IRES-dependent translation during infection. Altogether, we found that the stem-loop I of SVA is an essential element for IRES-dependent translation activity and viral replication.

Exploring Multi-Level Composition and Efficient MCM Schemes for an Energy-Efficient Wavelet Haar Architecture

This work explores multi-level fixed-point Haar transform compositions combined with MCM (multiple constant multiplication) schemes for an energy-efficient hardware architecture. We investigate a set of six lower-level Haar transforms for composing a Haar-9 architecture. The multiple-level Haar transforms use as a base M=1, M=2, and M=3 resolution levels. The processing module (PM) of the Haar explores efficient MCM schemes. The architectures were described in VHDL and synthesized employing the ST 65nm CMOS cell library. The results show that Haar-II architecture presents the lower circuit area results since this architecture requires fewer arithmetic operators. However, the most energy-efficient Haar-9 hardware architecture employs a combination of two M=2 with five M=1 blocks with a efficient MCM architecture reduced to only two arithmetic operators.

Functional study of a role of N-terminal HA stem region of swine influenza A virus in virus replication

Swine influenza A virus (SIV) is both a pathogen of economic significance to the swine industry and a potential zoonotic organism that may be transmitted to humans. We described here the detailed characterization of a role of N-terminal B-loop and CD helix of HA2 in swine influenza A virus replication. Results of our experiments demonstrated that Hemagglutinin (HA) protein of swine influenza virus could tolerate some mutations in functionally conserved B-loop and CD helix. These mutations, however, have substantially attenuated influenza virus replication in both cell lines and porcine primary tracheal epithelial cells. Significantly, we found that some B-loop or CD helix mutations generated virus mutants that replicated in MDCK and ST cell lines but failed to replicate in primary tracheal epithelial cells, thereby suggesting that swine HA protein may function as a viral virulence and pathogenesis factor. The described mutations may be further explored as attenuated vaccine candidates that can effectively prevent or eliminate the spread of influenza virus within and between swine herds.

Isolation, characterization and transcriptome analysis of porcine deltacoronavirus strain HNZK-02 from Henan Province, China

Porcine deltacoronavirus (PDCoV), an emerging porcine enteropathogenic coronavirus, causes acute watery diarrhea and vomiting in piglets. Here, we isolated a strain of PDCoV from intestinal content of a piglet with severe watery diarrhea on a farm located in Henan Province, named PDCoV strain HNZK-02. Subsequently, the complete genomes of cell-cultured PDCoV HNZK-02 passage 5 and 15 were sequenced and analyzed. There was a continuous 3-nucleotide deletion and 7 amino acid changes in S genes when compared with the other reported PDCoVs. RNA sequencing (RNA-seq)-based transcriptome analysis was used to quantitatively identify differentially expressed genes after PDCoV infection in ST cells. In total, 523 differentially expressed genes (DEGs) were identified, including 62 upregulated genes and 457 downregulated genes. The 62 upregulated genes were associated with TNF signaling pathway, cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, IL-17 signaling, chemokine signaling pathway and NF-κB signaling pathway. The significant expressing changed genes, including three antiviral genes (Mx1, OASL, OAS1) and three inflammatory chemokine related genes (CCL5, CXCL8, CXCL10) were further validated using quantitative real-time RT-PCR (qRT-PCR) assay. It showed the consistent expression patterns of the candidate genes with those from RNA-seq. Our results demonstrated that PDCoV infection activates NF-κB signaling pathway and leads to the expression of inflammatory factors, which may be related to TLRs but TLR2 is not a critical factor.In general, these results can help us to confirm the molecular regulation mechanism and also provide us a comprehensive resource of PDCoV infection.

Low‐power fast Fourier transform hardware architecture combining a split‐radix butterfly and efficient adder compressors

Fast Fourier transform (FFT) is the most common low-complexity implementation of the discrete Fourier transform, intensively employed to process real-world signals in smart sensors for the internet of things. Butterflies play a central role as the FFT computing core data path since it calculates complex terms employing several multipliers. A low-power FFT hardware architecture combining split-radix decimation-in-time butterfly and 5-2 adder compressors (ACs) is proposed and implemented. The circuits are described in Verilog hardware description language and synthesized using the Cadence Genus synthesis tool. The circuits are mapped onto a 65-nm CMOS ST standard cell library. Results reveal that the proposed FFT hardware architecture using the split-radix butterfly is 13.28% more power efficient than the radix-4 one. The results further show that, by combining 5-2 AC within the split-radix butterfly, our proposal saves up to 43.1% of the total power dissipation considering the whole FFT hardware architecture, compared with the state-of-the-art radix-4 butterfly employing the adder automatically selected by the logic synthesis tool.

Molecular Characteristics, Antigenicity, Pathogenicity, and Zoonotic Potential of a H3N2 Canine Influenza Virus Currently Circulating in South China.

Canine influenza viruses (CIVs) could be a source of influenza viruses which infect humans because canine are important companion pets. To assess the potential risk of H3N2 CIVs currently circulating in southern China to public health, biological characteristics of A/canine/Guangdong/DY1/2019 (CADY1/2019) were detected. CADY1/2019 bound to both avian-type and human-type receptors. CADY1/2019 had a similar pH value for HA protein fusion to human viruses, but its antigenicity was obviously different from those of current human H3N2 influenza viruses (IVs) or the vaccine strains recommended in the North hemisphere. CADY1/2019 effectively replicated in the respiratory tract and was transmitted by physical contact among guinea pigs. Compared to human H3N2 IV, CADY1/2019 exhibited higher replication in MDCK, A549, 3D4/21, ST, and PK15 cells. Sequence analysis indicated that CADY1/2019 is an avian-origin virus, and belongs to the novel clade and has acquired many adaptation mutations to infect other mammals, including human. Taken together, currently circulating H3N2 CIVs have a zoonotic potential, and there is a need for strengthening surveillance and monitoring of their pathogenicity.

Downregulation of miR-218 by porcine reproductive and respiratory syndrome virus facilitates viral replication via inhibition of type I interferon responses

Porcine reproductive and respiratory syndrome virus (PRRSV) is a devastating pathogen in the swine industry worldwide. miRNAs are reported to be involved in virus–host interaction. Here, we used high-throughput sequencing and miRNA inhibitors to screen possible miRNAs that can inhibit PRRSV infection on its target cell, porcine alveolar macrophages. We observed that miR-218 was downregulated upon virus infection, and knockdown of miR-218 significantly enhanced PRRSV replication. Overexpression of miR-218 resulted in a decrease in PRRSV replication, and this overexpression did not alter viral genomic RNA levels, but rather increased antiviral interferon signaling. Further analysis revealed that miR-218 regulated PRRSV replication by directly targeting porcine suppressor of cytokine signaling 3 (SOCS3), a JAK2 kinase inhibitor. Knockdown of the endogenous SOCS3 expression led to augmentation of type I interferon genes and resulted in decreased PRRSV replication, and vice versa. During PRRSV infection in vivo and in vitro, cellular miR-218 expression was downregulated and SOCS3 expression was upregulated, further supporting the inverse correlation between miR-218 and SOCS3 expression. The data on SOCS3 depletion in combination with miR-218 inhibition suggested that the antiviral activity of miR-218 required the SOCS3-mediated signaling pathway. Similarly, miR-218 negatively regulated PRRSV replication in Marc-145 cells, as well as the replication of porcine epidemic diarrhea virus and transmissible gastroenteritis virus in Vero and ST cells respectively. Taken together, these results demonstrate that PRRSV-induced miR-218 downregulation serves to inhibit the type I interferon response and may provide a novel therapeutic target for treatment of PRRSV and other viral infections.

AR regulates porcine immature Sertoli cell growth via binding to RNF4 and miR-124a.

Sertoli cells are the only somatic cells in the seminiferous epithelium which directly contact with germ cells. Sertoli cells exhibit polarized alignment at the basal membrane of seminiferous tubules to maintain the microenvironment for growth and development of germ cells, and therefore play a crucial role in spermatogenesis. Androgens exert their action through androgen receptor (AR) and AR signalling in the testis is essential for maintenance of spermatogonial numbers, blood-testis barrier integrity, completion of meiosis, adhesion of spermatids and spermiation. In the present study, we demonstrated that AR gene could promote the proliferation of immature porcine Sertoli cells (ST cells) and the cell cycle procession, and accelerate the transition from G1 phase into S phase in ST cells. Meanwhile, miR-124a could affect the proliferation and cell cycle procession of ST cells by targeting 3'-UTR of AR gene. Furthermore, AR bound to the RNF4 via AR DNA-binding domain (DBD) and we verified that RNF4 was necessary for AR to regulate the growth of ST cells. Above all, this study suggests that AR regulates ST cell growth via binding to RNF4 and miR-124a, which may help us to further understand the function of AR in spermatogenesis.

Long non-coding RNAs are associated with Seneca Valley virus infection

Sporadic outbreaks of Seneca Valley virus (SVV) have been detected in recent years causing huge economic losses to the pig industry. SVV infection can lead to redness and fever of the mouth, nose or hoof wall, ulcerative injury and inflammation in pigs. Although long non-coding RNAs (lncRNAs) have been shown to play an important role in antiviral and inflammatory regulation, how lncRNAs regulate and induce SVV infection inflammation remains unclear. Here, we found the differential expression of 1332 lncRNAs and 3299 mRNAs in SVV-infected ST cells using RNA-seq. Functional annotation analysis revealed that regulated transcripts are mainly involved in signaling pathways related to host immunity and inflammatory responses. We identified lnc-MSTRG.18940.1 as an important immune regulator in SVV infection. Lnc-MSTRG.18940.1 silencing specifically inhibited SVV replication and the production of inflammatory factors TNF-α, IL-1, IL-6, and IL-8. Our findings aid to a better understanding of host responses to SVV infection and provide new directions for understanding the potential association between lncRNAs and SVV pathogenesis.