Simultaneous expression of three reporter proteins from a porcine reproductive and respiratory syndrome virus-based vector.

The mechanism of discontinuous transcription for the synthesis of a series of sub-genomic mRNAs to express the structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV) potentially allows for the simultaneous expression of multiple foreign genes. This can occur by insertion of multiple novel independent transcription units between the ORF sequences of the PRRSV genome. Here, an expression cassette consisting of a red fluorescent protein (RFP) gene flanked at its 3' end by transcription-regulating sequences (TRS) and an expression cassette consisting of an iLOV gene flanked at its 5' end by TRS, was constructed. The resulting expression cassette containing a RFP and an iLOV gene were introduced between ORF1b and 2 as well as ORF7 and 3'UTR, respectively, in an infectious PRRSV cDNA clone. Transfection of the resulting clone (pGX-12RFP-73iLOV) into cells resulted in the recovery of a recombinant virus (rGX-12RFP-73iLOV). Simultaneous expression of RFP and iLOV was observed in MARC-145 cells infected with rGX-RFP-iLOV. To test the ability of the PRRSV genome to express all three reporter genes simultaneously, an expression cassette containing the Gluc gene and one containing the iLOV gene were also inserted in between ORF1b and 2 as well as ORF7 and 3'UTR, respectively. This was performed in a recently obtained infectious PRRSV cDNA clone carrying a RFP gene in nsp2. Transfection of the construct (pGX-R-Gluc-iLOV) carrying the three reporter genes into cells allowed the rescue of the recombinant reporter virus (rGX-R-Gluc-iLOV) which showed similar growth characteristics to the parental virus but yielded 100-fold less infectious viruses. Fluorescence microscopy of cells infected with rGX-R-Gluc-iLOV demonstrated the presence of both RFP and iLOV genes. Gluc activities in supernatants harvested at different time points from cells infected with recombinant viruses carrying Gluc showed increased levels of Gluc activity as the infection progressed. This indicated that Gluc gene as well as its activity were acceptable parameters to monitor viral propagation. Our results indicate that it is possible to introduce at least three foreign proteins simultaneously in a PRRSV-based vector and such studies will prove invaluable in our future understanding of these viruses.

miR-4299 inhibits tumor progression in pancreatic cancer through targeting ADAM17.

Pancreatic cancer (PC) is one of the most aggressive malignant tumors in human beings. Tumor capacity of evading immune-mediated lysis is a critical step in PC malignant progression. We aimed to evaluate the underlying regulatory mechanism of miR-4299 in the proliferation, metastasis, apoptosis, and immune escape in PC. miR-4299 and ADAM17 expressions in PC tissues and cell lines were detected using qRT-PCR. MTT assay and flow cytometry were used to detect cell viability and apoptosis, respectively. A luciferase reporter gene assay was conducted to confirm the targeted relationship between miR-4299 and ADAM17. Xenograft tumors in nude mice were used to detect tumorigenesis in vivo. PC cells were co-cultured with NK cells for determining the immune escape ability. NKG2D-positive rate of NK cells was detected using flow cytometry; NK cell-killing ability was detected using MTT assay. miR-4299 was downregulated in PC tissues and cell lines. miR-4299 inhibited PC cell proliferation and invasion, promoted cell apoptosis, and reduced PC tumor growth in vivo. ADAM17 3'UTR directly bound to miR-4299. ADAM17 overexpression could reverse miR-4299 effects on PC cell viability, invasion, apoptosis, and immune escape. miR-4299 exerted suppressive effects on PC cell proliferation, invasion, and immune escape via targeting ADAM17 expression. This study revealed a novel miR-4299/ADAM17 axis-modulating PC progression and proposed to concern the immune regulatory mechanism of miRNAs in PC development.

The Emergence and Pathogenesis of Recombinant Viruses Associated with NADC34-like Strains and the Predominant Circulating Strains of Porcine Reproductive and Respiratory Syndrome Virus in Southern China.

Since its recent appearance in China, the NADC30-like strains of porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) have caused an expanding epidemic, and this has further expanded the genetic diversity of PRRSV. In this study, three NADC30-like strains-GXFCG20210401, GXQZ20210403 and GXNN20210506-were isolated from pig serum samples obtained in Guangxi, and their genomes were sequenced. A comparative analysis of the whole genomes showed that the three strains were most similar to NADC30 (88.3-88.7%). In particular, the non-structural protein coding regions (nsp1, nsp4-5, nsp7-8 and nsp9) showed the highest similarities to JXA1, and the ORF2a-ORF5 regions showed the highest similarities to NADC34. The three strains had same discontinuous deletions of 111+1+19 amino acids in the nsp2 region, which were similar to the NADC30-like strains. Phylogenetic tree analysis based on the ORF5 gene showed that the three PRRSV isolates were divided into lineage 1.5 along with the representative NADC34-like strains, but they were classified as NADC30-like strains with respect to the whole genome and nsp2 evolutionary trees. Recombinant analysis revealed complex recombination patterns in the genomes of the three strains, which likely originated from multiple recombination events among JXA1-like, NADC30-like and NADC34-like strains. The results from animal experiments showed that the GXQZ20210403 strain was 20% lethal to piglets and caused more severe clinical reactions than GXFCG20210401, and both recombinant strains were similar in terms of pathogenicity to the previously reported NADC34 strains. This study demonstrates that NADC34-like strains of PRRSV have been circulating in the southern provinces of China and have exchanged genomes with several other indigenous strains. In addition, differences in recombination patterns may cause different clinical pathogenicity and indicate the importance of the surveillance and preventive control of recombinant strains.

Construction and characterization of a full-length infectious clone of Getah virus in vivo.

Getah virus (GETV) is a mosquito-borne virus of the genus Alphavirus in the family Togaviridae and, in recent years, it has caused several outbreaks in animals. The molecular basis for GETV pathogenicity is not well understood. Therefore, a reverse genetic system of GETV is needed to produce genetically modified viruses for the study of the viral replication and its pathogenic mechanism. Here, we generated a CMV-driven infectious cDNA clone based on a previously isolated GETV strain, GX201808 (pGETV-GX). Transfection of pGETV-GX into BHK-21 â€‹cells resulted in the recovery of a recombinant virus (rGETV-GX) which showed similar growth characteristics to its parental virus. Then three-day-old mice were experimentally infected with either the parental or recombinant virus. The recombinant virus showed milder pathogenicity than the parental virus in the mice. Based on the established CMV-driven cDNA clone, subgenomic promoter and two restriction enzyme sites (BamHI and EcoRI) were introduced into the region between E1 protein and 3'UTR. Then the green fluorescent protein (GFP), red fluorescent protein (RFP) and improved light-oxygen-voltage (iLOV) genes were inserted into the restriction enzyme sites. Transfection of the constructs carrying the reporter genes into BHK-21 â€‹cells proved the rescue of the recombinant reporter viruses. Taken together, the establishment of a reverse genetic system for GETV provides a valuable tool for the study of the virus life cycle, and to aid the development of genetically engineered GETVs as vectors for foreign gene expression.

Emergence and Phylogenetic Analysis of a Getah Virus Isolated in Southern China.

Getah virus (GETV) has caused many outbreaks in animals in recent years. Monitoring of the virus and its related diseases is crucial to control the transmission of the virus. In the summer of 2018, we conducted routine tests on clinical samples from different pig farms in Guangxi province, South China, and isolated and characterized a GETV strain, named GX201808. Cytopathic effects were observed in BHK-21 cells inoculated with GX201808. The expression of E2 protein of GETV could be detected in virus-infected cells by indirect immunofluorescence assays. Electron microscopic analysis showed that the virus particles were spherical and ~70 nm in diameter with featured surface fibers. The multistep growth curves showed the virus propagated well in the BHK-21 cells. Molecular genetic analysis revealed that GX201808 belongs to Group 3, represented by Kochi-01-2005 isolated in Japan in 2005, and it clustered closely with the recently reported Chinese strains isolated from pigs, cattle, and foxes. A comparison of the identities of nucleotides and amino acids in the coding regions demonstrated that the GX201808 showed the highest amino acid identity (99.6%) with the HuN1 strain, a highly pathogenic isolate resulting in an outbreak of GETV infection in swine herds in Hunan province in 2017. In the present study, GETV was identified and isolated for the first time in Guangxi province of southern China, suggesting that future surveillance of this virus should be strengthened.

Long Intergenic Nonprotein Coding RNA 0152 Promotes Hepatocellular Carcinoma Progression by Regulating Phosphatidylinositol 3-Kinase/Akt/Mammalian Target of Rapamycin Signaling Pathway through miR-139/PIK3CA.

Hepatocellular carcinoma (HCC) ranks as the fifth most common cancer worldwide, and it is the primary histologic subtype of liver cancer, with high incidence and poor prognosis. Recently, numerous long noncoding RNAs have been reported to be associated with the tumorigenesis of HCC; however, the underlying mechanisms of long intergenic nonprotein coding RNA 0152 (LINC00152) action in HCC are poorly understood. Herein, we identified a significant up-regulation of LINC00152 in both HCC tissues and cell lines. Functional studies showed that knockdown of LINC00152 inhibited cell proliferation, migration, and invasion, but promoted cell apoptosis, indicating its oncogenic functions in HCC tumorigenesis. Mechanistically, LINC00152 functioned as an efficient miR-139 sponge, thereby releasing the suppression of PIK3CA (a target gene of miR-139). Anti-miR-139 rescued the inhibition of cell proliferation, migration, and invasion induced by LINC00152 knockdown. Similarly, PIK3CA-overexpressing plasmid also reversed miR-139-mediated biological functions in HCC cells. Taken together, our study revealed a crucial regulatory network of LINC00152/miR-139/PIK3CA axis in the tumorigenesis of HCC, implying that LINC00152 may be a biomarker and novel therapeutic target for further clinical therapy of HCC.