A Recombinant Porcine Reproductive and Respiratory Syndrome Virus Stably Expressing a Gaussia Luciferase for Antiviral Drug Screening Assay and Luciferase-Based Neutralization Assay.

The reverse genetics system is a valuable tool in the virological study of RNA viruses. With the availability of reverse genetics, the porcine reproductive and respiratory syndrome virus (PRRSV) has been utilized as a viral vector for the expression of foreign genes of interest. Here, we constructed a full-length cDNA clone of a highly pathogenic PRRSV (HP-PRRSV) TA-12 strain. Using this cDNA clone, we generated a reporter virus expressing a gaussia luciferase (Gluc) via an additional subgenomic RNA between ORF7 and 3'UTR. This reporter virus exhibited similar growth kinetics to the wild-type (WT) virus and remained genetically stable for at least ten passages in MARC-145 cells. In cells infected with this reporter virus, the correlation between the expression levels of Gluc in culture media and the virus titers suggested that Gluc is a good indicator of the reporter virus infection. With this reporter virus, we further established the Gluc readout-based assays for antiviral drug screening and serum neutralizing antibody detection that exhibited comparable performance to the classical assays. Taken together, we established a reverse genetics system of HP-PRRSV and generated a novel reporter virus that could serve as a valuable tool for antiviral drug screening and serum neutralizing antibody detection.

One-Step Assembly of a Porcine Epidemic Diarrhea Virus Infectious cDNA Clone by Homologous Recombination in Yeast: Rapid Manipulation of Viral Genome With CRISPR/Cas9 Gene-Editing Technology.

Porcine epidemic diarrhea virus (PEDV), a swine enteric coronavirus causing acute diarrhea in piglets, is one of the major threatens to the pork industry globally. Reverse genetics is a valuable tool for the virological study and vaccine development for coronaviruses. Due to the large size and unstable problem in Escherichia coli of coronavirus genome, construction and manipulation of reverse genetics system for coronaviruses remain laborious and time-consuming. In this study, a reverse genetics system of the genotype II PEDV strain HM was generated using the transformation-associated recombination (TAR) technology in yeast within 1 week. The rescued virus (rPEDV) exhibited similar growth properties to the wild-type virus in vitro. With this PEDV infectious cDNA clone, CRISPR/Cas9 technology and homologous recombination were combined to generate a recombinant virus rPEDV-EGFP in which the ORF3 gene was swapped with an EGFP gene. The reporter virus displayed similar growth properties to the parental virus rPEDV and remained stable during serial passage in vitro. Of note, the strategies of construction and manipulation of PEDV infectious cDNA clone are extremely simple and efficient, which could be applied for other RNA viruses and DNA viruses.

Porcine TRIM35 positively regulate TRAF3-mediated IFN-ß production and inhibit Japanese encephalitis virus replication.

Tripartite motif 35 (TRIM35) protein is a ubiquitin E3 ligase that mediates interferon-beta (IFN-ß) production via regulating ubiquitination of multiple adaptor proteins in innate immune signaling pathways. Here, we cloned the porcine TRIM35 (porTRIM35) gene and analyzed its involvement in IFN-ß expression as well as the antiviral response against Japanese encephalitis virus (JEV). The full-length porTRIM35 gene encoded a 493-amino acid protein and exhibited 79.6%-89.5% sequence similarity with its orthologues in humans, mice, monkeys and rabbits. porTRIM35 possessed typical structural features of TRIMs, including a RING domain, a B-box domain, a coiled-coil domain and a PRY/SPYR domain. Exogenous overexpression of porTRIM35 significantly up-regulated the mRNA expression level of IFN-ß in swine testicular (ST) cell in response to poly(I:C) stimulation, whereas knockdown endogenous expression of porTRIM35 lead to a decrease in the expression level of IFN-ß. Mechanically, porTRIM35 directly interacted with porcine TNF-receptor associated factor 3 (TRAF3) and catalyzed its Lys63-linked polyubiquitination, thereby leading to the up-regulation of IFN-ß production. Meanwhile, we demonstrated that the RING and PRY/SPRY domains were essential for the E3 ligase activity of porTRIM35. In response to JEV infection, the endogenous expression of porTRIM35 was markedly inhibited at the mRNA level, while exogenous expression of porTRIM35 significantly elevated the expression of IFN-ß induced by JEV infection and reduced viral titers in ST cells, suggesting that porTRIM35 is a negative regulator for JEV replication. These data demonstrate the importance of porTRIM35 in IFN-ß expression as well as the antiviral response against JEV replication.

Growth, antioxidant capacity, intestinal morphology, and metabolomic responses of juvenile Oriental river prawn (Macrobrachium nipponense) to chronic lead exposure.

Understanding the mechanisms of metal toxicity to organisms farmed for food may suggest mitigation strategies. We determined the 24-, 48-, 72-, and 96-h median lethal concentrations of lead in juvenile oriental river prawn (Macrobrachium nipponense). The prawns were then exposed to sub-lethal concentrations (13.13 and 26.26 µg/L) of lead for 60 days and growth, antioxidant enzyme activity, intestinal morphology, and metabolite profiles were assessed. Prawns exposed to 26.26 µg/L but not to 13.13 µg/L lead exhibited lower weight gain than controls. The lead burden in muscle was 0.067 and 0.25 µg/g of dry weight exposed to 13.13 and 26.26 µg/L, respectively. Levels of glutamic oxaloacetic transaminase and glutamic-pyruvic transaminase were not altered following exposure. Exposure increased malondialdehyde activity in the hepatopancreas and decreased superoxide dismutase and glutathione peroxidase activities. Catalase activity first increased and then decreased as lead concentrations increased. Some intestinal epithelial cells disassociated from the basement membrane in prawns exposed to 13.13 µg/L lead. Intestinal epithelial cells in prawns exposed to 26.26 µg/L lead separated completely from the basement membrane. Gas chromatography-mass spectrometry metabolomics assays showed the 13.13-µg/L exposure did not elicit significant metabolic alterations. Exposure to 26.26 µg/L lead differentially up-regulated 58 metabolites and down-regulated 21 metabolites. The metabolites identified were involved in galactose, purine, glutathione, and carbon metabolism, biosynthesis of amino acids and steroids, and neuroactive ligand-receptor interaction. These data indicate that chronic lead exposure can adversely affect growth, increase accumulation in muscle, impair intestinal morphology, and induce oxidant stress or neurotoxicity-related effects in M. nipponense.