The Significance of the 98th Amino Acid in GP2a for Porcine Reproductive and Respiratory Syndrome Virus Adaptation in Marc-145 Cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the pig industry. Marc-145 cells are widely used for PRRSV isolation, vaccine production, and investigations into virus biological characteristics. Despite their significance in PRRSV research, Marc-145 cells struggle to isolate specific strains of the North American virus genotype (PRRSV-2). The involvement of viral GP2a, GP2b, and GP3 in this phenomenon has been noted. However, the vital amino acids have not yet been identified. In this study, we increased the number of blind passages and successfully isolated two strains that were previously difficult to isolate with Marc-145 cells. Both strains carried an amino acid substitution in GP2a, specifically phenylalanine to leucine at the 98th amino acid position. Through a phylogenetic and epidemiologic analysis of 32 strains, those that were not amenable to isolation widely exhibited this mutation. Then, by using the PRRSV reverse genetics system, IFA, and Western blotting, we identified the mutation that could affect the tropism of PRRSV-2 for Marc-145 cells. Furthermore, an animal experiment was conducted. Through comparisons of clinical signs, mortality rates, and viral load in the organs and sera, we found that mutation did not affect the pathogenicity of PRRSV-2. In conclusion, our study firmly establishes the 98th amino acid in GP2a as a key determinant of PRRSV-2 tropism for Marc-145 cells.

Transcriptome Analysis Revealed the Mechanism of Inhibition of Saprophytic Growth of Sparassis latifolia by Excessive Oxalic Acid.

Sparassis latifolia, a highly valued edible fungus, is a crucial medicinal and food resource owing to its rich active ingredients and pharmacological effects. Excessive oxalic acid secreted on a pine-sawdust-dominated substrate inhibits its mycelial growth, and severely restricts the wider development of its cultivation. However, the mechanism underlying the relationship between oxalic acid and slow mycelial growth remains unclear. The present study reported the transcriptome-based response of S. latifolia induced by different oxalic acid concentrations. In total, 9206 differentially expressed genes were identified through comparisons of three groups; 4587 genes were down-regulated and 5109 were up-regulated. Transcriptome analysis revealed that excessive oxalic acid mainly down-regulates the expression of genes related to carbohydrate utilization pathways, energy metabolism, amino acid metabolism, protein synthesis metabolism, glycan biosynthesis, and signal transduction pathways. Moreover, genes encoding for wood-degrading enzymes were predominantly down-regulated in the mycelia treated with excessive oxalic acid. Taken together, the study results provide a speculative mechanism underlying the inhibition of saprophytic growth by excessive oxalic acid and a foundation for further research on the growth of S. latifolia mycelia.

Excessive Oxalic Acid Secreted by Sparassis latifolia Inhibits the Growth of Mycelia during Its Saprophytic Process.

Sparassis latifolia is an edible and medicinal mushroom in Asia commercially cultivated on substrates containing pine sawdust. Its slow mycelial growth rate greatly increases the cultivation cycle. In this study, we mainly studied the role of oxalic acid (OA) secreted by S. latifolia in its saprophytic process. Our results show that crystals observed on the mycelial surface contained calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) according to X-ray diffraction (XRD). Vegetative mycelia secreted large amounts of OA during extended culture periods. However, high concentrations of OA decreased the mycelial growth rate significantly. Moreover, the degradation of lignocellulose was significantly inhibited under high concentrations of OA. These changes could be attributed to the significantly decreased activities of lignocellulose-degrading enzymes. In conclusion, by establishing a link between OA secretion by the mycelium and the slow growth rate of its saprophytic process, this work provides fundamental information for shortening the cultivation cycle of S. latifolia.

De novo transcriptome assembly and comprehensive assessment provide insight into fruiting body formation of Sparassis latifolia.

The genes associated with fruiting body formation of Sparasis latifolia are valuable for improving mushroom breeding. To investigate this process, 4.8 × 108 RNA-Seq reads were acquired from three stages: hyphal knot (SM), primordium (SP), and primordium differentiation (SPD). The de novo assembly generated a total of 48,549 unigenes, of which 71.53% (34,728) unigenes could be annotated by at least one of the KEGG (Kyoto Encyclopedia of Genes and Genomes), GO (Gene Ontology), and KOG (Eukaryotic Orthologous Group) databases. KEGG and KOG analyses respectively mapped 32,765 unigenes to 202 pathways and 19,408 unigenes to 25 categories. KEGG pathway enrichment analysis of DEGs (differentially expressed genes) indicated primordium initiation was significantly related to 66 pathways, such as "Ribosome", "metabolism of xenobiotics by cytochrome P450", and "glutathione metabolism" (among others). The MAPK and mTOR signal transduction pathways underwent significant adjustments during the SM to SP transition. Further, our research revealed the PI3K-Akt signaling pathway related to cell proliferation could play crucial functions during the development of SP and SPD. These findings provide crucial candidate genes and pathways related to primordium differentiation and development in S. latifolia, and advances our knowledge about mushroom morphogenesis.

Trehalose induced by reactive oxygen species relieved the radial growth defects of Pleurotus ostreatus under heat stress.

Trehalose is a nonreducing disaccharide, and it plays an intracellular protective role in organisms under various stress conditions. In this study, the trehalose synthesis and its protective role in Pleurotus ostreatus were investigated. As a signal in metabolic regulation, reactive oxygen species (ROS) accumulated in the mycelia of P. ostreatus under heat stress (HS). Furthermore, mycelial growth was significantly inhibited, and the malondialdehyde (MDA) level significantly increased under HS. First, exogenous addition of H2O2 inhibited mycelial growth and elevated the MDA level, while N-acetyl cysteine (NAC) and vitamin C (VC) reduced the MDA level and recovered mycelial growth under HS by scavenging ROS. These results indicated that the mycelial radial growth defect under HS might be partly caused by ROS accumulation. Second, adding NAC and VC to the media resulted in rescued trehalose accumulation, which indicated that ROS has an effect on inducing trehalose synthesis. Third, the mycelial growth was recovered by addition of trehalose to the media after HS, and the MDA level was reduced. This effect was further verified by the overexpression of genes for trehalose-6-phosphate synthase (TPS) and neutral trehalase (NTH), which led to increased and reduced trehalose content, respectively. In addition, adding validamycin A (NTH inhibitor) to the media promoted trehalose accumulation and the recovered mycelial growth after HS. In conclusion, trehalose production was partly induced by ROS accumulation in the mycelia under HS, and the accumulated trehalose could promote the recovery of growth after HS, partly by reducing the MDA level in the mycelia.