The mechanistic target of rapamycin complex 1 (mTORC1) couples nutrient sufficiency to cell growth. mTORC1 is activated by exogenously acquired amino acids sensed through the GATOR–Rag guanosine triphosphatase (GTPase) pathway, or by amino acids derived through lysosomal degradation of protein by a poorly defined mechanism. Here, we revealed that amino acids derived from the degradation of protein (acquired through oncogenic Ras-driven macropinocytosis) activate mTORC1 by a Rag GTPase–independent mechanism. mTORC1 stimulation through this pathway required the HOPS complex and was negatively regulated by activation of the GATOR-Rag GTPase pathway. Therefore, distinct but functionally coordinated pathways control mTORC1 activity on late endocytic organelles in response to distinct sources of amino acids.

Piscine reovirus (PRV) is the causative agent of heart and skeletal muscle inflammation (HSMI), which is detrimental to Atlantic Salmon (AS) aquaculture, but so far has not been cultivatable, which impedes studying the disease and developing a vaccine. Homogenates of head kidney and red blood cells (RBC) from AS in which PRV-1 had been detected were applied to fish cell lines. The cell lines were from embryos, and from brain, blood, fin, gill, gonads, gut, heart, kidney, liver, skin, and spleen, and had the shapes of endothelial, epithelial, fibroblast, and macrophage cells. Most cell lines were derived from the Neopterygii subclass of fish, but one was from subclass Chondrostei. Cultures were examined by phase contrast microscopy for appearance, and by quantitative polymerase chain reaction (qPCR) for PRV-1 RNA amplification and for the capacity to transfer any changes to new cultures. No changes in appearance and Ct values were observed consistently or transferable to new cultures. Therefore, 31 cell lines examined were unable to support PRV-1 amplification and are described as belonging to the non-supportive PRV-1 invitrome. However, these investigations and cell lines can contribute to understanding PRV-1 cellular and host tropism, and the interactions between virus-infected and bystander cells.

Vacuolization is an often observed characteristic of virus infection on cell lines during cell based virus detection and isolation protocols and is commonly considered as a cytopathic effect (CPE) resulting from virus infection. However, caution should be exercised when vacuolization is used to indicate CPE as in addition to being induced by viruses, vacuolization in cells can occur spontaneously, as a response to cellular damage, during natural sequestration of materials and fluids, and fusion of many smaller vesicles formed by cellular processes such as autophagy and material transport. In vitro, additional vacuolization inducing factors during virus isolation may include use of antibiotic and antifungal, tissue preserving agents (RNA Later), temperature, and medium levels. Therefore, the goal of this research is to examine the capacity of these factors to induce cellular vacuolization during fish virus isolation procedures. Four cell lines used were EPC, CHSE-214 and two Atlantic salmon cell lines Asimf20 (Atlantic Salmon Intestinal Myofibroblast) and ASP309 (Atlantic Salmon Pituitary). Amphotericin B at concentrations between 2.5 to 10 μg/mL induced vacuoles in all cell lines except EPC. RNA Later at dilutions of 0.01 to 0.001 (v/w) induced vacuoles in all cell lines. Vacuoles were more likely to appear in cell lines when incubated at lower temperature (4 o C) and lower infection volumes. Additionally, the effects of vacuolization inducing agents on fish virus stability and replication was examined with two fish viruses, viral hemorrhagic septicemia virus (VHSV) and Chum salmon reovirus (CSV). RNA Later reduced the titre of enveloped VHSV by one log while having no observable effect on non-enveloped CSV. Due to the multifactorial induction of vacuoles in cells, care must be taken during virus isolation procedures to control or reduce alternative factors that may be causing vacuoles formation in cells to prevent misinterpretation of results.