Accelerated and Improved Quantification of Lymphocytic Choriomeningitis Virus (LCMV) Titers by Flow Cytometry

Lymphocytic choriomeningitis virus (LCMV), a natural murine pathogen, is a member of the Arenavirus family, may cause atypical meningitis in humans, and has been utilized extensively as a model pathogen for the study of virus-induced disease and immune responses. Historically, viral titers have been quantified by a standard plaque assay, but for non-cytopathic viruses including LCMV this requires lengthy incubation, so results cannot be obtained rapidly. Additionally, due to specific technical constraints of the plaque assay including the visual detection format, it has an element of subjectivity along with limited sensitivity. In this study, we describe the development of a FACS-based assay that utilizes detection of LCMV nucleoprotein (NP) expression in infected cells to determine viral titers, and that exhibits several advantages over the standard plaque assay. We show that the LCMV-NP FACS assay is an objective and reproducible detection method that requires smaller sample volumes, exhibits a ∼20-fold increase in sensitivity to and produces results three times faster than the plaque assay. Importantly, when applied to models of acute and chronic LCMV infection, the LCMV-NP FACS assay revealed the presence of infectious virus in samples that were determined to be negative by plaque assay. Therefore, this technique represents an accelerated, enhanced and objective alternative method for detection of infectious LCMV that is amenable to adaptation for other viral infections as well as high throughput diagnostic platforms

LCMV-NP FACS analysis of inactivated virus.

<p>A 1×10⁶ PFU/ml stock of LCMV Arm was inactivated either by exposure to 1 Joule UV irradiation or by incubation at 57°C for 45 minutes. 3-fold serial dilutions were produced for inactivated samples as well as untreated LCMV Arm, incubated with Vero cells for 48 hours, and the percentage LCMV-NP positive cells was determined. Virus titers are represented in PFU/ml. All experiments were performed in duplicate and curves represent compilation of data for at least 3 individual experiments.</p

Comparison of detection methods for LCMV viral titers.

1<p>The limit of detection (LOD) equals +/−3× the SD of the mean for uninfected samples.</p

Further optimization of the LCMV-NP FACS assay.

<p>(<b>A</b>) The minimum virus exposure time required to detect LCMV-NP was ascertained by incubating 3-fold serial dilutions of a 1×10⁶ PFU/ml LCMV Arm stock with Vero cells for 2, 4, 6, 8, and 24 hours and subsequent determination of LCMV-NP expression by FACS analysis. (<b>B</b>) The effect of prolonged incubation of virus with Vero cells on LCMV-NP FACS assay sensitivity was determined by extending the 48 hour incubation of 1×10⁶ PFU/ml LCMV Arm stock serial dilutions by 24 hour increments; representative curves for 24, 48 and 72 hour incubations are shown. Virus titers are represented in PFU/ml. All experiments were performed in duplicate and curves represent compilation of data for at least 3 individual experiments.</p

Hypoxia and hypoxia-inducible factors as regulators of T cell development, differentiation, and function

Oxygen is a molecule that is central to cellular respiration and viability, yet there are multiple physiologic and pathological contexts in which cells experience conditions of insufficient oxygen availability, a state known as hypoxia. Given the metabolic challenges of a low oxygen environment, hypoxia elicits a range of adaptive responses at the cellular, tissue, and systemic level to promote continued survival and function. Within this context, T lymphocytes are a highly migratory cell type of the adaptive immune system that frequently encounters a wide range of oxygen tensions in both health and disease. It is now clear that oxygen availability regulates T cell differentiation and function, a response orchestrated in large part by the hypoxia-inducible factor transcription factors. Here, we discuss the physiologic scope of hypoxia and hypoxic signaling, the contribution of these pathways in regulating T cell biology, and current gaps in our understanding. Finally, we discuss how emerging therapies that modulate the hypoxic response may offer new modalities to alter T cell function and the outcome of acute and chronic pathologies