Abstract
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.
Highlights
Lymphocytic choriomeningitis virus (LCMV), an enveloped bisegmented RNA virus and natural murine pathogen, is the prototypic member of Arenaviridae, a family that includes human pathogenic viruses such as Lassa, Junin, Machupo and Whitewater Arroyo that can cause severe viral hemorrhagic fevers in humans
In order to be useful experimentally and allow for direct comparison with previous studies, an effective LCMV viral detection method must produce a determinant of viral titers that can be directly related to PFU/ml, the standard measure for virus concentration utilized in the field for more than 50 years
Optimal incubation conditions were determined by varying the length of exposure of Vero cells to virus within the context of a 48 hour overall incubation time, which has been demonstrated in previous experiments to produce a reliable standard curve for LCMV-NP FACS analysis
Summary
Lymphocytic choriomeningitis virus (LCMV), an enveloped bisegmented RNA virus and natural murine pathogen, is the prototypic member of Arenaviridae, a family that includes human pathogenic viruses such as Lassa, Junin, Machupo and Whitewater Arroyo that can cause severe viral hemorrhagic fevers in humans. Since viral titers are determined by visually counting plaques that develop as a result of viral destruction of a cell monolayer, this method requires prolonged incubation of virus with target cells, especially for non-cytopathic viruses such as LCMV. One potential drawback of quantification of viral titers in tissues and serum by Real-time RT-PCR is that the number of viral RNA copies present cannot be directly correlated with infectious virus, in light of the well-characterized presence of defective interfering virions in LCMV infection [12,13]. We have adapted this methodology to develop a diagnostic approach that accurately quantifies titers of infectious LCMV, provides both enhanced sensitivity and expedience, and can be employed effectively as a surrogate method for analyzing infected tissue samples and viral stocks routinely measured by the plaque assay
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