A method for the rapid isolation of virus from cultured cells

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Why?

A common method of recovering large quantities of virions from infected cell cultures has been accomplished by several freeze-thaw (F-T) cycles, whereby infected cells are disrupted and intracellular virus particles are released into the culture medium (1). Using the F-T technique, virus particles suspended in culture medium are generally stored at -70°C for virus analysis or for future rounds of infection. Although the F-T method is effective for releasing virions from infected cell cultures, it is time consuming and the frequent temperature changes may affect subsequent virus titers. In addition, the resulting cell debris may contain a considerable number of virus particles that still remain attached to cell membranes.

A hypotonic environment increases the water pressure inside cells through uptake of water to maintain the ionic balance in and outside of cells. Plasma membranes of eukaryotic cells eventually lyse due to increasing cell volume, resulting in the cell contents being released into the hypotonic environment. Early work in the diagnosis of avian DNA viruses revealed that the combination of the water lysis method and the F-T method was effective in isolating large quantities of adenovirus virions from chicken embryonic kidney cells (2). In the present study, we have found water lysis without F-T cycling to be a much simpler method for obtaining infectious avian metapneumovirus [AMPV; also known as turkey rhinotracheitis virus, which is a nonsegmented, single- stranded, negative-sense RNA virus belonging to Paramyxoviridaefamily (3)].

How?

Cell Culture and Virus Infection

All cell culture reagents were purchased (Gibco; Invitrogen, Carlsbad, CA, USA). Two different immortal cell lines, avian turkey turbinate [TT-1 (4)] and mammalian African green monkey kidney cells (Vero; provided by Dr. D.A. Halvorson, University of Minnesota, St. Paul, MN, USA) were subjected to hypotonic burst and an analysis of virion recovery. Both immortal TT-1 and Vero cells were grown in high-glucose DMEM, enriched with 10% fetal calf serum (FCS), 1% penicillin-streptomycin solution, and 2 mM L-glutamine (Gibco; Invitrogen). For virus infection, both cell lines were seeded at 2 × 106 cells/10 cm culture dish. After overnight incubation, culture medium was replaced with fresh DMEM containing reduced FCS (2%) and cells were infected with stock virus strain AMPV/MN2A strain at an optimal multiplicity of infection (MOI) of 0.1. At 1 h post-infection (p.i.), medium containing virus was replaced with 10 ml fresh medium (containing 2% FCS). At 72 h p.i., the culture medium containing mature infectious virions (virus medium) was collected and stored separately on ice.

Water Lysis of Virus-infected Cultures

To perform the hypotonic burst, 5 ml of double distilled water (DW) was used to lyse the AMPV-infected cells during a 15-min incubation at room temperature. Cells were scraped and transferred to a 14 ml polystyrene tube. DW-treated cells were mixed by repeated pipeting to ensure complete cell lysis. Cell debris and nuclei were collected by centrifugation at 3000 ×g for 15 min. The resulting DW supernatant (virus DW), which contained virions that were previously trapped inside infected cells following replication, was removed by pipeting and mixed with equal volumes (5 ml) of 2× DMEM containing 4% FCS, 2% penicillin-streptomycin solution, and 4 mM L-glutamine in order to restore the tonicity to that of the original virus stock. For F-T controls (virus F-T), AMPV-infected cells were subjected to three F-T cycles, then medium containing virus was clarified by centrifugation at 3000 ×g for 15 min.

Titration of AMPV

Virus titers and the total number of virus particles for virus medium, virus DW, or virus F-T were determined by infection of Vero cells in 96-well plates at 10-fold serial dilutions. At 72 h p.i., AMPV titers were determined by plaque enumeration scoring for cytopathic effect (CPE) and by immunofluorescent assay (IFA) using hyperimmune turkey AMPV antisera as the primary antibody and fluorescein isothiocyanate-labeled goat anti-turkey IgG as the secondary antibody