Abstract
Routine methods for virus detection in clinical specimens rely on a variety of sensitive methods, such as genetic, cell culture and immuno-based assays. It is imperative that the detection assays would be reliable, reproducible, sensitive and rapid. Isolation of viruses from clinical samples is crucial for deeper virus identification and analysis. Here we introduce a rapid cell-based assay for isolation and detection of viruses. As a proof of concept several model viruses including West Nile Virus (WNV), Modified Vaccinia Ankara (MVA) and Adenovirus were chosen. Suspended Vero cells were employed to capture the viruses following specific antibody labeling which enables their detection by flow cytometry and immuno-fluorescence microscopy assays. Using flow cytometry, a dose response analysis was performed in which 3.6e4 pfu/mL and 1e6 pfu/mL of MVA and WNV could be detected within two hours, respectively. When spiked to commercial pooled human serum, detection sensitivity was slightly reduced to 3e6 pfu/mL for WNV, but remained essentially the same for MVA. In conclusion, the study demonstrates a robust and rapid methodology for virus detection using flow cytometry and fluorescence microscopy. We propose that this proof of concept may prove useful in identifying future pathogens.
Highlights
Viruses are intracellular obligate infectious parasites that rely on host machineries for replication and propagation
The procedure of cell preparation for fluorescence microscopy analysis was carried out essentially as described in Section 2.4 with the following minor changes: after virus capture by Vero cells, the cells were centrifuged at 7800× g for 5 min to remove unbound viruses, and pellets were re-suspended in 2%fetal bovine serum (FBS)-MEM and either seeded in Labtek dishes and incubated for 24 h or immediately processed for immuno-fluorescence microscopy; cells were spotted on glass slides and air dried
As an initial proof of concept, we tested this methodology for its ability to detect three different viruses: Modified Vaccinia Ankara (MVA), West Nile Virus (WNV) and Adenovirus
Summary
Viruses are intracellular obligate infectious parasites that rely on host machineries for replication and propagation. Methods for detection of viruses mainly rely on replicating viruses in cells [4,5,6] or other methodologies such as bead based capture assays [7,8,9,10,11,12,13]. Viruses 2020, 12, 1165 beads (SO-magnetic), or poly(methyl vinyl ether-maleic anhydride), are widely used for virus capture and proved to be effective in concentrating diverse viruses for downstream detection assays, such as PCR, ELISA and immunoblotting [10,11,12,13,14,15,16]. The assay is based on virus capture by suspended Vero cells instead of beads. The infectivity of the viruses retained after attachment to the cells
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