Human Metapneumovirus: Laboratory Methods for Isolation, Propagation, and Plaque Titration

The isolation and propagation from patient-derived hMPV and the standardization of a practical, reliable, and inexpensive method of detection and quantification of hMPV were carried out, without the additional use of antibodies that had not been reported previously. These results offer some important insights for future studies of cellular and molecular biology of hMPV.

The aim of this study was to isolate and propagate hMPV from patients, and to establish a method to quantify the virus by plaque assay. Method: As part of a Latin American respiratory virus surveillance study, 12 nasal secretion samples - hMPV-positive by direct fluorescence - were inoculated on LLC-MK2 cells to isolate the virus. The supernatants were re-inoculated and the cytopathic effect and syncytium formation were evaluated daily; the infection was confirmed by immunofluorescence and RT-PCR. A protocol to titrate the harvested virus was established inoculating serial dilutions on LLC-MK2 cells, and agarose was then added as an overlay. After different time periods, the monolayers were fixed and stained with Naphthol blue/black or crystal violet and finally the viral titer was obtained.

Eight out of 12 hMPV-positive respiratory samples were positive for the isolation and confirmed by RT-PCR and immunofluorescence, but the cytopathic effect and syncytium formation were observed only in 5 cultures. One out of 8 viral isolates was used for propagation and plaque assay standardization. We found that incubation for 7 days in the semisolid overlay yielded plaques with appropriate size and shape to be counted, although crystal violet staining showed slightly larger plaques than those seen with Naphthol blue/black staining. Conclusions: The isolation and propagation from patient-derived hMPV and the standardization of a practical, reliable, and inexpensive method of detection and quantification of hMPV were carried out, without the additional use of antibodies that had not been reported previously. These results offer some important insights for future studies of cellular and molecular biology of hMPV.