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

Abstract

The human metapneumovirus (hMPV) is an important viral agent associated with severe infections of the upper and lower airways, especially in young children and immunosuppressed subjects. Nevertheless, in vitro studies of hMPV are very difficult due to the little knowledge we have on its laboratory manipulation. Objective: 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. Results: 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.

Actin cytoskeleton dynamics affect replication of Human Metapneumovirus

“…Isolation of the A2 hMPV viral strain in this study showed initial CPE signs at the third blind passage, at 14 dpi, characterized by cell rounding and refractive cells clusters, consistent with previous reports [7,17]. Conversely, other authors have described that hMPV can appear as a syncytium [5,6,18], similar to RSV, and this difference may be related to the cell lines that were infected (e.g. LLC-MK2, tMK, MNT-1, A549) or to the viral genotype [19][20][21].…”

“…The isolation of A 2 hMPV viral strain, showed the first signs of CPE in the third blind passage, at 14 d.p.i, characterized by cell rounding and formation of refractive corsages of cells, similarly to what has been previously reported (Abiko et al, 2007; Boivin et al, 2002; Chan et al, 2003; Deffrasnes et al, 2005; Kinder et al, 2020). In contrast, other authors have described that hMPV can appear as a syncytium (Bernal et al, 2019; Sato et al, 2017; Van Den Hoogen et al, 2001; Yang et al, 2016) such as RSV (member of the same family) and this difference in CPE may be related to the cell lines that were infected (e.g. LLC-MK2) or to the viral genotype (Jumat et al, 2014; Nao et al, 2019; Reina et al, 2007; Tollefson et al, 2010; Williams et al, 2005; Zhang et al, 2012b).…”

A new emerging zoonotic virus of concern: the 2019 novel Coronavirus (COVID-19)

“…At the same, as this new virus, is a concern for the world (at least it has already had a global economic impact), Latin America and even Colombia, among other regions and countries, Colombia faces an ongoing epidemic of dengue with over 130,000 cases, 1% of them severe, including more than 100 associated-deaths. When considering respiratory tract infections, multiple infectious agents are actively circulating, and will be increasing in the next few months in the region, including, those under surveillance, such as Influenza A (including AH1N1), Influenza B, human metapneumovirus, respiratory syncytial virus, adenovirus, parainfluenza 1, 2, 3 and 4, rhinovirus, bocavirus and enterovirus, among others [38][39][40] .…”