Studies of culture conditions and environmental stability of human metapneumovirus

Abstract

Human metapneumovirus (HMPV) is a paramyxovirus that is a leading cause of acute respiratory disease. HMPV is difficult to cultivate and limited published data describe the in vitro growth characteristics of the virus and its ability to replicate in different cell lines. Stability of HMPV to different temperatures or environmental conditions has not been described. Nosocomial infections due to HMPV have been reported, and thus the survival of infectious particles on environmental surfaces is important. We tested multiple cell lines for the ability to support HMPV replication both in the presence and absence of exogenous trypsin. The most permissive monkey kidney epithelial cells were LLC-MK2 and Vero, while the most permissive human airway epithelial cell line was BEAS-2B. LLC-MK2 cells were tolerant of trypsin and thus remain an ideal cell line for HMPV cultivation. Spinoculation significantly increased the infectivity of HMPV for cells in monolayer culture. Infectious virus was very stable to repeat freeze-thaw cycles, ambient room temperature, or 4 degrees C, while incubation at 37 degrees C led to degradation of virus titer. Finally, nonporous materials such as metal or plastic retained infectious virus for prolonged periods, while virus deposited on tissue and fabric rapidly lost infectivity. These findings provide guidance for laboratories attempting to culture HMPV and relevant information for infection control policies.

Figure 1

Replication of HMPV in different cell lines in the presence and absence of trypsin. A. Titer of HMPV grown in the indicated lines was calculated by plaque assay on the same cell lines, using medium supplemented with 5 µg/ml of trypsin. Data from three experiments performed in triplicate; error bars represent the SEM. B. Titer of HMPV grown in the indicated lines using medium supplemented with trypsin ranging from 0 to 5 µg/ml. Experiment performed in triplicate; error bars represent the SD. LOD = limit of detection.

Figure 2

Kinetics of HMPV replication at different MOI. LLC-MK2 cell monolayers were inoculated with the indicated MOI and harvested for plaque titration at the indicated time points. A. Titer of HMPV in the cell fraction. B. Titer of HMPV in the supernatant. $ = p<0.05, * = p<0.005 compared to MOI = 1, Student’s t test. Experiment performed in triplicate; error bars represent the SD.

Figure 3

Effects of different virus adsorption methods on the efficiency of HMPV infection. BEAS-2B cells were infected with HMPV at a MOI of 0.31, 0.62, or 1.24 pfu/cell by standard or spin inoculation. At 24 h post-infection, infected cells were identified by indirect immunostaining using flow cytometry. Values represent the mean percentage of infected cells for three independent experiments performed in triplicate; error bars represent the SEM.

Figure 4

Stability of HMPV under varying environmental conditions. A. Viral aliquots were incubated at room temperature, 4°C, or 37°C and harvested for plaque titration at the indicated time points. * = p<0.05 compared to 4°C, student’s t test. Data from three experiments performed in triplicate; error bars represent the SEM. B. Aliquots of virus were adsorbed to material surfaces, incubated and recovered for plaque titration at the indicated time points. Experiment performed in triplicate; error bars represent the SD. LOD = limit of detection.