Results and conclusion
The cell lines evaluated for the ability to support virus replication included rat epithelial L2 and RLE, mouse epithelial LA4, mouse macrophage RAW 267.4 and J774A.1, and primate epithelial A549, BS-C-1, and HEp-2. All were inoculated with PVM on day 0 (MOI = 0.02, 10
4 pfu per 5 × 10
6 cells). On day 7, virus titer in the culture supernatants was determined by standard plaque assay [
12]. Although pneumoviruses maintain strict host-pathogen specificity
in vivo, we determined that PVM replicated to a limited extent in vitro (< 10
3 pfu/ml supernatant) in each of the aforementioned cell lines. The mouse monocyte/macrophage RAW 264.7 cell line (established from a tumor induced by Abelson murine leukemia virus) generated the highest virus titers (10
4 pfu/ml) under culture conditions described. Cells of the RAW 264.7 line also support replication of other unrelated virus pathogens, including murine hepatitis virus and Japanese encephalitis virus [
15‐
17].
To evaluate the kinetics of virus replication and production of proinflammatory mediators in the RAW 264.7 cell line, cells at 50% confluence were inoculated with PVM (MOI 0.1) on day 0 and harvested on days 2 – 5 thereafter. RAW 264.7 is a semi-adherent cell line, and is not well-suited for plaque assays. Here, virus replication was examined qualitatively on western blot of cellular homogenates probed with PVM-specific antisera [Figure
1B]. Virus was first detected in infected cultures on day 3 post-inoculation, and then in increasing amounts through day 5. No immunoreactive PVM N protein was detected in uninfected control cultures.
Virus replication was also examined quantitatively by Q-RT-PCR using the virus SH gene as a target sequence [
13], [Figure
1C]. PVM replication was readily detected in inoculated RAW 264.7 cells, reaching ~4 × 10
5 copies per microgram total RNA on day 5 of infection. No copies of the virus SH gene were detected in uninfected cells.
RAW 264.7 cells respond to infection with PVM by producing a variety of proinflammatory mediators. Transcription of interferon-β in response to virus infection was detected by Q-RT-PCR [Figure
1D]. Cytokines MIP-2, TNF-α, MIP-1α, and MIP-1β were detected in culture supernatants by ELISA [Figure
1E]. Interestingly, MIP-1α and MIP-2 are among the most prominent mediators detected in BAL fluid of infected mice; MIP-1α levels correlate directly with the severity of pneumovirus disease in both PVM and hRSV infection [
18,
19]. In parallel to our findings, hRSV replicates in the human monocytic THP-1 cell line [
20], and several groups have provided evidence consistent with hRSV and bovine RSV (bRSV) replication in alveolar macrophages, although this point remains controversial [
21‐
25]. Furthermore, hRSV infection of the human monocytic U937 cell line was associated with production of the proinflammatory mediator, platelet-activating factor (PAF) [
26].
In summary, PVM has recently emerged as a useful novel model for the study respiratory disease in mice [
7,
27‐
30]; this has provided significant incentive toward identifying tissue culture systems for virus propagation. The mouse RAW 264.7 cell line supports efficient replication of PVM in vitro and responds to infection by augmenting production of cytokines implicated in the pathogenesis of respiratory disease. Use of this
ex vivo model of PVM infection will permit further study of biological responses associated with virus infection and the cellular and molecular level.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
KDD, IMMS, BVM and CAB performed experimental work. JBD and HFR conceived of the study, coordinated the research, and wrote and edited the manuscript. All authors read and approved the final manuscript.