Efficient replication of pneumonia virus of mice (PVM) in a mouse macrophage cell line

Abstract

Pneumonia virus of mice (PVM; family Paramyxoviridae, subfamily Pneumovirinae) is a natural respiratory pathogen of rodent species and an important new model for the study of severe viral bronchiolitis and pneumonia. However, despite high virus titers typically detected in infected mouse lung tissue in vivo, cell lines used routinely for virus propagation in vitro are not highly susceptible to PVM infection. We have evaluated several rodent and primate cell lines for susceptibility to PVM infection, and detected highest virus titers from infection of the mouse monocyte-macrophage RAW 264.7 cell line. Additionally, virus replication in RAW 264.7 cells induces the synthesis and secretion of proinflammatory cytokines relevant to respiratory virus disease, including tumor necrosis factor-α (TNF-α), interferon-β (IFN-β), macrophage inflammatory proteins 1α and 1β (MIP-1α and MIP-1β) and the functional homolog of human IL-8, mouse macrophage inflammatory peptide-2 (MIP-2). Identification and characterization of a rodent cell line that supports the replication of PVM and induces the synthesis of disease-related proinflammatory mediators will facilitate studies of molecular mechanisms of viral pathogenesis that will complement and expand on findings from mouse model systems.

Priming of the Respiratory Tract with ImmunobioticLactobacillus plantarumLimits Infection of Alveolar Macrophages with Recombinant Pneumonia Virus of Mice (rK2-PVM)

“…In previous studies, we demonstrated that PVM replicates in mouse macrophage cell lines and also in primary peritoneal macrophages isolated from mice and challenged ex vivo (50,51). However, we demonstrate here for the first time that PVM infects both CD11c ϩ MHC-II ϩ DCs and CD11c ϩ Siglec-F ϩ AMs in vivo and that AMs support both active replication and production of infectious virions.…”

“…Equal numbers of cells from all of the BAL fluid samples were plated. After 2 h, the nonadherent cells were removed and the adherent macrophages remaining were cultured for an additional 48 h. After 48 h, the culture supernatant was collected and dialyzed against 10 6 volumes of complete RPMI with 2% instead of 10% FBS across a 50-kDa membrane to remove low-molecular-weight cytokines prior to evaluation in a TCFD 50 assay as described in the next section. In some experiments, airway macrophages were isolated from mice that were control or L. plantarum primed as described above but not virus infected.…”

“…Although rK2-PVM is significantly (ϳ300 times) less virulent than parent PVM strain J3666 (Fig. 2C), fluorescence units (TCFD 50 ) correlated directly with the standard measure of absolute virus copy number (absolute number of copies of virus SH per absolute copy of virus glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) over a 10 4 range of virus concentrations (replication and clearance, days 3 to 11; Fig. 2D).…”

“…While the mechanisms underlying the observed antiviral response remain to be fully elucidated, they may involve Lactobacillus-mediated induction of antiviral cytokines in target AMs, notably, type I IFNs, which may have an impact on target cell susceptibility and/or virus clearance. 50 /ml identified from supernatants of cultures in panels E and F. n ϭ 3 to 5 cultures from individual mice; P Ͻ 0.01. All P values were determined by Mann-Whitney U test.…”

Pneumoviruses infect eosinophils and elicit MyD88-dependent release of chemoattractant cytokines and interleukin-6

“…Among these findings, several groups have detected immunoreactive eosinophil degranulation products in bronchial washings of patients with the most severe forms of RSV infection, 11,12 and we and others have reported direct recruitment of eosinophils to the lungs of PVM-infected mice. [13][14][15] RSV and PVM infections of their respective target cells result in the production of numerous proinflammatory cytokines, including the eosinophil chemoattractants RANTES and macrophage inflammatory protein-1␣/CCL3, [16][17][18][19] and RSV-infected target cells can activate eosinophils directly by virus-induced expression of ICAM-1. 20 Most intriguing, children who have recovered from severe RSV infection frequently go on to develop prolonged postinfectious wheezing 21 ; likewise, mice that have recovered from acute PVM infection experience persistent respiratory dysfunction with features similar to those described in mouse allergic asthma models.…”