Tobacco mosaic virus in the lungs of mice following intra-tracheal inoculation

Abstract

Plant viruses are generally considered incapable of infecting vertebrates. Accordingly, they are not considered harmful for humans. However, a few studies questioned the certainty of this paradigm. Tobacco mosaic virus (TMV) RNA has been detected in human samples and TMV RNA translation has been described in animal cells. We sought to determine if TMV is detectable, persists, and remains viable in the lung tissues of mice following intratracheal inoculation, and we attempted to inoculate mouse macrophages with TMV. In the animal model, mice were intratracheally inoculated with 10(11) viral particles and were sacrificed at different time points. The virus was detected in the mouse lungs using immunohistochemistry, electron microscopy, real-time RT-PCR and sequencing, and its viability was studied with an infectivity assay on plants. In the cellular model, the culture medium of murine bone marrow derived macrophages (BMDM) was inoculated with different concentrations of TMV, and the virus was detected with real-time RT-PCR and immunofluorescence. In addition, anti-TMV antibodies were detected in mouse sera with ELISA. We showed that infectious TMV could enter and persist in mouse lungs via the intratracheal route. Over 14 days, the TMV RNA level decreased by 5 log(10) copies/ml in the mouse lungs and by 3.5 log(10) in macrophages recovered from bronchoalveolar lavage. TMV was localized to lung tissue, and its infectivity was observed on plants until 3 days after inoculation. In addition, anti-TMV antibody seroconversions were observed in the sera from mice 7 days after inoculation. In the cellular model, we observed that TMV persisted over 15 days after inoculation and it was visualized in the cytoplasm of the BMDM. This work shows that a plant virus, Tobacco mosaic virus, could persist and enter in cells in mammals, which raises questions about the potential interactions between TMV and human hosts.

Figure 1. Lung sections from water-inoculated mouse (A and C), and TMV-inoculated mouse at day 3 (B and D) after intratracheal inoculation.

Note the absence of inflammation in the lungs of control mice, whereas inflammatory infiltrates in interalveolar walls composed in part by macrophages were observed in lungs from TMV-inoculated mice. Hematoxylin-eosin staining was used. Magnification, 200X (A and B) and 400X (C and D). Arrows indicate the inter-alveolar walls inflammation.

Figure 2. Detection of TMV antigen by immunohistochemistry in lungs of TMV-inoculated mice.

No immunodetection was observed in lung from a water-inoculated mouse (A), whereas cytoplasmically immunopositive cells located in inflammatory infiltrates present in interalveolar walls, most likely macrophages, were detected at day 1 (B) and day 3 (C) after intratracheal inoculation. Polyclonal rabbit anti-TMV antibody was used at a dilution of 1∶500, with hemaxylin counterstain. Magnification, 400X. Arrows show macrophages positive for TMV antigen detection.

Figure 3. Anti-TMV antibody testing in mouse serum samples.

Detection of anti-TMV total antibodies in serum samples of 10 TMV-inoculated and 5 control mice at day 0 (just before the operation), and 13 TMV-inoculated and 8 control mice at day 7 and day 14 after intratracheal inoculation. *Statistically significant.

Figure 4. TMV RNA quantification in mouse lungs by real-time RT-PCR.

A: TMV coat protein gene system; B: TMV replicase gene system. Control, non-inoculated mice.

Figure 5. TMV-like particles observed by negative coloration on electron microscopy in the homogenate of one inoculated lung sample 1 day (A) and 3 days (B) post-inoculation.

Arrows indicate TMV-like particles.

Figure 6. Infectivity test for TMV RNA-positive lung samples on Nicotiana benthamiana.

A: Leaves of a non-inoculated plant; B: Leaves of a plant inoculated with a TMV RNA positive sample (TMV-inoculated mouse number 1 at day 3). Arrows show signs of systemic infection with discoloration and deformation of young leaves.

Figure 7. Infectivity test for TMV RNA-positive lung samples on Nicotiana tabacum Xanthi.

A: Leaf of a non-inoculated plant; B: Leaf of a plant inoculated with a TMV RNA positive sample (TMV-inoculated mouse number 2 at day1); C: Leaf of a plant inoculated with a TMV RNA positive sample (TMV-inoculated mouse number 1 at day 3). On inoculated leaves, arrows show local necrotic lesions induced by the virus in the plant cells.

Figure 8. TMV RNA quantification by real-time PCR in bronchoalveolar lavage macrophages of TMV-inoculated and control mice.

The PCR systems targets the coat protein gene.

Figure 9. Immunofluorescence analysis of bone marrow derived macrophages (BMDM).

BMDM were observed at day 1 (A and B), day 7 (C and D) and 14 (E and F) after inoculation with TMV. Non-inoculated cells (A, C, E) and TMV-inoculated cells (B, D, E) were analyzed using a mouse monoclonal anti-TMV antibody diluted at 1∶25 with goat anti-mouse 555 antibodies (red). Actin is stained with phallacidin-488 (green) and nuclei are stained with DAPI (blue). Arrows show TMV antigen (red) detected in BMDM cytoplasm. BMDM were observed by confocal microscopy with an oil immersion objective of 63X and a digital zoom of 2.

Figure 10. TMV RNA quantification by real-time PCR in bone marrow-derived macrophages (BMDM).

Quantification of the TMV coat protein encoding RNA region in BMDM culture medium (A) and within cells (B) inoculated with 0, 10⁷,10⁹ or10¹¹TMV particles during 15 days.