This study examined the potential involvement of new isolates of TTMV, identified in pleural effusion samples, in the pathogenesis of severe pneumonia in children. The main findings are: 1) TTMV-LY can deeply colonise lungs; 2) alveolar epithelial cells, where efficient replication occurred, were permissive to the TTMV-LY isolates; and 3) TTMV-LY infection modulates the innate immune response of pulmonary cells by inducing the production of inflammatory mediators.
PPEs result from an inflammation of the pleura space as a consequence of infection by bacterial pathogens [11
]. Bacteria are frequently described in pleural fluids, and were also confirmed in this study in pleural effusion samples, but the presence of a viral origin remains poorly documented [12
]. This study is the first to reveal the presence of an anellovirus in parapneumonic effusions. Anelloviruses are characterised by extreme genetic diversity, a high prevalence in various populations and a wide distribution in body fluids. This ubiquity, together with the absence of suitable in vitro
culture systems, has hampered progress in the investigation of this group of viruses [14
In previous molecular epidemiological studies, unequivocal demonstration that anelloviruses are implicated in the pathogenesis and severity of disease is still lacking or is poorly demonstrated [15
]. Here, we found the presence of full-length TTMV genome in nasal aspirates and pleural effusions in children hospitalised with severe pneumonia. In this cohort, as in a Chinese cohort of 216 nasal aspiration samples of children suffering from LRTI (data not shown), no correlation has been observed between the severity of the disease and the presence of TTMV, the age or the sex of the patients. However, the presence of TTMV complete genome linked to the viral replication may indicate its role in the pathogenesis of pneumonia.
This study reports the isolation of three new full-length TTMV genomes (TTMV-LY) in children with severe pneumonia. As a first step, the biological properties of these new isolates of TTMV-LY were investigated. We first transfected the full-length genome of the three TTMV-LY viruses to ascertain whether the viral replication takes place in HEK293T cells, as already demonstrated [17
], and subsequently in alveolar epithelial A549 cells, wich act as a model of type II pneumocytes [19
]. We described for the first time that TTMV, like TTV, was able to replicate in cell lines after transfection of its genome and thus lead to the production of infectious virions. We observed that the viral infection was more efficient in alveolar cells than in kidney cells, suggesting a better tropism of the TTMV-LY genome in pulmonary cells and a strengthened role in the pathogenesis of LRTI. We hypothesised that, like porcine circoviruses and papillomaviruses, the potential pathogenicity of TTMV could be restricted to some species or a group of species [20
]. Indeed, in this study, high genetic TTMV diversity was observed only among different patients, but the same full-length genome was systematically found in the different samples taken from the same patient. All three genomes, moreover, belonged to the same phylogenetic branch of Betatorquevirus
(group I). We investigated the conserved 5′UTR genome sequence of the three TTMV-LY genome sequences to determine if specific replication patterns were present. No assumed origin of replication (_AGT_TTACA) was identified, but an insertion of 200 bases forming a stem-loop structure in the conserved 5′-UTR was detected in the TTMV-LY2. As previously described, this secondary structure could be involved in the viral pathogenicity as enhancer and promoter elements [22
]. Despite this secondary structure presented by TTMV-LY2, no gain in the replication yield was observed compared to the other TTMV-LYs. However, the effects of such genetic modifications could be expressed in another biological manner, in response to the interaction with the host cell, as observed for many viruses.
As a second step, we investigated the response of the host A549 cells to TTMV-LY infection by measuring the production of soluble mediators implicated in the inflammation process. Alveolar type II epithelial cells maintain alveolar integrity by forming the alveolar barrier, producing surfactants and repairing injured type I epithelium [23
]. Moreover, they are an integral part of the lung innate immunity, acting to intensify the function of dendritic cells and alveolar macrophages by cytokine secretion [24
]. No cytopathic effect was observed in A549 cells, but infection with TTMV-LY induced a complex innate immune response, differently modulated by the three isolates. Thus, TTMV-LY infection resulted in the production of not only pro-inflammatory cytokines, including IFN-γ as previously described [26
], but significant levels of IL-10 or IL-13 anti-inflammatory cytokines were also measured, corroborating that TTMV-LY may modify innate immune balance, as already demonstrated for TTV [27
]. The fact that different isolates of TTMV can impact differently on the production of soluble mediators is not surprising in view of what has been observed with other viral infections [28
], and may reflect their recognition by different receptors and/or interaction through different molecular pathways. IL-12 seems to play a central role in the response to TTMV-LY infection. This cytokine, enhancing the proliferation and cytotoxicity of activated T-cells and natural killer cells, is thought to be a key factor in the regulation of the host defence against many intracellular pathogens [30
]. We may assume that TTMV-LY-mediated lung inflammation may result from the abundant secretion of IL-12, although further studies would be needed to prove this.
Taken together, these results suggest that TTMV-LY isolates can invade the alveolar cells, where they replicate and induce innate immune imbalance in the respiratory tract. Further studies aimed at understanding the physiopathological implications of TTMV as an aetiological agent in respiratory diseases are necessary, as required for every newly discovered pathogen. These investigations would allow to us learn about the presumed need to enlarge the range of researched pathogens to the anelloviruses in the case of PPE, in order to improve the efficiency of diagnosis and treatment of this pathology.