Establishment of Myotis myotis cell lines--model for investigation of host-pathogen interaction in a natural host for emerging viruses

Abstract

Bats are found to be the natural reservoirs for many emerging viruses. In most cases, severe clinical signs caused by such virus infections are normally not seen in bats. This indicates differences in the virus-host interactions and underlines the necessity to develop natural host related models to study these phenomena. Due to the strict protection of European bat species, immortalized cell lines are the only alternative to investigate the innate anti-virus immune mechanisms. Here, we report about the establishment and functional characterization of Myotis myotis derived cell lines from different tissues: brain (MmBr), tonsil (MmTo), peritoneal cavity (MmPca), nasal epithelium (MmNep) and nervus olfactorius (MmNol) after immortalization by SV 40 large T antigen. The usefulness of these cell lines to study antiviral responses has been confirmed by analysis of their susceptibility to lyssavirus infection and the mRNA patterns of immune-relevant genes after poly I:C stimulation. Performed experiments indicated varying susceptibility to lyssavirus infection with MmBr being considerably less susceptible than the other cell lines. Further investigation demonstrated a strong activation of interferon mediated antiviral response in MmBr contributing to its resistance. The pattern recognition receptors: RIG-I and MDA5 were highly up-regulated during rabies virus infection in MmBr, suggesting their involvement in promotion of antiviral responses. The presence of CD14 and CD68 in MmBr suggested MmBr cells are microglia-like cells which play a key role in host defense against infections in the central nervous system (CNS). Thus the expression pattern of MmBr combined with the observed limitation of lyssavirus replication underpin a protective mechanism of the CNS controlling the lyssavirus infection. Overall, the established cell lines are important tools to analyze antiviral innate immunity in M. myotis against neurotropic virus infections and present a valuable tool for a broad spectrum of future investigations in cellular biology of M. myotis.

Figure 1. Newly established immortalized M. myotis cell lines from different tissues MmBr - brain; MmTo - tonsil; MmPca - peritoneal cavity; MmNep - nasal epithelium; MmNol - nervus olfactorius.

(A) Morphology of a 24 h culture of immortalized M. myotis cell lines. (B) Expression of SV40T transcripts in different M. myotis cell lines. Note the very low expression in MmTo. (C) Expression of SV40T protein visualized by immunofluorescence and (D) by western blot using anti-SV40T monoclonal antibody. Note the absence of SV40T protein in MmTo cell line.

Figure 2. PRRs distribution patterns in the established unstimulated M. myotis cell lines.

The mRNA expression levels of TLR3, RIG-1 and MDA5 in MmBr, MmTo, MmPca, MmNep and MmNol were determined by qRT-PCR (n = 3). The expression level was shown as a related fold and normalized against β-actin. The expression level of different genes in MmBr showed the lowest expression and was presented as one fold.

Figure 3. Comparative analysis of the expression patterns of antiviral molecules after poly I:C stimulation.

The established immortalized M. myotis cell lines were transfected with poly I:C (10 µg/mL) by lipofectamine 2000. The unstimulated cells were used as blank control. Twenty four hours post transfection, the mRNA expression levels of TLR3, ISG56, ISG43, Mx1 and IFIT3 were measured by qRT-PCR (n = 3). The expression level was shown as a related fold and normalized against β-actin. The expression level of different molecules of blank group in individual cell line was presented as one fold.

Figure 4. Susceptibility of M. myotis cell lines to lyssavirus infection.

Immortalized cells (third passage, morphology and cell density visualized in bright filed) were infected with GFP fused RABV at a MOI of 10, and the propagation of RABV was visualized by fluorescence microscope at 24 hpi. Note the low amount of viral antigen positive cells in MmBr in contrast to the other 4 cell lines.

Figure 5. Susceptibility of the established immortalized M. myotis cell lines to lyssaviruses (EBLV-1, EBLV-2 and RABV) infection.

(A) Cell lines were infected with lyssaviruses at a MOI of 0.1, and virus replication levels were measured by qRT-PCR at 24 hpi (n = 2). The viral RNA level was shown as a related fold and normalized against β-actin. The viral replication levels of EBLV-1, EBLV-2 and RABV were lowest in MmBr and presented as one fold, respectively. (B) Viral growth was analysed by immunofluorescence using anti-rabies monoclonal antibody at 72 hpi. Green: lyssavirus infected cell, Blue: nuclei stained with DAPI. (C) To further confirm the susceptibility, MmBr and MmNol cell lines were infected with EBLV-1 at a MOI of 0.01, and viral replication levels were measured by qRT-PCR over 72 hpi (n = 2). – changed: viral supernatant was changed with fresh medium at 1 hpi. – Not changed: viral supernatant was not changed during the whole infection process.

Figure 6. Comparative analysis of the expression patterns of antiviral molecules in MmBr and MmTo during RABV infection.

MmBr and MmTo were infected with RABV at a serial MOI of 0.01, 0.1 and 1.0, respectively. (A) The expression patterns of PRRs: TLR3, RIG-1 and MDA5 in the infected cells were investigated by qRT-PCR at 24 hpi (n = 2). (B) The expression patterns of IFN induced genes: ISG56, ISG43, Mx1 and IFIT3 were measured by qRT-PCR at 24 hpi (n = 2). The expression level was shown as a related fold and normalized against β-actin. The expression level of different molecules in blank group (MOI: 0) in both cell lines MmBr and MmTo was presented as one fold, respectively.

Figure 7. The mRNA expression patterns of CD14 and CD68 in immortalized M. myotis cell lines.

Note the absence of these two monocyte lineage markers in four of the five cell lines.