Understanding Host-Virus Interactions: Assessment of Innate Immune Responses in Mastomys natalensis Cells after Arenavirus Infection

Abstract

Mastomys natalensis is the natural host of various arenaviruses, including the human-pathogenic Lassa virus. Homologous arenaviruses, defined here as those having M. natalensis as a natural host, can establish long-lasting infection in M. natalensis, while these animals rapidly clear arenaviruses having another rodent species as a natural host (heterologous viruses). Little is known about the mechanisms behind the underlying arenavirus-host barriers. The innate immune system, particularly the type I interferon (IFN) response, might play a role. In this study, we developed and validated RT-PCR assays to analyse the expression of M. natalensis interferon-stimulated genes (ISGs). We then used these assays to study if homologous and heterologous viruses induce different IFN responses in M. natalensis cells. Infection experiments were performed with the homologous Lassa and Morogoro viruses and the related but heterologous Mobala virus. Compared to the direct induction with IFN or Poly(I:C), arenaviruses generally induced a weak IFN response. However, the ISG-expression profiles of homologous and heterologous viruses were similar. Our data indicate that, at least in M. natalensis cells, the IFN system is not a major factor in the virus-host barrier for arenaviruses. Our system provides a valuable tool for future in vivo investigation of arenavirus host restrictions at the level of the innate immune response.

Keywords: Lassa virus; Mastomys natalensis; arenavirus; innate immune response; interferon type I response; multiplex RT-PCR assay; natural reservoir animal.

Figure 1

Changes in ISG expression after stimulation with IFN-α or IFN-β. MasKECs (A) or MΦs (B) were stimulated for 16 h with 500 U/mL IFN-α (clear bars) or IFN-β (black bars). Cells without stimulation were used for later normalisation. Cellular RNA was harvested, depleted of DNA, and used for RT-PCR analysis of ISG expression levels. For the RT-PCR-based assays, 10 ng of overall extracted RNA was used. Normalisation was performed with the ΔΔCt method with the housekeeping gene Ppil4. Genes were marked with an asterisk (*) if no signal Ct value for the controls was obtained due to too low concentrations in unstimulated cells. The previously determined cut-off Ct values (Table 2) were used to calculate the fold change of ISG expression of these genes. The dashed line indicates the ISG level of untreated control cells. The experiment was performed in triplicate. Shown are the mean and standard deviation.

Figure 2

Changes in ISG expression after stimulation of MΦs with Poly(I:C). MΦs were stimulated with 1 µg/mL Poly(I:C) or PBS for the untreated controls for 2, 4, 6.5, 16, or 24 h. Cellular RNA was isolated and depleted of DNA. For the RT-PCR-based assays, 10 ng of overall extracted RNA were used. Normalisation was performed with the ΔΔCt method with the housekeeping gene Ppil4. Genes were marked with an asterisk (*) if no signal Ct value for the controls was obtained due to low concentrations in unstimulated cells. The previously determined cut-off Ct values (Table 2) were used to calculate the fold change of ISG expression of these genes. (#) no Ct values were obtained for the experimental or control-treated cells, and a calculation of the fold change of expression was not feasible. The dashed line indicates the ISG level of untreated control cells. The experiment was performed in triplicate. Shown are the mean and standard deviation.

Figure 3

Changes in MΦ ISG expression after infection with RVFV or CHIKV. MΦs were infected with either RVFV or CHIKV with an MOI of 1 and incubated for 24 h. RNA was isolated and depleted of DNA. For the RT-PCR-based assays, 10 ng of overall extracted RNA was used. Normalisation was performed with the ΔΔCt method with the housekeeping gene Ppil4. Genes were marked with an asterisk (*) if no signal Ct value for the controls was obtained due to too low concentrations in uninfected cells. The previously determined cut-off Ct values (Table 2) were used to calculate the fold change of ISG expression of these genes. The dashed line indicates the normal ISG level of untreated control cells. The experiment was performed in triplicate. Shown are the mean and standard deviation.

Figure 4

Changes in ISG expression after infection with different arenaviruses. MΦ were infected with either MORV, MOBV, or LASV with an MOI of 1 and incubated for 24 h. RNA was isolated and depleted of DNA. For the RT-PCR-based assays, 10 ng of overall extracted RNA was used. Normalisation was performed with the ΔΔCt method with the housekeeping gene Ppil4. Genes were marked with an asterisk (*) if no signal Ct value for the controls was obtained due to too low concentrations in uninfected cells. The previously determined cut-off Ct values (Table 2) were used to calculate the fold change of ISG expression of these genes. (#) no Ct values were obtained for the experimental and control-treated cells, and a calculation of the fold change of expression was not feasible. The dashed line indicates the normal ISG level of untreated control cells. The experiment was performed in triplicate. Shown are the mean and standard deviation.

Figure 5

Infection of stimulated MΦ with LASV. MΦ were stimulated with 500 U/mL IFN-α, or IFN-β or Poly(I:C) and sixteen hours post-stimulation, they were infected with LASV with an MOI of 0.1. The cell culture supernatant was collected 24 or 48 h post-infection (h. p. i.) and used for immunofocus assay. The virus titres are shown relative to their uninfected controls. The experiment was performed in triplicate. Shown are the mean and standard deviation. Differences in virus titres were analysed with one-way ANOVA and Bonferroni’s multiple comparison test. *, p < 0.033; ***, p < 0.001.