Influenza A Virus Inhibits RSV Infection via a Two-Wave Expression of IFIT Proteins

Abstract

Influenza viruses and respiratory syncytial virus (RSV) are respiratory viruses that primarily circulate worldwide during the autumn and winter seasons. Seasonal surveillance has shown that RSV infection generally precedes influenza. However, in the last four winter seasons (2016-2020) an overlap of the morbidity peaks of both viruses was observed in Israel, and was paralleled by significantly lower RSV infection rates. To investigate whether the influenza A virus inhibits RSV, human cervical carcinoma (HEp2) cells or mice were co-infected with influenza A and RSV. Influenza A inhibited RSV growth, both in vitro and in vivo. Mass spectrometry analysis of mouse lungs infected with influenza A identified a two-wave pattern of protein expression upregulation, which included members of the interferon-induced protein with the tetratricopeptide (IFITs) family. Interestingly, in the second wave, influenza A viruses were no longer detectable in mouse lungs. In addition, knockdown and overexpression of IFITs in HEp2 cells affected RSV multiplicity. In conclusion, influenza A infection inhibits RSV infectivity via upregulation of IFIT proteins in a two-wave modality. Understanding the immune system involvement in the interaction between influenza A and RSV viruses will contribute to the development of future treatment strategies against these viruses.

Keywords: IFITs; Influenza A; RSV; double infection.

Figure 1

An overview of the percentage of influenza-positive versus RSV-positive samples in winter seasons 2013–2020. (A) The monthly percentage of positive cases of infection with respiratory viruses (RSV, and/or influenza) from 2013 to 2020 among hospitalized patients. (B) The monthly percentage of positive cases of infection with respiratory viruses (RSV, and/or influenza) from 2013 to 2020 among non-hospitalized patients. The black line represents the infection patterns for RSV and the gray area represents influenza infection patterns. The percentage of patients bearing each virus was calculated each month in relation to the total number of samples tested for the presence of that virus. RSV’s peaks from 2016–2020 were compared to those from 2013–2016 in hospitalized/non-hospitalized patients (p < 0.001 one-way ANOVA followed by post-hoc Bonferroni t-test).

Figure 2

Co-infection of HEp2 cells with influenza A/H3N2 and RSV. HEp2 cells (1 × 10⁶) were infected with influenza A/H3N2 (6 × 10⁵ PFU) for 3 h, and then with RSV (6 × 10⁵ PFU), and vice versa. RSV-infected cells served as control. RNA was extracted from supernatant samples collected at predefined time points (24–144 h). Quantitative real-time polymerase chain reaction (qPCR) was performed to test for viral quantity. The black line indicates infection with RSV, the red line indicates infection with influenza A/H3N2 followed by RSV, and the blue line indicates infection with RSV followed by influenza A/H3N2. The data presented are an average of three independent experiments ± mean standard deviation * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 3

Viral loads in mouse lungs co-infected with influenza A (A/PR/8/1934) and RSV. (A) Experimental design scheme. Balb/c mice (2.5 weeks old) were intranasally infected with influenza A/PR/8/1934 (4 × 10² PFU/mL) at predefined time points (1, 2, 3, 5, 6, 8, 10, and 12 days) pre-RSV infection (6 × 10⁶ PFU/mL). Mice were sacrificed four days after RSV infection. Mice that were infected with RSV only were sacrificed four days later and served as the control group. qPCR analysis was performed to test for viral quantity. (B) Mouse lungs were homogenized using the Spex centri prep 8000-D Mixer (Mill). RNA was then extracted and the amount of virus was determined by qPCR. Left Y axis presents RSV percent compared to control group (blue columns), right Y axis presents influenza A/PR/8/1934 RNA copies/mL (Red line). The data presented are an average of three independent experiments ± mean standard deviation. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 4

Mass spectrometry analyses of influenza-infected mouse lungs (A/PR/8/1934) versus healthy (non-infected) mouse lungs. Plot of antiviral gene expression levels in influenza A/PR/8/1934-infected cells (2-fold change influenza A/PR/8/1934 vs. healthy) with expression patterns similar to that of IFIT1 (correlation ≥ 0.6, Tibco Spotfire V.7.7.0. software). The color scale (y axis) for each gene is independent, based on the distribution of the protein’s expression levels across the samples. The samples plotted (x axis) are the healthy, non-infected cells and the post-influenza A/PR/8/1934 infection samples, collected on day 1–3, day 6, day 8, day 10, and day 12.

Figure 5

IFIT1–3 and IFI44 protein expression pattern. Mass spectrometry analyses of influenza-infected mice lungs (A/PR/8/1934) versus healthy (non-infected). The data presented are an average fold change of each protein normalized to an average fold change of the protein in the healthy mice.

Figure 6

IFIT1–3 and IFI44 mRNAs expression pattern. (A) Agarose gel of IFIT1–3 and IFI44 mRNAs of healthy mice and mice infected with influenza A. Balb/c mice (2.5 weeks old) were intranasally infected with influenza A/PR/8/1934 (4 × 10² PFU/mL). Mice were sacrificed at various time points (1, 2, 3, 5, 6, 8, 10, and 12 days) post-infection. Non-infected mice served as the control group. Mouse lungs were homogenized using the Spex centri prep 8000-D Mixer (Mill). RNA was then extracted. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to test for IFIT1–3 or IFI44 mRNAs levels. (B) Relative quantification of IFIT1–3 and IFI44 mRNAs levels were normalized to the geometric mean of housekeeping gene GAPDH. The data presented are an average of three independent experiments ± mean standard deviation. The data presented is an average of three independent experiments ± mean standard deviation. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 7

Infection of IFIT1–3- and IFI44-silenced HEp2 cells with RSV. (A–D) Lentiviral vectors carrying shRNA targeting IFIT1, IFIT2, IFIT3, or IFI44 were infected into HEp2 cells. Following selection by puromycin, whole-cell extracts were harvested after 5 days of infection. Western blot analysis using antibodies specific for (a) IFIT1, (b) IFIT2, (c) IFIT3, or (d) IFI44. The bars on the graph present the quantification of IFIT1–3, IFIT4, IFIT1–3, and IFI44 expression levels (32.97%, 46.88%, 53.37%, and 34.20%, respectively) as compared to their levels in untreated control cells. Error bars represent S.E.* p < 0.05, ** p < 0.01 and *** p < 0.001. (E) shIFIT1/shIFIT2/shIFIT3/shIFI44-silenced HEp2 cells (1 × 10⁶) were infected with RSV (6 × 10⁵ PFU, 3 h). Supernatant samples were collected at predefined time points (24–144 h) post infection and RNA was extracted. The control growth curve (black line) is RSV-infected HEp2 cells (W.T) and is the same data represented in Figure 2. qPCR analysis was performed to test for viral quantity. The data presented are an average of the independent experiments ± mean standard deviation. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 8

Infection of HEp2 -IFIT1/ IFIT2/ IFIT3/ IF44 overexpressing (O/E) cells with RSV. (A–D). HEp2 cells were transduced with a lentivirus system carrying IFIT1, IFIT2, IFIT3, or IFI44 pHAGE-DsRED(−)eGFP(+). Western blots analysis showing overexpression of IFIT1–3 and IFI44 expression (29.031%, 21%, 19.8%, and 24.20%, respectively) in infected as compared to untreated control cells. Error bars represent S.E. ** p < 0.01, *** p < 0.001. (E) IFIT1/ IFIT2/IFIT3/IF44- O/E HEp2 cells (1 × 10⁶) were infected with RSV (6 × 10⁵ PFU, 3 h). Supernatant was collected at predefined time points thereafter (24–144 h) and RNA was extracted. The control growth curve (black line) is RSV-infected HEp2 cells (W.T) and is the same data represented in Figure 2. qPCR analysis was performed to determine viral load. The data presented are an average of three independent replicates ± mean standard deviation. * p < 0.05, ** p < 0.01 and *** p < 0.001.