Influenza A virus interferes with respiratory syncytial virus in mice and reconstituted human airway epithelium

Abstract

Epidemiological studies suggest that respiratory syncytial virus (RSV) and influenza A virus (IAV) might interfere with each other. Viral interference mainly relies on interferon production elicited by a first virus that reduces the replication of a second virus. In this paper, we first investigated the interactions between RSV-A2 and influenza A(H1N1)pdm09 in BALB/c mice infected with each single virus or both viruses simultaneously or sequentially before, at the peak of interferon elicited by each virus, or after that peak. IAV reduced by almost 3.0 logs the replication of RSV administered at the peak of interferon induced by influenza, but the opposite was not true. However, IAV-infected mice challenged with RSV or the vehicle lost more weight and had a lower survival rate compared to single infections. Interferon expression, cytokine levels, and pulmonary inflammation were almost similar between groups. Disease worsening was attributed to an aggravation of IAV-induced pulmonary congestion following intranasal instillation of fluid (with or without RSV). In human airway epithelia, IAV also interfered with RSV replication. Viral interference was dependent on the timing and sequence of infections but not on differential interferon susceptibilities. Overall, our results help to understand the mechanisms of the interaction between two major respiratory viruses.IMPORTANCERespiratory syncytial and influenza viruses may interfere with each other based on epidemiological studies. It is suggested that a first virus may induce the production of interferon and interfere with the replication of a second unrelated virus. Our data showed that the influenza A virus interferes with respiratory syncytial virus replication in mouse lungs, but the opposite was not observed. In reconstituted human airway epithelia, viral interference was dependent on the timing and sequence of infections but not on differential interferon susceptibilities. Understanding the mechanisms of interaction between respiratory viruses may help the development of prophylactic or therapeutic modalities.

Keywords: human airway epithelium; influenza virus; interferon; mice; respiratory syncytial virus; viral interference.

Fig 1

Kinetics of interferon (IFN) expression in the lungs of mice infected with respiratory syncytial virus (RSV) and influenza A virus (IAV). Interferon (IFN)-α and -β (type I) and IFN-λ2/3 (type III) expressions were measured in lung homogenates by RT-ddPCR daily after RSV (A) and IAV (B) infection. Results are expressed as the mean of the ratio of IFN mRNAs over that of the 18S housekeeping gene (both in copies per µL) ± standard error of the mean of three mice per group from a single experiment.

Fig 2

Timelines of single infections and coinfections with respiratory syncytial virus (RSV) and influenza A virus (IAV) in mice and human airway epithelia (HAEs). Mice and HAEs were infected with each single virus (RSV in orange and IAV in blue). In coinfection experiments, RSV and IAV were added either simultaneously (Simult) or sequentially with a 1- [Seq(1d)] or 4-day [Seq(4d)] interval. In mice, body weight changes and clinical signs of infection were monitored daily for 15 days. Lungs were collected 4 days after the last infection to determine viral RNA loads, interferon (IFN) expression, cytokine protein levels, and histopathological evaluations. In HAEs, apical washes were collected daily (viral RNA loads), while basolateral medium was taken (IFN protein levels) and replaced by 500 µL of fresh media every 2 days. HAEs were lysed with RNA extraction buffer 5 days after the last infection, except for the Seq(1d) group (4 days after the second viral challenge) (interferon-stimulated gene expression).

Fig 3

Viral RNA load in the lungs of mice infected with respiratory syncytial virus (RSV), influenza A virus (IAV), or both viruses. Mice were infected with RSV, IAV, or both viruses simultaneously or sequentially with a 1- [Seq(1d)] or 4-day [Seq(4d)] interval. Viral RNA loads were determined in mouse lungs by RT-qPCR on day 4 after infection with RSV (A) or IAV (B). Results are expressed as the mean of the Log₁₀ of the total viral RNA copies in lung homogenates ± standard error of the mean of four to 11 mice per group from three independent experiments. * in orange, compared with RSV alone; * in blue, compared with IAV alone; *P ≤ 0.05; **P ≤ 0.01.

Fig 4

Body weight changes and survival rates of mice infected with respiratory syncytial virus (RSV), influenza A virus (IAV), or both viruses. Mice received intranasal administration of (A) the vehicle (minimum essential medium; MEM), RSV or IAV alone, or both viruses simultaneously on day 0; (B) the vehicle, RSV, or IAV, followed by the vehicle, or both viruses with a 1-day interval [Seq(1d)]; (C) the vehicle, RSV, or IAV, followed by the vehicle, or both viruses with a 4-day interval [Seq(4d)]. Body weight changes and survival rates were recorded daily from days 0 to 14 post-infection (p.i.). Results are expressed as the mean of the percentage of the weight over that of day 0 ± standard error of the mean of eight to 19 mice per group from three independent experiments. * in orange, compared with RSV alone; * in blue, compared with IAV alone; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig 5

Expression of interferon (IFN) in the lungs of mice infected with respiratory syncytial virus (RSV), influenza A virus (IAV), or both viruses. Mice received intranasally the vehicle (mock), RSV, IAV, or both viruses simultaneously or sequentially with a 1- [Seq(1d)] or 4-day [Seq(4d)] interval. IFN-α (A), IFN-β (B), and IFN-λ2/3 (C) mRNAs were determined in lung homogenates by RT-ddPCR on day 4 after the last infection. Results are expressed as the ratio of IFN mRNAs over that of the 18S housekeeping gene (both in copies per mL) ± standard error of the mean of four to eight mice per group from three independent experiments. *, compared with single or simultaneous infection; ^#, compared with sequential coinfection; *^{, #}P ≤ 0.05; **^{, ##}P ≤ 0.01; ***^{, ###}P ≤ 0.001.

Fig 6

Production of cytokines in the lungs of mice infected with respiratory syncytial virus (RSV), influenza A virus (IAV), or both viruses. Mice received intranasally the vehicle (mock), RSV, IAV, or both viruses simultaneously or sequentially with a 1- [Seq(1d)] or 4-day [Seq(4d)] interval. Production of IFN-γ (A), IL-1α (B), IL-1β (C), IL-6 (D), IL-10 (E), and TNF-α (F) was determined in lung homogenates by magnetic microbead immunoassays on day 4 after the last infection. Results are expressed as the mean of the protein concentration in pg/mL ± standard error of the mean of four to 11 mice per group from three independent experiments. *, compared with single or simultaneous infection; ^#, compared with sequential coinfection; *^{, #}P ≤ 0.05; **^{, ##}P ≤ 0.01; ***^{, ###}P ≤ 0.001.

Fig 7

Viral interference between respiratory syncytial virus (RSV) and influenza A virus (IAV) in nasal human airway epithelia (HAEs). Nasal HAEs were infected with RSV, IAV, or both viruses simultaneously or sequentially with a 1- [Seq(1d)] or 4-day [Seq(4d)] interval. Viral RNA loads were determined in apical washes by RT-qPCR on a daily basis. Days post-infection (p.i.) represent the time after infection with either RSV (A) or IAV (B). Results are expressed as the mean of the Log₁₀ of viral RNA copies per mL ± standard error of the mean of three to five nasal HAE inserts from two independent experiments. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 compared to single infection.

Fig 8

Interferon (IFN) protein production in nasal human airway epithelia (HAEs) infected with respiratory syncytial virus (RSV) and influenza A virus (IAV). IFN-β (A), IFN-λ1 (B), and IFN-λ2 (C) protein production was measured in basolateral medium by magnetic microbead immunoassays daily for 5 days post-infection (p.i.). Results are expressed as the mean amount of IFN proteins in pg per mL ± standard error of the mean of two to four nasal HAE inserts from two independent experiments.

Fig 9

Interferon-stimulated gene (ISG) expression in nasal human airway epithelia (HAEs) infected with respiratory syncytial virus (RSV), influenza A virus (IAV), or both viruses. Nasal HAEs were infected with RSV, IAV, or both viruses simultaneously or sequentially with a 1- [Seq(1d)] or 4-day [Seq(4d)] interval. Non-infected (NI) HAEs were used in parallel. Expression of four ISGs [OAS1 (A), IFITM3 (B), ISG15 (C), and MxA (D)] was measured in cell lysates by RT-ddPCR on day 5 after the last infection, except for the Seq(1d) groups (4 days after the second viral challenge). Results are expressed as the mean of the ratio of ISG mRNAs over that of 18S housekeeping gene (both in copies per µL) ± standard error of the mean of three to five nasal HAE inserts from two independent experiments. *, compared with single or simultaneous infection; ^#, compared with sequential coinfection; *^{, #}P ≤ 0.05; **^{, ##}P ≤ 0.01; ***^{, ###}P ≤ 0.001.

Fig 10

Susceptibility of respiratory syncytial virus (RSV) and influenza A virus (IAV) to recombinant interferon (IFN) proteins in nasal human airway epithelia (HAEs). Nasal HAEs were infected with RSV and IAV in the presence of IFN-α2a, IFN-β, or IFN-λ2. Infected and untreated HAEs were used in parallel. The viral RNA loads of RSV (A) and IAV (B) were determined in apical washes by RT-qPCR daily for 5 days post-infection (p.i.). Results are expressed as the mean of the Log₁₀ of viral RNA copies per mL ± standard error of the mean of three nasal HAE inserts in one experiment. A value of 60 copies/mL corresponding to the detection limit of the assays was attributed to samples with undetectable RNA levels. *P ≤ 0.05; **P ≤ 0.01 compared to untreated HAEs.