Investigating Viral Interference Between Influenza A Virus and Human Respiratory Syncytial Virus in a Ferret Model of Infection

Abstract

Epidemiological studies have observed that the seasonal peak incidence of influenza virus infection is sometimes separate from the peak incidence of human respiratory syncytial virus (hRSV) infection, with the peak incidence of hRSV infection delayed. This is proposed to be due to viral interference, whereby infection with one virus prevents or delays infection with a different virus. We investigated viral interference between hRSV and 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09) in the ferret model. Infection with A(H1N1)pdm09 prevented subsequent infection with hRSV. Infection with hRSV reduced morbidity attributed to infection with A(H1N1)pdm09 but not infection, even when an increased inoculum dose of hRSV was used. Notably, infection with A(H1N1)pdm09 induced higher levels of proinflammatory cytokines, chemokines, and immune mediators in the ferret than hRSV. Minimal cross-reactive serological responses or interferon γ-expressing cells were induced by either virus ≥14 days after infection. These data indicate that antigen-independent mechanisms may drive viral interference between unrelated respiratory viruses that can limit subsequent infection or disease.

Figure 1.

Virus shedding among ferrets infected with 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09), followed at intervals of 3, 7, or 11 days by human respiratory syncytial virus (hRSV). A, Experimental plan and outcomes. Ferrets were infected via the intranasal route with A(H1N1)pdm09 and then challenged at various intervals (3, 7, or 11 days later) with hRSV, or vice versa. Control ferrets were not infected with the primary infecting virus. Virus shedding in nasal wash specimens was assessed every second day after primary infection and daily after challenge. B–E, Ferrets underwent primary infection with 103.5 50% tissue culture infectious doses of A(H1N1)pdm09, 3.100 followed by challenge with 105 plaque-forming units of hRSV strain Long 3 (C), 7 (D), or 11 (E) days later. Control animals were infected with hRSV alone (B). Quantitative reverse-transcription polymerase chain reaction analysis was used to detect the A(H1N1)pdm09 hemagglutinin gene (filled) and the hRSV N gene (striped) in viral RNA recovered from nasal wash samples. The lower dotted lines indicate the limit of detection of infectious A(H1N1)pdm09, and the upper dotted lines indicate the limit of detection of infectious hRSV, as defined in Materials and Methods.

Figure 2.

Neutralizing antibody responses following challenge virus infection, and kinetics of virus shedding following challenge infection. A, Ferrets were infected via the intranasal route with either 10^3.5 50% tissue culture infectious doses (TCID₅₀) of 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09) or 10⁵ plaque-forming units (PFU) of human respiratory syncytial virus (hRSV), and sera, collected on the days indicated, was assayed for neutralizing antibodies to the infecting virus, using hemagglutination inhibition (HI) or ViroSpot (VS) microneutralization (MN) assays. Data are geometric mean titers (GMTs) and 95% confidence intervals from 4 ferrets. B, After undergoing primary infection with either A(H1N1)pdm09 or hRSV, ferrets were challenged 3, 7, or 11 days later with the alternate virus. Control animals in each experiment received only the challenge virus. Sera were collected 14 days after challenge, and neutralizing antibodies to the challenge virus were measured by the HI assay, for influenza virus, and by the VS MN assay, for hRSV. Primary and challenge infections are as follows: 10^3.5 TCID₅₀ of A(H1N1)pdm09 (primary infection) and 10⁵ PFU of hRSV Long (challenge infection; Bi and Bii), 10⁵ PFU of hRSV Long (primary infection) and 10^3.5 TCID₅₀ of A(H1N1)pdm09 (challenge infection; Biii and Biv), and 10⁶ PFU of hRSV Long/A2 (primary infection) and 10^3.5 TCID₅₀ of A(H1N1)pdm09 (challenge infection; Bv and Bvi). Fold changes (Bi, Biii, and Bv) were calculated by dividing the titer of the serum sample collected 14 days after challenge by the titer of the serum sample collected prior to primary infection. Horizontal lines indicate the median of each group, samples above the dotted line are positive for seroconversion. Titers (Bii, Biv, and Bvi) were measured in serum samples collected 14 days after challenge, with horizontal lines indicating GMT, and samples above the dotted line considered seropositive. Closed circles and open circles indicate animals that did or did not, respectively, shed detectable challenge virus, as determined by quantitative reverse-transcription polymerase chain reaction analysis of viral RNA from nasal wash (NW) samples. For statistical analysis, titers or fold changes were compared between test and control groups, using 1-way Kruskal-Wallis analysis of variance with the Dunn multiple comparison test. *P < .05 and **P < .01. C, The kinetics of shedding was analyzed for all ferrets that shed challenge virus in Figures 1 and 3. Data from ferrets obtained at the 3-day, 7-day, and 11-day intervals were pooled into the test group. The number of days from challenge inoculation to the peak level of challenge virus shedding (Ci and Ciii) and the number of days the challenge virus was shed (Cii and Civ) was determined for each ferret in the indicated groups. Horizontal lines indicate median values. The number of days of virus shedding were compared between test and control groups, using the Mann-Whitney test. *P < .05 and **P < .01.

Figure 3.

Virus shedding among ferrets infected with human respiratory syncytial virus (hRSV), followed at intervals of 3, 7, or 11 days by 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09). A and D, Control ferrets infected with A(H1N1)pdm09 alone. B, C, and E, Ferrets underwent primary infection with 105 plaque-forming units (PFU) of hRSV strain Long following by challenge with 103.5 50% tissue culture infectious doses of A(H1N1)pdm09 3 (B), 7 (E), or 11 (C) days later. Note that control animals in panel A were in the same experiment as the test ferrets in the 3-day interval (B) and 11-day interval (C) groups, whereas control animals in panel D were in the same experiment as the test ferrets in the 7-day interval group (E). Quantitative reverse-transcription polymerase chain reaction analysis was used to detect the A(H1N1)pdm09 hemagglutinin gene (filled) and the hRSV N gene (striped) in viral RNA from nasal wash (NW) samples. The lower dotted lined indicate the limit of detection of infectious A(H1N1)pdm09, and the upper dotted lines indicate the limit of detection of infectious hRSV.

Figure 4.

Virus shedding among ferrets infected with human respiratory syncytial virus (hRSV), followed at an interval of 3 days by 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09). A, Control ferrets infected with A(H1N1)pdm09 alone. B and C, Ferrets underwent primary infection with 106 plaque-forming units (PFU) of hRSV strain Long (B) or strain A2 (C), followed by challenge with 103.5 50% tissue culture infectious doses of A(H1N1)pdm09 3 days later. Quantitative reverse-transcription polymerase chain reaction analysis was used to detect the A(H1N1)pdm09 hemagglutinin gene (filled) and the hRSV N gene (striped) in viral RNA obtained from nasal wash (NW) samples. The lower dotted lines indicate the limit of detection of infectious A(H1N1)pdm09, and the upper dotted lines indicate the limit of detection of infectious hRSV.

Figure 5.

Expression of inflammatory mediator genes in messenger RNA from nasal wash samples from ferrets after infection with 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09) or human respiratory syncytial virus (hRSV). Ferrets were infected with 10⁵ plaque-forming units (PFU) of hRSV strain Long or 10^3.5 50% tissue culture infectious doses of A(H1N1)pdm09 (n = 4 ferrets/virus). Nasal wash specimens were collected after challenge from ferrets on days 2 and 6 after infection, and mRNA was assayed for the indicated genes, using quantitative polymerase chain reaction (qPCR) assays. For each graph, qPCR data are expressed as fold changes relative to values for nasal wash specimens from uninfected animals and normalized to ATF4 and GAPDH housekeeping genes. In panel A, expression of N is shown for hRSV-infected ferrets, and expression of M is shown for influenza virus–infected ferrets. For statistical analyses, inflammatory mediators were compared between (1) hRSV-infected and A(H1N1)pdm09-infected animals sampled on day 2 after infection, (2) hRSV-infected and A(H1N1)pdm09-infected animals sampled on day 6/7 after infection, and (3) A(H1N1)pdm09-infected animals sampled on day 2 and hRSV-infected animals sampled on day 6 after infection. Fold changes were compared between viruses, using the Mann-Whitney U test. IFN, interferon; IL-1, interleukin 1; IL-6, interleukin 6; IL-8, interleukin 8; IL-10, interleukin 10; IL-12p40, interleukin 12p40; IL-17, interleukin 17; MCP-1, monocyte chemoattractant protein 1; TNF-α, tumor necrosis factor α. *P < .05, **P < .01, ***P < .001, and ****P < .0001.

Figure 6.

Assessment of cross-reactive immunological responses to with 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09) and human respiratory syncytial virus (hRSV). Ferrets were infected with A(H1N1)pdm09 (black circles), hRSV strain Long (white circles), or hRSV strain A2 (black triangles), and retropharyngeal lymph nodes (LNs) and sera were collected 17 and 14 days after infection, respectively. A and B, Single-cell suspensions of LNs were restimulated in vitro with live hRSV (A), A(H1N1)pdm09, or concanavalin A (ConA; B). The number of interferon γ (IFN-γ)–producing cells was determined by an enzyme-linked immunospot (ELISpot) assay. C–E, Sera were tested for antibodies to A(H1N1)pdm09, by a hemagglutination inhibition (HI) assay (C); for total serum antibody binding to hRSV F protein, by an enzyme-linked immunosorbent assay (ELISA; D); and for neutralizing serum antibody to hRSV Long or A2, by a ViroSpot microneutralization (MN) assay (E). Data were obtained from 2 ferrets per group.