Infection with different human influenza A subtypes affects the period of susceptibility to secondary bacterial infections in ferrets

Abstract

It is well-established that influenza virus infections predispose individuals to secondary bacterial infections (SBIs), which may result in a range of clinical outcomes from relatively mild (e.g. sinusitis and otitis media) to severe (e.g. pneumonia and septicaemia). The most common bacterial pathogen associated with SBI following influenza virus infections is Streptococcus pneumoniae(SPN). Of circulating human seasonal influenza viruses, influenza A viruses (IAV) of both the A(H1N1)pdm09 and A(H3N2) subtypes are associated with severe disease but have differing hospitalisation and complication rates. To study the interplay of these two IAV subtypes with SBI, we used a ferret model of influenza infection followed by secondary challenge with a clinical strain of SPN to determine the severity and the period of susceptibility for SBI. Ferrets challenged with SPN 5 days after infection with A(H3N2) or A(H1N1)pdm09 viruses developed severe disease that required euthanasia. When the time between viral infection and bacterial challenge was extended, A/H1N1pdm09-infected animals remained susceptible to SBI- for up to 10 days after the viral infection. For A(H3N2)- but not A(H1N1)pdm09-infected ferrets, susceptibility to SBI-associated disease could be extended out to 16 days postviral infection. While caution should be taken when extrapolating animal models to human infections, the differences between A(H3N2) and A(H1N1)pdm09 strains in duration of susceptibility to SBI observed in the ferret model, may provide some insight regarding the higher rates of SBI-associated disease associated with some strains of A(H3N2) viruses in humans.

Keywords: Influenza A virus; Streptococcus pneumoniae; community acquired pneumonia; ferrets; septicemia; viral bacterial coinfections.

Figure 1.

Clinical signs and survival in animals inoculated with SPN at various times following infection with A(H1N1)pdm09 strain A/Perth/265/2009 or A(H3N2) strain A/Sydney/1234/2019. Ferrets were infected via the intranasal route with either A/Perth/265/2009 (10³ TCID₅₀) or A/Sydney/1234/2019 (10⁵ TCID₅₀) in 500 µl of PBS. At 5 (circle), 10 (square), or 16 (triangle) days after viral infection, ferrets were inoculated with 10³ CFU of SPN in 50 µl of PBS. Animals were monitored daily for clinical signs. (A) and (B) Heat maps show the percentage of animals experiencing clinical signs. Amongst these are (i) reduced activity, (ii) respiratory signs, which include nasal discharge, laboured breathing, coughing, and sneezing, (iii) dehydration, (iv) loss of weight, (v) elevated temperatures, (vi) changes in posture, such as hunched disposition, and (vii) loss of coat or body condition. Survival following bacterial infection is shown in (C) and (D). Symbols represent the percentage of surviving animals.

Figure 2.

Viral and bacterial burden in animals challenged with SPN at various times following A/H1N1pdm09 and A/H3N2 infection. Ferrets were infected with A/Perth/265/2009 (10³ TCID₅₀) or A/Sydney/1234/2019 (10⁵ TCID₅₀) in 500 µl of PBS. (A) and (B) Influenza virus titres in the URT were enumerated using a TCID₅₀ assay. Symbols represent the mean virus titre (± SD). (C) and (D) At 5, 10, or 16 days after viral infection, all animals except for IAV alone were challenged with 10³ CFU of SPN in 50 µl PBS. Note that a control group (SPN alone) was included in which animals were challenged with SPN in the absence of prior IAV infection. Following bacterial inoculation, nasal washes were collected daily. Bacterial titres in nasal wash samples were determined on HBA plates. (E), (F), (G), and (H) Following euthanasia, lungs and blood were harvested and bacterial titres determined. Bars represent the mean bacterial titre (± SD) and symbols show titres in individual animals. The average number of lung lobes with detectable bacteria per group are shown above bars in (C) and (D). Skull and bones symbol (**) represents the time point where an animal was euthanised. Statistical significance was determined using a Mann–Whitney Student's t-test compared to SPN alone controls.

Figure 3.

Bacterial burden in animals infected with different A(H3N2) IAV strains 5 days prior to SPN challenge. Ferrets were infected with either A/Udorn/307/1972 or A/Sydney/1234/2019 (10⁵ TCID₅₀) in 500 µl PBS and monitored daily. A total of 5 days after viral infection, ferrets were inoculated with 10³ CFU of SPN in 50 µl of PBS. (A) Survival over time following bacterial inoculation. (B) Nasal wash samples were taken daily and the bacterial load determined on HBA plates. (C) Following euthanasia, blood was collected and the bacterial load determined. Bars represent the mean bacterial titre (± SD) and symbols show titres in individual animals.