Respiratory infections by HMPV and RSV are clinically indistinguishable but induce different host response in aged individuals

Abstract

Background: Human metapneumovirus and respiratory syncytial virus can cause severe respiratory diseases, especially in infants, young children, and the elderly. So far it remains unclear why infections in the elderly become life threatening despite the presence of neutralizing antibodies in the serum, and to which extent double infections worsen the clinical course.

Methods: Young and aged BALB/c-mice were infected with RSV or/and HMPV. Appearance of the mice was observed during course of infection. On day 5 p.i. animals were dispatched by cervical dislocation and levels of TNF-α and NF-κB were determined.

Results: The observation of activity, weight and appearance of the different mice showed no differences among the tested groups. Despite this, the immunologic response depends on the animals' age and the virus they were infected with. In young animals, NF-κB levels were elevated if infected with HMPV and HMPV/RSV but remained low in RSV infections, whereas in aged animals the opposite was observed: solely RSV-infected animals showed elevated levels of NF-κB. TNF-α was slightly elevated in HMPV-infected young and old animals, but only in young animals this elevation was significant.

Conclusions: Contrary to other studies, no weight loss or change in activity despite productive lung infection with the different viruses were observed. This may be due to the weaker anaesthesia or the lesser volume of virus solution used, leading to less stress in the animals. The observed differences in TNF-α and NF-κB elevation lead to the assumption that young and old individuals have different mechanisms to react against the viruses.

Figure 1. Daily food consumption of infected animals.

Daily food-consumption of 4–6 weeks (A) and 19 month (B) old BALB/c mice before and after infection with RSV, HMPV or double infection: 4–6 weeks and 19 months old BALB/c mice were infected with 2×10⁷ geq RSV or HMPV, or co-infected with 1×10⁷ geq of each virus. Untreated animals, animals anesthetized and treated with cell culture supernatant or PBS or only anesthetized served as controls. The food consumption was recorded at the beginning of the experiment, on the day of inoculation and at the end of the experiment. Values were standardized referring to the values before infection (n = 3–5, values are shown as mean ± standard deviation). + significanty different to the corresponding value before infection (b.i.) (p<0.05).

Figure 2. Weight change of infected animals.

Weight change of 4–6 weeks and 19 month old BALB/c-mice before and after infection with RSV, HMPV or double infection: 4–6 weeks and 19 months old BALB/c mice were infected with 2×10⁷ geq RSV or HMPV, or co-infected with 1×10⁷ geq of each virus. Untreated animals, animals anesthetized and treated with cell culture supernatant or PBS or only anesthetized served as controls. Animals were weighed daily. All values were set in relation to the weight of the animals on day 1. The red line marks the day of infection (n = 5, values are shown as mean).

Figure 3. Viral load of lungs after infection.

Viral load of lungs obtained from 4–6 weeks and 19 months old BALB/c mice after infection with RSV, HMPV or double infection: 4–6 weeks and 19 months old BALB/c mice were infected with 2×10⁷ geq RSV or HMPV, or co-infected with 1×10⁷ geq of each virus. Untreated animals, animals anesthetized and treated with cell culture supernatant or PBS or only anesthetized served as controls. Mice were killed by cervical dislocation and the lungs were removed, homogenised and viral RNA was extracted. The number of genome equivalents was estimated by qRT-PCR (n = 3–5, values are shown as mean ± standard deviation).

Figure 4. TNF-α expression in the lung after infection.

Amount of TNF-α in the lungs of 4–6 weeks (A) und 19 months (B) old BALB/c mice after infection with RSV, HMPV or double infection: 4–6 weeks and 19 months old BALB/c mice were infected with 2×10⁷ geq RSV or HMPV, or co-infected with 1×10⁷ geq of each virus. Untreated animals, animals anesthetized and treated with cell culture supernatant or PBS or only anesthetized served as controls. Mice were killed by cervical dislocation and the lungs were removed and homogenised. The amount of TNF-α was determined by ELISA. Values were standardized referring to non treated controls (n = 3–5, values are shown as mean ± standard deviation). * significantly different to untreated animals (p<0.05). # signifcantly different to cell culture infected animals (p<0.05).

Figure 5. NF-κB expression in the lung after infection.

Amount of NF-κB in the lungs of 4–6 weeks (A) und 19 months (B) old BALB/c mice after infection with RSV, HMPV or double infection: 4–6 weeks and 19 months old BALB/c-mice were infected with 2×10⁷ geq RSV, or HMPV, or co-infected with 1×10⁷ geq of each virus. Untreated animals, animals anesthetized and treated with cell culture supernatant or PBS or only anesthetized served as controls. Mice were killed by cervical dislocation and the lungs were removed, homogenised. The amount of NF-κB was determined by ELISA. Values were standardized referring to non treated controls (n = 3–5, values are shown as mean ± standard deviation). * significantly different to untreated animals (p<0.05). # signifcantly different to cell culture infected animals (p<0.05). + significantly different to hMPV and hMPV/RSV-infected animals (p<0.05).

Figure 6. Soluble Collagen Expression in the infected lung.

Amount of soluble collagens in the lungs of 4–6 weeks (A) und 19 months (B) old BALB/c mice after infection with RSV, HMPV or double infection: 4–6 weeks and 19 months old BALB/c mice were infected with 2×10⁷ geq RSV, or HMPV, or co-infected with 1×10⁷ geq of each virus. Untreated animals, animals anesthetized and treated with cell culture supernatant or PBS or only anesthetized served as controls. Mice were killed by cervical dislocation and the lungs were removed and homogenised. The amount of soluble collagens was determined by a collagen assay. Values were standardized referring to non treated controls (n = 3–5, values are shown as mean ± standard deviation). * significantly different to untreated animals (p<0.05).