Co-infection of SARS-CoV-2 and influenza virus causes more severe and prolonged pneumonia in hamsters

Abstract

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently a serious public health concern worldwide. Notably, co-infection with other pathogens may worsen the severity of COVID-19 symptoms and increase fatality. Here, we show that co-infection with influenza A virus (IAV) causes more severe body weight loss and more severe and prolonged pneumonia in SARS-CoV-2-infected hamsters. Each virus can efficiently spread in the lungs without interference by the other. However, in immunohistochemical analyses, SARS-CoV-2 and IAV were not detected at the same sites in the respiratory organs of co-infected hamsters, suggesting that either the two viruses may have different cell tropisms in vivo or each virus may inhibit the infection and/or growth of the other within a cell or adjacent areas in the organs. Furthermore, a significant increase in IL-6 was detected in the sera of hamsters co-infected with SARS-CoV-2 and IAV at 7 and 10 days post-infection, suggesting that IL-6 may be involved in the increased severity of pneumonia. Our results strongly suggest that IAV co-infection with SARS-CoV-2 can have serious health risks and increased caution should be applied in such cases.

Figure 1

Effect of co-infection on body weight and virus replication. (A) Body weight changes in Syrian hamsters during infection. Hamsters (n = 6 per group) were intranasally inoculated with 1 × 10⁵ PFU of IAV (PR8), 3 × 10⁵ PFU of SARS-CoV-2, or a mixture of both viruses (co-infection). The hamsters of the mock-infected group were administered DMEM. Mean percentage increase or decrease in body weight at 0 dpi ± SD is shown at each consecutive dpi. P values were calculated using Tukey’s multiple-comparison test (*P < 0.05; between SARS-CoV-2 and SARS-CoV-2/IAV). (B) SARS-CoV-2 viral RNA was detected by RT-qPCR for SARS-CoV-2N gene in the lung homogenate at 4 and 7 dpi. N gene copies per lung weight (mg) were recorded at 4 and 7 dpi for each treatment. (C) IAV viral RNA was determined by RT-qPCR for IAV M gene in lung homogenate at 4 and 7 dpi. M gene copies per lung weight (mg) were recorded at 4 and 7 dpi for each treatment. P values were calculated using Student’s t-test. (n.s.: not significant).

Figure 2

Assessment of pneumonia during infection using micro-CT. Lung abnormalities during infection were observed until 10 dpi using micro-CT. (A) CT images showed pneumonia caused by each virus at 4, 7, and 10 dpi. (B) CT severity score exhibited level of lung abnormalities. (C) Mean of CT severity score ± SD (n = 4 per group). Observation was performed every 2 days until 10 dpi. P values were calculated using Tukey’s multiple-comparison test (*P < 0.05; between SARS-CoV-2 and SARS-CoV-2/IAV).

Figure 3

Histopathological analysis of lungs of infected hamsters. Lung sections were stained with hematoxylin and eosin (HE). Leftmost, second left, second right, and rightmost columns show representative sections of IAV, SARS-CoV-2, co-infected, and mock-infected hamsters, respectively. (A–J) Low-magnification images of the lung sections. (K–T) High-magnification images of (A–J). Arrows and inset in (L) and (N) indicate bronchointerstitial pneumonia (IAV pattern). White arrowheads in (O, S, and T) indicate alveolar macrophages. Black arrowheads in (P and Q) indicate type 2 pneumocytes. Scale bars: 4 mm (A), 200 μm (K), 100 μm (L, N), 50 μm (M, O–T), and 20 μm (inset in L, N).

Figure 4

Immunohistochemical analysis of IAV NP and SARS N antigens in the tissue of IAV and SARS-CoV-2-infected hamsters, respectively. (A, B) Representative sections of tissue from the Mock-infected hamster stained with anti-SARS-CoV-2N antibody. (C–J) Representative sections of tissue from the IAV-infected hamster stained with anti-IAV NP antibody. (K–R) Representative sections of tissue from the SARS-CoV-2-infected hamster stained with anti-SARS-CoV-2N antibody. Lung are shown in (A–C, E, G, K, M and O). Trachea are shown in (D, F, H, L, N and P). Nasal mucosa are shown in (I, Q). Sections of the vomeronasal organ are shown in (J and R). Scale bars: 100 μm (A, B, I, Q) and 200 μm (C–H, J–P, R).

Figure 5

Immunohistochemical analysis of IAV NP and SARS N antigens in the tissue of co-infected hamsters. (A–I) Sections of the co-infected hamster at 4 dpi. (A–F) Representative serial sections of bronchi. (G) Representative coronal section of the head stained with HE. Nasal respiratory epithelium and vomeronasal organ are shown in (H and I), respectively. Sections in (A, C, E, and H) were stained with anti-IAV NP antibody. Sections in (B, D, F, and I) were stained with anti-SARS-CoV-2N antibody. Scale bars: 200 μm (A–F), 3 mm (G), and 100 μm (H, I).

Figure 6

Measurement of inflammatory and anti-inflammatory cytokines and chemokines in the serum. Inflammatory and anti-inflammatory cytokines and chemokines were quantified by ELISA. Data are presented as the mean value ± SD (n = 3 per group). P values were calculated using Tukey’s multiple-comparison test (*P < 0.05; **P < 0.01).