Influenza A and Respiratory Syncytial Virus Trigger a Cellular Response That Blocks Severe Acute Respiratory Syndrome Virus 2 Infection in the Respiratory Tract

Abstract

Background: Multiple viruses cocirculate and contribute to the burden of respiratory disease. Virus-virus interactions can decrease susceptibility to infection and this interference can have an epidemiological impact. As humans are normally exposed to a community of cocirculating respiratory viruses, experimental coinfection studies are necessary to understand the disease mechanisms of multipathogen systems. We aimed to characterize interactions within the respiratory tract between severe acute respiratory syndrome virus 2 (SARS-CoV-2) and 2 major respiratory viruses: influenza A virus (IAV), and respiratory syncytial virus (RSV).

Methods: We performed single infections and coinfections with SARS-CoV-2 combined with IAV or RSV in cultures of human bronchial epithelial cells. We combined microscopy with quantification of viral replication in the presence or absence of an innate immune inhibitor to determine changes in virus-induced pathology, virus spread, and virus replication.

Results: SARS-CoV-2 replication is inhibited by both IAV and RSV. This inhibition is dependent on a functional antiviral response and the level of inhibition is proportional to the timing of secondary viral infection.

Conclusions: Infections with other respiratory viruses might provide transient resistance to SARS-CoV-2. It would therefore be expected that the incidence of coronavirus disease 2019 (COVID-19) may decrease during periods of high circulation of IAV and RSV.

Keywords: human airway epithelium; influenza A virus; respiratory syncytial virus; severe acute respiratory syndrome virus 2; virus coinfection.

Figure 1.

Morphological changes in air-liquid interface cultures of human bronchial cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), and respiratory syncytial virus (RSV) in single and coinfections. Representative images of histological sections of cultures infected at different hours postinfection (hpi) with the indicated viruses and stained with hematoxylin and eosin. Images are representative of a minimum of 2 independent experiments. Arrows indicate ciliated cells (CC), basal cells (BC), and goblet cells (GC). Scale bar represents 20 μm.

Figure 2.

Replication kinetics of SARS-CoV-2, IAV, and RSV in single and coinfections of air-liquid interface cultures of human bronchial cells. A, SARS-CoV-2 titers in single SARS-CoV-2 infections (solid black circles, white background) and simultaneous SARS-CoV-2/IAV coinfections (solid black circles, red background). B, SARS-CoV-2 titers in single SARS-CoV-2 infections (solid black circles, white background) and simultaneous SARS-CoV-2/RSV coinfections (solid black circles, cyan background). C, IAV titers in single IAV infections (solid red circles, white background) and simultaneous SARS-CoV-2/IAV coinfections (solid red circles, background). D, RSV titers in single RSV infections (solid cyan circles, white background) and simultaneous SARS-CoV-2/RSV coinfections (solid cyan circles, black ground). Individual titers are shown as circles. Bars represent the mean of 9 values. Error bars represent the standard deviation. Data are combined titers from 3 independent experiments. Statistical significance was tested using Mann-Whitney U tests and separate tests were carried out for individual time points. * P <.05, *** P <.001. Abbreviations: FFU, focus forming units; IAV, influenza A virus; PFU: plaque forming units; RSV, respiratory syncytial virus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 3.

Virus staining in air-liquid interface cultures of human bronchial cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), and respiratory syncytial virus (RSV) in single and coinfections. Representative images of histological sections of cultures infected at different hours postinfection (hpi) with the indicated viruses and stained with antibodies against SARS-CoV-2 nucleocapsid (red), IAV nucleoprotein (green), and RSV fusion protein (yellow). Nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI). Images are representative of a minimum of 2 independent experiments. Scale bar represents 20 μm.

Figure 4.

Expression of interferon-stimulated genes in air-liquid interface cultures of human bronchial cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), and respiratory syncytial virus (RSV) in single and coinfections. A, Representative light microscopy images of IFITM3, ISG15, and MxA expression detected by immunohistochemistry at 48 hours postinfection. Air-liquid interface cultures were mock infected, infected with SARS-CoV-2 only, IAV only, RSV only, and coinfected with SARS-CoV-2 and IAV or with SARS-CoV-2 and RSV. Positive immunostaining is colored brown. Scale bar represents 20 μm. B, C, and D, Bar plots showing quantification of staining signal by IFITM3, ISG15, and MxA, respectively.

Figure 5.

Replication kinetics of SARS-CoV-2, IAV, and RSV in air-liquid interface cultures of human bronchial cells coinfected simultaneously with SARS-CoV-2 and IAV or SARS-COV-2 and RSV in the presence (purple bars) or absence of BX795 (grey bars). A, SARS-CoV-2 titers in coinfections with IAV. B, SARS-CoV-2 titers in coinfections with RSV. C, IAV titers in coinfections with SARS-CoV-2. D, RSV titers in coinfections with SARS-CoV-2. SARS-CoV-2 titers are shown in black, IAV in red, and RSV in cyan. Individual titers are shown as circles. Bars represent the mean of 9 values. Error bars represent the standard deviation. Data are combined titers from 3 independent experiments. Statistical significance was tested using Mann-Whitney U tests and separate tests were carried out for individual time points. * P <.05, ** P <.01, *** P <.001. Abbreviations: FFU, focus forming units; IAV, influenza A virus; PFU: plaque forming units; RSV, respiratory syncytial virus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 6.

Morphological changes and virus spread in air-liquid interface cultures of human bronchial cells coinfected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (IAV) or SARS-CoV-2 and respiratory syncytial virus (RSV) in the presence or absence of BX795. Left column shows representative images of histological sections stained with hematoxylin and eosin of cultures coinfected with the indicated viruses at 120 hours postinfection with the indicated viruses. Right column shows representative immunofluorescence images of histological sections stained with antibodies against SARS-CoV-2 nucleocapsid (red), IAV nucleoprotein (green), and RSV fusion protein (yellow). Nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI). White arrows indicate cells positive for both SARS-CoV-2 and RSV antigens. Images are representative of 3 independent experiments. Scale bars represent 20 μm.