In a recent study published in Nature Microbiology, researchers examined virus-virus interactions using human lung cells co-infected with two viruses that cause co-circulating respiratory infections, respiratory syncytial virus (RSV) and influenza A virus (IAV).
Study: Coinfection by influenza A virus and respiratory syncytial virus produces hybrid virus particles. Image credit: Kateryna Kon/Shutterstock
background
Intracellular pathogens such as viruses generally exhibit tropism, where they show affinity for selected cell types. Some cells and tissues may be co-infected by taxonomically different viruses, as several viruses may have an affinity for the same cell type.
Coinfections provide an ecological niche for viruses to interact with each other. These interactions include pseudotyping, where the surface proteins of one virus are incorporated into the other virus, or could lead to genomic rearrangements that could lead to entirely new strains with increased infectious potential. While some studies claim that disease outcomes are independent of co-infections, others indicate an increase in serious outcomes due to co-infections. However, the mechanisms of viral interactions during coinfections that determine disease outcomes remain unclear.
About the study
In the present study, Madin-Darby canine kidney (MDCK) cells and human squamous cell carcinoma (HEp-2) cells were used to culture stocks of hemagglutinin 1 neuraminidase 1 (H1N1) IAV and the A2 strain of RSV, respectively. Cultured human lung adenocarcinoma (A549) cells were infected with IAV and RSV individually and synchronously at a high multiplicity of infection (MOI).
Plaque assays were used to determine infectious titers of IAV and RSV in MDCK and HEp-2 cells, respectively. Immunofluorescence microscopy was used to evaluate single and co-infected cells to determine the effect of co-infections on viral protein localization and proportions of infected cells. Virions with IAV hemagglutinin and immunolabeled RSV fusion glycoproteins were examined by super-resolution confocal microscopy to determine whether mixing of the two viral glycoproteins resulted in budding viral particles incorporating IAV and RSV components.
In addition, cryo-electron tomography was performed to investigate the structural features of RSV and IAV filaments budding from co-infected cells. In addition, because the hybrid viral particles would contain both RSV and IAV surface glycoproteins, antibodies against IAV hemagglutinin and RSV fusion glycoprotein were used in neutralization assays against IAV and RSV viruses harvested from cells infected individually and synchronously.
The cellular receptor for IAV, sialic acid, was removed using neuraminidase, and cells were inoculated with IAV and RSV virus from singly or co-infected cells to determine whether the incorporation of RSV glycoproteins could expand the IAV receptor tropism of hybrid viral particles. Cells were immunostained for IAV and RSV nucleoproteins and quantified.
Additionally, human bronchial epithelial cells were co-infected with RSV and IAV, and infected paraffin-embedded cultures were immunostained for IAV hemagglutinin and nucleoprotein and whole RSV virion to determine whether other relevant biological systems form hybrid viral particles.
results
The results reported that in co-infected cells, IAV titers were equal or marginally higher and RSV titers were lower than in cells infected individually with the two viruses. In contrast, in cells coinfected with IAV and rhinovirus, IAV replication was inhibited. This suggested that the outcomes of coinfection depended on the type of virus involved and the subsequent virus-specific cellular responses.
Furthermore, the study showed that hybrid viral particles generated during coinfections contained structural, functional and genomic components of both parental viruses and were infectious. These hybrid viral particles showed evasion of IAV-targeted neutralization and the ability to infect neuraminidase-treated IAV receptor-negative cells, indicating modified antigenicity and broad tropism characteristics.
Neutralization assay using anti-RSV glycoprotein antibody demonstrated that entry of hybrid viral particles into cells is mediated by the RSV fusion glycoprotein, suggesting that IAV could recruit a viral glycoprotein unrelated as a functional envelope protein.
Although IAV infections are generally restricted to the upper respiratory tract, hybrid viral particles with structural and functional components of both viruses could allow IAV infections in lower respiratory tract regions. These results are indicative of the potential increase in pathogenicity and severity of the disease, and complications such as viral pneumonia.
Furthermore, since IAV undergoes high mutation rates, hybrid viral particles infecting the lower respiratory tract could result in the selection of viral particles with greater pathogenicity and greater tropism for lower respiratory tract regions. The results also showed that the hybrid viral particles persisted through several rounds of infection and aided the spread of IAV in refractory cell populations.
Co-infection of human bronchial epithelial cells demonstrated the formation of hybrid virus particles in biologically relevant tissues and indicated that because IAV and RSV circulate during the same season and share tropism for ciliated epithelial cells, the likelihood of coinfections and the in vivo generation of hybrid viral particles. is tall
Conclusions
Overall, the study demonstrated that RSV and IAV coinfections form hybrid viral particles that exhibit modified antigenicity and expanded tropism, and suggested the possibility of other hybrid viral particle formations from coinfections involving pleomorphic viruses such as RSV.
The authors believe that while the formation of hybrid virus particles depends on several factors other than structural compatibilities, such as overlaps in circulation season and geography and tropism, coinfections represent the risk of hybrid virus particles with broad tropism and greater immune evasion.
Journal reference:
- Haney, J., Vijayakrishnan, S., Streetley, J., Dee, K., Goldfarb, DM, Clarke, M., Mullin, M., Carter, SD, Bhella, D. and Murcia, PR (2022) . Coinfection by influenza A virus and respiratory syncytial virus produces hybrid virus particles. Microbiology of nature. doi: