In a recent study published in Nature Microbiology, researchers examined virus-virus interactions using human lung cells coinfected with two co-circulating respiratory infection-causing viruses, respiratory syncytial virus (RSV) and influenza A virus (IAV).
Intracellular pathogens such as viruses generally exhibit tropism, where they show affinity towards selected cell types. Some cells and tissues can be coinfected by taxonomically different viruses since diverse viruses can 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 surface proteins from one virus are incorporated into the other virus, or they could result in genomic rearrangements which could give rise to completely new strains with increased infection potential. While some studies claim that disease outcomes are independent of coinfections, others indicate an increase in severe outcomes due to coinfections. 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 cells (MDCK) and human epidermoid carcinoma cells (HEp-2) were used to grow virus stocks of hemagglutinin 1 neuraminidase 1 (H1N1) IAV and RSV strain A2, respectively. Cultured human lung adenocarcinoma cells (A549) were infected with IAV and RSV individually and synchronously at 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 assess single and coinfected cells to determine the effect of coinfections on viral protein localization and infected cell proportions. Virions with IAV haemagglutinin and RSV fusion glycoprotein immunolabels were examined using super-resolution confocal microscopy to determine whether the mixing of the two viral glycoproteins resulted in the budding viral particles incorporating components of IAV and RSV.
Additionally, cryo-electron tomography was performed to investigate the structural characteristics of the RSV and IAV filaments budding from the coinfected cells. Furthermore, since hybrid viral particles would contain surface glycoproteins of both RSV and IAV, antibodies against the IAV haemagglutinin and RSV fusion glycoprotein were used in neutralization assays against IAV and RSV viruses collected from cells infected individually and synchronously.
The cellular receptor for IAV, sialic acid, was removed using neuraminidase, and the cells were inoculated with IAV and RSV viruses from individually infected or coinfected cells to determine whether incorporation of RSV glycoproteins could expand the IAV receptor tropism of hybrid viral particles. Cells were immunostained for the IAV and RSV nucleoproteins and quantified.
Additionally, human bronchial epithelial cells were coinfected with RSV and IAV, and paraffin-embedded infected cultures were immunostained for IAV haemagglutinin and nucleoprotein and the whole RSV virion to determine whether other relevant biological systems formed hybrid viral particles.
The results reported that in coinfected cells, IAV titers were equal to or marginally higher and the RSV titers were lower than cells individually infected with the two viruses. In contrast, in cells coinfected with IAV and rhinovirus, IAV replication was inhibited. This suggested that coinfection outcomes were dependent on the type of viruses involved and the subsequent virus-specific cellular responses.
Furthermore, the study demonstrated the hybrid viral particles generated during coinfections contained structural, functional, and genomic components from both parental viruses, and were infectious. These hybrid viral particles exhibited evasion of IAV-targeted neutralization and the ability to infect neuraminidase-treated IAV receptor-negative cells, indicating modified antigenicity and wide tropism characteristics.
Neutralization assay using anti-RSV glycoprotein antibodies showed that the entry of hybrid viral particles into cells is mediated by the RSV fusion glycoprotein, which suggested that IAV could recruit an unrelated viral glycoprotein as the functional envelope protein.
Although IAV infections are generally restricted to the upper respiratory tracts, hybrid viral particles with structural and functional components of both viruses could enable IAV infections in the lower respiratory tract regions. These results are indicative of the potential increase in pathogenicity and disease severity, and complications such as viral pneumonia.
Additionally, since IAV experiences high mutation rates, hybrid viral particles infecting the lower respiratory tract could result in the selection of viral particles with increased pathogenesis and greater tropism for the lower regions of the respiratory tract. The results also showed that hybrid viral particles were maintained over multiple infection rounds, and aided the spread of IAV into refractory cell populations.
Coinfection of human bronchial epithelial cells demonstrated the formation of hybrid viral particles in biologically relevant tissues and indicated that since IAV and RSV both circulate during the same season and share tropism for ciliated epithelial cells, the probability of coinfections and in vivo generation of hybrid viral particles is high.
Overall, the study demonstrated that coinfections by RSV and IAV form hybrid viral particles that exhibit modified antigenicity and expanded tropism, and suggested the possibility of other such hybrid viral particle formations from coinfections involving pleomorphic enveloped viruses such as RSV.
The authors believe that while hybrid viral particle formation is dependent on various factors other than structural compatibilities, such as overlaps in circulation season and geography and tropism, coinfections pose the risk of hybrid virus particles with wide tropism and increased immune evasion.
- Haney, J., Vijayakrishnan, S., Streetley, J., Dee, K., Goldfarb, D. M., Clarke, M., Mullin, M., Carter, S. D., Bhella, D., & Murcia, P. R. (2022). Coinfection by influenza A virus and respiratory syncytial virus produces hybrid virus particles. Nature Microbiology. doi: https://doi.org/10.1038/s41564-022-01242-5 https://www.nature.com/articles/s41564-022-01242-5