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While it is now known that viruses can operate as collective infectious units, containing multiple particles delivered to the same host cell, mystery surrounds the underlying mechanisms.
The EU-supported Vis-a-Vis project has used social evolution theory to explore how viral interactions unfold during the processes of transmission and replication. Social evolution theory examines how microorganisms cooperate with those around them to perform multicellular behaviours, such as biofilm formation and quorum sensing.
“While this framework had been successfully applied to other microorganisms, including bacteria, we were among the first to apply it to viruses,” says Rafael Sanjuán, project coordinator.
The project demonstrated how viral units can be cooperative, with different genetic variants conferring complementary properties, or can accelerate infection by delivering multiple copies of a virus genome to the same cell.
But crucially, these units were also found to promote the emergence of ‘cheater viruses’ that exploit cooperators, compromising overall viral fitness.
Three viruses
The Vis-a-Vis team worked with three viruses: vesicular stomatitis virus (VSV), an enterovirus and a baculovirus. While it had long been known that baculoviruses transmit together in so-called occlusion bodies, collective transmission had not been explored in the other two.
Prior to the project, the team investigated genetic variation in VSV and discovered the same sets of mutations in different host cells, suggesting collective transmission. Around the same time, it was discovered by others that enteroviruses could be transmitted as large pools of virions held inside lipid vesicles.
“This first sparked my interest in group transmission and its implications for viral fitness and evolution,” notes Sanjuán from the University of Valencia, the project host. “By choosing such different viruses – a negative-strand RNA virus, a positive-strand RNA virus and a large DNA virus that infects insects – we hoped to gain generalised insights.”
Fluorescently tagged viruses were followed using real-time quantitative microscopy, to measure the fitness of the virions. Experimental evolution was used to study the fitness consequences of collective transmission after multiple infection cycles. Additionally, massive parallel sequencing and site-directed mutagenesis led to the identification of mutations responsible for specific traits, such as enterovirus transmission in vesicles.
Pros and cons of co-infection
The team found that co-infection by multiple virions accelerated the early replication of VSV, helping the virus evade innate immune responses, resulting in a stronger infection. This was observed by the team in many other viruses (including adenoviruses, vaccinia viruses and respiratory syncytial viruses), suggesting that viral replication is an intrinsically cooperative process.
“Co-infection has pros and cons for viruses. While it can accelerate infection, providing a fitness advantage over competing viruses and antiviral responses, it can also produce ‘cheaters’ – defective interfering particles. With most of their genomes deleted, cheaters are individually unable to productively infect cells, but can thrive in cells coinfected with a functional virus, to the detriment of the latter,” adds Sanjuán.
Indeed, the team used simulations and experiments to show how a defective virus, unable to counter interferon production, could damage the transmissive ability of nearby viruses by alerting the host to infection, causing uninfected cells to boost their immunity. They found that the scale of compromised infection depends on physical parameters such as the viscosity of the transmission medium, cell motility and virion size.
In line with Hamilton’s rule, the team also discovered that the pools of enterovirus particles, collectively transmitted inside lipid vesicles, tend to be ‘siblings’, which tends to prevent the emergence of cheater viruses.
Boosting the evolution of virology
Understanding how virus-virus interactions affect viral transmission and virulence helps lay the foundations for new approaches in virology.
“Recent evidence shows that virions of the Delta SARS-CoV-2 variant often aggregate, which our research suggests could have important consequences for viral infectivity,” says Sanjuán. “We are proud that our approach has inspired further research, such as whether cheater particles could be synthetically designed to function as antivirals against pathogens such as influenza or COVID-19.”
Currently within another EU-funded project EVADER, the team is investigating entry mechanisms used by zoonotic viruses, with early results suggesting these could also possibly be determined by virus-virus interactions.