This doctoral thesis, directed by Rafael Sanjuán, analyzes the genetic basis and evolutionary implications related to Collective membrane-associated virus transmission using coxsackie enterovirus B3 (CVB3) as an experimental model. The results of the research have been published in the journals Cell Reports and Molecular Biology and Evolution. The thesis was defended on April 27, 2021.

Viruses are responsible for numerous diseases. Thanks to their fabulous adaptive capacity and ease of transmission, they are very adept at jumping from one species to another, which is why many viruses that infect animals can cause zoonoses with considerable pandemic potential. Studying and better understanding viruses is essential for health and epidemiology protocols, as well as in the search for new antiviral drugs. In recent years it has been discovered that viruses can be transmitted as a group from one host to another in multiple ways, changing the classic virology paradigm "one infectious unit = one viral particle." One of the most ubiquitous forms of group infection is membrane-associated transmission. However, both the genetic basis of this process and its evolutionary implications are still a mystery awaiting resolution. The central objective of this doctoral thesis, entitled "Collective transmission of membrane-associated enteroviruses: genetic bases and evolutionary implications" is to illuminate these unknowns to try to better understand the evolutionary and genetic bases underlying this group transmission mechanism, using CVB3 as a study model. Cells infected by this virus release membranous structures that contain between 8 and 21 infectious particles on average. To test whether the virus encodes this process in its genome, a directed evolution experiment was performed in which the fraction of viruses that are transmitted freely or in groups were repeatedly selected. CVB3 responded to this selection regime in a reproducible manner through a series of mutations that altered the virus's ability to utilize this form of viral transmission. On the other hand, one of the main effects of group transmission is the consequent increase in the multiplicity of infection (MOI), which can favor viral cooperation events, but also the emergence of cheating viruses, such as defective interfering particles. To better understand this process, the genetic diversity of groups of membrane-associated CVB3 virions was examined. In most cases, these structures turned out not to promote the cotransmission of different viral genetic variants from one cell to another, so they mostly contain viral particles whose genomes are highly related. Finally, increasing MOI may also have effects on the efficiency of viral infection that are independent of the associated genetic diversity. When comparing the per capita efficiency of the virus, it was observed that the cost of dispersion is not compensated by productivity, but that the infection and release of viral progeny in group-infected cells appears to be accelerated.

J. V. Bou's thesis was carried out in the Experimental Virus Evolution group of I²SysBio, under the supervision of Rafael Sanjuán (Professor of Genetics, UV). During the development of the thesis J. V. Bou has enjoyed a contract within a Prometeo project (Generalitat Valenciana). The panel was made up of Pilar Domingo Calap (I²SysBio, UV-CSIC), Alberto Marina (IBV, CSIC) and Francisco Javier López Labrador (FISABIO), who rated the thesis as outstanding.