The study of giant viruses has become even more complex due to the discovery of small viruses capable of infecting them, such as the virophage. The first virophage, called sputnik, is about 50 nm in size and approximately 18 kbp, with circular double-stranded DNA, and it was found to be associated with a strain of mimivirus [
15]. The virophages are unable to multiply in the absence of giant viruses. Their replication occurs in the giant virus factory and can be deleterious to viral replication, resulting in a decrease in amoebae lysis [
15,
71]
. Since their discovery, dozens of new virophages have been isolated and classified in a new viral family called
Lavidaviridae [
72‐
80]. It is believed that the virophage can mediate lateral gene transfer between giant viruses. Furthermore, they have been shown to be able to integrate into giant viruses and host cell genomes. These findings strongly suggest that amoeba, virophages, and giant viruses seem to co-evolve with each other [
15,
81,
82]. The discovery of new virophages led to the description of some interesting interactions between virophages, giant virus and hosts. In 2014, a virophage named zamilon was isolated, which, unlike the virophages described to date, was not able to replicate in factories of mimiviruses from lineages A, but only in mimivirus factories from lineages B and C [
76]. Its host specificity aroused the curiosity of Levasseur and collaborators, who studied the genetic basis of this host specificity [
83]. It was observed that strains of the mimivirus lineage A, resistant to the zamilon virophage, contain the insertion of a repeated zamilon sequence in its genome. These repetitions were named mimivirus virophage resistance elements (MIMIVIREs). By analyses of the surrounding sequences the authors observed that the MIMIVIRE system presents nuclease and helicase proteins, which may play a vital role in the degradation of foreign nucleic acids, suggesting that this locus can be related to the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system, although it is not homologous to this system [
84]. Interestingly, the silencing of the MIMIVIRE genes restored zamilon’s ability to infect the factories of mimivirus lineage A. As a result of which, the researchers proposed that the MIMIVIRE system acts as a viral defense mechanism against virophages [
83]. Recently, additional biological demonstrations enabled further characterization of the MIMIVIRE system defense mechanism. It was demonstrated that a mimivirus gene of unknown function, called R349, one of the MIMIVIRE system components that contains four repeats homologous to the virophage sequence, has a key function in the MIMIVIRE system defense mechanism. The deletion of the R349 gene in mimivirus lineage A restored the replication of zamilon. In addition, it was observed that a mimivirus isolate of lineage A, lacking 3 of 4 repeats of R349 gene, was susceptible to zamilon infection [
85]. Considering the above mentioned, these results reinforce the role of the MIMIVIRE as a nucleic-acid-based immunity defense system against virophage infection, confirming the importance of the R349 gene in the MIMIVIRE system. This study revealed an unprecedented type of host–virus interaction and reinforced that host amoeba, virophages, and giant viruses are coevolving. Another notable virophage–giant virus–host interaction is that which involves the marine protist
Cafeteria roenbergensis with the C. roenbergensis giant virus and its associated virophage, mavirus. Cafeteria roenbergensis virus (CroV) is distantly linked to mimiviruses that infect the phagotrophic biflagellate
Cafeteria roenbergensis [
72]. Mavirus was the second virophage discovered, isolated from water collected in Texas, USA [
73]. The mavirus virophage replicates in the viral factory of CroV; however, it was observed that the mavirus can enter into
C. roenbergensis independent of CroV by endocytosis and is able to inhibit the production of new CroV particles, increasing the survival of the host
C roenbergensis [
73]. In 2016, Fischer and Hackl discovered through the co-infection of a host population with CroV and mavirus that the virophage is able to integrate into the genome of
C. roenbergensis [
86]. They showed that the mavirus genome was integrated at different genome locations, and although the integrated virophage genes are not constitutively expressed, they can be activated by CroV infection, inducing the production of infectious mavirus particles and reactivating this virophage in the host cell. Although this was expected, the reactivation of the mavirus was not able to block the replication of CroV, and, consequently,
C. roenbergensis infected with CroV died anyway, releasing CroV and mavirus particles. In spite of this, they observed that the released mavirus decreased the spread of CroV in the protist population and its replication in another replication cycle, protecting the neighboring cells from being killed by the giant virus infection. The authors associated this virophage–giant virus–host interaction as an altruistic defense mechanism of the host, in which a host dies, releasing viral particles that are able to protect the neighboring host population [
86]. Another possibility is that this interaction acts as an adaptive immunity CRISPR-Cas system, in which the virophage genome is retained by the host and used to prevent subsequent attacks by the giant virus. Viral elements can be found in eukaryotic genomes; however, little is known about how they act and their functions [
87]. This study provided an example of a virophage that integrates into a cell genome, acting as an inducible antiviral defense system. It has been demonstrated that a green alga called
Bigelowiella natans contains virophages integrated into its genome, providing another possible example of a virophage-mediated host defense [
82]. In addition to these virophage integration studies, several peculiarities have been observed in the virophage–giant virus–host interactions. Among these was a study showing that the virophage sputnik and marseillevirus co-infection affected the replicative capacity of marseillevirus [
88]. Using a metagenomic approach, it was suggested that virophages reduce the mortality caused by the giant viruses of phototrophic algae, and through the use of a mathematical model, it was proposed that besides the direct interference in the multiplication of giant viruses, virophage infection can select viruses with reduced replicative capacity, contributing to the protection of the host cell population [
74,
89]. Based on this and other studies, it has been suggested that virophages are associate with the regulation of the population of amoebae and other protists in the environment [
90]. In 2018, a virophage was isolated and said to be associated with a mimivirus strain that infects
Saccamoeba spp., with the ability to induce a high reduction (~ 70%) in viral capsid production [
91]. The growing description of new virophage isolates and new interactions involving them has revealed that virophages, giant viruses and its, host make up a complex and unprecedented type of host–virus interaction and that there are probably still many interactions to be studied.