The genus Alphavirus, under the family Togaviridae, is a group of enveloped viruses packaging a positive-strand genomic RNA. It includes more than 30 species, most of which are transmitted by mosquitoes and infect a broad range of vertebrates. Unlike these mosquito-borne alphaviruses, the Eilat virus (EILV), phylogenetically grouped within the same clade, is featured by an inability to infect vertebrates. It is the first reported insect-specific alphavirus. Since the establishment of the reverse genetic system for EILV in 2012, significant efforts have been made in genome manipulation and understanding its host restriction mechanism. However, its potential as a biotechnological tool has yet to be fully realized, and the interplay between EILV and its mosquito host remains to be characterized.

Historically, alphaviruses have been engineered in multiple ways to develop gene expression systems. EILV’s host restriction property makes it an appealing expression platform since it provides a highly advantageous safety profile. We generated seven distinct chimeric viruses by replacing the EILV’s structural genes with those of other alphaviruses. These chimeras were readily rescued and achieved high titers in mosquito cells. We also explored the utility of EILV to express authentic antigens via double subgenomic (SG) RNA vectors. Four foreign genetic materials of varied lengths were introduced into the EILV genome, and the expressed heterologous antigens were readily detected in infected cells. By inserting an additional SG promoter into the chimeric genome containing the structural genes of the chikungunya virus (CHIKV), we developed a bivalent expression vector encoding CHIKV and Zika virus antigens simultaneously. In all, our work demonstrated the feasibility of constructing diverse EILV-based expression systems. These expression vectors can be applied as versatile biotechnological tools, such as vaccine candidates, diagnostic tools, and experimental tools.

In addition to being engineered as expression platforms, insect-specific EILV can be applied to contain the transmission of pathogenic alphaviruses. It is phylogenetically closely related to mosquito-borne alphaviruses and can induce superinfection exclusion. Like bacterial symbionts, insect-specific viruses may also alter mosquitoes’ immune systems and modulate their vectorial capacity. However, the interplay is uncharacterized between EILV and mosquitoes or mosquito cells. Here, by RNA sequencing, we found that EILV profoundly modified the host cell gene expression profile but only triggered a mild antiviral response. Two EILV-based chimeras, consisting of EILV’s nonstructural genes and the structural genes of CHIKV or Venezuelan equine encephalitis virus (VEEV), namely EILV/CHIKV (E/C) and EILV/VEEV (E/V), induced more intensive transcriptome alteration than EILV and activated different antiviral mechanisms in host cells. E/C robustly promoted antimicrobial peptide production, and E/V strongly potentiated the transcription of RNA interference pathway components. On these bases, we showed that the initial EILV or chimera infection could inhibit subsequent pathogenic alphavirus infection to varying extents.

Accumulating evidence shows that defective viral genomes (DVGs) are a key driver of virus-host interaction. Via Nanopore Direct RNA Sequencing, we found that EILV and its chimeras produced a high abundance of deletion DVGs during multiplication in mosquito cells. In contrast, few DVGs were observed during mosquito-borne CHIKV and Sindbis virus growth in mosquito and mammalian cells. The presence of minus-strand DVG replication intermediates in EILV-infected cells indicated the replication potential of corresponding DVGs. A subset of them was also encapsidated into virus particles and released extracellularly. The presence of numerous prematurely terminated plus-strand viral RNAs in infected cells suggested that DVG formation was associated with the low processivity of the EILV replication complex. Structural gene sequences also appeared to regulate DVG production, as evidenced by the variation in DVG populations between EILV and its chimeras. In conclusion, our study enhanced the application of EILV as a biological tool and provided insights into the interaction between EILV and mosquito cells.