Study On The Roles Of Exosome In Cell-to-Cell Transmission Of Foot-and-Mouth Disease Virus

Exosomes are extracellular vesicles that can be secreted by cells and originate from endosomes.Exosomes can mediate the intracellular transport of intracellular proteins,mRNA,and miRNA.These molecules can regulate the physiological activity of target cells to a certain extent.Some studies have reported that viruses or viral RNA can be transmitted between cells with the help of exosomes to escape recognition and clearance by the host immune system.Foot-and-mouth disease virus is a single-stranded positive-strand RNA virus.After binding to the receptor,the virus enters the cell and releases the viral RNA in the endosome.Exosomes originate from endosomes,and there is an intersection between the origin of exosomes and the replication cycle of viruses in space.First,we enrich the exosomes secreted by the cells after virus infection from the cell culture fluid.After obtaining exosome samples,we use transmission electron microscopy to observe exosomes,immunoblotting analysis of exosome markers,and nanosight instrument analysis of the sample size range,confirming that we obtained higher purity exosome samples.After incubating the exosome samples with cells,we observed that the cells developed cytopathies similar to FMDV infection,and the viral proteins could be detected in the cells.We found that the copy number of viruses in the exosomes decreased significantly as the exosomes suppressed,after treating cells with exosome inhibitors.These results indicate that exosomes can carry viral infectious components.Total RNA is extracted from exosomes secreted by cells infected with FMDV.Then the full-length genome of the virus can be amplified by PCR.Using RNA-FISH technology,we can observe the colocalization of viral RNA and exosome markers under confocal microscope.Subsequently,we constructed a subgenomic replicon of FMDV without structural proteins.Replicon transfer experiments confirmed that in the absence of viral structural protein,replicons can still enter exosomes and enter sensitive cells in a manner mediated by exosomes.These results indicated that the genomic RNA of the virus may be an infectious component of the virus carried by exosomes.In order to explore the situation of virus transmission through exosomes in the presence of neutralizing antibodies,we incubated the cells with exosome samples and nAb.And we found that the viral infectious components carried by the exosomes were still form plaque.After reinfecting the exosome samples with nAb,we found that the virus titer did not decrease significantly.These experimental results indicate that in the presence of nAb,FMDV still be transmitted via exosomes.Our study found that exosomes can carry infectious components of FMDV into target cells to cause cell infection,and the genomic RNA of the virus can be used as infectious components carried by exosomes.To a certain extent,the exosome-mediated transmission of infectious can escape the effect of nAb.We generally believe that foot-and-mouth disease virus spreads between cells by cleaving host cells to release viral particles.Our results show that FMDV can also spread the infectious components to target cells through the exosome pathway,causing infection of target cells.