Exosome-Mediated Transmission Of Classical Swine Fever Virus Between ST Cells

Classical swine fever(CSF) is a highly contagious and devastating swine disease caused by CSF virus(CSFV). It remains unclear how CSFV hijacks the host biosynthetic systems for the viral budding and dissemination. Accumulating evidence showed that virus-modified exosomes can mediate the intercellular transmission of viruses between susceptibale cells via delivery of viral genome or viral particles. However, it still remains to be defined if exosome can also mediate the transmission of CSFV between cells.In order to explore the role of exosome in CSFV dissemination and infection,Total exosome isolation kit together with Opti-prep gradient ultracentrifugation were applied to isolate and purify CSFV-modified exosomes from conditioned medium of CSFV strain GD53-infected ST cells. The obtained exosomes(GD53-exosome) were detected by electron microscopy, Western blot,Real-time PCR and titration assay. In addition, subgenomic replicon of GD53(GD53-SGR) and its stable cell lines were constructed, from which the released exosomes(SGR-exosome)were isolated and incubated with na?ve ST cells to test if exosome can mediate the release and transmission of subgenomic replicon RNA. GD53-exosome were observed to be 30-80 nm under electron microscope. Western blot showed that GD53-exosomes contain exosomal marker proteins TSG101 and Alix, but not CSFV proteins Core and Npro. Analysis of purified GD53-exosomes by Opti-prep gradient ultracentrifugations howed that both GD53-exosomes and infectious particles are present in the 12-21% Opti-prep gradient, and reach peak at 15% and 18%. Further, RT-PCR results showed that full-length viral RNAs are packaged in GD53-exosome, which also can infect ST cells. These findings suggest that CSFV-modified exosome can mediate the viral transmission between cells and establish productive infection. Interestingly, subgenomic replicons can also be transferred between cells through exosomes.In conclusion, this study describes the isolation and characterization of CSFV-modified exosomes from conditioned culture medium. These modified exosome containing viral genome RNA and exosomal maker proteins can mediate the intercellular transmission and infection of CSFV, which update our previous understanding of the present form of CSFV infectious particles. Overall, this study will provide new insights into further elucidate the detailed molecular mechanism of exosome-mediated transmission of CSFV.