The Roles Of Exosomes And Autophagy In Caprine Parainfluenza Virus Type 3 Infection

Caprine parainfluenza virus type 3(CPIV3)is a novel virus discovered in 2013,mainly causing severe respiratory diseases in goats.The pathogen spread fast and up to 56% infection rate was detected in goats,while that in sheep has not been reported.The disease brings new threat to the development of goat industry.Since the research is just started,the mechanisms of CPIV3 replication,infection,pathogenic and immune response are still unknown,theoretical support is absent in the disease prevention and control.Therefore,we investigated the prevalence of CPIV3 in sheep at first,and then studied the pivotal roles of exosomes and autophagy in CPIV3 replication,transmission and maintenance of intracellular homeostasis systematically.In order to survey the prevalence of CPIV3 in sheep of China,1163 serum samples collected in 2012 and 1863 serum samples in 2016-2017 were detected by bELISA and qRT-PCR.The antibody positive rates were 50.3%(585/1,163)and 64.9%(1,210/1,863),respectively,and the percent of pathogen was 21.5%(401/1,863).The results indicated that CPIV3 infection was wide spread in sheep.Evolutionary analysis of M gene and whole open reading frame sequences of 11 identified sheep CPIV3 strains showed that CPIV3 sequences were conversed with a high homology,shared closed relationship with goat-origin viruses.To identify the information materials loading into exosomes,ultracentrifugation combined with CD63 immunomagnetic bead method was employed to purified the exosomes from CPIV3 infected MDBK cells.The purified vesicles were consistent with the characteristics of exosomes by detection of molecular markers,electron microscopy,nanoparticle tracking assay and sucrose density-gradient centrifugation.The CPIV3 exosomes(CPIV3-exo)carried viral proteins and RNA which identified by protein spectrum,Western blot and qRT-PCR.Moreover,CPIV3 induced significant changes in miRNA expression profile and exosomes loading miRNAs was a selective process.Target gene prediction and analysis of differential miRNA revealed that the genes were closely involved in autophagy pathways.The function of CPIV3-exo was further analyzed.PKH67-labled CPIV3-exo was co-incubated with MDBK cells,and fluorescence observation showed the uptake process of exosome was actively.By detection of viral proteins and RNA,we found that CPIV3-exo mediated productive infection after the treatment of CPIV3-exo with MDBK cells,and the replication was more efficient than that of free virus,although it could be blocked by the neutralization of CPIV3 antibody positive serum.Subsequently,immunoblotting and laser confocal microscopy assay of autophagy marker molecular,LC3 B and p62,revealed that CPIV3-exo inhibited autophagy,intracellular IFN-? and oas1 were also significantly down-regulated by CPIV3-exo compared to CPIV3 infection MDBK cells.The results suggested that CPIV3-exo not only mediated productive infection,but also inhibited the autophagy and expression of cytokines for contributing the viral replication.Then,is autophagy associated with CPIV3 replication? With the induction of autophagy,the CPIV3 replication level was evaluated by Western blot and qRT-PCR,the intracellular and extracellular viral proteins were significantly decreased while RNA levels were increased.Further purification of the exosomes from the autophagy induced cells,the number of exosomes secreted by the cells declined by detection of the marker proteins,while the viral proteins and RNA loaded in exosomes increased.These results indicated that autophagy induction inhibited the secretion of exosomes and promoted the entry of viral components into MVBs.Moreover,western blot analysis showed the expression levels of CD63 and HSP70 in cells were significantly decreased and positively corrected with the titer of CPIV3.The results suggested the induction of autophagy reduced the replication level of CPIV3,and inhibited the formation and release of exosomes as well.To further explore the relationship of autophagy and exosome formation and release in CPIV3 infection,the cells infected with CPIV3 were treated with exosome inhibitor GW4869,we found that the viral proteins and RNA in exosomes were decreased,but CD63 and viral F protein were stored in the cells,and the two proteins were co-located.These results revealed that the release of exosomes was blocked and viral components were retained in MVBs.Western blot,fluorescence detection and electron microscopy results also showed that autophagy was promoted after inhibition of exosome secretion with CPIV3 infection.These results proved more evidences about the balance between autophagy and exosome pathways,and MVBs might be play a pivotal role in the balance-regulation.In conclusion,CPIV3 infection was a widespread disease in goats and sheep,and the strains shared high identity with each other.During the replication of CPIV3 in MDBK cells,a complex regulation network is present among virus,exosomes and autophagy.CPIV3 inhibited cell autophagy after infection,but promoted exosome secretion,which could mediate efficient productive infection after uptake by target cells.When autophagy was upregulated,CPIV3 replication was inhibited and exosome formation and release were blocked.Once exosome secretion was regulated negatively,induced autophagy was present in treated cells,while viral proteins and RNA were stored in MVBs and subsequently accumulated in cells with MVBs.This regulatory network showed an important role in maintaining the stability of cellular environment and the efficient replication and transmission of CPIV3.The above results are not only contributing to elucidate the infection and transmission mechanism of CPIV3,but also of great significance to further study the interaction of virus-host and reasonable prevention and control measures.