Study On The Role Of MiR-B10-3p In BoHV-5 Latency And Reactivation In Vitro

Bovine Herpesvirus 5(BoHV-5)is a member of the genus Varicellovirus of the subfamily Alphaherpesvirinae.It usually causes severe neurological disorder in the calves and resulted in enormous economic losses.After infection,BoHV-5 produces lysis replication in the oral or genital mucosa;then through the nerve endings,virus enters neurons within the trigeminal and olfactory cranial nerves and spreads via retrograde axonal transport to peripheral nervous system,where established latency with Latency-related gene;After reactivation induced by stress,produces the infectious virus particle.Establish latent infection in nerve cells and encod miRNAs are the tapical two characteristic features of ?-herpesviruses.As the small structure,excellent functional flexibility and lacking immunogenicity,miRNAs are a new regulator,play an important role in regulating gene expression of virus or host.High throughput sequencing and Northern Blot were used by us to identify at least 11 miRNAs in BoHV-5 infected bovine kidney cells(MDBK),BoHV-5-miR-B1 to BoHV-5-miR-B11,whose precursors miRNAs(pre-miRNAs)are processed into 16 mature miRNAs during lytic infection in vitro.However,the function of BoHV-5 miRNAs expressed during lytic infection in virus latency and reactivation remains to be elucidated.The high cost and long time-consuming of animal models and the lack of robust experimental models to study the key molecules involved in reactivation of BoHV-5 hinder the detection of the expression of miRNAs during the latency,little is known about the underlying molecular mechanisms by which the virus switches between lytic and latent infection.To investigate the role of bovine herpesvirus miRNA in different infection transitions,we performed the following studies: 1.Establish the model of BoHV-5 latency and reactivation in Kunming mice SCG in vitroFirst,Kunming mice Superior Cervical ganglion(SCG)cells were isolated and cultured in vitro,we verified that SCG supports effective infection of BoHV-5.In the presence of 100 ?m ACV and 125 IU/m L IFN-?,SCG infected BoHV-5 showed latency markers,accumulation of Latency-related gene(LR),detectable disruption of production gene,and presence of viral genome,and reactivated after the NGF deletion.2.Expression of miRNA in BoHV-5 infected in vitroBased on the model of BoHV-5 latency and reactivation in vitro,the accumulation of 16 miRNAs encoded by BoHV-5 was detected by stem-loop RT-PCR,the existence of miR-B10-3p and miR-B10-5p was identified by FISH.Our results demonstrate that the expression of BoHV-5 miRNAs is different during lytic and latent,and the model of BoHV-5 latency and reactivation in vitro is useful for studying miRNAs in latency.3.Role of miRNA-B10-3p in BoHV-5 infection in vitroMiR-B10-3p which has the highest readings at lytic infection and detected during latency will be follow-up study as a typical role of miRNAs in the life cycle of BoHV-5.Bacterial artificial chromosome was used to mutate the 3'UTR of UL39,a target site for miR-B10-3p,into seven bases.Compared with the wild type,the mutant lost the regulation of target genes showed the high copies of DNA during lytic infection,required the higher usage of antiviral drug in order to establish latency in vitro,and the genome showed greater activity and more efficient reactivation in latency.In conclusion,our results suggest that studies of the latency of BoHV-5 would benefit from the model of BoHV-5 latency and reactivation in vitro,and the model may be useful in studying the expression of miRNAs in different infection states.Study on the role of miR-B10-3p in BoHV-5 latency and reactivation in vitro reveals that,viral miRNA play an important role in switch infection states independently of the host.