The Effect And Mechanism Of Torovirus,Sapelovirus Or Teschovirus Infection On IPEC-J2 Cells

Torovirus,sapelovirus and teschovirus were found co-infection and all associated with diarrhea.There were more and more attention paid on their role in enterovirus prevalence.Torovirus,belonged to coronavirus,was studies for researching on changes of intestinal cell function from coronavirus that causing pig subclinical intestinal disease.Sapelovirus and teschovirus,as representative picornavirus leading clinical or subclinical diarrhea,were used for studying intestinal cell function changes from picornavirus that associated with clinical and subclinical symptoms.Lectin-like oxidized low-density lipoprotein-1(LOX-1),the receptor of Ox-LDL,involves in lipid metabolism,accelerates inflammation,affects cell division and the cytoskeleton,causes tissue injury and reflects the pathological state of the body.LOX-1 was also found to be upregulated in sapelovirus infected porcine intestine epithelial cell(IPEC-J2).Until now,there was little comparative research on enteroviruses of different species or genuses.And,there were no report on fuctional analysis of torovirus,sapelovirus or teschovirus infected on intestinal cells.Though verified to participate in inflammatory pathological changes and tumorigenesis,the role of LOX-1 in chronic inflammation from virus infection was still unknown.In this study,we focused on observing IPECJ2 cell pathological process from torovirus,sapelovirus and teschovirus infection,and exploring the role and the signal pathway of LOX-1 in the enteroviruses infected intestinal epithelial cell.1.The identification of torovirusPrevalence investigation showed that there were a torovirus infectivity of 5.5% in Shanghai area.Based on bovine torovirus full-length genome,we achieved the fulllength of porcine torovirus genome(28301 bp),giving more information on torovirus genomes.The bases insertion and deletion were mostly on non-structural genome ORF1 a.Compared with bovine torovirus and equine torovirus,porcine torovirus(PTo V)SH1 lost 28 bases and 152 bases,individually.When positive PTo V fecal sample regressed on Bama miniature pigs,virus presented a periodical excretion.Every 5 to 6 days,there was a virus production and excretion period.Cohabitation infection was observed when negative pigs was exposed to positive pigs for more than three days.The time fecal specimen collected,some pigs may be in no virus excretion period.It makes the actual piggery infectivity to behigher.2.The identification and isolation of teschovirusPorcine teschovirus(PTV)had an infectivity of more than 82.2% in commercial piggeries of Shanghai.Serotype classification based on VP1 showed that PTV 4,8,and 10 were all very popular,the new serotype clustered with PTV wild boar/WB2CTV/2011/HUN was also withhigh prevalence,but the existence of PTV 3 and another new serotype of PTV SH8 were quite less.We isolated a most popular strain named PTV4 SH1,owning thehighest similarity with the sequence identity of about 90% to PTV4 10BJ02.However,comparing to PTV4 10BJ02,VP2 of PTV4 SH1 lost 4 AA.The 5’ UTR of PTV SH1 lacked poly(C)than other strains,the same as PTV8 Jinlin/2003/02 and PTV8 Fuyu/2009/02.3.The effect of torovirus infection on IPEC-J2 cell mechanismPorcine torovirus was analyzed with proteases papain-like protease(PLpro)and 3C-like protease(3CLpro).PLpro was distributed mainly with nucleus,while 3CLpro localized predominately in cytoplasma.The proteases expression resulted in cell apoptosis,microfilament remodeling and dissolving,and cell cycle arrested at G2/M phases.In proteases overexpressed IPEC-J2 cells,cellular NO level increased,cytosol ROS level decreased but the ROS level in mitochondrial rose.PLpro and 3CLpro upregulated the m RNA level of inflammatory cytokines TNF-α,IFN-α,TGF-β,NF-κΒ and IL-1α and function genes of LOX-1,STAT2 and RIG-I.And,the expression of LOX-1 and STAT2 also increased.However,proteases suppressed the protein expression of RIG-I in IPEC-J2 cell.4.The effect of sapelovirus infection on IPEC-J2 cell mechanismSapelovirus infection resulted in IPEC-J2 cell progressive cytopathic effect.In the early stage of virus proliferation,little cells went apoptosis.After infected for 24 hours,more cells entered into late apoptosis or go dying.In the final stage of virus infection,cell cycle was arrested at G2/M phases,while the level of ROS and NO was in burst.During sapelovirus proliferation,host cell cytoskeleton was reorganized,stress fibre disrupted while microfilament aggregated forming clumps outlined the cell periphery.The transcription level of pro-inflammation cytokines also changed.TNF-α m RNA level elevated,and the transcription of IFN-α,TGF-β,NF-κΒ and IL-1α increased obviously(p<0.05).Function genes of LOX-1,STAT2 and RIG-I were also affected by sapelovirus.The m RNA level of LOX-1,STAT2 and RIG-I all upregulated significantly(p<0.01).Protein expression of the function genes changes progressively.Accompanied with virus proliferation,LOX-1 and STAT2 was increasing,while RIGI expression was downregulating.5.The effect of teschovirus infection on IPEC-J2 cell mechanismThe IPEC-J2 cell progressive changes from teschovirus were similar to sapelovirus.However,there were also discrepancy.The cytopathic effect of teschovirus was weaker and developed slower than sapelovirus.As a result,cell apoptosis progress,cytoskeleton rearrangement and cell cycle arrest were relevantly slow down.During teschovirus infection,the ROS level firstly increased gradually,later reduced,and then elevated rapidly.Besides,the transcription level of cytokines and function genes was lower in teschovirus infected IPEC-J2 cell.However,the protein expression level of the function genes was higher than sapelovirus.6.The effect of LOX-1 overexpression on IPEC-J2 cell mechanismLOX-1 takes a role in the physiology of IPEC-J2 cell.Overexpression of LOX-1 in IPEC-J2 cell induced cell death,led to cytoskeleton reorganization,arrest cell cycle,and accelerated NO formation as well as ROS production.In LOX-1 overexpressed cells,the transcription level of inflammatory cytokines TNF-α,IFN-α,TGF-β,NF-κΒ and IL-1α increased,and function genes RIG-I and STAT2 on both m RNA and expression level also upregulated.Inoculation of sapelovirus and teschovirus on LOX-1 overexpressed IPEC-J2 cells,virus production increased.Coexpressing LOX-1 with PLpro/3CLpro in IPEC-J2 cells,the transcription of PLpro/3CLpro also enhanced.Above all,after infected by enteroviruses from different species or genuses,intestinal cell display similar fuctional changes.The proliferation of torovirus,sapelovirus and teschovirus in IPEC-J2 cells all resulted in cellular ROS level increased,cytoskeleton rearranged,cell cycle arrested and the transcription of inflammatory cytokines and functional genes LOX-1,STAT2 and RIG-I elevated.There were also a certain of discrepancies.The similarity and discrepancy gave us evidences on digging the different pathogenesis of enteroviruses,and also paved us the way for exploring the effect of synergism or antagonism while enteroviruses coinfection.LOX-1 was verified to play a role in entervirues infection.LOX-1 prensented the similar cell functional changes,and accelerate the proliferation of torovirus,sapelovirus and teschovirus.