Coxsackievirus B3 Infection Triggers Autophagy through 3 Pathways of Endoplasmic Reticulum Stress

Objective Autophagy is a highly conserved intracellular degradation pathway. Many picornaviruses induce autophagy to benefit viral replication, but an understanding of how autophagy occurs remains incomplete. In this study, we explored whether coxsackievirus B3(CVB3) infection induced autophagy through endoplasmic reticulum(ER) stress. Methods In CVB3-infected HeLa cells, the specific molecules of ER stress and autophagy were detected using Western blotting, reverse transcription polymerase chain reaction(RT-PCR), and confocal microscopy. Then PKR-like ER protein kinase(PERK) inhibitor, inositol-requiring protein-1(IRE1) inhibitor, or activating transcription factor-6(ATF6) inhibitor worked on CVB3-infected cells, their effect on autophagy was assessed by Western blotting for detecting microtubule-associated protein light chain 3(LC3). Results CVB3 infection induced ER stress, and ER stress sensors PERK/eIF2α, IRE1/XBP1, and ATF6 were activated. CVB3 infection increased the accumulation of green fluorescent protein(GFP)-LC3 punctuation and induced the conversion from LC3-Ⅰ to phosphatidylethanolamine-conjugated LC3-1(LC3-Ⅱ). CVB3 infection still decreased the expression of mammalian target of rapamycin(mTOR) and p-mTOR. Inhibition of PERK, IRE1, or ATF6 significantly decreased the ratio of LC3-Ⅱ to LC3-Ⅰ in CVB3-infected HeLa cells. Conclusion CVB3 infection induced autophagy through ER stress in HeL a cells, and PERK, IRE1, and ATF6 a pathways participated in the regulation of autophagy. Our data suggested that ER stress may inhibit mTOR signaling pathway to induce autophagy during CVB3 infection.

Exosomal let-7a-5p derived from human umbilical cord mesenchymal stem cells alleviates coxsackievirus B3-induced cardiomyocyte ferroptosis via the SMAD2/ZFP36 signal axis

Viral myocarditis(VMC)is one of the most common acquired heart diseases in children and teenagers.However,its pathogenesis is still unclear,and effective treatments are lacking.This study aimed to investigate the regulatory pathway by which exosomes alleviate ferroptosis in cardiomyocytes(CMCs)induced by coxsackievirus B3(CVB3).CVB3 was utilized for inducing the VMC mouse model and cellular model.Cardiac echocardiography,left ventricular ejection fraction(LVEF),and left ventricular fractional shortening(LVFS)were implemented to assess the cardiac function.In CVB3-induced VMC mice,cardiac insufficiency was observed,as well as the altered levels of ferroptosis-related indicators(glutathione) peroxidase 4(GPX4),glutathione(GSH),and malondialdehyde(MDA).However,exosomes derived from human umbilical cord mesenchymal stem cells(hucMSCs-exo)could restore the changes caused by CVB3 stimulation.Let-7a-5p was enriched in hucMSCs-exo,and the inhibitory ffect of hucMSCs-exoa-ie-pmimo on CVB3-induced ferroptosis was higher than that of hucMSCs-exommie N(NC:negative control).Mothers against decapentaplegic homolog 2(SMAD2)increased in the VMC group,while the expression of zinc-finger protein 36(ZFP36)decreased.Let-7a-5p was confirmed to interact with SMAD2 messenger RNA(mRNA),and the SMAD2 protein interacted directly with the ZFP36 protein.Silencing SMAD2 and overexpressing ZFP36 inhibited the expression of ferroptosis-related indicators.Meanwhile,the levels of GPX4,solute carrier family 7,member 11(SLC7A11),and GSH were lower in the SMAD2 overexpression plasmid(oe-SMAD2)+let-7a-5p mimic group than in the oe-NC+let-7a-5p mimic group,while those of MDA,reactive oxygen species(ROS),and Fe^(2+)increased.In conclusion,these data showed that ferroptosis could be regulated by mediating SMAD2 expression.Exo-let-7a-5p derived from hucMSCs could mediate SMAD2 to promote the expression of ZFP36,which further inhibited the ferroptosis of CMCs to alleviate CVB3-induced VMC.

Coxsackievirus B3 HFMD animal models in Syrian hamster and rhesus monkey

Coxsackievirus B3(CVB3)is the pathogen causing hand,foot and mouth disease(HFMD),which manifests across a spectrum of clinical severity from mild to severe.However,CVB3-infected mouse models mainly demonstrate viral myocarditis and pancreatitis,failing to replicate human HFMD symptoms.Although several enteroviruses have been evaluated in Syrian hamsters and rhesus monkeys,there is no comprehensive data on CVB3.In this study,we have first tested the susceptibility of Syrian hamsters to CVB3 infection via different routes.The results showed that Syrian hamsters were successfully infected with CVB3 by intraperitoneal injection or nasal drip,leading to nasopharyngeal colonization,acute severe pathological injury,and typical HFMD symptoms.Notably,the nasal drip group exhibited a longer viral excretion cycle and more severe pathological damage.In the subsequent study,rhesus monkeys infected with CVB3 through nasal drips also presented signs of HFMD symptoms,viral excretion,serum antibody conversion,viral nucleic acids and antigens,and the specific organ damages,particularly in the heart.Surprisingly,there were no significant differences in myocardial enzyme levels,and the clinical symptoms resembled those often associated with common,mild infections.In summary,the study successfully developed severe Syrian hamsters and mild rhesus monkey models for CVB3-induced HFMD.These models could serve as a basis for understanding the disease pathogenesis,conducting pre-trial prevention and evaluation,and implementing post-exposure intervention.

Pathogenesis of coxsackievirus B3-induced myocarditis： role of macrophage migration inhibitory factor

Background Macrophage migration inhibitory factor （MIF） is an upstream regulator in immune and inflammatory responses.However,its role in viral myocarditis remains unknown.In this study,we investigated the role of the MIF in coxsackievirus B3 （CVB3）-induced myocarditis.Methods Mice were randomized into two groups receiving either Eagle＇s minimal essential medium （EMEM,control group） or virus solution （infected group）.Subsets of mice in the infected group were sacrificed on days 3,7,14 and 28 after inoculation.Expression of MIF was detected using an enzyme-linked immunosorbent assay （ELISA）,reverse transcription polymerase chain reaction and immunohistochemistry.A neutralizing antibody （Ab） to MIF was injected intraperitoneally from day 0 to 7 after inoculation.Disease severity was estimated by histopathology of the heart and by the heart weight to body weight ratio,and the interleukin-1β （IL-1β） and tumor necrosis factor α （TNF-α） in the myocardium were measured by ELISA on day 14.Results The serum MIF concentration and expression levels of myocardial MIF mRNA and protein were significantly elevated in mice on days 7 and 14 post-infection.The survival rate was markedly higher and disease severity was obviously less in mice treated with anti-MIF Ab.Furthermore,MIF blockade significantly decreased the IL-1β and TNF-α in the myocarditic heart.Conclusion These results demonstrate that MIF is an important naturally occurring inflammatory cytokine in CVB3-induced myocarditis,and anti-MIF Ab may lessen the inflammatory response.

MicroRNA-324-3p Plays A Protective Role Against Coxsackievirus B3-Induced Viral Myocarditis

Viral myocarditis(VM) is an inflammatory disease of the myocardium associated with heart failure, which is caused by common viral infections. A majority of the infections are initiated by coxsackievirus B3(CVB3). Micro RNAs(mi RNAs)have a major role in various biological processes, including gene expression, cell growth, proliferation, and apoptosis, as well as viral infection and antiviral immune responses. Although, mi RNAs have been found to regulate viral infections,their role in CVB3 infection remains poorly understood. In the previous study, mi RNA microarray results showed that mi R-324-3 p expression levels were significantly increased when cells and mice were infected with CVB3. It was also found that miR-324-3p downregulated TRIM27 and decreased CVB3 replication in vitro and in vivo. In vitro, analysis of downstream signaling of TRIM27 revealed that, miR-324-3p inhibited CVB3 infection, and reduced cytopathic effect and viral plaque formation by reducing the expression of TRIM27. In vivo, miR-324-3p decreased the expression of TRIM27,reduced cardiac viral replication and load, thereby strongly attenuating cardiac injury and inflammation. Taken together,this study suggests that miR-324-3p targets TRIM27 to inhibit CVB3 replication and viral load, thereby reducing the cardiac injury associated with VM.

Antiviral Effects of Aqueous Extract from Spatholobus suberectus Dunn.against Coxsackievirus B3 in Mice

Objective： To investigate the antiviral effects of the aqueous extract of Spatholobus suberectus Dunn. （A.E.）, a Chinese medicinal herb, against coxsackievirus B3 （CVB3）. Methods： The antiviral effects of A.E. against CVB3 in vitro （primarily cultured myocardial cells） and in vivo （BALB/c mice） were determined. Serum pharmacological method was also adopted by in vitro experiments. The effects of A.E. inhibiting the CVB3 mRNA expression were compared by RT-PCR in mice in vivo. Results： A.E. exhibited obvious antiviral effects in vivo, and serum samples obtained from the rats with oral administration of A.E. （10 μg/mL, 5 μg/mL） reduced the virus titers in the infected myocardial cells （3.00±0.70, 3.55±0.52, P〈0.01）. Meanwhile, the viral myocarditis induced by CVB3 was inhibited significantly by A.E., and the 15-day mortality was reduced to 40% and 45% （P〈0.01） in mice treated with A.E. at doses of 50 mg/kg and 100 mg/kg, respectively, while the 30-day mortality was decreased to 45% and 50%, respectively （P〈0.01）. Moreover, the mRNA expression of Coxsackie virus B3 was significantly inhibited by A.E. Conclusion： Aqueous extract of Spatho/obus suberectus Dunn. （A.E.） has inhibitory effect on CVB3 both in vitro and in vivo.

Expression Profile and Function Analysis of Long Non-coding RNAs in the Infection of Coxsackievirus B3

The roles of IncRNAs in the infection of enteroviruses have been barely demonstrated.In this study,we used coxsackievirus B3(CVB3),a typical enterovirus,as a model to investigate the expression profiles and functional roles of IncRNAs in enterovirus infection.We profiled IncRNAs and mRNA expression in CVB3-infected HeLa cells by IncRNA-mRNA integrated microarrays.As a result,700 differentially expressed IncRNAs(431 up-regulated and 269 down-regulated)and 665 differentially expressed mRNAs(299 up-regulated and 366 down-regulated)were identified in CVB3 infection.Then we performed IncRNA-mRNA integrated pathway analysis to identify potential functional impacts of the differentially expressed mRNAs,in which IncRNA-mRNA correlation network was built.According to IncRNA-mRNA correlation,we found that XLOC-001188,an IncRNA down-regulated in CVB3 infection,was negatively correlated with NFAT5 mRNA,an anti-CVB3 gene reported previously.This interaction was supported by qPCR detection following siRNA-mediated knockdown of XLOC-001188,which showed an increase of NFAT5 mRNA and a reduction of CVB3 genomic RNA.In addition,we observed that four most significantly altered IncRNAs,SNHG11,RP11-145F16.2,RP11-1023L17.1 and RP11-1021N1.2 share several common correlated genes critical for CVB3 infection,such as BRE and IRF2BP1.In all,our studies reveal the alteration of IncRNA expression in CVB3 infection and its potential influence on CVB3 replication,providing useful information for future studies of enterovirus infection.

The effect of astragalus membranaceus in combination with taurine on coxsackievirus B3 murine myocarditis

Objective To evaluate the effect of astragalus membranaceus (AM) in combination with taurine on coxasckievirus B3 (CVB3) murine myocarditis. Methods One hundred and sixty mice were infected with CVB3 and equally divided into four groups: normal saline (NS) treated group, AM treated group, taurine treated group and AM combined with taurine treated group. The treatment were carried out from day 1 to 7 after infection. Ten mice were extracted from each group for observing mortality within day 21 postinfection, the rests were killed and the hearts were removed on day 8 (n=15) and 21 (all survival mice), respectively. Electrocardiograph (ECG) of each mouse was analysed before infection and repeated on the day of being killed. CVB3 RNA in murine myocardium was detected by in situ hybridization and myocardial lesions were observed by light microscope. Results Mortality was lower in drug treated groups than that in NS treated group, and it was the lowest in AM combined with taurine treated group, but all the differences were not significant (P>0.05). All drug treatments reduced the incidence of abnormal ECG changes in the acute phase of infection (P<0.05, vs NS treated group), but AM combined with taurine was the most effective in reducing the incidence of abnormal ECG (P<0.05, vs other groups). AM inhibited viral replication in myocardium at the early and late stage of murine myocarditis effectively and AM combined with taurine enhanced the effect of anti virus. All drugs decreased myocardial damage (P<0.05, vs control group), and the effect of AM combined with taurine was most significant (P<0.05). Conculsion The AM combined with taurine on CVB3 murine myocarditis is more effective than that of AM or taurine.

A Comparative Study on the Therapeutic Effect of Astragaloside (黄芪总皂甙) and Perindopril in Treating CVB_3-Infected Cardiomyocytes in Rats

Objective: To compare the therapeutic effects of Astragaloside and Perindopril on myocardial sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) activity and the SERCA type 2 mRNA level in Coxsackievirus B 3 (CVB 3) infected cardiomyocytes. Methods: Cultured cardiomyocytes of rats were divided into normal, model, Astragaloside and Perindopril groups. The model, Astragaloside and Perindopril groups were infected with CVB 3. Meanwhile, the Astragaloside and the Perindopril groups were treated with Astragaloside (10 μg/ml) and Perindopril (1.3 μg/ml) respectively. Cytopathic effect (CPE), cardiac troponin I ( cTnI) , the SERCA activity and mRNA level of the SERCA type 2 were observed after 96 hours. Results: The CPE and cTnI of model group were significantly higher than those of normal, Astragaloside and Perindopril groups ( P <0.01). The activity and the mRNA expression of myocardial SERCA of model group were significantly lower than those of normal, Astragaloside and Perindopril groups ( P <0.01-0.05). Compared with Astragaloside group, the CPE, cTnI of Perindopril group were higher and the activity and mRNA level of Perindopril group were lower. But there were no significant difference between the two groups ( P >0.05). Conclusion: Astragaloside and Perindopril were able to reverse the down regulations of cardiac SERCA activity and mRNA expression caused by virus infection to alleviate the cardiomyocyte injury.

Effect of Astragalus Membranaceus on Ca￣(2＋）Influx and CVB3 RNA Replication in Cultured Neonatal Rat Heart C

Here was investigated the effect of Radix Astragalus Membranaceus IAM) on Caz+ influxacross the myocardial plasma membrane and coxsackie virus B3 ( CVB3 ) -RNA replication in cultured neonatalrat heart cells infected with CVB3 . It was found that the Oa2+ intlux could be inhibited signiticantly by AM bothin heart cells intected with CVB3 for 48 hours and in normal control heart cells. In addition. the Caz+ intluxand the amounts of CVB3-RNA in rnyocytes simultaneously intected with CVB3 and treated with AM for 48hours were statistically decreased compared with that in CVB3-infected contrOI cells. These phenomena sug-gested that AM could exert the effects of decreasing the secondary Ca2+ damages, irnproving the abnormalmyocardial electric activity and inhibiting replication of CVB3-RNA in myocardium. Thus, it is a rationalchoice to treat patients with AM in viral myocarditis.

The nonstructural protein 2C of Coxsackie B virus has RNA helicase and chaperoning activities

RNA-remodeling proteins,including RNA helicases and chaperones,play vital roles in the remodeling of structured RNAs.During viral replication,viruses require RNA-remodeling proteins to facilitate proper folding and/or re-folding the viral RNA elements.Coxsackieviruses B3(CVB3)and Coxsackieviruses B5(CVB5),belonging to the genus Enterovirus in the family Picornaviridae,have been reported to cause various infectious diseases such as hand-foot-and-mouth disease,aseptic meningitis,and viral myocarditis.However,little is known about whether CVB3 and CVB5 encode any RNA remodeling proteins.In this study,we showed that 2C proteins of CVB3 and CVB5 contained the conserved SF3 helicase A,B,and C motifs,and functioned not only as RNA helicase that unwound RNA helix bidirectionally in an NTP-dependent manner,but also as RNA chaperone that remodeled structured RNAs and facilitated RNA strand annealing independently of NTP.In addition,we determined that the NTPase activity and RNA helicase activity of 2C proteins of CVB3 and CVB5 were dependent on the presence of divalent metallic ions.Our findings demonstrate that 2C proteins of CVBs possess RNA-remodeling activity and underline the functional importance of 2C protein in the life cycle of CVBs.

Effect of Prevention and Treatment of Murine Acute Viral Myocarditis and Protection of Lymphoid Organ Atrophy with Xinkang Oral Liquid (心康口服液)

ABSTRCAT Objective: The effect of prevention and treatment of Xinkang oral liquid (心康口服液, XKOL) on experimental coxsackievirus B 3 (CVB 3) myocarditis mice model were investigated. Methods: The mice were inoculated intraperitoneally with 0.3 ml of 10 5 TCID 50 of CVB 3 to induce acute viral myocarditis model. These mice were divided into model control group (Group A), prevention high dosage group (Group B) and prevention low dosage group (Group C), treatment high dosage group (Group D) and treatment low dosage group (Group E), respectively. In addition, XKOL control group (Group F) and normal control group (Group G) were not infected with CVB 3 intraperitoneally. The administration of XKOL in Group B and C began 2 days before virus infection. All animals were sacrificed on day 20 for evaluation. Results: Histological examination showed extensive myocardial necrosis and cell infiltration in most of Group A mice, but necrosis and cell infiltration were less severe in Group B,C,D and E mice. Thymus weight in Group B,C,D and E mice were heavier and less cell depletion occurred than those in Group A. Conclussion: The XKOL could effectively inhibit myocardial CVB 3 replication, reduce the myocardial inflammatory response, lower incidence rate of myocarditis and prevent the disease associated lymphoid organ atrophy in this animal models.