Antiviral Activity of trans-Hexenoic Acid against Coxsackievirus B and Enterovirus A71.

Enterovirus infections are life-threatening viral infections which occur mainly among children and are possible causes of viral outbreak. Until now, treatment and management of infections caused by members of the genus Enterovirus largely depended on supportive care, and no antiviral medications are currently approved for the treatment of most of these infections. The urgency of discovering new therapeutic options for the treatment of enterovirus infection is increasing. In the present study, we identified that trans-2-hexenoic acid (THA), a natural product from a dietary source, possesses antiviral activity against coxsackievirus B (CVB) and enterovirus A71 (EV-A71) in a dose-dependent manner. We found that THA possesses antiviral activity at 50% effective concentrations (EC50) of 2.9 µM and 3.21 µM against CVB3 and EV-A71 infections, respectively. The time of addition assay revealed that THA inhibits both CVB3 and EV-A71 replication at the entry stage of infection. Additional results from this study further suggest that THA inhibits viral replication by blocking viral entry. Given that THA has received approval as a food additive, treatment of enterovirus infections with THA might be a safe therapeutic option or could pave the way for semisynthetic manufacturing of more antiviral drugs in the future.

MiR-146a down-regulates inflammatory response by targeting TLR3 and TRAF6 in Coxsackievirus B infection.

Coxsackievirus B (CVB) is the major cause of human myocarditis and dilated cardiomyopathy. Toll-like receptor 3 (TLR3) is an intracellular sensor to detect pathogen's dsRNA. TLR3, along with TRAF6, triggers an inflammatory response through NF-κB signaling pathway. In the cells infected with CVB type 3 (CVB3), the abundance of miR-146a was significantly increased. The role of miR-146a in CVB infection is unclear. In this study, TLR3 and TRAF6 were identified as the targets of miR-146a. The elevated miR-146a inhibited NF-κB translocation and subsequently down-regulated proinflammatory cytokine expression in the CVB3-infected cells. Therefore, the NF-κB pathway can be doubly blocked by miR-146a through targeting of TLR3 and TRAF6. MiR-146a may be a negative regulator on inflammatory response and an intrinsic protective factor in CVB infection.

Coxsackievirus B3 induces the formation of autophagosomes in cardiac fibroblasts both in vitro and in vivo.

Coxsackievirus group B (CVB) is one of the common pathogens that cause myocarditis and cardiomyopathy. Evidence has shown that CVB replication in cardiomyocytes is responsible for the damage and loss of cardiac muscle and the dysfunction of the heart. However, it remains largely undefined how CVB would directly impact cardiac fibroblasts, the most abundant cells in human heart. In this study, cardiac fibroblasts were isolated from Balb/c mice and infected with CVB type 3 (CVB3). Increased double-membraned, autophagosome-like vesicles in the CVB3-infected cardiac fibroblasts were observed with electron microscope. Punctate distribution of LC3 and increased level of LC3-II were also detected in the infected cardiac fibroblasts. Furthermore, we observed that the expression of pro-inflammatory cytokines, IL-6 and TNF-α, was increased in the CVB3-infected cardiac fibroblasts, while suppressed autophagy by 3-MA and Atg7-siRNA inhibited cytokine expression. Consistent with the in vitro findings, increased formation of autophagosomes was observed in the cardiac fibroblasts of Balb/c mice infected with CVB3. In conclusion, our data demonstrated that cardiac fibroblasts respond to CVB3 infection with the formation of autophagosomes and the release of the pro-inflammatory cytokines. These results suggest that the autophagic response of cardiac fibroblasts may play a role in the pathogenesis of myocarditis caused by CVB3 infection.

MiR-10a* up-regulates coxsackievirus B3 biosynthesis by targeting the 3D-coding sequence.

MicroRNAs (miRNAs) are small non-coding RNAs that can posttranscriptionally regulate gene expression by targeting messenger RNAs. During miRNA biogenesis, the star strand (miRNA*) is generally degraded to a low level in the cells. However, certain miRNA* express abundantly and can be recruited into the silencing complex to regulate gene expression. Most miRNAs function as suppressive regulators on gene expression. Group B coxsackieviruses (CVB) are the major pathogens of human viral myocarditis and dilated cardiomyopathy. CVB genome is a positive-sense, single-stranded RNA. Our previous study shows that miR-342-5p can suppress CVB biogenesis by targeting its 2C-coding sequence. In this study, we found that the miR-10a duplex could significantly up-regulate the biosynthesis of CVB type 3 (CVB3). Further study showed that it was the miR-10a star strand (miR-10a*) that augmented CVB3 biosynthesis. Site-directed mutagenesis showed that the miR-10a* target was located in the nt6818-nt6941 sequence of the viral 3D-coding region. MiR-10a* was detectable in the cardiac tissues of suckling Balb/c mice, suggesting that miR-10a* may impact CVB3 replication during its cardiac infection. Taken together, these data for the first time show that miRNA* can positively modulate gene expression. MiR-10a* might be involved in the CVB3 cardiac pathogenesis.

Protease 2A induces stress granule formation during coxsackievirus B3 and enterovirus 71 infections.

BACKGROUND: Stress granules (SGs) are granular aggregates in the cytoplasm that are formed under a variety of stress situations including viral infection. Previous studies indicate that poliovirus, a member of Picornaviridae, can induce SG formation. However, the exact mechanism by which the picornaviruses induce SG formation is unknown. METHOD: The localization of SG markers in cells infected with coxsackievirus B3 (CVB3) or enterovirus 71 (EV71) and in cells expressing each viral protein was determined via immunofluorescence assays or plasmid transfection. Eight plasmids expressing mutants of the 2A protease (2A(pro)) of CVB3 were generated using a site-directed mutagenesis strategy. The cleavage efficiencies of eIF4G by CVB3 2A(pro) and its mutants were determined via western blotting assays. RESULTS: In this study, we found that CVB3 infection induced SG formation, as evidenced by the co-localization of some accepted SG markers in viral infection-induced granules. Furthermore, we identified that 2A(pro) of CVB3 was the key viral component that triggered SG formation. A 2A(pro) mutant with the G122E mutation, which exhibited very low cleavage efficiency toward eIF4G, significantly attenuated its capacity for SG induction, indicating that the protease activity was required for 2A(pro) to initiate SG formation. Finally, we observed that SGs also formed in EV71-infected cells. Expression of EV71 2A(pro) alone was also sufficient to cause SG formation. CONCLUSION: Both CVB3 and EV71 infections can induce SG formation, and 2A(pro) plays a crucial role in the induction of SG formation during these infections. This finding may help us to better understand how picornaviruses initiate the SG response.

Improving astaxanthin-loaded chitosan/polyvinyl alcohol/graphene oxide nanofiber membranes and their application in periodontitis.

Periodontitis is a chronic inflammatory disease primarily driven by host inflammation and plaque-induced immune responses. Controlling the host inflammatory response and improving the periodontal inflammatory microenvironment are crucial to promoting periodontal tissue regeneration. In this study, the blended nanofiber membranes previously prepared by our research group were improved, and we developed multifunctional chitosan/polyvinyl alcohol/graphene oxide/astaxanthin coaxial nanofiber membranes. Scanning electron microscopy showed that the prepared nanofibers had a smooth surface and a uniform diameter distribution. The mechanical property test results showed that the coaxial nanofiber membranes exhibited higher tensile strength compared to the blended nanofiber membranes, which increased from 4.50 ± 0.32 and 3.70 ± 0.45 MPa to 7.12 ± 0.22 and 5.62 ± 0.79 MPa respectively. Drug release studies indicated that the "shell-core" structure of coaxial nanofibers significantly reduced the initial burst release of astaxanthin (ASTA), with only 13.49 % and 10.71 % release in the first 24 h, and drug release lasted for over a week. Animal experiments confirmed that the coaxial nanofiber membranes loaded with ASTA promoted periodontal bone defect repair while inhibiting periodontal inflammation. In conclusion, the prepared coaxial nanofiber membranes are a promising sustained-release drug system for treating periodontitis.

Knockdown of Smox protects the integrity of the blood-brain barrier through antioxidant effect and Nrf2 pathway activation in stroke.

Once an ischemic stroke occurs, reactive oxygen species (ROS) and oxidative stress degrade the tight connections between cerebral endothelial cells resulting in their damage. The expression of antioxidant genes may be enhanced, and ROS formation may be reduced following Nrf2 activation, which is associated with protection against ischemic stroke. Overexpression of spermine oxidase (Smox) in the neocortex led to increased H2O2 production. However, how Smox impacts the regulation of the blood-brain barrier (BBB) through antioxidants has not been examined yet. We conducted experiments both in the cell level and in the transient middle cerebral artery occlusion (tMCAO) model to evaluate the effect of Smox siRNA lentivirus (si-Smox) knockdown on BBB protection against ischemic stroke. Mice treated with si-Smox showed remarkably decreased BBB breakdown and reduced endothelial inflammation following stroke. The treatment with si-Smox significantly elevated the Bcl-2 to Bax ratio and decreased the production of cleaved caspase-3 in the tMCAO model. Further investigation revealed that the neuroprotective effect was the result of the antioxidant properties of si-Smox, which reduced oxidative stress and enhanced CD31+ cells in the peri-infarct cortical areas. Of significance, si-Smox activated Nrf2 in both bEnd.3 cells and tMCAO animals, and blocking Nrf2 with brusatol diminished the protective effects of si-Smox. The study findings suggest that si-Smox exerts neuroprotective effects and promotes angiogenesis by activating the Nrf2 pathway, thus decreasing oxidative stress and apoptosis caused by tMCAO. As a result, si-Smox may hold potential as a therapeutic candidate for preserving BBB integrity while treating ischemic stroke.

Anisomycin inhibits Coxsackievirus B replication by promoting the lysosomal degradation of eEF1A1.

Group B Coxsackieviruses (CVB) are non-enveloped small RNA viruses in the genus Enterovirus, family Picornaviridae. CVB infection causes diverse conditions from common cold to myocarditis, encephalitis, and pancreatitis. No specific antiviral is available for the treatment of CVB infection. Anisomycin, a pyrrolidine-containing antibiotic and translation inhibitor, was reported to inhibit the replication of some picornaviruses. However, it is unknown if anisomycin can act as an antiviral against CVB infection. Here we observed that anisomycin showed potent inhibition on CVB type 3 (CVB3) infection with negligible cytotoxicity when applied at the early stage of virus infection. Mice infected with CVB3 showed markedly alleviated myocarditis with reduced viral replication. We found that CVB3 infection significantly increased the transcription of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1). CVB3 replication was suppressed by EEF1A1 knockdown, while elevated by EEF1A1 overexpression. Similar to the effect of CVB3 infection, EEF1A1 transcription was increased in response to anisomycin treatment. However, eEF1A1 protein level was decreased with anisomycin treatment in a dose-dependent manner in CVB3-infected cells. Moreover, anisomycin promoted eEF1A1 degradation, which was inhibited by the treatment of chloroquine but not MG132. We demonstrated that eEF1A1 interacted with the heat shock cognate protein 70 (HSP70), and eEF1A1 degradation was inhibited by LAMP2A knockdown, implicating that eEF1A1 is degraded through chaperone-mediated autophagy. Taken together, we demonstrated that anisomycin, which inhibits CVB replication through promoting the lysosomal degradation of eEF1A1, could be a potential antiviral candidate for the treatment of CVB infection.

The anti-inflammatory and osteogenic activity of chitosan/polyvinyl alcohol/graphene oxide/astaxanthin nanofibers membranes in vitro study.

Anti-inflammation and bone regeneration are the two major goals of periodontal therapy. We have demonstrated that chitosan (CS)/polyvinyl alcohol (PVA)/graphene oxide (GO)/astaxanthin (ASTA) nanofibers membranes prepared by electrospinning had favorable micro-morphology, good mechanical properties, and no cytotoxicity. In this study, CS/PVA/GO/ASTA nanofibers membranes were prepared to modulate both inflammatory response and osteogenic induction in vitro study. When the nanofibers membranes were co-cultured with RAW264.7 cells, glycoprotein nonmetastatic melanoma protein in the cells was highly expressed and RAW264.7 cells were polarized to M2 phenotype at the same time. In addition, following stimulation with nanofibers membranes, the messenger RNA (mRNA) and protein levels of Osteocalcin (OCN) and Runx2 in Bone marrow mesenchymal stem cells (BMSCs) were highly expressed. Taken together, these results suggested CS/PVA/GO/ASTA nanofibers membranes may promote the dissipation of inflammation and stimulate the differentiation of BMSCs into osteoblasts.

GPNMB plays an active role in the M1/M2 balance.

The phenotypic function of macrophages varies with the local microenvironment. Macrophages play an important role in the development of periodontitis. As one of the sources of GPNMB protein, the phenotype of macrophages is affected by GPNMB expression. In this study, activated macrophages were evaluated by flow cytometry, RT-qPCR and WB, and M2a macrophages had higher GPNMB expression than M0 and M1 macrophages. On this basis, a macrophage model with overexpression of GPNMB was established, and it was observed that GPNMB overexpression promoted the secretion of anti-inflammatory factors by macrophages and inhibited the secretion of pro-inflammatory factors by M1 macrophages.

Novel Antiviral Activity of Ethyl 3-Hydroxyhexanoate Against Coxsackievirus B Infection.

Coxsackievirus group B (CVB) is a member of the genus Enterovirus in the family Picornaviridae. CVB infection has been implicated as a major etiologic agent of viral myocarditis, dilated cardiomyopathy, meningitis, and pancreatitis among children and young adults. Until date, no antiviral agent has been licensed for the treatment of Coxsackievirus infection. In an effort to identify antiviral agents against diseases caused by the CVB, we found that ethyl 3-hydroxyhexanoate (EHX), a volatile compound present in fruits and food additives, is a potent antiviral compound. In this study, we demonstrated that EHX treatment significantly inhibits CVB replication both in vivo and in vitro. Furthermore, EHX possesses antiviral activity at 50% effective concentration (EC50) of 1.2 µM and 50% cytotoxicity (CC50) of 25.6 µM, yielding a selective index (SI) value as high as 20.8. Insights into the mechanism of antiviral activity of EHX showed that it acts at the step of viral RNA replication. Since EHX has received approval as food additives, treatment of CVB-related infections with EHX might be a safe therapeutic option and may be a promising strategy for the development of semi-synthetic antiviral drugs for viral diseases.

Circular RNA circ_0076631 promotes coxsackievirus B3 infection through modulating viral translation by sponging miR-214-3p.

Coxsackievirus B (CVB), a member of Enterovirus genus of Picornaviridae, is the leading pathogen of viral myocarditis and dilated cardiomyopathy. The pathogenesis of CVB-induced myocarditis has not been completely elucidated, and no specific antiviral measurement is available presently. Circular RNAs (circRNAs) have been reported to be able to modulate viral replication and infection through bridging over non-coding RNAs (ncRNAs) and coding messenger RNAs (mRNAs). To date, the role of circRNAs in CVB infection is largely unknown. In this study, we found that hsa_circ_0076631 (circ_0076631) significantly promoted CVB type 3 (CVB3) replication. Further study showed that the underneath mechanism was circ_0076631 indirectly interacting with CVB3 through sponging miR-214-3p, which targeted the 3D-coding region of CVB3 genome to suppress viral translation. Knocking down circ-0076631 caused a suppression of CVB3 infection; thus, circ-0076631 may be a potential target for anti-CVB therapy.

Destabilization of coxsackievirus b3 genome integrated with enhanced green fluorescent protein gene.

AIMS: To evaluate the stability of coxsackievirus B (CVB) genome integrated with the enhanced green fluorescent protein gene (egfp) and provide valuable information for the use of the recombinant CVB variant. METHODS: A CVB3 variant expressing eGFP was constructed by insertion of the egfp open-reading frame (ORF) at the 5' end of CVB3 ORF. The recombinant virus CVB3-eGFP was serially passaged in HeLa cells. The deletions in the CVB3-eGFP genome around egfp were examined by reverse transcription polymerase chain reaction and sequencing. RESULTS: Genomic deletions of CVB3-eGFP could be observed as early as the 2nd passage. Sequencing showed that the genomic deletions caused either viral ORF shifts or partial deletions of the viral VP4 coding sequence. The 6th passage of CVB3-eGFP was checked by plaque assay for eGFP expression. All plaque-like foci showed eGFP expression. eGFP expression was also viewed in HeLa cells infected with plaque-forming viruses. CONCLUSIONS: The insertion of egfp destabilized the CVB3 genome. The genomic deletions led to lethal mutations because of the termination of viral protein synthesis due to viral ORF shift and loss of partial viral gene. These findings imply that experimental data based on CVB integrated with the reporter gene should be interpreted with caution.

TAR DNA-Binding Protein 43 is Cleaved by the Protease 3C of Enterovirus A71.

Enterovirus A71 (EV-A71) is one of the etiological pathogens leading to hand, foot, and mouth disease (HFMD), which can cause severe neurological complications. The neuropathogenesis of EV-A71 infection is not well understood. The mislocalization and aggregation of TAR DNA-binding protein 43 (TDP-43) is the pathological hallmark of amyotrophic lateral sclerosis (ALS). However, whether TDP-43 was impacted by EV-A71 infection is unknown. This study demonstrated that TDP-43 was cleaved during EV-A71 infection. The cleavage of TDP-43 requires EV-A71 replication rather than the activated caspases due to viral infection. TDP-43 is cleaved by viral protease 3C between the residues 331Q and 332S, while mutated TDP-43 (Q331A) was not cleaved. In addition, mutated 3C which lacks the protease activity failed to induce TDP-43 cleavage. We also found that TDP-43 was translocated from the nucleus to the cytoplasm, and the mislocalization of TDP-43 was induced by viral protease 2A rather than 3C. Taken together, we demonstrated that TDP-43 was cleaved by viral protease and translocated to the cytoplasm during EV-A71 infection, implicating the possible involvement of TDP-43 in the pathogenesis of EV-A71infection.

Blocking of EGFR Signaling Is a Latent Strategy for the Improvement of Prognosis of HPV-Induced Cancer.

Human papillomavirus (HPV) is a double-stranded DNA (dsDNA) virus, and its high-risk subtypes increase cancer risks. However, the mechanism of HPV infection and pathogenesis still remain unclear. Therefore, understanding the molecular mechanisms and the pathogenesis of HPV are crucial in the prevention of HPV-related cancers. In this study, we analyzed cervix squamous cell carcinoma (CESC) and head and neck carcinoma (HNSC) combined data to investigate various HPV-induced cancer common features. We showed that epidermal growth factor receptor (EGFR) was downregulated in HPV-positive (HPV+) cancer, and that HPV+ cancer patients exhibited better prognosis than HPV-negative (HPV-) cancer patients. Our study also showed that TP53 mutation rate is lower in HPV+ cancer than in HPV- cancer and that TP53 can be modulated by HPV E7 protein. However, there was no significant difference in the expression of wildtype TP53 in both groups. Subsequently, we constructed HPV-human interaction network and found that EGFR is a critical factor. From the network, we also noticed that EGFR is regulated by HPV E7 protein and hsa-miR-944. Moreover, while phosphorylated EGFR is associated with a worse prognosis, EGFR total express level is not significantly correlated with prognosis. This indicates that EGFR activation will induce a worse outcome in HPV+ cancer patients. Further enrichment analysis showed that EGFR downstream pathway and cancer relative pathway are diversely activated in HPV+ cancer and HPV- cancer. In summary, HPV E7 protein downregulates EGFR that downregulates phosphorylated EGFR and inhibit EGFR-related pathways which in turn and consequently induce better prognosis.

miR-122 affects the viability and apoptosis of hepatocellular carcinoma cells.

OBJECTIVE. miR-122 is highly abundant in liver and a hepato-specific microRNA. There is evidence to show that miR-122 expression is down-regulated in human hepatocellular carcinoma (HCC). It is not known whether miR-122 affects the cellular behavior of hepatoma cells. The aim of this study was to investigate the effects of miR-122 on the viability and apoptosis of hepatoma cells. MATERIAL AND METHODS. The viability and apoptosis of Huh-7 and HepG2 cells treated with miR-122 or miR-122 antisense RNA (anti-miR-122) were analyzed by adenosine triphosphate (ATP)-based luminescent assay, annexin V-based flow cytometry, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) detection. The miR-122 coding genes in both cell lines were sequenced. RESULTS. Although two putative promoter sequences for the miR-122 gene at 18q21.31 were detected, the miR-122 coding sequence was missing in HepG2 cells, which might be the reason for the absence of miR-122 expression. There was no significant difference between the viabilities of HepG2 cells transfected with miR-122 and mock HepG2 cells (p >0.05). However, the viability of Huh-7 transfected with anti-miR-122 was significantly elevated at 24, 36, and 48 h posttransfection compared with that of mock cells (p <0.01). Both the flow cytometry and TUNEL assay showed that the apoptotic level of Huh-7 transfected with anti-miR-122 was significantly decreased at 48 h posttransfection (p <0.01). CONCLUSIONS. miR-122 down-regulated the viability but up-regulated the apoptosis of hepatoma cell Huh-7. The absence of miR-122 expression in HepG2 cells was due to the loss of the miR-122 coding sequence in chromosome 18. These results imply that aberrant expression of miR-122 may contribute to hepatocarcinogenesis.

Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB) Ameliorates the Inflammatory Response in Periodontal Disease.

Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a type I transmembrane protein that can modulate osteoblasts and bone mineralization. Periodontal disease (PD) is characterized by gum inflammation, alveolar bone resorption, and tooth loss. In this study, we found that GPNMB is highly expressed in inflamed periodontal tissue through microarray and immunohistochemistry (IHC) assays. The role of GPNMB in the pathogenesis of PD was evaluated with primary human periodontal ligament cells (hPDLCs) treated with lipopolysaccharide (LPS) and a GPNMB-expressing lentivirus (lenti-GP). In the hPDLCs treated with LPS and lenti-GP, the expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 was suppressed and that of IL-10 was upregulated. GPNMB significantly decreased apoptosis in the hPDLCs treated with LPS. GPNMB could upregulate the expression of Jumonji domain-containing protein 3 (Jmjd3), a histone 3 lysine 27 (H3K27) demethylase that is linked to the modulation of the inflammatory response and apoptosis. Taken together, our data find that GPNMB is highly expressed in gum tissue with PD and may be an anti-inflammatory player in the pathogenesis of PD.

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

The roles of lncRNAs 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 lncRNAs in enterovirus infection. We profiled lncRNAs and mRNA expression in CVB3-infected HeLa cells by lncRNA-mRNA integrated microarrays. As a result, 700 differentially expressed lncRNAs (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 lncRNA-mRNA integrated pathway analysis to identify potential functional impacts of the differentially expressed mRNAs, in which lncRNA-mRNA correlation network was built. According to lncRNA-mRNA correlation, we found that XLOC-001188, an lncRNA 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 lncRNAs, 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 lncRNA expression in CVB3 infection and its potential influence on CVB3 replication, providing useful information for future studies of enterovirus infection.

Aberrant PD-1 ligand expression contributes to the myocardial inflammatory injury caused by Coxsackievirus B infection.

Coxsackievirus group B (CVB) is considered as one of the most common pathogens of human viral myocarditis. CVB-induced myocarditis is mainly characterized by the persistence of the virus infection and immune-mediated inflammatory injury. Costimulatory signals are crucial for the activation of adaptive immunity. Our data reveal that the CVB type 3 (CVB3) infection altered the expression profile of costimulatory molecules in host cells. CVB3 infection caused the decrease of PD-1 ligand expression, partially due to the cleavage of AU-rich element binding protein AUF1 by the viral protease 3Cpro, leading to the exacerbated inflammatory injury of the myocardium. Moreover, systemic PD-L1 treatment, which augmented the apoptosis of proliferating lymphocytes, alleviated myocardial inflammatory injury. Our findings suggest that PD1-pathway can be a potential immunologic therapeutic target for CVB-induced myocarditis.

Mutations at nucleotides 573 and 579 within 5'-untranslated region augment the virulence of coxsackievirus B1.

Two mutants of coxsackievirus B1 (CVB1), CVB1c and CVB1e, with mutations in the stem-loop H of 5'-UTR were generated by site-directed mutagenesis. The A at nt579 of CVB1c was substituted by G. The U at nt573 and A at nt579 of CVB1e were substituted by A and G. The virulences of these mutants had been assessed by means of cytopathic effect (CPE), plaque formation, one-step growth curve, and 50% lethal dose (LD50) assays. The pathogenesis of these mutants was evaluated by attacking suckling Balb/c mice. Plaque assay and one-step growth curve showed that the replication of CVB1c and CVB1e on HeLa cells was significantly faster than that of their prototype CVB1n. Data of CPE assay, LD50, and pathological examination showed that CVB1c and CVB1e were more virulent than CVB1n. These data showed that mutation at nt579 (A-->G) alone and mutations at nt579 (A-->G) and nt573 (U-->A) together within 5'-UTR caused significant augment of the virulence and pathogenesis of coxsackievirus B1, and suggested that nt573 and nt579 might be molecular determinants for the virulence of coxsackievirus B1.