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. 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 (2Apro) of CVB3 were generated using a site-directed mutagenesis strategy. The cleavage efficiencies of eIF4G by CVB3 2Apro and its mutants were determined via western blotting assays. 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 2Apro of CVB3 was the key viral component that triggered SG formation. A 2Apro 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 2Apro to initiate SG formation. Finally, we observed that SGs also formed in EV71-infected cells. Expression of EV71 2Apro alone was also sufficient to cause SG formation. Both CVB3 and EV71 infections can induce SG formation, and 2Apro 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.

Objective: This study aimed to assess the efficacy and safety of Chinese herbal medicine (CHM) plus conventional western medicine (CWM) in comparison with CWM against COVID-19.Methods: We searched eight electronic databases and three trial registers spanning from January 1, 2020 to May 18, 2023. We included randomized controlled trials (RCTs) comparing the effectiveness and safety of CHM plus CWM and CWM against COVID-19 in our study. The Cochrane Risk of Bias tool 2.0 (RoB2) was applied to evaluate the methodological quality of the included RCTs. The Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) system was employed to assess the certainty of evidence. Statistical analysis was implemented in R version 4.1.2.Results: Our study included 50 RCTs involving 11,624 patients. In comparison with sole CWM, CHM plus CWM against COVID-19 significantly enhanced clinical effective rate (RR = 1.18, 95% CI [1.13, 1.22]), improved chest image (RR = 1.19, 95% CI [1.11, 1.28]), inhibited clinical deterioration (RR = 0.45, 95% CI [0.33, 0.60]), lowered mortality (RR = 0.53, 95% CI [0.40, 0.70]), and reduced the total score of TCM syndrome (SMD = −1.24, 95% CI [−1.82, −0.66]). SARS-CoV-2 nucleic acid conversion time (MD = −2.66, 95% CI [−3.88, −1.44]), duration of hospitalization (MD = −2.36, 95% CI [−3.89, −0.82]), and clinical symptom (fever, cough, fatigue, and shortness of breath) recovery times were shorter in CHM plus CWM groups than in CWM groups. Further, CHM plus CWM treatment was more conducive for some laboratory indicators returning to normal levels. No statistical difference was found in the incidence of total adverse reactions between the two groups (RR = 0.97, 95% CI [0.88, 1.07]). We assessed the risk of bias for 246 outcomes, and categorized 55 into “low risk”, 151 into “some concerns”, and 40 into “high risk”. Overall, the certainty of the evidence ranged from moderate to very low.Conclusions: Potentially, CHM listed in this study, as an adjunctive therapy, combining with CWM is an effective and safe therapy mode for COVID-19. However, more high-quality RCTs are needed to draw more accurate conclusions.Clinical trial registration: https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=293963.

Coxsackievirus B3 (CVB3) is a primary causative agent of viral myocarditis (VMC), which can lead to both acute and chronic cardiac inflammation accompanied by progressive heart failure and arrhythmias. Although CVB3 has been implicated in various forms of programmed cell death, whether it triggers necroptosis and the underlying mechanisms remains unclear. This study aimed to investigate the role and mechanism of CVB3-induced necroptosis and its effect on viral replication. Using both in vitro and in vivo models, we demonstrated that CVB3 infection significantly upregulates the expression of key necroptotic markers RIP1 and RIP3 in HeLa cells and mouse myocardial tissues. This upregulation was accompanied by elevated intracellular reactive oxygen species (ROS) levels and suppression of the Nrf2/HO-1 antioxidant pathway. Intervention with the necroptosis inhibitor Necrostatin-1 (Nec-1) or the ROS scavenger N-acetylcysteine (NAC) markedly attenuated cell death, suppressed viral replication, and ameliorated myocardial injury and inflammatory responses in infected mice. Mechanistically, CVB3 inhibits the Nrf2/HO-1 pathway, thereby inducing substantial ROS accumulation that promotes necroptosis. This effect can be reversed by NAC treatment. Our study reveals a novel mechanism through which CVB3 induces ROS-dependent necroptosis via the suppression of the Nrf2/HO-1 pathway, providing new insights into the pathogenesis of viral myocarditis and suggesting potential therapeutic strategies.

Viral myocarditis (VMC), predominantly driven by Coxsackievirus B3 (CVB3) infection and the resultant excessive immune response, lacks effective treatments and specific antiviral drugs in clinical practice. Chlorogenic acid (CGA) has been proven to have significant antiviral and anti-inflammatory properties. This study evaluated the potential and mechanism of action of CGA against CVB3-induced viral myocarditis. Our research results showed that CGA significantly alleviated myocardial tissue damage in vivo. This protective effect was accompanied by effective inhibition of myocardial inflammatory responses and viral replication. Further in vitro experiments confirmed that CGA significantly inhibited the replication of CVB3 in a dose-dependent manner, and its inhibitory effect mainly targeted the replication stage of the viral life cycle. Mechanistically, CGA treatment correlates with reduced ZBP1 expression and accelerated ZBP1 degradation involving the ubiquitin–proteasome pathway, accompanied by suppressed activation of PANoptosis markers. These findings suggest that CGA alleviates CVB3-induced myocarditis through concerted antiviral and anti-inflammatory effects, with ZBP1-mediated PANoptosis as a potential contributing mechanism.