Coxsackievirus B3 Research Articles

Antibody-dependent enhancement and neutralization against CVB4 investigated in vitro and in silico through an agent-based model.

The infection with coxsackievirus B4 (CVB4) can be enhanced in vitro by antibodies directed against the viral capsid protein VP4. In peripheral blood mononuclear cells, antibody-dependent enhancement (ADE) of CVB4 infection leads to the production of interferon alpha (IFN-α). To investigate ADE of CVB4-induced production of IFN-α, an agent-based model was constructed with enhancing and neutralizing antibodies. The model recapitulates viral neutralization and ADE in silico. The enhancing and neutralizing activities of serum samples were evaluated in vitro to confront the model predictions with experimental results. Increasing the incubation time of CVB4 with serum samples improves virus neutralization in silico as well as in vitro. It also results in ADE at lower antibody numbers in silico, which is confirmed in vitro with IFN-α production at lower serum concentrations. Furthermore, incubation of CVB4 with serum at a low temperature does not induce IFN-α production in vitro. Thus, taken together our results suggest that enhancing antibodies bind cryptic epitopes, more accessible with longer incubation time and at higher temperature due to changes in capsid conformation, consistent with previous results indicating that enhancing antibodies are anti-VP4 antibodies.

Parkin plays a crucial role in acute viral myocarditis by regulating mitophagy activity.

Rationale: Parkin (an E3 ubiquitin protein ligase) is an important regulator of mitophagy. However, the role of Parkin in viral myocarditis (VMC) remains unclear. Methods: Coxsackievirus B3 (CVB3) infection was induced in mice to create VMC. Cardiac function and inflammatory response were evaluated by echocardiography, histological assessment, and molecular analyses. AAV9 (adeno-associated virus 9), transmission electron microscopy (TEM) and western blotting were used to investigate the mechanisms by which Parkin regulates mitophagy and cardiac inflammation. Results: Our data indicated that Parkin- and BNIP3 (BCL2 interacting protein 3 like)-mediated mitophagy was activated in VMC mice and neonatal rat cardiac myocytes (NRCMs) infected with CVB3, which blocked autophagic flux by inhibiting autophagosome-lysosome fusion. Parkin silencing aggravated mortality and accelerated the development of cardiac dysfunction in CVB3-treated mice. While silencing of Parkin did not significantly increase inflammatory response through activating NF-κB pathway and production of inflammatory cytokines post-VMC, the mitophagy activity were reduced, which stimulated the accumulation of damaged mitochondria. Moreover, Parkin silencing exacerbated VMC-induced apoptosis. We consistently found that Parkin knockdown disrupted mitophagy activity and inflammatory response in NRCMs. Conclusion: This study elucidated the important role of Parkin in maintaining cardiac function and inflammatory response by regulating mitophagy activity and the NF-κB pathway during acute VMC. Although the functional impact of mitophagy remains unclear, our findings suggest that Parkin silencing may accelerate VMC development.

Antiviral activity of sulphated specialized metabolites from sea urchin Clypeaster humilis: in vitro and in silico studies.

Chemical investigations of the sea urchin Clypeaster humilis has led to separation of twelve compounds including one new sulfonic acid derivative (7R) tridec-1-en-7-yl hydrogen sulphate (1), first isolated from natural source, pyridine-3-yl methane sulfonate (2), and first isolated from marine organisms, boldine (12), in addition to nine known compounds (3-11), which were isolated for the first time from the genus Clypeaster. Their structures were elucidated based on spectroscopic analyses (1D and 2D NMR), HR-ESI-MS as well as comparison with the previously reported data. The antiviral activity of the crude extract and sulphated compounds were evaluated using MTT colorimetric assay against Coxsackie B4 virus. The crude extract and compound 1 showed very potent antiviral activity with a percentage of inhibition equal to 89.7 ± 0.53% and 86.1 ± 0.92%, respectively. Results of the molecular docking analysis of the isolated compounds within Coxsackie Virus B4 (COX-B4) X-ray crystal structure and quantum chemical calculation for three sulphated compounds are in a consistent adaptation with the in vitro antiviral results. The pharmacokinetic properties (ADME) of isolated compounds were determined.

Hsa-circ-ACSL1 Enhances Apoptosis and Autophagy in Myocarditis Cardiomyocytes Through the miR-7-5p/XBP1 Axis.

Viral myocarditis (VMC) is a common cardiovascular disease, and circular RNAs (circRNAs) have been identified to play an important role in the pathophysiology of cardiovascular disease. However, the clinical significance, biological functions, and regulatory mechanisms of circRNAs in VMC remain poorly understood. Therefore, this study explored the biological functions and regulatory mechanisms of circ-ACSL1 in VMC. The animal and cell models of VMC were established by infecting BABL/C mice and interleukin-2 cells with coxsackievirus B3 (CVB3). Pro-inflammatory factors, markers of myocardial injury, apoptosis, and autophagy were detected to evaluate the degree of myocardial inflammation and myocardial injury after altering circ-ACSL1, microRNA-7-5p (miR-7-5p), and X-box binding protein 1 (XBP1) expression alone or in combination. Knocking down circ-ACSL1 could inhibit inflammation, autophagy, and apoptosis in VMC animals and cells. Mechanistically, circ-ACSL1 targeted miR-7-5p to regulate the downstream target XBP1. In addition, depleting miR-7-5p rescued the therapeutic effect of depleting circ-ACSL1. Overexpression of circ-ACSL1 aggravated VMC; however, this effect was saved by knocking down XBP1. By competitively absorbing miR-7-5p, circ-ACSL1 increases XBP1 expression and aggravates myocardial inflammation. Meaningfully, VMC treatment may benefit from circ-ACSL1 as a potential biomarker for precise diagnosis and as a potential therapeutic target.

Novel intertypic recombination of Echovirus 11 in the Enterovirus species B.

Enteroviruses (EVs), single-stranded, positive-sense RNA viruses, can be classified into four species (A-D), which have previously been linked to a diverse range of disease manifestations and infections affecting the central nervous system. In the Enterovirus species B (EV-B), Echovirus type 11 (E11) has been observed to occasionally circulate in Taiwan, which was responsible for an epidemic of enterovirus infections in 2018. Here, 48 clinical specimens isolated in 2003, 2004, 2009, and 2018 were collected for the high-throughput sequencing. Notably, we identified 2018 Taiwanese strains having potential recombinations in the 3D gene, as well as one 2003 strain having a double recombination with E6 and Coxsackievirus B5 in the P2 and P3 regions, respectively. Additionally, one amino acid signature mutated from the Histidine (H) in throat swab specimens to the Tyrosine (Y) in cerebral spinal fluid specimens was detected at position 1496 (or 57) of the genomic coordinate (or 3A gene) to further demonstrate intra-host evolution in different organs. In conclusion, this study identifies potential intertypic recombination events and an intra-host signature mutation in E11 strains, isolated during a 2018 neurological disease outbreak in Taiwan, contributing to our understanding of its evolution and pathogenesis.

Bioactive potentials of the truffle mushrooms Tirmania nivea, Tirmania pinoyi and Tuber indicum

Background Truffles are ectomycorrhizal wild mushrooms that have ethnomycological importance as a folklore remedy used to treat different skin and eye diseases and they are currently known as expensive food and potential sources of bioactive compounds. Objective This study aims to investigate the in vitro bioactive potential of the ethyl acetate extracts of three truffle species Tirmania nivea, Tirmania pinoyi, and Tuber indicum collected from Egypt, Saudi Arabia, and China. Results and discussion Inhibition of α-glucosidase was investigated as an indication of the antidiabetic potential of extracts. Tuber indicum extract caused 21.7% inhibition at a concentration of 100ppm. On the other hand, inhibition of nitric oxide is evaluated as a key way to regulate inflammation. Tuber indicum extract at a concentration of 100μg/ml achieved the highest inhibition (50.2%) and had the lowest IC50 (86.0±0.09μg/ml) among tested extracts, while Tirmania pinoyi extract achieved the lowest inhibition (21.7%) with the highest IC50 (104.1±0.12μg/ml). Cell migration was used to evaluate the wound healing activity of extracts. Tirmania pinoyi extract at a concentration of 100μg/ml caused 74.71% wound closure followed by 73.43% and 62.38% by Tuber indicum and Tirmania nivea extracts, respectively. Finally, investigating the antiviral potential of extracts against coxsackie virus B3 revealed that Tirmania pinoyi extract showed the highest virucidal effect and pretreatment antiviral activity while Tuber indicum extract achieved the highest post-treatment antiviral activity. Results achieved by these truffles encourage further investigations to be used as functional foods or as sources of bioactive compounds.

Complement components regulates ferroptosis in CVB3 viral myocarditis by interatction with TFRC

BackgroundDysregulated cell death machinery and an excessive inflammatory response in Coxsackievirus B3(CVB3)-infected myocarditis are hallmarks of an abnormal host response. Complement C4 and C3 are considered the central components of the classical activation pathway and often participate in the response process in the early stages of virus infection. MethodsIn our study, we constructed a mouse model of CVB3-related viral myocarditis via intraperitoneal injection of Fer-1 and detected myocarditis and ferroptosis markers in the mouse myocardium. Then, we performed co-IP and protein mass spectrometry analyses to explore which components interact with the ferroptosis gene transferrin receptor (TFRC). Finally, functional experiments were conducted to verify the role of complement components in regulating ferroptosis in CVB3 infection. ResultsIt showed that the ferroptosis inhibitor Fer-1 could alleviate the inflammation in viral myocarditis as well as ferroptosis. Mechanistically, during CVB3 infection, the key factor TFRC was activated and inhibited by Fer-1. Fer-1 effectively prevented the consumption of complement C3 and overload of the complement product C4b. Interestingly, we found that TFRC directly interacts with complement C4, leading to an increase in the product of C4b and a decrease in the downstream complement C3. Functional experiments have also confirmed that regulating the complement C4/C3 pathway can effectively rescue cell ferroptosis caused by CVB3 infection. ConclusionsIn this study, we found that ferroptosis occurs through crosstalk with complement C4 in viral myocarditis through interaction with TFRC and that regulating the complement C4/C3 pathway may rescue ferroptosis in CVB3-infected cardiomyocytes.

Analysis of the improvement effect of Astragalus extract on oxidative stress injury in viral myocarditis through STAT3/IL-6.

The objective of this study was to investigate the improvement effect of Astragalus (AS) extract on oxidative stress (OS) and inflammatory response of myocarditis (MYO) cells through the STAT3/IL-6 axis. For this purpose, The MYO model cells prepared by intervening cardiomyocyte HL-1 with Coxsackievirus B3 (CVB3) were divided into four groups: model group, as well as high- (H-), medium- (M-) and low-dose (L-) AS groups treated by 80, 40, and 20 μg/mL AS, respectively. Conventionally cultured cells were set as the normal group. Cell multiplication and apoptosis, as well as levels of Myocardial injury markers (cTnT, BNP and CK), inflammatory cytokines (ICs; TNF-α, IL-1β and IL-6) and OS indices (SOD, GSH-Px and MDA), were measured. STAT3/IL-6 pathway expression was also observed. Results showed that the model group presented decreased cell multiplication than the normal group, but with increased myocardialinjury, apoptosis rate, Caspase3 protein, ICs and OS reaction (P < 0.05); In the three AS-intervened groups, enhanced cell multiplication, while reduced myocardialinjury, apoptosis rate, ICs and OS response were observed, especially in H-AS group (P < 0.05). Besides, STAT3 and IL-6 concentrations, statistically increased in the model group, were reduced by AS intervention (P < 0.05). Colivelin, a specific activator of STAT3, further aggravated the apoptosis, inflammatory reaction and OS response of MYO cells (P < 0.05), but its impacts on MYO cells could be reversed by AS. In conclusion, AS can ameliorate MYO, and its mechanism is related to the inhibition of cellular inflammatory response and OS response through the STAT3/IL-6 axis.

M2 macrophage exosome-derived lncRNA AK083884 protects mice from CVB3-induced viral myocarditis through regulating PKM2/HIF-1α axis mediated metabolic reprogramming of macrophages

Viral myocarditis (VM) is a clinically common inflammatory disease. Accumulating literature has indicated that M2 macrophages protect mice from Coxsackievirus B3 (CVB3)-induced VM. However, mechanisms that underlie M2 macrophages alleviating myocardial inflammation remain largely undefined. We found that M2 macrophage-derived exosomes (M2-Exo) can effectively attenuate VM. The long non-coding RNA (lncRNA) AK083884 in M2-Exo was found to be involved in the regulation of macrophage polarization by exosome lncRNA sequencing combined with in vitro functional assays. M2-Exo-derived AK083884 promotes macrophage M2 polarization and protects mice from CVB3-induced VM. Furthermore, we identified pyruvate kinase M2 (PKM2) as a protein target binding to AK083884 and found that PKM2 knockdown could promote macrophages to polarize to M2 phenotype. Intriguingly, functional assay revealed that downregulation of AK083884 promotes metabolic reprogramming in macrophages. In addition, co-immunoprecipitation was performed to reveal AK083884 could interact with PKM2 and inhibition of AK083884 can facilitate the binding of PKM2 and HIF-1α. Collectively, our findings uncovered an important role of M2-Exo-derived AK083884 in the regulation of macrophage polarization through metabolic reprogramming, identified a new participant in the development of VM and provided a potential clinically important therapeutic target.

PHENOTYPIC AND GENETIC DIVERSITY OF ENTEROVIRUSES IN THE REPUBLIC OF BELARUS, 2003-2022

Enteroviruses (EV) are widespread viral agents that regularly cause epidemic outbreaks of diseases that are characterized by a variety of clinical manifestations. The aim of this work was to study the phenotypic and genotypic diversity of non-poliomyelitis EVs that circulated from 2003 to 2022 in our country during epidemic upsurges and decreases of enteroviral morbidity. It was established that during this period there was a circulation of 44 different types of EVs belonging to the species Enterovirus B (97.8%), Enterovirus A (1.88%), Enterovirus C (0.9%), Enterovirus D (0.4 %). Among Enterovirus B viruses, Coxsackievirus B5, ECHO 30, and ECHO11 prevailed. During the years of epidemic rises of morbidity, the ECHO 30, ECHO 9 and ECHO 11 viruses were significantly more often identified (p&lt; 0.001), while in the years of decreases Coxsackievirus B1, B2, B3 and B4 were detected more often (p&lt; 0.001). ECHO 6 and Coxsackievirus B5 viruses were registered both during periods of epidemic ups and downs and were characterized by the maximum intratype genetic diversity, significantly greater than ECHO30, ECHO6 and ECHO 11 (p&lt; 0.05). During the observation period, 9 genetic variants of ECHO 30, 6 – of ECHO 9, 3 – of ECHO 11, 6 – of ECHO 6 and 13 – of Coxsackievirus B5 were identified. An analysis of the genetic diversity of ECHO 30, 9, 11, 6, Coxsackie B5 viruses showed that the upsurges of enteroviral morbidity was accompanied by the maximum number of detected genetic variants and some of them were new to the country's population. The data obtained indicate the multifactorial nature of the formation of the epidemic process and a significant contribution to its formation of the genetic diversity of pathogens.

GPR15-mediated T cell recruitment during acute viral myocarditis facilitated virus elimination and improved outcome

Viral myocarditis is characterized by infiltration of mononuclear cells essential for virus elimination. GPR15 has been identified as a homing receptor for regulatory T cells in inflammatory intestine diseases, but its role in inflammatory heart diseases is still elusive. Here we show that GPR15 deficiency impairs coxsackievirus B3 elimination, leading to adverse cardiac remodeling and dysfunction. Delayed recruitment of regulatory T cells in GPR15-deficient mice was accompanied by prolonged persistence of cytotoxic and regulatory T cells. In addition, RNA sequencing revealed prolonged inflammatory response and altered chemotaxis in knockout mice. In line, we identified GPR15 and its ligand GPR15L as an important chemokine receptor–ligand pair for the recruitment of regulatory and cytotoxic T cells. In summary, the insufficient virus elimination might be caused by a delayed recruitment of T cells as well as delayed interferon-γ expression, resulting in a prolonged inflammatory response and an adverse outcome in GPR15-deficient mice.

Antiviral Mechanisms of Saucerneol from Saururus chinensis against Enterovirus A71, Coxsackievirus A16, and Coxsackievirus B3: Role of Mitochondrial ROS and the STING/TKB-1/IRF3 Pathway.

Enterovirus A71 (EV71), coxsackievirus A16 (CVA16), and coxsackievirus B3 (CVB3) are pathogenic members of the Picornaviridae family that cause a range of diseases, including severe central nervous system complications, myocarditis, and pancreatitis. Despite the considerable public health impact of these viruses, no approved antiviral treatments are currently available. In the present study, we confirmed the potential of saucerneol, a compound derived from Saururus chinensis, as an antiviral agent against EV71, CVA16, and CVB3. In the in vivo model, saucerneol effectively suppressed CVB3 replication in the pancreas and alleviated virus-induced pancreatitis. The antiviral activity of saucerneol is associated with increased mitochondrial ROS (mROS) production. In vitro inhibition of mROS generation diminishes the antiviral efficacy of saucerneol. Moreover, saucerneol treatment enhanced the phosphorylation of STING, TBK-1, and IRF3 in EV71- and CVA16-infected cells, indicating that its antiviral effects were mediated through the STING/TBK-1/IRF3 antiviral pathway, which was activated by increased mROS production. Saucerneol is a promising natural antiviral agent against EV71, CVA16, and CVB3 and has potential against virus-induced pancreatitis and myocarditis. Further studies are required to assess its safety and efficacy, which is essential for the development of effective antiviral strategies against these viruses.

Acetylation of FOXO1 activates Bim expression involved in CVB3 induced cardiomyocyte apoptosis

Viral myocarditis (VMC) is the major reason for sudden cardiac death among both children and young adults. Of these, coxsackievirus B3 (CVB3) is the most common causative agent of myocarditis. Recently, the role of signaling pathways in the pathogenesis of VMC has been evaluated in several studies, which has provided a new perspective on identifying potential therapeutic targets for this hitherto incurable disease. In the present study, in vivo and in vitro experiments showed that CVB3 infection leads to increased Bim expression and triggers apoptosis. In addition, by knocking down Bim using RNAi, we further confirmed the biological function of Bim in apoptosis induced by CVB3 infection. We additionally found that Bim and forkhead box O1 class (FOXO1) inhibition significantly increased the viability of CVB3-infected cells while blocking viral replication and viral release. Moreover, CVB3-induced Bim expression was directly dependent on FOXO1 acetylation, which is catalyzed by the co-regulation of CBP and SirTs. Furthermore, the acetylation of FOXO1 was an important step in Bim activation and apoptosis induced by CVB3 infection. The findings of this study suggest that CVB3 infection induces apoptosis through the FOXO1 acetylation-Bim pathway, thus providing new insights for developing potential therapeutic targets for enteroviral myocarditis.

Broad-spectrum virucidal activity of a hydroalcoholic extract of grape pomace

Grape pomace is a by-product of winemaking often disposed of before full exploitation. Indeed, this natural product holds great potential for sustainable utilization. In this study, dried grape pomace was reprocessed to assess its potential as a valuable source of antiviral metabolites. The antiviral activity of the extract was assessed against Herpes Simplex virus type-2, Feline Calicivirus, Coxsackievirus B5, and Influenza type-A virus, revealing potent broad virucidal effect. Indeed, it abolished viral infection when the extract was incubated with viruses before or during infection, whereas no significant toxic effects were observed up to 3 mg/mL.UHPLC-MS/MS analysis identified quercetin, gallic acid, and ethyl gallate as its primary constituents. These did not fully account for its virucidal action, suggesting the presence of additional active components and/or synergistic effect of more compounds.Thus, grape pomace may be exploited as a sustainable source for the preparation of environmentally friendly broad-spectrum virucidal disinfectants.

Investigating the virulence of coxsackievirus B6 strains and antiviral treatments in a neonatal murine model

Coxsackievirus B6 (CVB6), a member of the human enterovirus family, is associated with severe diseases such as myocarditis in children. However, to date, only a limited number of CVB6 strains have been identified, and their characterization in animal models has been lacking. To address this gap, in this study, a neonatal murine model of CVB6 infection was established to compare the replication and virulence of three infectious-clone-derived CVB6 strains in vivo. The results showed that following challenge with a lethal dose of CVB6 strains, the neonatal mice rapidly exhibited a series of clinical signs, such as weight loss, limb paralysis, and death. For the two high-virulence CVB6 strains, histological examination revealed myocyte necrosis in skeletal and cardiac muscle, and immunohistochemistry confirmed the expression of CVB6 viral protein in these tissues. Real-time PCR assay also revealed higher viral loads in the skeletal and cardiac muscle than in other tissues at different time points post infection. Furthermore, the protective effect of passive immunization with antisera and a neutralizing monoclonal antibody against CVB6 infection was evaluated in the neonatal mouse model. This study should provide insights into the pathogenesis of CVB6 and facilitate further research in the development of vaccines and antivirals against CVBs.

Down-regulated TAB1 suppresses the replication of Coxsackievirus B5 via activating the NF-κB pathways through interaction with viral 3D polymerase

Coxsackievirus Group B type 5 (CVB5), an important pathogen of hand-foot-mouth disease, is also associated with neurological complications and poses a public health threat to young infants. Among the CVB5 proteins, the nonstructural protein 3D, known as the Enteroviral RNA-dependent RNA polymerase, is mainly involved in viral genome replication and transcription. In this study, we performed immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry (LC–MS/MS) to identify host proteins that interacted with CVB5 3D polymerase. A total of 116 differentially expressed proteins were obtained. Gene Ontology analysis identified that the proteins were involved in cell development and cell adhesion, distributed in the desmosome and envelope, and participated in GTPase binding. Kyoto Encyclopedia of Genes and Genomes analysis further revealed they participated in nerve diseases, such as Parkinson disease. Among them, 35 proteins were significantly differentially expressed and the cellular protein TGF-BATA-activated kinase1 binding protein 1 (TAB1) was found to be specifically interacting with the 3D polymerase. 3D polymerase facilitated the entry of TAB1 into the nucleus and down-regulated TAB1 expression via the lysosomal pathway. In addition, TAB1 inhibited CVB5 replication via inducing inflammatory factors and activated the NF-κB pathway through IκBα phosphorylation. Moreover, the 90-96aa domain of TAB1 was an important structure for the function. Collectively, our findings demonstrate the mechanism by which cellular TAB1 inhibits the CVB5 replication via activation of the host innate immune response, providing a novel insight into the virus-host innate immunity.

Antiviral functionalization of cellulose using tannic acid and tannin-rich extracts.

Due to seasonally appearing viruses and several outbreaks and present pandemic, we are surrounded by viruses in our everyday life. In order to reduce viral transmission, functionalized surfaces that inactivate viruses are in large demand. Here the endeavor was to functionalize cellulose-based materials with tannic acid (TA) and tannin-rich extracts by using different binding polymers to prevent viral infectivity of both non-enveloped coxsackievirus B3 (CVB3) and enveloped human coronavirus OC43 (HCoV-OC43). Direct antiviral efficacy of TA and spruce bark extract in solution was measured: EC50 for CVB3 was 0.12 and 8.41 μg/ml and for HCoV-OC43, 78.16 and 95.49 μg/ml, respectively. TA also led to an excellent 5.8- to 7-log reduction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infectivity. TA functionalized materials reduced infectivity already after 5-min treatment at room temperature. All the tested methods to bind TA showed efficacy on paperboard with 0.1 to 1% (w/v) TA concentrations against CVB3 whereas material hydrophobicity decreased activities. Specific signatures for TA and HCoV-OC43 were discovered by Raman spectroscopy and showed clear co-localization on the material. qPCR study suggested efficient binding of CVB3 to the TA functionalized cellulose whereas HCoV-OC43 was flushed out from the surfaces more readily. In conclusion, the produced TA-materials showed efficient and broadly acting antiviral efficacy. Additionally, the co-localization of TA and HCoV-OC43 and strong binding of CVB3 to the functionalized cellulose demonstrates an interaction with the surfaces. The produced antiviral surfaces thus show promise for future use to increase biosafety and biosecurity by reducing pathogen persistence.

Effect of Chitosan (Chi)-C Terminal 30 Amino Acids of Clostridium Perfringens Enterotoxin (CPE30)-pVP1 Nanoparticles on Rats with Viral Myocarditis

Coxsackie B3 virus (CVB3) is the most common pathogen of viral myocarditis (VMC), and it is necessary to study an efficient vaccine to prevent the VMC. In this research, chitosan (chi)-C-terminal 30 amino acid (CPE30) was prepared by chemical coupling, and then chi-CPE30-pcDNA3.1-VP1 plasmid (pVP) complex particles were formed by co-aggregation method. The biological characteristics of the chi-CPE30-pVP1 complex particles were analyzed. It was immunized into SD rats intranasally at different time points as a vaccine together with other by-products (such as chi-pVP1, chi-CPE30-pcDNA3.1, and chi-pcDNA3.1). 100 μg of plasmid was inoculated each time, with 4 times in total, and the specific antibody level and cellular immune response of all rats were detected. It was revealed that based on the coupling effect of ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS) chemical coupling reagent, nearly 70% of CPE30 was coupled to chi, and the efficiency of chi-CPE30 to wrap DNA was close to 100%. After a certain concentration of pVP1 solution was added, the chi-CPE30-pVP1 composite particles were obtained, and the surface of the chi-CPE30-pVP1 composite was scanned as spherical particles. When used as a vaccine, the composite particles can induce high serum immunoglobulin G (IgG) and mucosal IgA antibody levels in rats. Meantime, the specific lymphocyte proliferation test confirmed that chi-CPE30-pVP1 effectively induced the proliferative response of CVB specific lymphocytes in the spleen and mesenteric lymph nodes (MLN). After the rats were infected with 3LD50CVB3, it was found that the weight of rats changed slightly under the action of chi-CPE30-pVP1 vaccine (P &lt; 0.05). The creatine kinase and creatine kinase-myoglobin binding (CK-MB) levels of rats in this group were lower than those of chi-pVP1 rats and control group (P &lt; 0.05). Applying the prepared chi-CPE30-pVP1 vaccine to immunize rats in this research could provide a new immune method for the molecular design of new vaccines and the prevention and treatment of CVB3 infection.

Antiviral Effect of Triple Combination of VP1 Ligands against Coxsackievirus B3 Infection in Newborn Mice

The Coxsackie B viruses (CVB), are distributed worldwide and cause a broad spectrum of diseases. Currently, clinically effective antivirals for the treatment of these infections do not exist. It was found that the unsatisfactory effectiveness of enteroviral inhibitors in vivo has been associated with the rapid development of drug resistance and that explains why effective antivirals have failed in monotherapy trials. Combination therapy with two or more drugs has the potential to successfully inhibit viral infection more effectively than either drug alone. It could achieve greater effects and avoid side effects using lower drug concentrations, and thus, they could prevent the rapid development of drug resistance. In this work, we examine the combined treatment effect of three enterovirus replication inhibitors – pleconaril, pocapavir, and vapendavir, which are VP1 hydrophobic pocket ligands. All of them have passed clinical double-blind trials as monotherapy courses, but none of them have been approved for clinical application, because of slide effects. We evaluate the effectiveness of the combination pleconaril/pocapavir/vapendavir (PPV) applied via CAA treatment scheme on Coxsackievirus B3 (CVB3) infection in newborn mice.

Optimization of RT-PCR methods for enterovirus detection in groundwater

Enteroviruses (EVs), which belong to the Picornaviridae family, infect individuals asymptomatically or cause mild symptoms (fever, runny nose, cough, skin rash, sneezing, mouth blister). Severe cases can cause various diseases, such as acute hemorrhagic conjunctivitis, aseptic meningitis, or myocarditis, especially in infants. These viruses can be transmitted via the fecal-oral route via contaminated water. In this study, we established a polymerase chain reaction (PCR) method for detecting EVs in water sample using Coxsackievirus B5 (CV-B5) and Echovirus 30 (E-30), which belong to species B of the four species of EVs (EV-A to D). Several methods have been investigated and compared for the detection of EVs, including real-time reverse transcription (RT) polymerase chain reaction and conventional RT-PCR. The most sensitive primer sets were selected, and the PCR conditions were modified to increase sensitivity. We also quantified the detection limits of real-time and conventional RT-PCR. The detection limits of conventional RT-PCR were detected in 105–106 copy/mL for CV-B5 and 106–107 copy/mL for E-30, respectively. This optimized method for detecting EVs is expected to contribute substantially to the investigation of EV outbreaks in water samples.