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@#Abstract: Objective To genotype and analyze whole genomic features of Coxsackievirus B3 (CVB3) isolated in Tianjin, to improve evolution information of CVB3 virus in Tianjin, and to provide basis for surveillance and early warning of related diseases. Methods Viral RNA was extracted from five CVB3 strains isolated in Tianjin, whole genome sequence of the virus was amplified by RT-PCR and sequenced by next-generation sequencing method, and phylogenetic and recombinant analysis were carried out. Results The open reading frame 1(ORF) of the five CVB3 strains contained 6 555 nucleotides and encoded 2 185 amino acids, and ORF2 was composed of sequences encoding 68 amino acids. The nucleotide sequence similarity ranged from 78.3%-100%, and the amino acid sequence similarity ranged from 95.7%-100%. Compared with the CVB3 prototype strain, the nucleotide sequence similarity of the five viruses was between 78.2%-79.1%, and the similarity of amino acid sequences was 94.9%-95.3%. All five viruses exhibited a T151A mutation on the VP2 protein. Additionally, the encephalitis isolate showed a K158E mutation on the VP2 protein, while one of the sewage isolates had a C234T mutation in 5' noncoding region. The five strains belonged to two different genotypes, among which the encephalitis isolate in 2016 belonged to the D genotype, while the sewage isolates in 2021 belonged to the E genotype. This is also the first report of E genotype CVB3 in northern China. The CVB3 strain may have recombinant events in non-structural protein regions, in which encephalitis isolate may recombine with a Coxsackievirus B5 (CVB5) strain, while sewage isolates may have recombinant events with a strain of ECHO virus 18 (E18). Conclusions The CVB3 isolates in Tianjin belong to D and E genotypes, and recombination events may exist in non-structural protein region of the viral genome. The results of CVB3 virus genome analysis in sewage suggests presence of CVB3 infection in the population of Tianjin, and its epidemic dominant genotype may have changed.
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Two new ursane triterpenoids along with twelve known compounds were isolated from 80% ethanol extract of Agastache rugosa (Fisch. et. Mey.) O. Kuntze by using silica gel column, MCI column, ODS column and HPLC. The structures of the new compounds were identified as 2α,3α-dihydroxy-24-nor-urs-4(23),12(13)-dien-28-oic acid (1) and 2α,3α-dihydroxy-24-nor-urs-4(23),12(13),20(30) -trien-28-oic acid (2) by HR-ESI-MS, NMR and ECD spectral data, named agasursacid A and agasursacid B. In addition, compounds 3, 4, 6, 8 showed anti-coxsackievirus B3 (CVB3) activities with a IC50 as 4.77, 1.59, 11.11 and 25.87 μmol·L-1, resepectively.
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Objective:To investigate the serotypes and epidemic characteristics of non-polio enteroviruses (NPEV) in acute flaccid paralysis (AFP) cases in Henan Province in 2021.Methods:Fecal specimens of 529 AFP cases reported in Henan Province in 2021 were collected for virus isolation. The VP1 regions of NPEV were sequenced. MEGA5.1 software was used for sequence alignment and a phylogenetic tree was constructed as well. The epidemiological data were organized and statistically analyzed using Excel2016 and SPSS26 software.Results:A total of 30 strains of NPEV were isolated from the fecal specimens of 529 AFP cases, with an isolation rate of 5.67% (30/529). They were belonged to group A and group B with 15 strains in each group, and no group C or group D viruses were isolated. Group A contained six serotypes and was dominated by coxsackievirus A2 (CVA2) and CVA6. Group B contained tree serotypes and was dominated by CVB3. In the population distribution, the separation rate of NPEV was the highest among children under 5 years old, which was 76.67% (23/30), and the ratio of male to female was 1.51∶1. In the regional distribution, group A viruses were mainly distributed in the central, southern and southwestern parts of Henan Province with CVA2 and CVA4 being the most widely distributed, while group B viruses were relatively concentrated, mainly distributed in the central, northern and southwestern parts of Henan Province with CVB3 being the predominant. In terms of time distribution, NPEV could be isolated throughout the year except from January to February, showing the epidemic characteristics of high incidence in spring and summer and low incidence in autumn and winter. The peak of group A virus infection was in May and the peak period of group B virus infection was from June to July.Conclusions:CVB3 was the main serotype of NPEV isolated in Henan Province in 2021. The pathogenic spectrum and regional distribution of NPEV had changed significantly compared with those in 2018-2019. In order to provide reference for the diagnosis and surveillance of AFP and maintain the polio-free status in Henan Province, much attention should be paid to the current epidemic trend of NPEV.
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Abstract Although different etiological agents can cause acute meningoencephalitis, this syndrome is usually associated with viruses. Among these, enteroviruses play a significant role. Here, we describe a fatal case of meningoencephalitis in a previously healthy teenager. Real-time RT-PCR and cell culture assays were performed with serum and cerebrospinal fluid (CSF) from a clinically diagnosed meningoencephalitis case that occurred in Rio de Janeiro State, Brazil. Coxsackievirus B2 (CVB2) was identified. Phylogenetic analysis revealed that the identified CVB2 was genetically related to strains known to cause neurological diseases. This case highlights the importance of continuous laboratory surveillance of central nervous system infections.
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Humans , Adolescent , Meningoencephalitis/diagnosis , Phylogeny , BrazilABSTRACT
Objective To analyze the genetic characteristics of VP1 3'region of human coxsack-ievirus B2 (CV-B2) strains isolated from Yunnan province. Methods RT-PCR and gene sequencing were performed to analyze the VP1 3'region of 15 CV-B2 strains isolated from acute flaccid paralysis ( AFP) cases during 2005 to 2006, healthy children in 2013 and hand, foot and mouth disease (HFMD) cases in 2014 in Yunnan province. CV-B2 VP1 gene reference sequences were downloaded from the Genbank. Nucleotide (nt) and amino acid (aa) diversities were calculated by MEGA5. 2 software and a phylogenetic tree was constructed. Genetic and molecular epidemiological characteristics of CV-B2 strains circulating in Yunnan province were analyzed. Results A total of 15 CV-B2 strains were isolated, which were one from 232 AFP cases in 2005, one from 240 AFP cases in 2006, 12 from 400 healthy children in 2013 and one from 500 HFMD cases in 2014. Phylogenetic analysis of the 15 CV-B2 strains in Yunnan province and those down-loaded from the GenBank showed that CV-B2 could be genetically divided into five genotypes. The prototype strain Ohio-1 and one strain (01-1) isolated in Taiwan in 1988 belonged to genotype 1. Strains isolated in France in 2006, 2007 and 2010 belonged to genotype 2. Strains isolated in Yunnan, Shandong, Henan, Fu-jian and Taiwan belonged to genotype 3. Strains isolated in Russia, Yunnan AFP cases in 2005 and 2006 and India belonged to genotype 4. Strains isolated in Taiwan, Shandong and New South Wales, Australia be-longed to genotype 5. Different genotypes distributed in different countries/areas with some confined within specific countries/areas. Conclusions The 12 strains isolated from healthy children and one from HFMD cases in Yunnan province belonged to genotype 3, while the two strains isolated from AFP cases belonged to genotype 4. Diversities in nt and aa sequences between the strains isolated from the healthy children and HFMD case were only 0. 76％ and 0. 03％, respectively, indicating that they might come from the same transmission source. However, the nt and aa diversities between the isolates of genotype 3 ( from healthy children and HFMD case) and genotype 4 (from AFP cases in 2005 and 2006) were 15. 11％-15. 22％ and 2. 76％-2. 72％, respectively. Correlation of CV-B2 with AFP and HFMD was worthy of further study.
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Objective To investigate the molecular characteristics of immune response signaling molecules induced by transfection of coxsackievirus B2 ( CVB2 ) structural proteins into epithelial cells. Methods Recombinant eukaryotic expression plasmids containing the coding regions of CVB2 structural proteins VP1-VP4 were constructed and then transfected into 16HBE cells. Culture supernatants and cell ly-sates of the transfected 16HBE cells were collected. Expression of signaling molecules involved in innate im-mune responses in transfected 16HBE cells at mRNA level was detected by RT-Q-PCR. The proliferation of T cells co-cultured with culture supernatants and cell lysates of the transfected 16HBE cells was analyzed by ELISPOT. Results Expression of innate immunity-related signaling molecules such as TGF-β-activated ki-nase ( TAK) , NF-κB-inducing kinase ( NIK) , IκB kinase α ( IKKα) and IFN-β at mRNA level was up-regulated in 16HBE cells transfected with CVB2 structural proteins VP1-VP4. Both culture supernatants and cell lysates of the transfected 16HBE cells enhanced the proliferation of T cells. Conclusions CVB2 struc-tural proteins VP1-VP4 could enhance the expression of innate immunity-related signaling molecules to var-ying degrees and promote the activation of adaptive immunity.
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Objective@#To study the molecular epidemiology and genetic variation of coxsackievirus B5 (CV-B5) in certain areas in China.@*Methods@#MEGA 6.0 software was used to analyze the complete VP1 region of CV-B5 isolated strains from certain areas of China by retrieving the GenBank nucleotide database. Besides, the phylogenetic tree was constructed, the homology of nucleotide and amino acids were calculated and the rate of evolution was estimated.@*Results@#A total of 189 Chinese CV-B5 isolated strains were included in this study. Most of Chinese CV-B5 isolated strains belonged to genotype C, accounted for 90.5%. Compared with the genotype A, the homology of nucleotide sequences and amino acid sequences of complete VP1 region of 189 Chinese isolated strains were 79.8%-82.8% and 92.6%-97.9%, respectively; moreover, the nucleotide and the amino acid homology of 189 Chinese CV-B5 isolated strains among themselves ranged from 80.3% to 100.0% and ranged from 91.5% to 100.0%, respectively. The estimated rate of evolution of the CV-B5 was 4×10-3 substitutions/site/year.@*Conclusions@#The majority of CV-B5 isolated strains belonged to genotype C, and subgenotype C1 and C2 were co-circulating together in certain areas of China.
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Objective To detect the expression of Nrf2 in mice with viral myocarditis and to investigate the changes and effects of Nrf2 after puerarin (Pue) treatment.Methods A total of 130 BALB/C male mice aged 4 weeks were randomly divided into control group,VMC group,Nrf2 activator group and Pue group (20 mice in each group) with different concentrations.The models were made with Coxsackie B3 virus (CVB3).The mice were sacrificed on day 0,4,7,14 and 28 respectively,and blood and myocardial samples were harvested.Cardiomyocyte apoptosis was detected by flow cytometry.The expression changes of Nrf2,HO-1,Fas,TGF-beta 1 mRNA were detected by real-time PCR and Western blot respectively.Statistical software SPSS19.0 was used to analyze the results.The measurement data was expressed mean ± standard deviation.The paired samples were tested with mean t test.The group data were analyzed with two-way ANOVA.A P value of less than 0.05 was considered to indicate statistical significance.Correlation analysis was performed with Spearman's correlation test.Results Nrf2 mRNA and Nrf2 protein were expressed in all groups.The correlations between Nrf2 and HO-1,Fas and TGF-beta-1 were analyzed according to CPDT or Pue,and the results were consistent with each other.It showed that the relationship between Nrf2 and HO-1,Fas and TGF-beta-1 did not change with intervention measures.The transcription and protein expression of HO1 in CPDT and Pue groups were significantly increased,and were positively correlated with Nrf2 (r =0.969,P ＜0.01).At a certain dose gradient (＜ 45 mg/kg),the transcription and protein expression of HO-1 were dose-dependent;the decreased cardiomyocyte apoptosis was observed in both CPDT and Pue group,while Nrf2 and Fas were negatively correlated (r =-0.968,P ＜ 0.01);at a certain dose gradient,the expression of TGF-beta 1 in CPDT and Pue group decreased with the increase of dose,and Nrf2 and TGF-beta 1 were negatively correlated (r =-0.753,P ＜ 0.01).Conclusion The increased expression of Nrf2 in VMC is involved in the occurrence and development of VMC.Nrf2 has antioxidant effect in VMC by up-regulating the antioxidant enzyme HO-1,has the anti-myocardial APO effect by inhibiting the Fas/FasL signaling pathway,and inhibits myocardial fibrosis by suppressing the expression of TGF-beta 1 protein and transcription.The therapeutic effect of Pue on VMC is to activate Nrf2 to produce antioxidant,anti-apoptotic and anti-fibrotic effects.
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Objective@#To illuminate the gene characteristics and clinical characterization of Coxsackievirus B5 (CV-B5) strains isolated from patients with sevre hand, foot and mouth disease (HFMD) in Qingdao city.@*Methods@#A total of 1 844 patients of HFMD were consecutively admitted to Qingdao Women and Children's Hospital from 2013 to 2014. Information of the study population described above was collected retrospectively. The samples were collected from at least 1 site (throat swab, cerebrospinal fluid), which viral nucleic acid extracted and the entire VP1 gene sequences of CV-B5 isolates were amplified and sequenced, then the homology and phylogeny analysis were conducted by MEGA7.0. The prototype Faulkner strain and other VP1 amino acid sequences were derived from the GenBank database.@*Results@#A total of 8 CV-B5 positive cases were obtained, including 4 males and 4 females; 6 severe hospitalized cases and 2 outpatients. The age of 6 hospitalized patients ranged from 3 to 48 months, with a median of 26 months. For the six inpatients, fever, convulsions vomiting, diarrhea and rash were the main clinical manifestation, and all combined with viral encephalitis. Compared with the prototype strain Faulkner, in the VP1 region,the nucleotide and the amino acid homologies was 77.3%-78.8% and 95.5%-97.0% respectively. Five out of the six severe cases with substitution of serine (S) to asparagine (N) at amino acid site 95 in the VP1 region. The sequences of 8 CV-B5 strains were classified into genogroup D.@*Conclusion@#Hand, foot and mouth disease associated with CV-B5 virus infection can result in nervous system involvement and the main complication was viral encephalitis. The CV-B5 strains associated with severe hand, foot and mouth disease had high nucleotide homology and present a certain regional aggregation.
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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 HeLa cells, and PERK, IRE1, and ATF6a pathways participated in the regulation of autophagy. Our data suggested that ER stress may inhibit mTOR signaling pathway to induce autophagy during CVB3 infection.
Subject(s)
Humans , Activating Transcription Factor 6 , Metabolism , Autophagy , Coxsackievirus Infections , Metabolism , Endoplasmic Reticulum Stress , Endoribonucleases , Metabolism , Enterovirus B, Human , HeLa Cells , Protein Serine-Threonine Kinases , Metabolism , Signal Transduction , eIF-2 Kinase , MetabolismABSTRACT
Objective:To investigate the immunomodulation of CCK8 on the Coxsackievirus B ( CVB )-attacked human peripheral blood plasmacytoid dendritic cells(pDC). Methods:Peripheral blood mononuclear cells of healthy volunteers were separated by Ficoll-Hypaque gradient density centrifugation. The pDC was separated and divided into five groups,which were the control group, CVB attacked group,the group of CCK8 treated after CVB attack,the group of PGE2 treated after CVB attack and the group of CCK8+PGE2 treated after CVB attack. 100-time TCID50 of CVB was applied for the attack on pDC. Real-time PCR and Immunofluorescence technique were employed to detect the expression of CCK1R/CCK2R mRNA and protein. Then,the expression levels of costimulatory molecules such as CD80,CD86,HLA-DR ligand,and the chemokine receptor CCR7 were evaluated by Flow Cytometry Analysis. The supernatants of pDCs were collected, and the content of IFN-α was determined by Enzyme-linked Immunosorbent Assay. Results:CCK1R and CCK2R were co-expressed in human peripheral blood pDC,and both were significantly upregulated after CVB attack in vitro. Expression of CD80,CD86,HLA-DR and IFN-α were decreased in the CVB+CCK8 group compared with the CVB group,which suggested that CCK8 may reduce the CVB activation of pDC. Whereas expression of CD80,CD86,HLA-DR,CCR7 and IFN-α were increased in the CVB+PGE2 group compared with the CVB group,which suggested that PGE2 may increase the CVB activation of pDC in vitro. Conclusion:CCK8 repressed the CVB-attacked pDC,while PGE2 activated the CVB-attacked pDC.
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Group B coxsackieviruses (CVBs) are a group of common human pathogens producing various clinical symptoms. Although the virology of CVB is well known, there is limited information on viral pathogenesis and the relationship between clinical symptoms and viral phenotype, particularly for CVB type 2 (CVB2). In 2004 in Korea, two CVB2 strains were isolated: CB2/04/279 from stool of an acute myocarditis patient with heart failure and CB2/04/243 from an aseptic meningitis patient. In this study, a high degree of homology was observed between the CB2/04/279 and CB2/04/243 full genome sequences. The two Korean CVB2 isolates had 93.1% homology compared to 82.1%–82.5% nucleotide sequence identity with the cardiovirulence-associated reference CVB strain Ohio-1 (CVB/O). CVB2-induced pathogenesis was analyzed, focusing on virus-induced pathology of various tissues in 4-week-old BALB/c inbred male mice. Myocarditis developed and extensive pancreatic inflammation was observed in all mice infected with CB2/04/279 or CVB/O, but not in animals infected with CB2/04/243. This is the first report of the full-genomic sequence and pathogenesis of the CVB2 strain isolated from an acute myocarditis patient in Korea.
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Animals , Humans , Male , Mice , Base Sequence , Enterovirus , Genome , Heart Failure , Inflammation , Korea , Meningitis, Aseptic , Myocarditis , Pathology , Phenotype , VirologyABSTRACT
The chemical property of cinnamaldehyde is unstable in vivo, although early experiments have shown its obvious therapeutic effects on viral myocarditis (VMC). To overcome this problem, we used cinnamaldehyde as a leading compound to synthesize derivatives. Five derivatives of cinnamaldehyde were synthesized: 4-methylcinnamaldehyde (1), 4-chlorocinnamaldehyde (2), 4-methoxycinnamaldehyde (3), α-bromo-4-methylcinnamaldehyde (4), and α-bromo-4-chlorocinnamaldehyde (5). Neonatal rat cardiomyocytes and HeLa cells infected by coxsackievirus B3 (CVB3) were used to evaluate their antiviral and cytotoxic effects. In vivo BALB/c mice were infected with CVB3 for establishing VMC models. Among the derivatives, compound 4 and 5 inhibited the CVB3 in HeLa cells with the half-maximal inhibitory concentrations values of 11.38 ± 2.22 μM and 2.12 ± 0.37 μM, respectively. The 50% toxic concentrations of compound 4 and 5-treated cells were 39-fold and 87-fold higher than in the cinnamaldehyde group. Compound 4 and 5 effectively reduced the viral titers and cardiac pathological changes in a dose-dependent manner. In addition, compound 4 and 5 significantly inhibited the secretion, mRNA and protein expressions of inflammatory cytokines TNF-α, IL-1β and IL-6 in CVB3-infected cardiomyocytes, indicating that brominated cinnamaldehyde not only improved the anti-vital activities for VMC, but also had potent anti-inflammatory effects in cardiomyocytes induced by CVB3.
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Animals , Humans , Mice , Rats , Cytokines , HeLa Cells , Interleukin-6 , Myocarditis , Myocytes, Cardiac , RNA, Messenger , Therapeutic UsesABSTRACT
12-N-m-Cyanobenzenesulfonyl matrinic butyl methyl ether is a potent anti-coxsackievirus B3(CVB3) agent bearing a novel structure skeleton. Taking this compound as a lead, totally 15 novel target compounds have been synthesized and evaluated for the anti-CVB3 activities using CPE method. Structureactivity relationship (SAR) demonstrated that the shorten-length of 11-side chain was not helpful for keeping the good anti-virus activity. Among the newly synthesized compounds, compound c1 displayed a good anti-CVB3 activity with the IC50 of 7.1 μmol·L-1 and SI of 35.5, similar to that of the lead. The SAR results provided useful information for further optimization of these compounds in the molecular structure.
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Objective To investigate the serotypes of human enterovirus B ( HEV-B) species cau-sing hand, foot and mouth disease ( HFMD) and to analyze the genetic characteristics of VP1 region in cox-sackievirus B2 ( CVB2 ) and coxsackievirus B5 ( CVB5 ) strains circulating in Anyang area during 2011 to 2015. Methods Real-time RT-PCR and semi-nested RT-PCR were performed to identify coxsackievirus A16 (CVA16), enterovirus 71 (EV71) and other serotypes of enterovirus in order to obtain the complete etiologic composition of HFMD. The numbers of HEV-B serotypes and the percentages of specimens positive for every serotype in all enterovirus-positive specimens were calculated. As CVB2 and CVB5 were the pre-dominant serotypes of HEV-B species, five pairs of primers targeting the VP1 regions of CVB2 and CVB5 were designed to obtain the complete nucleotide sequences of CVB2 and CVB5 VP1 regions. The phylogenet-ic trees were constructed based on the VP1 sequences obtained in this study and those submitted to GenBank by using MEGA7. 0 and BioEdit7. 2. The selection pressures on VP1 regions of CVB2 and CVB5 strains cir-culating in China in recent years were evaluated with the online program of DataMonkey. Results A total of 57 specimens that belonged to 14 serotypes of HEV-B species were detected in Anyang area from 2011 to 2015. The 14 serotypes of HEV-B species accounted for 56% of all serotypes of enterovirus and the speci-mens positive for HEV-B species accounted for 3. 06% of all enterovirus-positive specimens. The HFMD ca-ses caused by most of the HEV-B serotypes were sporadic cases. Small outbreaks of HFMD could also be caused by some serotypes of HEV-B such as CVB2 and CVB5. The complete sequences of VP1 region were obtained from 8 CVB2 strains and 9 CVB5 strains. The phylogenetic trees based on the VP1 sequences dem-onstrated that the CVB2 strains were classified into four genotypes ( A-D) . The mean evolutionary distances between different genotypes ranged from 0. 191 to 0. 208 and the similarities in nucleotide sequences ranged from 79. 7% to 85. 8%. The CVB5 strains were classified into 6 genotypes (A-F). The mean evolutionary distances and the similarities in nucleotide sequences between different genotypes of CVB5 strains ranged from 0. 170 to 0. 285 and 76. 0% to 86. 8%, respectively. Strains of different genotypes varied significantly in the residues on positons 157 and 263 in the VP1 region of CVB2 strains and on positions 75, 90 and 95 in the VP1 region of CVB5 strains. All of the CVB2 strains isolated in Anyang area belonged to D genotype and located intensively in one lineage. The CVB5 strains circulated in Anyang area belonged to F genotype and located in two lineages. The selection pressures on CVB2 strains of D genotype and CVB5 strains of F geno-type circulating in China in recent years were 0. 037 and 0. 036, respectively. Six positively selected amino acid sites were found in the VP1 region of CVB5 strains, but no positively selected amino acid site was found in the VP1 region of CVB2 strains. Conclusion HEV-B species was an essential component of the etiologic spectrum of HFMD in Anyang area during 2011 to 2015, of which CVB5 and CVB2 were the predominant se-rotypes. The VP1 region of CVB5 was more complex and active than that of CVB2 over the course of evolution.
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Objective: This study aimed to investigate whether interleukin-28A (IL-28A) plays a role in murine myocarditis induced by coxsackievirus B3 (CVB3), and to explore its possible mechanism involved. Methods: Male BALB/c mice both infected and not infected by CVB3 were randomly divided into four groups (n = 40), untreated or treated with different doses of IL-28A for 4 days, and then sacrificed on days 4 and 7 post-infection. The heart samples were collected for histopathologic examination. Cardiac viral load was determined by a plaque assay. Additionally, immunoblot analysis, TUNEL assay, and immunohistochemistry were performed to examine the expression of signal transducer, activator of transcription 1 and 2 (STAT1 and STAT2), CVB3-induced apoptosis and the expression of Bcl-2, BAX and Caspase-3. Results: Compared to uninfected mice, the CVB3 infected mice exhibited higher mortality rate (p < 0.001), apparent inflammation and myocardial lesion (p < 0.01), and higher cardiac viral load (p < 0.01). After CVB3 infection, IL-28A treated mice presented no death (p < 0.001), reduced inflammation and myocardial lesion (p < 0.01), and lower viral load (p < 0.01) compared to untreated mice. Besides, treatment with IL-28A markedly increased the expressions of STAT1 and STAT2, and inhibited CVB3-induced apoptosis in myocardial cells with increased ratio of Bcl-2/BAX. Conclusion: The antiviral and myocyte protective effects of IL-28A in CVB3-inducedmyocarditis are regulated by STAT1 and STAT2. .
Subject(s)
Animals , Male , Mice , Antiviral Agents/therapeutic use , Coxsackievirus Infections , Interleukins/metabolism , Myocarditis/virology , Apoptosis , /immunology , /metabolism , Coxsackievirus Infections/drug therapy , Coxsackievirus Infections/immunology , Coxsackievirus Infections/metabolism , Immunoblotting , Immunohistochemistry , In Situ Nick-End Labeling , Interleukins/immunology , Mice, Inbred BALB C , Myocarditis/immunology , Myocarditis/metabolism , /immunology , /metabolism , STAT1 Transcription Factor/immunology , STAT1 Transcription Factor/metabolism , /immunology , /metabolism , Viral Load , /immunology , /metabolismABSTRACT
Objective To study the role of Ginsenoside Re and Rb3 on mice with viral myocarditis (VMC) and explore the mechanisms. Methods BALB/C mice were infected by coxsackievirus B3 ( CVB3) to establish VMC model. The mice were divided into control group,virus group and Ginsenoside Re and Rb3 treatment group(treatment group). On day 5,day 10,day 20 after infection,the level of serum crea-tine phosphokinase-MB( CK-MB) was detected. Then myocardial sections stained with Masson′s trichrome were used to observe the distribution of mice myocardial collagen fibers, quantify collagen volume fraction (CVF),and detect the levels of superoxide dismutase(SOD). Results (1)The level of CK-MB peaked on day 5,and decreased afterwards[day 5:(463. 68 ± 47. 62) U/L; day 10:(588. 81 ± 56. 09) U/L; day 20:(340. 48 ± 58. 22) U/L,respectively]. While the levels of CK-MB in treatment group were lower than those in virus group[day 5:(378. 69 ± 56. 02) U/L;day 10:(452. 56 ± 67. 78) U/L; day 20:(327. 13 ± 47. 20) U/L,respectively] in the same point. There were significant differences between groups on day 5 and day 10 (P<0. 01). (2) In viral group,the blue staining degree gradually increased with time in myocardial sections stained with Masson′s trichrome,especially in myocardial necrosis area. Compared with treatment group,CVF increased significantly in virus group on day 10 and day 20 ( day 10:6. 52% ± 2. 34% vs. 8. 94% ± 1. 67%;day 20:7. 00% ± 1. 53% vs. 10. 46% ± 1. 74%,P<0. 01). The levels of SOD in myocardial sections in virus group were lower than those of control group[day 5:(48. 83 ± 17. 74) U/L;day 10:(61. 41 ± 14. 58) U/L;day 20:(66. 26 ± 18. 97) U/L,respectively,P<0. 05],but in treatment group,the level of SOD could beimprovedsignificantly[day5:(72. 07 ±24. 85)U/L;day10:(83. 22 ±19. 52)U/L;day20:(92. 00 ± 20. 46) U/L, respectively, P < 0. 05]. Conclusion Because of the inhibition of oxygen radicals and oxidative stress, Ginsenoside Re and Rb3 can protect myocardial tissue. Ginsenoside Re and Rb3 can effectively reduce the extent of myocardial fibrosis. The mechanism may be related with the reduced peroxide level in vivo.
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Chrysin is a 5,7-dihydroxyflavone and was recently shown to potently inhibit enterovirus 71 (EV71) by suppressing viral 3C protease (3Cpro) activity. In the current study, we investigated whether chrysin also shows antiviral activity against coxsackievirus B3 (CVB3), which belongs to the same genus (Enterovirus) as EV71, and assessed its ability to prevent the resulting acute pancreatitis and myocarditis. We found that chrysin showed antiviral activity against CVB3 at 10 muM, but exhibited mild cellular cytotoxicity at 50 muM, prompting us to synthesize derivatives of chrysin to increase the antiviral activity and reduce its cytotoxicity. Among four 4-substituted benzyl derivatives derived from C(5) benzyl-protected derivatives 7, 9-11 had significant antiviral activity and showed the most potent activity against CVB3 with low cytotoxicity in Vero cells. Intraperitoneal injection of CVB3 in BALB/c mice with 1x106 TCID50 (50% tissue culture infective dose) of CVB3 induced acute pancreatitis with ablation of acinar cells and increased serum CXCL1 levels, whereas the daily administration of 9 for 5 days significantly alleviated the pancreatic inflammation and reduced the elevation in serum CXCL1 levels. Collectively, we assessed the anti-CVB3 activities of chrysin and its derivatives, and found that among 4-substituted benzyl derivatives, 9 exhibited the highest activity against CVB3 in vivo, and protected mice from CVB3-induced pancreatic damage, simultaneously lowering serum CXCL1 levels.
Subject(s)
Animals , Mice , Acinar Cells , Enterovirus , Inflammation , Injections, Intraperitoneal , Myocarditis , Pancreatitis , Vero CellsABSTRACT
Objective To analyze the complete genome sequence of a coxsackievirus B 3 (CVB3) strain KM06/2009 and its genetic variation , evolution and cardiovirulence .Methods Eight clones with overlapped gene fragments covering the complete viral genome ( excluding the poly-A tail) were obtained by RT-PCR and sequenced .The nucleotide ( nt ) and amino acid ( aa ) sequences of the eight clones were aligned with sequences of other known CVB 3 clinical strains .Phylogenetic and pairwise alignment analyses based on the genome and complete VP 1 regions were conducted by using Mega 4.1,RDP3 and SimPlot3.5.1 softwares.The RNA secondary structure of CVB3 stem loopⅡ (SLⅡ) was determined by using Mfold web server.Results The complete genome of CVB3 strain KM06/2009 was 7401 nt in length, consisting of 743 nt and 100 nt on 5′untranslated region (UTR) and 3′UTR,respectively.The entire open reading frame contained 6558 nt, encoding a 2185 aa polyprotein.There was no nucleotide deletion or insertion in the coding region,but some changes of amino acid were unique .KM06/2009 strain showed 81.4%and 95.7%identities in nucleotide and amino acid sequences respectively as compared with CVB 3 prototype Nancy strain.It shared 88.4%-98.1%nucleotide and 98.1%-99.4%amino acid homology with the other Chinese clinical strains isolated at the same period .KM06/2009 strain and CVB3 GA strain without cardiovirulence shared 80.7%homology in nucleotide and 96.4% in amino acid.The phylogenetic analysis indicated that the significant spatial and temporal clustering was detected in CVB 3 isolate.CVB3 KM06/2009 strain showed a strong cardiovirulence tendency as indicated by the RNA secondary structure of CVB 3 SL Ⅱ. Conclusion CVB3 KM06/2009 isolate showed a strong cardiovirulence tendency in comparison with other CVB3 clinical isolates based on the bioinformatics analysis .
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Objective To explore the expression of interleukin-17A(IL-17A) in the myocardium of mice with experimental viral myocarditis (VM) and its clinical significance.Methods Fifty-five mice were randomly divided into myocarditis group(n =45) and control group(n =10).Mice in the myocarditis group were inoculated intraperitoneally with 0.1 mL Eagle's solution containing coxsackievirus B3 (CVB3) to establish VM models.However,mice in the control mice were treated with 0.1 mL Eagle's solution.Ten infected mice were sacrificed on day 7,14 and 28 after inoculation,and the control mice were killed on day 28 postinoculation.Myocardial histopathology was determined with hematoxylin and eosin stain.The levels of myocardial IL-17A mRNA and protein expressions were detected by real time quantitative polymerase chain reaction(RQ-PCR) and immunohistochemistry.Serum IL-17A concentration was examined using enzyme linked immunosorbent assay(ELISA).In addition,the relationship between myocardial IL-17A protein levels as well as serum IL-17A concentration and myocardial histopathological scores were analyzed statistically in myocarditis group.Results The levels of myocardial IL-17A mRNA and protein and serum IL-17A concentration on day 7 and 14 after inoculation in myocarditis group increased significantly compared with those in control group(all P ＜ 0.01).Myocardial IL-17A protein levels and serum IL-17A concentration showed significantly positive correlation with the myocardial histopathologic scores in VM group,respectively (r =0.67,0.74,all P ＜ 0.05).Conclusions IL-17A may play a pivotal role in the pathogenesis of VM and is associated with the severity of VM.It can serve as a novel indicator for VM.