Virus B3 Research Articles

Spirulina maxima L-asparaginase: Immobilization, Antiviral and Antiproliferation Activities.

L-asparaginase (L-AsnA) enzyme has gained significant attention in the food, biocatalysts and pharmaceutics industry. It (L-AsnA) has been widely used in food processing industries as a promising acrylamide mitigating agent and as a therapeutic agent in the treatment of certain human cancers. Based on US Patent (4,433,054; 1984), L-asparaginase (L-AsnA) enzyme is immobilized by admixing the active enzyme on the polysaccharide to be in a gel form. The storage stability of immobilized L-AsnA enzyme and its anti-proliferation and antiviral activity were determined. In the present study, S. maxima was cultured at large scales (300 liter) for the production of enough extracellular L-asparaginase (L-AsnA) using modified (high N concentration) Zarrouk medium as we reported in a previous study. L-AsnA was immobilized on natural polymers, as agar cake beads, agarose pieces and gelatin blocks, in order to evaluate the efficiency of physical entrapment techniques. Anti-proliferation properties of L-AsnA against lung carcinoma A549, hepatocellular carcinoma Hep-G2 and prostate carcinoma PC3 human cancer cell lines were assessed by the MTT cell viability method. In addition, the antiviral activity against Coxsackie B3 (CSB3) Virus was assessed. The highest L-AsnA immobilized activity and immobilization yield were achieved with agar cakes bead. The purified S. maxima L-AsnA showed good antiviral activity against Coxsackie B3 (CSB3) Virus in a dose-dependent manner with an IC50 value 17.03 μg/ml. The antiviral mode of action is presumably due to their capability of inhibiting attachment, blocking the adsorption and penetration event of the viral replication cycle with 89.24%, 72.78% and 72.78%, respectively. Also, S. maxima L-AsnA showed anti-proliferation effect against lung carcinoma A549, hepatocellular carcinoma Hep-G2 and prostate carcinoma PC3 human cancer cell lines, with an IC50 of 22.54, 24.65 and 56.61 μg/ml, respectively. It is interesting to favor L-asparaginase of S. maxima which showed antiviral activity and anti-proliferation effect against different types of human cell lines. Thus, S. maxima microalgae might be a good source for L-AsnA enzymes and can be immobilized on natural polymers.

Antiviral, Cytotoxic and Antioxidant Effects of Tanacetum Vulgare L. Crude Extract In Vitro.

Due to the high prevalence of viral infections having no specific treatment and the constant emergence of resistant viral strains, searching for effective antiviral compounds is crucial. The present study explores in vitro the antiviral activity of ethanolic extract from aerial parts of. The aim of the current study was to evaluate antiviral activity of ethanolic extract from herbaceous plant. The crude aqueous ethanolic extract from aerial parts of. The results show that the extract has the lowest toxicity on the MDBK cell line and similar cytotoxicity in Hep-2, whereas in the MDCK cells it has more than twice the highest toxicity. Testing the antiviral activity of. The crude extract from aerial parts of the medicinal plant.

Lithium chloride confers protection against viral myocarditis via suppression of coxsackievirus B3 virus replication

Viral myocarditis (VMC) is a type of inflammation affecting myocardial cells caused by viral infection and has been an important cause of dilated cardiomyopathy (DCM) worldwide. Type B3 coxsackievirus (CVB3), a non-enveloped positive-strand RNA virus of the Enterovirus genus, is one of most common agent of viral myocarditis. Till now, effective treatments for VMC are lacking due to lack of drugs or vaccine. Lithium chloride (LiCl) is applied in the clinical management of manic depressive disorders. Accumulating evidence have demonstrated that LiCl, also as an effective antiviral drug, exhibited antiviral effects for specific viruses. However, there are few reports of evaluating LiCl's antiviral effect in mice model. Here, we investigated the inhibitory influence of LiCl on the CVB3 replication in vitro and in vivo and the development of CVB3-induced VMC. We found that LiCl significantly suppressed CVB3 replication in HeLa via inhibiting virus-induced cell apoptosis. Moreover, LiCl treatment in vivo obviously inhibited virus replication within the myocardium and alleviated CVB3-induced acute myocarditis. Collectively, our data demonstrated that LiCl inhibited CVB3 replication and negatively regulated virus-triggered inflammatory responses. Our finding further expands the antiviral targets of LiCl and provides an alternative agent for viral myocarditis.

The State of The Jak/Stat Pathway Affects the Sensitivity of TumorCells to Oncolytic Enteroviruses

A test of the sensitivity of seven colon cancer cell lines to a panel of 12 nonpathogenic human enteroviruses revealed significant differences in the ability of tumor cells to become infected and replicate different viral strains. Among the factors that can affect the sensitivity of cells to viruses are differences in the state of the mechanisms of antiviral protection, associated with a reaction to type I interferons. Using the two colon cancer cell lines CaCo2 and LIM1215 as a model, significant differences were revealed in the ability of cells to defend themselves against virus infection after 16 hours of treatment with 1000 units/mL of interferon-alpha. To study the effect of the state of the interferon response system, represented by the Jak/STAT signaling pathway, on the sensitivity of cells to different strains of enteroviruses, HEK293T cell lines were used. These are capable of supporting replication of each of the tested enteroviruses, as well as maintaining the ability to protect against viral infection after the treatment with interferon. Using the CRISPR/Cas9 system, HEK293T sublines with knockouts of the IFNAR1 and STAT2 genes were obtained. The sensitivity of control and knockout cells to infection with five strains of enteroviruses and the vesicular stomatitis virus was analyzed. It was noted that knockout of the IFNAR1 and STAT2 genes resulted in an increased sensitivity to all tested viruses. In knockout cells, the levels of reproduction of the vaccine derived of poliovirus type 1, Echoviruses 7 and 30, and Coxsackie viruses B5 and A7 were also significantly increased in comparison with the control HEK293T cells. Thus, deficiencies in the Jak/STAT signaling pathway in tumor cells lead to an overall increase in the sensitivity to oncolytic viruses.

Assessment of in vitro bioactivities of Pis v 1 (2S albumin) and Pis v 2.0101 (11S globulin) proteins derived from pistachio (Pistacia vera L.)

We studied the biological activity of Pis v 1 (2S albumin) and Pis v 2.0101 (11S globulin) against Coxsackie viruses (CV) B2, B3, B4 and B5 as well as their cytotoxicity against six cell lines. Results showed that the two allergens of pistachio significantly reduce the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) against four Gram-positive and four Gram-negative bacteria as well as two fungi. Moreover, they exerted significant in vitro cytotoxic activities against Human ovarian carcinoma (A2780), human colon carcinoma (HTC), human glioblastoma (U-87-MG), human breast adenocarcinoma (MCF-7), and human prostate cell lines (PC3 and DU-145). Our findings revealed that the highest antimicrobial effect of the allergens was found against M. loteus (MIC = 10 µg/mL). The lowest IC50 value was found for Pis v 2.0101 allergen (11S globulin) against CV-B4 (30.20 ± 1.01 µg/mL). Furthermore, in vitro cytotoxicity assay showed IC50 values ranging from 21.09 ± 0.55 to 34.52 ± 0.90 and 26.34 ± 0.88 to 38.63 ± 0.80 µg/mL for Pis v 2.0101 (11S globulin) and Pis v 1 (2S albumin), respectively. As these allergens presented considerable effects against biofilm-related infections, CV-B2-5, and several cell lines, they might be employed as alternative/complementary treatments after further preclinical and clinical studies.

Design and synthesis of novel Imidazo[2,1-b]thiazole derivatives as potent antiviral and antimycobacterial agents

A series of novel acyl-hydrazone (4a-d) and spirothiazolidinone (5a-d, 6a-d) derivatives of imidazo[2,1-b]thiazole were synthesized and evaluated for their antiviral and antimycobacterial activity. The antituberculosis activity was evaluated by using the Microplate Alamar Blue Assay and the antiviral activity was evaluated against diverse viruses in mammalian cell cultures. According to the biological activity studies of the compounds, 5a-c displayed hope promising antitubercular activity, 6d was found as potent for Coxsackie B4 virus, 5d was found as effective against Feline corona and Feline herpes viruses. Consequently, the obtained results displayed that, 5a-d and 6d present a leading structure for future drug development due to its straightforward synthesis and relevant bioactivity.

Advancement of Mechanisms of Coxsackie Virus B3-Induced Myocarditis Pathogenesis and the Potential Therapeutic Targets.

Viral myocarditis is a cardiac disease caused by Group B Coxsackie virus of Enterovirus genus in the Picorna viridae family. It causes heart failure in children, young and adults. Ten Percent (10%) of acute heart failure and 12% of sudden deaths in young and adults who are less than 40 years is due to this viral myocarditis. If treatment action is not taken earlier, the viral disease can develop into chronic myocarditis and Dilated Cardiomyopathy which lead to congestive heart failure. And these eventually result in a reduced cardiac function which finally brings the victim to death. The only treatment option of the disease is heart transplantation once the acute stage of disease develops to chronic and Dilated Cardiomyopathy. Currently, there is a limitation in daily clinical treatments and even some available treatment options are ineffective. Therefore, focusing on search for treatment options through investigation is imperative. Recent studies have reported that biological molecules show a promising role. But their mechanism of pathogenesis is still unclear. A detailed study on identifying the role of biological molecules involved in Coxsackie B3 virus induced myocarditis and their mechanisms of pathogenesis; compiling and disseminating the findings of the investigation to the scientific communities contribute one step forward to the solution. Therefore, this review is aimed at compiling information from findings of current studies on the potential therapeutic role of micro RNA, cytokines and chemokines on the mechanism of pathogenesis of Coxsackie virus B3- induced myocarditis to give brief information for scholars to conduct a detailed study in the area.

Investigation on seco-prezizaane sesquiterpenes from fruits of Illicium lanceolatum and their neuroprotection activity

Ten seco-prezizaane sesquiterpenes were isolated from the water-soluble fraction of the fruit of Illicium lanceolatum using the combined methods of silica gel column chromatography,Sephadex LH-20 column chromatography,and RP-preparative HPLC. They were elucidated as majusanol E( 1),2α-hydroxycycloparviflorolide( 2),2β-hydroxy-3,6-dedioxypseudoanisatin( 3),majusanol A( 4),merrillianone( 5),cycloparvifloralone( 6),3α-hydroxycycloparvifloralone( 7),1,2-dehydrocycloparvifloralone( 8),henrylactone C( 9),and( 11) 7,14-ortholactone-3α-hydroxyfloridanolide( 10) according to the NMR data. All compounds were obtained from this plant for the first time. Neuroprotection activity,anti-Coxsackie B3 virus,and anti-H3 N2 virus experiments were carried out to test their bioactivities. The bioassay results showed that compounds 1,4,6,7,9 and 10 displayed weak protective effects of the damage of nerve SH-SY5 Y cell induced by monosodium glutamate.

COXSACKIE B2 VIRUS: FROM A SOCCER GAME TO FULMINANT MYOCARDITIS

SESSION TITLE: Wednesday Medical Student/Resident Case Report Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: 10/23/2019 09:45 AM - 10:45 AM INTRODUCTION: The presentation of viral myocarditis ranges from no symptoms to devastating illness.1 This is an unfortunate case of Coxsackie B2 virus induced Fulminant Myocarditis (FM); which despite aggressive medical and mechanical circulatory support progressed to fatality. CASE PRESENTATION: A 20-year-old male patient was transferred to our hospital with respiratory failure and shock. He presented to his local hospital with dyspnea, fever, myalgias and arthralgias that started after an indoor soccer game two weeks earlier. Prior to admission he was treated with inhalers, steroids and antibiotics without improvement. The TTE showed an LVEF 30% and Chest CT Scan showed bilateral consolidations and multiple nodules surrounded by ground glass opacities. Mechanical ventilation, vasopressors and inotropes were initiated prior to transferring him to our hospital. Upon arrival he required paralysis and pronation to improve his hypoxia. He was started on broad spectrum antimicrobials. However, the initial infectious workup was negative. Further workup obtained due to persistent fever revealed portal vein thrombosis which was treated with heparin. Hypercoagulability, malignancy and rheumatology tests were unrevealing. Expanded infectious workup was most notable for positive Coxsackie B2 serology with titers that increased 4-fold during hospitalization to 1:320, consistent with viral myocarditis. TEE showed persistent LV dysfunction, global LV wall motion abnormalities, increased septal wall thickness, severe mitral regurgitation and moderate tricuspid regurgitation. After receiving diuretics, vasopressors and inotropes he improved and was extubated. He subsequently decompensated requiring reintubation and resumption of inotropes. ECMO was initiated with biventricular assist device support. Heart transplant was considered but his hospital course was complicated by severe deconditioning and went on to develop GI bleeds, acute renal failure, fungemia, pulmonary abscess status post decortication and contained pulmonary artery rupture. On ICU day #97, his illness was deemed not survivable and his family withdrew care. DISCUSSION: Coxsackie B serotypes are among the more common causes of viral myocarditis, which may evolve into FM.2 FM is defined as 2-4 weeks of acute illness, followed by cardiogenic shock, needing hemodynamic support; and myocardial inflammatory infiltrates on histology. FM has a dramatic and progressive clinical course, with a severely impaired LVEF.3 It often requires prolonged hemodynamic support and referral for heart transplant.4 In general, in-hospital outcomes are poor; with increased morbidity and up to 32% reported mortality.5 CONCLUSIONS: Coxsackie B induced Fulminant Myocarditis portends increased morbidity and mortality, even after aggressive medical and mechanical circulatory support. Reference #1: Kindermann et al. J Am Coll Cardiol. 2012;59:779–92. Reference #2: Mehta et al. Case Reports in Infectious Diseases. 2018;ID 4258296:1-4. Reference #3: Ammirati et al. Circulation. 2017;136:529-45. DISCLOSURES: No relevant relationships by Stefanie DiGiandomenico, source=Web Response No relevant relationships by Tirsa Ferrer Marrero, source=Web Response No relevant relationships by Javeria Haque, source=Web Response No relevant relationships by Christopher Roberts, source=Web Response No relevant relationships by Gabriel Ryan, source=Web Response no disclosure on file for Jonathon Truwit; No relevant relationships by Krista Tuomela, source=Web Response No relevant relationships by Jane Wainaina, source=Web Response

Epidemiology of measles cases and phylogenetic analysis of the virus circulated in the Federation of Bosnia and Herzegovina during 2018: implications for elimination efforts.

Aim To present combined measles cases data and phylogenetic analysis of the virus circulated in 2018 in the Federation of Bosnia and Herzegovina (FB&H, the entity of Bosnia and Herzegovina), in order to analyse endemic transmission patterns of circulating strains and its implications for elimination efforts. Methods The data were derived from epidemiological case investigations and laboratory diagnoses based on serology, molecular detection and genotyping of the measles virus. Results During 2018 16 measles cases were reported in FB&H, of which five were classified as laboratory confirmed cases, one was an epidemiologically linked case and 10 were clinically compatible cases. Among them 12 (75.00%) cases were unvaccinated or had unknown vaccination status. The most affected population was up to 14 years of age (13/16; 81.25%). None of the cases was fully vaccinated. Viruses of other genetic lineages had been introduced in FB&H in the recent period. Two virus lineages of genotype B3 were identified. Phylogenetic analysis indicated the presence of a unique sequence of measles B3 virus in FB&H (Sarajevo). Conclusion Further strengthening of measles surveillance system and renewed efforts to increase vaccination levels are necessary to prevent disease and for elimination setting.

Antiviral potential of green synthesized silver nanoparticles of Lampranthus coccineus and Malephora lutea.

BackgroundViral and microbial infections constitute one of the most important life-threatening problems. The emergence of new viral and bacterial infectious diseases increases the demand for new therapeutic drugs.PurposeThe objective of this study was to use the aqueous and hexane extracts of Lampranthus coccineus and Malephora lutea F. Aizoaceae for the synthesis of silver nanoparticles, and to investigate its possible antiviral activity. In addition to the investigation of the phytochemical composition of the crude methanolic extracts of the two plants through UPLC-MS metabolomic profiling, and it was followed by molecular docking in order to explore the chemical compounds that might contribute to the antiviral potential.MethodsThe formation of SNPs was further confirmed using a transmission electron microscope (TEM), UV-Visible spectroscopy and Fourier transform infrared spectroscopy. The antiviral activity of the synthesized nanoparticles was evaluated using MTT assay against HSV-1, HAV-10 virus and Coxsackie B4 virus. Metabolomics profiling was performed using UPLC-MS and molecular docking was performed via Autodock4 and visualization was done using the Discovery studio.ResultsThe early signs of SNPs synthesis were detected by a color change from yellow to reddish brown color. The TEM analysis of SNPs showed spherical nanoparticles with mean size ranges between 10.12 nm to 27.89 nm, and 8.91 nm 14.48 nm for Lampranthus coccineus and Malephora lutea aqueous and hexane extracts respectively. The UV-Visible spectrophotometric analysis showed an absorption peak at λmax of 417 nm.The green synthesized SNPs of L. coccineus and M. lutea showed remarkable antiviral activity against HSV-1, HAV-10, and CoxB4 virus. Metabolomics profiling of the methanolic extract of L. coccineus and M. lutea resulted in identifying 12 compounds. The docking study predicted the patterns of interactions between the compounds of L. coccineus and M. lutea with herpes simplex thymidine kinase, hepatitis A 3c proteinase, and Coxsackievirus B4 3c protease, which was similar to those of the co-crystal inhibitors and this can provide a supposed explanation for the antiviral activity of the aqueous and nano extracts of L. coccineus and M. lutea.ConclusionThese results highlight that SNPs of L. coccineus and M. lutea could have antiviral activity against HSV-1, HAV-10, and CoxB4 virus.

Calpain regulates CVB3 induced viral myocarditis by promoting autophagic flux upon infection

Calpains are calcium-activated neutral cysteine proteases. The dysregulation of calpain activity has been found to be related to cardiovascular diseases, for which calpain inhibition is used as a treatment. Viral myocarditis (VMC) is primarily caused by Coxsackievirus group B3 virus infection (CVB3). CVB3 virus infection induces autophagy and hijacks this process to facilitate its replication. In this study, we found that calpain was significantly activated in hearts affected by VMC. However, pharmacologically inhibiting calpain aggravated VMC symptoms in mice due to myocardial inflammation and cardiac dysfunction. The inhibition of calpain activity in vitro led to the accumulation of LC3-II and increased levels of p62/SQSTM1 protein expression, suggesting that autophagic flux was impaired by calpain inhibition. These effects of calpain inhibition were also observed in capn4-specific myocardial knockout mice in vivo. Furthermore, our results provided evidence that calpain inhibition in VMC, unlike other cardiovascular diseases, exacerbated the disease symptom by impairing CVB3-induced autophagic flux, which may subsequently reduce virus autolysosome degradation. Our findings indicated that calpain inhibition may not be a good treatment for VMC disease in a clinical setting.

Synthesis and evaluation of some new 1,3,4-oxadiazoles bearing thiophene, thiazole, coumarin, pyridine and pyridazine derivatives as antiviral agents.

In an attempt to produce heterocyclic compounds based on 1,3,4-oxadiazole derivatives with potential antiviral activity, synthesis of compound 1 [2-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)acetonitrile] was performed through the reaction of cyanoacetic acid hydrazide with carbon disulfide in alcoholic potassium hydroxide. Compound 1 has an activating methylene group, so it was directed toward some specific reactions. Thus, aryldiazonium chlorides reacted with compound 1 affording hydrazono derivatives 2a-c. Also, aromatic aldehydes reacted with compound 1 to produce compounds 3a,b. Furthermore, cyclic ketones were subjected to the synthesis of fused thiophene derivatives 4a,b via reaction with compound 1 in the presence of elemental sulfur. In addition, 1,3,4-oxadiazole derivative 1, when reacted with isothiocyanates, salicylaldehyde or 1,3-dicarbonyl compounds, formed thiazole derivatives 5a,b, coumarin derivative 6 and alkenyl derivatives 7a,b resp. Compound 7b underwent cyclization to afford pyridine derivative 8. Arylhydrazono derivatives 9a,b were produced through the reaction of compound 7a with aryldiazonium chlorides. Products 9a,b underwent cyclization to produce pyridazine derivatives 10a,b. Finally, 1,3,4-oxadiazole derivative 1 was directed toward reaction with hydrazine derivatives, bromoacetophenone and ethylchloroacetate affording compounds 11a,b, 12 and 13, resp. Fused thiophene derivatives 14a,b were produced via reaction of compounds 4a,b with a mixture of malononitrile and ethylorthoformate. Antiviral activity of the synthesized products showed that 5-(4-amino-3-ethyl-2-thioxo-2,3-dihydrothiazol-5-yl)-1,3,4-oxadiazole-2(3H)-thione (5a) and 5-(4-amino-3-phenyl-2-thioxo-2,3-dihydrothiazol-5-yl)-1,3,4-oxadiazole-2(3H)-thione (5b) acted as the most active agents against Feline herpes virus, Feline corona virus, Herpes simplex virus-1 and Herpes simplex virus-2, whereas compound 2-(5-(2-phenylhydrazono)-4,5-dihydro-1,3,4-oxadiazol-2-yl)acetonitrile (11b) was the most effective against Vaccinia virus, Herpes simplex virus (TK-KOS-ACVr), Coxsackie virus B4 and Vesicular stomatitis virus.

Protective effect of puerarin on viral myocarditis and the underlying mechanism

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 HO-1 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. Key words: Viral myocarditis; BALB/c mice; Coxsackievirus B3; Nrf2; Puerarin

The Ectodomain of the Vaccinia Virus Glycoprotein A34 Is Required for Cell Binding by Extracellular Virions and Contains a Large Region Capable of Interaction with Glycoprotein B5

An interaction between the orthopoxvirus glycoproteins A34 and B5 has been reported. The transmembrane and ectodomain of A34 are sufficient for interaction with B5, localization of B5 to the site of intracellular wrapping, and subsequent incorporation into the envelope of released extracellular virions. Several mutagenic approaches were undertaken to better define the B5 interaction domain on A34. A set of C-terminal truncations in A34 identified residues 1 to 80 as sufficient for interaction with B5. Additional truncations identified residues 80 to 130 of A34 as sufficient for interaction with B5. To better understand the function of this region, a set of recombinant viruses expressing A34 with the full, partial, or no B5 interaction site (residues 1 to 130, 1 to 100, and 1 to 70, respectively) was constructed. All the recombinants expressing truncations of A34 incorporated B5 into extracellular virions but had a small-plaque phenotype similar to that of a virus with the A34R gene deleted (vΔA34R). Further characterization indicated that the small-plaque phenotype exhibited by these viruses is due to a combination of abrogated actin tail formation, reduced cell binding, and a defect in polyanion-induced nonfusogenic dissolution. Taken together, these results suggest that residues 80 to 130 of A34 are not necessary for the proper localization and incorporation of B5 into extracellular virions and, furthermore, that the C-terminal residues of A34 are involved in cell binding and dissolution.IMPORTANCE Previous studies have shown that the vaccinia virus glycoproteins A34 and B5 interact, and in the absence of A34, B5 is mislocalized and not incorporated into extracellular virions. Here, using a transient-transfection assay, residues 80 to 130 of the ectodomain of A34 were determined to be sufficient for interaction with B5. Recombinant viruses expressing A34 with a full, partial, or no B5 interaction site were constructed and characterized. All of the A34 truncations interacted with B5 as predicted by the transient-transfection studies but had a small-plaque phenotype. Further analysis revealed that all of the recombinants incorporated detectable levels of B5 into released virions but were defective in cell binding and extracellular virion (EV) dissolution. This study is the first to directly demonstrate that A34 is involved in cell binding and implicate the ectodomain in this role.

The mature EV71 virion induced a broadly cross-neutralizing VP1 antibody against subtypes of the EV71 virus.

Enterovirus 71 (EV71) has emerged as a neurological virus causing life-threatening diseases in young children and infants. Although EV71 vaccines in development have presented promising results in several clinical trials, the identified key antigen for improving the broad protective efficacy of EV71 vaccines has not been well investigated. In this report, we show that different multiplicities of infection (MOIs) of the B4(E59) virus significantly affect EV71 vaccine production in a serum-free microcarrier bioreactor system. The antigens produced from high MOIs of 10−1 and 10−2 exhibited higher yield and more infectious full particle (FP) contents in the EV71 vaccines than those produced with low MOIs of 10−4 and 10−6, leading to better cross-neutralizing efficacy. The C4(E36) neutralization results showed that only antisera raised from EV71 FPs provided substantial neutralizing titers against C4(E36), whereas empty particles (EPs) of EV71 conferred no efficacy. Competitive ELISA showed that anti-FP mainly binds to FPs and that 20% of antibodies bind to EPs, whereas most anti-EP binds EPs, with only 10% antibodies binding to FPs. VP1-adsorbed anti-FP lost most of the virus neutralization efficiency, suggesting that the VP1 subunit of FP is the major immunogenic antigen determining the ability of the EV71 vaccine to elicit cross-neutralizing antibodies against EV71 virus subtypes. These findings demonstrate that the high-MOI production approach is significantly correlated with FP productivity, thereby improving the cross-neutralization efficacy of an EV71 vaccine and providing the basis for a better vaccine design against widespread EV71 viruses.

Relation of Coxsackie B3 and B4 Viral Infections for Development of type 1 Diabetes Mellitus in Children: A Case-Control Study

Background: Human Type 1 diabetes (T1D), previously called Juvenile-onset diabetes is one of the most common chronic, multifactorial diseases of autoimmune origin with a strong genetic component, affecting about 542 000 children in the world and represents about 5-10% of all cases of diabetes. Human enteroviruses (HEVs), particularly Coxsackie B viruses (CVBs), might trigger the onset of type 1 diabetes (T1D). Objectives: Find out any relation between the Coxsackie virus type B3 & B4 infections in addition to GAD65 autoantibodies and the development of T1DM Patients and Methods: A matched case-control study was conducted and sixty cases and 120 controls were enrolled in the study.Enzyme-Linked Immunoassay (ELIZA) technique was used to detect IgM and IgG in serum against the Coxsackie B3 , B4 and GAD65 (Glutamic Acid Decarboxylase 65) autoantibodies of both cases and controls. Qualitative detection of the RNA of the Coxsackie B3 & B4 viruses in the cases and controls by the conventional PCR method using suitable primers in both cases and control. Molecular detection of the CB3 and CB4 RNA was done using according to the manufacturers’ instruction. Results:The following risk factors were found to be independently associated with illness, it were significantly associated with illness and at higher risk of T1DM : CB4 IgM Positivity (OR 47 [95% = 6.1-364.1], p = 0.0002).), CB4 RNA Positive (OR 39.6 [95%CI= 5.1 – 309], p = 0.0004 IgG Antibodies against both CVB3 and GAD65 (OR 32.9 [95%CI = 4.2 - 258.7], p = 0.0009). , GAD65 IgG Positivity (OR 11.8 [95% CI = 4.4 – 31.2], p = 0.001).and IgM Antibodies against both CB4 andGAD65 (OR 8.8 [95%CI = 2.7 – 28.2], p = 0.0002). other risk factors like CB3 IgM Positivity (OR 1.7 [95%CI = 0.6 – 4.5] p= 0.2),CB3 IgG Positivity((OR 1.3 [95%CI = 0.7-2.4], p=0.3).CB4 IgG Positivity(OR 2.1 [95%CI = 0.9-44], p = 0.06) and IgM -Antibodies against both CB3andGAD65 ((OR 1.3 [95%CI = 0.3 – 5.0], p = 0.6) with a moderately increased risk of illness, but these were not statistically significant. CB3 RNA Positive (OR 0.8 [95%CI= 0.3 -1.9] p = 0.6), GAD65 IgM Positivity (OR 0.7 [95%CI = 0.4 – 1.5], p = 0.7), IgG Antibodies against both CB4-GAD65 (OR 0.9 [95%CI = 2.9 – 8.8], p = 0.05) was not associated with illness as these were not statistically significant with the decreased risk of illness and has protective role against infection with T1D. Conclusions: We propose that children aged less than 17 years are at risk of T1D infection if exposure to CB4 whereas CB3 has protective role.

Cell-type specificity and functional redundancy of RIG-I-like receptors in innate immune sensing of Coxsackievirus B3 and encephalomyocarditis virus

The contributions of RIG-I and MDA5 receptors to sensing viruses of the Picornaviridae family were investigated. The picornaviruses encephalomyocarditis virus (EMCV) and Coxsackievirus B3 (CVB3) are detected by both MDA5 and RIG-I in bone marrow derived macrophages. In macrophages from wild type mice, type I IFN is produced early after infection; IFNβ synthesis is reduced in the absence of each sensor, while IFNα production is reduced in the absence of MDA5. EMCV and CVB3 do not replicate in murine macrophages, and their detection is different in murine embryonic fibroblasts (MEFs), in which the viruses replicate to high titers. In MEFs RIG-I was essential for the expression of type I IFNs but contributes to increased yields of CVB3, while MDA5 inhibited CVB3 replication but in an IFN independent manner. These observations demonstrate functional redundancy within the innate immune response to picornaviruses.

Fully printed one-step biosensing device using graphene/AuNPs composite

Driven by the growing need of simple, cost efficient and flexible sensing systems, we have designed here a fully printed Reduced Graphene Oxide (rGO) based impedimetric sensor for one step sensing of DNA. The DNA sensor was fabricated by stamping of layered rGO and rGO/gold nanoparticles/single stranded DNA (rGO/AuNPs/ssDNA) composites over PET substrates using wax-printing technique. rGO works as an excellent working electrode, while the AuNPs create a suitable environment for ssDNA immobilization. Counter and reference electrodes were previously screen-printed on the plastic substrate, making thus a compact and highly integrated sensing platform. The change in electron transfer resistance after hybridization with a target ssDNA specific of Coxsackie B3 virus was monitored using electrochemical impedance spectroscopy (EIS), finding a linear response in the range of concentrations 0.01–20 µM. The novel, simple and straightforward one-step printing process for fabrication of a biosensing device developed keeps in mind the growing need of large scale device manufacturing. The successful proof-of-concept for the detection of DNA hybridization can be extended to other affinity biosensors, taking advantage of the integration of the bioreceptor on the sensor surface. Such ready-to-use biosensor would lead to a one-step electrochemical detection.

Scenedesmus obliquus: Antioxidant and antiviral activity of proteins hydrolyzed by three enzymes.

PurposeTo obtain protein hydrolysates from fresh water green algae Scenedesmus obliquus by three different enzymes and evaluate its antioxidant and antiviral activity.MethodsEnzymatic hydrolysates of green algae Scenedesmus obliquus protein were prepared by treatment with: 1.2% solution of pepsin, trypsin or papain. Protein was extracted from S. obliquus by three different extraction methods. Protein extracts and hydrolysates were assessed from stained gels following SDS–PAGE of samples. Antioxidant activity of protein hydrolysates was investigated.ResultsS. obliquus cells and protein extracts were rich in Arg, Lys, Asp, Ala, and His. Protein hydrolyzed by papain (Sd1pa) and protein hydrolyzed by trypsin (Sd2Try) induced highest antioxidant activity based on 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical-scavenging (41.41% and 40.62%) respectively, and on 2,2′-azinobis 3-ethyl-benzothiazoline-6-sulphonate (ABTS) radical (87.03% and 45.12%) respectively, at 150 µg/ml. The inhibitory effect and mode of action of protein hydrolysates were evaluated against Coxsackie B3 virus (CVB3). Protein hydrolyzed by papain (Sd2pa) and protein hydrolyzed by pepsin (Sd1pep) at 100 µg/ml exhibited antiviral activity (66.2% and 57.6%, respectively), against (CVB3) from all protein hydrolysates.ConclusionS. obliquus protein hydrolysates have a potential as antioxidative neutraceutical ingredients and a potential therapeutic agent against CVB3.