Displaying publications 41 - 60 of 87 in total

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  1. Expression of surface-bound nonstructural 1 (NS1) protein of influenza virus A H5N1 on Lactobacillus casei strain C1
    Tan TS, Syed Hassan S, Yap WB
    Lett Appl Microbiol, 2017 Jun;64(6):446-451.
    PMID: 28370088 DOI: 10.1111/lam.12738
    The study aimed to construct a recombinant Lactobacillus casei expressing the nonstructural (NS) 1 protein of influenza A virus H5N1 on its cell wall. The NS1 gene was first amplified and fused to the pSGANC332 expression plasmid. The NS1 protein expression was carried out by Lact. casei strain C1. PCR screening and DNA sequencing confirmed the presence of recombinant pSG-NS1-ANC332 plasmid in Lact. casei. The plasmid was stably maintained (98·94 ± 1·65%) by the bacterium within the first 20 generations without selective pressure. The NS1 was expressed as a 49-kDa protein in association with the anchoring peptide. The yield was 1·325 ± 0·065 μg mg(-1) of bacterial cells. Lactobacillus casei expressing the NS1 on its cell wall was red-fluorescently stained, but the staining was not observed on Lact. casei carrying the empty pSGANC332. The results implied that Lact. casei strain C1 is a promising host for the expression of surface-bound NS1 protein using the pSGANC332 expression plasmid.

    SIGNIFICANCE AND IMPACT OF THE STUDY: The study has demonstrated, for the first time, the expression of nonstructural 1 (NS1) protein of influenza A virus H5N1 on the cell wall of Lactobacillus casei using the pSGANC332 expression plasmid. Display of NS1 protein on the bacterial cell wall was evident under an immunofluorescence microscopic observation. Lactobacillus casei carrying the NS1 protein could be developed into a universal oral influenza vaccine since the NS1 is highly conserved among influenza viruses.

    Matched MeSH terms: Viral Nonstructural Proteins/genetics; Viral Nonstructural Proteins/metabolism*
  2. Sequences of E/NS1 gene junction from four dengue-2 viruses of northeastern Thailand and their evolutionary relationships with other dengue-2 viruses
    Zin K, Morita K, Igarashi A
    Microbiol. Immunol., 1995;39(8):581-90.
    PMID: 7494497
    We determined the 240-nucleotide sequences of the E/NS1 gene junction of four dengue-2 viruses by the primer extension dideoxy chain termination method. These viruses were isolated from dengue patients with different clinical severities in Nakhon Phanom, Northeastern Thailand in 1993. The results were compared with the 52 published dengue-2 sequences of the same gene region. Sequence divergence of four new isolates varied from 4.17% to 5.42% compared with dengue-2 prototype New Guinea C strain whereas it varied from 5.42% to 6.67% and from 6.67% to 7.09% when compared with Jamaica 1409 strain and PR159/S1 strain, respectively. All nucleotide substitutions were found at the 3rd position of the codons which were silent mutations. All 56 isolates studied were classified into five genotypic groups by constructing the dendrogram. The results indicated that four new isolates from Northeastern Thailand belong to genotype II of dengue virus serotype 2, and were most closely related to prototype New Guinea C strain. We also observed the variation in nucleotide and amino acid sequences among clusters of isolates (Thailand-1980, Malaysia-1989 and Thailand-1993) which were obtained from the dengue patients with different clinical severities. The significance of these genetic differences have been discussed in terms of the possible correlation between genetic variability and virulence.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics*
  3. Fulltext Cordycepin Inhibits Virus Replication in Dengue Virus-Infected Vero Cells
    Panya A, Songprakhon P, Panwong S, Jantakee K, Kaewkod T, Tragoolpua Y, et al.
    Molecules, 2021 May 23;26(11).
    PMID: 34071102 DOI: 10.3390/molecules26113118
    Dengue virus (DENV) infection causes mild to severe illness in humans that can lead to fatality in severe cases. Currently, no specific drug is available for the treatment of DENV infection. Thus, the development of an anti-DENV drug is urgently required. Cordycepin (3'-deoxyadenosine), which is a major bioactive compound in Cordyceps (ascomycete) fungus that has been used for centuries in Chinese traditional medicine, was reported to exhibit antiviral activity. However, the anti-DENV activity of cordycepin is unknown. We hypothesized that cordycepin exerts anti-DENV activity and that, as an adenosine derivative, it inhibits DENV replication. To test this hypothesis, we investigated the anti-DENV activity of cordycepin in DENV-infected Vero cells. Cordycepin treatment significantly decreased DENV protein at a half-maximal effective concentration (EC50) of 26.94 μM. Moreover, DENV RNA was dramatically decreased in cordycepin-treated Vero cells, indicating its effectiveness in inhibiting viral RNA replication. Via in silico molecular docking, the binding of cordycepin to DENV non-structural protein 5 (NS5), which is an important enzyme for RNA synthesis, at both the methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, was predicted. The results of this study demonstrate that cordycepin is able to inhibit DENV replication, which portends its potential as an anti-dengue therapy.
    Matched MeSH terms: Viral Nonstructural Proteins/metabolism
  4. Fulltext Doxycycline Interferes with Zika Virus Serine Protease and Inhibits Virus Replication in Human Skin Fibroblasts
    Chong Teoh T, J Al-Harbi S, Abdulrahman AY, Rothan HA
    Molecules, 2021 Jul 16;26(14).
    PMID: 34299596 DOI: 10.3390/molecules26144321
    Zika virus (ZIKV) represents a re-emerging threat to global health due to its association with congenital birth defects. ZIKV NS2B-NS3 protease is crucial for virus replication by cleaving viral polyprotein at various junctions to release viral proteins and cause cytotoxic effects in ZIKV-infected cells. This study characterized the inhibitory effects of doxycycline against ZIKV NS2B-NS3 protease and viral replication in human skin cells. The in silico data showed that doxycycline binds to the active site of ZIKV protease at a low docking energy (-7.8 Kcal/mol) via four hydrogen bonds with the protease residues TYR1130, SER1135, GLY1151, and ASP83. Doxycycline efficiently inhibited viral NS2B-NS3 protease at average human temperature (37 °C) and human temperature with a high fever during virus infection (40 °C). Interestingly, doxycycline showed a higher inhibitory effect at 40 °C (IC50 = 5.3 µM) compared to 37 °C (9.9 µM). The virus replication was considerably reduced by increasing the concentration of doxycycline. An approximately 50% reduction in virus replication was observed at 20 µM of doxycycline. Treatment with 20 µM of doxycycline reduced the cytopathic effects (CPE), and the 40 µM of doxycycline almost eliminated the CPE of human skin cells. This study showed that doxycycline binds to the ZIKV protease and inhibits its catalytic activity at a low micro-molecular concentration range. Treatment of human skin fibroblast with doxycycline eliminated ZIKV infection and protected the cells against the cytopathic effects of the infection.
    Matched MeSH terms: Viral Nonstructural Proteins/antagonists & inhibitors*; Viral Nonstructural Proteins/metabolism; Viral Nonstructural Proteins/chemistry
  5. Nucleotide sequences of the nonstructural protein NS1 gene of three dengue-2 viruses, M1, M2 and M3, isolated in Malaysia from patients with dengue haemorrhagic fever, dengue shock syndrome and dengue fever, respectively
    Fong MY, Koh CL, Samuel S, Pang T, Lam SK
    Nucleic Acids Res, 1990 Mar 25;18(6):1642.
    PMID: 2139210
    Matched MeSH terms: Viral Nonstructural Proteins
  6. Fulltext Antiviral cationic peptides as a strategy for innovation in global health therapeutics for dengue virus: high yield production of the biologically active recombinant plectasin peptide
    Rothan HA, Mohamed Z, Suhaeb AM, Rahman NA, Yusof R
    OMICS, 2013 Nov;17(11):560-7.
    PMID: 24044366 DOI: 10.1089/omi.2013.0056
    Dengue virus infects millions of people worldwide, and there is no vaccine or anti-dengue therapeutic available. Antimicrobial peptides have been shown to possess effective antiviral activity against various viruses. One of the main limitations of developing these peptides as potent antiviral drugs is the high cost of production. In this study, high yield production of biologically active plectasin peptide was inexpensively achieved by producing tandem plectasin peptides as inclusion bodies in E. coli. Antiviral activity of the recombinant peptide towards dengue serotype-2 NS2B-NS3 protease (DENV2 NS2B-NS3pro) was assessed as a target to inhibit dengue virus replication in Vero cells. Single units of recombinant plectasin were collected after applying consecutive steps of refolding, cleaving by Factor Xa, and nickel column purification to obtain recombinant proteins of high purity. The maximal nontoxic dose (MNTD) of the recombinant peptide against Vero cells was 20 μM (100 μg/mL). The reaction velocity of DENV2 NS2B-NS3pro decreased significantly after increasing concentrations of recombinant plectasin were applied to the reaction mixture. Plectasin peptide noncompetitively inhibited DENV2 NS2B-NS3pro at Ki value of 5.03 ± 0.98 μM. The percentage of viral inhibition was more than 80% at the MNTD value of plectasin. In this study, biologically active recombinant plectasin which was able to inhibit dengue protease and viral replication in Vero cells was successfully produced in E. coli in a time- and cost- effective method. These findings are potentially important in the development of potent therapeutics against dengue infection.
    Matched MeSH terms: Viral Nonstructural Proteins/antagonists & inhibitors; Viral Nonstructural Proteins/metabolism
  7. Fulltext Evaluation of commercially available diagnostic tests for the detection of dengue virus NS1 antigen and anti-dengue virus IgM antibody
    Hunsperger EA, Yoksan S, Buchy P, Nguyen VC, Sekaran SD, Enria DA, et al.
    PLoS Negl Trop Dis, 2014 Oct;8(10):e3171.
    PMID: 25330157 DOI: 10.1371/journal.pntd.0003171
    Commercially available diagnostic test kits for detection of dengue virus (DENV) non-structural protein 1 (NS1) and anti-DENV IgM were evaluated for their sensitivity and specificity and other performance characteristics by a diagnostic laboratory network developed by World Health Organization (WHO), the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and the Pediatric Dengue Vaccine Initiative (PDVI). Each network laboratory contributed characterized serum specimens for the panels used in the evaluation. Microplate enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT formats) were represented by the kits. Each ELISA was evaluated by 2 laboratories and RDTs were evaluated by at least 3 laboratories. The reference tests for IgM anti-DENV were laboratory developed assays produced by the Armed Forces Research Institute for Medical Science (AFRIMS) and the Centers for Disease Control and Prevention (CDC), and the NS1 reference test was reverse transcriptase polymerase chain reaction (RT-PCR). Results were analyzed to determine sensitivity, specificity, inter-laboratory and inter-reader agreement, lot-to-lot variation and ease-of-use. NS1 ELISA sensitivity was 60-75% and specificity 71-80%; NS1 RDT sensitivity was 38-71% and specificity 76-80%; the IgM anti-DENV RDTs sensitivity was 30-96%, with a specificity of 86-92%, and IgM anti-DENV ELISA sensitivity was 96-98% and specificity 78-91%. NS1 tests were generally more sensitive in specimens from the acute phase of dengue and in primary DENV infection, whereas IgM anti-DENV tests were less sensitive in secondary DENV infections. The reproducibility of the NS1 RDTs ranged from 92-99% and the IgM anti-DENV RDTs from 88-94%.
    Matched MeSH terms: Viral Nonstructural Proteins; Viral Nonstructural Proteins/immunology*
  8. Fulltext Dengue infection and miscarriage: a prospective case control study
    Tan PC, Soe MZ, Si Lay K, Wang SM, Sekaran SD, Omar SZ
    PLoS Negl Trop Dis, 2012;6(5):e1637.
    PMID: 22590658 DOI: 10.1371/journal.pntd.0001637
    Dengue is the most prevalent mosquito borne infection worldwide. Vertical transmissions after maternal dengue infection to the fetus and pregnancy losses in relation to dengue illness have been reported. The relationship of dengue to miscarriage is not known.
    Matched MeSH terms: Viral Nonstructural Proteins/blood
  9. Fulltext The diagnostic sensitivity of dengue rapid test assays is significantly enhanced by using a combined antigen and antibody testing approach
    Fry SR, Meyer M, Semple MG, Simmons CP, Sekaran SD, Huang JX, et al.
    PLoS Negl Trop Dis, 2011 Jun;5(6):e1199.
    PMID: 21713023 DOI: 10.1371/journal.pntd.0001199
    BACKGROUND: Serological tests for IgM and IgG are routinely used in clinical laboratories for the rapid diagnosis of dengue and can differentiate between primary and secondary infections. Dengue virus non-structural protein 1 (NS1) has been identified as an early marker for acute dengue, and is typically present between days 1-9 post-onset of illness but following seroconversion it can be difficult to detect in serum.
    AIMS: To evaluate the performance of a newly developed Panbio® Dengue Early Rapid test for NS1 and determine if it can improve diagnostic sensitivity when used in combination with a commercial IgM/IgG rapid test.
    METHODOLOGY: The clinical performance of the Dengue Early Rapid was evaluated in a retrospective study in Vietnam with 198 acute laboratory-confirmed positive and 100 negative samples. The performance of the Dengue Early Rapid in combination with the IgM/IgG Rapid test was also evaluated in Malaysia with 263 laboratory-confirmed positive and 30 negative samples.
    KEY RESULTS: In Vietnam the sensitivity and specificity of the test was 69.2% (95% CI: 62.8% to 75.6%) and 96% (95% CI: 92.2% to 99.8) respectively. In Malaysia the performance was similar with 68.9% sensitivity (95% CI: 61.8% to 76.1%) and 96.7% specificity (95% CI: 82.8% to 99.9%) compared to RT-PCR. Importantly, when the Dengue Early Rapid test was used in combination with the IgM/IgG test the sensitivity increased to 93.0%. When the two tests were compared at each day post-onset of illness there was clear differentiation between the antigen and antibody markers.
    CONCLUSIONS: This study highlights that using dengue NS1 antigen detection in combination with anti-glycoprotein E IgM and IgG serology can significantly increase the sensitivity of acute dengue diagnosis and extends the possible window of detection to include very early acute samples and enhances the clinical utility of rapid immunochromatographic testing for dengue.
    Matched MeSH terms: Viral Nonstructural Proteins/blood
  10. Vitamin D is a potential inhibitor of COVID-19: In silico molecular docking to the binding site of SARS-CoV-2 endoribonuclease Nsp15
    Shalayel MH, Al-Mazaideh GM, Aladaileh SH, Al-Swailmi FK, Al-Thiabat MG
    Pak J Pharm Sci, 2020 Sep;33(5):2179-2186.
    PMID: 33824127
    Novel coronavirus disease (COVID-19) has become a pandemic threat to public health. Vaccines and targeted therapeutics to prevent infections and stop virus proliferation are currently lacking. Endoribonuclease Nsp15 plays a vital role in the life cycle, including replication and transcription as well as virulence of the virus. Here, we investigated Vitamin D for its in silico potential inhibition of the binding sites of SARS-CoV-2 endoribonuclease Nsp15. In this study, we selected Remdesivir, Chloroquine, Hydroxychloroquine and Vitamin D to study the potential binding affinity with the putative binding sites of endoribonuclease Nsp15 of COVID-19. The docking study was applied to rationalize the possible interactions of the target compounds with the active site of endoribonuclease Nsp 15. Among the results, Vitamin D was found to have the highest potency with strongest interaction in terms of LBE, lowest RMSD, and lowest inhibition intensity Ki than the other standard compounds. The investigation results of endoribonuclease Nsp15 on the PrankWeb server showed that there are three prospective binding sites with the ligands. The singularity of Vitamin D interaction with the three pockets, particularly in the second pocket, may write down Vitamin D as a potential inhibitor of COVID-19 Nsp15 endoribonuclease binding sites and favour addition of Vitamin D in the treatment plan for COVID-19 alone or in combination with the other used drugs in this purpose, which deserves exploration in further in vitro and in vivo studies.
    Matched MeSH terms: Viral Nonstructural Proteins/metabolism
  11. Fulltext A new paradigm for Aedes spp. surveillance using gravid ovipositing sticky trap and NS1 antigen test kit
    Lau SM, Chua TH, Sulaiman WY, Joanne S, Lim YA, Sekaran SD, et al.
    Parasit Vectors, 2017 Mar 21;10(1):151.
    PMID: 28327173 DOI: 10.1186/s13071-017-2091-y
    BACKGROUND: Dengue remains a serious public health problem in Southeast Asia and has increased 37-fold in Malaysia compared to decades ago. New strategies are urgently needed for early detection and control of dengue epidemics.

    METHODS: We conducted a two year study in a high human density dengue-endemic urban area in Selangor, where Gravid Ovipositing Sticky (GOS) traps were set up to capture adult Aedes spp. mosquitoes. All Aedes mosquitoes were tested using the NS1 dengue antigen test kit. All dengue cases from the study site notified to the State Health Department were recorded. Weekly microclimatic temperature, relative humidity (RH) and rainfall were monitored.

    RESULTS: Aedes aegypti was the predominant mosquito (95.6%) caught in GOS traps and 23% (43/187 pools of 5 mosquitoes each) were found to be positive for dengue using the NS1 antigen kit. Confirmed cases of dengue were observed with a lag of one week after positive Ae. aegypti were detected. Aedes aegypti density as analysed by distributed lag non-linear models, will increase lag of 2-3 weeks for temperature increase from 28 to 30 °C; and lag of three weeks for increased rainfall.

    CONCLUSION: Proactive strategy is needed for dengue vector surveillance programme. One method would be to use the GOS trap which is simple to setup, cost effective (below USD 1 per trap) and environmental friendly (i.e. use recyclable plastic materials) to capture Ae. aegypti followed by a rapid method of detecting of dengue virus using the NS1 dengue antigen kit. Control measures should be initiated when positive mosquitoes are detected.

    Matched MeSH terms: Viral Nonstructural Proteins/analysis*; Viral Nonstructural Proteins/genetics; Viral Nonstructural Proteins/immunology
  12. Fulltext Thioguanine-based DENV-2 NS2B/NS3 protease inhibitors: Virtual screening, synthesis, biological evaluation and molecular modelling
    Hariono M, Choi SB, Roslim RF, Nawi MS, Tan ML, Kamarulzaman EE, et al.
    PLoS One, 2019;14(1):e0210869.
    PMID: 30677071 DOI: 10.1371/journal.pone.0210869
    Dengue virus Type 2 (DENV-2) is predominant serotype causing major dengue epidemics. There are a number of studies carried out to find its effective antiviral, however to date, there is still no molecule either from peptide or small molecules released as a drug. The present study aims to identify small molecules inhibitor from National Cancer Institute database through virtual screening. One of the hits, D0713 (IC50 = 62 μM) bearing thioguanine scaffold was derivatised into 21 compounds and evaluated for DENV-2 NS2B/NS3 protease inhibitory activity. Compounds 18 and 21 demonstrated the most potent activity with IC50 of 0.38 μM and 16 μM, respectively. Molecular dynamics and MM/PBSA free energy of binding calculation were conducted to study the interaction mechanism of these compounds with the protease. The free energy of binding of 18 calculated by MM/PBSA is -16.10 kcal/mol compared to the known inhibitor, panduratin A (-11.27 kcal/mol), which corroborates well with the experimental observation. Results from molecular dynamics simulations also showed that both 18 and 21 bind in the active site and stabilised by the formation of hydrogen bonds with Asn174.
    Matched MeSH terms: Viral Nonstructural Proteins/antagonists & inhibitors
  13. Fulltext Correlation of host inflammatory cytokines and immune-related metabolites, but not viral NS1 protein, with disease severity of dengue virus infection
    Soe HJ, Manikam R, Raju CS, Khan MA, Sekaran SD
    PLoS One, 2020;15(8):e0237141.
    PMID: 32764789 DOI: 10.1371/journal.pone.0237141
    Severe dengue can be lethal caused by manifestations such as severe bleeding, fluid accumulation and organ impairment. This study aimed to investigate the role of dengue non-structural 1 (NS1) protein and host factors contributing to severe dengue. Electrical cell-substrate impedance sensing system was used to investigate the changes in barrier function of microvascular endothelial cells treated NS1 protein and serum samples from patients with different disease severity. Cytokines and metabolites profiles were assessed using a multiplex cytokine assay and liquid chromatography mass spectrometry respectively. The findings showed that NS1 was able to induce the loss of barrier function in microvascular endothelium in a dose dependent manner, however, the level of NS1 in serum samples did not correlate with the extent of vascular leakage induced. Further assessment of host factors revealed that cytokines such as CCL2, CCL5, CCL20 and CXCL1, as well as adhesion molecule ICAM-1, that are involved in leukocytes infiltration were expressed higher in dengue patients in comparison to healthy individuals. In addition, metabolomics study revealed the presence of deregulated metabolites involved in the phospholipid metabolism pathway in patients with severe manifestations. In conclusion, disease severity in dengue virus infection did not correlate directly with NS1 level, but instead with host factors that are involved in the regulation of junctional integrity and phospholipid metabolism. However, as the studied population was relatively small in this study, these exploratory findings should be confirmed by expanding the sample size using an independent cohort to further establish the significance of this study.
    Matched MeSH terms: Viral Nonstructural Proteins/blood*; Viral Nonstructural Proteins/immunology
  14. Fulltext Complete genome analysis and characterization of neurotropic dengue virus 2 cosmopolitan genotype isolated from the cerebrospinal fluid of encephalitis patients
    Ngwe Tun MM, Muthugala R, Nabeshima T, Soe AM, Dumre SP, Rajamanthri L, et al.
    PLoS One, 2020;15(6):e0234508.
    PMID: 32555732 DOI: 10.1371/journal.pone.0234508
    Dengue virus (DENV) infection remains a major public health concern in many parts of the world, including Southeast Asia and the Americas. Sri Lanka experienced its largest dengue outbreak in 2017. Neurological symptoms associated with DENV infection have increasingly been reported in both children and adults. Here, we characterize DENV type 2 (DENV-2) strains, which were isolated from cerebrospinal fluid (CSF) and/or serum of patients with dengue encephalitis. Acute serum and CSF samples from each patient were subjected to dengue-specific non-structural protein 1 (NS1) antigen test, IgM and IgG enzyme-linked immunosorbent assay (ELISA), virus isolation, conventional and real-time polymerase chain reaction (PCR), and next-generation sequencing (NGS). Among the 5 dengue encephalitis patients examined, 4 recovered and 1 died. DENV-2 strains were isolated from serum and/or CSF samples of 3 patients. The highest viral genome levels were detected in the CSF and serum of the patient who succumbed to the illness. A phylogenetic tree revealed that the DENV-2 isolates belonged to a new clade of cosmopolitan genotype and were genetically close to strains identified in China, South Korea, Singapore, Malaysia, Thailand, and the Philippines. According to the NGS analysis, greater frequencies of nonsynonymous and synonymous mutations per gene were identified in the nonstructural genes. The full genomes of serum- and CSF-derived DENV-2 from the same patient shared 99.7% similarity, indicating that the virus spread across the blood-brain barrier. This is the first report to describe neurotropic DENV-2 using whole-genome analysis and to provide the clinical, immunological, and virological characteristics of dengue encephalitis patients during a severe dengue outbreak in Sri Lanka in 2017.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics*
  15. Fulltext Fusion of protegrin-1 and plectasin to MAP30 shows significant inhibition activity against dengue virus replication
    Rothan HA, Bahrani H, Mohamed Z, Abd Rahman N, Yusof R
    PLoS One, 2014;9(4):e94561.
    PMID: 24722532 DOI: 10.1371/journal.pone.0094561
    Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1) and plectasin (PLSN) were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN) as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro) with half-maximal inhibitory concentration (IC50) 0.5±0.1 μM. The real-time proliferation assay (RTCA) and the end-point proliferation assay (MTT assay) showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities.
    Matched MeSH terms: Viral Nonstructural Proteins/antagonists & inhibitors; Viral Nonstructural Proteins/metabolism
  16. Fulltext Immunodominant IgM and IgG Epitopes Recognized by Antibodies Induced in Enterovirus A71-Associated Hand, Foot and Mouth Disease Patients
    Aw-Yong KL, Sam IC, Koh MT, Chan YF
    PLoS One, 2016;11(11):e0165659.
    PMID: 27806091 DOI: 10.1371/journal.pone.0165659
    Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). Unlike other enteroviruses that cause HFMD, EV-A71 is more frequently associated with severe neurological complications and fatality. To date, no effective licensed antivirals are available to combat EV-A71 infection. Little is known about the immunogenicity of viral non-structural proteins in humans. Previous studies have mainly focused on characterization of epitopes of EV-A71 structural proteins by using immunized animal antisera. In this study, we have characterized human antibody responses against the structural and non-structural proteins of EV-A71. Each viral protein was cloned and expressed in either bacterial or mammalian systems, and tested with antisera by western blot. Results revealed that all structural proteins (VP1-4), and non-structural proteins 2A, 3C and 3D were targets of EV-A71 IgM, whereas EV-A71 IgG recognized all the structural and non-structural proteins. Sixty three synthetic peptides predicted to be immunogenic in silico were synthesized and used for the characterization of EV-A71 linear B-cell epitopes. In total, we identified 22 IgM and four IgG dominant epitopes. Synthetic peptide PEP27, corresponding to residues 142-156 of VP1, was identified as the EV-A71 IgM-specific immunodominant epitope. PEP23, mapped to VP1 41-55, was recognized as the EV-A71 IgG cross-reactive immunodominant epitope. The structural protein VP1 is the major immunodominant site targeted by anti-EV-A71 IgM and IgG antibodies, but epitopes against non-structural proteins were also detected. These data provide new understanding of the immune response to EV-A71 infection, which benefits the development of diagnostic tools, potential therapeutics and subunit vaccine candidates.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics; Viral Nonstructural Proteins/immunology*
  17. Molecular epidemiology of Malaysian dengue 2 viruses isolated over twenty-five years (1968-1993)
    Fong MY, Koh CL, Lam SK
    Res. Virol., 1998 Nov-Dec;149(6):457-64.
    PMID: 9923022
    The limited sequencing approach was used to study the molecular epidemiology of 24 Malaysian dengue 2 viruses which were isolated between 1968 and 1993. The sequences of a 240-nucleotide-long region across the envelope/non-structural 1 protein (E/NS1) gene junction of the isolates were determined and analysed. Alignment and comparison of the nucleotide and deduced amino acid sequences of the isolates revealed that nucleotide changes occurred mostly at the third position of a particular codon and were of the transition (AG, CU) type. Five nucleotide changes resulted in amino acid substitutions. Pairwise comparisons of the nucleotide sequences gave divergence values ranging from 0 to 9.2%. At the amino acid level, the divergence ranged between 0 and 3.8%. Based on the 6% divergence as the cut-off point for genotypic classification, the isolates were grouped into two genotypes, I and II. Comparison of the nucleotide sequences of the Malaysian dengue isolates with those of the dengue viruses of other regions of the world revealed that members of genotypes I and II were closely related to viruses from the Indian Ocean and Western Pacific regions, respectively.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics*
  18. Fulltext The Use of NS1 Rapid Diagnostic Test and qRT-PCR to Complement IgM ELISA for Improved Dengue Diagnosis from Single Specimen
    Teoh BT, Sam SS, Tan KK, Johari J, Abd-Jamil J, Hooi PS, et al.
    Sci Rep, 2016 06 09;6:27663.
    PMID: 27278716 DOI: 10.1038/srep27663
    Timely and accurate dengue diagnosis is important for differential diagnosis and immediate implementation of appropriate disease control measures. In this study, we compared the usefulness and applicability of NS1 RDT (NS1 Ag Strip) and qRT-PCR tests in complementing the IgM ELISA for dengue diagnosis on single serum specimen (n = 375). The NS1 Ag Strip and qRT-PCR showed a fair concordance (κ = 0.207, p = 0.001). While the NS1 Ag Strip showed higher positivity than qRT-PCR for acute (97.8% vs. 84.8%) and post-acute samples (94.8% vs. 71.8%) of primary infection, qRT-PCR showed higher positivity for acute (58.1% vs. 48.4%) and post-acute (50.0% vs.41.4%) samples in secondary infection. IgM ELISA showed higher positivity in samples from secondary dengue (74.2-94.8%) than in those from primary dengue (21.7-64.1%). More primary dengue samples showed positive with combined NS1 Ag Strip/IgM ELISA (99.0% vs. 92.8%) whereas more secondary samples showed positive with combined qRT-PCR/IgM ELISA (99.4% vs. 96.2%). Combined NS1 Ag Strip/IgM ELISA is a suitable combination tests for timely and accurate dengue diagnosis on single serum specimen. If complemented with qRT-PCR, combined NS1 Ag Strip/IgM ELISA would improve detection of secondary dengue samples.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics*
  19. Fulltext Inhibition of SARS-CoV-2 3CL protease by the anti-viral chimeric protein RetroMAD1
    Chan LC, Mat Yassim AS, Ahmad Fuaad AAH, Leow TC, Sabri S, Radin Yahaya RS, et al.
    Sci Rep, 2023 Nov 17;13(1):20178.
    PMID: 37978223 DOI: 10.1038/s41598-023-47511-z
    COVID-19 results from SARS-CoV-2, which mutates frequently, challenging current treatments. Therefore, it is critical to develop new therapeutic drugs against this disease. This study explores the interaction between SARS-CoV-2 3CLpro and RetroMAD1, a well-characterized coronavirus protein and potential drug target, using in-silico methods. The analysis through the HDOCK server showed stable complex formation with a binding energy of -12.3, the lowest among reference drugs. The RetroMAD1-3CLpro complex underwent a 100 ns molecular dynamics simulation (MDS) in an explicit solvation system, generating various trajectories, including RMSD, RMSF, hydrogen bonding, radius of gyration, and ligand binding energy. MDS results confirmed intact interactions within the RetroMAD1-3CLpro complex during simulations. In vitro experiments validated RetroMAD1's ability to inhibit 3CLpro enzyme activity and prevent SARS-CoV-2 infection in human bronchial cells. RetroMAD1 exhibited antiviral efficacy comparable to Remdesivir without cytotoxicity at effective concentrations. These results suggest RetroMAD1 as a potential drug candidate against SARS-CoV-2, warranting further in vivo and clinical studies to assess its efficiency.
    Matched MeSH terms: Viral Nonstructural Proteins/metabolism
  20. Fulltext Computational Approach Towards Exploring Potential Anti-Chikungunya Activity of Selected Flavonoids
    Seyedi SS, Shukri M, Hassandarvish P, Oo A, Shankar EM, Abubakar S, et al.
    Sci Rep, 2016 Apr 13;6:24027.
    PMID: 27071308 DOI: 10.1038/srep24027
    Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes chikungunya infection in humans. Despite the widespread distribution of CHIKV, no antiviral medication or vaccine is available against this virus. Therefore, it is crucial to find an effective compound to combat CHIKV. We aimed to predict the possible interactions between non-structural protein 3 (nsP) of CHIKV as one of the most important viral elements in CHIKV intracellular replication and 3 potential flavonoids using a computational approach. The 3-dimensional structure of nsP3 was retrieved from the Protein Data Bank, prepared and, using AutoDock Vina, docked with baicalin, naringenin and quercetagetin as ligands. The first-rated ligand with the strongest binding affinity towards the targeted protein was determined based on the minimum binding energy. Further analysis was conducted to identify both the active site of the protein that reacts with the tested ligands and all of the existing intermolecular bonds. Compared to the other ligands, baicalin was identified as the most potential inhibitor of viral activity by showing the best binding affinity (-9.8 kcal/mol). Baicalin can be considered a good candidate for further evaluation as a potentially efficient antiviral against CHIKV.
    Matched MeSH terms: Viral Nonstructural Proteins/metabolism; Viral Nonstructural Proteins/chemistry*
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