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Virus-like particles of Macrobrachium rosenbergii nodavirus produced in bacteria

Goh ZH, Tan SG, Bhassu S, Tan WS

J Virol Methods, 2011 Jul;175(1):74-9.
PMID: 21536072 DOI: 10.1016/j.jviromet.2011.04.021

Macrobrachium rosenbergii nodavirus (MrNv) infects giant freshwater prawns and causes white tail disease (WTD). The coding region of the capsid protein of MrNv was amplified with RT-PCR and cloned into the pTrcHis2-TOPO vector. The recombinant plasmid was introduced into Escherichia coli and protein expression was induced with IPTG. SDS-PAGE showed that the recombinant protein containing the His-tag and myc epitope has a molecular mass of about 46 kDa and it was detected by the anti-His antibody in Western blotting. The protein was purified using immobilized metal affinity chromatography (IMAC) and transmission electron microscopic analysis revealed that the recombinant protein assembled into virus-like particles (VLPs) with a diameter of about 30±3 nm. The size of the particles was confirmed by dynamic light scattering. Nucleic acids were extracted from the VLPs and treatment with nucleases showed that they were mainly RNA molecules. This is the first report describing the production of MrNv capsid protein in bacteria and its assembly into VLPs.
RNA-binding region of Macrobrachium rosenbergii nodavirus capsid protein

Goh ZH, Mohd NAS, Tan SG, Bhassu S, Tan WS

J Gen Virol, 2014 Sep;95(Pt 9):1919-1928.
PMID: 24878641 DOI: 10.1099/vir.0.064014-0

White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20-29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein.
Fulltext Dystrophin gene expression and intracellular calcium changes in the giant freshwater prawn, Macrobrachium rosenbergii, in response to white spot symptom disease infection

Noor AF, Soo TCC, Ghani FM, Goh ZH, Khoo LT, Bhassu S

Heliyon, 2017 Dec;3(12):e00446.
PMID: 29322096 DOI: 10.1016/j.heliyon.2017.e00446

Background: Dystrophin, an essential protein functional in the maintenance of muscle structural integrity is known to be responsible for muscle deterioration during white spot syndrome virus (WSSV) infection among prawn species. Previous studies have shown the upregulation of dystrophin protein in Macrobrachium rosenbergii (the giant freshwater prawn) upon white spot syndrome virus (WSSV) infection. The literature has also suggested the important role of calcium ion alterations in causing such muscle diseases. Thus, the interest of this study lies within the linkage between dystrophin functioning, intracellular calcium and white spot syndrome virus (WSSV) infection condition.
Methods: In this study, the dystrophin gene from M. rosenbergii (MrDys) was first characterised followed by the characterization of dystrophin gene from a closely related shrimp species, Penaeus monodon (PmDys). Dystrophin sequences from different phyla were then used for evolutionary comparison through BLAST analysis, conserved domain analysis and phylogenetic analysis. The changes in mRNA expression levels of dystrophin and the alteration of intracellular calcium concentrations in WSSV infected muscle cells were then studied.
Results: A 1246 base pair long dystrophin sequence was identified in the giant freshwater prawn, Macrobrachium rosenbergii (MrDys) followed by 1082 base pair long dystrophin sequence in P. monodon (PmDys). Four conserved domains were identified from the thirteen dystrophin sequences compared which were classified into 5 different phyla. From the phylogenetic analysis, aside from PmDys, the characterised MrDys was shown to be most similar to the invertebrate phylum of Nematoda. In addition, an initial down-regulation of dystrophin gene expression followed by eventual up-regulation, together with an increase in intracellular calcium concentration [Ca2+]
i
were shown upon WSSV experimental infection.
Discussion: Both the functionality of the dystrophin protein and the intracellular calcium concentration were affected by WSSV infection which resulted in progressive muscle degeneration. An increased understanding of the role of dystrophin-calcium in MrDys and the interactions between these two components is necessary to prevent or reduce occurrences of muscle degeneration caused by WSSV infection, thereby reducing economic losses in the prawn farming industry from such disease.
Fulltext Tracking the virus-like particles of Macrobrachium rosenbergii nodavirus in insect cells

Hanapi UF, Yong CY, Goh ZH, Alitheen NB, Yeap SK, Tan WS

PeerJ, 2017;5:e2947.
PMID: 28194311 DOI: 10.7717/peerj.2947

Macrobrachium rosenbergii nodavirus (MrNv) poses a major threat to the prawn industry. Currently, no effective vaccine and treatment are available to prevent the spread of MrNv. Its infection mechanism and localisation in a host cell are also not well characterised. The MrNv capsid protein (MrNvc) produced in Escherichia coli self-assembled into virus-like particles (VLPs) resembling the native virus. Thus, fluorescein labelled MrNvc VLPs were employed as a model to study the virus entry and localisation in Spodoptera frugiperda, Sf9 cells. Through fluorescence microscopy and sub-cellular fractionation, the MrNvc was shown to enter Sf9 cells, and eventually arrived at the nucleus. The presence of MrNvc within the cytoplasm and nucleus of Sf9 cells was further confirmed by the Z-stack imaging. The presence of ammonium chloride (NH4Cl), genistein, methyl-β-cyclodextrin or chlorpromazine (CPZ) inhibited the entry of MrNvc into Sf9 cells, but cytochalasin D did not inhibit this process. This suggests that the internalisation of MrNvc VLPs is facilitated by caveolae- and clathrin-mediated endocytosis. The whole internalisation process of MrNvc VLPs into a Sf9 cell was recorded with live cell imaging. We have also identified a potential nuclear localisation signal (NLS) of MrNvc through deletion mutagenesis and verified by classical-NLS mapping. Overall, this study provides an insight into the journey of MrNvc VLPs in insect cells.
Cultural tightness-looseness and normative social influence in eight Asian countries: Associations of individual and collective norms with vaccination intentions

Shi J, Kim HK, Salmon CT, Tandoc EC, Goh ZH

Soc Sci Med, 2024 Jan;340:116431.
PMID: 38000175 DOI: 10.1016/j.socscimed.2023.116431

RATIONALE: Countries worldwide faced the same public health crisis that required promoting the same health behavior-vaccinations-during the COVID-19 pandemic. Thus, scholars have a unique opportunity to test behavioral change theories across countries with different cultural backgrounds.
OBJECTIVE: Employing the extended theory of social normative behavior, this study examines the influence of individual and collective norms on COVID-19 vaccination intention across eight Asian countries. We examine how cultural tightness-looseness, defined as the degree of a culture's emphasis on norms and tolerance of deviant behavior, shapes normative social influence on COVID-19 vaccination intention.
METHODS: We conducted a multicountry online survey (N = 2676) of unvaccinated individuals in China, Indonesia, Japan, Malaysia, Singapore, South Korea, Thailand, and Vietnam in May and June 2021, when COVID-19 vaccination mandates had not yet been implemented in those countries. We conducted hierarchical regression analyses with interaction terms for the total sample and then re-categorizied the eight countries as either "tight" (n = 1102) or "loose" (n = 1574) to examine three-way interactions between individual norms, collective norms, and cultural tightness-looseness.
RESULTS: Perceived injunctive norms exerted the strongest impact of all normative factors on vaccination intention. Collective injunctive norms' influence depended on both perceived injunctive and descriptive norms, which was larger when norms were lower (vs. higher). The interactive pattern between perceived and collective norms was more pronounced in countries with greater cultural tightness.
CONCLUSION: Our findings reveal nuanced patterns of how individual and collective social norms influence health behavioral decisions, depending on the degree of cultural tightness-looseness.
Fulltext Induction of humoral and cell-mediated immune responses by hepatitis B virus epitope displayed on the virus-like particles of prawn nodavirus

Yong CY, Yeap SK, Goh ZH, Ho KL, Omar AR, Tan WS

Appl Environ Microbiol, 2015 Feb;81(3):882-9.
PMID: 25416760 DOI: 10.1128/AEM.03695-14

Hepatitis B virus (HBV) is a deadly pathogen that has killed countless people worldwide. Saccharomyces cerevisiae-derived HBV vaccines based upon hepatitis B surface antigen (HBsAg) is highly effective. However, the emergence of vaccine escape mutants due to mutations on the HBsAg and polymerase genes has produced a continuous need for the development of new HBV vaccines. In this study, the "a" determinant within HBsAg was displayed on the recombinant capsid protein of Macrobrachium rosenbergii nodavirus (MrNV), which can be purified easily in a single step through immobilized metal affinity chromatography (IMAC). The purified protein self-assembled into virus-like particles (VLPs) when observed under a transmission electron microscope (TEM). Immunization of BALB/c mice with this chimeric protein induced specific antibodies against the "a" determinant. In addition, it induced significantly more natural killer and cytotoxic T cells, as well as an increase in interferon gamma (IFN-γ) secretion, which are vital for virus clearance. Collectively, these findings demonstrated that the MrNV capsid protein is a potential carrier for the HBV "a" determinant, which can be further extended to display other foreign epitopes. This paper is the first to report the application of MrNV VLPs as a novel platform to display foreign epitopes.