Sequence Analysis of the Malaysian Low Pathogenic Avian Influenza Virus Strain H5N2 from Duck.

The avian influenza viruses (AIV) of the H5 subtype have the ability to mutate from low pathogenic (LPAI) to highly pathogenic (HPAI), which can cause high mortality in poultry. Little is known about the pathogenic switching apart from the mutations at the haemagglutinin cleavage site, which significantly contributes to the virus virulence switching phenomenon. Therefore, this study aimed to compare the molecular markers in the haemagglutinin (HA), neuraminidase (NA), and matrix (M) genes of a locally isolated LPAI AIV strain H5N2 from Malaysia with the reference HPAI strains using bioinformatics approaches, emphasising the pathogenic properties of the viral genes. First, the H5N2 strain A/Duck/Malaysia/8443/2004 was propagated in SPF eggs. The viral presence was verified by haemagglutination assay, RT-PCR, and sequencing. Results showed successful amplifications of HA (1695 bp), NA (1410 bp), and M (1019 bp) genes. The genes were sequenced and the deduced amino acid sequences were analysed computationally using MEGA 11 and NetNGlyc software. Analysis of the HA protein showed the absence of the polybasic cleavage motif, but presence of two amino acid residues that are known to affect pathogenicity. There were also two glycosylation sites (glycosites) compared to the reference HPAI viruses, which had three or more at the HA globular head domain. No NA stalk deletion was detected but the haemadsorbing and active centres of the studied NA protein were relatively similar to the reference HPAI H5N2 isolates of duck but not chicken origins. Six NA glycosites were also identified. Finally, we observed a consistent M1 and M2 amino acid sequences between our LPAI isolate with the other HPAI H5N1 or H5N2 reference proteins. These data demonstrate distinct characteristics of the Malaysian LPAI H5N2, compared to HPAI H5N2 or H5N1 from ducks or chickens, potentially aiding the epidemiological research on genetic dynamics of circulating AIV in poultry.

Next-generation viral nanoparticles for targeted delivery of therapeutics: Fundamentals, methods, biomedical applications, and challenges.

INTRODUCTION: Viral nanoparticles (VNPs) are virus-based nanocarriers that have been studied extensively and intensively for biomedical applications. However, their clinical translation is relatively low compared to the predominating lipid-based nanoparticles. Therefore, this article describes the fundamentals, challenges, and solutions of the VNP-based platform, which will leverage the development of next-generation VNPs. AREAS COVERED: Different types of VNPs and their biomedical applications are reviewed comprehensively. Strategies and approaches for cargo loading and targeted delivery of VNPs are examined thoroughly. The latest developments in controlled release of cargoes from VNPs and their mechanisms are highlighted too. The challenges faced by VNPs in biomedical applications are identified, and solutions are provided to overcome them. EXPERT OPINION: In the development of next-generation VNPs for gene therapy, bioimaging and therapeutic deliveries, focus must be given to reduce their immunogenicity, and increase their stability in the circulatory system. Modular virus-like particles (VLPs) which are produced separately from their cargoes or ligands before all the components are coupled can speed up clinical trials and commercialization. In addition, removal of contaminants from VNPs, cargo delivery across the blood brain barrier (BBB), and targeting of VNPs to organelles intracellularly are challenges that will preoccupy researchers in this decade.

Virus-like Particles of Nodavirus Displaying the Receptor Binding Domain of SARS-CoV-2 Spike Protein: A Potential VLP-Based COVID-19 Vaccine.

Since the outbreak of the coronavirus disease 2019 (COVID-19), various vaccines have been developed for emergency use. The efficacy of the initial vaccines based on the ancestral strain of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) has become a point of contention due to the emergence of new variants of concern (VOCs). Therefore, continuous innovation of new vaccines is required to target upcoming VOCs. The receptor binding domain (RBD) of the virus spike (S) glycoprotein has been extensively used in vaccine development due to its role in host cell attachment and penetration. In this study, the RBDs of the Beta (ß) and Delta (δ) variants were fused to the truncated Macrobrachium rosenbergii nodavirus capsid protein without the protruding domain (CΔ116-MrNV-CP). Immunization of BALB/c mice with the virus-like particles (VLPs) self-assembled from the recombinant CP showed that, with AddaVax as an adjuvant, a significantly high level of humoral response was elicited. Specifically, mice injected with equimolar of adjuvanted CΔ116-MrNV-CP fused with the RBD of the ß- and δ-variants increased T helper (Th) cell production with a CD8+/CD4+ ratio of 0.42. This formulation also induced proliferation of macrophages and lymphocytes. Overall, this study demonstrated that the nodavirus truncated CP fused with the SARS-CoV-2 RBD has potential to be developed as a VLP-based COVID-19 vaccine.

Genomic analysis and biological characterization of a novel Schitoviridae phage infecting Vibrio alginolyticus.

Vibrio alginolyticus is a Gram-negative bacterium commonly associated with mackerel poisoning. A bacteriophage that specifically targets and lyses this bacterium could be employed as a biocontrol agent for treating the bacterial infection or improving the shelf-life of mackerel products. However, only a few well-characterized V. alginolyticus phages have been reported in the literature. In this study, a novel lytic phage, named ΦImVa-1, specifically infecting V. alginolyticus strain ATCC 17749, was isolated from Indian mackerel. The phage has a short latent period of 15 min and a burst size of approximately 66 particles per infected bacterium. ΦImVa-1 remained stable for 2 h at a wide temperature (27-75 °C) and within a pH range of 5 to 10. Transmission electron microscopy revealed that ΦImVa-1 has an icosahedral head of approximately 60 nm in diameter with a short tail, resembling those in the Schitoviridae family. High throughput sequencing and bioinformatics analysis elucidated that ΦImVa-1 has a linear dsDNA genome of 77,479 base pairs (bp), with a G + C content of ~ 38.72% and 110 predicted gene coding regions (106 open reading frames and four tRNAs). The genome contains an extremely large virion-associated RNA polymerase gene and two smaller non-virion-associated RNA polymerase genes, which are hallmarks of schitoviruses. No antibiotic genes were found in the ΦImVa-1 genome. This is the first paper describing the biological properties, morphology, and the complete genome of a V. alginolyticus-infecting schitovirus. When raw mackerel fish flesh slices were treated with ΦImVa-1, the pathogen loads reduced significantly, demonstrating the potential of the phage as a biocontrol agent for V. alginolyticus strain ATCC 17749 in the food. KEY POINTS: • A novel schitovirus infecting Vibrio alginolyticus ATCC 17749 was isolated from Indian mackerel. • The complete genome of the phage was determined, analyzed, and compared with other phages. • The phage is heat stable making it a potential biocontrol agent in extreme environments.

Expression of Influenza M2e-NP Recombinant Fusion Protein in Escherichia coli BL21 (DE3) and Its Binding to Antibodies.

The current influenza vaccines only confer protection against the circulating influenza subtypes, therefore universal vaccines are needed to prevent upcoming influenza outbreaks caused by emerging influenza subtypes. The extracellular domain of influenza A M2 protein (M2e) is highly conserved among different subtypes of influenza A viruses, and it is able to elicit protective immunity against the viruses. The influenza nucleoprotein (NP) was used to display the M2e in this study due to its promising T-cell response and adjuvanticity. The M2e gene was fused to the 5'-end of the NP gene and then cloned into pRSET B vector. The DNA sequencing analysis revealed six point mutations in the M2e-NP fusion gene, including one mutation in the M2e peptide and five mutations in the NP. The mutations were reverted using PCR site-directed mutagenesis. The recombinant plasmids (pRSET B-M2e-NP and pRSET B-mM2e-NP) were introduced into Escherichia coli (E. coli) BL21 (DE3) for protein expression. The mutated and non-mutated proteins were subsequently expressed and named mM2e-NP and M2e-NP, respectively. The expression of mM2e-NP and M2e-NP was not affected by the mutations. The binding of anti-M2e antibody to the purified native mM2e-NP and M2e-NP also remained active. However, when the anti-NP antibody was tested, the signal produced by mM2e-NP was very weak. The results implied that the amino acid changes in the NP had adversely impacted on the conformation of mM2e-NP and subsequently affected the antibody binding. In light of the remarkable antibody binding to the M2e-NP fusion protein, this study highly recommends the potential of M2e-NP as a universal influenza vaccine candidate.

Quartz crystal microbalance-based biosensing of hepatitis B antigen using a molecularly imprinted polydopamine film.

Quartz crystal microbalance (QCM)-based biosensors are highly attractive as rapid diagnostic devices for detecting infectious diseases. However, the fabrication of QCM-based biosensors often involves tedious processes due to the poor stability of the biological recognition elements. In this work, the simple self-polymerisation of dopamine was used to functionalise the QCM crystal surface with a molecularly imprinted polydopamine (MIPDA) sensing film for detecting the hepatitis B core antigen (HBcAg), a serological biomarker of hepatitis B. Recognition cavities that complemented the size and shape of HBcAg were observed on the QCM crystal surface after functionalisation with the MIPDA film. The MIPDA-QCM biosensor showed a selective affinity for HBcAg, recording frequency responses up to 7.8 folds larger towards HBcAg compared to human serum albumin at the same analyte concentrations. The biosensor response was enhanced by using the optimal concentrations of 10 mg mL-1 of dopamine and 1 mg mL-1 of template for MIPDA film formation, resulting in a low detection limit (0.88 µg mL-1) that enables the detection of clinically relevant titres of HBcAg. The detection process could be completed within 10 min after sample loading without additional steps for signal amplification, highlighting the practical advantages of the MIPDA-QCM biosensor for point-of-care detection of hepatitis B.

Challenges and Complications of Poly(lactic-co-glycolic acid)-Based Long-Acting Drug Product Development.

Poly(lactic-co-glycolic acid) (PLGA) is one of the preferred polymeric inactive ingredients for long-acting parenteral drug products that are constituted of complex formulations. Despite over 30 years of use, there are still many challenges faced by researchers in formulation-related aspects pertaining to drug loading and release. Until now, PLGA-based complex generic drug products have not been successfully developed. The complexity in developing these generic drug products is not just due to their complex formulation, but also to the manufacturing process of the listed reference drugs that involve PLGA. The composition and product attributes of commercial PLGA formulations vary with the drugs and their intended applications. The lack of standard compendial methods for in vitro release studies hinders generic pharmaceutical companies in their efforts to develop PLGA-based complex generic drug products. In this review, we discuss the challenges faced in developing PLGA-based long-acting injectable/implantable (LAI) drug products; hurdles that are associated with drug loading and release that are dictated by the physicochemical properties of PLGA and product manufacturing processes. Approaches to overcome these challenges and hurdles are highlighted specifically with respect to drug encapsulation and release.

Potentially Toxic Metals in the High-Biomass Non-Hyperaccumulating Plant Amaranthus viridis: Human Health Risks and Phytoremediation Potentials.

Human health risk and phytoremediation of potentially toxic metals (PTMs) in the edible vegetables have been widely discussed recently. This study aimed to determine the concentrations of four PTMs, namely Cd, Fe, Ni, and Zn) in Amaranthus viridis (leaves, stems, and roots) collected from 11 sampling sites in Peninsular Malaysia and to assess their human health risk (HHR). In general, the metal levels followed the order: roots > stems > leaves. The metal concentrations (µg/g) in the leaves of A. viridis ranged from 0.45 to 2.18 dry weight (dw) (0.05−0.26 wet weight (ww)), 74.8 to 535 dw (8.97−64.2 ww), 2.02 to 7.45 dw (0.24−0.89 ww), and 65.2 to 521 dw (7.83−62.6 ww), for Cd, Fe, Ni, and Zn, respectively. The positive relationships between the metals, the plant parts, and the geochemical factions of their habitat topsoils indicated the potential of A. viridis as a good biomonitor of Cd, Fe, and Ni pollution. With most of the values of the bioconcentration factor (BCF) > 1.0 and the transfer factor (TF) > 1.0, A. viridis was a very promising phytoextraction agent of Ni and Zn. Additionally, with most of the values of BCF > 1.0 and TF < 1.0, A. viridis was a very promising phytostabiliser of Cd and Fe. With respect to HHR, the target hazard quotients (THQ) for Cd, Fe, Pb, and Zn in the leaves of A. viridis were all below 1.00, indicating there were no non-carcinogenic risks of the four metals to consumers, including children and adults. Nevertheless, routine monitoring of PTMs in Amaranthus farms is much needed.

Bio-nanogate manipulation on electrode surface as an electrochemical immunosensing strategy for detecting anti-hepatitis B surface antigen.

The diagnosis of hepatitis B virus (HBV) and monitoring of the vaccination efficiency against HBV require real-time analysis. The presence of antibody against hepatitis B virus surface antigen (anti-HBsAg) as a result of HBV infection and/or immunization may indicate individual immune status towards HBV. This study investigated the ability of a bio-nanogate-based displacement immunosensing strategy in detecting anti-HBsAg antibody, via nonspecific-binding between polyamidoamine dendrimers encapsulated gold nanoparticles (PAMAM-Au) and the 'antigenic determinant' region (aD) of HBsAg. For this purpose, maltose binding protein harbouring the aD region (MBP-aD) was synthesized as a bioreceptor and immobilized on the screen-printed carbon electrode (SPCE). Following that, PAMAM-Au was deposited on MBP-aD, forming the 'gate' and was used as a monitoring agent. Under optimal conditions, the high specificity of anti-HBsAg antibody towards MBP-aD displaced PAMAM-Au causing the decrement of anodic peak in differential pulse voltammetry (DPV) analysis. The signal changes were proportionally related to the concentration of anti-HBsAg antibody, in a range of 1 - 1000 mIU/mL with a limit of detection (LOD) of 2.5 mIU/mL. The results also showed high specificity and selectivity of the immunosensor platform in detecting anti-HBsAg antibody both in spiked buffer and human serum samples.

Immunological Analysis of Nodavirus Capsid Displaying the Domain III of Japanese Encephalitis Virus Envelope Protein.

Japanese encephalitis virus (JEV) is the pathogen that causes Japanese encephalitis (JE) in humans and horses. Lethality of the virus was reported to be between 20-30%, of which, 30-50% of the JE survivors develop neurological and psychiatric sequelae. Attributed to the low effectiveness of current therapeutic approaches against JEV, vaccination remains the only effective approach to prevent the viral infection. Currently, live-attenuated and chimeric-live vaccines are widely used worldwide but these vaccines pose a risk of virulence restoration. Therefore, continuing development of JE vaccines with higher safety profiles and better protective efficacies is urgently needed. In this study, the Macrobrachium rosenbergii nodavirus (MrNV) capsid protein (CP) fused with the domain III of JEV envelope protein (JEV-DIII) was produced in Escherichia coli. The fusion protein (MrNV-CPJEV-DIII) assembled into virus-like particles (VLPs) with a diameter of approximately 18 nm. The BALB/c mice injected with the VLPs alone or in the presence of alum successfully elicited the production of anti-JEV-DIII antibody, with titers significantly higher than that in mice immunized with IMOJEV, a commercially available vaccine. Immunophenotyping showed that the MrNV-CPJEV-DIII supplemented with alum triggered proliferation of cytotoxic T-lymphocytes, macrophages, and natural killer (NK) cells. Additionally, cytokine profiles of the immunized mice revealed activities of cytotoxic T-lymphocytes, macrophages, and NK cells, indicating the activation of adaptive cellular and innate immune responses mediated by MrNV-CPJEV-DIII VLPs. Induction of innate, humoral, and cellular immune responses by the MrNV-CPJEV-DIII VLPs suggest that the chimeric protein is a promising JEV vaccine candidate.

Regulation of Proteolytic Activity to Improve the Recovery of Macrobrachium rosenbergii Nodavirus Capsid Protein.

The causative agent of white tail disease (WTD) in the giant freshwater prawn is Macrobrachium rosenbergii nodavirus (MrNV). The recombinant capsid protein (CP) of MrNV was previously expressed in Escherichia coli, and it self-assembled into icosahedral virus-like particles (VLPs) with a diameter of approximately 30 nm. Extensive studies on the MrNV CP VLPs have attracted widespread attention in their potential applications as biological nano-containers for targeted drug delivery and antigen display scaffolds for vaccine developments. Despite their advantageous features, the recombinant MrNV CP VLPs produced in E. coli are seriously affected by protease degradations, which significantly affect the yield and stability of the VLPs. Therefore, the aim of this study is to enhance the stability of MrNV CP by modulating the protease degradation activity. Edman degradation amino acid sequencing revealed that the proteolytic cleavage occurred at arginine 26 of the MrNV CP. The potential proteases responsible for the degradation were predicted in silico using the Peptidecutter, Expasy. To circumvent proteolysis, specific protease inhibitors (PMSF, AEBSF and E-64) were tested to reduce the degradation rates. Modulation of proteolytic activity demonstrated that a cysteine protease was responsible for the MrNV CP degradation. The addition of E-64, a cysteine protease inhibitor, remarkably improved the yield of MrNV CP by 2.3-fold compared to the control. This innovative approach generates an economical method to improve the scalability of MrNV CP VLPs using individual protease inhibitors, enabling the protein to retain their structural integrity and stability for prominent downstream applications including drug delivery and vaccine development.

M2e-Based Influenza Vaccines with Nucleoprotein: A Review.

Discovery of conserved antigens for universal influenza vaccines warrants solutions to a number of concerns pertinent to the currently licensed influenza vaccines, such as annual reformulation and mismatching with the circulating subtypes. The latter causes low vaccine efficacies, and hence leads to severe disease complications and high hospitalization rates among susceptible and immunocompromised individuals. A universal influenza vaccine ensures cross-protection against all influenza subtypes due to the presence of conserved epitopes that are found in the majority of, if not all, influenza types and subtypes, e.g., influenza matrix protein 2 ectodomain (M2e) and nucleoprotein (NP). Despite its relatively low immunogenicity, influenza M2e has been proven to induce humoral responses in human recipients. Influenza NP, on the other hand, promotes remarkable anti-influenza T-cell responses. Additionally, NP subunits are able to assemble into particles which can be further exploited as an adjuvant carrier for M2e peptide. Practically, the T-cell immunodominance of NP can be transferred to M2e when it is fused and expressed as a chimeric protein in heterologous hosts such as Escherichia coli without compromising the antigenicity. Given the ability of NP-M2e fusion protein in inducing cross-protective anti-influenza cell-mediated and humoral immunity, its potential as a universal influenza vaccine is therefore worth further exploration.

Chimeric Virus-Like Particles of Prawn Nodavirus Displaying Hepatitis B Virus Immunodominant Region: Biophysical Properties and Cytokine Response.

Hepatitis B is a major global health challenge. In the absence of an effective treatment for the disease, hepatitis B vaccines provide protection against the viral infection. However, some individuals do not have positive immune responses after being vaccinated with the hepatitis B vaccines available in the market. Thus, it is important to develop a more protective vaccine. Previously, we showed that hepatitis B virus (HBV) 'a' determinant (aD) displayed on the prawn nodavirus capsid (Nc) and expressed in Spodoptera frugiperda (Sf9) cells (namely, Nc-aD-Sf9) self-assembled into virus-like particles (VLPs). Immunisation of BALB/c mice with the Nc-aD-Sf9 VLPs showed significant induction of humoral, cellular and memory B-cell immunity. In the present study, the biophysical properties of the Nc-aD-Sf9 VLPs were studied using dynamic light scattering (DLS) and circular dichroism (CD) spectroscopy. Enzyme-linked immunosorbent assay (ELISA) was used to determine the antigenicity of the Nc-aD-Sf9 VLPs, and multiplex ELISA was employed to quantify the cytokine response induced by the VLPs administered intramuscularly into BALB/c mice (n = 8). CD spectroscopy of Nc-aD-Sf9 VLPs showed that the secondary structure of the VLPs predominantly consisted of beta (ß)-sheets (44.8%), and they were thermally stable up to ~52 °C. ELISA revealed that the aD epitope of the VLPs was significantly antigenic to anti-HBV surface antigen (HBsAg) antibodies. In addition, multiplex ELISA of serum samples from the vaccinated mice showed a significant induction (p < 0.001) of IFN-γ, IL-4, IL-5, IL-6, IL-10, and IL-12p70. This cytokine profile is indicative of natural killer cell, macrophage, dendritic cell and cytotoxic T-lymphocyte activities, which suggests a prophylactic innate and adaptive cellular immune response mediated by Nc-aD-Sf9 VLPs. Interestingly, Nc-aD-Sf9 induced a more robust release of the aforementioned cytokines than that of Nc-aD VLPs produced in Escherichia coli and a commercially used hepatitis B vaccine. Overall, Nc-aD-Sf9 VLPs are thermally stable and significantly antigenic, demonstrating their potential as an HBV vaccine candidate.

Hepatitis B Virus-Like Particle: Targeted Delivery of Plasmid Expressing Short Hairpin RNA for Silencing the Bcl-2 Gene in Cervical Cancer Cells.

Gene therapy research has advanced to clinical trials, but it is hampered by unstable nucleic acids packaged inside carriers and there is a lack of specificity towards targeted sites in the body. This study aims to address gene therapy limitations by encapsidating a plasmid synthesizing a short hairpin RNA (shRNA) that targets the anti-apoptotic Bcl-2 gene using truncated hepatitis B core antigen (tHBcAg) virus-like particle (VLP). A shRNA sequence targeting anti-apoptotic Bcl-2 was synthesized and cloned into the pSilencer 2.0-U6 vector. The recombinant plasmid, namely PshRNA, was encapsidated inside tHBcAg VLP and conjugated with folic acid (FA) to produce FA-tHBcAg-PshRNA VLP. Electron microscopy revealed that the FA-tHBcAg-PshRNA VLP has an icosahedral structure that is similar to the unmodified tHBcAg VLP. Delivery of FA-tHBcAg-PshRNA VLP into HeLa cells overexpressing the folate receptor significantly downregulated the expression of anti-apoptotic Bcl-2 at 48 and 72 h post-transfection. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay demonstrated that the cells' viability was significantly reduced from 89.46% at 24 h to 64.52% and 60.63%, respectively, at 48 and 72 h post-transfection. As a conclusion, tHBcAg VLP can be used as a carrier for a receptor-mediated targeted delivery of a therapeutic plasmid encoding shRNA for gene silencing in cancer cells.

Molecular detection and characterisation of Domestic Cat Hepadnavirus (DCH) from blood and liver tissues of cats in Malaysia.

BACKGROUND: A new domestic cat hepadnavirus (DCH, family Hepadnaviridae) was first reported from whole blood samples of domestic cats in Australia in 2018, and from cat serum samples in Italy in 2019. The pathogenesis of DCH is unknown, but it was reported in cats with viraemia (6.5-10.8%), chronic hepatitis (43%) and hepatocellular carcinoma (28%). Recent reports suggest that DCH resembles the human hepatitis B virus (HBV) and its related hepatopathies. This study aims to detect and characterize DCH among domestic cats in Malaysia. A cross-sectional study was performed on 253 cats, of which 87 had paired blood and liver samples, entailing whole-genome sequencing and phylogenetic analysis of DCH from a liver tissue sample. RESULTS: Among the 253 cats included in this study, 12.3% of the whole blood samples tested positive for DCH. The detection rate was significantly higher in pet cats (16.6%, n = 24/145) compared to shelter cats (6.5%, n = 7/108). Liver tissues showed higher a DCH detection rate (14.9%, n = 13/87) compared to blood; 5 out of these 13 cats tested positive for DCH in their paired liver and blood samples. Serum alanine transaminase (ALT) was elevated (> 95 units/L) in 12 out of the 23 DCH-positive cats (52.2%, p = 0.012). Whole-genome sequence analysis revealed that the Malaysian DCH strain, with a genome size of 3184 bp, had 98.3% and 97.5% nucleotide identities to the Australian and Italian strains, respectively. The phylogenetic analysis demonstrated that the Malaysian DCH genome was clustered closely to the Australian strain, suggesting that they belong to the same geographically-determined genetic pool (Australasia). CONCLUSIONS: This study provided insights into a Malaysian DCH strain that was detected from a liver tissue. Interestingly, pet cats or cats with elevated ALT were significantly more likely to be DCH positive. Cats with positive DCH detection from liver tissues may not necessarily have viraemia. The impact of this virus on inducing liver diseases in felines warrants further investigation.

Loop-Mediated Isothermal Amplification (LAMP) as a Promising Point-of-Care Diagnostic Strategy in Avian Virus Research.

Over the years, development of molecular diagnostics has evolved significantly in the detection of pathogens within humans and their surroundings. Researchers have discovered new species and strains of viruses, while mitigating the viral infections that occur, owing to the accessibility of nucleic acid screening methods such as polymerase chain reaction (PCR), quantitative (real-time) polymerase chain reaction (qPCR) and reverse-transcription qPCR (RT-qPCR). While such molecular detection methods are widely utilized as the benchmark, the invention of isothermal amplifications has also emerged as a reliable tool to improvise on-field diagnosis without dependence on thermocyclers. Among the established isothermal amplification technologies are loop-mediated isothermal amplification (LAMP), recombinant polymerase amplification (RPA), strand displacement activity (SDA), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA) and rolling circle amplification (RCA). This review highlights the past research on and future prospects of LAMP, its principles and applications as a promising point-of-care diagnostic method against avian viruses.

Assessments of the Ecological and Health Risks of Potentially Toxic Metals in the Topsoils of Different Land Uses: A Case Study in Peninsular Malaysia.

Human activities due to different land uses are being studied widely in many countries. This study aimed to determine the ecological risks and human health risk assessments (HHRA) of Cd, Pb, Ni, Cu, and Zn in the topsoils of six land uses in Peninsular Malaysia. The ranges of the potentially toxic metals (PTMs) in the soils (mg/kg, dry weight) of this study were 0.24-12.43 for Cd (mean: 1.94), 4.66-2363 for Cu (mean: 228), 2576-116,344 for Fe (mean: 32,618), 2.38-75.67 for Ni (mean: 16.04), 7.22-969 for Pb (mean: 115) and 11.03-3820 for Zn (mean: 512). For the ecological risk assessments, the potential ecological risk index (PERI) for single metals indicated that the severity of pollution of the five metals decreased in the following sequence: Cd > Cu > Pb > Zn > Ni. It was found that industry, landfill, rubbish heap, and mining areas were categorized as "very high ecological risk". For HHRA, the land uses of industry, landfill and rubbish heap were found to have higher hazard quotient (HQ) values for the three pathways (with the order: ingestion > dermal contact > inhalation ingestion) of the five metals for children and adults, when compared to the mining, plantation, and residential areas. The values for both the non-carcinogenic (Cd, Cu, Ni, and Zn), and carcinogenic risks (CR) for inhalation (Cd and Ni) obtained for children and adults in this study showed no serious adverse health impacts on their health. However, of public concern, the hazard index (HI), for Pb of children at the landfill (L-3) and the rubbish heap (RH-3) sites exceeded 1.0, indicating non-carcinogenic risk (NCR) for children. Therefore, these PERI and HHRA results provided fundamental data for PTMs pollution mitigation and environmental management in areas of different land uses in Peninsular Malaysia.

Virus-like Particle Vaccines: A Prospective Panacea Against an Avian Influenza Panzootic.

Epizootics of highly pathogenic avian influenza (HPAI) have resulted in the deaths of millions of birds leading to huge financial losses to the poultry industry worldwide. The roles of migratory wild birds in the harbouring, mutation, and transmission of avian influenza viruses (AIVs), and the lack of broad-spectrum prophylactic vaccines present imminent threats of a global panzootic. To prevent this, control measures that include effective AIV surveillance programmes, treatment regimens, and universal vaccines are being developed and analysed for their effectiveness. We reviewed the epidemiology of AIVs with regards to past avian influenza (AI) outbreaks in birds. The AIV surveillance programmes in wild and domestic birds, as well as their roles in AI control were also evaluated. We discussed the limitations of the currently used AI vaccines, which necessitated the development of a universal vaccine. We evaluated the current development of AI vaccines based upon virus-like particles (VLPs), particularly those displaying the matrix-2 ectodomain (M2e) peptide. Finally, we highlighted the prospects of these VLP vaccines as universal vaccines with the potential of preventing an AI panzootic.

Targeted delivery of 5-fluorouracil-1-acetic acid (5-FA) to cancer cells overexpressing epithelial growth factor receptor (EGFR) using virus-like nanoparticles.

Chemotherapy is widely used in cancer treatments. However, non-specific distribution of chemotherapeutic agents to healthy tissues and normal cells in the human body always leads to adverse side effects and disappointing therapeutic outcomes. Therefore, the main aim of this study was to develop a targeted drug delivery system based on the hepatitis B virus-like nanoparticle (VLNP) for specific delivery of 5-fluorouracil-1-acetic acid (5-FA) to cancer cells expressing epithelial growth factor receptor (EGFR). 5-FA was synthesized from 5-fluorouracil (5-FU), and it was found to be less toxic than the latter in cancer cells expressing different levels of EGFR. The cytotoxicity of 5-FA increased significantly after being conjugated on the VLNP. A cell penetrating peptide (CPP) of EGFR was displayed on the VLNP via the nanoglue concept, for targeted delivery of 5-FA to A431, HT29 and HeLa cells. The results showed that the VLNP displaying the CPP and harboring 5-FA internalized the cancer cells and killed them in an EGFR-dependent manner. This study demonstrated that the VLNP can be used to deliver chemically modified 5-FU derivatives to cancer cells overexpressing EGFR, expanding the applications of the VLNP in targeted delivery of chemotherapeutic agents to cancer cells overexpressing this transmembrane receptor.

Advances in the Diagnosis of Foot-and-Mouth Disease.

Foot-and-mouth disease (FMD) is a devastating livestock disease caused by foot-and-mouth disease virus (FMDV). Outbreaks of this disease in a country always result in conspicuous economic losses to livestock industry and subsequently lead to serious socioeconomic damages due to the immediate imposition of trade embargo. Rapid and accurate diagnoses are imperative to control this infectious virus. In the current review, enzyme-linked immunosorbent assay (ELISA)-based methods used in FMD diagnosis are extensively reviewed, particularly the sandwich, liquid-phase blocking, and solid-phase competition ELISA. The differentiation of infected animals from vaccinated animals using ELISA-based methods is also highlighted, in which the role of 3ABC polyprotein as a marker is reviewed intensively. Recently, more studies are focusing on the molecular diagnostic methods, which detect the viral nucleic acids based on reverse transcription-polymerase chain reaction (RT-PCR) and RT-loop-mediated isothermal amplification (RT-LAMP). These methods are generally more sensitive because of their ability to amplify a minute amount of the viral nucleic acids. In this digital era, the RT-PCR and RT-LAMP are progressing toward the mobile versions, aiming for on-site FMDV diagnosis. Apart from RT-PCR and RT-LAMP, another diagnostic assay specifically designed for on-site diagnosis is the lateral flow immunochromatographic test strips. These test strips have some distinct advantages over other diagnostic methods, whereby the assay often does not require the aid of an external device, which greatly lowers the cost per test. In addition, the on-site diagnostic test can be easily performed by untrained personnel including farmers, and the results can be obtained in a few minutes. Lastly, the use of FMDV diagnostic assays for progressive control of the disease is also discussed critically.