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Papillomaviruses (PVs), double-stranded circular DNA viruses, typically cause regressing papillomas (warts) on mucosal or keratinized epithelia of a wide spectrum of species. The viruses largely infect mammals, whereby PV infections in humans, bovines, and rabbits are extensively reported. However, studies on non-mammalian PVs, particularly avian ones, are relatively lacking and worthy of investigation. This study performed a meta-analysis post-systematic review based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement guidelines to evaluate the occurrences of avian papillomaviruses (APVs) in bird species and effective materials used for virus detection. The electronic databases Science Direct, Medline via PubMed, and Google Scholar were used to search for the journal articles. Upon article eligibility check, the QUADAS-2 was employed to assess the data. Of 1139 records, 31 were eligible for full-text review, but only 9 were significant for the final review. The results showed that APVs are highly prevalent among the Fringillidae family, with a proportion of 81%, followed by Laridae (30%) and Anatidae (13%). The pooled prevalence of APV in tissue samples was 38%, while in swab samples was 13%. Only one study reported positive APV from fecal materials (0.4%); hence, the reliability comparison between these three samples was not performed. This study concluded that APVs are most prevalent in the Fringillidae bird family, while tissues are the most suitable biological samples for APV screening and should be considered as a single sample material. From epidemiology, knowledge of APV incidences and distribution may assist in controlling papillomatosis in bird species.

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.

The ecological and children’s Health Risk Assessments (HRA) of Copper (Cu) in aquatic bodies ranging from rivers, mangrove, estuaries, and offshore areas were studied using the Cited Cu Data in The Sediments (CCDITS) from 125 randomly selected papers published from 1980 to 2022. The ecological and children’s HRA were assessed in all CCDITS. Generally, local point Cu sources (8%) and lithogenic sources were the main controlling factors of Cu concentrations. The present review revealed three interesting points. First, there were 11 papers (8%) documenting Cu levels of more than 500 mg/kg dw while China was the country with the highest number (26%) of papers published between 1980 and 2022, out of 37 countries. Second, with the Cu data cited from the literature not normally distributed, the maximum Cu level was higher than all the established guidelines. However, the median Cu concentration was lower than most of the established guidelines. The median values of the geoaccumulation index (Igeo) indicated a status of ‘unpolluted‘ and ‘moderate contamination’ for the contamination factor (CF), and ‘low potential ecological risk’ for the ecological risk (ER) of Cu. However, the Cu ER could be based at present on the above mentioned 8% of the literature in the present study. Third, the calculated hazard index (HI) values were found to be below 1, indicating no potential chance of Cu non–carcinogenic effects in both adults and children, except for children’s HI values from Lake Pamvotis of Greece, and Victoria Harbor in Hong Kong. Thus, regular monitoring (every 2 years), depending upon the available resources, is recommended to assess the ecological–health risk of Cu pollution in aquatic bodies to abate the risk of Cu exposure to children’s health and avoid injurious impacts on the biota. It can be concluded that there is always a need for the mitigation and management of a Cu exposure risk assessment that can be used successfully for screening purposes to detect important human health exposure routes. Consequently, any sediments contaminated with Cu require rapid sediment remediation techniques.

Monitoring the level of metal pollution in a water body, especially in polluted areas, is crucial. Gastropod shells have been used as a biomonitor for metal pollution. The goal of this study was to evaluate the utilisation of the mudflat snails, Telescopium telescopium shells, as biomonitoring materials for nickel (Ni) pollution in the intertidal area. The snails and their habitat surface sediments were sampled from 17 sites in Peninsular Malaysia. Up to 21 individuals from each site were sampled and dissected. In addition to the shells, six parts of the soft tissues (cephalic tentacle, foot, gill, muscle, mantle, and remaining soft tissues) were analysed for Ni. The snail shell was found to be a potential biomonitoring material for Ni pollution based on four positive points: (i) higher value of shell/soft tissue ratios (> 1.00); (ii) categorisation as a ‘microconcentrator’ based on bioaccumulation factor; (iii) significant correlation coefficients (at least P < 0.05) and significant influential total Ni levels in the sediments to the shell Ni; and (iv) higher precision of Ni in the shells based on the lowest value of the coefficient of variation of Ni. The described results indicated that the shell of T. telescopium would be suitable for assessing Ni pollution in the intertidal areas.

Biomonitoring of heavy metals is an important part of the ecotoxicological study. However, without the application of the metal data to reflect human wellbeing, it is considered the low impact on the society. In this study, the heavy metals in green mustard Brassica rapa var. parachinensis were collected from Sikamat (Negeri Sembilan in 2013), Kg. Sitiawan (Perak in 2016), and Ara Kuda (Penang in 2016) of Peninsular Malaysia, were determined. The samples are analyzed for the concentrations of Cu, Fe, Ni, Pb, and Zn by using the flame atomic absorption spectrophotometer. For the leafy edible part, the metal concentrations (mg/kg dry weight) ranged from 16.1-18.5 for Cu, 145-207 for Fe, 1.02-1.64 for Ni, 0.90-2.73 for Pb, and 74.0-203 for Zn. These metal data were assessed for human health risks. It was found that all the values of target hazard quotients for Cu, Fe, Ni, Pb, and Zn in both adults and children were less than 1.00. This exhibited there were no non-carcinogenic risks of the five metals through the intake of the Brassica from the present study. This could indicate that the accumulation of metals and pollution threshold is below the limit of causing hazardous effects to consumers. Nonetheless, regular assessment of health risks of heavy metals in this vegetable needs to be conducted from time to time these vegetables are prone to continuous heavy metal contamination.

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

There are many biomonitoring studies of nickel in the mollusks, but to understand how the nickel is accumulated in the human body and the health risk posed by the metal is not a straightforward formula. Therefore, the aim of this paper is to draw a conceptual model of nickel content in the bodies of invertebrate mollusks and their gastrointestinal absorption of nickel. This model is useful to understand how nickel accumulation in the tissues of the mollusks could affect the nickel oral ingestion pathways. This conceptual model can shed some light on the mechanisms of nickel that may cause various toxicological risks and target cancer slope formulation in the future.

The current study assessed the ecological–health risks of potentially toxic arsenic (As) and antimony (Sb) in the vegetable Centella asiatica, topsoils, and mangrove sediments sampled from Peninsular Malaysia. The As concentrations ranged from 0.21 to 4.33, 0.18 to 1.83, and 1.32 to 20.8 mg/kg dry weight, for the leaves, stems, and roots of the vegetable, respectively. The ranges of Sb concentrations were 0.31–0.62, 0.12–0.35, and 0.64–1.61 mg/kg dry weight, for leaves, stems, and roots of the vegetable, respectively. The children’s target hazard quotient (THQ) values indicated no non-carcinogenic risks of As and Sb in both leaves and stems, although children’s THQ values were higher than those in adults. The calculated values of estimated weekly intake were lower than established provisional tolerable weekly intake of As and Sb for both children and adult consumers. The carcinogenic risk (CR) values of As for children’s intake of leaves and stems of vegetables showed more public concern than those of adults. The levels of Sb and As in the topsoils were generally higher (although not significantly) than those in the mangrove sediments, resulting in a higher geoaccumulation index, contamination factor and ecological risk, hazard index, THQ, and CR values. This indicated that the anthropogenic sources of Sb and As originated from the land-based activities before reaching the mangrove near the coast. The CR of As signifies a dire need for comprehensive ecological–health risks exposure studies, as dietary intake involves more than just vegetable consumption. Therefore, risk management for As and Sb in Malaysia is highly recommended. The present findings of the ecological–health risks of As and Sb based on 2010–2012 samples can be used as an important baseline for future reference and comparison. Copyright © 2022 Yap, Tan, Cheng, Syazwan, Azrizal-Wahid, Krishnan, Go, Nulit, Ibrahim, Mustafa, Omar, Chew, Edward, Okamura, Al-Mutairi, Al-Shami, Sharifinia, Keshavarzifard, You, Bakhtiari, Bintal, Zakaly, Arai, Naji, Saleem, Abd Rahman, Ong, Subramaniam and Wong. Ministry of Higher Education, Malaysia, MOHE The present study was supported financially by the Fundamental Research Grant Scheme (FRGS), (Vote No.: 5524953) and by the Ministry of Higher Education, Malaysia.

Microbial ecotoxicology was initially proposed by [1], while the field of ecotoxicology was founded by René Truhaut [2]. The anthropogenic pollution caused by population growth is the main cause of environmental problem nowadays [3]. It answers to a growing demand in social-economy around the world as a result of the harm to the environment and human health posed by intensive anthropogenic activities [4]. Most of the 17 United Nations Sustainable Development Goals (UN-SDGs) have made the underlying link between environmental health and human health and well-being a top focus [5]. When it comes to the abundance and distribution of living species, the ecosystem nutrient cycling has always been a basic ecological element with four primary components (nutrient pool, producers, consumers, and decomposers) under the energy flow (Figure 1). The ecological distribution and abundance of living organisms in the presence of a single chemical pollution or a combination of organic and inorganic pollutants (Figure 1) are well documented in the literature. From the molecular [6,7] to population levels [8-10], investigations on marine mussels are widely documented. However, in the presence of emerging novel viruses including bacteriophages, plant and animal viruses, the mechanisms of nutrient cycles are poorly known (Figure 1). It is believed that the components of decomposers consisting of bacteria, viruses and other microorganisms, may have accelerated the decomposition processes, resulting in a higher rate of mortality among producers and consumers. The goal of the microbial Eco toxicological study is to perform both basic and applied research in order to develop tools that society may use to track the evolution of environmental quality, and rehabilitate damaged locations [11]. Microbial ecotoxicology, in addition to Eco toxicological investigations, can provide novel ecological indicators and insights into quantifying the influence of contaminants on 'ecosystem health' [12]. https://www.raftpubs.com/jvsr-veterinary-science-and-research/articles/jvsr_raft1017.php

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

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 (β) 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 β- 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.

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

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.

The purpose of this paper is to discuss Cu and Zn concentrations in Perna viridis soft tissues from the western and eastern parts of Johore Singapore Causeway. The polluted eastern portion of the Causeway had greater levels of Cu and Zn in different areas of the soft tissues of mussels than the western part. This indicated the eastern section of the semi-enclosed Causeway had higher Cu and Zn bioavailability than the western part. With some reports of high metal levels in the eastern part of the Causeway from 2015 to 2018, it is predicted that there will be a plausible constant source of anthropogenic metal contamination in the eastern part of the Causeway beyond 2030 if anthropogenic activities are not effectively controlled.

The objective of this study was to compare the cited data of Cd and Pb levels in the green-lipped mussels (Perna viridis) with established maximum limits of both metals by European Union (EU) legislation for animal feeds. It was found that most mussel populations from Malaysia (90% for Cd; 87.5% for Pb) and Hong Kong (96.2% for Cd; 100% for Pb) were found to have lower levels of Cd and Pb than the maximum limits for animal feeds by EU. Therefore, both toxic metals based on the feeds of mussel meats on the animals should not pose serious toxicological effects. However, the meat of P. viridis are potential animal feeds similarly to the fish meal. Considering the accumulation of metals in the long term, future biomonitoring of Cd and Pb should focus on animal feed materials on mussel-based origins. Keywords:Animal Feed; Mussels; Safety https://www.raftpubs.com/jvsr-veterinary-science-and-research/articles/jvsr_raft1016.php

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.

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.

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.

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 (β)-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 &lt, 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.

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.

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.

Infectious diseases are the ever-present threats to public health and the global economy. Accurate and timely diagnosis is crucial to impede the progression of a disease and break the chain of transmission. Conventional diagnostic techniques are typically time-consuming and costly, making them inefficient for early diagnosis of infections and inconvenient for use at the point of care. Developments of sensitive, rapid, and affordable diagnostic methods are necessary to improve the clinical management of infectious diseases. Quartz crystal microbalance (QCM) systems have emerged as a robust biosensing platform due to their label-free mechanism, which allows the detection and quantification of a wide range of biomolecules. The high sensitivity and short detection time offered by QCM-based biosensors are attractive for the early detection of infections and the routine monitoring of disease progression. Herein, the strategies employed in QCM-based biosensors for the detection of infectious diseases are extensively reviewed, with a focus on prevalent diseases for which improved diagnostic techniques are in high demands. The challenges to the clinical application of QCM-based biosensors are highlighted, along with an outline of the future scope of research in QCM-based diagnostics., Highlights • QCM-based biosensors are promising tools for the rapid detection of infections. • Discussion of strategies for detecting disease biomarkers in QCM platforms. • Review of reported QCM-based biosensors for prevalent infectious diseases. • Overview of research opportunities in QCM-based diagnostics. • Microfluidic systems can boost the applicability of QCM for point-of-care diagnosis.

In order to identify host factors that impact Bovine Herpes Virus Type 1 (BHV-1) infection we previously applied a genome wide CRISPR knockout screen with a library covering all bovine protein coding genes. We compiled a list of both pro-viral and anti-viral proteins involved in BHV-1 replication; here we provide further analysis of those that are potentially involved in viral entry into the host cell. These entry related factors include the cell surface proteins PVR and PVRL2, a group of enzymes directly or indirectly associated with the biosynthesis of Heparan Sulfate Proteoglycans (HSPG), and proteins that reside in the Golgi apparatus engaging in intra-Golgi trafficking. For the first time, we provide evidence that PVRL2 serves a receptor for BHV-1, mediating more efficient entry than the previously identified PVR. By knocking out two enzymes that catalyze HSPG chain elongation, HST2ST1 and GLCE, we demonstrated the significance of HSPG in BHV-1 entry. Another intriguing cluster of genes, COG1, COG2 and COG4-7 encodes for six subunits of the conserved oligomeric Golgi (COG) complex. MDBK cells lacking COG6 were less infectable by BHV-1 but release newly produced virions more efficiently as evidenced by fewer but bigger plaques compared to control cells, suggesting impaired HSPG biosynthesis. To facilitate candidate validation, we devised a one-step multiplex CRISPR interference (CRISPRi) system named CRISPR3i that enables quick and simultaneous deployment of three CRISPRs for efficient gene inactivation. Using CRISPR3i, we verified an additional 23 candidates, with many implicated in cellular entry.

Chimeric virus-like particles (VLPs) have been widely exploited for various purposes including their use as vaccine candidates, particularly due to their ability to induce stronger immune responses than VLPs consisting of single viral proteins. In the present study, VLPs of the Macrobrachium rosenbergii nodavirus (MrNV) capsid protein (Nc) displaying the hepatitis B virus “a” determinant (aD) were produced in Spodoptera frugiperda (Sf9) insect cells. BALB/c mice immunised with the purified chimeric Nc-aD VLPs elicited a sustained titre of anti-aD antibody, which was significantly higher than that elicited by a commercially available hepatitis B vaccine and Escherichia coli-produced Nc-aD VLPs. Immunophenotyping showed that the Sf9-produced Nc-aD VLPs induced proliferation of cytotoxic T-lymphocytes and NK1.1 natural killer cells. Furthermore, enzyme-linked immunospot (ELISPOT)analysis showed the presence of antibody-secreting memory B cells in the mice splenocytes stimulated with the synthetic aD peptide. The significant humoral, natural killer cell and memory B cell immune responses induced by the Sf9-produced Nc-aD VLPs suggest that they present good prospects for use as a hepatitis B vaccine candidate.

Hepatitis B core antigen with the mutation of Y132A (HBcAg-Y132A) was successfully expressed in Escherichia coli. The mutant HBcAg-Y132A forms dimers and is unable to self-assemble into virus-like particles (VLPs). Hence, it is a potential antigen used in the antibody-responsive biosensor for the detection of anti-HBcAg in patients infected with hepatitis B virus. The aim of this study was to establish a direct purification strategy to recover HBcAg-Y132A dimer from the E. coli feedstock using SepFast™ Supor DEAE pre-packed column. The performance of this anion exchange chromatography was optimized in terms of the buffer composition (for adsorption and elution steps) and the mode of elution (i.e., step or gradient). The highest adsorption of HBcAg-Y132A dimer in the DEAE column was achieved with the buffer composed of 50 mM Tris-HCl (pH 8.4). The step elution using 50 mM Tris-HCl elution buffer (pH 8.4) supplemented with 1 M NaCl resulted in 1.2-fold increase in the purity of HBcAg, as compared to the gradient elution mode. In addition, it was found that the optimized 3-step elution is not directly applicable to elute the self-assembled HBcAg VLPs, as only 24.7% of the particles were recovered due to the limitation of size effect.

Foot-and-mouth disease (FMD) is a major threat to the livestock industry worldwide. Despite constant surveillance and effective vaccination, the perpetual mutations of the foot-and-mouth disease virus (FMDV) pose a huge challenge to FMD diagnosis. The immunodominant region of the FMDV VP1 protein (residues 131–170) displayed on phage T7 has been used to detect anti-FMDV in bovine sera. In the present study, the functional epitope was further delineated using amino acid sequence alignment, homology modelling and phage display. Two highly conserved regions (VP1145–152 and VP1159–170) were identified among different FMDV serotypes. The coding regions of these two epitopes were fused separately to the T7 genome and displayed on the phage particles. Interestingly, chimeric phage displaying the VP1159–170 epitope demonstrated a higher antigenicity than that displaying the VP1131–170 epitope. By contrast, phage T7 displaying the VP1145–152 epitope did not react significantly with the anti-FMDV antibodies in vaccinated bovine sera. This study has successfully identified a smaller functional epitope, VP1159–170, located at the C-terminal end of the structural VP1 protein. The phage T7 displaying this shorter epitope is a promising diagnostic reagent to detect anti-FMDV antibodies in vaccinated animals.

The enhanced green fluorescent protein (EGFP) is widely used as a marker in life science. Currently, purifications of the EGFP mostly involve chromatographic methods, which are multistep, time-consuming and costly. In the present study, the recombinant EGFP expressed in Escherichia coli was purified using an economic aqueous two-phase system (ATPS). Short-chain aliphatic alcohol and organic salt were chosen as the phase-forming components owing to their recyclability and biodegradability, respectively. The partition behaviour of EGFP was evaluated under the varying conditions of ATPS, including types and concentrations of phase-forming components, feedstock concentration, and pH. In an optimal primary 2-g ATPS comprising 1-propanol and tripotassium citrate, a high recovery of EGFP (i.e., 92.1%) was attained in the salt-rich bottom phase. To facilitate the easy recovery of purified EGFP, a secondary ATPS was used to back extract the EGFP to a new top ethanol-rich top phase, and the ethanol was removed from the purified EGFP via evaporation. The 1-propanol and ethanol used in the primary and secondary ATPSs (i.e. 20-g system) were successfully recycled in three successive rounds of EGFP purification, yielding the average EGFP purification factor of 11.34 and 75.7% of EGFP yield. The purification of EGFP using the two stages of alcohol + salt ATPSs is efficient in terms of operation time, cost and process scalability.

Simple Summary Many of the existing screening methods of avian viruses depend on clinical symptoms and pathological gross examinations that still necessitate confirmatory microscopic testing. Confirmation of a virus is often conducted at centralized laboratories that are well-equipped with instruments for virus isolation, hemagglutinin inhibition, virus neutralization, ELISA, PCR and qPCR. These assays are known for their great accuracy and sensitivity, and hence are set as standard practices. Nevertheless, limitations arise due to the time, cost and on-site applicability. As the technology progresses, molecular diagnostics should be more accessible to isolated areas and even practicable for use by non-skilled personnel such as farmers and private breeders. One of the point-of-care diagnostic strategies to consider for such matters is loop-mediated isothermal amplification (LAMP). Abstract 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.

Multifunctional nanocarriers displaying specific ligands and simultaneously response to stimuli offer great potentials for targeted and controlled drug delivery. Several synthetic thermally-responsive nanocarriers have been studied extensively for hyperthermia incorporated chemotherapy. However, no information is available on the application of virus-like particle (VLP) in thermally-controlled drug delivery systems. Here, we describe the development of a novel multifunctional nanovehicle based on the VLP of Macrobrachium rosenbergii nodavirus (MrNVLP). Folic acid (FA) was covalently conjugated to lysine residues located on the surface of MrNVLP, while doxorubicin (Dox) was loaded inside the VLP using an infusion method. This thermally-responsive nanovehicle, namely FA-MrNVLP-Dox, released Dox in a sustained manner and the rate of drug release increased in response to a hyperthermia temperature at 43 °C. The FA-MrNVLP-Dox enhanced the delivery of Dox to HT29 cancer cells expressing high level of folate receptor (FR) as compared to CCD841CoN normal cells and HepG2 cancer cells, which express low levels of FR. As a result, FA-MrNVLP-Dox increased the cytotoxicity of Dox on HT29 cells, and decreased the drug’s cytotoxicity on CCD841CoN and HepG2 cells. This study demonstrated the potential of FA-MrNVLP-Dox as a thermally-responsive nanovehicle for targeted delivery of Dox to cancer cells rich in FR.

Macrobrachium rosenbergii nodavirus (MrNV) is a virus native to giant freshwater prawn. Recombinant MrNV capsid protein has been produced in Escherichia coli, which self-assembled into virus-like particles (VLPs). However, this recombinant protein is unstable, degrading and forming heterogenous VLPs. In this study, MrNV capsid protein was produced in insect Spodoptera frugiperda (Sf9) cells through a baculovirus system. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the recombinant protein produced by the insect cells self-assembled into highly stable, homogenous VLPs each of approximately 40 nm in diameter. Enzyme-linked immunosorbent assay (ELISA) showed that the VLPs produced in Sf9 cells were highly antigenic and comparable to those produced in E. coli. In addition, the Sf9 produced VLPs were highly stable across a wide pH range (2-12). Interestingly, the Sf9 produced VLPs contained DNA of approximately 48 kilo base pairs and RNA molecules. This study is the first report on the production and characterization of MrNV VLPs produced in a eukaryotic system. The MrNV VLPs produced in Sf9 cells were about 10 nm bigger and had a uniform morphology compared with the VLPs produced in E. coli. The insect cell production system provides a good source of MrNV VLPs for structural and immunological studies as well as for host-pathogen interaction studies. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:549-557, 2017.

The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus-like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti-myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high-performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent.

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.

The major health issues posed by hepatitis B virus (HBV) globally have always been the impetus for the development of novel diagnostic assays. Here, we report the synthesis of bio-conjugated polyacrylamide-based hydrogel (HBPAAm hydrogel) that responds to the presence of hepatitis B core antigen (HBcAg) by a weight change in the hydrogel. The HBPAAm hydrogel contained the affinity crosslinks formed by the bio-specific coupling between the bound HBcAg and its antibody (anti-HBc). In the presence of analyte (i.e., free HBcAg), the immobilized HBcAg-(anti-HBc) complexes in HBPAAm hydrogel were disrupted; the competitive binding of free HBcAg onto the bound anti-HBc in HBPAAm hydrogel triggered the swelling of hydrogel. The swelling of HBPAAm hydrogel corresponded to the concentration of free HBcAg. The bio-sensing property of HBPAAm hydrogel is highly specific, and the reusability of HBPAAm hydrogel for multiple runs of HBcAg sensing was demonstrated. The bio-conjugated hydrogel has great potential as a sensing component of the biosensor that allows the detection of HBV serological markers at a lower cost and a shorter time.

Bio-nanogate involves synthesized or natural molecules as a 'gate' towards bioreceptors and responds upon the presence of targeted analytes in nanoscale dimension. Development of bio-nanogate improves analyte selectivity and signal response across various types of biosensors. The versatility of PAMAM dendrimers to form conjugates with guest molecules, such as proteins can be utilized in forming a bio-nanogate. PAMAM interaction with peptide bioreceptor for antibody detection is of interest in this study. This study investigated the interaction of synthesized immunogenic 'a' determinant (aD) region of hepatitis B virus surface antigen (HBsAg) with PAMAM G4 and anti-HBsAg antibody, as a potential bio-nanogate for anti-HBsAg detection. The aD peptide fused with maltose binding protein (MBP), was confirmed with Western blotting. Nano-Differential Scanning Fluorimetry (nano-DSF) study revealed that the interaction of MBP-aD with anti-HBsAg indicated a higher thermal stability as compared to its interaction with PAMAM G4. Electrochemical impedance spectroscopy showed that a higher binding constant of MBP-aD interaction with anti-HBsAg (0.92 μM-1) was observed at maximum saturation, as compared with PAMAM G4 (0.07 μM-1). Thermodynamic parameters demonstrated that MBP-aD interacted with anti-HBsAg and PAMAM G4, through van der Waals and hydrogen bonding. These analyses suggest that the weak interaction of MBP-aD and PAMAM G4 may form a potential bio-nanogate. It is hypothesized that the presence of anti-HBsAg has a higher affinity towards MBP-aD which may displace PAMAM G4 in the anti-HBsAg detection system. This interaction study is crucial as an initial platform of using peptide-PAMAM as a bio-nanogate in an antibody detection system.

Eupatorin is a polymethoxy flavone extracted from Orthosiphon stamineus and was reported to exhibit cytotoxic effects on several cancer cell lines. However, its effect as an anti–breast cancer agent in vivo has yet to be determined. This study aims to elucidate the potential of eupatorin as an anti–breast cancer agent in vivo using 4T1 challenged BALB/c mice model. In this article, BALB/c mice (20-22 g) challenged with 4T1 cells were treated with 5 mg/kg or 20 mg/kg eupatorin, while the untreated and healthy mice were fed with olive oil (vehicle) via oral gavage. After 28 days of experiment, the mice were sacrificed and blood was collected for serum cytokine assay, while tumors were harvested to extract RNA and protein for gene expression assay and hematoxylin-eosin staining. Organs such as spleen and lung were harvested for immune suppression and clonogenic assay, respectively. Eupatorin (20 mg/kg) was effective in delaying the tumor development and reducing metastasis to the lung compared with the untreated mice. Eupatorin (20 mg/kg) also enhanced the immunity as the population of NK1.1+ and CD8+ in the splenocytes and the serum interferon-γ were increased. Concurrently, eupatorin treatment also has downregulated the expression of pro-inflammatory and metastatic related genes (IL-1β. MMP9, TNF-α, and NF-κB). Thus, this study demonstrated that eupatorin at the highest dosage of 20 mg/kg body weight was effective in delaying the 4T1-induced breast tumor growth in the animal model.

Macrobrachium rosenbergii nodavirus (MrNV) is a pathogen of freshwater prawns that poses a threat to food security and causes significant economic losses in the aquaculture industries of many developing nations. A detailed understanding of the MrNV virion structure will inform the development of strategies to control outbreaks. The MrNV capsid has also been engineered to display heterologous antigens, and thus knowledge of its atomic resolution structure will benefit efforts to develop tools based on this platform. Here, we present an atomic-resolution model of the MrNV capsid protein (CP), calculated by cryogenic electron microscopy (cryoEM) of MrNV virus-like particles (VLPs) produced in insect cells, and three-dimensional (3D) image reconstruction at 3.3 Å resolution. CryoEM of MrNV virions purified from infected freshwater prawn post-larvae yielded a 6.6 Å resolution structure, confirming the biological relevance of the VLP structure. Our data revealed that unlike other known nodavirus structures, which have been shown to assemble capsids having trimeric spikes, MrNV assembles a T = 3 capsid with dimeric spikes. We also found a number of surprising similarities between the MrNV capsid structure and that of the Tombusviridae: 1) an extensive network of N-terminal arms (NTAs) lines the capsid interior, forming long-range interactions to lace together asymmetric units; 2) the capsid shell is stabilised by 3 pairs of Ca2+ ions in each asymmetric unit; 3) the protruding spike domain exhibits a very similar fold to that seen in the spikes of the tombusviruses. These structural similarities raise questions concerning the taxonomic classification of MrNV., Macrobrachium rosenbergii nodavirus (MrNV) is an economically important pathogen of freshwater prawns. Determination of the structure of the MrNV virion by cryo-electron microscopy reveals a capsid protein that is highly divergent from other known nodaviruses but has a surprising resemblance to plant-infecting tombusviruses., Author summary The freshwater prawn Macrobrachium rosenbergii is widely cultivated for food. Production is threatened by Macrobrachium rosenbergii nodavirus (MrNV), the causative agent of white-tail disease. Outbreaks in hatcheries often result in mortality rates of up to 100% in larvae and post-larvae, leading to devastating economic losses and threatening food security. We describe the atomic structure of the MrNV capsid, solved by cryogenic electron microscopy and three-dimensional image reconstruction. Our analysis revealed surprising differences between the structure of MrNV and that of other known nodaviruses. Moreover, we observed several features in the MrNV capsid that have been previously described in virion structures of the plant-infecting tombusvirus family. Most notably, the MrNV capsid exhibits pronounced dimeric spikes on its surface, the topology of this region closely resembling tombusvirus capsid spikes. Known nodavirus structures have trimeric spikes and do not display the same protein fold. The MrNV capsid is stabilised by divalent cations and laced together by a network of N-terminal arms that line the interior of the virion. Our analysis raises questions about the taxonomic classification of MrNV as well as revealing the structure of the capsid of this important pathogen. These data have the potential to inform the development of future interventions to prevent white-tail disease.

Macrobrachium rosenbergii nodavirus (MrNv) causes white tail disease (WTD) in giant freshwater prawns, which leads to devastating economic losses in the aquaculture industry. Despite extensive research on MrNv, there is still no antiviral agent to treat WTD. Thus, the main aim of this study was to identify potential anti-MrNv molecules. A 12-mer phage-displayed peptide library was biopanned against the MrNv virus-like particle (VLP). After four rounds of biopanning, two dominant phages harbouring the amino acid sequences HTKQIPRHIYSA and VSRHQSWHPHDL were selected. An equilibrium binding assay in solution was performed to determine the relative dissociation constant ( K D r e l ) of the interaction between the MrNv VLP and the selected fusion phages. Phage-HTKQIPRHIYSA has a K D r e l value of 92.4±22.8 nM, and phage-VSRHQSWHPHDL has a K D r e l value of 12.7±3.8 nM. An in-cell elisa was used to determine the inhibitory effect of the synthetic peptides towards the entry of MrNv VLP into Spodoptera frugiperda (Sf9) cells. Peptides HTKQIPRHIYSA and VSRHQSWHPHDL inhibited the entry of the MrNv VLP into Sf9 cells with IC50 values of 30.4±3.6 and 26.5±8.8 µM, respectively. Combination of both peptides showed a significantly higher inhibitory effect with an IC50 of 4.9±0.4 µM. An MTT assay revealed that the viability of MrNv-infected cells increased to about 97 % in the presence of both peptides. A real-time RT-PCR assay showed that simultaneous application of both peptides significantly reduced the number of MrNv per infected cell, from 97±9 to 11±4. These peptides are lead compounds which can be further developed into potent anti-MrNv agents.

Skin cancer or cutaneous carcinoma, is a pre-eminent global public health problem with no signs of plateauing in its incidence. As the most common treatments for skin cancer, surgical resection inevitably damages a patient’s appearance, and chemotherapy has many side effects. Thus, the main aim of this study was to screen for a cell penetrating peptide (CPP) for the development of a targeting vector for skin cancer. In this study, we identified a CPP with the sequence NRPDSAQFWLHH from a phage displayed peptide library. This CPP targeted the human squamous carcinoma A431 cells through an interaction with the epidermal growth factor receptor (EGFr). Methyl-β-cyclodextrin (MβCD) and chlorpromazine hydrochloride (CPZ) inhibited the internalisation of the CPP into the A431 cells, suggesting the peptide entered the cells via clathrin-dependent endocytosis. The CPP displayed on hepatitis B virus-like nanoparticles (VLNPs) via the nanoglue successfully delivered the nanoparticles into A431 cells. The present study demonstrated that the novel CPP can serve as a ligand to target and deliver VLNPs into skin cancer cells.

Hepatitis B virus core antigen (HBcAg) is the major structural protein of hepatitis B virus (HBV). The presence of anti-HBcAg antibody in a blood serum indicates that a person has been exposed to HBV. This study demonstrated that the immobilization of HBcAg onto the gold nanoparticles-decorated reduced graphene oxide (rGO-en-AuNPs) nanocomposite could be used as an antigen-functionalized surface to sense the presence of anti-HBcAg. The modified rGO-en-AuNPs/HBcAg was then allowed to undergo impedimetric detection of anti-HBcAg with anti-estradiol antibody and bovine serum albumin as the interferences. Upon successful detection of anti-HBcAg in spiked buffer samples, impedimetric detection of the antibody was then further carried out in spiked human serum samples. The electrochemical response showed a linear relationship between electron transfer resistance and the concentration of anti-HBcAg ranging from 3.91ngmL-1 to 125.00ngmL-1 with lowest limit of detection (LOD) of 3.80ngmL-1 at 3σm-1. This established method exhibits potential as a fast and convenient way to detect anti-HBcAg.

Poly(oligo(ethylene glycol) methacrylate) (POEGMA), an inert polymer was grafted onto an anion exchange adsorbent for the exclusion of relatively larger hepatitis B virus-like particles (HB-VLPs) from the anion exchange ligand (Q) and at the same time this process allowed the selective adsorption of smaller size Escherichia coli host cell proteins (HCPs). The chain lengths of the POEGMA grafted were modulated by varying the amount of monomers used in the polymer grafting. The purification factor and yield of the HB-VLPs obtained from the flow-through of negative chromatography were 2.3 and 66.0 ± 3.1%, respectively, when shorter chain length of POEGMA (SQ) was grafted. Adsorbent grafted with longer chain of POEGMA (LQ) excluded some HCPs that are larger in size together with the HB-VLPs, reducing the purity of the recovered HB-VLPs. Further heat-treatment of the flow-through pool from SQ followed by centrifugation increased the purity of heat stable HB-VLPs to 87.5 ± 1.1%. Heat-treatment of the flow through sample resulted in thermal denaturation and aggregation of HCPs, while the heat stable HB-VLPs still remained intact as observed under a transmission electron microscope. The performance of the negative chromatography together with heat treatment in the purification of HB-VLPs is far better than the reported bind-and-elute techniques.

Immunoglobulins G (IgG) against hepatitis B core antigen (HBcAg) was successfully purified using a purification scheme comprising ammonium sulphate precipitation and SepFast™ MM AH-1 column chromatography. Ammonium sulphate precipitation performed at 40% saturation was optimum in terms of the recovered polyclonal IgG concentration (7.8 mg/ml) and the removal of albumin (72%). The yield, purity and purification factor achieved from this simple purification method were 99%, 94% and 7.8, respectively. The IgG recovered from ammonium sulphate precipitation was subjected to SepFast™ MM AH-1 column chromatography and the purity of IgG was further increased to 98%, corresponding to a purification factor of 8.1. Protein aggregation was also reduced significantly in the purified IgG sample. Furthermore, the salt content in the purified sample was reduced by 75% and therefore the need of desalting final product was eliminated. Enzyme-linked immunosorbent assay (ELISA) showed that the antigenicity of anti-HBcAg IgG obtained after these purification processes was maintained.

Virus-like particles composed of the core antigen of hepatitis B virus (HBcAg) have been shown to be an effective platform for the display of foreign epitopes in vaccine development. Heterologous sequences have been successfully inserted at both amino and carboxy termini as well as internally at the major immunodominant epitope. We used cryogenic electron microscopy (CryoEM) and three-dimensional image reconstruction to investigate the structure of VLPs assembled from an N-terminal extended HBcAg that contained a polyhistidine tag. The insert was seen to form a trimeric spike on the capsid surface that was poorly resolved, most likely owing to it being flexible. We hypothesise that the capacity of N-terminal inserts to form trimers may have application in the development of multivalent vaccines to trimeric antigens. Our analysis also highlights the value of tools for local resolution assessment in studies of partially disordered macromolecular assemblies by cryoEM.

Hepatitis B virus‐like particles expressed in Escherichia coli were purified using anion exchange adsorbents grafted with polymer poly(oligo(ethylene glycol) methacrylate) in flow‐through chromatography mode. The virus‐like particles were selectively excluded, while the relatively smaller sized host cell proteins were absorbed. The exclusion of virus‐like particles was governed by the accessibility of binding sites (the size of adsorbents and the charge of grafted dextran chains) as well as the architecture (branch‐chain length) of the grafted polymer. The branch‐chain length of grafted polymer was altered by changing the type of monomers used. The larger adsorbent (90 μm) had an approximately twofold increase in the flow‐through recovery, as compared to the smaller adsorbent (30 μm). Generally, polymer‐grafted adsorbents improved the exclusion of the virus‐like particles. Overall, the middle branch‐chain length polymer grafted on larger adsorbent showed optimal performance at 92% flow‐through recovery with a purification factor of 1.53. A comparative study between the adsorbent with dextran grafts and the polymer‐grafted adsorbent showed that a better exclusion of virus‐like particles was achieved with the absorbent grafted with inert polymer. The grafted polymer was also shown to reduce strong interaction between binding sites and virus‐like particles, which preserved the particles’ structure.

Cancers have killed millions of people in human history and are still posing a serious health problem worldwide. Therefore, there is an urgent need for developing preventive and therapeutic cancer vaccines. Among various cancer vaccine development platforms, virus-like particles (VLPs) offer several advantages. VLPs are multimeric nanostructures with morphology resembling that of native viruses and are mainly composed of surface structural proteins of viruses but are devoid of viral genetic materials rendering them neither infective nor replicative. In addition, they can be engineered to display multiple, highly ordered heterologous epitopes or peptides in order to optimize the antigenicity and immunogenicity of the displayed entities. Like native viruses, specific epitopes displayed on VLPs can be taken up, processed, and presented by antigen-presenting cells to elicit potent specific humoral and cell-mediated immune responses. Several studies also indicated that VLPs could overcome the immunosuppressive state of the tumor microenvironment and break self-tolerance to elicit strong cytotoxic lymphocyte activity, which is crucial for both virus clearance and destruction of cancerous cells. Collectively, these unique characteristics of VLPs make them optimal cancer vaccine candidates. This review discusses current progress in the development of VLP-based cancer vaccines and some potential drawbacks of VLPs in cancer vaccine development. Extracellular vesicles with close resembling to viral particles are also discussed and compared with VLPs as a platform in cancer vaccine developments.

pH-responsive virus-like nanoparticles (VLNPs) hold promising potential as drug delivery systems for cancer therapy. In the present study, hepatitis B virus (HBV) VLNPs harbouring His-tags were used to display doxorubicin (DOX) via nitrilotriacetic acid (NTA) conjugation. The His-tags served as pH-responsive nanojoints which released DOX from VLNPs in a controlled manner. The His-tagged VLNPs conjugated non-covalently with NTA-DOX, and cross-linked with folic acid (FA) were able to specifically target and deliver the DOX into ovarian cancer cells via folate receptor (FR)-mediated endocytosis. The cytotoxicity and cellular uptake results revealed that the His-tagged VLNPs significantly increased the accumulation of DOX in the ovarian cancer cells and enhanced the uptake of DOX, which improved anti-tumour effects. This study demonstrated that NTA-DOX can be easily displayed on His-tagged VLNPs by a simple Add-and-Display step with high coupling efficiency and the drug was only released at low pH in a controlled manner. This approach facilitates specific attachment of any drug molecule on His-tagged VLNPs at the very mild conditions without changing the biological structure and native conformation of the VLNPs.