HLA-B*57:01 typing in a Malaysian cohort: implications of abacavir hypersensitivity in people living with HIV.

Background: Abacavir (ABC) in combination with other antiretroviral drugs, is used to treat people living with HIV (PLWH). However, it is linked to a fatal hypersensitivity reaction in susceptible individuals, and is strongly associated with the HLA-B*57:01 allele. Materials & methods: A total of 152 patients, 50 PLWH and 102 HIV-1 negative patients, were assessed for the HLA-B*57:01 allele through a sequence-specific primer PCR. Results: All PLWH tested negative for the HLA-B*57:01 allele, but two HIV-negative patients were found to have HLA-B*57, with one of them expressing the HLA-B*57:01 allele. Conclusion: Given the low prevalence of this risk allele in the population, testing for the presence of HLA-B*57:01 in PLWH may not provide significant benefit for the reported population.

Plastomes of Garcinia mangostana L. and Comparative Analysis with Other Garcinia Species.

The two varieties of mangosteen (Garcinia mangostana L.) cultivated in Malaysia are known as Manggis and Mesta. The latter is preferred for its flavor, texture, and seedlessness. Here, we report a complete plastome (156,580 bp) of the Mesta variety that was obtained through a hybrid assembly approach using PacBio and Illumina sequencing reads. It encompasses a large single-copy (LSC) region (85,383 bp) and a small single-copy (SSC) region (17,137 bp) that are separated by 27,230 bp of inverted repeat (IR) regions at both ends. The plastome comprises 128 genes, namely, 83 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The plastome of the Manggis variety (156,582 bp) obtained from reference-guided assembly of Illumina reads was found to be nearly identical to Mesta except for two indels and the presence of a single-nucleotide polymorphism (SNP). Comparative analyses with other publicly available Garcinia plastomes, including G. anomala, G. gummi-gutta, G. mangostana var. Thailand, G. oblongifolia, G. paucinervis, and G. pedunculata, found that the gene content, gene order, and gene orientation were highly conserved among the Garcinia species. Phylogenomic analysis divided the six Garcinia plastomes into three groups, with the Mesta and Manggis varieties clustered closer to G. anomala, G. gummi-gutta, and G. oblongifolia, while the Thailand variety clustered with G. pedunculata in another group. These findings serve as future references for the identification of species or varieties and facilitate phylogenomic analysis of lineages from the Garcinia genus to better understand their evolutionary history.

Increased Prevalence and New Evidence of Multi-Species Chelonid Herpesvirus 5 (ChHV5) Infection in the Sea Turtles of Mabul Island, Borneo.

Fibropapillomatosis (FP) is a debilitating tumor disease affecting all species of sea turtles globally. The most probable etiological agent for FP is the chelonid herpesvirus 5 (ChHV5). A 2015-2016 field survey of the sea turtles at Mabul Island, Sabah, Malaysia, found three green turtles (Chelonia mydas) with FP tumors. However, the presence of ChHV5 was confirmed in 7.8% (9/115) green turtles and was absent (0/16) in the hawksbill (Eretmochelys imbricata) turtles, as determined through molecular approaches. Subsequent to this, we managed to conduct field sampling of sea turtles in November 2019, just prior to the pandemic lockdown. Here, we aim to determine the extent of ChHV5 infection, and whether the virus has spread to other species of sea turtles around Mabul Island after the first reports of ChHV5 and FP. A total of 69 tissue samples were obtained from green (63), hawksbill (5), and olive ridley (Lepidochelys olivacea) (1) turtles in November 2019. We observed only one green turtle that exhibited FP tumors. To determine the presence of ChHV5, viral DNA was isolated from all the tissue samples, and polymerase chain reaction (PCR) analysis targeting three highly conserved regions of the virus, i.e., the capsid protein gene, glycoprotein H gene, and glycoprotein B gene, was performed. Out of 63 green turtles, 27 were positive for the presence of the virus. The prevalence of ChHV5 in the green turtles showed an increase of 42.9% as compared to the previous sampling conducted in 2015-2016. Additionally, for the first time, three out of the five hawksbill turtles, and one olive ridley turtle, were also PCR-positive for the virus. In conclusion, this study reveals that there has been an increase in ChHV5 infection among turtles in Mabul Island over the last 3 years. ChHV5 should be considered a potential threat, and mitigation efforts should be taken to prevent the spread of infection among the endangered sea turtles of Mabul Island and surrounding islands within the Coral Triangle.

Improvements to the Rapid Detection of the Marine Pathogenic Bacterium, Vibrio harveyi, Using Loop-Mediated Isothermal Amplification (LAMP) in Combination with SYBR Green.

The common methods that are presently used to identify Vibrio harveyi include microscopic examination and biochemical, immunological and PCR-based assays. These methods require technical expertise, and can be time-consuming. A rapid method is required for the high-throughput screening of large number of samples. As such, we have developed a rapid, simple yet sensitive and specific detection method based on the use of the loop-mediated isothermal amplification (LAMP) of DNA. A set of six primers, i.e., two outer, two inner and two loop primers, was designed based on the in silico analysis of a large pool of 39 strains of the toxR gene sequence of V. harveyi. The addition of the loop primers decreased the reaction time of the LAMP by more than half. Furthermore, with the application of SYBR Green, the result can be obtained as quickly as in 10 to 15 min without the need of gel electrophoresis. The specificity of the method primers was then determined by performing LAMP with Vibrio and non-Vibrio samples. LAMP has a greater sensitivity than PCR reaction. The sensitivity of PCR was at 0.6 pg concentration of V. harveyi recombinant plasmid DNA standard, while LAMP was able to detect lower amounts even at 0.6 fg. The development of the LAMP assay will provide a valuable tool for the high-throughput rapid detection of V. harveyi contamination both in laboratories and in the field.

Heterologous Expression and Catalytic Properties of Codon-Optimized Small-Sized Bromelain from MD2 Pineapple.

Bromelain is a unique enzyme-based bioactive complex containing a mixture of cysteine proteases specifically found in the stems and fruits of pineapple (Ananas comosus) with a wide range of applications. MD2 pineapple harbors a gene encoding a small bromelain cysteine protease with the size of about 19 kDa, which might possess unique properties compared to the other cysteine protease bromelain. This study aims to determine the expressibility and catalytic properties of small-sized (19 kDa) bromelain from MD2 pineapple (MD2-SBro). Accordingly, the gene encoding MD2-SBro was firstly optimized in its codon profile, synthesized, and inserted into the pGS-21a vector. The insolubly expressed MD2-SBro was then resolubilized and refolded using urea treatment, followed by purification by glutathione S-transferase (GST) affinity chromatography, yielding 14 mg of pure MD2-SBro from 1 L of culture. The specific activity and catalytic efficiency (kcat/Km) of MD2-SBro were 3.56 ± 0.08 U mg-1 and 4.75 ± 0.23 × 10-3 µM-1 s-1, respectively, where optimally active at 50 °C and pH 8.0, and modulated by divalent ions. The MD2-SBro also exhibited the ability to scavenge the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) with an IC50 of 0.022 mg mL-1. Altogether, this study provides the production feasibility of active and functional MD2-Bro as a bioactive compound.

Mitochondrial genome of Garcinia mangostana L. variety Mesta.

Fruits of Garcinia mangostana L. (mangosteen) are rich in nutrients with xanthones found in the pericarp having great pharmaceutical potential. Mangosteen variety Mesta is only found in Malaysia, which tastes sweeter than the common Manggis variety in Southeast Asia. In this study, we report the complete mitogenome of G. mangostana L. variety Mesta with a total sequence length of 371,235 bp of which 1.7% could be of plastid origin. The overall GC content of the mitogenome is 43.8%, comprising 29 protein-coding genes, 3 rRNA genes, and 21 tRNA genes. Repeat and tandem repeat sequences accounted for 5.8% and 0.15% of the Mesta mitogenome, respectively. There are 333 predicted RNA-editing sites in Mesta mitogenome. These include the RNA-editing events that generated the start codon of nad1 gene and the stop codon of ccmFC gene. Phylogenomic analysis using both maximum likelihood and Bayesian analysis methods showed that the mitogenome of mangosteen variety Mesta was grouped under Malpighiales order. This is the first complete mitogenome from the Garcinia genus for future evolutionary studies.

Draft genome sequence data of a clinical Enterococcus faecalis isolate SHH039 from a patient with cholecystitis from a tertiary care hospital in Sabah, Malaysia.

An Enterococcus faecalis strain SHH039 was isolated from a 68 year old man who was hospitalised with cholecystitis. The genomic sequence of this isolate which had a size of 2,990,081 bp and 2,663 proteins with functional assignments is presented here. Analysis of the genome revealed Enterococcus faecalis with multiple antibiotic resistance genes which may be associated with acute cholecystitis. It may be not clear if the infection symptoms are the consequence of enterococci manifestation. However, this opportunistic organism may play a minor role in the disease.

First Evidence of Chelonid Herpesvirus 5 (ChHV5) Infection in Green Turtles (Chelonia mydas) from Sabah, Borneo.

Fibropapillomatosis (FP) of sea turtles is characterised by cutaneous tumours and is associated with Chelonid herpesvirus 5 (ChHV5), an alphaherpesvirus from the family Herpesviridae. Here, we provide the first evidence of ChHV5-associated FP in endangered Green turtles (Chelonia mydas) from Sabah, which is located at the northern region of Malaysian Borneo. The aims of our study were firstly, to determine the presence of ChHV5 in both tumour exhibiting and tumour-free turtles using molecular techniques and secondly, to determine the phylogeography of ChHV5 in Sabah. We also aim to provide evidence of ChHV5 infection through histopathological examinations. A total of 115 Green turtles were sampled from Mabul Island, Sabah. We observed three Green turtles that exhibited FP tumours and were positive for ChHV5. In addition, six clinically healthy turtles (with no presence of tumours) were also positive for the virus based on Polymerase Chain Reaction of three viral genes (Capsid protein gene UL18, Glycoprotein H gene UL22, and Glycoprotein B gene UL27). The prevalence of the ChHV5 was 5.22% in asymptomatic Green turtles. Epidermal intranuclear inclusions were identified in tumour lesions upon histopathological examination. In addition, phylogenetic analyses of the UL18, UL22, UL27, and UL30 gene sequences showed a worldwide distribution of the ChHV5 strain with no clear distinction based on geographical location suggesting an interoceanic connection and movement of the sea turtles. Thus, the emergence of ChHV5 in Green turtles in the waters of Sabah could indicate a possible threat to sea turtle populations in the future and requires further monitoring of the populations along the Bornean coast.

Whole genome sequence data of Chromobacterium violaceum WCH4, a human pathogenic strain from Sabah, Malaysia.

Chromobacterium violaceum is a gram-negative, facultative anaerobic bacillus which is commonly found in soil. It can cause mild diarrhoea upon infection but can progress, although rarely, to fatal multi-organ failure and death. Here we report the whole genome sequence data of Chromobacterium violaceum WCH4 strain, a pathogenic strain that was obtained from a 78 year old male patient suffering from an eye infection. This is a rare case of human infection of the bacteria. Blood culture report as well as 16S rRNA confirmed the presence of C. violaceum WCH4. DNA sequencing using the Illumina HiSeq 4000 system revealed a genome size of 4,637,406 bp with a GC-content of 64.89%. We identified 4,572 protein coding sequences (CDS), 78 transfer RNA (tRNA) genes, and 3 ribosomal RNA (rRNA) genes. The CDS included 1,261 hypothetical proteins and 3,311 proteins with functional assignments. We also identified seven putative genes involved in efflux pump and conferring multidrug antibiotic resistance. The genome data has been deposited at NCBI under the accession number JAFBBB000000000 and consist of full annotated genome and raw sequence data. Our data resource will assist in further downstream analysis and understanding of the mechanism of rare human infection caused by Chromobacterium violaceum WCH4 strain.

Soluble Expression and Catalytic Properties of Codon-Optimized Recombinant Bromelain from MD2 Pineapple in Escherichia coli.

Bromelain, a member of cysteine proteases, is found abundantly in pineapple (Ananas comosus), and it has a myriad of versatile applications. However, attempts to produce recombinant bromelain for commercialization purposes are challenging due to its expressibility and solubility. This study aims to express recombinant fruit bromelain from MD2 pineapple (MD2Bro; accession no: OAY85858.1) in soluble and active forms using Escherichia coli host cell. The gene encoding MD2Bro was codon-optimized, synthesized, and subsequently ligated into pET-32b( +) for further transformation into Escherichia coli BL21-CodonPlus(DE3). Under this strategy, the expressed MD2Bro was in a fusion form with thioredoxin (Trx) tag at its N-terminal (Trx-MD2Bro). The result showed that Trx-MD2Bro was successfully expressed in fully soluble form. The protein was successfully purified using single-step Ni2+-NTA chromatography and confirmed to be in proper folds based on the circular dichroism spectroscopy analysis. The purified Trx-MD2Bro was confirmed to be catalytically active against N-carbobenzoxyglycine p-nitrophenyl ester (N-CBZ-Gly-pNP) with a specific activity of 6.13 ± 0.01 U mg-1 and inhibited by a cysteine protease inhibitor, E-64 (IC50 of 74.38 ± 1.65 nM). Furthermore, the catalytic efficiency (kcat/KM) Trx-MD2Bro was calculated to be at 5.64 ± 0.02 × 10-2 µM-1 s-1 while the optimum temperature and pH were at 50 °C and pH 6.0, respectively. Furthermore, the catalytic activity of Trx-MD2Bro was also affected by ethylenediaminetetraacetic acid (EDTA) or metal ions. Altogether it is proposed that the combination of codon optimization and the use of an appropriate vector are important in the production of a soluble and actively stable recombinant bromelain.

Shared Signature of Recent Positive Selection on the TSBP1-BTNL2-HLA-DRA Genes in Five Native Populations from North Borneo.

North Borneo (NB) is home to more than 40 native populations. These natives are believed to have undergone local adaptation in response to environmental challenges such as the mosquito-abundant tropical rainforest. We attempted to trace the footprints of natural selection from the genomic data of NB native populations using a panel of ∼2.2 million genome-wide single nucleotide polymorphisms. As a result, an ∼13-kb haplotype in the Major Histocompatibility Complex Class II region encompassing candidate genes TSBP1-BTNL2-HLA-DRA was identified to be undergoing natural selection. This putative signature of positive selection is shared among the five NB populations and is estimated to have arisen ∼5.5 thousand years (∼220 generations) ago, which coincides with the period of Austronesian expansion. Owing to the long history of endemic malaria in NB, the putative signature of positive selection is postulated to be driven by Plasmodium parasite infection. The findings of this study imply that despite high levels of genetic differentiation, the NB populations might have experienced similar local genetic adaptation resulting from stresses of the shared environment.

Whole genome sequencing data of a clinical Enterococcus gallinarum strain EGR748 from Sabah, Malaysia.

Enterococcus gallinarum is a gram positive facultatively anaerobic bacteria that is typically found in mammalian intestinal tracts. It is generally not considered pathogenic to humans and is rarely reported. Here, we present the draft genome sequence data of Enterococcus gallinarum strain EGR748 isolated from a human clinical sample, and sequenced using the Illumina HiSeq 4000 system. The estimated whole genome size of the strain was 3,730,000 bp with a G + C content of 40.43%. The de novo assembly of the genome generated 55 contigs with an N50 of 208,509 bp. In addition, the Maximum Likelihood phylogenetic analysis based on the 16S rRNA sequence data accurately clustered EGR748 with other E. gallinarum strains. The data may be useful to demonstrate the capacity of this enterococcal species becoming the causal agents of nosocomial blood-stream infections. The genome dataset has been deposited at DDBJ/ENA/GenBank under the accession number JAABOR000000000.

Morphological Variability Identification of Harumanis Mango (Mangifera indica L.) Harvested from Different Location and Tree Age.

Harumanis is one of the main signatures of Perlis with regards to its delightful taste, pleasant aroma and expensive price. Harumanis authenticity and productivity had become the remarks among the farmers, entrepreneurs, consumers and plant breeders due to the existence of morphological characteristics variation among the fruits and high production cost. Assessment of Harumanis morphological characteristics of natural population and different tree ages may represent a possible source of important characteristics for development and breeding purposes of Harumanis. The aim of this study is to evaluate the morphological variation of Harumanis collected from different location in Perlis and tree age. A total of 150 Harumanis fruits from 50 trees with three different stages of development (young, middle-aged and old) were characterised using 11 traits; 10 quantitative and one qualitative morphological trait. The ANOVA analyses in combination with Dunn's pairwise and Kruskal-Wallis multiple comparison test able to point out the existence of environmental factor and age influence towards the significant different of identified morphological traits except for Total Soluble Solid (TSS) and pulp percentage. Five clusters of 50 Harumanis accessions reflect a grouping pattern which not according to neither geographical region nor age. The result of Principal Component Analysis (PCA) using the first two principal components (PCs) provided a good approximation of the data explaining 84.09% of the total variance which majorly contributed by parameters of weight, fruit dimensional characteristics, peel percentage and hue angle, h. Preliminary screening of important morphological characteristics which contribute to the phenotypic diversity of Harumanis is successfully achieved. The findings can be employed by the plant breeders and farmers for the establishment of standard grading of Harumanis and advancement of breeding crop of Harumanis in future.