Genetic characterization of dengue virus 4 complete genomes from East Java, Indonesia.

Dengue is an endemic arboviral disease with continuous transmission in Indonesia for more than five decades. A recent outbreak in Jember, East Java province, demonstrated the predominance of DENV-4, a serotype known for its low global spread and limited transmission. While epidemiological factors such as new serotype introduction and lacking herd immunity may explain its predominance, viral factors may also contribute. Using next-generation sequencing, we generated 13 representative complete genomes of DENV-4 responsible for the outbreak. Phylogenetic and evolutionary analyses on complete genomes were performed to understand the spatial and temporal dynamics of the viruses. Further analyses were done to study amino acid variations in DENV genes, as well as the potential events of recombination and selection pressure within the genomes. We revealed the DENV-4 genetic factors that may lead to its predominance in the 2019 Jember dengue outbreak. A combination of selection pressure and mutational genetic changes may contribute to the DENV-4 predominance in East Java, Indonesia. The possible intra-serotype recombination events involving the non-structural protein 5 (NS5) gene were also observed. Altogether, these genetic factors may act as additional factors behind the complex dengue outbreak mechanism.

Non-structural protein 1 and hematology parameters as predictors of dengue virus infection severity in Indonesia.

Dengue virus infection (DVI) remains a significant health challenge, and diagnosis must still be considered. Non-structural protein 1 (NS1) is a potential marker of the dengue virus that can help diagnose DVI. The study aimed to assess the role of NS1 as a predictor of the severity of DVI. We utilized Dengue PCR-confirmed samples and employed semi-quantitative NS1Ag ELISA for NS1 examination, adhering to the World Health Organization South-East Asia Region (WHO-SEARO) 2011 criteria for DVI. We included DVI patients from Indonesia aged 1-65 years. Secondary infections had more severe clinical conditions than primary infections. Leukocyte and platelet levels had a more significant effect on NS1 positivity (6.19 (1.9-30.2); p<0.001; 190 (11-417); p=0.015; respectively). Multivariate analysis revealed leukocytes as a more significant predictor of NS1 values than platelets, with an odds ratio of 5.38 contributing to 30.5% of the NS1 value variation. The NS1 value could distinguish undifferentiated fever and dengue fever in the children group with a sensitivity of 76.0% and specificity of 87.5% (p=0.015). The number of NS1(-) in the severe dengue hemorrhagic fever (DHF) group was higher than NS1(+). DENV-4 type and primary infection were dominant in this study, although they did not significantly differ from the NS1 value. NS1 value can be used as a predictor to determine the severity of DVI in children but not in the adult group. The levels of leukocytes and platelets influenced the NS1 value.

Experimental and natural infections of severe acute respiratory syndrome-related coronavirus 2 in pets and wild and farm animals.

The severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has spread globally and has led to extremely high mortality rates. In addition to infecting humans, this virus also has infected animals. Experimental studies and natural infections showed that dogs have a low susceptibility to SARS-CoV-2 infection, whereas domesticated cats and other animals in the family Felidae, such as lions, tigers, snow leopards, and cougars, have a high susceptibility to viral infections. In addition, wild white-tailed deer, gorillas, and otters have been found to be infected by SARS-CoV-2. Furry farm animals, such as minks, have a high susceptibility to SARS-CoV-2 infection. The virus appears to spread among minks and generate several new mutations, resulting in increased viral virulence. Furthermore, livestock animals, such as cattle, sheep, and pigs, were found to have low susceptibility to the virus, whereas chicken, ducks, turkeys, quail, and geese did not show susceptibility to SARS-CoV-2 infection. This knowledge can provide insights for the development of SARS-CoV-2 mitigation strategies in animals and humans. Therefore, this review focuses on experimental (both replication and transmission) in vitro, ex vivo, and in vivo studies of SARS-CoV-2 infections in pets and in wild and farm animals, and to provide details on the mechanism associated with natural infection.

The espD full gene as a potential biomarker in active pulmonary tuberculosis.

Background: Pulmonary tuberculosis (PTB) is still a major health problem worldwide. The espD has a potential to be a new biomarker because it is important for the espA, espC, and ESX-1 protein secretion system that are actively expressed in active multiplication of Mycobacterium tuberculosis complex. Methods: A total of 55 sputum samples and 41 culture isolates from newly diagnosed PTB patients at Dr. Soetomo Academic Hospital were collected from September 2016 to April 2019. The tested samples using polymerase chain reaction targeted 555 bp of espD gene and sequencing. Clone Manager Version 6 and NCBI BLAST were used to align the gene sequence against wild-type M. tuberculosis. The prediction of T-cell epitope in espD gene was detected by GENETYX. The three-dimensional (3D) structure of espD was modeled by SWISS-MODEL and I-TASSER and was visualized with PyMOL. Results: From 55 sputum samples, 43 (78.18%) showed positive results, and all culture isolates showed positive results. In addition, all sequenced samples showed 100% homolog with M. tuberculosis H37Rv gene without detected variant or mutation. There were four T-cell epitopes that could be obtained. The 3D model had a I-TASSER confidence score of 3.91 with estimated RMSD of approximately 14.5 Å. The structure consists of a main fold of a three-stranded antiparallel ß-sheet and a long α-helix surrounded by several minor secondary structures. Conclusions: This study provides a brief information about the sequence, epitope prediction, and 3D structure of EspD protein from M. tuberculosis strains in Indonesia.

In silico characterization of the GH5-cellulase family from uncultured microorganisms: physicochemical and structural studies.

BACKGROUND: Hydrolysis of cellulose-based biomass by cellulases produce fermented sugar for making biofuels, such as bioethanol. Cellulases hydrolyze the ß-1,4-glycosidic linkage of cellulose and can be obtained from cultured and uncultured microorganisms. Uncultured microorganisms are a source for exploring novel cellulase genes through the metagenomic approach. Metagenomics concerns the extraction, cloning, and analysis of the entire genetic complement of a habitat without cultivating microbes. The glycoside hydrolase 5 family (GH5) is a cellulase family, as the largest group of glycoside hydrolases. Numerous variants of GH5-cellulase family have been identified through the metagenomic approach, including CelGH5 in this study. University-CoE-Research Center for Biomolecule Engineering, Universitas Airlangga successfully isolated CelGH5 from waste decomposition of oil palm empty fruit bunches (OPEFB) soil by metagenomics approach. The properties and structural characteristics of GH5-cellulases from uncultured microorganisms can be studied using computational tools and software. RESULTS: The GH5-cellulase family from uncultured microorganisms was characterized using standard computational-based tools. The amino acid sequences and 3D-protein structures were retrieved from the GenBank Database and Protein Data Bank. The physicochemical analysis revealed the sequence length was roughly 332-751 amino acids, with the molecular weight range around 37-83 kDa, dominantly negative charges with pI values below 7. Alanine was the most abundant amino acid making up the GH5-cellulase family and the percentage of hydrophobic amino acids was more than hydrophilic. Interestingly, ten endopeptidases with the highest average number of cleavage sites were found. Another uniqueness demonstrated that there was also a difference in stability between in silico and wet lab. The II values indicated CelGH5 and ACA61162.1 as unstable enzymes, while the wet lab showed they were stable at broad pH range. The program of SOPMA, PDBsum, ProSA, and SAVES provided the secondary and tertiary structure analysis. The predominant secondary structure was the random coil, and tertiary structure has fulfilled the structure quality of QMEAN4, ERRAT, Ramachandran plot, and Z score. CONCLUSION: This study can afford the new insights about the physicochemical and structural properties of the GH5-cellulase family from uncultured microorganisms. Furthermore, in silico analysis could be valuable in selecting a highly efficient cellulases for enhanced enzyme production.

Universal capability of 3-ketosteroid Δ1-dehydrogenases to catalyze Δ1-dehydrogenation of C17-substituted steroids.

BACKGROUND: 3-Ketosteroid Δ1-dehydrogenases (KSTDs) are the enzymes involved in microbial cholesterol degradation and modification of steroids. They catalyze dehydrogenation between C1 and C2 atoms in ring A of the polycyclic structure of 3-ketosteroids. KSTDs substrate spectrum is broad, even though most of them prefer steroids with small substituents at the C17 atom. The investigation of the KSTD's substrate specificity is hindered by the poor solubility of the hydrophobic steroids in aqueous solutions. In this paper, we used 2-hydroxpropyl-ß-cyclodextrin (HBC) as a solubilizing agent in a study of the KSTDs steady-state kinetics and demonstrated that substrate bioavailability has a pivotal impact on enzyme specificity. RESULTS: Molecular dynamics simulations on KSTD1 from Rhodococcus erythropolis indicated no difference in ΔGbind between the native substrate, androst-4-en-3,17-dione (AD; - 8.02 kcal/mol), and more complex steroids such as cholest-4-en-3-one (- 8.40 kcal/mol) or diosgenone (- 6.17 kcal/mol). No structural obstacle for binding of the extended substrates was also observed. Following this observation, our kinetic studies conducted in the presence of HBC confirmed KSTD1 activity towards both types of steroids. We have compared the substrate specificity of KSTD1 to the other enzyme known for its activity with cholest-4-en-3-one, KSTD from Sterolibacterium denitrificans (AcmB). The addition of solubilizing agent caused AcmB to exhibit a higher affinity to cholest-4-en-3-one (Ping-Pong bi bi KmA = 23.7 µM) than to AD (KmA = 529.2 µM), a supposedly native substrate of the enzyme. Moreover, we have isolated AcmB isoenzyme (AcmB2) and showed that conversion of AD and cholest-4-en-3-one proceeds at a similar rate. We demonstrated also that the apparent specificity constant of AcmB for cholest-4-en-3-one (kcat/KmA = 9.25∙106 M-1 s-1) is almost 20 times higher than measured for KSTD1 (kcat/KmA = 4.71∙105 M-1 s-1). CONCLUSIONS: We confirmed the existence of AcmB preference for a substrate with an undegraded isooctyl chain. However, we showed that KSTD1 which was reported to be inactive with such substrates can catalyze the reaction if the solubility problem is addressed.

Application of microbial 3-ketosteroid Δ1-dehydrogenases in biotechnology.

3-Ketosteroid Δ1-dehydrogenase catalyzes the 1(2)-dehydrogenation of 3-ketosteroid substrates using flavin adenine dinucleotide as a cofactor. The enzyme plays a crucial role in microbial steroid degradation, both under aerobic and anaerobic conditions, by initiating the opening of the steroid nucleus. Indeed, many microorganisms are known to possess one or more 3-ketosteroid Δ1-dehydrogenases. In the pharmaceutical industry, 3-ketosteroid Δ1-dehydrogenase activity is exploited to produce Δ1-3-ketosteroids, a class of steroids that display various biological activities. Many of them are used as active pharmaceutical ingredients in drug products, or as key precursors to produce pharmaceutically important steroids. Since 3-ketosteroid Δ1-dehydrogenase activity requires electron acceptors, among other considerations, Δ1-3-ketosteroid production has been industrially implemented using whole-cell fermentation with growing or metabolically active resting cells, in which the electron acceptors are available, rather than using the isolated enzyme. In this review we discuss biotechnological applications of microbial 3-ketosteroid Δ1-dehydrogenases, covering commonly used steroid-1(2)-dehydrogenating microorganisms, the bioprocess for preparing Δ1-3-ketosteroids, genetic engineering of 3-ketosteroid Δ1-dehydrogenases and related genes for constructing new, productive industrial strains, and microbial fermentation strategies for enhancing the product yield. Furthermore, we also highlight the recent development in the use of isolated 3-ketosteroid Δ1-dehydrogenases combined with a FAD cofactor regeneration system. Finally, in a somewhat different context, we summarize the role of 3-ketosteroid Δ1-dehydrogenase in cholesterol degradation by Mycobacterium tuberculosis and other mycobacteria. Because the enzyme is essential for the pathogenicity of these organisms, it may be a potential target for drug development to combat mycobacterial infections.

Dengue Virus Serotype 4 Is Responsible for the Outbreak of Dengue in East Java City of Jember, Indonesia.

Outbreaks of dengue virus (DENV) in Indonesia have been mainly caused by the DENV serotype-1; -2; or -3. The DENV-4 was the least-reported serotype in Indonesia during the last five decades. We recently conducted a molecular epidemiology study of dengue in the Jember regency, East Java province, Indonesia. Dengue is endemic in the region and outbreaks occur annually. We investigated the clinical characteristics and etiology of dengue-like febrile illness in this regency to understand the disease dynamics. A total of 191 patients with clinical symptoms similar to dengue were recruited during an 11-month study in 2019-2020. Children accounted for the majority of cases and dengue burden was estimated in 41.4% of the cases based on NS1 antigen, viral RNA, and IgG/IgM antibody detection with the majority (73.4%) being primary infections. Secondary infection was significantly associated with a higher risk of severe dengue manifestation. All four DENV serotypes were detected in Jember. Strikingly, we observed the predominance of DENV-4, followed by DENV-3, DENV-1, and DENV-2. Genotype determination using Envelope gene sequence revealed the classification into Genotype I, Cosmopolitan Genotype, Genotype I, and Genotype II for DENV-1, -2, -3, and -4, respectively. The predominance of DENV-4 in Jember may be associated with a new wave of DENV infections and spread in a non-immune population lacking a herd-immunity to this particular serotype.

ß-Xylosidases: Structural Diversity, Catalytic Mechanism, and Inhibition by Monosaccharides.

Xylan, a prominent component of cellulosic biomass, has a high potential for degradation into reducing sugars, and subsequent conversion into bioethanol. This process requires a range of xylanolytic enzymes. Among them, ß-xylosidases are crucial, because they hydrolyze more glycosidic bonds than any of the other xylanolytic enzymes. They also enhance the efficiency of the process by degrading xylooligosaccharides, which are potent inhibitors of other hemicellulose-/xylan-converting enzymes. On the other hand, the ß-xylosidase itself is also inhibited by monosaccharides that may be generated in high concentrations during the saccharification process. Structurally, ß-xylosidases are diverse enzymes with different substrate specificities and enzyme mechanisms. Here, we review the structural diversity and catalytic mechanisms of ß-xylosidases, and discuss their inhibition by monosaccharides.

Sequence analysis of the cytochrome c oxidase subunit 1 gene of Sarcoptes scabiei isolated from goats and rabbits in East Java, Indonesia.

AIM: This study aimed to sequence the Cytochrome c oxidase (COX-1) gene sequence from mitochondrial DNA of Sarcoptes scabiei isolated from Lamongan goats and Mojokerto rabbits, align it with DNA isolated from Zi'gong rabbit (GenBank accession No. EU256389.1), and produce a phylogenetic analysis of S. scabiei COX-1 gene. MATERIALS AND METHODS: S. scabiei mites were obtained from goats and rabbits, and DNA was extracted using QIAamp DNA Mini Kit. The forward and reverse primer sequences were designed based on the DNA sequence of an S. scabiei COX-1 gene isolated from the Zi'gong rabbit (5'-TCTTAGGGGCTGGATTTAGTATG-3' and 5'-AGTTCCTCTACCAGTTCCAC-3', respectively). To confirm sequencing output, the sequence resulting from the reverse primer was inverted and aligned to the sequence from the forward primer using Clone Manager Professional Version 9 for Windows (Scientific & Educational Software; http://www.scied.com). This alignment was subsequently used to build a phylogenetic tree, using the Neighbor-Joining method, in the MEGA6 program (https://www.megasoftware.net/). RESULTS: Polymerase chain reaction (PCR) products from S. scabiei isolates from Lamongan goats and Mojokerto rabbits produced bands of around 290 bp with 2% agarose gel electrophoresis. Comparing the DNA sequences of the S. scabiei COX-1 gene with those isolated from Lamongan goats and Mojokerto rabbits showed 99% homology. CONCLUSION: PCR products of the S. scabiei COX-1 gene isolated from Lamongan goats and Mojokerto rabbits were around 290 bp long. The sequences had more than 99% homology. The sequences of the COX-1 gene of S. scabiei from Lamongan goats and Mojokerto rabbits were relatively close to the sequence of the gene in S. scabiei obtained from various hosts according to National Center for Biotechnology Information data.

The role and mechanism of microbial 3-ketosteroid Δ1-dehydrogenases in steroid breakdown.

3-Ketosteroid Δ1-dehydrogenases are FAD-dependent enzymes that catalyze the introduction of a double bond between the C1 and C2 atoms of the A-ring of 3-ketosteroid substrates. These enzymes are found in a large variety of microorganisms, especially in bacteria belonging to the phylum Actinobacteria. They play a critical role in the early steps of the degradation of the steroid core. 3-Ketosteroid Δ1-dehydrogenases are of particular interest for the etiology of some infectious diseases, for the production of starting materials for the pharmaceutical industry, and for environmental bioremediation applications. Here we summarize and discuss the biochemical and enzymological properties of these enzymes, their microbial sources, and their natural diversity. The three-dimensional structure of a 3-ketosteroid Δ1-dehydrogenase in connection with the enzyme mechanism is highlighted.

Structural basis of product inhibition by arabinose and xylose of the thermostable GH43 ß-1,4-xylosidase from Geobacillus thermoleovorans IT-08.

Complete degradation of the xylan backbone of hemicellulosic plant cell walls requires the synergistic action of endo-xylanases and ß-1,4-xylosidases. While endo-xylanases produce xylooligosaccharides from xylan, ß-1,4-xylosidases degrade the xylooligosaccharides into xylose monomers. The glycoside hydrolase family 43 ß-1,4-xylosidase from Geobacillus thermoleovorans IT-08 is a promising, heat stable catalyst for the saccharification of hemicellulosic material into simple fermentable sugars, but it is competitively inhibited by its products arabinose and xylose. As a first step to help overcome this problem, we elucidated crystal structures of the enzyme in the unliganded form and with bound products, at 1.7-2.0 Å resolution. The structures are very similar to those of other enzymes belonging to glycoside hydrolase family 43. Unexpectedly, the monosaccharides are bound in very different ways. Arabinose preferentially binds in subsite -1, while xylose exclusively interacts with subsite +1. These structures and sugar binding preferences suggest ways for improving the catalytic performance of the enzyme by rational mutational design.

Crystal structure and site-directed mutagenesis of 3-ketosteroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1 explain its catalytic mechanism.

3-Ketosteroid Δ(1)-dehydrogenases are FAD-dependent enzymes that catalyze the 1,2-desaturation of 3-ketosteroid substrates to initiate degradation of the steroid nucleus. Here we report the 2.0 Å resolution crystal structure of the 56-kDa enzyme from Rhodococcus erythropolis SQ1 (Δ(1)-KSTD1). The enzyme contains two domains: an FAD-binding domain and a catalytic domain, between which the active site is situated as evidenced by the 2.3 Å resolution structure of Δ(1)-KSTD1 in complex with the reaction product 1,4-androstadiene-3,17-dione. The active site contains four key residues: Tyr(119), Tyr(318), Tyr(487), and Gly(491). Modeling of the substrate 4-androstene-3,17-dione at the position of the product revealed its interactions with these residues and the FAD. The C1 and C2 atoms of the substrate are at reaction distance to the N5 atom of the isoalloxazine ring of FAD and the hydroxyl group of Tyr(318), respectively, whereas the C3 carbonyl group is at hydrogen bonding distance from the hydroxyl group of Tyr(487) and the backbone amide of Gly(491). Site-directed mutagenesis of the tyrosines to phenylalanines confirmed their importance for catalysis. The structural features and the kinetic properties of the mutants suggest a catalytic mechanism in which Tyr(487) and Gly(491) work in tandem to promote keto-enol tautomerization and increase the acidity of the C2 hydrogen atoms of the substrate. With assistance of Tyr(119), the general base Tyr(318) abstracts the axial ß-hydrogen from C2 as a proton, whereas the FAD accepts the axial α-hydrogen from the C1 atom of the substrate as a hydride ion.

Purification, crystallization and preliminary X-ray crystallographic analysis of 3-ketosteroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1.

3-Ketosteroid Δ(1)-dehydrogenase plays a crucial role in the early steps of steroid degradation by introducing a double bond between the C1 and C2 atoms of the A-ring of its 3-ketosteroid substrates. The 3-ketosteroid Δ(1)-dehydrogenase from Rhodococcus erythropolis SQ1, a 56 kDa flavoprotein, was crystallized using the sitting-drop vapour-diffusion method at room temperature. The crystals grew in various buffers over a wide pH range (from pH 5.5 to 10.5), but the best crystallization condition consisted of 2%(v/v) PEG 400, 0.1 M HEPES pH 7.5, 2.0 M ammonium sulfate. A native crystal diffracted X-rays to 2.0 Šresolution. It belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 107.4, b = 131.6, c = 363.2 Å, and contained eight molecules in the asymmetric unit. The initial structure of the enzyme was solved using multi-wavelength anomalous dispersion (MAD) data collected from a Pt-derivatized crystal.

Purification, crystallization and preliminary X-ray analysis of a thermostable glycoside hydrolase family 43 beta-xylosidase from Geobacillus thermoleovorans IT-08.

The main enzymes involved in xylan-backbone hydrolysis are endo-1,4-beta-xylanase and beta-xylosidase. beta-Xylosidase converts the xylo-oligosaccharides produced by endo-1,4-beta-xylanase into xylose monomers. The beta-xylosidase from the thermophilic Geobacillus thermoleovorans IT-08, a member of glycoside hydrolase family 43, was crystallized at room temperature using the hanging-drop vapour-diffusion method. Two crystal forms were observed. Bipyramid-shaped crystals belonging to space group P4(3)2(1)2, with unit-cell parameters a = b = 62.53, c = 277.4 A diffracted to 1.55 A resolution. The rectangular crystals belonged to space group P2(1), with unit-cell parameters a = 57.94, b = 142.1, c = 153.9 A, beta = 90.5 degrees , and diffracted to 1.80 A resolution.