Insight into the Enterovirus A71: A review.

Enterovirus A71 is a major causative pathogen of hand, foot and mouth disease. It has become a global public health threat, and is especially important for infants and young children in the Asian-Pacific countries. The enterovirus A71 is a non-enveloped virus of the Picornaviridae family having a single-stranded positive-sense RNA genome of about 7.4 kb which encodes the structural and nonstructural proteins. Currently there are no US FDA-approved vaccines or antiviral therapy available against enterovirus A71 infection. Although enterovirus A71 vaccines have been licenced in China, clinically approved vaccines for widespread vaccination programs are lacking. Substantial progress has recently been achieved on understanding the structure and function of enterovirus A71 proteins together with information on the viral genetic diversity and geographic distribution. The present review is intended to provide an overview on our current understanding of the molecular biology and epidemiology of enterovirus A71 which will aid the development of vaccines, therapeutics and other control strategies so as to bolster the preparedness for future enterovirus A71 outbreaks.

Current understanding of structure, function and biogenesis of yeast mitochondrial ATP synthase.

The yeast mitochondrial ATP synthase is a rotary molecular machine primarily responsible for the production of energy used to drive cellular processes. The enzyme complex is composed of 17 different subunits grouped into a soluble F1 sector and a membrane-embedded F0 sector. The catalytic head of the F1 sector and the membrane integrated motor module in the F0 sector are connected by two stalks, the F1 central stalk and the F0 peripheral stalk. Proton translocation through the F0 motor module drives the rotation of the subunit 910-ring that generates torque which is transmitted to the calaytic head through the γ subunit of the central stalk. The rotation of the γ subunit causes changes in conformation of the catalytic head which leads to the synthesis of ATP. Biogenesis of the enzyme involves modular assembly of polypeptides of dual genetic origin, the nuclear and the mitochondrial genomes. Most of the yeast ATP synthase subunits are encoded by the genome of the nucleus, translated on cytosolic ribosomes and imported into mitochondria. In the mitochondria, the enzyme forms a dimer which contributes to the formation of cristae, a characteristic of mitochondrial morphology. Substantial progress has recently been made on the elucidation of detailed stucture, function and biogenesis of yeast mitochondrial ATP synthase. The recent availability of high-resolution structure of the complete monomeric form, as well as the atomic model for the dimeric F0 sector, has advanced the understanding of the enzyme complex. This review is intended to provide an overview of current understanding of the molecular structure, catalytic mechanism, subunit import into mitochondria, and the subunit assembly into the enzyme complex. This is important as the yeast mitochondrial ATP synthase may be used as a model for understanding the corresponding enzyme complexes from human and other eukaryotic cells in physiological and diseased states.

Molecular insights into artemisinin resistance in Plasmodium falciparum: An updated review.

Malaria still poses a major burden on human health around the world, especially in endemic areas. Plasmodium resistance to several antimalarial drugs has been one of the major hindrances in control of malaria. Thus, the World Health Organization recommended artemisinin-based combination therapy (ACT) as a front-line treatment for malaria. The emergence of parasites resistant to artemisinin, along with resistant to ACT partner drugs, has led to ACT treatment failure. The artemisinin resistance is mostly related to the mutations in the propeller domain of the kelch13 (k13) gene that encodes protein Kelch13 (K13). The K13 protein has an important role in parasite reaction to oxidative stress. The most widely spread mutation in K13, with the highest degree of resistance, is a C580Y mutation. Other mutations, which are already identified as markers of artemisinin resistance, are R539T, I543T, and Y493H. The objective of this review is to provide current molecular insights into artemisinin resistance in Plasmodium falciparum. The trending use of artemisinin beyond its antimalarial effect is described. Immediate challenges and future research directions are discussed. Better understanding of the molecular mechanisms underlying artemisinin resistance will accelerate implementation of scientific findings to solve problems with malarial infection.

Seroprevalence of Chikungunya in an Asymptomatic Adult Population in North and South Sulawesi, Indonesia.

Chikungunya (CHIK) is an emerging and reemerging infectious disease of public health importance in Indonesia. Information on the asymptomatic and true burden of CHIK virus (CHIKV) infections is limited. We assayed 1,092 healthy population samples, collected in North and South Sulawesi between 2019 and 2020, for antibodies against CHIKV. Blood samples were screened by IgM and IgG ELISAs and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay. CHIKV IgG seroprevalence in North and South Sulawesi was 53.2% and 53.9%, respectively. The overall prevalence of anti-CHIKV IgM antibody was 12.9%. Molecular testing of blood donors revealed 0.66% (2/300) were positive for CHIKV qRT-PCR. Our study provides new insights into the CHIKV endemicity situation in the eastern part of Indonesia and warrants the need for further systematic surveillance considering there is no treatment or vaccine for CHIK infection.

Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis.

Successful detection of the first SARS-CoV-2 cases using the real-time polymerase chain reaction (real-time PCR) method reflects the power and usefulness of this technique. Real-time PCR is a variation of the PCR assay to allow monitoring of the PCR progress in actual time. PCR itself is a molecular process used to enzymatically synthesize copies in multiple amounts of a selected DNA region for various purposes. Real-time PCR is currently one of the most powerful molecular approaches and is widely used in biological sciences and medicine because it is quantitative, accurate, sensitive, and rapid. Current applications of real-time PCR include gene expression analysis, mutation detection, detection and quantification of pathogens, detection of genetically modified organisms, detection of allergens, monitoring of microbial degradation, species identification, and determination of parasite fitness. The technique has been used as a gold standard for COVID-19 diagnosis. Modifications of the standard real-time PCR methods have also been developed for particular applications. This review aims to provide an overview of the current applications of the real-time PCR technique, including its role in detecting emerging viruses such as SARS-CoV-2.

Effects of Methods and Durations of Extraction on Total Flavonoid and Phenolic Contents and Antioxidant Activity of Java Cardamom (Amomum compactum Soland Ex Maton) Fruit.

Free radicals contribute to the pathophysiology of degenerative diseases which increase mortality globally, including mortality in Indonesia. Amomum compactum Soland. Ex Maton fruit from the Zingiberaceae family, also known as Java cardamom, contains secondary metabolites that have high antioxidant activities. The antioxidant activity of the methanol extract of Java cardamom fruit correlates with its flavonoid and phenolic compound contents, which can be affected by different methods and durations of extraction. This study aimed to measure and compare the effects of extraction methods and durations on total flavonoid and phenolic contents (TFCs and TPCs) and subsequent antioxidant activities by the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical, ferric reducing antioxidant power (FRAP), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and cupric ion reducing antioxidant capacity (CUPRAC) assays. Methanol extracts of Java cardamom were produced by continuous shaking (CSE), microwave-assisted (MAE), or ultrasonic-assisted extractions (UAE) for three different durations. CSE for 360 min resulted in the highest TFCs (3.202 mg Quercetin Equivalent/g dry weight), while the highest TPCs (1.263 mg Gallic Acid Equivalent/g dry weight) were obtained by MAE for 3 min. Out of the investigated methods, MAE for 3 min resulted in the highest antioxidant activity results for the extracts. We conclude that the polyphenolic antioxidant yield of Java cardamom depends on two parameters: the method and the duration of extraction.

Association of FTO rs1421085 single nucleotide polymorphism with fat and fatty acid intake in Indonesian adults.

OBJECTIVE: Recent studies showed that genetic polymorphisms in the fat mass and obesity-associated gene (FTO) were associated with obesity and dietary intake. In this study of 71 adults in Jakarta, Indonesia, we investigated FTO rs1421085 association with body mass index (BMI), macronutrient intake, and fatty acid intake. The association was evaluated using linear regression analyses assuming co-dominant, dominant, recessive, over-dominant, and additive genetic models. RESULTS: Only individuals with the CC genotype had a considerably higher BMI (p < 0.001), which indicates a recessive genetic trait, but the incidence for this genotype is low (68 TT + TC vs. 3 CC). Individuals with the minor C allele had an estimated increase of fat intake by 3.45-4.06% across various genetic models (dominant: p < 0.010, over-dominant: p < 0.030, additive: p < 0.010). Subjects with TC/CC genotypes had increased dietary monounsaturated fatty acid (MUFA; 1.14%, p = 0.046) and saturated fatty acid (SAFA; 2.06%, p = 0.023) intakes, compared to those with the TT genotype. In conclusion, our study provided evidence for the association between FTO rs1421085 risk allele with higher BMI and individual preferences for consuming more fat, MUFA, and SAFA. This study highlights the important role of FTO gene in food preference, and its influence on body weight.

Identification and antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus-associated subclinical mastitis isolated from dairy cows in Bogor, Indonesia.

BACKGROUND AND AIM: Subclinical mastitis is an udder infection devoid of clinical symptoms, and Staphylococcus aureus is one of the bacteria causing this disease. This study aimed to identify and determine the prevalence and antibiotic susceptibility of methicillin-resistant S. aureus (MRSA)-associated subclinical mastitis isolated from dairy cows in Bogor, Indonesia. MATERIALS AND METHODS: S. aureus was isolated from subclinical mastitis milk specimens. All strains were confirmed by polymerase chain reaction-based detection of staphylococcal nuc, and MRSA was confirmed by the presence of mecA. Antibiotic susceptibility was determined using the disk diffusion method. RESULTS: From 86 milk samples, 49 isolates (57%) were confirmed as S. aureus. All S. aureus isolates were susceptible to tetracycline, gentamicin, chloramphenicol, erythromycin, and trimethoprim/sulfamethoxazole. Nine isolates were identified as MRSA (10.5%). CONCLUSION: In this study, we reported MRSA-associated subclinical mastitis in Bogor, Indonesia.

Allotopic expression of mitochondrial genes: Basic strategy and progress.

Allotopic expression of mitochondrial genes is a deliberate functional relocation of mitochondrial genes into the nucleus followed by import of the gene-encoded polypeptide from the cytoplasm into the mitochondria. For successful allotopic expression of a mitochondrial gene, several key aspects must be considered. These include the different codon dictionary used by the mitochondrial and nuclear genomes, different codon preferences between mitochondrial and nuclear-cytosolic translation systems, and the provision of an import signal to ensure that the newly translated protein in the cytosol is successfully imported into mitochondria. The allotopic expression strategy was first developed in yeast, a useful model organism for studying human and other eukaryotic cells. Currently, a number of mitochondrial genes have been successfully recoded and nuclearly expressed in yeast and human cells. In addition to its use in evolutionary and molecular biology studies, the allotopic expression strategy has been developed as a potential approach to treat mitochondrial genetic disorders. Substantial progress has been recently achieved, and the development of this technique for therapy of the mitochondrial disease Leber's hereditary optic neuropathy (LHON) has entered phase III clinical trials. However, a number of challenges remain to be overcome to accelerate the successful application of this technique. These include improvement of nuclear gene expression, import into mitochondria, processing, and functional integration of the allotopically expressed polypeptides into mitochondrial protein complexes. This review discusses the current basic strategy, progress, challenges, and prospects of the allotopic expression strategy for mitochondrial genes.

Molecular biology of coronaviruses: current knowledge.

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) late December 2019 in Wuhan, China, marked the third introduction of a highly pathogenic coronavirus into the human population in the twenty-first century. The constant spillover of coronaviruses from natural hosts to humans has been linked to human activities and other factors. The seriousness of this infection and the lack of effective, licensed countermeasures clearly underscore the need of more detailed and comprehensive understanding of coronavirus molecular biology. Coronaviruses are large, enveloped viruses with a positive sense single-stranded RNA genome. Currently, coronaviruses are recognized as one of the most rapidly evolving viruses due to their high genomic nucleotide substitution rates and recombination. At the molecular level, the coronaviruses employ complex strategies to successfully accomplish genome expression, virus particle assembly and virion progeny release. As the health threats from coronaviruses are constant and long-term, understanding the molecular biology of coronaviruses and controlling their spread has significant implications for global health and economic stability. This review is intended to provide an overview of our current basic knowledge of the molecular biology of coronaviruses, which is important as basic knowledge for the development of coronavirus countermeasures.

Pathogenic viruses: Molecular detection and characterization.

Pathogenic viruses are viruses that can infect and replicate within human cells and cause diseases. The continuous emergence and re-emergence of pathogenic viruses has become a major threat to public health. Whenever pathogenic viruses emerge, their rapid detection is critical to enable implementation of specific control measures and the limitation of virus spread. Further molecular characterization to better understand these viruses is required for the development of diagnostic tests and countermeasures. Advances in molecular biology techniques have revolutionized the procedures for detection and characterization of pathogenic viruses. The development of PCR-based techniques together with DNA sequencing technology, have provided highly sensitive and specific methods to determine virus circulation. Pathogenic viruses potentially having global catastrophic consequences may emerge in regions where capacity for their detection and characterization is limited. Development of a local capacity to rapidly identify new viruses is therefore critical. This article reviews the molecular biology of pathogenic viruses and the basic principles of molecular techniques commonly used for their detection and characterization. The principles of good laboratory practices for handling pathogenic viruses are also discussed. This review aims at providing researchers and laboratory personnel with an overview of the molecular biology of pathogenic viruses and the principles of molecular techniques and good laboratory practices commonly implemented for their detection and characterization.

Japanese encephalitis virus infection in non-encephalitic acute febrile illness patients.

Although Japanese encephalitis virus (JEV) is considered endemic in Indonesia, there are only limited reports of JEV infection from a small number of geographic areas within the country with the majority of these being neuroinvasive disease cases. Here, we report cases of JEV infection in non-encephalitic acute febrile illness patients from Bali, Indonesia. Paired admission (S1) and discharge (S2) serum specimens from 144 acute febrile illness patients (without evidence of acute dengue virus infection) were retrospectively tested for anti-JEV IgM antibody and confirmed by plaque reduction neutralization test (PRNT) for JEV infection. Twenty-six (18.1%) patients were anti-JEV IgM-positive or equivocal in their S2 specimens, of which 5 (3.5%) and 8 (5.6%) patients met the criteria for confirmed and probable JEV infection, respectively, based on PRNT results. Notably, these non-encephalitic JE cases were less likely to have thrombocytopenia, leukopenia, and lower hematocrit compared with confirmed dengue cases of the same cohort. These findings highlight the need to consider JEV in the diagnostic algorithm for acute febrile illnesses in endemic areas and suggest that JEV as a cause of non-encephalitic disease has likely been underestimated in Indonesia.

Evolutionary study and phylodynamic pattern of human influenza A/H3N2 virus in Indonesia from 2008 to 2010.

Influenza viruses are by nature unstable with high levels of mutations. The sequential accumulation of mutations in the surface glycoproteins allows the virus to evade the neutralizing antibodies. The consideration of the tropics as the influenza reservoir where viral genetic and antigenic diversity are continually generated and reintroduced into temperate countries makes the study of influenza virus evolution in Indonesia essential. A total of 100 complete coding sequences (CDS) of Hemagglutinin (HA) and Neuraminidase (NA) genes of H3N2 virus were obtained from archived samples of Influenza-Like Illness (ILI) surveillance collected from 2008 to 2010. Our evolutionary and phylogenetic analyses provide insight into the dynamic changes of Indonesian H3N2 virus from 2008 to 2010. Obvious antigenic drift with typical 'ladder-like' phylogeny was observed with multiple lineages found in each year, suggesting co-circulation of H3N2 strains at different time periods. The mutational pattern of the Indonesian H3N2 virus was not geographically related as relatively low levels of mutations with similar pattern of relative genetic diversity were observed in various geographical origins. This study reaffirms that the existence of a particular lineage is most likely the result of adaptation or competitive exclusion among different host populations and combination of stochastic ecological factors, rather than its geographical origin alone.

Laboratory biosafety for handling emerging viruses.

Emerging viruses are viruses whose occurrence has risen within the past twenty years, or whose presence is likely to increase in the near future. Diseases caused by emerging viruses are a major threat to global public health. In spite of greater awareness of safety and containment procedures, the handling of pathogenic viruses remains a likely source of infection, and mortality, among laboratory workers. There is a steady increase in both the number of laboratories and scientist handling emerging viruses for diagnostics and research. The potential for harm associated to work with these infectious agents can be minimized through the application of sound biosafety concepts and practices. The main factors to the prevention of laboratory-acquired infection are well-trained personnel who are knowledgable and biohazard aware, who are perceptive of the various ways of transmission, and who are professional in safe laboratory practice management. In addition, we should emphasize that appropriate facilities, practices and procedures are to be used by the laboratory workers for the handling of emerging viruses in a safe and secure manner. This review is aimed at providing researchers and laboratory personnel with basic biosafety principles to protect themselves from exposure to emerging viruses while working in the laboratory. This paper focuses on what emerging viruses are, why emerging viruses can cause laboratory-acquired infection, how to assess the risk of working with emerging viruses, and how laboratory-acquired infection can be prevented. Control measures used in the laboratory designed as such that they protect workers from emerging viruses and safeguard the public through the safe disposal of infectious wastes are also addressed.

Anticancer activity test of ethyl acetate extract of endophytic fungi isolated from soursop leaf (Annona muricata L.).

OBJECTIVE: To analyze anticancer activity of an ethyl acetate extract of endophytic fungi isolated from soursop leaf (Annona muricata L.). METHODS: Anticancer activity of fungal extracts was determined by observing its toxicity against MCF-7 (Michigan Cancer Foundation-7) cells in vitro by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay method. At an extract concentration of 100 µg/mL, 4 isolates out of 12 showed high activity against the cancer cell growth. The four isolates were then selected for further IC50 determination, by measuring the inhibition of cancer cell proliferation at extract concentration of 25 µg/mL, 50 µg/mL, 100 µg/mL, 200 µg/mL and 400 µg/mL. RESULTS: Results showed that isolate Sir-G5 had the highest anticancer activity with an IC50 of 19.20 µg/mL. The best isolates were screened again using a normal cell (Chang cells) to determine its toxicity against normal cells. Results indicated that the extracts do not affect the proliferation of normal cells. Molecular identification showed that the fungal isolate Sir-G5 has a close relationship with Phomopsis sp. CONCLUSIONS: The endophytic fungi isolated from soursop leaf has the potential to be used as a source of anticancer agents.

Primer development to obtain complete coding sequence of HA and NA genes of influenza A/H3N2 virus.

BACKGROUND: Influenza is an acute respiratory illness and has become a serious public health problem worldwide. The need to study the HA and NA genes in influenza A virus is essential since these genes frequently undergo mutations. This study describes the development of primer sets for RT-PCR to obtain complete coding sequence of Hemagglutinin (HA) and Neuraminidase (NA) genes of influenza A/H3N2 virus from Indonesia. The primers were developed based on influenza A/H3N2 sequence worldwide from Global Initiative on Sharing All Influenza Data (GISAID) and further tested using Indonesian influenza A/H3N2 archived samples of influenza-like illness (ILI) surveillance from 2008 to 2009. RESULTS: An optimum RT-PCR condition was acquired for all HA and NA fragments designed to cover complete coding sequence of HA and NA genes. A total of 71 samples were successfully sequenced for complete coding sequence both of HA and NA genes out of 145 samples of influenza A/H3N2 tested. CONCLUSIONS: The developed primer sets were suitable for obtaining complete coding sequences of HA and NA genes of Indonesian samples from 2008 to 2009.

Detection and identification of coxsackievirus B3 from sera of an Indonesian patient with undifferentiated febrile illness.

INTRODUCTION: Coxsackievirus B3 (CVB3) virus has been implicated as the causative agent of various outbreaks of clinical disease, including hand, foot, and mouth diseases, aseptic meningitis, acute myocarditis, and inflammatory cardiomyopathy. METHODOLOGY: Two hundred and nine undiagnosed cryopreserved specimens obtained from factory workers in Bandung, Indonesia, who displayed symptoms of acute febrile illness were gathered. Total RNA was isolated from serum and tested by conventional polymerase chain reaction (PCR) using Enterovirus genus-level primers and confirmed by sequencing. Concurrently, the virus was isolated in LLC-MK2 cells. RESULTS: CVB3 virus was identified in an archived specimen from a patient who presented with symptoms of fever, headache, myalgia, and nausea. Sequencing results of the VP1 region from both the clinical sample and tissue culture supernatant showed 97% homology to a CVB3 virus isolate from Taiwan. Virus propagation in LLC-MK2 cell culture exhibited severe cytopathic effects two days post-inoculation. CONCLUSIONS: We report the first case of CVB3 from an undifferentiated febrile illness specimen from Indonesia.

Impact of composted guava leaves and neem seeds on the growth and curcuminoid- and xanthorrhizol-yields of Curcuma zanthorrhiza RoxB / Impacto de folhas de goiabeira compostada e sementes de nim no crescimento e r endimento de curcuminóides e xanthorrizil de Curcuma zanthorrhiza RoxB

ABSTRACT: The compost from the waste of pharmaceutical industries, such as guava leaves (GL) and neem seeds (NS) is used in organic agriculture. Curcuma zanthorrhiza RoxB. is a widely recognized herbal medicine that grows natively in Indonesia. Curcuminoids and xanthorrhizol are the primary bioactive components of C. zanthorrhiza. In this study, we aimed to evaluate the impact of GL and NS compost on the growth and bioactive yields of C. zanthorrhiza. Treatments consisted of cow manure, GL compost, NS compost, GL+NS compost, or a no compost control, at 2 and 4 kg per plant. Results demonstrated that the NS and GL+NS compost applied with 4 kg per plant had increased fresh rhizome yield compared with the other treatments. Composted NS at 2 kg per plant increased the plant height and pseudo stem diameter traits compared with the control treatment. The compost application of GL+NS at 2 and 4 kg per plant significantly increased the leaf length and number of shoots. All treatments showed unchanged the leaf width and number of leaves. The compost application of GL and NS (2 kg per plant) showed higher production of curcuminoidsthan the control. The compost consisting of GL (2 kg per plant), NS (4 kg per plant), and GL+NS also increased the production of xanthorrhizol compared with the control treatment. Results illustrated the practical application of GL and NS composts from industrial pharmaceutical extraction waste for the organic farming cultivation of C. zanthorrhiza.

RESUMO: O composto dos resíduos das indústrias farmacêuticas, como folhas de goiaba (GL) e sementes de nim (NS), é usado na agricultura orgânica. Curcuma zanthorrhiza RoxB. é um medicamento fitoterápico amplamente reconhecido que cresce de forma nativa na Indonésia. Os curcuminóides e o xanthorrizol são os principais componentes bioativos de C. zanthorrhiza. Neste trabalho, objetivou-se avaliar o impacto do composto GL e NS no crescimento e produtividade bioativa de C. zanthorrhiza. Os tratamentos consistiram em esterco de vaca, composto GL, composto NS, composto GL + NS ou controle sem composto, em 2 e 4 kg por planta. Os resultados demonstraram que o composto NS e GL + NS aplicado com 4 kg por planta aumentou a produção de rizoma fresco, em comparação com os outros tratamentos. A NS compostada a 2 kg por planta aumentou as características de altura da planta e diâmetro do pseudoestêmico em comparação com o tratamento controle. A aplicação de composto de GL + NS em 2 e 4 kg por planta aumentou significativamente o comprimento das folhas e o número de brotações. Todos os tratamentos apresentaram alteração na largura e número de folhas. A aplicação de composto de GL e NS (2 kg por planta) apresentou maior produção de curcuminóide do que o controle. Os compostos constituídos por GL (2 kg por planta), NS (4 kg por planta) e GL + NS também aumentaram a produção de xanthorrizol em comparação com o tratamento controle. Os resultados ilustraram a aplicação prática de compostos GL e NS de resíduos de extração farmacêutica industrial para o cultivo orgânico de C. zanthorrhiza.

West Nile virus documented in Indonesia from acute febrile illness specimens.

We report the presence of West Nile virus in a cryopreserved, dengue-negative serum specimen collected from an acute fever case on Java in 2004-2005. The strain belongs to genotype lineage 2, which has recently been implicated in human outbreaks in Europe.