The evolution of chikungunya virus circulating in Indonesia: Sequence analysis of the orf2 gene encoding the viral structural proteins.

Chikungunya virus (CHIKV) is an arthropod-borne virus that has caused several major epidemics globally, including in Indonesia. Although significant progress has been achieved in understanding the epidemiology and genotype circulation of CHIKV in Indonesia, the evolution of Indonesian CHIKV isolates is poorly understood. Thus, our study aimed to perform phylogenetic and mutation analyses of the orf2 gene encoding its viral structural protein to improve our understanding of CHIKV evolution in Indonesia. Complete orf2 gene sequences encoding the viral structural proteins of Indonesian-derived CHIKV were downloaded from GenBank until August 31, 2022. Various bioinformatics tools were employed to perform phylogenetic and mutation analyses of the orf2 gene. We identified 76 complete sequences of orf2 gene of CHIKV isolates originally derived from Indonesia. Maximum likelihood trees demonstrated that the majority (69/76, 90.8%) of Indonesian-derived CHIKV isolates belonged to the Asian genotype, while seven isolates (9.2%) belonged to the East/Central/South African (ECSA) genotype. The Indonesian-derived CHIKV isolates were calculated to be originated in Indonesia around 95 years ago (1927), with 95% highest posterior density (HPD) ranging from 1910 to 1942 and a nucleotide substitution rate of 5.07 × 10-4 (95% HPD: 3.59 × 10-4 to 6.67 × 10-4). Various synonymous and non-synonymous substitutions were identified in the C, E3, E2, 6K, and E1 genes. Most importantly, the E1-A226V mutation, which has been reported to increase viral adaptation in Aedes albopictus mosquitoes, was present in all ECSA isolates. To our knowledge, our study is the first comprehensive research analyzing the mutation and evolution of Indonesian-derived CHIKV based on complete sequences of the orf2 genes encoding its viral structural proteins. Our results clearly showed a dynamic evolution of CHIKV circulating in Indonesia.

The origin and continuing adaptive evolution of chikungunya virus.

Chikungunya virus (CHIKV) is the responsible agent of chikungunya fever, a debilitating arthritic disease in humans. CHIKV is endemic in Africa and Asia, although transmission cycles are considerably different on these continents. Before 2004, CHIKV had received little attention, since it was only known to cause localised outbreaks in a limited region with no fatalities. However, the recent global reemergence of CHIKV has caused serious global health problems and shown its potential to become a significant viral threat in the future. Unexpectedly, the reemergence is more rapid and is geographically more extensive, especially due to increased intensity of global travel systems or failure to contain mosquito populations. Another important factor is the successful adaptation of CHIKV to a new vector, the Aedes albopictus mosquito. Ae. albopictus survives in both temperate and tropical climates, thus facilitating CHIKV expansion to non-endemic regions. The continuous spread and transmission of CHIKV pose challenges for the development of effective vaccines and specific antiviral therapies. In this review, we discuss the biology and origin of CHIKV in Africa as well as its subsequent expansion to other parts of the world. We also review the transmission cycle of CHIKV and its continuing adaptation to its mosquito vectors and vertebrate hosts. More-complete understanding of the continuous evolution of CHIKV may help in predicting the emergence of CHIKV strains with possibly greater transmission efficiency in the future.

Phylogenetic and immunoinformatic analysis of VP4, VP7, and NSP4 genes of rotavirus strains circulating in children with acute gastroenteritis in Indonesia.

Rotavirus is a major cause of diarrhea in Indonesian children. However, rotavirus vaccines have not been introduced in the national immunization program of Indonesia. Understanding the genetic diversity and conserved antigenic regions of circulating strains are therefore essential to assess the potential efficacy of rotavirus vaccines. We collected fecal samples from hospitalized children less than 5 years of age with acute diarrhea. Rotavirus genotyping was performed by reverse transcriptase polymerase chain reaction, followed by sequencing of the VP4, VP7, and NSP4 genes of representative strains. Phylogenetic analysis was performed to investigate their relationship with globally circulating strains. Conservational analysis, immunoinformatics, and epitope mapping in comparison to vaccine strains were also performed. The sequence analyses showed that differences of multiple amino acid residues existed between the VP4, VP7, and NSP4 antigenic regions of the vaccine strains and the Indonesian isolates. However, many predicted conserved epitopes with higher antigenicity were observed in the vaccine and Indonesian strains, conferring the importance of these epitopes. The identified epitopes showed a higher potential of rotavirus vaccine to be employed in Indonesia. It could also be helpful to inform the design of a peptide vaccine based on the conserved regions and epitopes in the viral proteins.

Norovirus and rotavirus infections in children less than five years of age hospitalized with acute gastroenteritis in Indonesia.

Rotaviruses and noroviruses are the most important viral causes of acute gastroenteritis in children. While previous studies of acute gastroenteritis in Indonesia mainly focused on rotavirus, here, we investigated the burden and epidemiology of norovirus and rotavirus disease. Children less than five years of age hospitalized with acute gastroenteritis were enrolled in this study from January to December 2015 at three participating hospitals. Rotavirus was detected by enzyme immunoassay (EIA), followed by genotyping by reverse transcription PCR (RT-PCR). Norovirus genogroups were determined by TaqMan-based quantitative RT-PCR. Among 406 enrolled children, 75 (18.47%), 223 (54.93%) and 29 (7.14%) cases were positive for norovirus, rotavirus and both viruses (mixed infections), respectively. Most cases clinically presented with fever, diarrhea, vomiting and some degree of dehydration. The majority (n = 69/75 [92%]) of the noroviruses identified belonged to genogroup II, and several genotypes were identified by sequencing a subset of samples. Among 35 samples tested for rotavirus genotype, the most prevalent genotype was G3P[8] (n = 30/35 [85.6%]). Our study suggests that the burden of norovirus diseases in Indonesian children should not be underestimated. It also shows the emergence of rotavirus genotype G3P[8] in Indonesia.

Understanding the Biology and Immune Pathogenesis of Chikungunya Virus Infection for Diagnostic and Vaccine Development.

Chikungunya virus, the causative agent of chikungunya fever, is generally characterized by the sudden onset of symptoms, including fever, rash, myalgia, and headache. In some patients, acute chikungunya virus infection progresses to severe and chronic arthralgia that persists for years. Chikungunya infection is more commonly identified in tropical and subtropical regions. However, recent expansions and epidemics in the temperate regions have raised concerns about the future public health impact of chikungunya diseases. Several underlying factors have likely contributed to the recent re-emergence of chikungunya infection, including urbanization, human travel, viral adaptation to mosquito vectors, lack of effective control measures, and the spread of mosquito vectors to new regions. However, the true burden of chikungunya disease is most likely to be underestimated, particularly in developing countries, due to the lack of standard diagnostic assays and clinical manifestations overlapping with those of other endemic viral infections in the regions. Additionally, there have been no chikungunya vaccines available to prevent the infection. Thus, it is important to update our understanding of the immunopathogenesis of chikungunya infection, its clinical manifestations, the diagnosis, and the development of chikungunya vaccines.

Prevalence of HIV Infection and Resistance Mutations in Patients Hospitalized for Febrile Illness in Indonesia.

HIV prevalence in Indonesia is increasing, and only 64% of infected individuals know their status. In a prospective cohort of 1,453 hospitalized patients with unexplained fever, 46 (3.2%) had HIV, including 15 (1.1%) patients without a prior HIV diagnosis. Among 31 subjects previously known to have HIV, 21 (68%) had been receiving combination antiretroviral therapy (cART) at the time of enrollment. Of 39 HIV cases with HIV RNA levels ≥ 100 copies/mL, sequencing for genotype analysis and resistance testing was successful in 30 (77%) subjects. The most common HIV subtypes were AE (90%) and B (10%). Five (16.7%) subjects had resistance mutations to nucleoside and non-nucleoside reverse transcriptase inhibitors, and all of them were on cART. No evidence of transmitted drug resistance was found in newly diagnosed individuals. Hospital-based screening may be an efficient method to expand HIV testing and identify a significant number of new cases. Access to care, close monitoring, expansion of anti-retroviral options, and ensuring availability of CD4 determinations, viral load testing, and genotyping are crucial to control of the epidemic in Indonesia.

Burden of severe rotavirus diarrhea in indonesia.

Globally, rotavirus is the leading cause of diarrhea-related hospitalizations and deaths among young children, but the burden of rotavirus disease in Indonesia is poorly documented. From January through December 2006, we conducted prospective surveillance (inpatient and outpatient) among children aged <5 years at 6 hospitals in 6 provinces of Indonesia, using standardized methodology. Of 2240 enrolled children hospitalized for diarrhea, 1345 (60%) were rotavirus positive. Of 176 children enrolled in outpatient clinics in 3 hospitals, 73 (41%) were rotavirus positive. Among children hospitalized for diarrhea, dehydration was more common among those who tested positive for rotavirus than among those who did not (91% vs 82%; P < .05), as was vomiting (86% vs 67%; P < .05). Children aged 6-23 months experienced 72% of all rotavirus episodes. Rotavirus prevalence increased slightly in the cool, dry season. The most commonly detected genotypes were G9 (30%) and P[6] (56%). G1P[6] and G9P[6] accounted for 34% and 21% of strains, respectively. A high proportion of genotype P[6] was detected, in combination with the common G types G1 and G9. Available rotavirus vaccines would likely be efficacious against the most common circulating strains, but continued monitoring of uncommon genotypes is needed.

Effect of cinnamon oil on icaA expression and biofilm formation by Staphylococcus epidermidis.

Staphylococcus epidermidis is notorious for its biofilm formation on medical devices, and novel approaches to prevent and kill S. epidermidis biofilms are desired. In this study, the effect of cinnamon oil on planktonic and biofilm cultures of clinical S. epidermidis isolates was evaluated. Initially, susceptibility to cinnamon oil in planktonic cultures was compared to the commonly used antimicrobial agents chlorhexidine, triclosan, and gentamicin. The MIC of cinnamon oil, defined as the lowest concentration able to inhibit visible microbial growth, and the minimal bactericidal concentration, the lowest concentration required to kill 99.9% of the bacteria, were determined using the broth microdilution method and plating on agar. A checkerboard assay was used to evaluate the possible synergy between cinnamon oil and the other antimicrobial agents. The effect of cinnamon oil on biofilm growth was studied in 96-well plates and with confocal laser-scanning microscopy (CLSM). Biofilm susceptibility was determined using a metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Real-time PCR analysis was performed to determine the effect of sub-MIC concentrations of cinnamon oil on expression of the biofilm-related gene, icaA. Cinnamon oil showed antimicrobial activity against both planktonic and biofilm cultures of clinical S. epidermidis strains. There was only a small difference between planktonic and biofilm MICs, ranging from 0.5 to 1% and 1 to 2%, respectively. CLSM images indicated that cinnamon oil is able to detach and kill existing biofilms. Thus, cinnamon oil is an effective antimicrobial agent to combat S. epidermidis biofilms.

Biofilm formation and antibiotic resistance of Klebsiella pneumoniae isolated from clinical samples in a tertiary care hospital, Klaten, Indonesia.

BACKGROUND: Klebsiella pneumoniae (K. pneumoniae) is a common cause of health-care associated infections (HAIs) and has high levels of antibiotic resistance. These bacteria are well-known for their ability to produce biofilm. The purpose of this study was to identify the antibiotic resistance pattern and biofilm-producing capacity of K. pneumoniae isolated from clinical samples in a tertiary care hospital in Klaten, Indonesia. METHODS: K. pneumoniae was isolated from inpatients in Soeradji Tirtonegoro Hospital Klaten from June 2017 to May 2018. Identification of K. pneumoniae isolate was done by analyzing colony morphology, microscopic examination, and by performing biochemical testing. Testing of antibiotics susceptibility and biofilm-producing capacity used the Kirby-Bauer disk diffusion method and adherence quantitative assays, respectively. RESULTS: A total of 167 (17.36%) K. pneumoniae isolates were isolated from 962 total clinical bacterial isolates during the study. Most of them were collected from patients aged more than 60 years old and were mainly obtained from respiratory specimens (51.50%). Most of K. pneumoniae isolates were extensively resistant to antibiotics. A more favorable profile was found only towards meropenem, amikacin, and piperacillin-tazobactam, showing 1.20%; 4.79% and 10.53% of resistance, respectively. The overall proportion of multidrug-resistant K. pneumoniae isolates was 54.49%. In addition, 148 (85.63%) isolates were biofilm producers, with 45 (26.95%) isolates as strong, 48 (28.74%) isolates as moderate, and 50 (29.94%) isolates as weak biofilm producers. CONCLUSION: Most of the K. pneumoniae isolates demonstrated resistance to a wide range of antibiotics and are biofilm producers.

Ica-expression and gentamicin susceptibility of Staphylococcus epidermidis biofilm on orthopedic implant biomaterials.

Ica-expression by Staphylococcus epidermidis and slime production depends on environmental conditions such as implant material and presence of antibiotics. Here, we evaluate biofilm formation and ica-expression of S. epidermidis strains on biomaterials involved in total hip- and knee arthroplasty [polyethylene (PE), polymethylmethacrylate (PMMA), stainless steel (SS)]. Ica-expression, assayed using real-time RT-PCR, was highest on PE as confirmed using confocal laser scanning microscopy. Yet biofilm formation by S. epidermidis was most extensive on SS, with less slime production. Ica-expression and slime production were minimal on PMMA. After 3 h of continued growth of 24 h old biofilms in the presence of gentamicin, biofilms on PE showed lower susceptibility to gentamicin, relative to the other materials, presumably as a result of the stronger ica-expression. A higher gentamicin concentration further decreased metabolic activity on all biomaterials. It is concluded that the level of biomaterial-induced ica-expression does not correlate with the amount of biofilm formed, but initially aids bacteria in surviving antibiotic attacks. Once antibiotic treatment has started however, also the antibiotic itself induces slime production and only if its concentration is high enough, killing results. Results suggest that biomaterial-associated infections in orthopedics by S. epidermidis on PE may be more difficult to eradicate than on PMMA or SS.

recA mediated spontaneous deletions of the icaADBC operon of clinical Staphylococcus epidermidis isolates: a new mechanism of phenotypic variations.

Phenotypic variation of Staphylococcus epidermidis involving the slime related ica operon results in heterogeneity in surface characteristics of individual bacteria in axenic cultures. Five clinical S. epidermidis isolates demonstrated phenotypic variation, i.e. both black and red colonies on Congo Red agar. Black colonies displayed bi-modal electrophoretic mobility distributions at pH 2, but such phenotypic variation was absent in red colonies of the same strain as well as in control strains without phenotypic variation. All red colonies had lost ica and the ability to form biofilms, in contrast to black colonies of the same strain. Real time PCR targeting icaA indicated a reduction in gene copy number within cultures exhibiting phenotypic variation, which correlated with phenotypic variations in biofilm formation and electrophoretic mobility distribution of cells within a culture. Loss of ica was irreversible and independent of the mobile element IS256. Instead, in high frequency switching strains, spontaneous mutations in lexA were found which resulted in deregulation of recA expression, as shown by real time PCR. RecA is involved in genetic deletions and rearrangements and we postulate a model representing a new mechanism of phenotypic variation in clinical isolates of S. epidermidis. This is the first report of S. epidermidis strains irreversibly switching from biofilm-positive to biofilm-negative phenotype by spontaneous deletion of icaADBC.