Whole-Genome Sequencing and Mutation Analyses of SARS-CoV-2 Isolates from Indonesia.

The SARS-CoV-2 infection that caused the COVID-19 pandemic has become a significant public health concern. New variants with distinct mutations have emerged, potentially impacting its infectivity, immune evasion capacity, and vaccine response. A whole-genome sequencing study of 292 SARS-CoV-2 isolates collected from selected regions of Indonesia between January and October 2021 was performed to identify the distribution of SARS-CoV-2 variants and common mutations in Indonesia. During January-April 2021, Indonesian lineages B.1.466.2 and B.1.470 dominated, but from May 2021, Delta's AY.23 lineage outcompeted them. An analysis of 7515 published sequences from January 2021 to June 2022 revealed a decline in Delta in November 2021, followed by the emergence of Omicron variants in December 2021. We identified C241T (5'UTR), P314L (NSP12b), F106F (NSP3), and D614G (Spike) mutations in all sequences. The other common substitutions included P681R (76.4%) and T478K (60%) in Spike, D377Y in Nucleocapsid (61%), and I82T in Membrane (60%) proteins. Breakthrough infection and prolonged viral shedding cases were associated with Delta variants carrying the Spike T19R, G142D, L452R, T478K, D614G, P681R, D950N, and V1264L mutations. The dynamic of SARS-CoV-2 variants in Indonesia highlights the importance of continuous genomic surveillance in monitoring and identifying potential strains leading to disease outbreaks.

Antibody response after a third dose mRNA-1273 vaccine among vaccinated healthcare workers with two doses of inactivated SARS-CoV-2 vaccine.

BACKGROUND: Health care workers (HCWs), a high-risk group for contracting COVID-19 disease, are being prioritized to receive COVID-19 vaccination. A third dose messenger RNA (mRNA) vaccine, mRNA-1273 (Moderna), after 2 doses of inactivated vaccine (CoronaVac), has been used to increase the level of protection against SARS-CoV-2 among Indonesian HCWs. However, data regarding antibody response after mRNA-1273 booster dose are limited. OBJECTIVE: To evaluate the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (anti-S) titers induced by the third mRNA-1273 vaccine among fully vaccinated HCWs with CoronaVac. RESULTS: A total of 90 HCWs with no history of SARS-CoV-2 infection and who had received the third dose of vaccination were included in this study. The mRNA-1273 vaccine booster was administered 6 months after completing primary vaccination with CoronaVac. After the third dose, the anti-S antibodies level significantly increased, from a median of 41.7 U/mL (interquartile range [IQR], 22.4-92.5) to 28 394 U/mL (IQR, 20 837-41 646) (p <0.0001). After the third dose, seropositivity with the anti-S antibodies level >210 U/mL was observed in all HCWs. Age was negatively associated with the anti-S antibodies level after the mRNA-1273 booster. CONCLUSION: The heterologous prime booster with CoronaVac and mRNA-1273 vaccine booster elicit a pronounced antibody response against SARS-CoV-2 infection.

Safety and Immunogenicity of a Single Dose of BNT162b2 COVID-19 mRNA Vaccine in a Warfarin-Treated Protein S Deficient Patient: A Case Report and Literature Review.

Patients with protein S (PS) deficiency possibly have a higher risk of developing severe COVID-19 disease. Therefore, vaccination against SARS-CoV-2 infections is recommended for PS-deficient patients. However, there are limited data regarding the safety and immunogenicity of the currently available COVID-19 mRNA vaccine in PS-deficient patients. We report a case of monitoring the antibody response of a 40-year-old female diagnosed with PS deficiency and on warfarin treatment following a single dose of BNT162b2 mRNA vaccine. Antibody against the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (anti-S) was measured on days 7, 14, and 21 after vaccination. Seroconversion was detected on day 21 but was possibly lower than the anti-S level previously reported in healthy individuals after receiving the first dose of the BNT162b2 mRNA vaccine. There were no local and systemic events reported up to 7 days in this patient after vaccination. This case highlights that the administration of the BNT162b2 vaccine had a favourable safety profile, and the second dose of the vaccine is required to provide the optimal protection against SARS-CoV-2 infection in PS-deficient patients.

Recurrence of Asymptomatic COVID-19 after Recovery among Healthcare Workers.

We describe five healthcare workers (HCWs) with a recurrence of asymptomatic SARS-CoV-2 infection at Siloam Teaching Hospital, Indonesia. All cases involved nurses, with an average age of 27 years. The RT-PCR assay confirmed the first and second infection episodes. All cases showed negative RT-PCR results in the period between two infection episodes. The median interval time between two infection episodes was 123 days, ranging from 92 to 158 days. The clinical outcomes for all cases were favourable, with no mortality observed among study cases. Further studies will be required to understand the true nature of this phenomenon.

Heterologous prime-boost with the mRNA-1273 vaccine among CoronaVac-vaccinated healthcare workers in Indonesia.

Purpose: This study was performed to investigate humoral immune response and adverse events upon the heterologous prime-boost with a single dose of the mRNA-1273 vaccine among fully CoronaVac-vaccinated, infection-naïve healthcare workers in Indonesia. Materials and Methods: One hundred twenty-five eligible healthcare workers were recruited from one hospital for this prospective cohort study. Blood collection was conducted twice, i.e., on 7 days before and 28 days after the booster vaccination. The titer of anti-SARS-CoV-2 receptor-binding domain (RBD) antibodies was quantified accordingly. The post-vaccination adverse event was recorded for both CoronaVac and mRNA-1273 vaccinations. Any breakthrough infection was monitored during the follow-up period. Wilcoxon matched-pairs signed rank test was used to test differences between groups. Results: A significant increase was observed in the titer of anti-SARS-CoV-2 RBD antibodies upon receiving the mRNA-1273 booster (geometric mean titers of 65.57 and 47,445 U/mL in pre- and post-booster, respectively), supporting the argument to use heterologous prime-boost vaccination to improve the protection against COVID-19 in a high-risk population. The mRNA-1273 vaccine, however, caused a higher frequency of adverse events than the CoronaVac vaccine. Nonetheless, the adverse events were considered minor medical events and temporary as all subjects were not hospitalized and fully recovered. Of note, no breakthrough infection was observed during the follow-up to 12 weeks post-booster. Conclusion: The heterologous prime-boost vaccination of healthcare workers with a single dose of the mRNA-1273 vaccine generated a significant elevation in humoral immune response towards RBD of SARS-CoV-2 and was associated with a higher frequency, but minor and transient, adverse events.

Genetic factors associated with patient-specific warfarin dose in ethnic Indonesians.

BACKGROUND: CYP2C9 and VKORC1 are two major genetic factors associated with inter-individual variability in warfarin dose. Additionally, genes in the warfarin metabolism pathway have also been associated with dose variance. We analyzed Single Nucleotide Polymorphisms (SNPs) in these genes to identify genetic factors that might confer warfarin sensitivity in Indonesian patients. METHODS: Direct sequencing method was used to identify SNPs in CYP2C9, VKORC1, CYP4F2, EPHX1, PROC and GGCX genes in warfarin-treated patients. Multiple linear regressions were performed to model the relationship warfarin daily dose requirement with genetic and non-genetic variables measured and used to develop a novel algorithm for warfarin dosing. RESULTS: From the 40 SNPs analyzed, CYP2C9 rs17847036 and VKORC1 rs9923231 showed significant association with warfarin sensitivity. In our study population, no significant correlation could be detected between CYP2C9*3, CYP2C9C-65 (rs9332127), CYP4F2 rs2108622, GGCX rs12714145, EPHX1 rs4653436 and PROC rs1799809 with warfarin sensitivity. CONCLUSIONS: VKORC1 rs9923231 AA and CYP2C9 rs17847036 GG genotypes were associated with low dosage requirements of most patients (2.05 ± 0.77 mg/day and 2.09 ± 0.70 mg/day, respectively). CYP2C9 and VKORC1 genetic variants as well as non-genetic factors such as age, body weight and body height account for 15.4% of variance in warfarin dose among our study population. Additional analysis of this combination could allow for personalized warfarin treatment in ethnic Indonesians.

Co-Occurrence of SARS-CoV-2 Infection and Inactivated SARS-CoV-2 Vaccination among Healthcare Workers.

The presented cases describe the concurrent SARS-CoV-2 infection and inactivated SARS-CoV-2 vaccination among eight healthcare workers (HCWs). These cases highlighted the importance of broad hospital screening during the COVID-19 vaccination campaign. Further study regarding the durability of antibody response induced by infection and first-dose vaccination is required to determine the appropriate time for giving a second dose of inactivated SARS-CoV-2 vaccine among these cases.

Antibody response to the inactivated SARS-CoV-2 vaccine among healthcare workers, Indonesia.

BACKGROUND: As healthcare workers (HCWs) are at high risk for infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), they have priority for receipt of the coronavirus disease 2019 (COVID-19) vaccine. The inactivated SARS-CoV-2 vaccine has been used in Indonesia to induce an antibody response against SARS-CoV-2 infection in HCWs. However, information regarding the kinetics of antibodies induced by this vaccine remains scarce. OBJECTIVE: To investigate the magnitude and durability of antibodies against the spike (S) protein (anti-S) in fully-vaccinated HCWs using an electrochemiluminescence immunoassay. RESULTS: Seroconversion of anti-S antibodies was observed among 159 (99.4%) of 160 HCWs without prior SARS-CoV-2 infection 14 days after full-dose vaccination. The level of anti-S antibodies decreased significantly by day 42 post-vaccination compared with day 14 post-vaccination, but persisted for up to 98 days post-vaccination. In contrast, vaccinated HCWs with prior SARS-CoV-2 infection had significantly higher, stable levels of anti-S antibodies compared with vaccinated HCWs without prior SARS-CoV-2 infection. CONCLUSION: The remarkable decline and lower level of anti-S antibodies among HCWs without prior SARS-CoV-2 infection may indicate the need for an additional booster dose of SARS-CoV-2 vaccine for protection against COVID-19. This study of antibody responses induced by the inactivated SARS-CoV-2 vaccine among HCWs may contribute to future policy decisions regarding vaccination.

Post-vaccination cases of COVID-19 among healthcare workers at Siloam Teaching Hospital, Indonesia.

BACKGROUND: Healthcare workers (HCWs) are at increased risk of exposure to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), compared with the general population. Therefore, they are given priority for the COVID-19 vaccine in the national COVID-19 vaccination campaign in Indonesia. However, while the daily number of new COVID-19 cases remains high, and data regarding the efficacy of the vaccine in healthcare settings remain unavailable, vaccinated HCWs remain at risk of COVID-19 infection and further transmission. OBJECTIVE: To identify cases of COVID-19 among vaccinated HCWs at Siloam Teaching Hospital, Indonesia via active and passive surveillance conducted by the hospital's COVID-19 infection prevention and control unit. RESULTS: Of 1040 HCWs who had received two doses of the COVID-19 vaccine, 13 (1.25%) tested positive for SARS-CoV-2 RNA on reverse transcriptase polymerase chain reaction between 2 and 11 days (median 5 days) after the second vaccination. CONCLUSION: Laboratory-confirmed COVID-19 among vaccinated HCWs soon after the second vaccination indicates that HCWs remain at risk of COVID-19. Therefore, the presence of symptoms soon after full vaccination cannot be considered as vaccine-related symptoms, and regular COVID-19 testing should be conducted among HCWs.

Genetic risk factors for colorectal cancer in multiethnic Indonesians.

Colorectal cancer is a common cancer in Indonesia, yet it has been understudied in this resource-constrained setting. We conducted a genome-wide association study focused on evaluation and preliminary discovery of colorectal cancer risk factors in Indonesians. We administered detailed questionnaires and collecting blood samples from 162 colorectal cancer cases throughout Makassar, Indonesia. We also established a control set of 193 healthy individuals frequency matched by age, sex, and ethnicity. A genome-wide association analysis was performed on 84 cases and 89 controls passing quality control. We evaluated known colorectal cancer genetic variants using logistic regression and established a genome-wide polygenic risk model using a Bayesian variable selection technique. We replicate associations for rs9497673, rs6936461 and rs7758229 on chromosome 6; rs11255841 on chromosome 10; and rs4779584, rs11632715, and rs73376930 on chromosome 15. Polygenic modeling identified 10 SNP associated with colorectal cancer risk. This work helps characterize the relationship between variants in the SCL22A3, SCG5, GREM1, and STXBP5-AS1 genes and colorectal cancer in a diverse Indonesian population. With further biobanking and international research collaborations, variants specific to colorectal cancer risk in Indonesians will be identified.

Antiviral interferons induced by Newcastle disease virus (NDV) drive a tumor-selective apoptosis.

Newcastle disease virus (NDV) strongly induces both type I and III antiviral interferons (IFNs-α/-ß and IFN-λ, respectively) in tumor cells while it induces mainly type III IFN in normal cells. Impairment of antiviral type I IFN signaling in tumor cells is thought to be the reason for effective oncolysis. However, there is lack of clarity why lentogenic strain NDV can also induce oncolysis. NDV infection caused apoptosis in normal and tumor cells as demonstrated with the caspase-3 enzyme activation and annexin-V detection. The apoptosis response was inhibited by B18R protein (a type I IFN inhibitor) in tumor cells i.e. A549 and U87MG, and not in normal cells i.e. NB1RGB and HEK293. Similarly, UV-inactivated medium from NDV infection was shown to induce apoptosis in corresponding cells and the response was inhibited in A549 and U87MG cells with the addition of B18R protein. Treatment with combination of IFNs-α/-ß/-λ or IFNs-α/-ß or IFN-λ in NB1RGB, HEK293, A549 and U87MG showed that caspase activity in IFNs-α/-ß/-λ group was the highest, followed with IFN-α/-ß group and IFN-λ group. This suggests that tumor-selectivity of NDV is mainly because of the cumulative effect of type I and III in tumor cells that lead to higher apoptotic effect.

Pre-S2 Start Codon Mutation of Hepatitis B Virus Subgenotype B3 Effects on NF-κB Expression and Activation in Huh7 Cell Lines.

A cross-sectional study on hepatitis B patients in Indonesia showed association of pre-S2 start codon mutation (M120 V) with cirrhosis and hepatocellular carcinoma (HCC), which was dissimilar from studies from other populations where pre-S2 deletion mutation was more prevalent. Different mutation patterns were attributed to different hepatitis B virus (HBV) subgenotypes in each population study. HBV surface proteins are reported to induce the activation of NF-κB, a transcriptional factor known to play an important role in the development of liver disease. This study aimed to see the effects of HBs variants in HBV subgenotype B3 on the expression and activation of NF-κB as one of the mechanisms in inducing advanced liver disease. HBV subgenotypes B3, each carrying wild-type (wt) HBs, M120 V, and pre-S2 deletion mutation were isolated from three HCC patients. HBs genes were amplified and cloned into pcDNA3.1 and were transfected using Lipofectamine into a Huh7 cell line. NF-κB activation was measured through IκB-α expression, which is regulated by NF-κB. RNA expressions for HBs, IκB-α, and NF-κB subunit (p50) were evaluated using real-time PCR. M120 V mutant had a significantly higher mRNA level compared with wt and pre-S2 deletion mutant; however, there were no significant differences in HBs protein expressions. The transcription level of p50 was higher in M120 V mutation compared with HBs wild-type and pre-S2 deletion mutant. NF-κB activation was higher in HBs wild-type compared with the two mutant variants. Pre-S2 mutations had no effect on the increment of NF-κB activation. However, M120 V mutation may utilize a different pathway in liver disease progression that involves high expression of NF-κB subunit, p50.

Characterization of protein-protein interactions: application to the understanding of peroxisome biogenesis.

With the approaching completion of the Pichia pastoris genome, a greater emphasis will have to be placed on the proteome and the protein-protein interactions between its constituents. This chapter discusses methods that have been used for the study of such interactions among both soluble and membrane-associated proteins in peroxisome biogenesis. The procedures are equally applicable to other cellular processes.

Alpha-fetoprotein gene polymorphisms and risk of HCC and cirrhosis.

BACKGROUND: Elevated level of alpha fetoprotein (AFP) is found in approximately 60% of hepatocellular carcinoma (HCC) cases. Other liver diseases including cirrhosis and chronic hepatitis are related with an increased level of AFP. The regulation of AFP gene expression has been relatively less studied although the gene has been suggested to play a role in HCC development. This study aimed at identifying genetic variations in AFP that might be associated with the presence of HCC and cirrhosis among ethnic Indonesians. METHODS: Direct DNA sequencing was carried out to sequence AFP promoter, exons, and 3' untranslated region (UTR) in DNA samples isolated from 119 HCC, 119 cirrhosis and 105 control subjects. For each sample serum AFP level was determined and association studies with single nucleotide polymorphisms (SNPs) and haplotypes were performed. RESULTS: In this study we identified 47 SNPs in the AFP gene. Statistically significant associations with HCC and cirrhosis were detected for six individual SNPs in the AFP promoter, AFP intron 1 and intron 2 (rs6834059, rs3796678, rs3796677, rs3796676, rs28532518 and rs4646038). Furthermore, we identified two SNPs in AFP intron 7 and 3'UTR, rs2298839 and rs10020432, which are associated with increased risk of cirrhosis. CONCLUSION: Genetic variants in the AFP gene may be associated with HCC and cirrhosis risk for ethnic Indonesians.

Peroxisome remnants in pex3delta cells and the requirement of Pex3p for interactions between the peroxisomal docking and translocation subcomplexes.

During peroxisomal matrix protein import, the peroxisomal targeting signal receptors recognize cargo in the cytosol and interact with docking and translocation subcomplexes on the peroxisomal membrane. Using immunoprecipitations of multiple protein components, we show that in Pichia pastoris the docking subcomplex consists of the unique peroxins Pex13p, Pex14p and Pex17p, whereas the putative translocation subcomplex has all three RING-finger peroxins, Pex2p, Pex10p and Pex12p, as unique constituents. We identify Pex3p as a shared component of both subcomplexes. In pex3delta cells, the unique constituents of the docking subcomplex interact as they do in wild-type cells, but the assembly of the translocation subcomplex is impaired and its components are present at reduced levels. Furthermore, several interactions detected in wild-type cells between translocation and docking subcomplex components are undetectable in pex3delta cells. Contrary to previous reports, pex3delta cells have peroxisome remnants that pellet during high-speed centrifugation, associate with membranes on floatation gradients and can be visualized by deconvolution microscopy using antibodies to several peroxins which were not available earlier. We discuss roles for Pex3p in the assembly of specific peroxisomal membrane protein subcomplexes whose formation is necessary for matrix protein import.