Background: The report of mutation sites ORF3a SARS CoV-2 in Indonesia is still limited. Some research showed that mutations in ORF3a protein might alter SARS-CoV-2 pathogenesis. Observation of new variants should be conducted as a risk monitoring framework.Materials and method: We assessed the impact of mutations in ORF3a protein by analyzing 3,751 SARS-CoV-2 DNA sequences from the GISAID database from March 2020 until July 2021. The whole-genome sequences were aligned using Clustal Omega Multiple Sequence Alignment from EMBL-EBI and analyzed using BioEdit version 7.2.5 software. The reference whole genome sequence was taken from the Genbank database with accession number NC045512. We excluded the samples containing N letters due to inaccurate reading. Effect of point mutations on protein structure was analyzed using PredictProtein (https://predictprotein.org) and Protein Variation Effect Analyzer (PROVEAN) v1.1.3. online software.Results: We identified five most frequent non-synonymous mutations in ORF3a protein of SARS-CoV-2 which were Q57H (58.04%), S26L (27.25%), S220I (10.37%), D155H (8.98%), and P104S (5.47%).Conclusion: These mutation data showed the phenomenon of amino acid changes in ORF3a SARS-CoV-2 in Indonesia until July 2021. The implication of this mutation needs to be determined in further studies.Keywords: Indonesia, mutations, non-synonymous, SARS-CoV-2, whole genome

Background: High mutation rates in HIV-1 could affect the accuracy of diagnostic tests. Therefore, recombinant antigen that has an immunodominant and conserved region from HIV-1 need to be developed to detect HIV-1 infection in Indonesia.Materials and methods: The recombinant antigens comprise of Gag (p24), Pol and Env (gp41). Each antigens was expressed in the Escherichia coli expression system and purified using Ni-NTA chromatography. The reactivity of purified antigen against HIV antibodies was tested against a group of 50 HIV-positive plasma samples and 45 HIV-negative plasma samples in a recombinant immunoblot assay (RIBA) platform test. Moreover, 21 of 50 HIV-positive samples and 3 of 45 HIV-negative samples were also tested using HIV blot 2.2 to compare RIBA with a commercial western blot kit. Ten HBV-positive and 10 HCV-positive plasma samples were used to check cross-reactivity with HIV recombinant proteins in RIBA.Results: All HIV-positive samples (100%) tested with RIBA were reactive towards Gag (p24), Pol, Env (gp41). Otherwise, 3 of 21 HIV-positive samples assayed with HIV blot 2.2 were not reactive to Pol protein. All HIV-negative samples tested with RIBA and 3 HIV-negative samples tested with HIV blot 2.2 did not produce any bands of HIV antigens. Few HBV and HCV samples showed reactivity towards HIV recombinant proteins.Conclusion: Each recombinant protein, Gag (p24), Pol, Env (gp41), could be expressed and purified, as well as had reactivity to HIV-positive samples in RIBA test. Therefore, RIBA can be used as a diagnostic test to detect HIV-1 infection in Indonesia.Keywords: diagnostic, HIV-1, immunodominant, recombinant immunoblot assay (RIBA)

Background: Indonesia is the second-highest country with hepatitis B cases in the South East Asian region. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 12 (Cas12) could be developed as a diagnostic tool to detect hepatitis B infection. This study was aimed to develop a diagnostic method for hepatitis B virus by designing CRISPR target-based single-guide RNA (sgRNA).Materials and method: The preCore/Core-gene sequences of hepatitis B virus were collected from the National Center for Biotechnology Information (NCBI) website. The selected sequence was submitted to Cas Designer and CRISPOR tools to design sgRNA. The resulting sgRNA was cloned in silico into an expression vector using Benchling software.Results: The 23-nucleotide sequence 5'- GTAGTCAGTTATGTCAATGTTAA-3’ had 30% GC content, 68.3 out-of-frame and 76 predicted efficiencies. This sequence had no mismatch based on analysis.Conclusion: This preliminary study will help design a CRISPR-based diagnostic kit for the detection of hepatitis B virus in Indonesia. However, further in vitro and in vivo studies are required to demonstrate its potential and efficiency.Keywords: CRISPR-Cas12b, diagnostic, HBV, sgRNA

AIDS is a severe immunodeficiency disease caused by HIV. Identification of new HIV infection in a population is required for the evaluation of intervention strategy of HIV-1 transmission. The avidity assay has been promoted for HIV-1 detection. Avidity assay is based on affinity strength of the epitopes of the HIV antigen against its specific corresponding antibodies. The binding of the antigen - the antibody formed in the initial phase of infection is relatively weak and easy to break with chaotropic reagents. In contrary, the antigen-antibody binding formation in long-term infection is strong and not easily broken by addition of chaotropic reagents. Commercial avidity assays are available, however the antigens used might not be compatible with the circulating HIV strains in Indonesia. AbstractThis research aimed to identify the most appropriate antigen candidate for avidity assay, three structural proteins from HIV were used i.e., p24, IDR-gp41 and ID2-Pol employed from the HIV strains circulating in Indonesia. The avidity assay was performed based on ELISA with pH 3 sodium citrate as chaotropic reagent. Serum samples was previously determined as positive and negative reactive by the Indonesian Red Cross. Each sample was tested in triplicates. The results of the avidity index were compared with the corresponding pattern of reactivity shown by Western Blotting. Comparative analysis of the avidity index using the IDR-Gp41 antigen showed correlation with increased value of avidity index with the completeness of the Western Blot reactivity pattern. This finding is not true in antigen ID2-Pol, and p24. Based on the results of the study, it can be concluded that IDR-Gp41 antigen has potential to be used in HIV avidity assay that is based on circulating strains of HIV in Indonesia. AbstrakAIDS merupakan penyakit imunodefisiensi berat yang disebabkan oleh HIV. Penentuan infeksi baru HIV-1 pada level populasi diperlukan guna evaluasi strategi intervensi pencegahan penularan HIV-1. Uji aviditas telah diajukan sebagai salah satu uji deteksi HIV-1. Prinsip uji aviditas adalah kekuatan afinitas epitope antigen HIV terhadap antibodi spesifik yang mengenali epitop tersebut. Ikatan antigen-antibodi yang terbentuk pada fase awal infeksi merupakan ikatan yang lemah dan mudah diputuskan dengan pemberian reagensia chaotropic. Pada fase infeksi lama, ikatan antigen-antibodi yang terbentuk merupakan ikatan yang kuat sehingga tidak mudah diputuskan oleh pemberian reagensia chaotropic. Uji aviditas komersial telah tersedia namun antigen yang digunakan belum tentu sesuai dengan galur HIV yang beredar di Indonesia. Pada penelitian ini digunakan 3 kandidat antigen yaitu p24, IDR-gp41 dan ID2-Pol dari galur HIV yang beredar di Indonesia, untuk menentukan kandidat yang sesuai. Uji aviditas dilakukan dengan prinsip ELISA dengan sodium sitrat pH 3 sebagai reagensia chaotropic. Sampel yang diujikan adalah sampel serum yang telah ditentukan reaktivitasnya sebagai positif dan negatif oleh Palang Merah Indonesia. Sampel diuji secara triplikat. Hasil indeks aviditas sampel dibandingkan dengan pola reaktivitasnya pada uji Western Blot. Analisis perbandingan menunjukkan bahwa peningkatan nilai indeks aviditas yang menggunakan antigen IDR-Gp41 berkorelasi dengan kelengkapan pola reaktivitas uji Western Blot. Hal ini tidak ditemukan pada pengujian menggunakan antigen IDR-Pol2, dan p24. Berdasarkan hasil penelitian, dapat disimpulkan bahwa antigen IDR-Gp41 berpotensi untuk digunakan lebih lanjut dalam pengembangan uji aviditas HIV berbasis galur HIV yang beredar di Indonesia.

Previous studies showed that mutations in the SARS-CoV-2 ORF3a protein can influence viral pathogenesis. Therefore, it is necessary to observe mutations, especially in the functional domain of the protein. We observed the presence of mutations in the ORF3a protein by analyzing 5,131 samples from the GISAID database since it was first discovered in March 2020 until November 2021. The sequence was aligned using Clustal Omega Multiple Sequence Alignment from EMBL-EBI and analyzed using BioEdit version 7.2.5 software using reference sequences NC045512. Samples having the letter N were omitted from the analysis. The effect of point mutations on proteins was analyzed using the Protein Variation Effect Analyzer (PROVEAN) v1.1.3 software. The functional domains of the ORF3a protein were visualized using RasWin software. We identified 312 mutations in the SARS-CoV-2 ORF3a protein. In addition, from 5,131 samples, 915 samples were found to be truncated in the C-terminal region of the protein. These non-synonymous mutations data in functional domains and truncated sequences indicate that amino acid changes in the ORF3a protein require further studies to determine the effect of viral pathogenicity in humans.