Experimental Models Point Mutations In Plasmodium falciparum pfatpase6 Gene Exposed to Recuring Artemisinin In Vitro

The aims of this research  to prove that repeated exposure of artemisinin can cause pfatpase6 gene mutation on Plasmodium falciparum in vitro. The research methods used culture In Vitro Plasmodium  falciparum of strain 2300 IC50 value determination test artemisinin, artemisinin repeated exposure test  (PO1, PO2, PO3 dan PO4) dose IC50, DNA extraction, gene amplification of pfatpase6 using Polymerase Chain Reaction (PCR) technique,  electrophoresis, PCR product purification, labeling DNA from PCR results, DNA precipitation of PCR product, application of product labeling on the sequencing machines, analysis of  the results of sequencing, and Data Analysis. The results of PCR pfatpase6 gene amplification include region 6 – 3216 for codon 89-1031 located in exon 1 and 2 Plasmodium falciparum 2300  by using five pairs of primers. Primer pair 1FR produce a long amplicon of 737 bp which covers of codon 89; primer pair 2FR produce a long amplicon of 813 bp which covers of codon 263, 431; primer pair 4FR produce a long amplicon of 700 bp which covers of codon 460, 465, 623; primer pair 5FR produce a long amplicon of 550 bp which includes of codon 683, 769; and primer pair 6FR produce a long amplicon of 876 bp which covers of codon 898, 1031.Multialigment pfatpase6 gene Plasmodium  falciparum of strains  Papua 2300 point mutations are obtained in the form of transition and transversion in treatment groups at the same nucleotide region 123, 2035, 2043, 2138 dan 2148. Conclusion of this research Artemisinin repeated exposure  can cause point mutations in pfatpase6 genes Plasmodium falciparum of strains  2300 in vitro Keyword: Artemisinin, Plasmodium falciparumof strain Papua 2300, pfatpase6 gene,  point mutatio

Profil Fenotipik Plasmodium falciparum Galur Papua 2300 Akibat Paparan Antimalaria Artemisinin in Vitro

The presence of the P. falciparum resistance and decreased of efficacy against artemisinin and its derivatives result in increasingly complex malaria issues. Malaria has become one of the currently unresolved world’s health problems due to the lack of new artemisinin replacement drugs. This study aimed to provide evidence that the repeated exposure of in vitro artemisinin may cause a change in P. falciparum Papua 2300 strain phenotypic. This study was conducted during the period of February to November 2013 in Biomedics Brawijaya University and the Faculty of Veterinary Medicine, Airlangga University. A post-test control only experimental design was used. In vitro cultures of P. falciparum Papua 2300 strain were treated by repeated artemisin in IC50 concentration and were observed for their viability and IC50 using probit analysis. The control group did not show any changes after IC50value and PO1 treatment. An increase in IC50 value was occurred after PO2. Repeated exposures of artemisinin in PO2, PO3 and PO4 had shorter viability periods than PO1. The viability of was stable after PO3 in this group. In conclusion, repeated exposures of artemisinin influence changes in IC50 value and viability period of P. falciparum Papua 2300 strain

Genetic characterization of H5N1 influenza viruses isolated from chickens in Indonesia in 2010

Since 2003, highly pathogenic H5N1 avian influenza viruses have caused outbreaks among poultry in Indonesia every year, producing the highest number of human victims worldwide. However, little is known about the H5N1 influenza viruses that have been circulating there in recent years. We therefore conducted surveillance studies and isolated eight H5N1 viruses from chickens. Phylogenic analysis of their hemagglutinin and neuraminidase genes revealed that all eight viruses belonged to clade 2.1.3. However, on the basis of nucleotide differences, these viruses could be divided into two groups. Other viruses genetically closely related to these two groups of viruses were all Indonesian isolates, suggesting that these new isolates have been evolving within Indonesia. Among these viruses, two distinct viruses circulated in the Kalimantan islands during the same season in 2010. Our data reveal the continued evolution of H5N1 viruses in Indonesia

Biological and Structural Characterization of a Host-Adapting Amino Acid in Influenza Virus

Two amino acids (lysine at position 627 or asparagine at position 701) in the polymerase subunit PB2 protein are considered critical for the adaptation of avian influenza A viruses to mammals. However, the recently emerged pandemic H1N1 viruses lack these amino acids. Here, we report that a basic amino acid at position 591 of PB2 can compensate for the lack of lysine at position 627 and confers efficient viral replication to pandemic H1N1 viruses in mammals. Moreover, a basic amino acid at position 591 of PB2 substantially increased the lethality of an avian H5N1 virus in mice. We also present the X-ray crystallographic structure of the C-terminus of a pandemic H1N1 virus PB2 protein. Arginine at position 591 fills the cleft found in H5N1 PB2 proteins in this area, resulting in differences in surface shape and charge for H1N1 PB2 proteins. These differences may affect the protein's interaction with viral and/or cellular factors, and hence its ability to support virus replication in mammals

Influenza A (H5N1) Viruses from Pigs, Indonesia

TOC summary: Pigs may serve as intermediate hosts in which this avian virus can adapt to mammals

Efficacy of the New Neuraminidase Inhibitor CS-8958 against H5N1 Influenza Viruses

Currently, two neuraminidase (NA) inhibitors, oseltamivir and zanamivir, which must be administrated twice daily for 5 days for maximum therapeutic effect, are licensed for the treatment of influenza. However, oseltamivir-resistant mutants of seasonal H1N1 and highly pathogenic H5N1 avian influenza A viruses have emerged. Therefore, alternative antiviral agents are needed. Recently, a new neuraminidase inhibitor, R-125489, and its prodrug, CS-8958, have been developed. CS-8958 functions as a long-acting NA inhibitor in vivo (mice) and is efficacious against seasonal influenza strains following a single intranasal dose. Here, we tested the efficacy of this compound against H5N1 influenza viruses, which have spread across several continents and caused epidemics with high morbidity and mortality. We demonstrated that R-125489 interferes with the NA activity of H5N1 viruses, including oseltamivir-resistant and different clade strains. A single dose of CS-8958 (1,500 µg/kg) given to mice 2 h post-infection with H5N1 influenza viruses produced a higher survival rate than did continuous five-day administration of oseltamivir (50 mg/kg twice daily). Virus titers in lungs and brain were substantially lower in infected mice treated with a single dose of CS-8958 than in those treated with the five-day course of oseltamivir. CS-8958 was also highly efficacious against highly pathogenic H5N1 influenza virus and oseltamivir-resistant variants. A single dose of CS-8958 given seven days prior to virus infection also protected mice against H5N1 virus lethal infection. To evaluate the improved efficacy of CS-8958 over oseltamivir, the binding stability of R-125489 to various subtypes of influenza virus was assessed and compared with that of other NA inhibitors. We found that R-125489 bound to NA more tightly than did any other NA inhibitor tested. Our results indicate that CS-8958 is highly effective for the treatment and prophylaxis of infection with H5N1 influenza viruses, including oseltamivir-resistant mutants

Produksi Dan Karakterisasi Antibodi Poliklonal Sebagai Bahan Pendeteksi Ekspresi Protein S Dari Virus Infectious Bronchitis Isolasi Lokal

Penyakit Infectious Bronchitis merupakan penyakit yang sangat menular dapat menimbulkan kerugian besar bagi peternakan ayam petelur maupun pedaging. Penyakit ini menimbulkan gangguan pernafasan dan gangguan ginjal. Penyakit ini disebabkan oleh virus lnfectious Bronchitis, yaitu virus RNA yang mempunyai tiga macam regio gen yaitu regio gen M, E dan S. Pencegahan penyakit Infectious Bronchitis ini dengan vaksinasi baik vaksin inaktif maupun vaksin aktif. Penggunaan vaksin aktif , secara tidak langsung dapat menimbulkan varian baru di lapangan. Hal ini disebabkan oleh adanya mutasi asam amino dari protein gen S (spike). Akibatnya vaksinasi berikutnya menjadi tidak efektif lagi. Untuk itu perlu dilakukan penelitian virus lnfectious Bronchitis di lapangan dengan pendekatan biologi molekuler , khususnya mutasi asam amino pada gen S. Salah satu bahan yang diperlukan untuk penelitian molekuler virus Infectious Bronchitis tersebut adalah antibodi poliklonal, baik dari virus lnfectious Bronchitis isolasi Lokal (belum diketahui serotipenya) dan virus Infectious Bronchitis yang ah diidentifikasi serotipenya (stok laboratorium). Sebanyak empat ekor kelinci disuntik dengan protein Virus Infectious bronchitis, setelah dua minggu dilakukan penyuntikan booster yang dicampur gan ajuvan komplit. Selang dua minggu berikutnya dilakukan penyuntikan boster dengan menggunakan ajuvan tidak komplit. Sedangkan penyuntikan boster ke tiga dilakukan dengan cara penyuntikan selama lima hari berturut- turut Kemudian diuji dengan metode ELISA dan setelah itu dilakukan permanenan antibodi poliklonal

Pengaruh Paparan Artemisinin terhadap Ekspresi Gen PArt pada Plasmodium falciparum Galur Papua 2300

no new therapeutic medicine to replace artemisinin. Even though the mechanism of artemisinin resistance has not been clearly understood, the resistance of P. falciparum towards the antimalaria artemisinin may occur due to the influence of by the internal factors of P. falciparum, including the induction of the protein-expressing gene expression. One of the genes is the Triptophan-rich Protein (PArt) gene that is important in the membrane-spanning protein and plays a role in protein folding to maintain hydrophobic contact.. This study aimed to prove that Triptophan-rich Protein overexspression in P. falciparum Papua 2300 strain may cause repeated artemisin exposure in vitro. This study was performed in a period from February to November 2013 in Infection and Tropical Diseases Hospital, Airlangga University. The design used was experimental study with post-test only control group design. In-vitro culture of P. falciparum Papua 2300 strain were divided into a control group (K) and treatment groups that were treated regularly with artemisinin, i.e. artemisinin exposure I (PO1), artemisinin exposure 2 (PO2) and artemisinin exposure 3 (PO3) using IC50 concentration. The Tryptophan-rich Protein gene expression level was detected using qRTPCR. The result showed that in vitro repeated artemisinin exposure in P. falciparum Papua 2300 strain relatively increased the expression level of the Tryptophan-rich Protein (PArt) genes (2ΔΔCT) when comparedwith control. In conclusion, in vitro artemisinin exposure may cause Tryptophan-rich Proteins (PArt) gene overexpression by P. falciparum Papua 2300 strain promoter

Konstruksi Seed Vaksin Bivalen Virus Influenza Melalui Metode Knockout Menggunakan Virus Newcastle Disease Dan Flu Burung H5N1 Di Indonesia

Virus flu burung H5NI merupakan virus zoonosis yang dikhawatirkan menjadi penyebab pandemi penyakit intluenza yang haru dengan korban yang tinggi. Ini disebabkan oleh sifat patogenisitas virus ang tinggi ketika menginLksi inang. transmisinya yang aerosol dan kemampuan melakukan mutasi yang memungkinkan timbulnya subtipe baru pada populasi yang naive. Belum ada bukti bahwa virus Flu Burung ini telah menginfeksi antar manusia, sehingga pengendalian pada hewan lebih diutamakan. Salah satu upaya untuk meneegah kejadian tersebut adalah melalui program vaksinasi. Kesiapsiagaan terhadap aneaman pandemi diw ujudkan dengan membuat dan menyiapkan seed vaksin terhadap virus flu burung H5N I . Selain itu. Necasde diseas(ND) merupakan penyakit strategis yang menimbulkan kerugian yang tinggi pada ekonomi peternakan unggas. Selama ini vaksinasi pada unggas yang clilakukan bersifat monovalen, sehingga mengakibakan biaya operasional peternakan yang tinggi, karena harus melakukan vaksinasi dua macam penyakit Dengan demikian, solusi yang efektif saat ini adalah perbaikan perbaikan konstruksi dari monovalen menjadi bi-t i valen. Vaksin konvensional 115N I yang ada merupakan seed vaksin yang berasal dari virus Flu Burung yang highl pothogenic avian (1-1PA1). Metode yang bisa digunakan adalah melalui penggunaan teknologi rever.ve genetic dalam menghasilkan seed vaksin terhadap virus H5N I . vang bersifat /ow pathogenic. Selanjutnya divabungkan dengan bagian virus ND yanu imunogenik pada virus flu tersebut. Salah satu metode dalam peng,gabungan ke dua virus tlu burunu dan ND - yang saat ini dikembangkan adalah teknologi knockout (KO). Metode ini merupakan suattt metode yang diraneang untuk menghasilkan secd vaksin dengan fenotipe yang dirancang di awal dan memiliki valensi lebih dari satu. Tahapan awal dalam menghasilkan seed vaksin dengan metode KO adalah pembentukan plasmid KO, yang terdiri dari: (1) tahapan penghilangan ORF (open readingfame) dan urutan penyandi dari gen P132 virus inlluenza yang digunztkan sebagai ba khone virus (2) tahapan digesti gen PB2 clan gen in.vrt (dalam hal ini gen l-1A virus flu burung 1-15N 1 serta fragmen gen l' dai virus ND isolat Indonesia. untuk menuhasilkan protein permukaan HA dan NI) sebagai antigen seed vaksin) oleh enzim restriksi; (3) insersi dan ligasi segmen gen insert yang akan diekspresikan pada gen PB2 dan plasmid ekspresi vRNA yakni pPol I: (4) transformasi pada bakteri E. coli: serta (5) analisis bioinformatik plasmid KO. Gen HA yanu, di insersikan pada penelitian ini adalah gen HA dari virus Ilu burung I-15N 1 dan fragmen uen I: dari ‘,irus ND yang diisolasi dari unggas di Indonesia, juga diinsersikan pada kerangka dasar virus flu. Gen HA ini telah dilakukan mutagenesis pada mullibovic anllno acicl di daerah cleavage site menjadi /ow pathogenic (Reviany et al.. 2017), sedangkan fragmen gen virus ND telah diuji serologis dan tilogentika molekuler terhadap virus ND yang berkembang di Indonesia. Pada penelitian ini, dilakukan identifikasi molekuler terhadap virus ND sebagai kandidat seecl vaksin bivalen untuk tahapan awal dari keseluruhan tahap penelitia

Induction of Plasmodium falciparum strain 2300 dormant forms by artemisinin

BACKGROUND The presence of Plasmodium falciparum resistance and decreased efficacy of artemisinin and its derivatives has resulted in the issue of malaria becoming increasingly complex, because there have been no new drugs as artemisinin replacements. The aims of this research were to evaluate in vitro changes in ultrastructural morphology of P. falciparum 2300 strain after exposure to artemisinin. METHODS The research used an experimental design with post test only control group. Cultures of P. falciparum 2300 strain in one control and one mutant group were treated by exposure to artemisinin at IC50 10-7 M for 48 hours. Ultrastructural phenotypic examination of ring, trophozoite and schizont morphology and developmental stage in the control and mutant group were done at 0, 12, 24, 36, 48 hours by making thin blood smears stained with 20% Giemsa for 20 minutes and examined using a microscope light at 1000x magnification. RESULTS Dormant forms occurred after 48 hours of incubation with IC50 10-7 M artemisinin in the control group. In the mutant group, dormant forms, trophozoites with blue cytoplasm and normal schizont developmental stages were seen. Ultrastructural phenotypic morphology at 0, 12, 24, 36, 48 hours showed that in the control group dormant formation already occurred with exposure to IC50 10-7 M, while in the mutant group dormant formation occurred only with exposure to IC50 2.5x10-5 M. CONCLUSION Exposure to artemisinin antimalarials in vitro can cause phenotypic morphological changes of dormancy in P. falciparum Papua 2300 strain