Analisis Bioinformatika dan Ekspresi Protein Rekombinan Hemagglutinin Domain Globular dari Virus H5N1 Indonesia pada Eschericia coli BL21 (DE3) sebagai Komponen Vaksin Subunit Influenza

Flu burung merupakan salah satu penyakit zoonosis yang patut diwaspadai di Indonesia, khususnya galur High Pathogenic Avian Influenza (HPAI) karena dapat mematikan jika menular kepada manusia. Penggunaan vaksin influenza pada unggas, merupakan langkah preventif terhadap evolusi virus yang berbahaya dan juga penyebarannya. Selama ini, Indonesia masih menggunakan seed vaksin impor yang berasal dari luar Indonesia. Namun, karena Indonesia merupakan negara yang berada di garis khatulistiwa, karakteristik virus bisa berbeda dengan virus dari nothern-hemispere maupun southern-hemispere. Mengingat hal tersebut, Indonesia harus mengembangkan vaksin influenza menggunakan galur virus lokal. Berbeda dengan vaksin whole virus, vaksin rekombinan memiliki keunggulan dari sisi kemurnian, kecepatan produksi, dan kesesuaian galur terhadap virus yang beredar saat diperlukan. Penelitian ini bertujuan untuk menganalisis sekuen hemagglutinin (HA) Indonesia dengan strain lainnya serta mengekspresikkan protein HA1 rekombinan pada Escherichia coli BL21 (DE3). Galur yang digunakan pada studi ini berasal dari virus H5N1 (A/Indonesia/05/05), khususnya bagian domain globular dari HA1. Sekuen HA1 bervariasi antara strain Indonesia dengan nothern-hemispere maupun southern-hemispere dan merupakan protein yang terpapar ke luar virus. Gen HA1 disisipkan pada vektor pET-28a, kemudian plasmid diisolasi menggunakan meoe manniatis, setelah itu diekspresikan dengan induksi 1 mM IPTG selama 4 jam. Protein HA1 telah berhasil diekspresikan secara intraseluler dan telah dikonfirmasi pada berat molekul 40 kDa menggunakan SDS-PAGE. Penelitian ini dapat digunakan untuk mengembangkan vaksin subunit yang lebih spesifik terhadap virus yang beredar di lapangan.

Laporan Kasus Avian Influenza di Dusun Cabean, Kelurahan Mangunsari, Kecamatan Sidomukti, Kota Salatiga Tahun 2019

Virus Avian Influenza (AI) dibedakan menjadi Low Pathogenic Avian Influenza (LPAI) dan High Pathogenic Avian Influenza (HPAI) dimana pada HPAI ditunjukkan dengan tingkat kematian unggas yang mencapai 100% dalam waktu yang singkat. Tujuan dari penelitian terhadap kematian unggas di Cabean, kelurahan Mangunsari, Kecamatan Sidomukti, Kota Salatiga adalah untuk mengidentifikasi penyebab kematian dan menentukan tindakan pengendalian. Observasi dilakukan pada tanggal 29 Juli 2019 hingga 3 Agustus 2019 dan analisis data dilakukan secara deskriptif. Informasi kematian mendadak pada unggas dilaporkan pemilik pada 29 Juli 2019. Dalam satu minggu kematian ayam kampung mencapai 38,75%, itik manila (entok) mencapai 90% dan angsa mencapai 100%. Dari hasil pemeriksaan berdasarkan informasi, nekropsi, rapid test dan laboratorium, dapat disimpulkan bahwa kematian mendadak pada unggas yang terjadi di Cabean disebabkan oleh virus Avian Influenza. Hasil investigasi diharapkan mampu memberikan kejelasan penyebab kematian unggas dan pemahaman kepada masyarakat terutama peternak unggas mengenai virus Avian Influenza, tindak pencegahan dan penanggulangannya.

Crystal structure of inhibitor-bound human MSPL that can activate high pathogenic avian influenza

Infection of certain influenza viruses is triggered when its HA is cleaved by host cell proteases such as proprotein convertases and type II transmembrane serine proteases (TTSP). HA with a monobasic motif is cleaved by trypsin-like proteases, including TMPRSS2 and HAT, whereas the multibasic motif found in high pathogenicity avian influenza HA is cleaved by furin, PC5/6, or MSPL. MSPL belongs to the TMPRSS family and preferentially cleaves [R/K]-K-K-R↓ sequences. Here, we solved the crystal structure of the extracellular region of human MSPL in complex with an irreversible substrate-analog inhibitor. The structure revealed three domains clustered around the C-terminal α-helix of the SPD. The inhibitor structure and its putative model show that the P1-Arg inserts into the S1 pocket, whereas the P2-Lys and P4-Arg interacts with the Asp/Glu-rich 99-loop that is unique to MSPL. Based on the structure of MSPL, we also constructed a homology model of TMPRSS2, which is essential for the activation of the SARS-CoV-2 spike protein and infection. The model may provide the structural insight for the drug development for COVID-19.

Avian influenza infections in poultry farms in Egypt, a continuous challenge: Current problems related to pathogenesis, epidemiology, and diagnosis

This study's main objective was to update avian influenza (AI) epidemiological situation, including molecular characterization reassortment analysis and genotyping of circulating AI virus (AIV) subtypes in Egyptian poultry farms between 2017 and 2019. As a necessity for such work, improved diagnostic tools were developed for AIV detection. Subtype H9N2 infections were detected in 27 out of 39 examined farms and were frequently mixed with high pathogenic avian influenza (HPAI)AIV H5N8 in 22/39 farms. Next-generation and Sanger sequencing helped to define novel reassortant HPAIV H5N2 and low pathogenic avian influenza (LPAIV) H9N2 in Egypt. Systematic reassortment analysis confirmed at least seven genotypes of HPAI H5NX viruses of clade 2.3.4.4b and three genotypes of LPAIV H9N2 circulating in Egypt. Several internal genes of AIVs previously detected in wild birds in Egypt were represented in the genome of novel reassortants of both HP H5Nx and H9N2 viruses suggesting local reassortment processes.

Crystal structure of inhibitor-bound human MSPL that can activate high pathogenic avian influenza

Viral infection is triggered when a surface envelope glycoprotein, hemagglutinin (HA), is cleaved by host cell proteins of the transmembrane protease serine (TMPRSS) family. The extracellular region of TMPRSS-2, -3, -4, and MSPL are composed of LDLA, SRCR, and SPD domains. MSPL can cleave the consensus multibasic (R-X-X/R-R) and monobasic (Q(E)-T/X-R) motifs on HA, while TMPRSS2 or -4 only cleaves monobasic motifs. To better understand HA cleavage mediated by MSPL, we solved the crystal structure of the extracellular region of human MSPL in complex with the furin inhibitor. The structure revealed that three domains are gathered around the C-terminal α-helix of the SPD domain. The furin inhibitor structure shows that the side chain of P1-Arg inserts into the highly conserved S1 pocket, whereas the side chain of P2-Lys interacts with the Asp/Glu-rich 99 loop that is unique to MSPL. We also constructed a homology model of TMPRSS2, which is identified as an initiator of SARS-CoV-2 infection. The model suggests that TMPRSS2 is more suitable for Ala/Val residues at the P2 site than Lys/Arg residues.