Wahidi, Budi Rianto (2019) Ekspresi Gen dan Virulensi Koi Herpes Virus (KHV) pada Ikan Nila (Oreochromis niloticus) Berdasarkan Karakter Genetik dan Morfologi. Doctor thesis, Universitas Brawijaya.
Abstract
Koi Herpesvirus (KHV) merupakan virus yang menginfeksi ikan budidaya air tawar terutama Cyprinidae. Gejala klinis ikan yang terinfeksi KHV diantaranya ikan berenang ke permukaan, bergerak kehilangan arah dan warna ujung lembaran insang pucat dan terdapat bercak-bercak putih. Tujuan dilakukan penelitian ini adalah 1) Menganalisa ekspresi gen pada ikan nila berpengaruh terhadap virulensi KHV. 2) Menjelaskan karakter genetik dan morfologi KHV yang menginfeksi ikan nila berkaitan dengan fungsi fisiologis baik secara imunologi maupun molekuler. Metode penelitian yang digunakan adalah deskriptif. Sampel ikan nila ukuran 7 - 10 cm diambil pada lokasi budidaya nila dan merupakan daerah suspect KHV. Sampel diperiksa klinis dan dipilih yang menunjukkan hasil positif PCR terhadap KHV, selanjutnya diuji kerusakan organnya secara makroskopis (patologi anatomi) maupun mikroskopis (histopatologi dan scoring). Pengamatan karakterisasi genetik dengan uji PCR dilanjutkan dengan sekuensing untuk melihat perubahan gen KHV maupun kekerabatan sampel KHV dengan menggunakan pohon filogenetik Pengamatan morfologi virion KHV dilakukan dengan pemeriksaan TEM. Pemeriksaan HSP70 secara kualitatif dilakukan untuk mengetahui peran serta fungsi HSP70 terhadap virulensi KHV pada ikan nila. Pengamatan organ eksternal ikan nila menunjukkan terdapat bercak putih pada insang dan perubahan warna kulit/diskolorisasi yang berbeda dengan ikan nila normal. Berdasarkan karakterisasi genetik, ekspresi gen TK terdeteksi pada ikan nila dan hasil analisis sekuen dan pohon filogenetik menunjukkan bahwa genotype KHV pada ikan nila termasuk dalam varian Asian genotype. Analisa karakterisasi morfologi KHV menunjukkan virion KHV dengan ukuran diameter 150 – 170 nm berada pada insang. Pada ikan nila, diduga proses replikasi virus paling banyak terjadi pada organ insang, selain itu partikel virus juga terdistribusi pada organ lain, berupa terbentuknya intranuclear inclusion body, pembengkakan sel, nekrosis, hipertropi, edema, hemoragi, inflamasi, kongesti, degenerasi dan apoptosis dengan derajat kerusakan yang berbeda untuk masing-masing organ. Keberadaan HSP70 sebagai inflammatory respon terdeteksi pada insang, dimana insang diyakini sebagai jalur masuk utama (major portal entry) KHV pada tubuh ikan. Berdasarkan hasil penelitian membuktikan bahwa terkait ekspresi gen TK sebagai penyandi gen virulensi KHV pada ikan nila (Oreochromis niloticus) secara fisiologis dibuktikan dengan adanya ekspresi gen TK pada 409 bp yang berkorelasi dengan ekspresi protein stress pada ikan nila dengan terinduksinya HSP70. Isolat KHV NBJT_1 dan NBJT_2 yang yang menginfeksi ikan nila termasuk dalam cluster asian genotype. Selain fungsinya sebagai inflammatory respon, HSP juga berfungsi sebagai fasilitator transportasi KHV dan terlibat setidaknya dalam satu tahap replikasi DNA. Infeksi KHV pada ikan nila dalam penelitian ini tidak menyebabkan kematian (virulen), akan tetapi ikan nila tersebut dapat bersifat carrier.
English Abstract
Koi Herpesvirus (KHV) is a virus that infects freshwater fish, especially Cyprinidae. Clinical symptoms of infected KHV fish include fish swimming to the surface, moving to lose direction and color of the tip of the pale gill sheet and white spots. The purpose of this study is 1) Analyzing gene expression in tilapia influences the virulence of KHV. 2) Describe the genetic and morphological characters of KHV that infect tilapia related to physiological functions both immunologically and molecularly. The research method used is descriptive. Samples of tilapia measuring 7-10 cm were taken at the location of tilapia cultivation and were areas suspected of KHV. Samples were examined clinically and selected which showed positive PCR results against KHV, then tested for organ damage macroscopically (anatomical pathology) and microscopic (histopathology and scoring). Observation of genetic characterization by PCR test followed by sequencing to see changes in KHV gene and kinship of KHV samples using phylogenetic trees Observation of KHV virion morphology was carried out by TEM examination. Qualitative HSP70 examination was carried out to determine the role and function of HSP70 on the virulence of KHV in tilapia. Observation of the external organs of tilapia shows white spots on the gills and changes in skin color / discoloration that are different from normal tilapia. Based on genetic characterization, TK gene expression was detected in tilapia and the results of sequence and phylogenetic tree analysis showed that the KHV genotype in tilapia was included in the Asian genotype variant. Morphological characterization analysis of KHV showed that KHV virions with diameters of 150 - 170 nm were in the gills. In tilapia, it is suspected that the virus replication process occurs mostly in gill organs, besides that virus particles are also distributed to other organs, in the form of intranuclear inclusion body, cell swelling, necrosis, hypertrophy, edema, hemorrhage, inflammation, congestion, degeneration and apoptosis with different degrees of damage for each organ. The presence of HSP70 is an inflammatory response detected in the gills, where the gills are believed to be the main entrance (major portal entry) of KHV in the fish body. Based on the results of the study prove that the expression of TK genes as encoding the KHV virulence gene in tilapia (Oreochromis niloticus) is physiologically proven by the presence of TK gene expression at 409 bp which correlates with protein stress expression in tilapia with HSP70 induction. Isolates of KHV NBJT_1 and NBJT_2 which infect tilapia are included in the Asian genotype cluster. In addition to its function as an inflammatory response, HSP also functions as a facilitator of KHV transport and is involved in at least one stage of DNA replication. KHV infection in tilapia in this study did not cause virulence, but the tilapia can be carrier.
Other obstract
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| Item Type: | Thesis (Doctor) |
|---|---|
| Identification Number: | DIS/639.964/WAH/e/2019/061902115 |
| Uncontrolled Keywords: | FISH--DISEASES |
| Subjects: | 600 Technology (Applied sciences) > 639 Hunting, fishing & conservation > 639.9 Conservation of biological resources > 639.96 Control of injuries, diseases, pests > 639.964 Diseases |
| Divisions: | S2/S3 > Doktor Ilmu Perikanan dan Kelautan, Fakultas Perikanan dan Ilmu Kelautan |
| Depositing User: | Budi Wahyono Wahyono |
| Date Deposited: | 09 Sep 2019 04:28 |
| Last Modified: | 09 Sep 2019 04:28 |
| URI: | http://repository.ub.ac.id/id/eprint/172548 |
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