Malays. Appl. Biol.(2010) 39(2): 7-13
TISSUE CULTURE ISOLATION, ELECTRON MICROSCOPIC CHARACTERIZATION AND PCR-DETECTION OF A BETANODAVIRUS ISOLATED FROM DISEASED ASIAN SEA BASS Lates calcarifer
MAUIDA, F.H.1, HASSAN, M.D.*1, SURI, A.S.2 and BEJO, M.H.2
*1Aquatic Animal Health Unite, Faculty of Veterinary Medicine, University Putra Malaysia 43400 Serdang, Selangor.Email:
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2Dept of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, University Putra Malaysia 43400 Serdang, Selangor.
ABSTRACT
A viral agent was successfully isolated from Asian sea bass juveniles (Letes calcarifer) exhibiting clinical signs of viral nervous necrosis (VNN) in southern Malaysia on February 2008. Eyes and brains of diseased fish were pooled, extracted and filtered. The filtrates were inoculated on SSN-1 cells and incubated at 25°C. Cytopathic effect (CPE) recognized as rounded cells continued to aggregate and the vacuolation. Electron micrographs of the infected SSN-1 cells revealed icosahedral nucleocapsid virions with 22-28 nm in diameter. Viral harvest was resistant to chloroform and iodine treatments, which indicated that it was naked and contained RNA genome. Identification using reverse transcription polymerase chain reaction (RT-PCR) and sequence analysis of PCR product was conducted and gave a single PCR product at 460 bp in agarose gel, followed by nested PCR with specific primers for PCR products at 220 bp. Partial nucleotide sequence of the nervous necrosis virus coat protein gene showed 94.0-96.1% homology to the nucleotide sequences of coat protein gene from nervous necrosis virus isolated from other countries in the Southeast Asia.
ABSTRAK
Sejenis virus telah berjaya diasingkan daripada ikan Siakap (Letes calcarifer) di Malaysia selatan pada bulan Februari 2008. Virus tersebut menunjukkan ciri-ciri serta gejala klinikal yang menyerupai virus nervous necrosis (VNN). Mata dan otak ikan yang sakit itu disatukan dan disaring. Para filtrates diinokulasi pada SSN-1 sel, dan diinkubasi pada 25°C. Kesan sitopatik (CPE) menunjukkan sel bulat, terus agregat dan penghasilan vacuolation. Mikrograf elektron dari sel yang dijangkiti- 1 SSN mendedahkan virion nukleokapsid ikosahedral, berdiameter 22-28 nm. Virus tersebut dapat bertahan terhadap kloroform dan yodium dan mengandungi genom RNA. Pengenalan menggunakan asai transkripsi terbalik rangkaian polimerasi (RT- PCR) memberikan hasil PCR tunggal pada 460 bp pada gel agarosa, diikuti dengan nested PCR dengan primer khusus untuk produk PCR yang didapati di kedudukan 220 bp. Analisis jujukan nukleotida dari gen protein VNN virus menunjukkan homologi 94.0-96.1% dengan urutan nukleotida gen selubung protein dari VNN dari negara-negara lain di Asia Tenggara.
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