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SEQUENCE ANALYSIS OF MEGALOCYTIVIRUS GENOMIC DNA ISOLATED FROM VARIOUS GROUPER AND SEA BASS ON PST 1 FRAGMENT AREA

Indah Mastuti*, Ketut Mahardika
Institute for Mariculture Research and Development
Singaraja-Bali, Indonesia.
kmahardika@yahoo.com

Megalocytivirus is one of piscine iridovirus that cause mass mortality in marine finfish culture. Formerly, member of Megalocytivirus were classified in to genus of Ranavirus. Recently, genus Megalocytivirus were established based on genes encoding ATPase and Major capsid protein (MCP). Amplification using primer set (1F-1R) to detect Megalocytivirus have been developed based on Pst I fragment of RSIV genomic DNA. In north Bali, outbreaks of Megalocytivirus have been reported on humpback grouper (Cromileptes altivelis), tiger grouper (Epinephelus fuscoguttatus), and coral trout (Plectropomus leopardus), sea bass (Lates calcarifer). PCR test using PST1 (1F and 1R) primer set confirmed that the fished positively infected by Iridovirus. The PCR product of genome template sequenced to describe relationship of Megalocytivirus from the groupers and seabass.

Viral DNA were isolated from the spleen of diseased fish. PCR for detection Iridovirus was performed using primer set 1-F (5'CTCAAACACTCTGGCTCATC-3') and 1-R (5'-GCACCAACACATCTCCTATC-3') which amplify 570 bp of PST1 fragment of RSIV genome. Purified PCR products (QIAquick PCR purification kit) were directly sequenced for forward and reverse direction. PCR product sequencing by ABI Prism was undertaken by private company. Nucleotide sequences were assembled using BioEditSequence Alignment Editor with 85% minimum percent match. Assembled sequences were used to retrieve related sequences from BLAST. Multiple alignment and phylogenetic tree construction were performed using program MEGA 5.1: Molecular Evolutionary Genetics Analysis. Phylogenetic tree was constructed based on distance by neighbor joining method and the clade robustness was assessed by bootstrap with 1000 replicates.

Phylogenetic tree revealed that viral genome of all samples were belong to Megalocytivirus. Phylogenetic tree also indicated that Iridovirus from humpback grouper (C. altivelis) and Iridovirus from coral trout P. leopardus were closely related. While Iridovirus from Iridovirus from tiger grouper (E. fuscoguttatus) was closely related to L. calcarifer. We tentatively divided the member of Megalocytivirus into three groups: Group I, Group II and Group III. Iridovirus from humpback grouper and coral trout were included in to Group I. While Iridovirus from tiger grouper and sea bass were included in to group II (Figure 3)




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