반복염기서열에 의한 넙치 이리도 바이러스의 다양성분석

Abstract

We examined the various sizes of amplicon in PCR with a single set of primers derived from an open reading frame, ORF-2, in the megalocytivirus obtained from olive flounder (Paralichthys olivaceus) cultured in Korea. From sequence analysis of the multiple amplicons, we demonstrated the presence of distinct genomic polymorphisms in the ORF-2 gene with differing numbers of repetitive element of 60 amino acids or 69 amino acids. 2-step PCR with the cloned plasmid templates of three different sizes of produced amplicon and mixed templates of these three cloned plasmids did not produce any PCR product with a length different from the original templates. Moreover, we did not observe the disappearance of any different lengths of amplicon in PCR with isolated from tissue homogenate incubated more than 10 minutes. These results implied that the amplicons are not derived from artifacts of PCR and viral DNAs used as template are present in viral particles rather than as a naked nucleic acid. FLIV-1 after challenge experiments in two different host species, olive flounder and rock bream (Oplegnathus fasciatus), and in vitro inoculation experiment in primary rock bream embryo cell displayed the same feature of polymorphism in the ORF-2 gene as those in the original sample. In monitoring experiments, the same pattern of polymorphism in ORF-2 region were found in comparison of each other 14 FLIV isolates in marine fishes from 2003 to 2013. Additionally, from 2006 to 2009, 16 FLIV isolates obtained from shellfish known as a suspected reservior of viruses released from infected fish also showed the same polymorphism in ORF-2 region except 2 and 3 isolates showing a single and double bands respectively in gel electrophoresis after PCR. Based on the MCP gene sequences and characterized pattern of repeating sequence in ORF-2 region, FLIVs (14 and 16 isolates obtained from fish and shellfish of this study and reported from other laboratories repectively) were further grouped to 6 and 3 different sub-subgroups respectively (FLIV sub-subgroup 1-6, and FLIV-1, 2 and 3 type). Out of 30 isolates, 25 isolates were grouped in FLIV-1 type and assumed as major repeating sequence type in FLIVs in Korea. In comparison of these sub-subgroups distinguished by nucleotide sequence of different genomic region in FLIV, we founde the relationship of groups between FLIV-1 type and FLIV sub-subgroup 1, 3, 5, 6 and FLIV-2 type and FLIV sub-subgroup 4, 5 and FLIV-3 type and FLIV sub-subgroup 2. Although TRBIV reported in China and CH-1 obtained from imported fish from china can be distinguished as a seperated sub-subgroup 2, in FLIV-type grouping manner, TRBIV was a member of FLIV-2 type and different from CH-1 of FLIV-3 type. Consequently, fish can be infected by a single or multiple types of particle in FLIV, and the genomic polymorphism might be formed by replication of each different polymorphic type of viral particle that has the same potential to produce the viral genomic DNA for progeny virus.