1. Introduction Phylogenetic clustering of 10 prevalent virulence genes in Orientia tsutsugamushi isolates from humans. Eun-Ju Kim, Hyuk Chu, Sang-Hee Park, Young-Sill Choi, Soo-Kyoung Shim, Kyu-Jam Hwang, Mi-Yeoun Park* Division of Zoonoses, Center for Immunology & Pathology, National Institute of Health, Korea Center for Disease Control & Prevention, 194, Tongil-Lo, Eunpyeong-Gu, Seoul, 122-701, Korea O. tsutsugamushi is an antigenically diverse bacteria that is the etiological agent of scrub typhus. Four prototype strains, Gilliam, Karp, Kato and Boryong have been classified by the 56-kDa protein sequences. In this study, we analyzed the 56 kDa protein sequences of isolates from different area. Moreover, we clustered for comparison of pathogenic relationship between 56 kDa protein sequence and many virulence factors among the O. tsutsugamushi. Materials and Methods Bacteria O. tsutsugamushi Boryong, Kuroki, Pajoo, Yongworl, and Yonchon and 3 isolates, #18, #24 and #32. (3 isolates were isolated from patient blood in different locations; Daejeon, Wonju and Mujoo). phylogenetic analysis Sequences of close relatives were aligned with the newly determined sequences using the ClustalX software program, and an unrooted neighbor-joining tree was constructed using TreeExplorer (Tamura; http://evolgen.biol.metro-.ac.jp/TE/TE_man.html). Results Table 1. PCR primers and annealing temperatures Fig. 1. Agarose gel electrophoresis of PCR products of 10 genes of O. tsutsugamushi Boryong. Lanes:M, 100bp marker; 1, virB11; 2, sodB; 3, secF; 4, purC, 5, tpiA; 6, mviN; 7, mdh; 8, corC, 9, aas;10, hscA. Fig. 2. Phylogenetic tree derived from various gene sequence data of O. tsutsugamushi strains. Bootstrap values, based on 1000 replications, at the nodes of the tree display the significance of these nodes. Horizontal bar represents sequence divergence. 56-kDa protein sequence of O. tsutsugamushi strains were used based on sequence from GenBank accession numbers M33267 (Gillam), M33004 (Karp), M63382 (Kato), L04956 (Boryong), AF430142 (Pajoo), AF430141 (Yongworl), and U19903 (Yonchon). VirB11 gene sequence of R. felis and R. typhi were used from GenBank accession numbers CP000053 and AE017197, respectively. All figures show the phylogenetic tree. (a), O. tsutsugamushi strains with 56-kDa protein sequence; (B), virB11; (c), sodB; (d), secF; (e), purC; (f), tpiA; (g), mviN; (h), mdh; (i), corC; (j), aas; (k), hscA Conclusion References 1. O. tsutsugamushi Boryong and Kuroki strains and isolates #24 and #32 were divided into one group in virB11, sodB, secF, purC, tpiA, mviN, mdh, corC, aas and hscA. 2. The isolate #18 was Boryong strain by 56 kDa protein sequence, but it was clustered in other group by virulence factors. 3. There are slightly different sequences between Boryong (pathogenic) and Kuroki (non-pathogenic) strain in sodB, purC, tpiA, corC, aas genes. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389-3402. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876-4882. Shirai, A., Robinson, D. M., Brown, G. W., Gan, E. & Huxsoll, D. L. (1979). Antigenic analysis by direct immunofluorescence of 114 isolates of Rickettsia tsutsugamushi recovered from febrile patients in rural Malaysia. Jpn J Med Sci Biol 32, 337-344.