Phylogenetic analysis of the 56-kDa type-specific protein genes of Orientia tsutsugamushi in Central Korea

Abstract

There are several antigenic variants of Orientia tsutsugamushi. The 56-kDa type-specific antigen (TSA) is responsible for the antigenic variation. Nucleotide sequences of the 56-kDa TSA obtained from 44 eschar samples of Korean scrub typhus patients and from 40 representative strains retrieved from the GenBank database were analyzed phylogenetically. Clinical patient data were assessed based on the genotyping results. Of the 44 nucleotide sequences, 32 (72.7%) clustered with the Boryong genotype, which is the major genotype in Korea. Eleven nucleotide sequences (25%) clustered with the Kawasaki genotype, not identified in Korea until 2010. One nucleotide sequence was consistent with the Karp genotype. The clinical course of the patients infected with each genotype showed no differences. Diagnostic performance of the immunofluorescence assay (IFA) using the 56-kDa TSA from Gilliam, Karp and Boryong as test antigens were not different for the Boryong and Kawasaki genotypes. Although Boryong is still the predominant genotype, the results suggest that Kawasaki genotype is quite prevalent in Chungbuk province of Korea.

Keywords: 56-kDa Type-Specific Antigen; Boryong Genotype; Kawasaki Genotype; Orientia tsutsugamushi.

Fig. 1

Positions of the primers designed to obtain the entire open reading frame (ORF) sequences of the 56-kDa type-specific protein. The ORF is represented by the heavy line.

Fig. 2

Phylogenetic tree showing the nucleotide sequences of the 56-kDa type-specific protein genes of Orientia tsutsugamushi isolates from this study compared with nucleotide sequences from selected strains available in GenBank. Samples from the present study are identified by their strain name (e.g., CBNU-1). Reference strains from GenBank database are identified by their accession number. The nucleotide sequences were aligned using CLUSTAL V (13) and phonogram was generated by the neighborjoining method using the tree drawing program embedded within the Lasergene sequence analysis software package (DNASTAR 5.0, Madison, WI, USA) and viewed using NJPlot (14). The scale bars represent the number of substitutions per nucleotide. Branch labels record the stability of the branches over 1000 bootstrap replicates. Only bootstrap values > 600% are shown in the tree. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches.

Fig. 3

IFA titers of the patients infected with the Kawasaki and Boryong genotypes. Paired sera were obtained from each patient and IFA was done based upon the 56-kDa type-specific antigen from the Gilliam, Karp and Boryong genotype. The sensitivity of the IFA in Kawasaki genotype was not different to that in Boryong genotype suggesting cross-reactivity among genotypes.