五例恙虫病东方体感染基因型及其基因变异

目的旨在了解浙闽地区人群恙虫病东方体感染的基因型，并分析其基因变异。 方法从浙闽地区5例散发恙虫病发热期患者血中分离病原体并进行细胞培养；提取感染细胞DNA，巢式PCR扩增完整恙虫病东方体56-kD型特异性抗原（TSA）基因和热休克蛋白基因（groESL）并测序；采用MEGA 7.0软件，进行序列比对和系统发育分析。 结果病原学确认5例恙虫病患者，并分离到恙虫病东方体菌株。序列比对表明，5菌株中有2株的56-kD TSA基因和groESL基因100%一致，另2菌株的此二个基因序列一致性亦为100%，分别将两组菌暂时命名为浙江-1型和浙江-2型。56-kDa TSA基因比对和系统发育分析表明，浙江-1型和浙江-2型分别与台湾-A基因型和Gilliam-C基因型亲缘较近（98.45%和98.50%），但有明显变异；另1株菌Wuj/2014与台湾-A基因型高度相似（99.94%）。56-kDa TSA基因各基因型支系的时间树分析表明，台湾-A基因型、浙江-1型和浙江-2型3支系与祖先的分歧时间相对其他原型株晚，尤其是浙江-2型，说明这些基因型或亚型在恙虫病东方体的进化过程中出现较晚。 结论本研究病例所感染恙虫病东方体基因型不同，可能为未被认识的新的基因亚型，迫切需要进行全基因组测序确认，并探讨其基因变异与人恙虫病严重程度间的关系。

Gene genotypes and variation of 5 cases with Orientia tsutsugamushi infection

ObjectiveTo investigate the genotype of Orientia tsutsugamushi (O. tsutsugamushi) infection in Zhejiang and Fujian provinces, southeastern China and to analyze the gene variation. MethodsStrains of O. tsutsugamushi were isolated from the blood of 5 scrub typhus patients of febrile phase and then cell culture were carried out. DNA from the infected cells were extracted and full-length 56-kD type-specific antigen (TSA) gene and full-length heat shock protein gene (groESL) were amplified. The gene products were sequenced. Multiple sequence alignment and phylogenetic analyses were conducted by Mega 7.0 software. ResultsFive febrile patients with O. tsutsugamushi infection were confirmed etiologically and five O. tsutsugamushi strains were isolated. The sequence alignment of 56-kD TSA gene and groESL gene showed that 2 among the 5 strains had 100% identity; while other two strains also had 100% identity of these two genes. These two groups were temporarily named Zhejiang-1 and Zhejiang-2. The 56-kDa TSA gene alignment and phylogenetic analysis showed that Zhejiang-1 was closely related to Taiwan-A genotype (98.45%) and Zhejiang-2 was closely related to Gilliam-C genotype (98.50%), but there were obvious variations. The other 1 strain of Wuj/2014 was significantly similar to Taiwan-A genotype (99.94%). Based on 56-kDa TSA gene, the timetree based on the RelTime method showed that Taiwan-A genotype, Zhejiang-1 and especially Zhejiang-2 genotype were later than other prototypes on the divergence time from their ancestors of each genotypic clade. These genotypes or subtypes emerged later in evolutionary process of O. tsutsugamushi. ConclusionsThere may be unrecognized novel O. tsutsugamushi variants among the patients in this study. The relationship between genetic variation and severity of scrub typhus also should be discussed.