目的 对衢州市2017年1例曾有孟加拉国旅行史的发热患者血清标本进行基孔肯雅病毒（CHIKV）全基因组序列测定及遗传进化分析。方法 无菌采集患者血液，分离血清后采用实时荧光定量PCR检测CHIKV和登革热病毒核酸，反转录PCR扩增CHIKV全基因组靶向基因片段并测序，采用生物信息学软件（Contig Express、SeqMan、Clustalx V2.0、BioEdit V7.0、GENEDOC和MEGA X）分析全基因序列。结果 患者血清为CHIKV核酸阳性。测序数据拼接处理后获得CHIKV全基因组序列（QZ0823株），包含11 787 bp核苷酸，非结构蛋白主要结构位点未发现变异，结构蛋白缺乏E1中的适应性突变A226V和E2中可能影响CHIKV神经毒力的新取代K252Q突变，不存在增强埃及伊蚊的适应性突变E1：K211E和E2：V264A。全基因组序列进化分析显示，QZ0823与孟加拉国、印度、巴基斯坦流行株彼此紧密聚集，具有密切的进化相关性。结论 衢州市输入性基孔肯雅病毒QZ0823全基因组和结构基因C-E3-E2-6K-E1核苷酸序列均符合CHIKV特征，与孟加拉国流行株同属东中南非洲遗传谱系（ECSA）。
Objective To perform whole-genome sequencing and phylogenetic analyses of Chikungunya virus (CHIKV) from the serum sample of a febrile patient who had traveled to Bangladesh in Quzhou, 2017. Methods Blood samples were collected aseptically from the patient. The serum was separated for detection of nucleic acids of CHIKV and Dengue virus by RT-PCR. The genome-wide target gene fragments of CHIKV were amplified by RT-PCR and then sequenced. The whole-genome sequence was analyzed by bioinformatic programs:Contig Express, SeqMan, Clustalx V2.0, BioEdit V7.0, GENEDO and MEGA X. Results The patient's serum was positive for CHIKV nucleic acids. The sequencing data were assembled to obtain the whole-genome sequence of CHIKV strain QZ0823, containing 11 787 bp nucleotides. No mutation was found in major structural sites of non-structural proteins. For structural proteins, E1 lacked the adaptive mutation A226V; E2 lacked the new substitution mutation K252Q that might affect the neurovirulence of CHIKV; there were no Aedes aegypti adaptive mutations E1:K211E or E2:V264A. The whole genome-based phylogenetic analysis showed that QZ0823 was tightly clustered with the epidemic strains in Bangladesh, India, and Pakistan, suggesting a close evolutionary relationship. Conclusion The nucleotide sequences of whole genome and structural genes C-E3-E2-6K-E1 of the imported virus in Quzhou show obvious features of CHIKV. QZ0823 and the epidemic strains in Bangladesh are derived from the Eastern, Central, and Southern African genetic lineage.
[1] Burt FJ,Rolph MS,Rulli NE,et al. Chikungunya:a re-emerging virus[J]. Lancet,2012,379(9816):662-671. DOI:10.1016/S0140-6736(11)60281-X.
[2] Weaver SC,Lecuit M. Chikungunya virus and the global spread of a mosquito-borne disease[J]. N Engl J Med,2015,372(13):1231-1239. DOI:10.1056/NEJMra1406035.
[3] Ross RW. The Newala epidemic:III. The virus:isolation,pathogenic properties and relationship to the epidemic[J]. J Hyg (Lond),1956,54(2):177-191. DOI:10.1017/S0022172400044442.
[4] Pastorino B,Muyembe-Tamfum JJ,Bessaud M,et al. Epidemic resurgence of chikungunya virus in democratic Republic of the Congo:identification of a new central African strain[J]. J Med Virol,2004,74(2):277-282. DOI:10.1002/jmv.20168.
[5] Hapuarachchi HC,Bandara KBAT,Sumanadasa SDM,et al. Re-emergence of chikungunya virus in South-east Asia:virological evidence from Sri Lanka and Singapore[J]. J Gen Virol,2010,91(4):1067-1076. DOI:10.1099/vir.0.015743-0.
[6] Zheng K,Li JD,Zhang QF,et al. Genetic analysis of chikungunya viruses imported to mainland China in 2008[J]. Virol J,2010,7:8. DOI:10.1186/1743-422X-7-8.
[7] Li XF,Jiang T,Deng YQ,et al. Complete genome sequence of a chikungunya virus isolated in Guangdong,China[J]. J Virol,2012,86(16):8904-8905. DOI:10.1128/JVI.01289-12.
[8] Sun Y,Yan JY,Mao HY,et al. Characterization of the complete genome of chikungunya in Zhejiang,China,using a modified virus discovery method based on cDNA-AFLP[J]. PLoS One,2013,8(12):e83014. DOI:10.1371/journal.pone.0083014.
[9] Lu X,Li XB,Mo ZY,et al. Chikungunya emergency in China:microevolution and genetic analysis for a local outbreak[J]. Virus Genes,2014,48(1):15-22. DOI:10.1007/s11262-013-0991-2.
[10] 赵瑞芳,余新,郑楷增. 浙江省衢州市一起基孔肯雅热疫情流行病学调查[J]. 中国媒介生物学及控制杂志,2018,29(5):505-507. DOI:10.11853/j.issn.1003.8280.2018.05.022.
[11] 杨瑞军,王晓光,黄世腾,等. 浙江省衢州市首例输入性基孔肯雅热病毒基因特征分析[J]. 疾病监测,2018,33(10):875-878. DOI:10.3784/j.issn.1003-9961.2018.10.019.
[12] Schwartz O,Albert ML. Biology and pathogenesis of chikungunya virus[J]. Nat Rev Microbiol,2010,8(7):491-500. DOI:10.1038/nrmicro2368.
[13] Singh RK,Tiwari S,Mishra VK,et al. Molecular epidemiology of chikungunya virus:mutation in E1 gene region[J]. J Virol Methods,2012,185(2):213-220. DOI:10.1016/j.jviromet. 2012.07.001.
[14] Tsetsarkin KA,Vanlandingham DL,McGee CE,et al. A single mutation in chikungunya virus affects vector specificity and epidemic potential[J]. PLoS Pathog,2007,3(12):e201. DOI:10.1371/journal.ppat.0030201.
[15] Schuffenecker I,Iteman I,Michault A,et al. Genome microevolution of chikungunya viruses causing the Indian Ocean outbreak[J]. PLoS Med,2006,3(7):e263. DOI:10.1371/journal.pmed.0030263.
[16] Agarwal A,Sharma AK,Sukumaran D,et al. Two novel epistatic mutations (E1:K211E and E2:V264A) in structural proteins of chikungunya virus enhance fitness in Aedes aegypti[J]. Virology,2016,497:59-68. DOI:10.1016/j.virol.2016.06.025.
[17] Powers AM,Brault AC,Tesh RB,et al. Re-emergence of chikungunya and o'nyong-nyong viruses:evidence for distinct geographical lineages and distant evolutionary relationships[J]. J Gen Virol,2000,81(2):471-479. DOI:10.1099/0022-1317-81-2-471.
[18] Tsetsarkin KA,Chen RB,Weaver SC. Interspecies transmission and chikungunya virus emergence[J]. Curr Opin Virol,2016,16:143-150. DOI:10.1016/j.coviro.2016.02.007.
[19] Yergolkar PN,Tandale BV,Arankalle VA,et al. Chikungunya outbreaks caused by African genotype,India[J]. Emerg Infect Dis,2006,12(10):1580-1583. DOI:10.3201/eid1210.060529.
[20] Rodas JD,Kautz T,Camacho E,et al. Genetic characterization of northwestern Colombian chikungunya virus strains from the 2014-2015 epidemic[J]. Am J Trop Med Hyg,2016,95(3):639-646. DOI:10.4269/ajtmh.16-0091.
[21] Sun SY,Xiang Y,Akahata,W,et al. Structural analyses at pseudo atomic resolution of chikungunya virus and antibodies show mechanisms of neutralization[J]. eLife,2013,2:e00435. DOI:10.7554/eLife.00435.
