2018年福建省首例疑似狂犬病病例的病毒分离鉴定及G基因序列分析

Isolation,identification and G gene sequence analysis of rabies from the first suspected patient in Fujian Province,China in 2018

目的对2018年福建省首例疑似狂犬病病例进行病毒分离鉴定及G基因序列分析。方法收集2018年福建晋江送检的疑似狂犬病患者的血液、唾液及脑脊液样本,进行实验室诊断,昆明乳鼠颅内接种唾液及脑脊液样本,酶联免疫法检测狂犬病病毒(rabies virus,RABV)抗原;免疫荧光灶抑制试验(rapid fluorescent focus inhibition test,RFFIT)检测血液样本中抗RABV中和抗体;提取接种唾液的乳鼠脑悬液RNA,RT-PCR扩增G基因序列,将其与国内外疫苗株、福建省及相邻省分离的街毒株序列构建NJ(Neighbor-Joining)分子系统进化树,并进行同源性分析。结果血液样本中未检出抗RABV中和抗体;经乳鼠颅内接种及RT-PCR法,仅唾液样本检测出RABV,确诊患者为狂犬病病例,新分离的街毒株命名为18FJ01。该毒株G基因开放阅读框全长1575 bp,编码524个氨基酸,属基因Ⅰ型RABV,与山东省CTN-33疫苗株的亲缘关系最近,与国内PV、aG、HEP-Flury及ERA疫苗株亲缘关系相对较远;与福建邻近省份街毒株核苷酸及氨基酸序列的同源性分别为84.3%~97.8%和92.9%~98.8%,与国内外疫苗株核苷酸及氨基酸序列的同源性分别为80.1%~93.2%和88.9%~96.9%。结论成功对该病例进行了病毒分离及鉴定,该毒株与CTN-33疫苗株同源性较高。

Objective To isolate and identify rabies virus from the first suspected patient in Jinjiang Ciy,Fujian Province,China in 2018 and analyze the sequence of glycoprotein(G)gene.Methods The blood,saliva and cerebrospinal fluid samples of suspected patients with rabies submitted from Fujian Province were subjected to laboratory test.Kunming suckling mice were inoculated i.c.with the saliva and cerebrospinal fluid samples,and determined for rabies virus(RABV)antigen in brain tissue by ELISA,and for neutralizing antibody against RABV in blood by rapid fluorescent focus inhibition test(RFFIT).RNAs were extracted from the brain suspension of suckling mice inoculated with saliva,with which the G gene sequence was amplified by RT-PCR and compared with those of vaccine strains at home and abroad as well as street virus strains isolated from Fujian and neighboring provinces to construct a phylogenetic tree by Neighbor-Joining(NJ)method and analyze the homology.Results No neutralizing antibody against RABV was detected in blood samples.RABV was only detected in saliva samples by intracerebral inoculation test in suckling mice and RT-PCR,confirming the patient as that with rabies.The newly isolated street strain was named as 18 FJ01,of which the open reading frame of G gene,at a full-length of 1575 bp,encoded 524 amino acids.The strain belonged to rabies genotypeⅠ,which showed the closest relationship to vaccine strain CTN-33 isolated in Shandong Province and distant relationship to domestic vaccine strains PV,aG,HEP-Flury and ERA.The homologies of nucleotide and amino acid sequences of the isolated strain were 84.3%~97.8%and 92.9%~98.8%to those of street strains isolated in the neighboring provinces,while were 80.1%~93.2%and 88.9%~96.9%to the vaccine strains at home and abroad,respectively.Conclusion Virus strain was successfully isolated from the patient and identified,which showed high homology to vaccine strain CTN-33.