中国急性弛缓性麻痹病例脊髓灰质炎疫苗病毒分离株中优势株的基因特征及形成原因浅析

Molecular Characterizations and Reasons for the Formation of the Preponderant Vaccine Related Polioviruses Strains Isolated From AFP Cases in China

目的研究从中国1996～2002年急性弛缓性麻痹（AFP）病例的粪便标本中分离到的脊髓灰质炎（脊灰）疫苗病毒中优势株的基因特征，以及分析优势株形成的原因，从而为中国维持无脊灰状态、疫苗免疫策略的转变提供科学依据。方法对1996-2002年省级疾病预防控制中心（CDC）脊灰实验室送检的所有脊灰病毒分离株，通过统计学方法确定优势株，然后使用聚合酶链反应．限制性酶切片段长度多态性分析（PCR—RFLP）和酶联免疫吸附试验（ELISA）两种方法对优势株进行型内鉴定。型内鉴定显示与疫苗病毒有差异者，进行VP1区基因核苷酸序列的测定和分析。结果中国1996～2002年从AFP病例中分离的脊灰疫苗病毒Ⅱ型比Ⅰ型和Ⅲ型多，差异有显著的统计学意义，是优势病毒株。在Ⅱ型脊灰病毒中，PCR．RFLP型内鉴定方法检测出Ⅱ型疫苗变异脊灰病毒70株，ELISA型内鉴定方法检测出Ⅱ型双反应（DRV）株13株，全部排除脊灰野病毒。在总计82株Ⅱ型变异脊灰病毒中（1株病毒既是疫苗变异株、也是DRV株），nt2909位点是个突变热点（69／83，83．1％），其中14株变化形式为T2909A，54株变化形式为T2909C，1株变化形式为T2909G；4株Ⅱ型疫苗衍生脊灰病毒（VDPV）被鉴定出来，其在VP1区的抗原性与Sabin株相比并无大的差别。结论中国1996～2002年从AFP病例中分离的脊灰病毒中，Ⅱ型脊灰病毒一直是优势株，但Ⅱ型病毒在VP1区抗原性以及核苷酸序列上与Ⅱ型疫苗病毒相比，并无很大改变。因此，Ⅱ型病毒成为优势株的原因应该在于Ⅱ型病毒本身的一些特性，例如在Ⅱ型疫苗病毒基因组中决定减毒的位点比Ⅰ型疫苗病毒少，以及Ⅱ型脊灰疫苗病毒在人体肠道内复制的时间比Ⅰ型疫苗病毒长等。这是中国Ⅰ型脊灰疫苗病毒比Ⅱ型脊灰疫苗病毒少的两个重要原因。但Ⅱ型脊灰疫苗病毒显著多于Ⅲ型脊灰疫苗病毒的原因尚需进一步深入研究。

Objective To investigate the gene characterizations of the preponderant vaccine related Polioviruses strains isolated from acute flaccid paralysis （AFP） cases in China during 1996 to 2002 ,and analyze the reasons for the formation, provide the theoretical basis for maintaining the ＂polio-free＂status, and the switching use of polio vaccine. Methods Determining the preponderant strains among vaccine related poliovirus strains sent by provincial polio laboratories during 1996 to 2002 by statistical methods,then PCR-RFLP and ELISA methods were used for intratypic differentiating （ITD） the polioviruses and VP1 capsid genes were determined and analyzed with all the aberrant strains. Results During 1996 to 2002,the number of type 2 polioviruses is larger than type 1 and type 3 isolated in AFP cases,and there were significant differences between the number of type 2 and type 1 or type 3, so type 2 poliovirus is preponderant. Among type 2 polioviruses,70 strains were identified as typical strains by PCR-RFLP ITD method, 13 strains were identified as Non-Sabin like （NSL） viruses or Double-reactive viruses （DRV） by ELISA and no wild polioviruses were detected. In total 82 types, 2 aberrant strains （1 strain is both atypical strain and DRV）,nt 2909 is a Mutational hotspot（69/83, 83.1% ）,14 of T2909A, 54 of T2909C,and 1 of T2909G. 4 Vaccine-derived Polioviruses （VDPVs） were identified, and their antigenic properties in VP1 region are similar compared with Sabin 2 strain. Conclusion During 1996 to 2002, type II poliovirus is preponderant at all times among the polioviruses isolated from AFP cases,but the antigenic determinants and the nucleotide acids in VP1 region of type Ⅱ polioviruses are of no changes compared with type 2 vaccine strain. The reasons that type Ⅱ poliovirus is the preponderant one lies in the inherent characteristics, such as less neurovirulence determinant sites of type 2 oral polio vaccine （OPV） genome than type 10PV genomes,and the survival time of type 2 strains in human guts is the longest,etc. These are the most important reasons for the number of type 1 polioviruses is less than type 2. But more studies should be investigated on the number of type 2 polioviruses is more than type 3.