2009～2010年北京儿童腹泻中轮状病毒新VP4基因亚型的研究

Investigation of a Novel VP4 Subgenotype of Rotavirus in Children with Diarrhea in Beijing during 2009～2010

P[8]b基因亚型是国内外新近发现的A组人轮状病毒(HRV)VP4基因的一种新亚型,本研究旨在建立有效鉴别HRV P[8]a和P[8]b基因亚型及P[4]和P[6]基因型的VP4基因打点杂交分型方法,并运用此方法对2009～2010年首都儿科研究所附属儿童医院门诊及住院腹泻患儿中P[8]b基因亚型HRV的流行情况及其G/P基因组合情况进行研究。通过对GenBank序列数据库可检索到的国内外HRV各种P基因型及亚型的VP4基因序列应用相关软件进行基因分析,在不同基因型别间核苷酸变异密集而相同P基因型内核苷酸高度保守的的位置设计各型别探针,并分别以本实验室上传GenBank的北京HRV地方株P[4]和P[6]基因型及P[8]a和P[8]b亚型的VP4基因作为相应型别探针的合成引物的设计模板及探针经PCR合成的合成模板,合成地高辛素标记的DNA探针。经测序验证所建立的VP4基因杂交分型方法结果可靠。对门诊88例(55%,88/160)及住院79例(70.5%,79/112)HRV腹泻患儿的P分型结果显示P[8]a亚型仍为主要型别,前者为96.6%(85/88),而后者为62.0%(49/79);P[8]b亚型在住院HRV感染腹泻患儿中占较高比例(27.9%,22/79),虽然其在门诊HRV感染患儿中也存在,但仅占2.3%(2/88);另外单纯P[4]基因型HRV感染仅在住院腹泻患儿中检测到1例(1.3%,1/79),而P[6]基因型在门诊及住院HRV感染腹泻患儿中均未检测到;本组标本中HRV P[8]b亚型主要与G9基因型组合。本研究表明G9P[8]b型HRV在北京腹泻儿童中有流行。

P[8]b is a newly discovered sub-genotype for VP4 gene of group A human rotaviruses （HRV） worldwide. This study was to develop an effective method to identify P[8]a, P[8]b, P[4] and P[6] （sub） genotypes of VP4 genes of HRV and to investigate the prevalence of P[8]b sub-genotype and its G/P combinations of HRV in outpatient and inpatient children with diarrhea in Children＇s Hospital affiliated to Capital Institute of Pediatrics from 2009 to 2010. By analyzing the collected nucleotide sequences of VP4 gene for all known P genotypes of HRV including P[8]b subtype from GenBank and using softwares of DNAS- tar and MegAlign to align and analyze multiple sequences, probes for P[8]a, P[8]b, P[4] and P[6] （sub） genotypes in the corresponding regions which are highly divergent among genes from different genotypes and conserved within genes of VP4s in same genotypes were designed. Then four sets of primers for PCR amplified DIG labeled probes were designed and corresponding DIG-labeled specific P genotype probes were synthesized with PCR by using VP8~ genes of Beijing field HRV strains representing P-genotypes P[8]a, P[8]b, P[-4] and P[-6], respectively, as templates. Dot-blot hybridization was developed based on cDNA of VP4 genes. The dot-blot hybridization assay for P genotyping was reliable which was confirmed by se- quencing of RT-PCR products of VP4 genes amplified from corresponding clinical samples. P genotyping for VP4 genes from 88 HRV positive specimens from the Outpatient Department （550/00, 88/160） and 79 HRV positive specimens from the hospitalized （70.5%, 79/112） children with diarrhea indicated that P[82 a subtype was still the most prevalent sub-genotype, which was 96.6 % （85/88） and 62.0% （49/79） respectively. The positive rate for P[8]b subtypes in hospitalized children with HRV diarrhea was higher （27.9%, 22/79） than that of in outpatient （2.3%, 2 / 88） HRV infected children. HRV with P[4] genotype was only found in one of the hospitalized children （1.3%, 1 / 79）, and HRV with P[6] genotype was not detected from specimens either from outpatient or inpatient. G9P[8]b was the predominant combination among the P[8]b subtype of HRV positive specimens in this study. The results in this study indicated that G9P[8]b HRV circulated in children with diarrhea in Beijing.