2012年重庆单中心腹泻患儿感染诺如病毒及其基因型别和重组的研究

目的 了解重庆地区门诊腹泻患儿感染诺如病毒（NV）流行株的变迁及基因重组情况。方法 采集2012年1~12月就诊于重庆医科大学附属儿童医院的疑似病毒性腹泻患儿的粪便标本。采用JVl2/JVl3、GⅠ SKF /GⅠ SKR(COG2F/GⅡ SKR)两对引物，对NV基因组的部分RNA依赖的RNA聚合酶区和衣壳蛋白的N/S区分别进行RT-PCR核酸检测，所有阳性产物进行回收纯化、测序，用DNAstar和MEGA 5.05软件对序列分别进行比对和构建进化树。并将疑似重组株的标本再用JVl2/GⅠ SKR(JVl2/GⅡ SKR)进行PCR扩增，用SimPlot软件对序列进行重组鉴定。结果 384例腹泻患儿粪便标本进入本文分析，男248例，女136例，年龄（13.1±14.4）个月。①NV阳性84/384例（21.9%），<60月龄组NV阳性构成比为96.4%（81/84）。NV在6、8和9月份检出率较高，3和4月份检出率最低。②84例NV阳性标本capsid的N/S区基因片段测序显示，GⅡ.4 2006b型37例，GⅡ.4 New Orleans 2009型1例，GⅡ.4 Sydney 2012型27例，GⅡ.3型12例，GⅡ.6型3例， GⅡ.13型3例，GⅡ.5型1 例；1~7月份 GⅡ.4 2006b型为主要流行株，8~12月GⅡ.4 Sydney 2012型为主要流行株；③共检出44株重组株，分别是GⅡ.e/GⅡ.4 Sydney 2012型27株、GⅡ.7/GⅡ.6型1株、GⅡ.22/GⅡ.5型1株、GⅡ.12/GⅡ.3型12株，GⅡ.16/GⅡ.13型3株。结论 重庆地区NV重组现象非常明显，2012年8~12月NV优势株逐渐由GⅡ.4 2006b型转为GⅡ.e/GⅡ.4 Sydney 2012型的重组株，并检出GⅡ.22/GⅡ.5型和GⅡ.16/GⅡ.13型2种新型重组株。

The genotype and recombination of norovirus in children with diarrhea caused by viral infection in Chongqing, 2012

Objective To investigate the genetic variation and recombination of norovirus(NV) in children with acute diarrhea in Chongqing. Methods Diarrhea specimens in children with suspected viral diarrhea from January 2012 to December 2012 at the clinical laboratory center of the Affiliated Children's Hospital of Chongqing Medical University were collected. Two regions of NV genome(partial RdRp and capsid N/S region) were respectively amplified by primers of JVl2/JVl3 and GⅠ SKF /GⅠ SKR(COG2F/GⅡ SKR) using RT-PCR．All positive samples were purified and sequenced.The obtained sequences were aligned by DNAstar followed by phylogenetic analysis using MEGA 5.05 software. Some potential recombinant genomes of NV were amplified by PCR using primers JVl2/GⅠ SKR(JVl2/GⅡ SKR) when phylogenetic analysis indicated incongruent clustering for partial sequences of the RdRp and capsid genes in the same samples and recombination analysis was conducted using the SimPlot program. Results A total of 384 pediatric outpatients (248 males and 136 females) were enrolled in the present study, aged from <1 month to 121 months with a mean of (13.1 ± 14.4) months. ①84 of 384 stool specimens were detected as NV-positive(21.9%).The constituent ratio of NV was 96.4%（81/84）in children younger than 60 months.The isolation rates were higher in June, August and September, and the lower ones were in March and April. ②84 positive specimens were classified based on the capsid sequence:37 strains were GⅡ.4 2006b，1 was GⅡ.4 New Orleans 2009，27 were GⅡ.4 Sydney 2012，12 were GⅡ.3，3 were GⅡ.6，3 were GⅡ.13， and 1 was GⅡ.5. GⅡ.4 2006b was the predominant genotype from January to June,while GⅡ.4 Sydney 2012 was the predominant genotype from August to December.③Phylogenetic and Simplot analyses showed that 44 recombinant strains(RdRp/capsid) were detected: 27 were GⅡ.e/GⅡ.4 Sydney 2012 recombinants,1 wasGⅡ.7/GⅡ.6 recombinant, 1 was GⅡ.22/GⅡ.5 recombinant, 12 were GⅡ.12/GⅡ.3 recombinants, 3 were GⅡ.16/GⅡ.13 recombinants. Conclusion The NV recombinant in Chongqing region was at common frequency,after August 2012, the NV predominant strains of GⅡ.4 2006 gradually shifted to the recombination strains of GⅡ.e/GⅡ.4 Sydney 2012,and this is the first report of the detection of GⅡ.22/GⅡ.5 and GⅡ.16/GⅡ.13 novel recombinant NV.