镇江地区诺如病毒分子流行病学分析及其与肠道菌群关系的初步研究

发布时间：2018-04-22 14:04

本文选题：诺如病毒 + NoV　；参考：《江苏大学》2017年硕士论文

【摘要】：目的:诺如病毒(Noroviruses,NoVs)是镇江地区食源性疾病及突发公共卫生事件的常见病原体,极易引起人群中的暴发流行。为明确NoV在镇江地区的分子流行病学特征及其与肠道菌群的关系,本研究从分子生物学、生物信息学、基因组学等方面出发,系统分析本地区NoV的分子流行病学特征、进化、重组及对肠道菌群关系的影响。方法:1.为了分析NoV不同基因型在镇江的流行,本研究采用实时荧光定量PCR(q RT-PCR)和逆转录PCR(RT-PCR)相结合的方法。收集2015.1-2016.12镇江市食源性疾病常规腹泻患者的粪便样本及突发公共卫生事件的病例样本,使用NoV GI/GII双重PCR试剂盒进行q RT-PCR检测,再基于NoV基因组衣壳(Capsid)蛋白区设计引物实施RT-PCR和测序,进行遗传分型。2.扩增RNA依赖的RNA聚合酶(Rd Rp)区并测序,结合Capsid区结果,检索Gen Bank进行相似性搜索。应用Mega 6.06软件邻近归并法(Neighbor-joining,N-J)基于核苷酸构建进化树,进行系统发育分析。同时运用RDP 4.0软件针对Rd Rp区和Capsid区序列分析镇江地区NoV的系统进化和重组。3.提取样本总DNA,设计针对16S v4-v5区的特定引物进行基因扩增,得到扩增片段后,加接头,采用Hiseq 2500测序平台,通过拼接得到较长序列,基于16S r RNA基因进行种群、丰度及群落结构等多样性分析,阐明NoV感染与肠道菌群的关系。结果:1.感染状况:2015年-2016年,共采集粪便及肛拭样本1605份,NoV阳性率5.30%(85/1605),其中GI组检出率为0.87%(14/1605),GII组检出率为4.55%(73/1605),有2份样本同时感染GI/GII组,GI组和GII组检出率差异有统计学意义(P0.05)。男女NoV的检出率差异无统计学意义(P0.05)。NoV的流行高峰主要集中在11月至次年4月,以丹徒区的检出率最高。本地区0岁~10岁的学龄儿童以及60岁以上的老年人主要感染NoV GII组,其他年龄组GI组和GII组均可感染。GII组在10岁~20岁的中青年中阳性率最高,达17.80%,相对于其它年龄组,差异有统计学意义(P0.05)。GI组的感染差异无统计学意义。2.NoV基因型:2015年NoV的基因型以GII.17所占比例最高为63.89%,与其它基因型的差异具有统计学意义,其它基因型为:GI.3、GII.3、GII.4、GII.13。2016年以GII.4 Sydney_2012所占比例最高为35%,其它基因型为:GI.3、GI.4、GI.5、GI.6、GI.7、GI.8、GII.2、GII.3、GII.14、GII.17。3.进化与重组:系统进化分析结果进一步证实镇江地区的31株GII.17型,有3株和1978年-2002年的GII.17型在同一进化分支上,其他28株均为GII.17型变异株,与以往的GII.17型具有同源性,但存在一定的变异。还发现2个重组事件,GII.P12_GII.3型的ZJ086/2016/CHN和ZJ096/2016/CHN株等是由加拿大分离的U02030/Toronto/CA/GII.3株和英国分离的X86557/Lordsdale/UK/GII.4株重组而成;GII.P16_GII.2型的ZJ152/2016/CHN株由GII.P2_GII.2型ZJ124/2016/CHN株与GII.P16_GII.13型的ZJ105/2015/CHN株重组而成,重组位点为NoV重组的热点区域:ORF1-ORF2交界区域。4.与肠道菌群关系:NoV感染人群可操作运算分类单位(Operational Taxonomic Units,OTU)大于正常人群。在门分类水平上,NoV感染者的变形菌门数比正常人群显著减少,厚壁菌门、拟杆菌门数显著增多;在属分类水平上,埃希菌属、假单胞菌属显著减少,链球菌属增多,拟杆菌属显著增多,另有未定位生物显著增多。NoV感染者与正常人群有统计学意义的肠道菌属达43种之多,其中单胞菌属、小细菌属、红球菌属等在NoV感染人群显著降低,拟杆菌属、韦荣球菌属、纤毛菌属、厌氧菌等显著升高,并发现NoV感染者常导致多种菌属的显著增多。结论:1.NoV是镇江地区食源性疾病及突发公共卫生事件的常见病原体。本地区NoV的流行具有时间、地区、人群特征,NoV的流行高峰主要集中在11月至4月,以丹徒区的检出率最高,感染者以NoV GII组为主。GII组在10岁~20岁的中青年中阳性率最高,GI组的感染无明显年龄差异。2.镇江地区存在多种NoV基因型,并且处在动态变化中,主要流行GII.17和GII.4基因型,其中GII.17基因型为新型变异株,GII.4基因型为最新的GII.4Sydney_2012株。3.本地区分到的NoV发现2个重组事件,分别是GII.P12_GII.3型由GII.3与GII.4型重组而成;GII.P16_GII.2型由GII.P2_GII.2与GII.P16_GII.13重组而成。4.基于16S r RNA基因的高通量序列测定发现,NoV感染会伴随多种肠道菌群的改变。
[Abstract]:Objective: Noroviruses (NoVs) is a common pathogen of food borne diseases and public health emergencies in Zhenjiang. It is very easy to cause the outbreak in the population. To clarify the molecular epidemiological characteristics of NoV in Zhenjiang and the relationship with the intestinal flora, the research from molecular biology, bioinformatics, genomics and so on. In order to analyze the molecular epidemiological characteristics, evolution, recombination and influence on the relationship of intestinal flora in the region of NoV, 1. in order to analyze the prevalence of different NoV genotypes in Zhenjiang, this study uses real-time fluorescence quantitative PCR (Q RT-PCR) and reverse transcriptase PCR (RT-PCR) to collect food borne diseases in 2015.1-2016.12 of Zhenjiang. Routine diarrhea patients' fecal samples and cases of public health emergencies were detected by NoV GI/GII double PCR kit for Q RT-PCR, and then RT-PCR and sequencing were designed based on NoV genome capsid (Capsid) protein region, and RNA polymerase (Rd) region was sequenced by genetic typing.2. amplification RNA dependence and sequenced. Fruit, retrieving Gen Bank for similarity search. Using the Mega 6.06 software adjacent merging method (Neighbor-joining, N-J) to construct the phylogenetic tree based on nucleotides to carry out phylogenetic analysis. At the same time, the RDP 4 software is used to analyze the system intake of NoV in Zhenjiang region and the total DNA of the.3. extraction samples for the Rd Rp region and Capsid region sequence. The specific primers of the region were amplified, and after the amplified fragment was amplified, the Hiseq 2500 sequencing platform was used to get a long sequence by splicing. The population, abundance and community structure based on the 16S R RNA gene were analyzed to clarify the relationship between the NoV infection and the intestinal flora. Results: 1. infection status: 2015 -2016, collecting fecal and feces. 1605 samples of anal swab, NoV positive rate 5.30% (85/1605), the detection rate of group GI was 0.87% (14/1605), the detection rate of GII group was 4.55% (73/1605), 2 samples were simultaneously infected with GI/GII group, GI and GII group detection rates were statistically significant (P0.05). The prevalence peak of male and female NoV was not statistically significant (P0.05) was mainly concentrated in November. By April the next year, the detection rate in the Dantu area was the highest. The school age children aged 0 and over 60 years old in the region were mainly infected with the NoV GII group. The positive rate of the GI group and the GII group in the other age groups was the highest in the group.GII group at the age of 10 years old, reaching 17.80%. Compared with its age group, the difference was statistically significant (P0.05).GI group. The difference in infection was not statistically significant.2.NoV genotypes: the highest proportion of the genotype of NoV in 2015 was 63.89%, and the difference between the genotype and other genotypes was statistically significant. The other genotypes were GI.3, GII.3, GII.4, and the highest proportion of GII.4 Sydney_2012 in GII.13.2016, and the other genotypes were GI.3, GI.4, GI.5, GI.5. II.2, GII.3, GII.14, GII.17.3. evolution and recombination: phylogenetic analysis further confirmed that 31 strains of GII.17 in Zhenjiang, 3 and GII.17 in the year -2002 were in the same evolutionary branch, and the other 28 were GII.17 variant, which were identical with the previous GII.17 type, but there were some variations. 2 recombination events, G, were found, G. The ZJ086/2016/CHN and ZJ096/2016/CHN strains of type II.P12_GII.3 were reorganized by the U02030/Toronto/CA/GII.3 strain isolated from Canada and the X86557/Lordsdale/UK/GII.4 strain isolated from the UK. The GII.P16_GII.2 type ZJ152/2016/CHN strain was reorganized from the ZJ124/2016/CHN strain of GII.P2_GII.2 type to the ZJ105/2015/CHN strain of the GII.P16_GII.13 type. Point is the hot area of NoV recombination: the relationship between.4. and intestinal flora in the ORF1-ORF2 junction area: the operation classification unit of NoV infected people (Operational Taxonomic Units, OTU) is larger than the normal population. At the portal classification level, the number of strains of the NoV infected persons is significantly less than that of the normal population, and the number of the thick wall bacteria and the bacteriobacteria increased significantly; in the genus At the level of classification, the genus and Pseudomonas genera were significantly reduced, Streptococcus increased, and Pseudomonas increased significantly, and there was a significant increase in the number of.NoV infected individuals with more than 43 species in the normal population. Among them, monomonas, small bacteria, and rhodotonas sp. were significantly reduced in NoV infected people. Genus, wehonia, cilium, anaerobes and other significant increases, and found that NoV infection often leads to a significant increase in the number of bacteria. Conclusion: 1.NoV is a common pathogen of food borne diseases and public health emergencies in Zhenjiang. The epidemic of NoV in the region has time, ground area, and population characteristics. The peak of NoV is mainly concentrated in November. In April, the detection rate in Dantu area was the highest. The positive rate of NoV GII group in group.GII was the highest in group.GII and ~20 years old, and there was no obvious age difference in GI group. There was a variety of NoV genotypes in.2. Zhenjiang region, and in dynamic changes, the main prevalent GII.17 and GII.4 genotypes, and GII.17 genotypes as new variant, GII. The 4 genotype was the latest GII.4Sydney_2012 strain.3. locally identified by NoV, which was found to be a recombination of the GII.P12_GII.3 type from GII.3 to GII.4, and the GII.P16_GII.2 type from GII.P2_GII.2 to GII.P16_GII.13 and.4. based on 16S R, found that the infection would accompany a variety of intestinal flora. Change.

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.5;R181.3

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