【作者】 张敏；

【导师】 夏雪山； 冯悦；

【作者基本信息】 昆明理工大学 ， 微生物学， 2023， 硕士

【摘要】 诺如病毒(Norovirus,简称Novs)成为引起全球急性病毒性腹泻(AGE)爆发性疫情的主要病原体。每年因为感染该病毒而造成全球经济负担约60亿美元、超过219万人住院和2.2万人死亡。诺如病毒是杯状病毒科诺如病毒属的一种无包膜单股正链RNA病毒,全长7500bp左右。2013年Novs分类工作组(NCWG)正式确立该病毒的命名规则,由VP1基因和Rd Rp基因来共同命名,截至2019年,Novs共有12个基因组(GI-GX,GNA1和GNA2)和49个基因型。GII.4是全球主要传播的类型,且每隔2-3年就会产生新的变异株,其次是GII.3,然后依次是GII.2、GII.17、GII.6、GI.3、GII.13、GI.4、GII.12和GII.14。在我国,Novs不同的基因型引起了不同省市的多起爆发性疫情,包括广东、浙江、江苏、四川、北京和上海等地,其中GII.4、GII.17和GII.2在我国引起大范围的传播,传染速度快,传播面积广。且Novs的感染在我国有很明显的季节性,绝大多数Novs感染在10月至次年3月达到高峰。尽管我国已经建立起了对于Novs的监测系统,但检测地区多集中于沿海,仍然缺乏对于西南地区的监测。昆明市特殊的地理位置和气候,且该病毒传染性强,传播范围广,变异速度快,因此,了解该市Novs的流行和传播情况是很有必要的。首先,本论文为了研究Novs的流行变化情况,我们随机选取了2021-2022年云南省的249例样本。利用RT-PCR的方法针对该病毒VP1基因和Rd Rp基因进行了扩增测序分析,扩增成功249例样本。结果表明Novs基因型出现了GII.4[P16](47.77%)、GII.4[P31](18.22%)、GII.6[P7](18.22%)、GII.3[P12](4.86%)、GII.3[P25](2.83%)、GII.17[P17](2.83%)、GII.2[P16](0.40%)、GII.4[P12](0.40%)、GII.7[P7](2.43%)、GII.15[P15](0.81%)、GII.13[P17](0.40%)、GII.13[P16](0.40%)、和GII.13[P21](0.40%),共13种基因型,并出现了两例没有准确分型的样品。呈现出以GII.4[P16]、GII.4[P31]、GII.6[P7]为主的流行特征。此外,我们对VP1基因和Rd Rp基因分型做了统计研究。对于VP1基因来说,出现了GII.4(65.99%)、GII.6(18.22%)、GII.3(7.69%)、GII.17(2.83%)、GII.7(2.43%)、GII.13(1.21%)、GII.15(1.21%),呈现出以GII.4、GII.6、GII.3为主的流行特征。对于Rd Rp基因来说,出现了P16(48.58%)、P7(20.65%)、P31(17.81%)、P12(5.26%)、P17(3.24%)、P25(2.83%)、P15(1.21%)、P21(0.40%),呈现出以P16、P7、P31为主的流行特征。其次,Novs病毒感染患儿引起了腹泻、呕吐、高烧、颈强直,抽搐,畏光以及反应迟钝等临床症状,为了了解该病毒基因型与发生的临床症状之间的关系,我们收集到了61例发生临床严重症状(高烧n=13,颈强直n=12,抽搐n=57,畏光n=1以及反应迟钝n=16)的患者,并对其进行了分型研究。结果发现GII.4Sydney 2012[P16]中出现抽搐(31.5%)、呕吐(23.3%)、腹泻(21.9%)、发热大于38℃(7.5%)、颈强直(6.8%);GII.4 Sydney 2012[P31]基因型中出现抽搐(33.3%)、腹泻(28.6%)、呕吐(9.5%)、颈强直(9.5%)和反应迟钝(9.5%);GII.6[P7]中出现抽搐(42.9%)、呕吐(28.6%)、反应迟钝(14.3%)、发热大于38℃(14.3%);GII.3[P12]中出现腹泻、呕吐和抽搐各占33.3%。最后,我们对于未能准确分型的2例样品进行了全长扩增以及重组分析,发现其中899号样本在ORF2和ORF3处发生了重组,基因组核苷酸差异为13.8%-16.8%,VP1基因分型为GII.4 Sydney 2012,Rd Rp基因分型为P7,疑似产生了新的重组体。综上所述,本研究发现Novs病毒在昆明地区广泛传播,引起患儿腹泻、呕吐、高烧等各种临床症状,且流行毒株的基因型也发生了变化,并发现新的重组亚型。因此,本研究首次将昆明市患儿Novs病毒的基因型与临床症状进行了深入分析,为了解云南省由Novs病毒引起儿童病毒性胃肠炎的流行情况提供了理论支持,并为临床诊断提供了方向。更多还原

【Abstract】 Norovirus(Novs)has become the main pathogen causing the global acute viral diarrhea(AGE)outbreak.Every year,the global economic burden caused by infection with the virus is approximately $6 billion,with over 2.19 million people hospitalized and 20.02 million deaths.Norovirus,referred to as Novs for short,is a non enveloped single stranded positive RNA virus of the genus Konoru,calicivirus,with a total length of about 7500 bp.In 2013,the Novas Classification Working Group(NCWG)officially established the naming rules for this virus,which is named jointly by the VP1 gene and the Rd Rp gene.As of 2019,Novas has a total of 12 genomes(GI-GX,GNA1,and GNA2)and 49 genotypes.In the United States,Europe,and Japan,up to 93% of non bacterial gastroenteritis outbreaks and 60-85% of gastroenteritis outbreaks are associated with Novs.In Africa,the overall prevalence of norovirus is very high,and genotype II and GII.4 are dominant.In recent years,GII.4 [P16],GII.2 [P16],and GII.12 [P16] have become newly emerging epidemic genotypes worldwide.GII.4 is still the main type of transmission worldwide,and new mutants are generated every 2-3years,followed by GII.3,followed by GII.2,GII.17,GII.6,GI.3,GII.13,GI.4,GII.12,and GII.14.In China,different genotypes of Novs have caused multiple outbreaks in different provinces and cities,including Guangdong,Zhejiang,Jiangsu,Sichuan,Beijing,and Shanghai.Among them,GII.4,GII.17,and GII.2 have caused widespread transmission in China,with rapid transmission speed and wide spread area.In addition,Novs has a significant seasonality in China,with the vast majority of Novs diseases peaking from October to March of the next year.Although China has established a monitoring system for Novs,the detection areas are mostly concentrated in the coastal areas,and there is still a lack of monitoring for the southwest region.Due to the special geographical location and climate of Kunming City,as well as the strong infectivity,wide range of transmission,and rapid mutation rate of the virus,it is necessary to understand the prevalence and transmission of Novs in this province.This study took the onset of acute viral gastroenteritis in children from 2012 to 2022 as a starting point,and conducted a Novs sampling test on them using RT-PCR.By establishing a phylogenetic tree,we can understand the prevalence and transmission of different genotypes of the virus in the child population,as well as the relationship between clinical symptoms and genotypes.After analysis,The results are shown as follows :Firstly,in order to study the epidemiological changes of Novs,we randomly selected 249 samples from Kunming City from 2021-2022.RT-PCR was used to amplify and sequence the VP1 gene and Rd Rp gene of the virus,and 249 samples were successfully amplified.The results showed that there were GII.4[P16](47.77%)、GII.4[P31](18.22%)、GII.6[P7](18.22%)、GII.3[P12](4.86%)、GII.3[P25](2.83%)、GII.17[P17](2.83%)、GII.2[P16](0.40%)、GII.4[P12](0.40%)、GII.7[P7](2.43%)、GII.15[P15](0.81%)、GII.13[P17](0.40%)、GII.13[P16](0.40%)、and GII.13[P21](0.40%),a total of 13 genotypes,Two samples that were not accurately typed appeared.Presents a popular feature dominated by GII.4 [P16],GII.4 [P31],and GII.6 [P7].In addition,we conducted statistical studies on VP1 gene and Rd Rp genotyping.For the VP1 gene,there are GII.4(65.99%)、GII.6(18.22%)、GII.3(7.69%)、GII.17(2.83%)、GII.7(2.43%)、GII.13(1.21%)and GII.15(1.21%),presenting the main epidemic characteristics of GII.4,GII.6,and GII.3.For the Rd Rp gene,there were P16(48.58%),P7(20.65%),P31(17.81%),P12(5.26%),P17(3.24%),P25(2.83%),P15(1.21%)and P21(0.40%),presenting the main epidemiological characteristics of P16,P7,and P31.Secondly,children infected with Novs virus caused clinical symptoms such as diarrhea,vomiting,high fever,neck stiffness,convulsions,photophobia,and delayed response.In order to understand the relationship between the virus genotype and the clinical symptoms,we collected 61 patients with clinically severe symptoms(high fever n=13,neck stiffness n=12,convulsion n=57,photophobia n=1,and delayed response n=16)and conducted a typing study on them.The results showed that convulsions(31.5%),vomiting(23.3%),diarrhea(21.9%),fever above 38 ℃(7.5%),and neck stiffness(6.8%)were observed in GII.4 [P16];The GII.4 [P31] genotype showed seizures(33.3%),diarrhea(28.6%),vomiting(9.5%),neck stiffness(9.5%),and delayed response(9.5%);In GII.6 [P7],convulsions(42.9%),vomiting(28.6%),delayed reactions(14.3%),and fever greater than 38℃(14.3%)were observed;In GII.3[P12],diarrhea,vomiting,and convulsions each accounted for 33.3%.Finally,we conducted full-length amplification and recombination analysis on two samples that could not be accurately typed,and found that sample 899 had undergone recombination at ORF2 and ORF3,with a genomic nucleotide difference of 13.8% to16.8%.The VP1 genotype was GII.4 Sydney,and the Rd Rp genotype was P7,producing a suspected new recombinant.In summary,this study found that Novs virus is widely spread in Yunnan Province,causing various clinical symptoms such as diarrhea,vomiting,and high fever in children.The genotype of the epidemic virus strain has also changed,and new recombinant subtypes have been found.Therefore,this study conducted an in-depth analysis of the genotype and clinical symptoms of Novs virus in Kunming City for the first time,providing theoretical support for understanding the prevalence of viral gastroenteritis in children caused by Novs virus in Yunnan Province,and providing direction for clinical diagnosis.更多还原