【作者】 王文；

【导师】 张永振； 杨占秋； 覃新程；

【作者基本信息】 中国疾病预防控制中心 ， 病原生物学， 2017， 博士

【摘要】 冠状病毒是有包膜的单股正链RNA病毒,是目前已知的RNA病毒中基因组最大的。冠状病毒主要感染呼吸道、肠道、肝脏和神经系统,可以引起人和动物多种疾病,是非常重要的病原体。在2002/2003年SARS冠状病毒引起的新发传染病暴发流行前,兽医、养殖业对冠状病毒的重视程度高于公共卫生对冠状病毒的关注。SARS冠状病毒新发传染病的暴发使得人们对冠状病毒高度重视,全世界的科学家致力于SARS冠状病毒的溯源研究和新型冠状病毒的发现工作。在过去的十几年间,在人和动物中发现了大量的新型冠状病毒。2012年MERS冠状病毒在中东的暴发流行使得冠状病毒再次成为关注的焦点。动物中很有可能存在新的未被识别的冠状病毒存在跨种间传播的风险。目前已知的冠状病毒分为4属,其中Alpha和Beta属病毒的宿主主要为哺乳动物,Gamma和Delta属病毒主要发现于鸟类与禽类。啮齿动物包括约2277种,可传播60多种已知的人类和家畜传染病。啮齿动物生活在人类或家养动物的附近,一旦接触被啮齿动物的唾液、尿液和粪便污染的食物和物品以及分泌物形成的气溶胶,增加了病原体的跨种间传播风险。鼠等啮齿动物是RNA病毒重要的储存宿主,但是迄今为止只发现啮齿动物携带一种冠状病毒,即鼠科冠状病毒,包括鼠肝炎病毒(Murine hepatitis virus,MHV)和大鼠涎泪腺炎冠状病毒(Ratsialodacryoadenitiscoronavirus,RCoV)。为了探索冠状病毒在啮齿动物中的多样性及其进化,我们2011-2013年在浙江省的龙泉市、文成县和鹿城区采集了 1,465只啮齿动物,通过逆转录PCR的方法检测冠状病毒RNA,使用深度测序、基因组步移和RACE等方法扩增冠状病毒的全基因组序列。在浙江省龙泉市和温州市鹿城区的褐家鼠中发现了一种新型冠状病毒,命名为Lucheng Rn rat coronavirus(LRNV);在龙泉市的罗赛鼠、黄胸鼠和社鼠中发现了鼠科冠状病毒的新种Longquan R1 rat coronavirus(LRLV),在龙泉市的黑线姬鼠、褐家鼠和罗赛鼠中发现了Betacoronavirus 1 的新种 Longquan Aa mouse coronavirus(LAMV)。这是鼠科冠状病毒发现60年后首次报道啮齿动物携带其它类型的冠状病毒,并首次发现啮齿动物携带Alpha属的冠状病毒,这些数据表明啮齿动物是冠状病毒重要的宿主。系统发生分析表明LAMV and LRLV都属于Beta冠状病毒的A组,该组包括在人、家畜和野生动物中发现的冠状病毒。有意思的是,褐家鼠携带的LRNV在3CLpro和RdRp基因树中形成单独的分支,并与Alpha冠状病毒聚集在一起,在Hel、ExoN、NendoU和O-MT进化树中形成单独分支,并位于Alpha冠状病毒的根部位置,而在N和S基因树中形成了更分化的一支,LRNV在基因结构上含有许多独特的辅助蛋白基因,表明可能是起源于重组。此外,在LRLV中也发现了种内重组事件。重组是冠状病毒进化的重要方式之一,也是产生遗传多样性的重要机制之一。由此可见,这些数据表明啮齿动物可能携带未知的冠状病毒并在Beta冠状病毒A分支和Alpha冠状病毒的起源进化中起重要的作用。登革病毒是人类最重要的新发传染病病原体之一,对世界大约三分之一的人口构成威胁。虽然二战后该疾病在中国消失了 30年,但是登革热自70年代末以来每年都有报道。然而,由于暴发通常高度地域化,因此并没有引起人们的广泛关注。此外,由于人口流动性增加,在中国其它气候条件不适宜该病原体增殖/传播的省份也有了登革热输入病例的报道。因此,更好地了解这些非流行地区的登革热流行病学将对其预防和控制具有重大的意义。登革热输入病例对非疫区,尤其是埃及伊蚊广泛分布的地区,可造成威胁人类生命健康的重大公共卫生问题。在本研究中,我们在温州市和武汉市(这两个地区在过去很少有登革热的报道)共收集了 20例登革热病例,其中温州市5例,武汉市15例。感染的患者表现为典型的登革热临床症状,包括高烧(100%)、头痛(100%)、头晕(45%)、肌痛(50%)、恶心和呕吐(40%)、皮疹(40%)以及出血点(25%)。系统发生分析显示,从四名患者中获得的病毒基因组序列均为DENV-1,并且与来自印度次大陆的毒株有最近的亲缘关系,表明该登革病毒在两地间的传播。值得注意的是,尽管大多数患者(18/20)有发病前曾到过国内或国外登革热流行地区的旅游史,表明这些病例可能是输入性病例,但包括一名7岁儿童在内的其他两名患者并没有近期曾到过登革热流行地区的旅行史,提示是登革病毒的本地传播。因此,本研究对中国登革热的流行病学、控制和预防有重要意义,并将与其它非流行情况下的病毒传播直接相关。更多还原

【Abstract】 Coronaviruses(CoVs)are single-stranded,positive-sense and enveloped RNA viruses,owning the largest genome among known RNA viruses.Coronaviruses are the etiological agents of respiratory,enteric,hepatic,and neurological diseases in animals and humans.However,until the emergence of severe acute respiratory syndrome(SARS)in China in 2002/2003,coronaviruses had been of greater concern to agriculture than public health.Since the discovery of SARS-CoV intense scientific efforts have focused on characterizing additional CoVs in animals and humans.As a consequence,the number of novel CoVs in both humans and animals has increased rapidly over the past ten years.MERS-CoVs are heightened global attention again.It is most likely that there are extremely large unidentified coronaviruses circulating in animals that could pose great risk of cross-species transmission.CoVs were classified into four distinct groups,Alpha and Beta coronavirus are mainly harbored by mammals;Gamma and Delta coronavirus are mainly identified in birds.Rodentia(rodents)is the largest,ubiquitous and most diverse order of mammals with approximately 2277 species throughout the world,representing some 42%of all mammalian species,and may be the largest zoonotic source of human infectious diseases(at least 60 pathogenic agents).For rodents that live in close proximity to humans or domestic animals there is an increased cross-species transmission risk following exposure to rodent saliva,urine,faeces and aerosol.Although rodents are important reservoirs for RNA viruses,to date,only one species of rodent coronavirus(CoV)has been identified,murine coronavirus,which include the Murine hepatitis virus(MHV)and rat sialodacryoadenitis coronavirus(RCoV).Herein,we describe a new CoV,denoted Lucheng Rn rat coronavirus(LRNV),and two novel variants of Betacoronavirus termed Longquan Aa mouse coronavirus(LAMV)and Longquan R1 rat coronavirus(LRLV),that were identified in a survey of 1,465 rodents sampled in China during 2011-2013.Importantly,this is the first description of rodent coronaviruses for over 60 years since murine coronavirus were idendified,and suggests that rodents maybe important natural reservoir hosts of CoV.These data indicate that rodents are an important natural reservoir host of CoV.Phylogenetic analysis revealed that LAMV and LRLV fell into lineage A of the genus Betacoronavirus,which included CoVs discovered in humans and domestic and wild animals.In contrast,LRNV harbored by Rattus norvegicus formed a distinct lineage within the genus Alphacoronavirus in the 3CLpro and RdRp,in Hel,ExoN,NendoUand O-MT gene trees,LRNV occupy a deep branch at the root of Alphacoronavirus,but formed a more divergent lineage in the N and S gene trees,indicative of a recombinant origin.LRNV contains many unique accessory protein genes in the gene structure,suggesting that it may be origined from recombination.Additional recombination events were identified in LRLV,Recombination is an important tool for the evolution of coronavirus,an important mechanism for the generation of genetic diversity.In sum,these data indicated that rodents may carry additional unidentified Co Vs.Dengue is one of the most important emerging viruses of humans,posing a threat to approximately one-third of the world’s population.Although the disease had disappeared for 30 years in China after the World War II,dengue has reported each year since the end of 1970s.However,because outbreaks have usually been highly localized,they have received little attention.Moreover,because of increased population mobility,imported dengue cases have been recorded in Chinese provinces with less permissive climates.Consequently,a better understanding of dengue epidemiology in these non-endemic regions will be enormous benefit to its prevention and control.Imported cases of dengue virus(DENY)can pose an important public health risk to non-endemic areas,especially those where the Aedes aegypti mosquito vector is established.We documented 20 dengue cases in the Chinese cities of Wenzhou(5 cases)and Wuhan(15 cases)where dengue has only rarely been reported in the past.The infected individuals presented with clinical signs typical of dengue,including high fever(100%),headache(100%),dizziness(45%),myalgia(50%),nausea and vomiting(40%),rash(40%),and patechiae(25%).Phylogenetic analysis revealed that the viral genome sequences recovered from four patients were all DENV-1,and closely related to those from the Indian subcontinent,indicative of viral traffic between these two localities.Notably,although most(18/20)patients had a recent travel history in endemic areas in and outside of China,indicating that these cases were likely imported,two patients,including a seven year old child,had not recently travelled to dengue endemic areas,suggestive of autochthonous viral transmission.In sum,our study presents an important contribution to the epidemiology,control and prevention of dengue in China,and will be direct relevance to the virus transmission in other non-endemic situations.更多还原