鼠疫噬菌体研究概述

鼠疫噬菌体是一种特异性很强的鼠疫耶尔森菌（Yersinia pestis,鼠疫菌）的细菌病毒,一直被用来进行鼠疫病原体的鉴定。鼠疫噬菌体的早期研究主要以如何在自然界分离、生物学特性、诊断和治疗鼠疫,近年来,随着微生物基因组和蛋白质组学技术的发展,揭示了鼠疫噬菌体在鼠疫菌进化过程中所发生的作用。现就鼠疫噬菌体的研究进展,尤其是基因组的结构、与宿主菌在致病和免疫相关研究做一些介绍,为研发新型分子生物学技术和鼠疫治疗药物提供帮助。     

小肠结肠炎耶尔森菌基因组中前噬菌体基因结构特征分析

目的了解小肠结肠炎耶尔森菌基因组中前噬菌体和噬菌体元件的携带情况及基因组结构特征。方法利用PHAST软件,对NCBI数据中18株已完成全基因组测序完成图的小肠结肠炎耶尔森菌的前噬菌体区域进行预测,并与参考噬菌体基因组序列进行比对,分析小肠结肠炎耶尔森菌前噬菌体基因的分布及其基因组特征。结果 18株小肠结肠炎耶尔森菌全基因组序列中前噬菌体携带率100%,共预测到141个前噬菌体区域,其中71个是具有完整噬菌体特征的前噬菌体,其余70个属于噬菌体元件。非致病性小肠结肠炎耶尔森菌LC20预测到的噬菌体区域最多,含有9个完整的前噬菌体和7个噬菌体元件,在一定程度上解释LC20菌株染色体基因组大于其余菌株,且含有更多重组及SNP位点的原因。截取71个完整前噬菌体区域的序列信息,与112株NCBI公布的噬菌体基因组序列进行聚类分析显示,共分为两个大簇。预测的前噬菌体与已知噬菌体之间不存在共有的核心基因组。部分前噬菌体区域含有毒力相关基因。结论小肠结肠炎耶尔森菌基因组中的前噬菌体具有基因组多样性,为噬菌体和宿主菌的协同进化研究奠定基础。     

野生型鼠疫噬菌体YP060的分离和生物学特性鉴定

目的 从鼠疫疫源地鼠巢中分离1株野生型鼠疫噬菌体(YP060)并分析其生物学特性。方法 采用双层琼脂平皿法从云南省鼠疫疫源地鼠巢中分离鼠疫噬菌体;描述其形态特征;了解裂解能力、宿主谱、最佳感染复数、一步生长特性;分析不同温度和pH值、紫外线、氯仿对噬菌体的敏感性。结果 YP060形态呈蝌蚪形,头部为正多面体结构,带有一伸缩的尾鞘;对鼠疫疫苗株EV76的最佳感染复数为0.1;潜伏期为50 min,暴发期为80 min;宿主谱评价显示,YP060仅裂解鼠疫疫苗株;YP060在30~50℃具有较强的热稳定性;pH值5~10范围内均有较强的裂解活性;对紫外线比较敏感;对氯仿不敏感,5%浓度的氯仿对其活性基本没有影响。结论 本研究为国内首次从鼠疫疫源地鼠巢中分离到鼠疫噬菌体YP060,该噬菌体具有窄宿主谱和较强的生物学裂解特性。     

鼠疫菌基因组与脉冲场电泳图型

目的利用PFGE图形来了解鼠疫菌基因组重排现象。方法PFGE分析并与全基因组序列进行比较。结杲计算PFGE各条带大小可初步判断鼠疫菌基因组序列重排结构。结论利用PFGE图型来判断鼠疫菌基因组序列重排结构,可以大大节约测序所需花费的成本。     

福氏痢疾杆菌2b血清型菌株基因组中SfII和SfX前噬菌体整合位点及排列方式研究

目的 研究福氏痢疾杆菌2b血清型菌株中SfII和SfX前噬菌体整合位点及排列方式.方法 根据2b血清型菌株基因组中SFII和SfX前噬菌体可能的整合位点及排列方式,设计引物,对50株2b血清型菌株进行PCR扩增,并对扩增产物测序,根据扩增和测序结果判断噬菌体的整合位点和排列方式.结果 在2b血清型菌株中,SfII和SfX前噬菌体前后相连,整合于宿主菌染色体proA和yaiC基因间的thrW tRNA基因上;以SfII在前,SfX在后,或SfX在前,SfII在后两种方式排列;而以后一种方式为主,在共50株检测菌株中,有49株为此方式.结论 福氏痢疾杆菌血清型相关噬菌体的整合位点及排列方式,对研究福氏痢疾杆菌血清型转换及血清型相关噬菌体的整合机制具有重要意义.     

福氏痢疾杆菌2b血清型菌株基因组中SfII和SfX前噬菌体整合位点及排列方式研究

目的 研究福氏痢疾杆菌2b血清型菌株中SfII和SfX前噬菌体整合位点及排列方式.方法 根据2b血清型菌株基因组中SFII和SfX前噬菌体可能的整合位点及排列方式,设计引物,对50株2b血清型菌株进行PCR扩增,并对扩增产物测序,根据扩增和测序结果判断噬菌体的整合位点和排列方式.结果 在2b血清型菌株中,SfII和SfX前噬菌体前后相连,整合于宿主菌染色体proA和yaiC基因间的thrW tRNA基因上;以SfII在前,SfX在后,或SfX在前,SfII在后两种方式排列;而以后一种方式为主,在共50株检测菌株中,有49株为此方式.结论 福氏痢疾杆菌血清型相关噬菌体的整合位点及排列方式,对研究福氏痢疾杆菌血清型转换及血清型相关噬菌体的整合机制具有重要意义.     

福氏痢疾杆菌2b血清型菌株基因组中SfII和SfX前噬菌体整合位点及排列方式研究

目的 研究福氏痢疾杆菌2b血清型菌株中SfII和SfX前噬菌体整合位点及排列方式.方法 根据2b血清型菌株基因组中SFII和SfX前噬菌体可能的整合位点及排列方式,设计引物,对50株2b血清型菌株进行PCR扩增,并对扩增产物测序,根据扩增和测序结果判断噬菌体的整合位点和排列方式.结果 在2b血清型菌株中,SfII和SfX前噬菌体前后相连,整合于宿主菌染色体proA和yaiC基因间的thrW tRNA基因上;以SfII在前,SfX在后,或SfX在前,SfII在后两种方式排列;而以后一种方式为主,在共50株检测菌株中,有49株为此方式.结论 福氏痢疾杆菌血清型相关噬菌体的整合位点及排列方式,对研究福氏痢疾杆菌血清型转换及血清型相关噬菌体的整合机制具有重要意义.     

鼠疫菌3种常见质粒的结构特征分析

目的研究鼠疫菌基因组中3种常见质粒的基因构成和分布特征。方法应用编码基因序列相似性比对和生物信息学软件自动分析2种方法,分别对国际上已完成全基因组测序的9株鼠疫菌的鼠毒素质粒(pMT1)、低钙反应质粒(pCD1)和鼠疫菌素质粒(pPCP1)进行整体结构的比较分析。结果 pMT1质粒上存在基因大片段的重新排列,整条质粒被划分成4个主要的基因模块。CO92、KIM和91001菌株分属于东方型、中世纪型和田鼠型3个不同的生物型,他们的p MT1质粒结构各有特点;Nepal 516等其余6株菌均是古典型鼠疫菌,他们的pMT1质粒结构基本一致,且与其他3个生物型鼠疫菌株的p MT1质粒结构不同。pCD1质粒被划分成3个主要的基因模块,但整体结构在不同菌株中并无差异,只有IS100在质粒中的插入位置各不相同。pPCP1质粒上所有编码基因的排列顺序和方向在各菌株中完全相同。结论鼠疫菌pCD1和pPCP1质粒的基因结构比较稳定,而pMT1质粒结构在鼠疫菌的长期进化过程中发生了一定程度的变异。     

人类肠道病毒的特征和研究进展

噬菌体和真核生物病毒是最具代表性的肠道病毒,其中CrAss噬菌体是人类病毒组的核心,人内源性逆转录病毒是人类基因组中稳定的遗传标志。噬菌体的溶原转化增强了其自身和宿主菌的进化优势,使其逐渐与宿主基因融为一体。本综述通过对肠道病毒的分类与特性、细菌-病毒-宿主的相互作用、研究肠道病毒的挑战与解决措施进行探讨,旨在给人类肠道病毒的研究提供证据。研究者应趋利避害,利用噬菌体特异性杀灭细菌的特性,研发相应的抗菌药物,使其成为肠道的守护军。病毒宏基因组测序的过程中会遇到许多已知、未知的挑战,本综述在讨论常规实验室经验的基础上,采用模拟社区模拟肠道的生态环境,减少环境因素对病毒检测的影响。     

鼠疫研究与控制措施

从自然进化和基因突变入手，阐述鼠疫菌的基因分型和致病性，认为这种突变正好也是各疫源地菌株实现“本土化”，形成不同类型鼠疫自然疫源地的过程。在动物问鼠疫监测中，以生境及其鼠迹、鼠洞探查和每月地毯式的搜检自毙鼠为基本出发点，另加一定数量的鼠类及指示动物的血清学监测模式；鼠疫菌与其特定区域的土壤动物、其他微生物、植物群落所具有的不同时空活动特点和生物学特性，以及共同组成的复杂营养食物链网络和在各自能流、物流、基因传递及其协同进化中的角色等，都将是鼠疫自然疫源地空间结构研究的所属范畴。建立以鼠疫噬菌体为病原学和血清学的协同诊断技术。     

鼠疫耶尔森菌和假结核耶尔森菌基因鉴别方法的建立及评价

目的 建立鼠疫耶尔森菌和假结核耶尔森菌基因鉴别方法。方法 依据鼠疫菌、假结核菌特有的基因组序列["疫岛(PeI)"和"假岛(PsI)"], 与已公布的12株鼠疫菌和4株假结核菌全基因序列进行比对, 设计特异性的引物, 对鼠疫菌、假结核菌和其他肠道细菌进行鉴定。结果 用52株鼠疫菌、57株假结核菌和其他肠道菌株进行验证, 结果显示, 5对鼠疫菌的鉴定引物中, 2对(PeI2和PeI11)仅在52株鼠疫菌中扩出目的条带, 另3对引物(PeI1、PeI3和PeI12)除鼠疫菌外在部分假结核菌株中也扩出目的条带;5对假结核菌鉴定引物中, 1对引物(PsI1)在52株鼠疫菌和57株假结核菌株中扩出目的条带, 4对引物(PsI7、PsI16、PsI18和 PsI19)仅在57株假结核菌株中均扩出目的条带, 在鼠疫菌中未扩出目的条带。结论 用鼠疫菌和假结核菌共有的PsI1序列、鼠疫菌特有的PeI2和PeI11序列及假结核菌特有的PsI7、PsI16、PsI18和 PsI19序列组成的基因鉴别方法, 可以用于鼠疫菌和假结核菌的基因快速鉴别。     

青海高原藏系绵羊鼠疫流行病学和病原学特征分析

目的 分析青海高原藏系绵羊鼠疫流行病学和病原学特征。方法 汇总1975－2009年青海省藏系绵羊鼠疫背景资料,分析其地区、时间和人间分布及感染途径和传播的生态学因素,并对藏系绵羊鼠疫分离的14株菌进行生化试验、毒力测定、毒力因子鉴定、质粒分析、差异片段(DFR)分型等研究。结果 1975－2009年从青海省藏系绵羊体内分离的鼠疫菌共14株。由藏系绵羊作为传染源引起人间鼠疫10起,鼠疫病例25例,死亡13例。首发病例均有剥食鼠疫病死藏羊史,其次为接触鼠疫病例而感染;以腺鼠疫为首发病例,由腺鼠疫继发为肺鼠疫、败血型鼠疫病例多且病死率高。藏系绵羊动物鼠疫及人间鼠疫几乎均发生于甘南生态区,与其独特的生态地理景观密切相关。藏羊动物鼠疫与藏羊引发的人间鼠疫几乎一致,其中11月(旱獭入蛰后)从藏羊体内分离的鼠疫菌株数及藏羊作为传染源引发的人间鼠疫病例数最多,构成了青海高原藏羊鼠疫流行时间明显滞后于旱獭鼠疫的特点。分离的14株鼠疫菌均为青藏高原型,其毒力因子及毒力检测均显示为强毒菌。鼠疫菌基因型DFR分析显示,玉树、治多县分离的菌株均为5型,囊谦县分离的2株菌分别为5型和7型,德令哈市分离的菌株为8型。结论 藏系绵羊可感染鼠疫并作为传染源引发人间鼠疫,分离的菌株均具备青藏高原鼠疫病原体特性,并具有青海鼠疫流行演变新特点。     

A review of methods for subtyping Yersinia pestis: From phenotypes to whole genome sequencing

Numerous subtyping methods have been applied to Yersinia pestis with varying success. Here, we review the various subtyping methods that have been applied to Y. pestis and their capacity for answering questions regarding the population genetics, phylogeography, and molecular epidemiology of this important human pathogen. Methods are evaluated in terms of expense, difficulty, transferability among laboratories, discriminatory power, usefulness for different study questions, and current applicability in light of the advent of whole genome sequencing.     

Comparison of serological and bacteriological methods in the confirmation of plague infections

A recent, defined outbreak of bubonic plague in a remote area of the central highlands of Viet-Nam provided an opportunity to undertake studies of antibody production during the course of infections with Pasteurella pestis (Yersinia pestis). The haemagglutination (HA) test of Chen & Meyer, modified to a microtechnique, was used in studies of patients with clinical plague, and in studies of unvaccinated, asymptomatic contacts of plague patients.     

Further development of raccoon poxvirus-vectored vaccines against plague (Yersinia pestis)

In previous studies, we demonstrated protection against plague in mice and prairie dogs using a raccoon pox (RCN) virus-vectored vaccine that expressed the F1 capsular antigen of Yersinia pestis. In order to improve vaccine efficacy, we have now constructed additional RCN-plague vaccines containing two different forms of the lcrV (V) gene, including full-length (Vfull) and a truncated form (V307). Mouse challenge studies with Y. pestis strain CO92 showed that vaccination with a combination of RCN-F1 and the truncated V construct (RCN-V307) provided the greatest improvement (P = 0.01) in protection against plague over vaccination with RCN-F1 alone. This effect was mediated primarily by anti-F1 and anti-V antibodies and both contributed independently to increased survival of vaccinated mice.     

Pleiotropic effects of the lpxM mutation in Yersinia pestis resulting in modification of the biosynthesis of major immunoreactive antigens

Deletion mutants in the lpxM gene in two Yersinia pestis strains, the live Russian vaccine strain EV NIIEG and a fully virulent strain, 231, synthesise a less toxic penta-acylated lipopolysaccharide (LPS). Analysis of these mutants revealed they possessed marked reductions in expression and immunoreactivity of numerous major proteins and carbohydrate antigens, including F1, Pla, Ymt, V antigen, LPS, and ECA. Moreover, both mutants demonstrated altered epitope specificities of the antigens as determined in immunodot-ELISAs and immunoblotting analyses using a panel of monoclonal antibodies. The strains also differed in their susceptibility to the diagnostic plague bacteriophage L-413C. These findings indicate that the effects of the lpxM mutation on reduced virulence and enhanced immunity of the Y. pestis EV ΔlpxM is also associated with these pleiotropic changes and not just to changes in the lipid A acylation.     

Efficacy and safety of a modified vaccinia Ankara (MVA) vectored plague vaccine in mice

The efficacy and safety of plague vaccines based on the modified vaccinia Ankara (MVA) viral vector was evaluated. MVA recombinants were constructed expressing Yersinia pestis antigens under the translational control of the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) and/or fused to the tissue plasminogen activator (tPA) secretory signal. A MVA/Y. pestis recombinant that expressed a truncated version of the low-calcium response V antigen (MVA/IRES/tPA/V₃₀₇), conferred significant protection (87.5–100%) against intranasal or intraperitoneal challenge with CO92 (encapsulated) or Java 9 (non-encapsulated) strains of Y. pestis, respectively. In contrast, a MVA/Y. pestis recombinant that expressed the full-length V antigen provided only 37.5% protection against challenge with CO92 or Java 9 strains, respectively. Interestingly, a MVA/Y. pestis recombinant that expressed the capsular protein (F1) did not elicit significant antibody titers but still conferred 50% and 25% protection against CO92 or Java 9 challenge, respectively. The MVA/Y. pestis recombinant viruses did not demonstrate any mortality or morbidity in SCID mice. Based on their safety and efficacy in mice, these MVA/Y. pestis recombinants are candidates for further development as biodefense and public health vaccines.     

中国幽门螺杆菌基因组多样性与种群结构分析

目的 了解中国幽门螺杆菌（Helicobacter pylori,HP）基因组特征及种群结构。方法 利用中国不同地域不同疾病分离的10株HP的基因组序列,并整合公共数据库中其他地域的HP基因组数据,通过比较基因组和生物信息学方法分析中国HP的基因组与种群结构特征。结果 中国HP核心基因为1 203个。菌株特异基因为19～32个,这些基因可能与中国HP在不同地域、不同疾病宿主中的适应性进化有关。基因组变异较大区域主要集中在编码限制修饰系统的基因和编码四型分泌系统的基因。基于核心基因组单核苷酸多态性（SNP）的种群分析确定中国菌株均属于hpEastAsia群,hspEAsia亚群,且不同地域菌株具有地域聚集性特点。在3株中国HP基因组序列中发现了前噬菌体序列,携带噬菌体组装所需的必要元件。结论 基于核心基因组SNP分析中国菌株均属于hpEastAsia群,hspEAsia亚群,且具有地域聚集性。为深入挖掘中国不同地域不同疾病相关HP的遗传特征及研究噬菌体在HP进化与致病中的作用奠定了基础。     

Legionella pneumophila: Population genetics, phylogeny and genomics

Legionella pneumophila is a human pathogen that was recognized only about 30 years ago. It is the causative agent of Legionnaires’ disease, a severe pneumonia that is transmitted through inhalation of aerosols of contaminated water. Shortly after its discovery, the ability of Legionella to multiply intracellularly in fresh water protozoa was discovered. This long lasting co-evolution between the eukaryotic host and Legionella has led to the selection of a panoply of virulence factors, which allow to exploit important cellular processes during infection. Compelling evidence for the importance of protozoa in the evolution of this bacterium comes from analysis of complete genome sequences. A key feature of the L. pneumophila genomes is the presence of a high number and wide variety of eukaryotic like proteins and protein domains probably acquired through horizontal gene transfer and/or convergent evolution.In the last years several different typing methods aiming in investigating the molecular epidemiology of L. pneumophila have been developed. Furthermore, the access to whole genome sequences of several L. pneumophila strains allowed to apply large scale comparative genomic studies using DNA arrays. A higher genetic diversity among environmental isolates with respect to clinical isolates and the presence of specific clones of L. pneumophila overrepresented in human disease or causing legionellosis world wide, were identified. Further studies analyzing the natural populations of Legionella more in detail will allow to gain a better understanding of the population structure and the ecological diversity of this species. This review describes the latest observations about the structure of L. pneumophila populations, the techniques used to study the molecular epidemiology and evolution of L. pneumophila, the knowledge gained from genome analysis, and discusses future perspectives.     

Development of a vaccinia virus based reservoir-targeted vaccine against Yersinia pestis

Yersinia pestis, the causative organism of plague, is a zoonotic organism with a worldwide distribution. Although the last plague epidemic occurred in early 1900s, human cases continue to occur due to contact with infected wild animals. In this study, we have developed a reservoir-targeted vaccine against Y. pestis, to interrupt transmission of disease in wild animals as a potential strategy for decreasing human disease. A vaccinia virus delivery system was used to express the F1 capsular protein and the LcrV type III secretion component of Y. pestis as a fusion protein. Here we show that a single dose of this vaccine administered orally, generates a dose-dependent antibody response in mice. Antibody titers peak by 3 weeks after administration and remain elevated for a minimum of 45 weeks. Vaccination provided up to 100% protection against challenge with Y. pestis administered by intranasal challenge at 10 times the lethal dose with protection lasting a minimum of 45 weeks. An orally available, vaccinia virus expressed vaccine against Y. pestis may be a suitable vaccine for a reservoir targeted strategy for the prevention of enzootic plague.