一株A型口蹄疫流行毒株全序列的测定及其全长感染性克隆的构建

【目的】测定一株A型口蹄疫流行毒株的全基因组序列,并构建其全长感染性克隆。【方法】参照已公布的A型口蹄疫病毒序列设计引物,将分离的口蹄疫病毒株A/Sea-97/CHA/2014全基因组分为4个重叠的片段进行RT-PCR扩增,并对其进行序列测定与分析。利用酶切连接法将4个基因片段依次克隆至p Blue Script SKhdv载体中,构建该流行毒株的全长c DNA克隆p QAHN。pQAHN经NotⅠ线性化后转染表达T7 RNA聚合酶的BSR/T7细胞,拯救病毒。【结果】口蹄疫病毒全基因组序列测定结果表明该毒株基因组全长8 171 bp[不包括poly(C)区段和poly(A)尾巴],开放阅读框为6 996 bp,编码2 332个氨基酸,5′和3′非编码区分别为1 091 bp和95 bp。VP1系统发生树分析表明该毒株与A/GDMM/CHA/2013毒株亲缘关系最近,相似性为99.1%。线化全长质粒转染BSR/T7细胞68 h后可观察到典型的细胞病变。拯救病毒的间接免疫荧光、RT-PCR和序列测定结果表明成功拯救出了具有感染性的FMDV。拯救病毒与亲本病毒的噬斑表型及生长曲线试验表明二者具有相似的生长表型和增殖能力。【结论】该研究为我国口蹄疫病原生态分布、分子流行病学调查以及A型FMD新型疫苗的研究提供了有益的材料。     

型内嵌合口蹄疫病毒全长感染性cDNA克隆的构建

【目的】近年来,O型口蹄疫的不断暴发严重危害了我国畜牧业的发展,其病原——O型口蹄疫病毒已演化出3种谱系:中国型猪毒系、泛亚系和缅甸98系。其中中国型猪毒系病毒高度嗜猪,对养猪业危害最大。目前应用的疫苗已不能有效保护中国型猪毒系变异株的流行,这给我国猪口蹄疫的防控带来了极大的困难。为了进一步发展免疫原性好、抗原谱广的猪O型口蹄疫疫苗候选株,本研究以O/HN/93现用疫苗毒株的感染性克隆为骨架,用流行的新猪毒系病毒的部分VP3和VP1基因(主要是替换VP1蛋白上的B-C环和G-H环)替换疫苗毒株的相应部分,构建了嵌合的FMDV全长cDNA克隆。【方法】线化的嵌合全长质粒和表达T7 RNA聚合酶的真核质粒pcDNAT7P共转染BHK-21细胞,体内转录拯救嵌合病毒。【结果】嵌合全长质粒转染BHK-21细胞36h后,出现明显的FMDV致细胞病变效应。对收获的病毒分别用RT-PCR、间接免疫荧光、电子显微镜观察结果证实成功拯救到嵌合的FMDV。拯救的病毒乳鼠致病性试验结果表明该拯救病毒对乳鼠的致病力减弱。该嵌合病毒的成功拯救为研制口蹄疫新型疫苗等奠定了基础。     

表达O型口蹄疫病毒 VP1基因的重组病毒BHV-1的构建与鉴定

摘要：【目的】为了构建表达口蹄疫病毒（O/China/99）VP1基因的牛疱疹病毒1型,将人工合成的口蹄疫病毒VP1基因插入到巨细胞病毒（CMV）启动子之下构建gE基因缺失转移载体。【方法】利用磷酸钙介导转染法将该转移载体与亲本病毒BHV-1/gE-/LacZ+的基因组DNA共转染牛鼻甲细胞后收获增殖的病毒。通过筛选白色病毒蚀斑,得到重组病毒BHV-1/gE-/VP1。【结果】PCR检测结果表明VP1基因已经插入到了重组病毒BHV-1/gE-的基因组中,间接免疫荧光试验和Western blot证实了BHV-1/gE-/VP1中的VP1基因在感染的细胞中获得了表达。【结论】本研究成功的构建了表达口蹄疫病毒VP1基因的重组病毒BHV-1/gE-/VP1,为研制口蹄疫及其他重要牛传染病的BHV-1病毒载体疫苗奠定了基础。     

中国禽源新城疫病毒(NDV)流行株F和HN基因的遗传演化和变异频率

【目的】新城疫(ND)是中国流行最严重的疫病之一,对家禽业可造成巨大的经济损失,疫苗防控是控制ND的重要措施。新城疫病毒(NDV)流行株的遗传演化一直是研究NDV的焦点。本文利用分子信息学手段,通过比较近20年间NDV流行株不同基因型F和HN基因的分子特征和遗传变异频率,解析免疫压力下NDV的演化规律。【方法】利用Lasergene 7.1和MEGA5.1软件,选取本实验室89株NDV分离株,结合从Gen Bank下载的364株NDV流行株以及15株NDV经典毒株的基因序列,对其进行系统发育、分子特征和替代频率分析。【结果】系统发育表明,NDV已经演化为15个基因型。一致性比较显示,NDV流行株相同基因型之间核苷酸(氨基酸)高度同源,而不同基因型之间差异较大且存在明显的氨基酸变异积累。NDV基因型的分布与时间、地域密切相关,VII d亚型为中国NDV优势流行株。为评估NDV变异的频率,以Go/GD/QY/1997株(中国较早发生的基因VII亚型)为参照,1997-2015年间NDV的F/HN基因的年平均核苷酸(氨基酸)替代率为2.31×10~(-3)(2.26×10~(-3))/3.37×10~(-3)(2.35×10~(-3))。其中,1997-2001年(未使用基因VII型疫苗)F/HN基因核苷酸年平均替代率为4.72×10~(-3)/8.28×10~(-3);2002-2015年(疫苗使用后)为1.6×10~(-3)/1.84×10~(-3),显示出基因VII型疫苗在控制NDV变异速度方面具有明显的效果。【结论】生物信息学分析证实:研制出与NDV流行毒株相匹配的新型疫苗是控制当前NDV变异的关键。     

新甲型H1N1(2009)病毒的早期分子特征

摘要：【目的】本世纪首次流感大流行的病原属于甲型H1N1流感病毒,在遗传特性和抗原等方面都有别于人群中流行多年的季节性H1N1流感病毒。为了深入了解病毒的遗传特性,跟踪病毒的演化趋势,及时发现具有流行病学意义的变异株,本研究对早期分离的甲型H1N1(2009)病毒的分子特性进行了详细分析。【方法】通过GenBank的流感资源中心下载相关毒株的基因组信息, 序列分析采用DNAStar软件包的EditSeq和MegAlign比较与病毒致病性和宿主特异性相关的氨基酸变化情况。以A/California/07/2009（H1N1）作为新甲型H1N1(2009)的代表株进行详细的分子特征分析。【结果】A/California/07/2009不具备高致病性流感病毒的分子特征;病毒编码的11个蛋白大部分保留有猪流感病毒的分子特征,同时也具有一些禽和人流感病毒的特征;PB1-F2在11aa,57aa和87aa后发生断裂,具有古典猪H1N1和人H1N1双重特点,这是甲型H1N1（2009）病毒一个特有的分子特征。【结论】首次详细分析了新甲型H1N1（2009）病毒的分子特征。随着病毒在人群中的进一步适应和持续存在,这些分子特征将发生变化,应该特别关注这些变化对病毒的传播力和致病性的影响。     

2009−2015年中国甲型H1N1流感病毒血凝素基因进化分析

【目的】为了解中国地区2009?2015年甲型H1N1流感病毒流行态势,分析血凝素(Hemagglutinin,HA)基因的变异情况及其遗传进化特征。【方法】汇集国家流感中心2009?2015年流感周报的流感流行数据,分析甲型H1N1流感的流行病学特征;从全球共享禽流感数据倡议组织数据库及美国国家生物技术中心数据库下载甲型H1N1流感病毒HA基因序列,采用生物学软件进行系统进化和遗传特性的分析。【结果】2009?2015年全国共发生4次甲型H1N1流感的流行高峰。2009?2015年毒株与参考毒株A/California/07/2009(H1N1)的HA基因同源性逐年降低。遗传进化分析显示同一年份的毒株在系统进化树上基本呈现集中分布,2011年的毒株独立形成2个分支。分子特征表现为HA基因的4个抗原决定簇氨基酸位点均有变异,其中Ca区的203位、Sa区的163位和Sb区的185位氨基酸位点逐渐替换为新的氨基酸。除2010年与2012年,其他年份的毒株通过不同模型均得到正向压力选择HA氨基酸位点240。【结论】甲型H1N1流感在中国地区成为主要流行的亚型之一。HA基因与其编码的氨基酸逐年变异,未来进一步的流感监测能力还需加强。     

3A蛋白104–115位氨基酸缺失口蹄疫A型标记病毒的构建

【目的】构建一株含3A非结构蛋白104–115位氨基酸缺失的口蹄疫A型标记病毒,分析其生物学特性和发展标记疫苗的潜力。【方法】采用融合PCR技术,在当前流行毒株A/Sea-97/CHA/2014全长感染性克隆p QAHN中引入3A104–115位氨基酸的缺失,构建全长重组质粒。全长质粒经NotI线化后转染表达T7RNA聚合酶的稳定细胞系,拯救标记病毒。RT-PCR、序列分析、间接免疫荧光和Western blotting鉴定标记病毒。噬斑表型和一步生长曲线分析标记病毒的生物学特性,并用实验室开发的针对3A优势表位(AEKNPLE)的阻断ELISA方法分析其区分亲本和标记病毒感染的动物。【结果】成功拯救到一株含3A 104–115位氨基酸缺失的口蹄疫A型标记病毒,3A表位的缺失没有影响标记病毒的噬斑表型和一步生长曲线。3A单抗阻断ELISA可以明显区分标记病毒和亲本病毒感染的动物。【结论】本研究构建的3A蛋白104–115位氨基酸缺失的标记病毒可以作为发展口蹄疫鉴别诊断疫苗的候选毒株,用于我国未来口蹄疫A型的有效防控。     

山东地区犬冠状病毒的分子流行病学调查及其基因型多样性分析

【目的】了解分析山东地区健康犬及腹泻犬中犬冠状病毒(CCoV)的分子流行病学及其基因型。【方法】采集2017年以来山东省宠物市场、流浪犬中心、犬舍、各地宠物医院等的健康和腹泻犬只的肛拭子样品,采用PCR方法检测CCoV及其CCoV-Ⅰ和CCoV-Ⅱ(CCoV-Ⅱa和CCoV-Ⅱb)基因分型情况,并对扩增出的M、S全基因进行测序分析。【结果】199份样品中,共检出CCoV阳性样品79份,健康犬中的检出率为40.2%(33/82),腹泻犬中的检出率为39.3%(46/117)。健康犬中CCoV-Ⅰ型与CCoV-Ⅱ型阳性检出率分别为78.8%(26/33)和51.5%(17/33);腹泻犬中CCoV-Ⅰ型与CCoV-Ⅱ型阳性检出率分别为39.13%(18/46)和80.4%(37/46)。基因型阳性比率分析表明,健康犬中单独感染CCoV-I型的比率最高,为48.5%(16/33);腹泻犬中单独感染CCoV-Ⅱa比率最高,为47.8%(22/46)。测序分析获得48株M基因序列,遗传进化分析表明,12株CCoV-Ⅰ型毒株中除1株外,其余均从健康犬中分离。36株CCoV-Ⅱ型毒株中除7株来自健康犬外,其余均为腹泻犬中获得。获得的3株S全基因均来自腹泻犬且均属于CCoV-Ⅱa亚型。【结论】山东地区犬群中CCoV检出率较高,说明CCoV广泛流行,健康犬和腹泻犬中均存在多重感染情况,健康犬中主要流行CCoV毒株为CCoV-Ⅰ型,腹泻犬中主要流行CCoV-Ⅱa型。     

我国2002−2016年间鸡传染性支气管炎病毒基因组序列重组分析

【目的】传染性支气管炎病毒(Infectious bronchitis virus,IBV)主要引起鸡群呼吸道与肾脏疾病,是影响养禽业重要的病毒性病原之一。了解我国IBV的流行及基因重组情况。【方法】收集GenBank中我国2002?2016年间分离的92株IBV S1基因及55株IBV基因组序列,并对这些序列进行比对分析。【结果】IBV S1基因序列分析结果表明,2002?2016年间我国流行的92株IBV可以分为13个基因型,包括QX、4/91、Mass、tl/CH/LDT3/03、CK/CH/LSC/99I、TW-I、TW-II、TC07-2、Ck/CH/LDL/97I、N1/62-associated、Arkansas、New-I及一个新鉴定的基因分支New-II。值得注意的是,属于美国相关基因分支的ck/CH/LSD/110712已在我国出现。RDP4方法进行重组分析显示,新出现的基因分支New-II病毒S1基因来源于tl/CH/LDT3/03型与QX型毒株重组,我国2002?2016年间流行的55株IBV中有52株IBV基因组存在重组事件,其中25个IBV分离株基因组中发现有疫苗型(Mass,tl/CH/LDT3/03及4/91型等)病毒基因组片段的重组,这一结果在SimPlot分析中进一步得到确认。【结论】根据生物信息学分析结果,证明流行于我国的IBV基因型众多,疫苗毒株基因频繁参与了IBV基因重组,导致IBV新的基因型或变异株出现,提示在防控IB时要注意合理使用IBV疫苗。     

2017年河北省蛋鸡养殖场H9N2亚型禽流感病毒的遗传演化和抗原性分析

【背景】H9N2亚型禽流感病毒在鸡群中广泛流行,引起巨大损失。【目的】了解河北省蛋鸡养殖场H9N2亚型禽流感病毒(avian influenza virus,AIV)的基因序列和抗原性的变异情况,为该病原的科学防控提供理论依据。【方法】于2017年从河北省部分蛋鸡养殖场分离鉴定出7株H9N2亚型AIV,对其HA基因进行序列测定,并进行遗传演化、关键氨基酸位点及抗原性分析。【结果】7株分离毒株HA基因同源性在95.5%?97.2%之间;与2016年前的流行毒株相比,分离病毒HA裂解位点均为典型低致病性AIV特征,在受体结合区域出现变异,潜在糖基化位点无明显差异;抗原分析结果显示分离毒株与早期分离株相比抗原性发生了变异,形成了新的抗原群;抗原性相关位点分析显示,分离毒株在9个位点发生了较为明显的突变,可能是导致抗原性变异的分子基础。【结论】河北省蛋鸡养殖场H9N2亚型AIV中的流行毒株在关键功能区发生基因突变,并且抗原性发生变异,提示应持续监测H9N2亚型AIV的遗传变异情况,并及时更换疫苗株。     

Evaluation of Infectivity,Virulence and Transmission of FDMV Field Strains of Serotypes O and A Isolated In 2010 from Outbreaks in the Republic of Korea

Since the early 2000s outbreaks of foot-and-mouth disease (FMD) have been described in several previously FMD-free Asian nations, including the Republic of Korea (South Korea). One outbreak with FMD virus (FDMV) serotype A and two with serotype O occurred in South Korea in 2010/2011. The causative viruses belonged to lineages that had been spreading in South East Asia, far East and East Asia since 2009 and presented a great threat to the countries in that region. Most FMDV strains infect ruminants and pigs, as it happened during the outbreaks of FMDV serotype O in South Korea. Contrastingly, the strain of serotype A affected only ruminants. Based upon these findings, the intention of the work described in the current report was to characterize and compare the infectivity, virulence and transmission of both strains under laboratory conditions in cattle and pigs, by direct inoculation and contact exposure. As expected, FMDV serotype O was highly virulent in both cattle and swine by contact exposure and direct inoculation. Surprisingly, FMDV serotype A was highly virulent in swine, but was less infectious in cattle by contact exposure to infected swine or cattle. Interestingly, similar quantities of aerosolized FMDV RNA were detected during experiments with viruses of serotypes O and A. Specific virus-host interaction of A/SKR/2010 could affect the transmission of this strain to cattle, and this may explain in part the limited spread of the serotype A epizootic.     

Characterization of Foot-And-Mouth Disease Viruses (FMDVs) from Ugandan Cattle Outbreaks during 2012-2013: Evidence for Circulation of Multiple Serotypes

To investigate the foot-and-mouth disease virus (FMDV) serotypes circulating in Uganda’s cattle population, both serological and virological analyses of samples from outbreaks that occurred during 2012–2013 were performed. Altogether, 79 sera and 60 oropharyngeal fluid (OP)/tissue/oral swab samples were collected from herds with reported FMD outbreaks in seven different Ugandan districts. Overall, 61/79 (77%) of the cattle sera were positive for antibodies against FMDV by PrioCHECK FMDV NS ELISA and solid phase blocking ELISA detected titres ≥ 80 for serotypes O, SAT 1, SAT 2 and SAT 3 in 41, 45, 30 and 45 of these 61 seropositive samples, respectively. Virus neutralisation tests detected the highest levels of neutralising antibodies (titres ≥ 45) against serotype O in the herds from Kween and Rakai districts, against SAT 1 in the herd from Nwoya district and against SAT 2 in the herds from Kiruhura, Isingiro and Ntungamo districts. The isolation of a SAT 2 FMDV from Isingiro was consistent with the detection of high levels of neutralising antibodies against SAT 2; sequencing (for the VP1 coding region) indicated that this virus belonged to lineage I within this serotype, like the currently used vaccine strain. From the Wakiso district 11 tissue/swab samples were collected; serotype A FMDV, genotype Africa (G-I), was isolated from the epithelial samples. This study shows that within a period of less than one year, FMD outbreaks in Uganda were caused by four different serotypes namely O, A, SAT 1 and SAT 2. Therefore, to enhance the control of FMD in Uganda, there is need for efficient and timely determination of outbreak virus strains/serotypes and vaccine matching. The value of incorporating serotype A antigen into the imported vaccines along with the current serotype O, SAT 1 and SAT 2 strains should be considered.     

中国蓝舌病病毒流行株血清型实时荧光定量RT-PCR检测方法的建立与应用

【背景】蓝舌病病毒(Bluetongue virus,BTV)是一种严重危害反刍动物的虫媒病毒,我国存在12种血清型BTV (BTV-1、-2、-3、-4、-5、-7、-9、-12、-15、-16、-21和-24)的流行。【目的】建立12种血清型BTV的RT-qPCR定型方法,为BTV的诊断与流行病学研究提供技术保障。【方法】根据我国流行BTV基因节段2 (Seg-2)序列设计引物和TaqMan探针,对引物的特异性与敏感性进行评估;以12种血清型BTV毒株和核酸阳性血液样本验证建立的血清型RT-qPCR检测方法;将其应用于库蠓与动物血液样本中BTV的定型。【结果】建立的BTV血清型RT-qPCR检测方法具有良好的特异性与灵敏性,反应的扩增效率(E)值90.3%,相关系数(R2)值在0.991-0.999之间,对12种血清型BTV核酸的检测下限在25-48拷贝之间。对165株BTV的RT-qPCR定型结果与病毒的Seg-2测序鉴定结果一致;对194份采集于哨兵动物的BTV核酸阳性血液样本的RT-qPCR定型结果与感染动物上分离BTV的血清型一致。采用建立的方法,从2019年云南省师宗县与景洪市采集的库蠓与牛血液样本中鉴定出6种血清型的BTV(BTV-1、-2、-4、-5、-16和-24)。【结论】研究建立的12种BTV血清型RT-qPCR定型方法具有特异、敏感和省时的优点,可用于媒介与动物感染BTV的血清型定型,具有良好的应用与推广价值。     

鸭疫里默氏杆菌流行菌株的分离鉴定及生物学特性

【背景】鸭疫里默氏杆菌(Riemerella anatipestifer,RA)是引起雏鸭、鹅、火鸡等多种家禽、野禽发生传染性浆膜炎的病原,在全世界范围内广泛存在,危害养禽业发展,造成严重的经济损失。【目的】了解国内RA的流行现状,探究该菌的生物学特性,更好地指导防控鸭疫里默氏杆菌病。【方法】对2020-2021年从山东省、河北省、广东省、山西省等地分离的RA疑似菌株进行PCR、生化和血清型鉴定,并根据药物敏感性结果分析耐药现象,根据半数致死量(median lethal dose,LD₅₀)测定结果分析致病力差异。【结果】共鉴定78株RA分离株,其中,血清1型4株、2型21株、10型11株、6型3株、7型17株,1株有交叉凝集现象,21株血清型未定型;药敏结果显示78株分离株对多粘菌素B、磷霉素、克林霉素等的耐药性最强,对头孢拉定、多西环素、呋喃唑酮、氟苯尼考等抗生素最为敏感,并且78株分离株均存在多重耐药性,其中77株耐药5重及以上;动物试验结果显示,RA的分离株致病力普遍较强,而且不同地区分离株、同地区不同分离株之间均存在差异,数株RA分离株的LD₅₀在10⁴-10⁷ CFU不等。【结论】RA在国内流行表现了强致病力及严重耐药情况,本研究结果为更好地预防控制、临床用药及后续致病机制研究提供了参考依据。     

苏北地区规模化猪场产肠毒素大肠杆菌的分离鉴定及灭活疫苗效果评估

[目的]产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)是引起仔猪腹泻的重要病原菌,本研究通过调查苏北地区规模化猪场ETEC的流行情况,分析其生物学特性,研制具有免疫保护效果的优势血清型菌株的灭活疫苗,以期对苏北地区ETEC的防控提供参考。[方法]从苏北地区规模化猪场采集3-30日龄的仔猪新鲜粪样、肛拭子及小肠组织样,分离出ETEC,对分离菌株进行血清型鉴定、耐药性测定、小鼠致病力测定;最后通过动物免疫试验研究优势血清型菌株灭活疫苗对小鼠的免疫保护效果。[结果]从21个规模化猪场采集病料562份,通过PCR鉴定及测序得到141株ETEC;血清凝集试验鉴定出85株菌的O抗原血清型,其中08、0101和0128为优势血清型,占定型菌株的61.2%(52/85),其他血清型包括09、03、020、0148、0149等;分析141株ETEC对14种常见抗生素的耐药情况,得出分离株对新霉素、红霉素、四环素、庆大霉素、强力霉素、阿莫西林、甲氧苄啶/磺胺甲恶唑高度耐药,耐药率均高达80%以上;对恩诺沙星敏感性较高,敏感率达50.4%(71/141);对多粘菌素B和头孢噻肟中介耐药,占比分别为66%(93/141)和51.8%(73/141);多重耐药现象严重,其中10重耐药的菌株占比最大,为19%(27/141);小鼠攻毒试验测得08血清型强毒株YC-6的半数致死量(median lethal dose,LD50)为1.4×10^7 CFU/只,最低致死量(minimum lethal dose,MLD)为3×10^7 CFU/只;08血清型强毒株YC-6和0101血清型强毒株LYG-3制备的单价灭活疫苗对小鼠的保护率均达到100%,因此利用08血清型强毒株YC-6和0101血清型强毒株LYG-3研制二价灭活疫苗,结果显示该二价疫苗对感染不同血清型ETEC小鼠的保护率在83%以上。[结论]本研究通过对苏北地区ETEC的流行病学调查,得出其优势血清型,并研制出针对对优势血清型免疫保护效果较好的二价灭活疫苗,给临床ETEC的监测和防控提供参考。     

The molecular epidemiology of Foot-and-Mouth Disease virus serotypes A and O from 1998 to 2004 in Turkey

Background

Foot-and-Mouth Disease (FMD) causes significant economic losses in Turkish livestock. We have analysed the genetic diversity of the 1D sequences, encoding the hypervariable surface protein VP1, of Turkish isolates of serotype A and O collected from 1998 to 2004 in order to obtain epidemiological and immunological information.

Results

The 1D coding region of 33 serotype O and 20 serotype A isolates, obtained from outbreaks of FMD between 1998 and 2004, was sequenced. For serotype A, we confirmed the occurrence of the two subtypes IRN99 and IRN96. These subtypes are most divergent within the region encoding the immuno-dominant GH-loop. Also a close relationship to Foot-and-Mouth Disease virus (FMDV) serotype A isolates obtained from outbreaks in Iraq and Iran were detected and a clustering of isolates collected during the same period of time were found. The analysis of the deduced amino-acid sequences of these subtypes revealed evidence of positive selection in one site and one deletion, both within the GH-loop region. By inferring the ancestral history of the positively selected codon, two potential precursors were found. Furthermore, the structural alignment of IRN99 and IRN96 revealed differences between the tertiary structures of these subtypes. The similarity plot of the serotype O isolates suggested a more homogeneous group than the serotype A isolates. However, phylogenetic analysis revealed two major groups, each further divided in subgroups, of which some only consisted of Turkish isolates. Positively selected sites and structural differences of the Turkish isolates analysed, were not found.

Conclusion

The sequence and structural analysis of the IRN99 strains is indicative of positive selection suggesting an immunological advantage compared to IRN96. However, results of antigenic comparison reported elsewhere do not substantiate such a conclusion. There is evidence that IRN99 was introduced to Turkey, in all probability from Iran. Since, a member of the IRN96 lineage was included as a component of the FMDV vaccine produced since 2000, the outbreaks caused by IRN96 strains in 2004 could be due to incomplete vaccine coverage. The Turkish type O strains, all with a VP1 structure similar to the O1/Manisa/69 vaccine, appear in several sublineages. Whether these sublineages reflect multiple samplings from a limited number of outbreaks, or if they reflect cross-boundary introductions is not clear.     

广州地区GⅡ.17型诺如病毒GZ-L343毒株病毒样颗粒的构建与免疫特征的鉴定

【背景】人源诺如病毒是急性胃肠炎暴发的主要原因,GII.4是过去几十年的主要流行基因型。2014/2015年出现的GII.17型变异株是中国首例导致大规模暴发的非GII.4流行株。通过对来自华南地区的诺如病毒GII.17型毒株的完整基因组序列进行分析,证实了该GII.17型突变株与先前确定的GII型变异株不同。【目的】制备广州地区GII.17型诺如病毒GZ-L343的病毒样颗粒,并系统表征其免疫原性及功能特性。【方法】借助杆状病毒表达系统制备GII.17-GZ-L343的病毒样颗粒,并通过氯化铯梯度超速离心对其进行纯化,制备抗血清并对其免疫功能进行评价。【结果】聚丙烯酰胺凝胶电泳和蛋白质免疫印迹结果表明所得蛋白分子量大小约为58kDa;透射电镜结果表明病毒样颗粒直径约为30nm;酶联免疫吸附测定结果显示该病毒样颗粒具有较好的免疫原性;唾液组织血型抗原的体外受体结合测定表明,该病毒样颗粒与部分A型、B型、O型及AB型分泌及非分泌血型样本存在阳性结合;效价测定结果表明免疫所得血清效价在104以上;交叉反应结果表明该抗血清与异型病毒样颗粒不存在交叉反应。此外,体外阻断结果表明,该抗血清仅能阻...     

Characteristics of the Shiga-toxin-producing enteroaggregative Escherichia coli O104:H4 German outbreak strain and of STEC strains isolated in Spain

A Shiga-toxin-producing Escherichia coli (STEC) strain belonging to serotype O104:H4, phylogenetic group B1 and sequence type ST678, with virulence features common to the enteroaggregative E. coli (EAEC) pathotype, was reported as the cause of the recent 2011 outbreak in Germany. The outbreak strain was determined to carry several virulence factors of extraintestinal pathogenic E. coli (ExPEC) and to be resistant to a wide range of antibiotics. There are only a few reports of serotype O104:H4, which is very rare in humans and has never been detected in animals or food. Several research groups obtained the complete genome sequence of isolates of the German outbreak strain as well as the genome sequences of EAEC of serotype O104:H4 strains from Africa. Those findings suggested that horizontal genetic transfer allowed the emergence of the highly virulent Shiga-toxin-producing enteroaggregative E. coli (STEAEC) O104:H4 strain responsible for the outbreak in Germany. Epidemiologic investigations supported a linkage between the outbreaks in Germany and France and traced their origin to fenugreek seeds imported from Africa. However, there has been no isolation of the causative strain O104:H4 from any of the samples of fenugreek seeds analyzed. Following the German outbreak, we conducted a large sampling to analyze the presence of STEC, EAEC, and other types of diarrheagenic E. coli strains in Spanish vegetables. During June and July 2011, 200 vegetable samples from different origins were analyzed. All were negative for the virulent serotype O104:H4 and only one lettuce sample (0.6%) was positive for a STEC strain of serotype O146:H21 (stx1, stx2), considered of low virulence. Despite the single positive case, the hygienic and sanitary quality of Spanish vegetables proved to be quite good. In 195 of the 200 samples (98%), <10 colony-forming units (cfu) of E. coli per gram were detected, and the microbiological levels of all samples were satisfactory (<100 cfu/g). The samples were also negative for other pathotypes of diarrheagenic E. coli (EAEC, ETEC, tEPEC, and EIEC). Consistent with data from other countries, STEC belonging to serotype O157:H7 and other serotypes have been isolated from beef, milk, cheese, and domestic (cattle, sheep, goats) and wild (deer, boar, fox) animals in Spain. Nevertheless, STEC outbreaks in Spain are rare.     

Spatial trend of Foot and Mouth Disease virus (FMDV) serotypes in cattle and buffaloes, Pakistan

The present study describes the frequency of Foot and Mouth Disease (FMD) virus serotypes (O, A and Asia-1) in major regions (all provinces) of Pakistan using Indirect Sandwich ELISA. Also, spatial distribution of various FMD serotypes and their comparison is discussed. A total of 590 samples (Epithelial tissue) have been analyzed during a period of five years (2005–2009). Out of 590 samples, 180 were found positive, giving an overall confirmation of FMDV about 33.2 %. Of the prevalent serotypes, FMDV ‘O’ serotype caused most outbreaks (20.7 %), followed by serotype A (6.6 %) and serotype Asia-1 (4.6 %) while there was no positive case of type ‘C’. The study clearly showed that the disease was more frequent in the agro-climatic zones than in hilly areas. Based on the data of 590 samples (>50 outbreaks), the overall prevalence of FMDV in cattle and buffaloes in Pakistan was 33.2 %, while in cattle alone, it was 37.1 %, higher than in buffalo (28.7 %). There were eight cases of mixed serotypes infection, indicating the presence of endemic state of disease. Another significant feature was the change over time. In phase-I (2005–2007), there was an overall prevalence of 29.4 %, while the occurrence of the serotype O, A and Asia-1 was 20.4 %, 2.9 % and 4.7 %, respectively. During phase-II (2008-2009), the overall prevalence was 59.21 %, while those of serotype O, A and Asia-1 were 22.4 %, 31.6 % and 4.0 %, respectively. This clearly indicated a shift from serotype O to A, which may help to explain the occurrence of more severe outbreaks, despite vaccination.