霍乱弧菌山梨醇发酵相关基因序列特征分析

目的 比较霍乱弧菌流行株与非流行株山梨醇发酵相关基因的差异，为采用分子生物学方法快速区分两类菌株提供理论依据。方法采用基因测序的方法，分别对42株霍乱弧菌(01群埃尔托型33株、0139群9株)流行株和非流行株的糖发酵激活蛋白、外周质麦芽糖结合蛋白、外周质磷酸盐结合蛋白和外周质氨基酸结合蛋白编码基因进行序列比较。结果糖发酵激活蛋白编码基因在霍乱弧菌流行株与非流行株共发现三个位置有单个碱基的差异，即第106、150和378位核苷酸(在流行株分别为A、A和T，而在非流行株分别为G、G和C)。第106位碱基的差异导致了氨基酸的不同(编码蛋白的第36个氨基酸在流行株和非流行株分别为苏氨酸和丙氨酸)。外周质麦芽糖结合蛋白和外周质磷酸盐结合蛋白编码基因各发现两个碱基的规律变化，外周质氨基酸结合蛋白编码基因未发现一致性碱基改变。结论在这些基因中存在着多个单核苷酸多态性，可能会成为快速区分两类菌株的重要依据。糖发酵激活蛋白第36位氨基酸的改变，可能会引起该蛋白活性的改变。     

中国部分甲型肝炎病毒流行株结构蛋白区基因特征分析

目的分析中国部分甲型肝炎(甲肝)病毒(Hepatitis A Virus,HAV)流行株结构蛋白VP3~VP1编码区核苷酸及氨基酸序列特点,并比较与甲肝减毒活疫苗(Hepatitis A Attenuated Live Vaccine,Hep A-L)标本序列差别。方法收集39份甲肝病例急性期血清样本及2份市售Hep A-L标本,经核酸提取、逆转录及巢式聚合酶链反应,测得结构-非结构蛋白VP3~VP1-2A编码区序列,进行基因亲缘关系、氨基酸序列同源性等分析。结果检测到的部分HAV流行株序列均为IA亚型,其在结构-非结构蛋白VP3~VP1-2A编码区核苷酸和氨基酸序列同源性分别为94.8%~100%和99.3%~100%,与检测的2份Hep A-L标本的核苷酸和氨基酸序列同源性为90.6%~91.9%和99.1%~99.4%。其中的1条序列在VP3-56位,发生丝氨酸(Serine,Ser)→脯氨酸(Proline,Pro)替代;3条序列在VP1-112位,发生苏氨酸(Threonine,Thr)→异亮氨酸(Isoleucine,Ile)替代,靠近中和抗原位点VP1-114位;所有序列在已发表的中和抗原位点处氨基酸无变化。选择压力分析表明,检测到的部分HAV流行株序列在VP3~VP1编码区处于负向选择。检测的2份Hep A-L标本序列均为IB亚型,在已发表的中和抗原位点处氨基酸无变化。结论HAV的结构蛋白编码区核苷酸序列存在一定差异,但其氨基酸序列相对保守。检测到的3条HAV流行株序列在中和抗原位点附近发生氨基酸替代,值得进一步研究。     

浙江省麻疹病毒流行株血凝素基因的变异及其与国内外流行株的差异

目的 探讨浙江省麻疹病毒(measles virus,MV)流行株血凝素基因的变异情况,及其与国内外流行株的差异.方法 选取浙江省1999-2011年分离保存的麻疹病毒流行株33株,提取病毒核酸,采用RT-PCR法扩增其血凝素(H)基因全序列,并与GenBank上下载的中国麻疹疫苗株沪191,以及95株国内外各基因型麻疹毒株的H基因序列进行比较分析.结果 从浙江省1999-2011年麻疹患者咽拭子标本中分离麻疹病毒株33株,与1993-2007年我国其他地区麻疹流行株构建H基因系统进化树,结果表明33株浙江麻疹流行株均属于H1a亚型,流行株分布无明显时间和地域特征.以疫苗株沪191为参考株,我国2003-2011年H1a亚型流行株H基因的核苷酸、氨基酸平均变异率以及进化率分别为5.15％、4.44％和5.81％,分别高于1965-1993年(4.75％、3.86％和5.30％),以及1994-2002年(4.80％、4.08％和5.37％)流行株平均变异率与进化率;3个时间段流行株H基因非同义替换率与同义替换率的比值(dn/ds)分别为0.19、0.21和0.23,表明1993-2011年我国H1a型麻疹流行株尚未受到明显的正向选择作用.我国2003-2011年H1a亚型毒株与沪191之间,在H基因上共存在24个稳定的氨基酸变异位点;H1a亚型流行株与A基因型疫苗株间差异最大,遗传距离为0.053,氨基酸差异数为26 ～28个,而B3、D4型代表株与沪191间稳定的氨基酸差异仅为15个.另外,H1a型流行株与B、D基因型毒株间差异明显,组间遗传距离分别为0.074和0.071,氨基酸差异数达27～33个.结论 麻疹病毒H1a亚型流行株与疫苗株以及其他基因型麻疹病毒株,在H基因上存在明显差异,且该差异随时间累积而增大,应该引起足够的重视.     

新疆维吾尔自治区2003～2004年麻疹野病毒分离株基因特征分析

目的了解新疆维吾尔自治区流行的麻疹野病毒的基因型或亚型特征,为消除麻疹提供科学依据。方法对2003~2004年用健康产婴脐血单个核细胞分离的麻疹野病毒进行分子流行病学分析。采用逆转录-聚合酶链反应对麻疹病毒核蛋白(N)基因羧基(COOH)末端676个核苷酸片段进行扩增,并测定扩增产物的核苷酸序列。通过分析COOH末端450个核苷酸序列构建基因亲缘关系树,并分析毒株变异情况。结果2003年和2004年各分离麻疹病毒3株(XJ03-26、XJ03-27、XJ03-74;XJ04-146、XJ04-150、XJ04-152),除XJ03-26株与沪191株核苷酸差异<1%,属疫苗相关株A基因型外,其余5株均为H1基因型。其中XJ03-27、XJ03-74、XJ04-150、XJ04-152与H1a参考株China9322的核苷酸差异为0.5%~1.6%,同属于H1a亚型;XJ04-146与H1b参考株China9475的核苷酸同源性为98.7%,属于H1b亚型。4株H1a毒株分属两组(即XJ03-27与XJ04-150,XJ03-74与XJ04-152),每组内毒株N基因COOH末端450个核苷酸序列几近相同或无差异。结论新疆维吾尔自治区2003~2004年流行的麻疹病毒以H1a亚型为主,亦存在H1b亚型。新疆维吾尔自治区流行的麻疹野病毒间存在较大基因变异,不同年份存在相同麻疹病毒的持续循环传播,需加强麻疹疫苗的预防接种及病毒学监测。     

新疆地区2003-2004年麻疹野病毒分离株基因特征分析

目的了解新疆地区流行的麻疹野病毒基因型和亚型特征。方法对2003-2004年用健康产婴儿脐带血单个核细胞分离的麻疹野病毒进行分子流行病学分析。采用逆转录-聚合酶链反应对麻疹病毒N基因C末端676个核苷酸片段进行扩增，并对扩增产物进行核苷酸序列测定。通过分析C末端450个核苷酸序列构建基因亲缘关系树，并分析毒株变异情况。结果2003年和2004年各分离麻疹病毒3株（XJ03-26、XJ03-27、XJ03-74、XJ04-146、XJ04-150、XJ04-152），除XJ03-26株与沪191株核苷酸差异〈1％，属疫苗相关株A基因型外，其余5株均为H1基因型。其中XJ03-27、XJ03-74、XJ04-150和XJ04-152与H1a参考株China9322的核苷酸差异为0．5％～1．6％，同属于H1a亚型；XJ04-146与H1b参考株China9475的核苷酸同源性为98．7％，属于H1b亚型。4株H1a毒株分属两组（即XJ03-27与XJ04-150，XJ03-74与XJ04-152），每组内毒株N基因碳末端450个核苷酸序列几近相同无差异，但两组间毒株却存在较大变异，核苷酸差异达6．1％（27个核苷酸差异）。结论新疆地区2003-2004年流行的麻疹病毒以H1a亚型为主，亦存在H1b亚型。     

霍乱弧菌4种新类型tcpA基因发现及序列分析

目的研究中国霍乱弧菌（VC）毒力协同调节菌毛A亚单位基因（tcpA）的多态性.方法分别进行聚合酶链反应及限制性片段长度多态性分析、型特异引物PCR分型、基因测序及序列分析.结果 200株O1群埃尔托型（EVC）产毒株和100株O139群VC产毒株的tcpA基因均与EVC国际标准株N16961株为同一类型（t2型）.50株EVC非产毒株中只有3株携带tcpA基因,1株为t2型,另2株为2种新类型.20株O139群VC非产毒株均不携带tcpA基因.20株非O1非O139群VC中只有2株携带tcpA基因,且为2种新类型.4种新类型tcpA基因与国外发现的4种类型比较,基因序列及推测的氨基酸序列同源性分别为58.3%～77.5%和61.0%～85.5%;5′端约102bp基因序列基本一致,推测的氨基酸序列完全一致;3′端约210bp基因序列变异最大,推测的氨基酸序列变异也最大.抗原表位分析,C末端差异最大.结论建立快速准确区分不同类型tcpA基因的方法,并发现4种新类型.     

吉林省2001～2008年麻疹病毒血凝素蛋白基因特征和氨基酸变异分析

目的研究吉林省2001～2008年麻疹病毒代表株的血凝素蛋白基因（Hemagglutinin,HA）特征和氨基酸变异。方法从吉林省2001～2008年流行的麻疹野病毒中选取5株代表株,提取核糖核酸,用逆转录-聚合酶链反应扩增HA基因片段,对扩增产物进行核苷酸序列分析,并与基因库中的中国麻疹疫苗病毒株沪191以及所有23种麻疹野病毒基因型代表株的H基因序列,进行基因亲缘关系、同源性、氨基酸变异以及糖基化位点变异比较。结果吉林省5株麻疹病毒分离株均属于H1基因型H1a基因亚型,5株病毒H基因之间有6～17个核苷酸差异（0.3%～0.9%）,3～9个氨基酸差异（0.5%～1.5%）。与中国现行疫苗病毒株沪191H基因相比,有89～95个核苷酸差异（4.8%～5.1%）,25～30个氨基酸差异（4.1%～4.8%）。与H1a基因亚型代表株China93-4H基因相比,有9～17个核苷酸差异（0.5%～0.9%）,3～7个氨基酸差异（0.5%～1.2%）。吉林省5株麻疹病毒分离株的H蛋白均保留了4个糖基化位点,第5个糖基化位点在氨基酸第240位由丝氨酸突变成天冬酰胺,导致一个潜在糖基化位点NLS238～240的缺失。结论吉林省2001～2008年流行的5株麻疹病毒,均丢失了一个可能影响抗原性的糖基化位点。5株麻疹病毒的核苷酸、氨基酸差异均不大,但无论与H1a基因亚型代表株China93-4相比,还是与疫苗株S191相比,核苷酸和氨基酸的同源性均呈逐年递减趋势,吉林省麻疹野病毒H基因的变异仍在逐年增加、逐年积累。     

广东省流行的3株登革热2型病毒基因组全序列测定及分析

目的测定广东省3株登革热2型病毒的全序列,探讨其来源及基因型。方法运用RT PCR法扩增来自广东省的登革热2型GD09/93、GD05/98和GD19/2001病毒株的全序列,采用遗传距离法构建进化树。结果3株登革热2型病毒的全基因组长度均为10 723 nt,5′及3′端各有一非编码区,结构基因和非结构基因位于基因组97~10 269 nt之间,共编码3391个氨基酸,读码结构完全相同。GD05/98与GD09/93、GD05/98与GD19/2001、GD09/93与GD19/2001之间的碱基序列同源性分别为93.3%、92.4%和97.6%,氨基酸序列同源性分别为96.7%、96.5%和98.5%。结论3株登革热2型病毒均对乳鼠致病,与对乳鼠不致病的参考株（DEN2-04株）比较共有18个氨基酸位点的差异引起极性或电荷变化,PrM-134、NS2A-153、NS4B-102所带电荷的变化对抗原性影响较大。GD05/98株与泰国分离株同为Ⅱ基因型,GD09/93和GD19/2001株与印度尼西亚、澳大利亚、台湾株分在Ⅳ基因型;表明我国登革热2型病毒有不同的基因型,而不同时期也存在同一种基因型传播。     

浙江省野生动物鼬獾狂犬病毒全基因组序列测定分析

目的 测定浙江省分离的2株野生动物鼬獾狂犬病毒株全基因组序列,从分子水平进行遗传变异特征分析,了解狂犬病毒在浙江省的流行和变异情况.方法 RT-PCR测定鼬獾狂犬病毒株全基因组核苷酸序列,并进行基因序列和编码蛋白相似性比较及种系发生分析.结果 测序获得2株鼬獾狂犬病毒全基因组核苷酸序列信息:基因组全长11 923 nts,leader长58 nts,由5个编码区组成:NP(1353 nts)、PP(894 nts)、MP(609 nts)、GP(1575 nts)、LP(6386 nts),N-P-M-G间隔序列长2、5、5 nts;G-L基因间伪基因ψ长423 nts;trailer长70 nts.核酸BLAST及多序列比对显示,浙江省鼬獾狂犬病毒株全基因组序列的组成和结构符合弹状病毒科狂犬病毒属特征;鼬獾病毒株负链RNA基因组5个基因编码氨基酸的长度没有变异,编码区基因没有发生重组,编码蛋白仅表现较少的序列变化,多数只发生碱基的替代;中国病毒株之间特别是同种动物狂犬病毒之间各个基因区域核苷酸与氨基酸序列相似性最高,鼬獾狂犬病毒基因组序列相似性在氨基酸水平明显高于核苷酸水平,蛋白质编码基因的核苷酸变异大多属于同义突变.结论 鼬獾狂犬病毒与研究中选择的代表性疫苗株或者街毒株的变异位点和变异类型相似,多序列相似性比较和N基因种系发生分析显示,鼬獾狂犬病毒均属于基因1型,具有中国地域性特点,2株野生动物鼬獾狂犬病毒极有可能是存在于自然界中固有的街毒株.     

吉林省2013年肠道病毒71型VP1编码区基因特征分析

目的为了解吉林省2013年肠道病毒71型(EV71)流行株基因学特征。方法对吉林省2013年手足口病来源的EV71阳性分离物进行VP1编码区逆转录-聚合酶链反应(RT-PCR)扩增,并对扩增产物进行核苷酸序列测定,使用Mega4和Bioedit软件对扩增序列进行基因亲缘关系和氨基酸位点变异分析。结果吉林省2013年分离到的7株EV71同属于C4a基因亚型,并且在VP1编码区第22位氨基酸位点均由谷氨酰胺(Q)突变为组氨酸(H),这一结果与2008年安徽阜阳株突变一致;有2株在VP1编码区第289位点由丙氨酸(A)突变为缬氨酸(V)。结论吉林省2013年的7株EV71流行株与C4a代表株位于同一分支,属于C4a基因亚型,且与2008年阜阳株(EU703813-Fuyang-08/2-C4a)亲缘关系最近,而且发生了氨基酸位点变异。     

Investigation of the 1994-5 Ukrainian Vibrio cholerae epidemic using molecular methods.

Thirty-seven Vibrio cholerae and four non-cholera Vibrio isolates from Ukraine, including strains from the epidemic of 1994-5, were analysed by molecular methods. Results from PFGE and ribotyping indicated that all Ukrainian toxigenic V. cholerae were closely related to each other and to an isolate from a patient from Pakistan. A non-toxigenic river water strain obtained during the height of the epidemic was more distantly related to these V. cholerae strains, while the Vibrio parahaemolyticus isolates and Vibrio alginolyticus isolate were not closely related to V. cholerae or each other. ERIC- and REP-PCR allowed the differentiation of strains identical by other methods. The results obtained confirm that the epidemic Ukrainian strains are most closely related to seventh pandemic strains from Asia and support a hypothesis that the Ukrainian epidemic of 1994-5 was caused by toxigenic environmental strains surviving since the time of the 1991 Ukrainian epidemic or before.     

B lymphocyte migration to the bone marrow of humans is not random

This research is a result of analysing a total of 20 independently isolated immunoglobulin gene sequences from a defined population of mature B cells found in the bone marrow of five healthy adults. Each individual exhibited at least two identical gene sequences in our sample. Each variable immunoglobulin sequence is the product of the recombination of three gene segments, V, D and J genes. All sequences sampled contained a specific V gene, V5-51. Comparing the particular J and D genes expressed together with V5-51 as well as the junctional modifications between these genes established the relatedness of the sequences. The likelihood of finding at least two out of k identical gene sequences, if they occur randomly, is found and compared to the experimentally determined sequences. We conclude that it is unlikely that the B cells containing the related sequences found in the bone marrow is by chance. This suggests that this particular population of B cells migrate to the bone marrow in a co-ordinated fashion.     

Phenotypic and molecular characterization of Vibrio cholerae O1 isolated in Samutsakorn,Thailand before,during and after the emergence of V. cholerae O139.

Seventy clinical strains of Vibrio cholerae O1 isolated from 1982-96 in Samutsakorn, a port city 30 km southwest of Bangkok where cholera occurs at low levels with regular seasonality, were characterized to investigate if there were any differences among the O1 strains isolated before, during and after the 0139 epidemic. Pulsed-field gel electrophoresis (PFGE) typing, ribotyping and southern blot hybridization with a cholera toxin probe (CT genotyping) demonstrated several genotypes among O1 strains isolated before the emergence of V. cholerae 0139. However, O1 strains isolated during and after the advent of 0139 showed identical ribotypes which were distinctly different from the types identified in strains isolated before the emergence of 0139. Ribotypes identified in strains during and after the advent of 0139 were also demonstrated by O1 strains isolated immediately before the emergence of 0139. Considering the seasonality of cholera in Samutsakorn, the identical ribotype and CT genotype and the closely related PFGE types shown by all O1 strains isolated during and after the appearance of 0139 is remarkable and suggest that the V. cholerae O1 strain may reemerge from an environmental source. A subgroup of V. cholerae O1 strains isolated before the emergence of the 0139 epidemic had a ribotype identical to a type demonstrated by 0139 strains isolated in Thailand. Our results support similar findings in Bangladesh and India that a distinct O1 strain appeared during the 0139 epidemic. However, compared with the apparent identical strain which replaced 0139 in Bangladesh and India, the emerged O1 strain in Samutsakorn showed a different ribotype and CT genotype.     

Molecular identification of pathogenicity genes and ERIC types in Vibrio cholerae O1 epidemic strains from Mozambique

The phenotypic and genotypic profiles of the V. cholerae strains causing the Mozambican 1997-8 epidemic were characterized to provide a reference for comparison with other epidemic strains. A total of 75 strains of V. cholerae O1 isolated in different provinces, were analysed. Strains were characterized by PCR for detecting toxin genes (ctxA, zot and ace), virulence associated genes (tcpA. nanH, hlyA and torR) and ERIC sequences. All V. cholerae strains were serotype O1, Ogawa, biotype El Tor. MIC testing showed a high proportion of strains multi-resistant to drugs (100% to cotrimoxazole and 52% to tetracycline) and susceptibility to ciprofloxacin. The isolates contained two intact copies of the CTX genetic element and all other genes tested. PCR of restricted DNA revealed two ERIC types: the first in provincial isolates, also predominant in other African epidemic strains, and the second in Maputo isolates (the national capital).     

Preferential association of the heat-stable enterotoxin gene (stn) with environmental strains of Vibrio cholerae belonging to the O14 serogroup

Toxigenic Vibrio cholerae O1 and O139 serogroups have the capacity of causing epidemic and pandemic cholera but are infrequently found in the environment. The other serogroups are abundant in aquatic environments but do not possess the virulence genes necessary for causing the disease. Of the 559 environmental strains of V. cholerae, collected during different periods from environmental samples in Calcutta, 9 (1.6%) harboured the heat-stable enterotoxin gene (stn). Six of the 9 strains belonged to the O14 serogroup. Thus, V. cholerae strains carrying the stn gene revealed preferential association with the O14 serogroup. Three of the six strains harboured the tcpA gene of the E1 Tor type, which is an unusual feature among environmental V. cholerae strains. A strain that possessed the E1 Tor type tcpA also had the CTX prophage. Pulsed field gel electrophoresis (PFGE) revealed that the stn gene positive O14 strains of V. cholerae were not clonal.     

山东省分离的霍乱弧菌毒力基因的研究

目的：了解山东省不同年代、不同地区收集的霍乱菌株携带的毒力基因情况、分布特点和变迁。方法：应用分子杂交、PCR技术对霍乱弧菌的CTX基因元件中的ctxA、zot基因，VPI毒力岛中的tcpA、tcpH、aldA、toxT、acfB基因，TLC因子中的cri、orf2—3基因，RTX基因簇中的rtxA、rtxC，以及toxR基因进行了检测。结果：用原位杂交检测，180株霍乱弧菌中有168株携带CTX遗传单元中的ctxA、zot、RS1基因，阳性率均为93．33％，表现出高度的一致性。用打点杂交检测不同时期的28株01群霍乱弧菌，所有被检菌都具有ToxR、RTX相关基因，在1980～1987年间分离的O1群霍乱弧菌流行株中，有92．86％携带编码TLC因子的基因，而1994～2000年间分离的流行株菌株中编码TLC因子的基因携带率仅有7．14％。Southern杂交结果显示，48株O1群霍乱弧菌可产生大小为9．4kb和6．5kb左右的两条ctxA杂交带，用zot基因探针进行杂交，11株O1群霍乱弧菌可产生与ctxA杂交带同样大小的条带。1株O139霍乱弧菌具有检测的所有毒力基因，但其ctxA（zot）杂交带与O1群霍乱弧菌有差异。结论：目前已知的霍乱弧菌毒力基因在山东省分离到的菌株中有广泛的分布，并且随时间的推移而发生变迁。     

从海、水产品中检出3株霍乱弧菌的报告

目的:了解奉贤区海水产品中霍乱弧菌的污染状况,为霍乱防治工作提供实验依据。方法:依据《霍乱防治手册》第五版进行霍乱弧菌分离培养,生化及血清学鉴定,同时用PCR方法进行检测。结果:从20件样品中检出霍乱弧菌3株,其中1株为O1群霍乱弧菌稻叶27L型,另外2株分别为O1群霍乱弧菌小川9L型和15d型。结论:本次检出的3株霍乱弧菌均为非流行株,但仍需加强对海水产品的主动监测,预防霍乱流行株的出现而导致霍乱的散发和暴发流行。     

Surveillance methodology for Vibrio cholerae in environmental samples

The purpose of this study was to examine the prevalence of Vibrio cholerae in environmental water samples by using a series of biochemical tests. A total of 223 V. cholerae-like bacteria were isolated from TCBS agar after spreading the alkaline peptone water enriched sewer (n = 21) and water (n = 16) samples. All oxidase positive isolates were subjected to confirmation for V. cholerae by seven other biochemical tests and polymerase chain reaction. Only 74.2% isolates were found to be V. cholerae by PCR using primers against an outer membrane protein (ompW) gene, out of which only 2 isolates were positive for cholera toxin (ctxAB) gene. Among the various biochemical tests studied, arginine hydrolysis, arabinose fermentation and string test showed 92 - 100% sensitivity and 42 - 67% specificity. Eight isolates including the toxigenic ones, showed agglutination with V. cholerae O1 antiserum. The present study showed that no biochemical test is 100% specific for V. cholerae. However, a few tests, if performed in a sequence after growing the alkaline peptone water enriched samples onto TCBS media can be used for screening of V. cholerae from the environmental samples. This study also showed that most of the environmental isolates are non-O1/non-O139 and the chances of presence of toxigenic V. cholerae are very rare in the environment.     

Comparison of pulsed-field gel electrophoresis and ribotyping for subtyping of Vibrio cholerae O139 isolated in Thailand.

Pulsed-field gel electrophoresis (PFGE) of Cpo I-digested genomic DNA and ribotyping (Bgl I) were applied to 60 Vibrio cholerae strains including 48 V. cholerae O139 from Thailand to compare their value in differentiating strains of the present V. cholerae O139 epidemic. PFGE patterns were divided into groups A and B representing five and four subtypes, respectively, while ribotyping showed four different patterns. PFGE group B subtypes were only presented among O139 isolates from Thailand, whereas four O139 strains from Bangladesh and India showed identical PFGE group A subtypes observed in O139 isolates from Thailand. Two nontoxigenic O139 isolates from Thailand showed different and unique PFGE types as did five V. cholerae non-O139 isolates containing a gene virulence complex found in V. cholerae O139. These results indicate that PFGE (Cpo I) can resolve recent evolutionary divergence within V. cholerae O139 and offers a useful supplementary tool for following the progressing V. cholerae O139 epidemic.