埃及伊蚊电压依赖性钠离子通道3''端cDNA的克隆及序列分析

目的 扩增埃及伊蚊电压依赖性钠离子通道基因3'末端,并对其核苷酸序列进行分析.方法 提取埃及伊蚊雌蚊总RNA,以Trsa为反转录引物反转录成单链cDNA,继而设计阳性与阴性对照,采用巢式聚合酶链反应(PCR)和快速扩增cDNA 3'末端(3'RACE)技术,对埃及伊蚊电压依赖性钠离子通道基因3'末端进行PCR扩增、克隆与测序.结果 获得钠离子通道基因的3'端核苷酸序列共809 bp,包括编码区和非编码区poly(A)尾,编码区共编码208个氨基酸.相似性分析结果 显示编码区氨基酸序列与家蝇钠离子通道基因3'端氨基酸序列相似性为65%左右.结论 成功获得埃及伊蚊钠离子通道基因3'端,为进一步研究该基因的分子生物学特性奠定了基础.     

埃及伊蚊抗凝血因子Xa基因片段的克隆测序

目的：探讨我国登革热的重要媒介蚊种埃及伊蚊唾液中的抗凝血因子Xa基因。方法：用兼并引物PCR方法从我国的埃及伊蚊基因组DNA扩增抗凝血因子Xa基因片段,克隆入测序T载体进行测序。结果：扩增出抗凝血因子Xa基因片段长度约300bp,测序后发现与报道的埃及伊蚊的cDNA基因相应序列多了一段63bp内含子,并有几个碱基和氨基酸序列变化。结论：我国海南岛的埃及伊蚊抗凝血因子Xa基因有一定差异,可能对应的是一个新的基因型。     

小鼠抵抗素基因及其反义核酸真核表达体系的构建与鉴定

目的构建载有小鼠抵抗素（resistin）基因及其反义核酸的重组真核表达质粒,为下一步进行resistin生物功能研究打基础。方法用resistin基因mRNA编码区序列特异引物,从小鼠脂肪组织中．通过RT—PCR的方法合成resistin cDNA,T4DNA连接酶将resistin cDNA克隆于pGEM-T载体,经双酶切及测序鉴定克隆成功后再亚克隆于pcDNA3．1^（＋）或pcDNA3．1^（-）真核表达载体,并测序鉴定。结果PCR产物长度与resistin cDNA理论长度363 bp相符;重组pGEM—T被EcoR Ⅰ和Xba Ⅰ内切酶切为约3000bp和355bp两个片段,测序结果表明插入pGEM-T的DNA片段的核苷酸序列与小鼠resistin基因mRNA编码区序列完全一致。重组pcDNA3．1^（＋）和pcDNA3．1^（-）测序结果表明插入的DNA片段分别与小鼠resistin基因mRNA编码区序列和反义resistin基因mRNA编码区核苷酸序列一致。结论成功克隆载有resistin基因和载有resistin基因反义核酸的重组真核表达质粒。     

潍坊市9例重症与轻症手足口病患者肠道病毒71型5′非编码区、VP1基因特征分析

目的 探讨重症手足口病肠道病毒71型(enterovirus 71,EV71)5′非编码区(untranslated region, UTR)、VP1区是否存在潜在的毒力位点。 方法 分离培养不同临床症状手足口病患者粪便标本中EV71,提取病毒总RNA,逆转录PCR扩增5′-UTR、VP1区后测定基因序列,对两个区域核苷酸序列及VP1区编码氨基酸序列进行比对和同源性分析,构建VP1区及不同临床症状EV71病毒5′- UTR基因进化树。 结果 本实验共获得9株EV71毒株(5株来源于重症病例,4株来源于轻症),5′-UTR、VP1区核苷酸同源性均为94.5%~ 99.7%,VP1区氨基酸同源性为97.3%~99.9%。9株EV71毒株5′-UTR共有67个核苷酸突变位点,与4株轻症EV71毒株相比,5株重症EV71毒株VP1区编码氨基酸有2处发生替换(S283T、A289T),5′-UTR共有37个核苷酸位点发生突变。VP1区基因进化树显示9个EV71毒株均属于C4a基因亚型,并且两组不同临床症状毒株处于同一较小分支中。5′-UTR核苷酸序列的系统进化树显示临床表现不同的EV71株呈交错分布,相同临床表现不单独聚类。 结论 9株EV71毒株均属于C4a基因亚型,5′-UTR核苷酸突变和VP1区氨基酸替换可能影响病毒毒力,对EV71病毒的全基因组序列特征分析及与宿主之间的相互作用机制进行研究非常必要。     

新疆猪戊型肝炎病毒株CHN-XJ-SW33全基因组序列分析

目的测定从新疆南部地区分离的猪戊型肝炎病毒(HEV)株全基因组序列,并在此基础上分析猪HEV与人源HEV的关系。方法设计HEV基因4型通用PCR引物,用逆转录巢式聚合酶链反应法(RT-nPCR)分段扩增猪HEV株CHN-XJ-SW33的全基因组序列;用cDNA末端快速扩增法(RACE)扩增其末端序列;对扩增的目的片段进行克隆测序,并对拼接后的基因组进行序列比对和进化分析。结果除3′poly(A)尾外,CHN-XJ-SW33基因组全长为7 238 nt,由3个开放读码框(ORF1-3)组成,分别编码1 706、674和114个氨基酸。CHN-XJ-SW33全基因组序列与HEV基因1-3型病毒株同源性仅为72.1%~74.9%,而与HEV基因4型病毒株同源性高达82.8%~95.5%,其中与日本人源中国输入型HEV株JKO-ChiSai98C同源性最高,为95.5%。基因进化分析显示,CHN-XJ-SW33属于HEV4a基因亚型。结论猪HEV与人HEV在全基因组核苷酸序列上高度同源,提示基因4型HEV可能由猪传染给人。     

日本血吸虫新基因精氨酸酶的扩增及序列分析

目的获取并真核克隆日本血吸虫新基因精氨酸酶全长cDNA.方法对本实验室曾获得精氨酸酶基因利用生物信息学进行分析,发现其5'端尚缺一段序列;根据已知序列设计引物,用5'端单巢式PCR从尾蚴文库中扩增,以获得精氨酸酶基因完整的阅读框(ORF);用生物信息学技术对获得的精氨酸酶编码基因进行结构与功能的分析;并将新基因的完整编码阅读框克隆入真核表达载体pCDNA3.1.结果用5'端单巢式PCR从尾蚴文库中获得了精氨酸酶5'端所缺序列,得到其完整的ORF,其ORF长1 095 bp,编码364个氨基酸.利用生物信息学技术鉴定其为日本血吸虫精氨酸酶的完整cDNA序列.重组质粒经双酶切DCR及测序鉴定证明日本血吸虫精氨酸酶真核表达质粒构建成功.结论成功获得、识别、扩增并克隆日本血吸虫精氨酸酶编码基因的全长cDNA.     

三氧化二砷反式激活基因1的克隆化

目的 克隆三氧化二砷反式激活靶基因1（AsTP1）。方法 从构建的三氧化二砷差异表达的肝HepG2细胞、T淋巴细胞cDNA消减文库筛选，利用RT-PCR和生物信息学分析相结合的方法获得新基因AsTP1的编码序列，对其氨基酸序列进行分析比较，并对其进行克隆化研究。结果 AsTP1基因编码区为243个核苷酸（nt），编码产物为80个氨基酸残基（aa）。经核苷酸序列数据库（GenBank）和蛋白质一级结构序列数据库（SwissProt）同源序列的搜寻，与已知基因序列之间没有显著同源性，说明克隆的AsTP1基因属于未知功能新基因。结论 发现三氧化二砷反式激活靶基因AsTP1，为进一步研究三氧化二砷的反式激活作用和揭示慢性砷中毒的分子机制提供新线索。     

日本血吸虫酪蛋白激酶Ⅱβ亚基的序列分析

目的 发现并克隆日本血吸虫新基因。方法 对已获得的日本血吸虫表达序列标签(EST)进行同源性分析。识别血吸虫新基因；根据EST设计引物，用3′RACE从mRNA中扩增获得新基因全长，用生物信息学技术对获得的编码基因进行结构与功能分析，将新基因的完整编码阅读框克隆入原核表达载体pGEX一4T—1。结果 用3′RACE获得一个EST的3′端所缺序列，得到完整编码阅读框，其开放阅读框((ORF)654bp，编码217个氨基酸。利用生物信息学技术确定其为日本血吸虫酪蛋白激酶Ⅱβ亚单位的编码基因。重组质粒经双酶切及测序鉴定，证明日本血吸虫CKⅡβ原核表达质粒构建成功。结论 扩增并成功克隆日本血吸虫酪蛋白激酶Ⅱβ亚单位编码基因的全长cDNA。为进一步的功能研究打下基础。     

分子生物学方法在儿童流行性感冒监测中的应用

目的：建立一种能够快速，特异，有效地直接从临床标本中检测流行感冒（流感）病毒并进行病毒型和亚型鉴别的方法，同时了解近年来北京地区A3型流感病毒因凝素重链（HA1）区基因的变异特点。方法：根据编码A、B型流感病毒膜蛋白M基因的核苷酸序列设计两对引物，用于同一逆转录（RT）－PCR反应中（多重RT－PCR），根据A、B型毒株扩增产物的大小（分别为506bp和261bp）鉴别A、B型流感病毒，另根据编码A1和A3型流感病毒糖蛋白HA基因的核苷酸序列设计两对引物，与B型M基因基因引物用同一RT－PCR反应中，可区分A1、A3或B型病毒HA基因和B型病毒M基因又设计了3对引物作为外侧引物，进行巢式－PCR反应。根据第二次PCR的扩增产物在1．2％的琼脂糖凝胶电泳中的大小即可确定型别。经PCR扩增1996－2002年间分离的北京地区A3型流感病毒株HA1区基因，测序并进行序列分析，结果：用上述多重RT－PCR鉴定流感病毒分离株156株，与血凝抑制试验阳性符合率为100％，用多重巢式－PCR检测92份已经病毒分离确定为流感的儿科临床呼吸道标本，与病毒分离和血凝抑制试验阳性符合率分别为76．9％（A1型）、57．1％（A3型）、86．5％（B型），对5株1996－2002年间A3间分离株HA1区基因序列分析结果显示，不同年份的分离株HA1区基因具有较高的核苷酸和氨基酸同源性，而且年份越近的毒株之间同生越测。为流感监测提供更加快速的新方法，北京地区A3型流感毒分离株HA1区基因持续不断地发生点突变，糖基化位点不断增多，可能是导致其抗原漂移的原因。     

HBsAg与抗-HBs同时阳性者体内S基因序列分析

目的 报告乙型肝炎病毒（HBV）表面抗原（HBsAg）／表面抗体（抗－HBs )同时阳性患血清中S区编码碱基序列及基氨基酸序列的为异特点。方法 以adr亚型的HBV基因序列为依据，设计特异性多聚酶链反应（PCR）引物，自2例HBsAg/抗－HBs均阳性的患体内扩增的HBV全S基因片段，克隆入pGEMTasy质粒，DNA测序确定病毒的变异特点。结果 测序结果发现双阳患与HBsAg阳性患血清中的HBV全S基因比较，核苷酸序列有4个核苷酸位点（0．3％）的替换突变，表面抗原氨基酸序列无特异性替换突变，但HBV多聚酶的间隔区氨基酸序列有3个位点的特异性替换突变。结论 HBV长期携带体内有HBV准种共存：HBsAg与抗－HBs同时阳性的原因不能归因于病毒的变异，应归结于患免疫系统的个体化反应。     

First record of Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) in North American Aedes albopictus

Aedes albopictus collected in the East St. Louis, Illinois, area were found infected with the gregarine protozoan, Ascogregarina taiwanensis. Infection rates varied from 67 to 95% at 4 sites and 0 to 10% at 2 others. Lower infection levels were found in Ae. epactius (42%) and Culex restuans (one larva). Four mosquito species were cross-infected in the laboratory with gregarines isolated from field-collected hosts. Aedes atropalpus was 90% susceptible to A. taiwanensis (100% in Ae. albopictus), with abnormal development and some melanization of trophozoites and gametocysts. In Ae. aegypti and Cx. restuans, the experimental infection was much lower (12-56%) and exhibited abnormalities similar to the Ae. atropalpus infections. Ascogregarina oocysts recovered from both Ae. aegypti and Ae. atropalpus hosts were subsequently infective to Ae. albopictus. In Ae. triseriatus, A. taiwanensis infection was very low (25%, 1-2 trophozoites per larva); gametocysts were not observed nor were infectious oocysts obtained. We conclude that A. taiwanensis, newly introduced to the USA with Ae. albopictus, can develop in 4 indigenous mosquito species and can produce deleterious effects in at least 2, Ae. aegypti and Ae. atropalpus.     

Evaluation of surveillance devices for monitoring Aedes aegypti in an urban area of northeastern Peru

In this study, we assessed the efficacy of the American Biophysics Corporation Standard Professional (ABC-PRO) light trap, the Omni-Directional Fay-Prince trap (with and without CO2), and the Centers for Disease Control and Prevention Wilton trap as a means of evaluating populations of adult Aedes aegypti in an urban area of northeastern Peru. Efficacies of collections from each of the trap types were compared to backpack-aspirator collections and human-landing collections. Collections were conducted twice daily, 3 days per week, for 27 wk from July 2001 to July 2002. Backpack-aspirator collections yielded significantly more mosquitoes (1,764) than any of the other collecting methods with a mean of 21.80 mosquitoes collected per sampling period. This method was less specific for Ae. aegypti than were human-landing collections because only 28.3% of mosquitoes collected with backpack aspirators were Ae. aegypti. Human-landing collections yielded only 23% (554/2,411) of the total mosquitoes collected. However, more than 80% (445/554) of the mosquitoes collected by this method were Ae. aegypti. None of the trapping devices evaluated collected mosquitoes, specifically Ae. aegypti, as effectively as backpack-aspirator or human-landing collections. The ABC-PRO trap, which was the most effective device in collecting mosquitoes, particularly Ae. aegypti, collected less than 2% of the total mosquitoes (mean of 0.12 mosquitoes/sampling period), and less than 3% of total Ae. aegypti (mean of 0.11 Ae. aegypti/sampling period). We conclude that none of the trap devices evaluated in this study is an acceptable alternative to backpack-aspirator or human-landing collections for monitoring populations of adult Ae. aegypti in Peru.     

Subsoil drain sumps are a key container for Aedes aegypti in Cairns, Australia

The contribution of subterranean drain sumps to pupal and adult populations of Aedes aegypti is reported for the 1st time in Cairns, Australia. Pupal surveys were used to quantify the relative contribution of drain sumps to the total population of Ae. aegypti by concurrent survey of sump and water-bearing containers in yards of inner-city premises. A total of 854 mosquito pupae were collected, predominantly Ae. aegypti and Culex quinquefasciatus (26.3 and 69.8%, respectively). Drain sumps provided a relatively uncommon (n = 4) but productive source for pupal Ae. aegypti, producing 14.7% of the combined yard and drain sump population. Drain sumps in inner-city Cairns most commonly occurred in parking lots (52.6%). Subsequently, a sticky emergent adult trap (SEAT) was developed to provide a pragmatic method to assess production of Ae. aegypti by drain sumps. A total of 866 adult mosquitoes were trapped from 162 drain sumps over a 48-h exposure period, comprising Ae. aegypti and Cx. quinquefasciatus (21 and 79%, respectively). Advantages of the SEAT are an ability to rapidly count, identify, and sex mosquitoes and to provide specimens for molecular analysis where necessary. The treatment of water-bearing drain sumps is a critical element of control campaigns against Ae. aegypti.     

Susceptibility of laboratory and field-collected Aedes aegypti and Aedes albopictus to Bacillus thuringiensis israelensis H-14

Susceptibility levels of a few laboratory-cultured and dengue-endemic area field-collected strains of Aedes aegypti and Aedes albopictus to Bacillus thuringiensis israelensis (Bti) at different storage ages were studied. The susceptibility of laboratory-cultured World Health Organization (WHO) Bora Bora reference, Vector Control Research Unit (VCRU), and Fumakilla Malaysia Berhad (FMB) strains of Ae. aegypti to Bti was examined. The sensitivity to Bti decreased with storage age. The median lethal concentration (LC50) for Bti increased by 2-3 times after 2 years compared to a fresh sample (3-6 months of storage). However, after the 2-year storage period, Bti still provided very good efficacy against all laboratory-cultured susceptible strains of Ae. aegypti and Ae. albopictus. The observed 95% lethal concentration values were about 20 times lower than the recommended concentration (6,000 international toxic units (ITU)/liter). Results obtained from the study against the dengue-endemic area field-collected strains of Ae. aegypti and Ae. albopictus confirmed the effectiveness of the Bti after storage for 2 years (18-24 months). For Ae. aegypti, the Ujung Batu strain was the most susceptible to Bti, whereas the Sungai Nibong strain showed the most tolerance. Susceptibility of laboratory-cultured strains varied; the Air Itam strain of Ae. albopictus was the most susceptible to Bti, whereas the Kampung Serani strain was the most tolerant among the field strains. However, the laboratory strain of Ae. albopictus was more susceptible than all the field strains.     

Climate and the superimposed distribution of Aedes aegypti and Aedes albopictus on infestation of São Paulo State,Brazil

OBJECTIVE: To study the influence of climatic factors and superimposed distribution of Aedes aegypti and Aedes albopictus populations on their geographic dispersal in the state of S?o Paulo. METHODS: Data from government agencies on Ae. aegypti and Ae. albopictus foci and their distribution throughout counties of the state of S?o Paulo year by year, from 1985 to 1995, were obtained. Temperatures and rainfall data were also collected. Two indicators were used: infestation and rate. RESULTS AND CONCLUSIONS: The analysis indicated that the lower the temperature, the slower the geographic dispersal of Ae. aegypti population. This factor predominantly influenced the macro regional dispersal patterns of the species in the state of S?o Paulo. There was no clear influence of temperature on the dispersal of Ae. albopictus. The influence of rainfall on dispersal patterns was seen only for Ae. aegypti in a state area. No influence was detected on the prior presence of Ae. albopictus on the establishment of Ae. aegypti.     

Larvicidal activity of Tagetes erecta against Aedes aegypti

The aim of this study was to evaluate the activity of essential oil from Tagetes erecta against 3rd instars of Aedes aegypti and to determine the amounts of larvicidal thiophenes in all plant tissues. The oil obtained by steam distillation and analyzed by gas chromatography/mass spectrometry showed 14 compounds. The main compounds were piperitone (45.72%), D-limonene (9.67%), and piperitenone (5.89%). The essential oil was active against larvae of Ae. aegypti, with LC50 of 79.78 microg/ml and LC90 of 100.84 microg/ml. The larvicidal thiophene contents were higher in the roots and flowers as demonstrated by high-performance liquid chromatography analysis. Thus, T. erecta constitutes a good source of varied compounds showing larvicidal activity against Ae. aegypti.     

A comparison of seven traps used for collection of Aedes albopictus and Aedes aegypti originating from a large tire repository in Harris County (Houston), Texas

Among 7 traps tested, significantly higher (P < 0.01) mean numbers of Aedes albopictus (269) and Aedes aegypti (55) females were collected within the Mosquito Magnet Liberty trap compared with the remaining traps. The second highest mean captures for both species were obtained from omnidirectional Fay-Prince (77 Ae. albopictus) and Dragonfly (13 Ae. aegypti) traps, which were not significantly different (P > 0.01) from an experimental moving-target trap that produced mean captures of 40 Ae. albopictus and 6 Ae. aegypti (alpha = 0.01). In terms of Ae. albopictus capture, no significant differences (P > 0.01) existed between Dragonfly, CDC without light (CDC -), and CDC with light (CDC +) captures, which were significantly different (P < 0.01) from Mosquito Deleto. No statistical significance existed between moving-target, omnidirectional, CDC +, CDC -, and Mosquito Deleto traps in terms of Ae. aegypti capture (P > 0.01), individual trap positions, or number of Ae. albopictus and Ae. aegypti females collected throughout the 21-day test (P > 0.05). Mosquito Magnet Liberty collected 7,208 Ae. albopictus, 1467 Ae. aegypti, and 13 other species representing 5 genera, which comprised the largest total (9662) and percentage (62.5%) of mosquitoes collected by all traps combined. Omnidirectional and moving-target traps captured 1941 and 1050 Ae. albopictus, 138 and 220 Ae. aegypti, and 2171 (14.0%) and 1397 (9.0%) of the total mosquitoes captured by all traps, with 8 and 10 species representing 5 genera, respectively, included in these collections. The Dragonfly captured 476 Ae. albopictus, 376 Ae. aegypti, and 1008 total specimens (6.5%) representing 8 species and 4 genera in these collections. CDC + and CDC - traps collected nearly identical numbers of Ae. albopictus (431, 450) and Ae. aegypti (71, 71) with 537 (3.4%) and 551 (3.5%) total specimens, respectively. Eight species representing 5 genera were captured from CDC +, whereas CDC - captured 6 species representing 4 genera. Mosquito Deleto captured 118 mosquitoes, including 19 Ae. albopictus and 62 Ae. aegypti females (0.7%), with 6 species representing 4 genera. Battery-powered traps with contrasting color schemes and movement worked considerably better than stationary CDC miniatures without color or movement. Omnidirectional Fay-Prince and moving-target traps without octenol captured Ae. albopictus and Ae. aegypti females as frequently as some commercial traps. Additionally, costs incurred per mosquito trapped, future trap design, and important consumer-centered issues are briefly discussed.     

Interspecific mating between Louisiana strains of Aedes albopictus and Aedes aegypti in the field and laboratory

Interspecific mating between Aedes albopictus males and Ae. aegypti females was detected in the field using mark-release-recapture techniques. By 3 days after the release of virgin Ae. aegypti females into a field site containing only Ae. albopictus, 100% of the captured females were inseminated. Laboratory investigations indicated that male Ae. albopictus were very proficient at inseminating Ae. aegypti females and that Ae. aegypti males rarely inseminated Ae. albopictus females, especially if Ae. aegypti females were available. Most of the Ae. aegypti females inseminated by Ae. albopictus males contained only small amounts of dead sperm in their spermathecae, while inseminated females from the converse interspecific mating and from intraspecific matings contained only large amounts of live sperm. The results are discussed in relation to the decline in Ae. aegypti densities observed since the introduction of Ae. albopictus into the southern USA.