在蚊子(埃及斑蚊)感染引起的表型测定为登革热感染病例的议定书“

Published 7/04/2007
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Summary

一旦一个基因被确定为潜在的顽固性登革热病毒,它必须进行评估,它在防止病毒感染的蚊子作用。该协议说明登革热的蚊子感染的程度如何,可以检测。成长的文化,膜喂养蚊子人体血液中的病毒,并化验在蚊子中肠病毒滴度的技术演示。

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Das, S., Garver, L., Ramirez, J. R., Xi, Z., Dimopoulos, G. Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination. J. Vis. Exp. (5), e220, doi:10.3791/220 (2007).

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Abstract

这一程序的目的是感染登革热病毒的伊蚊,在实验室条件和研究感染病毒在蚊子的组织水平和动态。该协议是经常用于研究蚊子病毒的相互作用,特别是确定新的宿主因素,能够确定载体能力。在整个实验必须在BSL2实验室进行。类似恶性疟原虫感染,在任何时候都必须佩戴适当的服装,包括手套和白大褂。实验结束后,所接触到的病毒的所有材料需要75%的乙醇,然后再进行正常洗涤漂白处理。需要抛弃他们之前蒸压的所有其他材料。

Protocol

A.传播病毒在C6/36细胞线。

  1. 细胞生长至80%,在75 厘米 2烧瓶汇合;
  2. 删除3-4毫升的容量瓶中剩余的媒体;
  3. 取0.5毫升股票病毒,到上述单元格中添加1.5%细胞的病毒颗粒的多重感染。慢慢地在室温下摇晃15分钟烧瓶。
  4. 5%CO 2和37 ° C孵育45分钟
  5. 添加30毫升的媒体,孵育5天。

B.准备病毒和血液的混合物

  1. 用刮板的细胞分离,细胞和媒体转移到50 ml锥形管;
  2. 离心10分钟;在800克收集上清液,细胞沉淀,但保留上清1毫升;
  3. 冻结上述细胞沉淀在干燥的CO 2,然后在37℃水浴,重复两到三次中解冻;
  4. 800克为10分钟,离心取上清液,并结合从第3步收集上清;
  5. 收集与编制配子体文化感染与恶性疟原虫的按蚊蚊子相同的步骤(步骤3)整个人类的血液;
  6. 结合上述整个人体血液中病毒从第4步上清的平等,并加入人血清(整个体积的10%)。
  7. 30分钟,在37℃水浴孵育上述混合物对人体血液和登革热病毒

C.喂养蚊子

此过程一直在恶性疟原虫在蚊子感染的部分描述几乎是相同的。在第7天,我们检测肠病毒感染,吸血后第14天的唾液感染。在与病毒接触的所有材料的处理,首先用75%乙醇,然后用10%漂白粉。

D.含量的蚊子组织中的病毒滴度

  1. 蚊子组织清扫术前两三天,增长C6/36细胞在24孔板的细胞达到80%,在检测时进行一天的汇合;
  2. 蚊子麻醉在4 ° C时,75%的乙醇浸泡1分钟,无菌牺牲浮出水面。蚊子肠或唾液腺解剖解剖显微镜下用无菌水冲洗,前两次。在此步骤中,所有下面的程序需要在无菌的环境,如生物安全柜中进行。这些组织被转移到管内含有150μL媒体和匀浆一个Kontes颗粒杵电机为90秒;
  3. 10匀浆液加入到990μL媒体1:100稀释;
  4. 使进一步的1:10,1:100和1:1000稀释介质,在96孔板;
  5. 媒体在24孔板中删除,并添加100μL上述四个稀释,每年每个相应的井;
  6. 板块缓慢摇动15分钟,在室温下孵育45分钟,然后在5%,CO 2和37℃;
  7. methycellulose叠加(1%)加1ml到每口井;
  8. 板在37 ° C的5%的CO 2为5天;
  9. 从这里开始的步骤并不需要无菌的环境,但应在任何时候都采取与任何其他的病原体护理。丢弃从每个板块methycellulose覆盖和污点去除多余媒体
  10. 添加甲醇/丙酮固定液(1:1)1ml到​​每口井。在4 ° C为1小时保持;
  11. 倒掉固定液,用1X PBS清洗一次;
  12. 1:1000稀释的5%烂醉的对抗病毒的抗体;
  13. 200μL稀释高免液添加到每个井,37孵育° C为1小时;
  14. 删除高免1 × PBS液和洗板
  15. 准备在5%烂醉的(5G脱脂牛奶100毫升为1 × PBS)1:1000稀释的羊抗鼠共轭(嘉里建设猫#074-1806),然后每孔加入200μl,孵育37℃1小时;
  16. 准备基板如下:1 3' -二氨基terrahydrochloride片剂(西格玛猫#D5905),添加20毫升1 × PBS,溶解后,再加入8μL30%的氢过氧化物酶
  17. 每孔加入200μL上述基板。让我们在室温下站10分钟
  18. 取出的基材,并停止加入蒸馏水1ml到每口井的反应
  19. 倒入水和杂交板干
  20. 计数的病毒粒子

Materials

Name Type Company Catalog Number Comments
Incubator with 5% CO2, 37oC
50 ml conical tubes plastic
human serum and blood O+
pipette tip for tissue culture
pipettman
Pasteur pipetts glass, sterile
water bath 37C
centrifuge
parafilm
circulating water bath
glass membrane feeders with rubber tubing to fit feeders
mosquito cups
75% ethanol
10% bleach
Tissue-culture plates 6-well, 24-well and 96-well plates
Petri dish glass
Slides
fine-tip forceps
PBS 1X, sterile
dissecting microscope Microscope
C6/36 cells cells For virus propagation
C6/36 medium MEM with 10% heat inactivated FBS, 1% L-glutamine and non-essential amino acid and 1% (v/v) Penicillin-Streptomycin.
3’-Diaminobenzidine terrahydrochloride Sigma-Aldrich D5905 1 tablet in 20 ml of 1 X PBS, dissolve and then add 8 μl of 30% hydrogen peroxidase
30% hydrogen peroxidase
A. aegypti Animal mosquito

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Comments

6 Comments

  1. Do you normally culture your Ae. albopictus (C6/36) cells at 37C? Conventionally we culture at around ²8C; what is the reason for keeping yours so warm?

    Reply
    Posted by: Anonymous
    May 15, 2008 - 2:06 PM
  2. Hi KellyI'm from the Dimopoulos Lab - we actually keep the C6/36 cells at 3² C, not 37 C (that was probably a typo). We have found that they grow fine at lower temperatures (²7 C), but we have been getting weird results with plaque assays done at that temperature, so it may affect how the virus replicates in them.RegardsShuzhen

    Reply
    Posted by: Anonymous
    May 15, 2008 - 3:15 PM
  3. I watched with interest your demo. The one step I was waiting to see is missing! You do not show in your video how exactly you remove the methylcellulose overlay and blot excess medium before adding the fixative.

    Reply
    Posted by: Anonymous
    January 16, 2009 - 3:15 PM
  4. Hi, I have a suggestion. After you kill the mosquitŒs with 70% ethanol, picking them up individually with forceps to wash them looks a bit tedious. How about putting them in a cell strainer so you can pick up and wash a bunch together?

    Reply
    Posted by: Anonymous
    May 6, 2010 - 10:47 PM
  5. Where can I get the hyperimmune fluid?

    Reply
    Posted by: Anonymous
    September 9, 2010 - 8:32 PM
  6. Do you guys keep the C6/36 cell in the incubator with CO² or not?

    Reply
    Posted by: Anonymous
    February 10, 2012 - 5:35 PM

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