福建艾滋病病毒的分离和微量全血分离法的建立  

Isolation of HIV - 1 strains in Fujian and the development of microseparation of the whole blood specimens

作　　者：严延生[1] 王惠榕[1] 吴守丽[1] 陈舸[1] 翁育伟[1] 郑兆双[1] 郑健[1] 

机构地区：[1]福建省卫生防疫站,福州市350000

出　　处：《中国性病艾滋病防治》2001年第2期70-73,共4页Chinese Journal of Std & Aids Prevention and Control

基　　金：福建省科技项目(编号:20002164)

摘　　要：目的 从HIV/AIDS病例血液中分离HIV并进行微量全血分离方法的研究。方法 采集20份在福建发现的HIV-1感染者肝素抗凝血,分离外周血单核细胞（PBMC）,与健康人PBMC共培养,使用含神经氨酸酶（NA）的细胞培养液进行HIV-1的分离,建立微量全血分离法,通过检测p24抗原、间接免疫荧光试验（IFA）等确定病原分离结果。用MT4细胞感染试验分析病毒表型,并通过测定病毒分离株 DNA的 C2-V3区的核苷酸序列鉴定亚型。结果 从 20例HIV/AIDS病例 PBMC标本中分离到 18株HIV-1,分离率达90％.其中对HIV感染者的分离率为84.6％（11/13）,对AIDS患者的分离率为100％（7/7）。预刺激和同时刺激PBMC共培养对分离结果没有明显影响,可从10～125μl感染者全血中分离出病毒,与大量法分离比较,结果没有明显差异。18株病毒分离株只有1株可在MT4细胞中稳定传代,其它为M嗜性株;HIV-1A亚型1株,B亚型3株,E亚型13株,E亚型基因离散率为13.724±3.595。结论 添加 NA可能有助于提高 HIV的分离率,微量全血分离法可用于病毒的分离,福建病毒分离株主要为HIV-1E亚型的M嗜性慢低复制株。Objective: To isolate HIV - 1 from the blood specimen of HIV- infected persons, and develop microsepara-tion of the whole blood specimen. Methods: PBMCs from 20 heparinized blood specimens of HIV - 1 infected persons were collected to isolate the virus with PBMC cocultivation by adding neuramindase. The supemantant of cocultures were detected by P24 antigen capture assay and IFA test was also used to identify the isolates. Cell tropism of the HIV- 1 isolates was tested by using MT4 cell lines, and nucleotide sequence analysis of C2 - V3 region of the isolates was done to identify subtypes. Results: Eighteen HIV - 1 isolates were obtained from 20 HIV/AIDS specimens, and the isolation rate could reached 90% , of which the isolation rate from HIV - 1 infected specimens was 84. 6% and that from AIDS patients was 100% . PBMC from healthy person prestimulated with PHA - P or simultaneously stimulated when added to the coculture seemed to have no negative effect on the isolation. Ten to 125μl of the whole blood were enough for the isolation. Of the isolates, only one strain was able to infect MT4 cells successively, and the others showed only a transient infection in MT4. Sequence analysis showed three subtypes in these isolates, that was A(1), B(3)and E(13), except one not being tested. Gene divergence of E subtype was 13.724 ± 3.595. Conclusion: Addition of neuramindase into coculture medium may be useful for raising HIV isolation rate. Small volume of whole blood separation can be used for HIV isolation. Most of HIV- 1 isolates in Fujian are E subtype with characteristics of M tropic and slow/low replication.

关 键 词：HIV-1 病毒分离 艾滋病 微量全血标本 细胞嗜性 亚型鉴定 

分 类 号：R512.91[医药卫生—内科学]