Department of Medicine, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia.
Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia.
J Virol Methods. 2014 Apr;199:39-45. doi: 10.1016/j.jviromet.2013.12.004. Epub 2014 Jan 18.
Alkhumra hemorrhagic fever virus (AHFV) is a novel flavivirus identified first in Saudi Arabia. In this study, successful propagation of AHFV in the brains of newborn Wistar rats is described and the median rat lethal dose (RLD50) is determined. AHFV-RNA-positive human sera diluted 1:10 were injected intracerebrally into 16, ≤24h old rats. Post-inoculation, the rats were observed daily for 30 days. Brains of moribund rats were tested for AHFV-RNA using RT-PCR and cultured in LLC-MK2 cells. The titer of the isolated virus was determined and expressed in median tissue culture infectious dose (TCID50). To determine the RLD50, AHFV brain suspension was 10-fold diluted serially and each dilution was inoculated in the cerebral hemispheres of 10 rats for a total of 90 rats. Three days post-inoculation, the rats developed tremor, irritability, convulsion, opisthotonus, and spastic paresis starting in the hind limbs and ascending to involve the whole body. All infected rats died within 3-7 days with histopathologically confirmed meningoencephalitis. AHFV-RNA was detected in the brains of all infected rats and the virus titer was 10(9.4) RLD50/ml. The virus titer in LLC-MK2 was 10(8.2) TCID50/ml. In conclusion, AHFV was propagated successfully to high titers in the brains of newborn Wistar rats.
阿尔及利亚出血热病毒（AHFV）是一种新型黄病毒，最初在沙特阿拉伯被发现。本研究描述了 AHFV 在新生 Wistar 大鼠脑中的成功繁殖，并确定了中位大鼠致死剂量（RLD50）。将稀释度为 1:10 的 AHFV 阳性人血清通过脑内途径注射到 16 小时内≤24 小时的新生大鼠中。接种后，每天观察大鼠 30 天。通过 RT-PCR 检测濒死大鼠的大脑中的 AHFV-RNA，并在 LLC-MK2 细胞中培养。测定分离病毒的滴度，并以中位数组织培养感染剂量（TCID50）表示。为了确定 RLD50，将 AHFV 脑悬液进行 10 倍系列稀释，每个稀释度接种于 10 只大鼠的大脑半球，共 90 只大鼠。接种后 3 天，大鼠出现震颤、易怒、抽搐、角弓反张和痉挛性轻瘫，从后肢开始并逐渐累及全身。所有感染的大鼠均在 3-7 天内死亡，组织病理学证实为脑膜脑炎。所有感染的大鼠均检测到 AHFV-RNA，病毒滴度为 10(9.4)RLD50/ml。在 LLC-MK2 中的病毒滴度为 10(8.2)TCID50/ml。总之，AHFV 在新生 Wistar 大鼠的大脑中成功地繁殖到高滴度。
