Propagation and titration of Alkhumra hemorrhagic fever virus in the brains of newborn Wistar rats

• Published in: Journal of virological methods Vol. 199; pp. 39 - 45
• Main Authors: Madani, Tariq A., Kao, Moujahed, Abuelzein, El-Tayeb M.E., Azhar, Esam I., Al-Bar, Hussein M.S., Abu-Araki, Huda, Bokhary, Rana Y., Ksiazek, Thomas G.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2014
• Subjects: Alkhumra hemorrhagic fever virus; Animals; Animals, Newborn; Brain - virology; Encephalitis Viruses, Tick-Borne - growth & development; Encephalitis Viruses, Tick-Borne - isolation & purification; Encephalitis Viruses, Tick-Borne - pathogenicity; Encephalitis, Arbovirus - pathology; Encephalitis, Arbovirus - virology; Female; Humans; Lethal Dose 50; Median rat lethal dose; Propagation; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Viral Load; Virus Cultivation - methods; Wistar rats; PBS; RLD50; Propagation; LLC-MK2; AHFV; TCID50; EMEM; Wistar rats; FITC; IFAT; RT-PCR; FCS; CPE; Median rat lethal dose; ICTV; Alkhumra hemorrhagic fever virus
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2013.12.004

•Alkhumra hemorrhagic fever virus was successfully propagated in the brains of newborn Wistar rats.•The virus caused neurological manifestations followed by death in all inoculated rats.•The median rat lethal dose (RLD50) in the brain suspension was 109.4 RLD50/ml.•The median tissue culture infective dose (TCID50) of the virus in LLC-MK2 was 108.2 TCID50/ml.•Histopathology confirmed meningoencephalitis. 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 109.4 RLD50/ml. The virus titer in LLC-MK2 was 108.2 TCID50/ml. In conclusion, AHFV was propagated successfully to high titers in the brains of newborn Wistar rats.