Molecular cloning, sequencing, expression and purification of Alkhumra hemorrhagic fever virus capsid protein in Saudi Arabia

Abstract

Introduction: Alkhumra hemorrhagic fever virus (AHFV) is a newly discovered virus in the Flaviviridae family. It was discovered in 1995 among animal handlers in Saudi Arabia. AHFV spreads through close contact with infected animals and tick bites. Symptoms range from fever and flu-like symptoms to hemorrhagic manifestations, and rarely, encephalitis. The Saudi Arabian Ministry of Health has reported 604 cases so far. There are no approved vaccines, antiviral therapies, or routine screening systems for AHFV. This lack of preventive measures makes it challenging to predict future outbreaks or re-emergence in endemic regions of Saudi Arabia.

Methodology: We cloned, sequenced, analyzed, expressed, and purified the recombinant AHFV capsid protein (CP) using the PET-28a (+) vector. The CP gene was amplified through reverse transcriptase polymerase chain reaction (RT-PCR) and cloned into a vector. The expression and purification processes were carried out in E. coli.

Results: The sequence of the CP gene was deposited in GenBank (Accession number OR785375) and designated as AHFV-SIAU-1-KSA. Sequence analysis revealed similarities with other AHFV isolates obtained from humans, animals, and ticks. Phylogenetic analysis showed that the AHFV CP gene formed distinct clusters with other AHFV genomes collected at different time intervals. The expressed protein was successfully purified and analyzed by SDS-PAGE.

Conclusions: This study is the first to document the cloning, expression, and purification of the recombinant AHFV CP gene from Saudi Arabia. The purified protein will be used to develop a serological assay for routine screening of AHFV samples in humans, animals, and ticks in Saudi Arabia.