Serological surveillance of Crimean-Congo Hemorrhagic Fever Virus in animals using targeted Enzyme Immuno Assay

Crimean-Congo hemorrhagic fever (CCHF) is endemic to many countries including Pakistan and remains the second most important disease on the WHO's diseases priority list.With a mortality rate of 10 -40%, there is a dire need to develop indigenous diagnostic methods for surveillance of asymptomatic infections in animals.The current study employed CCHFV Nucleoprotein (NP), for the development of a targeted Enzyme Immuno Assay (EIA) and was tested on 1034 animal serum samples collected throughout the country.The CCHFV-NP was purified using Fast Protein Liquid Chromatography (FPLC) and confirmed through western blotting.In vivo immunogenicity of the purified antigen was tested in BALB/c mice and was subsequently used for establishing a sensitive in-house indirect Enzyme Immuno Assay.CCHFV immunized rabbit serum and CCHFV positive human serum were used as positive controls while uninfected rabbit and human sera and untransformed BL21 lysate were used as negative controls.Finally, the assay was used to test Asymptomatic, domestic and wild animal serum samples collected from all four provinces including CCHFV endemic and non-endemic regions.Our results indicate that from Baluchistan 10% (20/200), Khyber Pakhtunkhwa 9.3% (14/150), Sindh 4% (8/201), Kashmir 2% (1/50) and Punjab 13.6% (55/434) samples were found to be positive.These findings suggest a potential disease risk in the mentioned regions, some of which had not previously reported any human cases or outbreaks.The assay can be further used for screening animals for potential disease risk in the country and to mitigate the required diseases response strategies.