Antigen-capture enzyme-linked immunosorbent assay for detection and quantification of Crimean-Congo hemorrhagic fever virus in the tick, Hyalomma truncatum

Abstract

A viral antigen-capture ELISA was compared to a viral plaque-assay on human cell monolayers for detection and quantification of Crimean-Congo hemorrhagic fever virus in triturated experimentally infected Hyalomma truncatum ticks. In suspensions of ticks exposed as larvae to viremic mice, the ELISA detected 13% positive as compared to 3% (n = 721) positive by plaque-assay. Adult ticks inoculated with virus and sampled up to 102 days later were 84% positive by ELISA compared to 36% (n = 273) positive by plaque-assay. The two tests detected similar proportions of positive ticks in the two weeks immediately after viral inoculation; however, the ELISA was positive in 100% of inoculated adult ticks from 18-102 days post-inoculation while the plaque-assay was positive in 33% (n = 135) of the same specimens. CCHF viral antigen was detected in 10% (n = 101) of first-generation progeny tested as unfed larva pools by ELISA, yet no virus was detected by plaque-assay, indicating that either non-infective viral fragments or very low levels of live virus were detected by ELISA in these tick progeny. As detected by plaque assay, virus inoculated unfed adult ticks were virtually all infected by day 8 post-inoculation; by day 21 post-inoculation only 33% were detected as positive. A cohort of these ticks were allowed to blood feed from day 21-31 post-inoculation. When assayed after feeding all female ticks and nearly 50% of male ticks were detected as virus-positive. This indicates that the virus likely persisted in the unfed ticks below the level of detectability of the plaque-assay and increased in the blood fed ticks up to a detectable level. The ELISA however, detected 100% of ticks as virus-positive from day 14 post-inoculation throughout the remainder of the study, regardless of feeding status (day 102 post-inoculation). These results indicate that antigen-detection ELISA is more sensitive in detecting CCHF virus in ticks than plaque-assay. Since an infected tick remains antigen-positive by ELISA for possibly the remainder of its life, this assay will be a major improvement in field surveys and vector competency studies of ticks for CCHF virus.