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Significance: Kyasanur forest disease (KFD), a re-emerging tick-borne viral disease, causes severe hemorrhagic fever in humans and nonhuman primates. KFD virus (KFDV) is a member of the genus Flavivirus. KFD is now increasingly reported outside its endemic zone in India. Rapid and specific detection of the KFDV plays a critical role in containment of the outbreak. The diagnosis of KFD currently relies on real-time RT-PCR, nested RT-PCR, end point RT-PCR, and serodiagnostic assay. These assays are tedious, time-consuming, and cannot be used as a routine screening platform. Objective: The present study was aimed to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for molecular diagnosis of KFD. Design: The gene amplification reaction was accomplished by incubation at a constant temperature of 63 °C for 60 min. Results: The limit of detection of RT-LAMP assay was 10 copies. KFD RT-LAMP assay was successfully evaluated with diverse host samples including humans, monkeys, and tick. The assay correctly picked up different KFD isolates indicating its applicability for divergent strains. Comparative evaluation of RT-LAMP assay with quantitative TaqMan real-time RT-PCR revealed 100% concordance. No cross-reaction with related flavi and other hemorrhagic fever viruses was observed, indicating its high specificity. Conclusion and relevance: The RT-LAMP test developed in this study will serve as a rapid, sensitive alternate detection method for KFDV infection and would be useful for high throughput screening of clinical samples in resource limited areas during outbreaks.

Crimean-Congo hemorrhagic fever (CCHF) is an important tick borne zoonotic viral disease of humans. CCHF virus causes sporadic cases of severe illness across a huge geographic area across Africa to Europe to Asia including India. CCHF has emerged as a major public health concern in western Indian states including Gujarat and Rajasthan, where regular human cases were reported since the year 2011. Human serve as the dead-end host, and they gain infection via infected tick bite, in close contact with ruminants and from slaughter house. Currently, the detection of this fatal infection is limited to BSL-4 laboratory which is scarce even in developed economies. Thus, a safe, sensitive assay for early immunodiagnosis is crucial for disease management and containing the outbreak. In this study, the conserved recombinant nucleoprotein was exploited as a safe, scalable alternate antigen for development of indirect IgM and IgG ELISA detection platform. The indirect ELISA was evaluated using suspected clinical samples collected from hotspot areas in India. Comparison with reference MAC ELISA and IgG ELISA revealed a correlation of 95% and 100% respectively. The results indicate that the developed IgM and IgG indirect ELISA has high sensitivity and specificity for detecting CCHFV antibodies among human. These assays are easy to perform and can be employed for high throughput screening of human samples for clinical diagnosis as well as serosurveillance. These assays are also amenable for conversion to low-cost point of care testing formats for application in resource limited settings.