Development of a quantitative real-time RT-PCR assay that differentiates between Kyasanur Forest disease virus and Alkhurma hemorrhagic fever virus

Kyasanur Forest disease virus (KFDV) and Alkhurma hemorrhagic fever virus (AHFV) are enveloped, positivestranded RNA viruses of clinical importance with complex enzootic life cycles involving hematophagous ticks which feed on small and large mammals. Humans and monkeys are dead-end hosts for these viruses. Recent trends in epidemiological data suggest both virus incidences are steadily increasing and their geographical distribution expanding out of previously known circulation regions. For the detection and discrimination of these two closely related flaviviruses, we have developed quantitative real-time RT-PCR assays with 100 % sensitivity and 100 % specificity for KFDV, 100 % sensitivity and 99.4 % specificity for AHFV as determined using 550 clinical samples collected between 2015-2018 from Western Ghats region of India. This rapid and sensitive assay will enable researchers to accurately diagnose the presence of the virus during viremia in human and animal blood samples, and also from tick specimens. Incorporation of these new tests into a routine diagnosis will help in the diagnosis of KFDV as well as AHFV in the endemic areas and also would provide an early warning of the spread of this virus to newer regions with similar epidemiology.