Development and evaluation of an assay for the detection of tick-borne encephalitis virus RNA via real-time PCR with reverse transcription.

BACKGROUND: Tick-borne encephalitis virus (TBEV, Orthoflavivirus encephalitidis) is an arbovirus of the family Flaviviridae. It is the etiological agent of tick-borne encephalitis (TBE), a severe disease affecting the central nervous system. Among arboviral infections, TBE represents the greatest burden in northern Eurasia, both in terms of emerging infection risk and mortality. Globalization and climate change increase the risk of TBEV introduction into nonendemic countries. They may also lead to the emergence of new viral variants featuring increased virulence for humans or altered antigenic characteristics. Hence, sensitive and specific TBEV detection methods are needed not only for diagnostics but also for One Health approach goals (surveillance and identification of viral sources in the environment).

METHODS: Here, we describe a newly developed reverse transcription PCR (RT‒PCR) assay for TBEV detection. The assay was developed and evaluated using armored RNA positive control particles (ARCs). The assay was evaluated using several sample types: (1) a panel of heterologous viral and bacterial RNA/DNA; (2) RNA from TBEV strains isolated in different years in various Russian regions; and (3) RNA from TBEV-positive and TBEV-negative ticks (collected in northwest Russia).

RESULTS: The limit of detection (LOD) of the assay is 103 copies/mL (20 copies/reaction) of TBEV RNA. The developed demonstrated 100% analytical specificity. The assay was compared with the two most commonly used Russian commercial kits for TBEV diagnostics.

CONCLUSIONS: The results indicate that the developed RT‒PCR assay is a reliable and competitive method for the detection of TBEV RNA, which establishes its value as a tool for diagnosing and monitoring the virus.