Identification of tick-borne encephalitis virus ribonucleic acid in tick suspensions and in clinical specimens by a reverse transcription-nested polymerase chain reaction assay

Background: Tick-borne encephalitis virus (TBEV) is a major human pathogenic flavivirus. Sensitive assays for the detection of viral RNA may be valuable both for the identification of virus in ticks as well as for diagnostic purposes. Objectives: (1) The development of a sensitive polymerase chain reaction (PCR) test system for the detection of TBEV-RNA and its application to the identification of infected ticks; and (2) evaluation of the PCR assay for diagnostic purposes, i.e., detection of TBE virus RNA in blood and in cerebrospinal fluid (CSF) of TBE patients. Study design: (1) Establishment of a TBEV-specific reverse transcription (RT)-nested PCR assay and evaluation of its sensitivity; (2) comparison of the PCR assay with that of virus isolation from tick suspensions; and (3) investigation of 105; serum and CSF samples from patients with serologically confirmed TBE by RT-nested PCR. Results: An RT-nested PCR assay was established with a detection limit of 100-1000 copies of TBEV RNA. All tick suspensions from which the virus could be isolated by inoculation of suckling mice also screened positive in the PCR assay. Of the 105 clinical samples investigated, only one serum and one CSF sample were positive by PCR assay, and these were both obtained very early in the course of the disease. Conclusions: The PCR assay described is valuable for the detection of TBEV in tick suspensions and can substitute for the usual virus isolation procedure in which suckling mice are inoculated. Its application for diagnostic purposes, however, does not seem to provide a significant improvement over serological diagnosis. Only in very rare cases, when a sample is drawn extremely early in the course of disease, may TBEV RNA be detected in serum or CSF before the appearance of specific IgM antibodies and thus allow an earlier diagnosis.