Tick-borne encephalitis (TBE), caused by tick-borne encephalitis virus (TBEV), is the most important tick-borne viral infection of the central nervous system in Europe. TBEV infection can range from an asymptomatic, febrile illness to a severe illness with neurological symptoms, but the reasons for wide range of clinical pictures in individuals are still largely unknown. In addition to host immune factors, pathogenesis is also influenced by the virus, but due to its short and low viremia, it is extremely difficult to obtain viral genome directly from clinical samples. The aim of the study was to analyse the genetic diversity of the European subtype of TBEV in Slovenia and the frequency of mutations in different clinical forms of TBE. We developed specific primers and a protocol for an amplicon sequencing that allows determination of the complete TBEV genome directly in clinical samples. We successfully obtained the complete TBEV genome from 94 blood samples, 3 from cerebrospinal fluid and 6 from urine samples. We found a statistically significant association between more frequent synonymous consensus mutations and a milder TBE course for protein E and found the same trend for protein NS5. The length of the 3'UTR did not differ between mild and severe disease. We validated 7 mutations previously reported in the literature. In addition, we described 105 new mutations specific for each clinical form: 7 specific for the abortive form, 65 specific for meningoencephalitis, and 33 specific for meningoencephalomyelitis. Of these, 92 mutations were synonymous and 9 were nonsynonymous, and 4 were found in untranslated regions. Most mutations were found in the NS5, NS3, and M proteins. This study represents a unique collection of first phase of TBE samples and is the first study to directly investigate the genetic variability of TBEV in clinical samples and discover a potential new virulence markers.
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