Tick-borne encephalitis is an important human arbovirus neuroinfection spread across the Northern Eurasia. Inhibitors of tick-borne encephalitis virus (TBEV) strain Absettarov, presumably targeting E protein n-octyl-β-d-glucoside (β-OG) pocket, were reported earlier. In this work, these inhibitors were tested in vitro against seven strains representing three main TBEV subtypes. The most potent compound, 2-[(2-methyl-1-oxido-5,6,7,8-tetrahydroquinazolin-4-yl)amino]-phenol, showed EC50 values lower than 22 µM against all the tested strains. Nevertheless, EC50 values for virus samples of certain strains demonstrated a substantial variation, which appeared to be consistent with the presence of E protein not only in infectious virions, but also in non-infectious and immature virus particles, protein aggregates, and membrane complexes.
Currently the only effective measure against tick-borne encephalitis (TBE) is vaccination. Despite the high efficacy of approved vaccines against TBE, rare cases of vaccine failures are well documented. Both host- and virus-related factors can account for such failures. In this work, we studied the influence of mouse strain and sex and the effects of cyclophosphamide-induced immunosuppression on the efficacy of an inactivated TBE vaccine. We also investigated how an increased proportion of non-infectious particles in the challenge TBE virus would affect the protectivity of the vaccine. The vaccine efficacy was assessed by mortality, morbidity, levels of viral RNA in the brain of surviving mice, and neutralizing antibody (NAb) titers against the vaccine strain and the challenge virus. Two-dose vaccination protected most animals against TBE symptoms and death, and protectivity depended on strain and sex of mice. Immunosuppression decreased the vaccine efficacy in a dose-dependent manner and changed the vaccine-induced NAb spectrum. The vaccination protected mice against TBE virus neuroinvasion and persistence. However, viral RNA was detected in the brain of some asymptomatic animals at 21 and 42 dpi. Challenge with TBE virus enriched with non-infectious particles led to lower NAb titers in vaccinated mice after the challenge but did not affect the protective efficacy.
X-ray imaging of virus particles at European XFEL could eventually allow solving their complete structure, potentially approaching resolution of other structural virology methods. To achieve this ambitious goal with today's technologies, several mL of purified virus suspension containing at least 10 to the 12 particles per mL are required. Such large amounts of concentrated suspension have never before been obtained for enveloped viruses. Tick-borne encephalitis virus (TBEV) represents an attractive model system for the development of enveloped virus purification and concentration protocols, given the availability of large amounts of inactivated virus material provided by vaccine manufacturing facilities. Here we present the development of a TBEV vaccine purification and concentration scheme combined with a quality control protocol allowing substantial amounts of highly concentrated non-aggregated suspension to be obtained. Preliminary single particle imaging experiments were performed for this sample at European XFEL, showing distinct diffraction patterns.
Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus, is common in Europe and Asia and causes a severe disease of the central nervous system. A promising approach in the development of therapy for TBEV infection is the search for small molecule antivirals targeting the flavivirus envelope protein E, particularly its β-n-octyl-d-glucoside binding pocket (β-OG pocket). However, experimental studies of candidate antivirals may be complicated by varying amounts and different forms of the protein E in the virus samples. Viral particles with different conformations and arrangements of the protein E are produced during the replication cycle of flaviviruses, including mature, partially mature, and immature forms, as well as subviral particles lacking genomic RNA. The immature forms are known to be abundant in the viral population. We obtained immature virion preparations of TBEV, characterized them by RT-qPCR, and assessed in vivo and in vitro infectivity of the residual mature virions in the immature virus samples. Analysis of the β-OG pocket structure on the immature virions confirmed the possibility of binding of adamantylmethyl esters of 5-aminoisoxazole-3-carboxylic acid in the pocket. We demonstrated that the antiviral activity of these compounds in plaque reduction assay is significantly reduced in the presence of immature TBEV particles.
Tick-borne encephalitis (TBE) is an emerging zoonosis that may cause long-term neurological sequelae or even death. Thus, there is a growing interest in understanding the factors of TBE pathogenesis. Viral genetic determinants may greatly affect the severity and consequences of TBE. In this study, nonstructural protein 1 (NS1) of the tick-borne encephalitis virus (TBEV) was tested as such a determinant. NS1s of three strains with similar neuroinvasiveness belonging to the European, Siberian and Far-Eastern subtypes of TBEV were studied. Transfection of mouse cells with plasmids encoding NS1 of the three TBEV subtypes led to different levels of NS1 protein accumulation in and secretion from the cells. NS1s of TBEV were able to trigger cytokine production either in isolated mouse splenocytes or in mice after delivery of NS1 encoding plasmids. The profile and dynamics of TNF-α, IL-6, IL-10 and IFN-γ differed between the strains. These results demonstrated the involvement of TBEV NS1 in triggering an immune response and indicated the diversity of NS1 as one of the genetic factors of TBEV pathogenicity.
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