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. 2010 Sep 15;405(1):243-52.
doi: 10.1016/j.virol.2010.06.014. Epub 2010 Jul 1.

The neurovirulence and neuroinvasiveness of chimeric tick-borne encephalitis/dengue virus can be attenuated by introducing defined mutations into the envelope and NS5 protein genes and the 3' non-coding region of the genome

Amber R Engel  1 , Alexander A Rumyantsev, Olga A Maximova, James M Speicher, Brian Heiss, Brian R Murphy, Alexander G Pletnev
Affiliations

The neurovirulence and neuroinvasiveness of chimeric tick-borne encephalitis/dengue virus can be attenuated by introducing defined mutations into the envelope and NS5 protein genes and the 3' non-coding region of the genome

Amber R Engel et al. Virology. .

Abstract

Tick-borne encephalitis (TBE) is a severe disease affecting thousands of people throughout Eurasia. Despite the use of formalin-inactivated vaccines in endemic areas, an increasing incidence of TBE emphasizes the need for an alternative vaccine that will induce a more durable immunity against TBE virus (TBEV). The chimeric attenuated virus vaccine candidate containing the structural protein genes of TBEV on a dengue virus genetic background (TBEV/DEN4) retains a high level of neurovirulence in both mice and monkeys. Therefore, attenuating mutations were introduced into the envelope (E(315)) and NS5 (NS5(654,655)) proteins, and into the 3' non-coding region (Delta30) of TBEV/DEN4. The variant that contained all three mutations (vDelta30/E(315)/NS5(654,655)) was significantly attenuated for neuroinvasiveness and neurovirulence and displayed a reduced level of replication and virus-induced histopathology in the brains of mice. The high level of safety in the central nervous system indicates that vDelta30/E(315)/NS5(654,655) should be further evaluated as a TBEV vaccine.

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Figures

Figure 1
Figure 1
Virus replication kinetics of TBEV/DEN4Δ30 or its derived mutants in the brains of suckling mice. Litters of 5-day-old Swiss mice were inoculated IC with 103 PFU of virus. Brains of at least three mice per group were harvested on odd days post-inoculation and quantitated for virus titers by immunofocus assay on Vero cells; the mean virus replication titers + SE are shown. Asterisks indicate that the vΔ30/E315/NS5654,655 mutant replicates significantly lower than the remaining viruses on the indicated day (unpaired t test or one-way ANOVA followed by Tukey post-hoc test, p< 0.05). The § indicates that only two mouse brains were harvested in the vΔ30/E315 group on day 9, as they were the only remaining animals in the group. The limit of detection was 2.0 log10 PFU/g. Lines end at the day when all animals succumbed to infection, with the exception of day 21 when the experiment was terminated.
Figure 2
Figure 2
Replication of TBEV/DEN4, TBEV/DEN4Δ30, and TBEV/DEN4Δ30 mutant derivatives in the brains of 3-week-old SCID mice following IP inoculation with 105 PFU of virus. On the indicated days, three mouse brains per group were harvested, and the virus titer of each mouse brain homogenate was determined by immunofocus assay on Vero cells. Mean virus titers are indicated + SE. Asterisks indicate that virus replication on indicated day is significantly different from remaining viruses (unpaired t test or one-way ANOVA followed by Tukey post-hoc test, p<0.05). Horizontal dashed line indicates limit of detection (1.7 log10 PFU/g) of the assay.
Figure 3
Figure 3
Neuroinflammation in the brain of mice infected with TBEV/DEN4 (A, D, and G), TBEV/DEN4Δ30 (B, E, and H), or vΔ30/E315/NS5654,655 (C, F, and I). Representative images of neuroinflammation in the brain on day 6 from mice IC inoculated with each virus are shown (from 25 sections per brain of each of three mice; H & E staining). The boxed areas in A – C (x 20) are shown in D – F at higher magnification (x 40); G – I show the boxed areas in D – F at higher magnification (x 100). Inflammatory foci are shown by arrows (D, E, G, and H) and the dashed circle (G). Abbreviations: Cx - cortex; CA1 – hippocampus; DG - dentate gyrus; Th - thalamus; Or - oriens layer of the hippocampus; Py - pyramidal layer of the hippocampus; Rad - radiatum layer of the hippocampus.
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